Background

Bacterial vaginosis (BV) is the most common vaginal disorder of reproductive-age women. Yet the cause of BV has not been established. To uncover key determinants of BV, we employed a multi-omic, systems-biology approach, including both deep 16S rRNA gene-based sequencing and metabolomics of lavage samples from 36 women. These women varied demographically, behaviorally, and in terms of health status and symptoms.

Principal Findings

16S rRNA gene-based community composition profiles reflected Nugent scores, but not Amsel criteria. In contrast, metabolomic profiles were markedly more concordant with Amsel criteria. Metabolomic profiles revealed two distinct symptomatic BV types (SBVI and SBVII) with similar characteristics that indicated disruption of epithelial integrity, but each type was correlated to the presence of different microbial taxa and metabolites, as well as to different host behaviors. The characteristic odor associated with BV was linked to increases in putrescine and cadaverine, which were both linked to Dialister spp. Additional correlations were seen with the presence of discharge, 2-methyl-2-hydroxybutanoic acid, and Mobiluncus spp., and with pain, diethylene glycol and Gardnerella spp.

Conclusions

The results not only provide useful diagnostic biomarkers, but also may ultimately provide much needed insight into the determinants of BV.

BACKGROUND

Observational studies have shown improvement in patients with type 2 diabetes mellitus after bariatric surgery.

METHODS

In this randomized, nonblinded, single-center trial, we evaluated the efficacy of intensive medical therapy alone versus medical therapy plus Roux-en-Y gastric bypass or sleeve gastrectomy in 150 obese patients with uncontrolled type 2 diabetes. The mean (±SD) age of the patients was 49 ± 8 years, and 66% were women. The average glycated hemoglobin level was 9.2 ± 1.5%. The primary end point was the proportion of patients with a glycated hemoglobin level of 6.0% or less 12 months after treatment.

RESULTS

Of the 150 patients, 93% completed 12 months of follow-up. The proportion of patients with the primary end point was 12% (5 of 41 patients) in the medical-therapy group versus 42% (21 of 50 patients) in the gastric-bypass group (P = 0.002) and 37% (18 of 49 patients) in the sleeve-gastrectomy group (P = 0.008). Glycemic control improved in all three groups, with a mean glycated hemoglobin level of 7.5 ± 1.8% in the medical-therapy group, 6.4 ± 0.9% in the gastric-bypass group (P<0.001), and 6.6 ± 1.0% in the sleeve-gastrectomy group (P = 0.003). Weight loss was greater in the gastric-bypass group and sleeve-gastrectomy group (−29.4 ± 9.0 kg and −25.1 ± 8.5 kg, respectively) than in the medical-therapy group (−5.4 ± 8.0 kg) (P<0.001 for both comparisons). The use of drugs to lower glucose, lipid, and blood-pressure levels decreased significantly after both surgical procedures but increased in patients receiving medical therapy only. The index for homeostasis model assessment of insulin resistance (HOMA-IR) improved significantly after bariatric surgery. Four patients underwent reoperation. There were no deaths or life-threatening complications.

CONCLUSIONS

In obese patients with uncontrolled type 2 diabetes, 12 months of medical therapy plus bariatric surgery achieved glycemic control in significantly more patients than medical therapy alone. Further study will be necessary to assess the durability of these results. (Funded by Ethicon Endo-Surgery and others; ClinicalTrials.gov number, NCT00432809.)

Tay Sachs disease (TSD) is a neurodegenerative disorder due to β-hexosaminidase A deficiency caused by mutations in the HEXA gene. The mutations leading to Tay Sachs disease in India are yet unknown. We aimed to determine mutations leading to TSD in India by complete sequencing of the HEXA gene. The clinical inclusion criteria included neuroregression, seizures, exaggerated startle reflex, macrocephaly, cherry red spot on fundus examination and spasticity. Neuroimaging criteria included thalamic hyperdensities on CT scan/T1W images of MRI of the brain. Biochemical criteria included deficiency of hexosaminidase A (less than 2% of total hexosaminidase activity for infantile patients). Total leukocyte hexosaminidase activity was assayed by 4-methylumbelliferyl-N-acetyl-β-D-glucosamine lysis and hexosaminidase A activity was assayed by heat inactivation method and 4-methylumbelliferyl-N-acetyl-β-D-glucosamine-6-sulphate lysis method. The exons and exon-intron boundaries of the HEXA gene were bidirectionally sequenced using an automated sequencer. Mutations were confirmed in parents and looked up in public databases. In silico analysis for mutations was carried out using SIFT, Polyphen2, MutationT@ster and Accelrys Discovery Studio softwares. Fifteen families were included in the study. We identified six novel missense mutations, c.340 G>A (p.E114K), c.964 G>A (p.D322N), c.964 G>T (p.D322Y), c.1178C>G (p.R393P) and c.1385A>T (p.E462V), c.1432 G>A (p.G478R) and two previously reported mutations. c.1277_1278insTATC and c.508C>T (p.R170W). The mutation p.E462V was found in six unrelated families from Gujarat indicating a founder effect. A previously known splice site mutation c.805+1 G>C and another intronic mutation c.672+30 T>G of unknown significance were also identified. Mutations could not be identified in one family. We conclude that TSD patients from Gujarat should be screened for the common mutation p.E462V.

