PLOS ONE Alerts: New Articles

Leuconostoc Mesenteroides Growth in Food Products: Prediction and Sensitivity Analysis by Adaptive-Network-Based Fuzzy Inference Systems

2013-05-21T21:00:00Z

by Hue-Yu Wang, Ching-Feng Wen, Yu-Hsien Chiu, I-Nong Lee, Hao-Yun Kao, I-Chen Lee, Wen-Hsien Ho

Background

An adaptive-network-based fuzzy inference system (ANFIS) was compared with an artificial neural network (ANN) in terms of accuracy in predicting the combined effects of temperature (10.5 to 24.5°C), pH level (5.5 to 7.5), sodium chloride level (0.25% to 6.25%) and sodium nitrite level (0 to 200 ppm) on the growth rate of Leuconostoc mesenteroides under aerobic and anaerobic conditions.

Methods

The ANFIS and ANN models were compared in terms of six statistical indices calculated by comparing their prediction results with actual data: mean absolute percentage error (MAPE), root mean square error (RMSE), standard error of prediction percentage (SEP), bias factor (B_f), accuracy factor (A_f), and absolute fraction of variance (R²). Graphical plots were also used for model comparison.

Conclusions

The learning-based systems obtained encouraging prediction results. Sensitivity analyses of the four environmental factors showed that temperature and, to a lesser extent, NaCl had the most influence on accuracy in predicting the growth rate of Leuconostoc mesenteroides under aerobic and anaerobic conditions. The observed effectiveness of ANFIS for modeling microbial kinetic parameters confirms its potential use as a supplemental tool in predictive mycology. Comparisons between growth rates predicted by ANFIS and actual experimental data also confirmed the high accuracy of the Gaussian membership function in ANFIS. Comparisons of the six statistical indices under both aerobic and anaerobic conditions also showed that the ANFIS model was better than all ANN models in predicting the four kinetic parameters. Therefore, the ANFIS model is a valuable tool for quickly predicting the growth rate of Leuconostoc mesenteroides under aerobic and anaerobic conditions.

Evaluation and Correction for Optical Scattering Variations in Laser Speckle Rheology of Biological Fluids

2013-05-21T21:00:00Z

by Zeinab Hajjarian, Seemantini K. Nadkarni

Biological fluids fulfill key functionalities such as hydrating, protecting, and nourishing cells and tissues in various organ systems. They are capable of these versatile tasks owing to their distinct structural and viscoelastic properties. Characterizing the viscoelastic properties of bio-fluids is of pivotal importance for monitoring the development of certain pathologies as well as engineering synthetic replacements. Laser Speckle Rheology (LSR) is a novel optical technology that enables mechanical evaluation of tissue. In LSR, a coherent laser beam illuminates the tissue and temporal speckle intensity fluctuations are analyzed to evaluate mechanical properties. The rate of temporal speckle fluctuations is, however, influenced by both optical and mechanical properties of tissue. Therefore, in this paper, we develop and validate an approach to estimate and compensate for the contributions of light scattering to speckle dynamics and demonstrate the capability of LSR for the accurate extraction of viscoelastic moduli in phantom samples and biological fluids of varying optical and mechanical properties.

SigB Is a Dominant Regulator of Virulence in Staphylococcus aureus Small-Colony Variants

2013-05-21T21:00:00Z

by Gabriel Mitchell, Alexandre Fugère, Karine Pépin Gaudreau, Eric Brouillette, Eric H. Frost, André M. Cantin, François Malouin

Staphylococcus aureus small-colony variants (SCVs) are persistent pathogenic bacteria characterized by slow growth and, for many of these strains, an increased ability to form biofilms and to persist within host cells. The virulence-associated gene expression profile of SCVs clearly differs from that of prototypical strains and is often influenced by SigB rather than by the agr system. One objective of this work was to confirm the role of SigB in the control of the expression of virulence factors involved in biofilm formation and intracellular persistence of SCVs. This study shows that extracellular proteins are involved in the formation of biofilm by three SCV strains, which, additionally, have a low biofilm-dispersing activity. It was determined that SigB activity modulates biofilm formation by strain SCV CF07-S and is dominant over that of the agr system without being solely responsible for the repression of proteolytic activity. On the other hand, the expression of fnbA and the control of nuclease activity contributed to the SigB-dependent formation of biofilm of this SCV strain. SigB was also required for the replication of CF07-S within epithelial cells and may be involved in the colonization of lungs by SCVs in a mouse infection model. This study methodically investigated SigB activity and associated mechanisms in the various aspects of SCV pathogenesis. Results confirm that SigB activity importantly influences the production of virulence factors, biofilm formation and intracellular persistence for some clinical SCV strains.