Background. Acral lentiginous melanoma (ALM) is a less-common form of melanoma in US, and it accounts for about 5% of all diagnosed melanomas in US. ALM is often overlooked until it is well advanced because of the lesion's location and its atypical appearance in the early stages. We present a case of ALM initially presented as a diabetic foot ulcer. Case Report. An 81-year-old man initially presented to the primary care clinic with a right foot diabetic ulcer. There was a large plantar, dark-colored ulcer that bled easy. Initial excision biopsy revealed Clark's Level IV ALM. Subsequent definitive wide excision and sentinel node biopsy confirmed ALM with metastasis to inguinal lymph nodes (stage IIIb). The treatment included wide margin excision of the lesion with en bloc amputations of 4th and 5th toes, followed by adjuvant chemotherapy. Discussion. The development of ALM may potentially relate to diabetes as a reported higher prevalence of diabetes with ALM patients. Conclusion. The difficulty in early diagnosing of ALM remains as a formidable challenge particularly in diabetic patients who commonly develop plantar foot ulcers due to the diabetic neuropathy. This case reiterates the importance of a thorough foot exam in such patients.

The assessment of macrophage response to nanoparticles is a central component in the evaluation of new nanoparticle designs for future in vivo application. This work investigates which feature, nanoparticle size or charge, is more predictive of non-specific uptake of nanoparticles by macrophages. This was investigated by synthesizing a library of polymer-coated iron oxide micelles, spanning a range of 30–100 nm in diameter and −23 mV to +9 mV, and measuring internalization into macrophages in vitro. Nanoparticle size and charge both contributed towards non-specific uptake, but within the ranges investigated, size appears to be a more dominant predictor of uptake. Based on these results, a protease-responsive nanoparticle was synthesized, displaying a matrix metalloproteinase-9 (MMP-9)-cleavable polymeric corona. These nanoparticles are able to respond to MMP-9 activity through the shedding of 10–20 nm of hydrodynamic diameter. This MMP-9-triggered decrease in nanoparticle size also led to up to a six-fold decrease in nanoparticle internalization by macrophages and is observable by T2-weighted magnetic resonance imaging. These findings guide the design of imaging or therapeutic nanoparticles for in vivo targeting of macrophage activity in pathologic states.

Background

This is a pilot cross sectional study using both quantitative and qualitative approach towards tutors teaching large classes in private universities in the Klang Valley (comprising Kuala Lumpur, its suburbs, adjoining towns in the State of Selangor) and the State of Negeri Sembilan, Malaysia. The general aim of this study is to determine the difficulties faced by tutors when teaching large group of students and to outline appropriate recommendations in overcoming them.

Findings

Thirty-two academics from six private universities from different faculties such as Medical Sciences, Business, Information Technology, and Engineering disciplines participated in this study. SPSS software was used to analyse the data. The results in general indicate that the conventional instructor-student approach has its shortcoming and requires changes. Interestingly, tutors from Medicine and IT less often faced difficulties and had positive experience in teaching large group of students.

Conclusion

However several suggestions were proposed to overcome these difficulties ranging from breaking into smaller classes, adopting innovative teaching, use of interactive learning methods incorporating interactive assessment and creative technology which enhanced students learning. Furthermore the study provides insights on the trials of large group teaching which are clearly identified to help tutors realise its impact on teaching. The suggestions to overcome these difficulties and to maximize student learning can serve as a guideline for tutors who face these challenges.

Background

We earlier reported a simple specific test for detection of anti-ovarian antibodies in infertile women and identified number of specific molecular and cellular targets of which human heat shock protein 90-beta (HSP90 beta) was found to be the most immunodominant. The present study focuses on prediction and validation of the immunodominant epitope/s of this protein using sera from infertile women having anti-HSP90 autoantibodies.

Methods

Delineation of the immunodominant epitopes of HSP90 beta was done by using epitope prediction algorithms and 10 peptides (EP1-EP10) were custom synthesized. Their immunoreactivity was measured by ELISA using sera from patients and controls. To determine the most immunodominant epitope, the results were subjected to statistical analysis. The immunoreactivity of the immunodominant peptides were confirmed by dot blots using sera from patients. A rabbit polyclonal antibody against the immunodominant epitope was generated and its immunoreactivity to the parent protein in ovarian extracts as well in oocytes and embryos was investigated.

Results

Experimentally and statistically, peptide EP6 (380-389) seems to be the major antigenic epitope for the serum antibody binding followed by EP1 (1-12) and EP8 (488-498). Predicted 3D structures of these peptides demonstrated that they exist in the loop conformation which is the most mobile part of the protein. Also, analysis of the sequences of HSP90 beta across several species reveals that EP6 peptide forms a part of a well conserved motif. The polyclonal antibody generated to the immunodominant epitope- EP6 confirms similar biochemical and cellular immunoreactivity as seen with the patients' sera having anti-HSP90 autoantibodies.

Conclusions

The decapeptide EP6 is a major immunogenic epitope of HSP90 followed by EP1 and EP8. Knowledge of binding epitopes on the autoantigen is necessary to understand the subsequent pathologic events. The study might generate new tools for the detection of disease-inducing epitopes and a possible therapeutic intervention.

Background

Gardnerella vaginalis is described as a common vaginal bacterial species whose presence correlates strongly with bacterial vaginosis (BV). Here we report the genome sequencing and comparative analyses of three strains of G. vaginalis. Strains 317 (ATCC 14019) and 594 (ATCC 14018) were isolated from the vaginal tracts of women with symptomatic BV, while Strain 409-05 was isolated from a healthy, asymptomatic individual with a Nugent score of 9.

Principal Findings

Substantial genomic rearrangement and heterogeneity were observed that appeared to have resulted from both mobile elements and substantial lateral gene transfer. These genomic differences translated to differences in metabolic potential. All strains are equipped with significant virulence potential, including genes encoding the previously described vaginolysin, pili for cytoadhesion, EPS biosynthetic genes for biofilm formation, and antimicrobial resistance systems, We also observed systems promoting multi-drug and lantibiotic extrusion. All G. vaginalis strains possess a large number of genes that may enhance their ability to compete with and exclude other vaginal colonists. These include up to six toxin-antitoxin systems and up to nine additional antitoxins lacking cognate toxins, several of which are clustered within each genome. All strains encode bacteriocidal toxins, including two lysozyme-like toxins produced uniquely by strain 409-05. Interestingly, the BV isolates encode numerous proteins not found in strain 409-05 that likely increase their pathogenic potential. These include enzymes enabling mucin degradation, a trait previously described to strongly correlate with BV, although commonly attributed to non-G. vaginalis species.

Conclusions

Collectively, our results indicate that all three strains are able to thrive in vaginal environments, and therein the BV isolates are capable of occupying a niche that is unique from 409-05. Each strain has significant virulence potential, although genomic and metabolic differences, such as the ability to degrade mucin, indicate that the detection of G. vaginalis in the vaginal tract provides only partial information on the physiological potential of the organism.

Background

Chronic lung infection with the bacterium Pseudomonas aeruginosa is one of the hallmarks of cystic fibrosis (CF) and is associated with worsening lung function, increased hospitalisation and reduced life expectancy. A virulent clonal strain of P. aeruginosa (Australian epidemic strain I; AES-I) has been found to be widespread in CF patients in eastern Australia.

Methods

Suppression subtractive hybridization (SSH) was employed to identify genetic sequences that are present in the AES-I strain but absent from the sequenced reference strain PAO1. We used PCR to evaluate the distribution of several of the AES-I loci amongst a collection of 188 P. aeruginosa isolates which was comprised of 35 AES-I isolates (as determined by PFGE), 78 non-AES-I CF isolates including other epidemic CF strains as well as 69 P. aeruginosa isolates from other clinical and environmental sources.

Results

We have identified a unique AES-I genetic locus that is present in all 35 AES-I isolates tested and not present in any of the other 153 P. aeruginosa strains examined. We have used this unique AES-I locus to develop a diagnostic PCR and a real-time PCR assay to detect the presence of P. aeruginosa and AES-I in patient sputum samples.

Conclusions

We have developed diagnostic PCR assays that are 100% sensitive and 100% specific for the P. aeruginosa strain AES-I. We have also shown that Whatman FTA® Elute cards may be used with PCR-based assays to rapidly detect the presence of P. aeruginosa strains in CF sputum.

Background

The identification of estrogen receptors alpha and beta and aromatase in the testis has highlighted the important role of estrogens in regulating spermatogenesis. There is a wealth of information on the deleterious effects of fetal and neonatal exposure of estrogens and xenoestrogens in the testis, including spermiation failure and germ cell apoptosis. However, very little is known about gene transcripts affected by exogenous estradiol exposure in the testis. The objective of the present study was to unveil global gene expression profiles and testicular cell number changes in rats after estradiol treatment.

Methods

17beta-estradiol was administered to adult male rats at a dose of 100 micrograms/kg body weight in saline daily for 10 days; male rats receiving only saline were used as controls. Microarray analysis was performed to examine global gene expression profiles with or without estradiol treatment. Real time RT-PCR was conducted to verify the microarray data. In silico promoter and estrogen responsive elements (EREs) analysis was carried out for the differentially expressed genes in response to estradiol. Quantitation of testicular cell number based on ploidy was also performed using flow cytometry in rats with or without estradiol treatment.

Results

We found that 221 genes and expressed sequence tags (ESTs) were differentially expressed in rat testes treated with estradiol compared to the control; the microarray data were confirmed by real time RT-PCR. Gene Ontology analysis revealed that a number of the differentially expressed genes are involved in androgen and xenobiotic metabolism, maintenance of cell cytoskeleton, endocytosis, and germ cell apoptosis. A total of 33 up-regulated genes and 67 down-regulated genes showed the presence of EREs. Flow cytometry showed that estradiol induced a significant decrease in 2n cells (somatic and germ cells) and 4n cells (pachytene spermatocytes) and a marked increase in the number of elongated and elongating spermatids.

Conclusions

This study provides a novel insight into the molecular basis for spermiation failure and apoptosis caused by 17beta-estradiol and it also offers new mechanisms by which adult exposure to environmental estrogens can affect spermatogenesis and fertility.

Antimicrobial peptides (AMPs) are gaining popularity as better substitute to antibiotics. These peptides are shown to be active against several bacteria, fungi, viruses, protozoa and cancerous cells. Understanding the role of primary structure of AMPs in their specificity and activity is essential for their rational design as drugs. Collection of Anti-Microbial Peptides (CAMP) is a free online database that has been developed for advancement of the present understanding on antimicrobial peptides. It is manually curated and currently holds 3782 antimicrobial sequences. These sequences are divided into experimentally validated (patents and non-patents: 2766) and predicted (1016) datasets based on their reference literature. Information like source organism, activity (MIC values), reference literature, target and non-target organisms of AMPs are captured in the database. The experimentally validated dataset has been further used to develop prediction tools for AMPs based on the machine learning algorithms like Random Forests (RF), Support Vector Machines (SVM) and Discriminant Analysis (DA). The prediction models gave accuracies of 93.2% (RF), 91.5% (SVM) and 87.5% (DA) on the test datasets. The prediction and sequence analysis tools, including BLAST, are integrated in the database. CAMP will be a useful database for study of sequence-activity and -specificity relationships in AMPs. CAMP is freely available at http://www.bicnirrh.res.in/antimicrobial.

Recent culture-independent studies have revealed that a healthy vaginal ecosystem harbors a surprisingly complex assemblage of microorganisms. However, the spatial distribution and composition of vaginal microbial populations have not been investigated using molecular methods. Here, we evaluated site-specific microbial composition within the vaginal ecosystem and examined the influence of sampling technique in detection of the vaginal microbiota. 16S rRNA gene clone libraries were prepared from samples obtained from different locations (cervix, fornix, outer vaginal canal) and by different methods (swabbing, scraping, lavaging) from the vaginal tracts of eight clinically healthy, asymptomatic women. The data reveal that the vaginal microbiota is not homogenous throughout the vaginal tract but differs significantly within an individual with regard to anatomical site and sampling method used. Thus, this study illuminates the complex structure of the vaginal ecosystem and calls for the consideration of microenvironments when sampling vaginal microbiota as a clinical predictor of vaginal health.

Background

Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia (CAH). Detection of underlying mutations in CYP21A2 gene encoding steroid 21-hydroxylase enzyme is helpful both for confirmation of diagnosis and management of CAH patients. Here we report a novel 9-bp insertion in CYP21A2 gene and its structural and functional consequences on P450c21 protein by molecular modeling and molecular dynamics simulations methods.

Methods

A 30-day-old child was referred to our laboratory for molecular diagnosis of CAH. Sequencing of the entire CYP21A2 gene revealed a novel insertion (duplication) of 9-bp in exon 2 of one allele and a well-known mutation I172N in exon 4 of other allele. Molecular modeling and simulation studies were carried out to understand the plausible structural and functional implications caused by the novel mutation.

Results

Insertion of the nine bases in exon 2 resulted in addition of three valine residues at codon 71 of the P450c21 protein. Molecular dynamics simulations revealed that the mutant exhibits a faster unfolding kinetics and an overall destabilization of the structure due to the triple valine insertion was also observed.

Conclusion

The novel 9-bp insertion in exon 2 of CYP21A2 genesignificantly lowers the structural stability of P450c21 thereby leading to the probable loss of its function.

Objectives

To test the hypothesis that in comparison with those with shorter risk duration, individuals with longer HIV risk duration would have reduced susceptibility to HIV-1 infection as measured by CCR5 expression, and to evaluate whether variation in CCR5 expression could be explained by known genetic polymorphisms.

Design and methods

A cross-sectional study of HIV-1 exposed but uninfected men who have sex with men. The risk duration was estimated from self-reported years since first receptive anal intercourse. CCR5 expression on peripheral blood CD4+ monocytes and T cells was determined by flow cytometry. The CCR5-Δ32 mutation and polymorphisms in the CCR5 promoter and CCR2 as well as the copy number of CCL3L1 were analyzed by polymerase chain reaction. Plasma levels of MIP-1α (CCL3), MIP-1β (CCL4) and RANTES (CCL5) were also measured. As risk duration varied with age, analyses were restricted to 67 individuals aged 30–49 years.

Results

Multiple linear regression analyses, adjusted for age and race, showed a significant negative association between HIV risk duration and CCR5 expression on monocytes (P = 0.01), and in a separate model, a similar negative association with CCR5 expression on T cells (P = 0.03). Low CCR5 expression was attributable mainly to CCR5-Δ32 heterozygosity and the CCR5-59029G allele.

Conclusions

We confirmed a role for reduced CCR5 expression in HIV-1 resistance. CCR5-Δ32 heterozygosity and the CCR5-59029G allele were significant predictors of low CCR5 expression. Individuals with high CCR5 expression who resisted infection despite long HIV risk duration form an interesting group within which to search for additional mechanisms of resistance to HIV infection.

Balarishta and Dhanvantara gutika are ayurvedic medicines prescribed in different diseases including rheumatism. These medicines were screened for anti-inflammatory activity against cotton pellet induced granuloma in albino rats. There was significant reduction in cotton pellet weight by both the tested drugs. Dhanvantara gutika significantly reduced the ascorbic acid in adrenal. Acid phosphatase, GPT and GOT activities were significantly reduced by Balarishta, Dhanvantara gutika and phenyl butazone in liver. In the serum acid phosphatase activity was significantly reduced by both the tested drugs and phenul butazone while GPT activity was lowered by Balarishta alone and GOT activity was reduced by Balarishta and Dhanvantara gutika. Phenyl butazgone reduced the activity of GPT.

Microtubules are dynamic structures whose proper rearrangement during the cell cycle is essential for the positioning of membranes during interphase and for chromosome segregation during mitosis. The previous discovery of a cyclin B/cdc2-activated microtubule-severing activity in M-phase Xenopus egg extracts suggested that a microtubule-severing protein might play an important role in cell cycle-dependent changes in microtubule dynamics and organization. However, the isolation of three different microtubule-severing proteins, p56, EF1α, and katanin, has only confused the issue because none of these proteins is directly activated by cyclin B/cdc2. Here we use immunodepletion with antibodies specific for a vertebrate katanin homologue to demonstrate that katanin is responsible for the majority of M-phase severing activity in Xenopus eggs. This result suggests that katanin is responsible for changes in microtubules occurring at mitosis. Immunofluorescence analysis demonstrated that katanin is concentrated at a microtubule-dependent structure at mitotic spindle poles in Xenopus A6 cells and in human fibroblasts, suggesting a specific role in microtubule disassembly at spindle poles. Surprisingly, katanin was also found in adult mouse brain, indicating that katanin may have other functions distinct from its mitotic role.