Phase Locking Asymmetries at Flexor-Extensor Transitions during Fictive Locomotion

2013-05-21T21:00:00Z

by David L. Boothe, Avis H. Cohen, Todd W. Troyer

The motor output for walking is produced by a network of neurons termed the spinal central pattern generator (CPG) for locomotion. The basic building block of this CPG is a half-center oscillator composed of two mutually inhibitory sets of interneurons, each controlling one of the two dominant phases of locomotion: flexion and extension. To investigate symmetry between the two components of this oscillator, we analyzed the statistics of natural variation in timing during fictive locomotion induced by stimulation of the midbrain locomotor region in the cat. As a complement to previously published analysis of these data focused on burst and cycle durations, we present a new analysis examining the strength of phase locking at the transitions between flexion and extension. Across our sample of nerve pairs, phase locking at the transition from extension to flexion (E to F) is stronger than at the transition from flexion to extension (F to E). This pattern did not reverse when considering bouts of fictive locomotion that were flexor vs. extensor dominated, demonstrating that asymmetric locking at the transitions between phases is dissociable from which phase dominates cycle duration. We also find that the strength of phase locking is correlated with the mean latency between burst offset and burst onset. These results are interpreted in the context of a hypothesis where network inhibition and intrinsic oscillatory mechanisms make distinct contributions to flexor-extensor alternation in half-center networks.

Investigation of Specificity Determinants in Bacterial tRNA-Guanine Transglycosylase Reveals Queuine, the Substrate of Its Eucaryotic Counterpart, as Inhibitor

2013-05-21T21:00:00Z

by Inna Biela, Naomi Tidten-Luksch, Florian Immekus, Serghei Glinca, Tran Xuan Phong Nguyen, Hans-Dieter Gerber, Andreas Heine, Gerhard Klebe, Klaus Reuter

Bacterial tRNA-guanine transglycosylase (Tgt) catalyses the exchange of the genetically encoded guanine at the wobble position of tRNAs^{His,Tyr,Asp,Asn} by the premodified base preQ₁, which is further converted to queuine at the tRNA level. As eucaryotes are not able to synthesise queuine de novo but acquire it through their diet, eucaryotic Tgt directly inserts the hypermodified base into the wobble position of the tRNAs mentioned above. Bacterial Tgt is required for the efficient pathogenicity of Shigella sp, the causative agent of bacillary dysentery and, hence, it constitutes a putative target for the rational design of anti-Shigellosis compounds. Since mammalian Tgt is known to be indirectly essential to the conversion of phenylalanine to tyrosine, it is necessary to create substances which only inhibit bacterial but not eucaryotic Tgt. Therefore, it seems of utmost importance to study selectivity-determining features within both types of proteins. Homology models of Caenorhabditis elegans Tgt and human Tgt suggest that the replacement of Cys158 and Val233 in bacterial Tgt (Zymomonas mobilis Tgt numbering) by valine and accordingly glycine in eucaryotic Tgt largely accounts for the different substrate specificities. In the present study we have created mutated variants of Z. mobilis Tgt in order to investigate the impact of a Cys158Val and a Val233Gly exchange on catalytic activity and substrate specificity. Using enzyme kinetics and X-ray crystallography, we gained evidence that the Cys158Val mutation reduces the affinity to preQ₁ while leaving the affinity to guanine unaffected. The Val233Gly exchange leads to an enlarged substrate binding pocket, that is necessary to accommodate queuine in a conformation compatible with the intermediately covalently bound tRNA molecule. Contrary to our expectations, we found that a priori queuine is recognised by the binding pocket of bacterial Tgt without, however, being used as a substrate.

Characterization of Neonatal Vocal and Motor Repertoire of Reelin Mutant Mice

2013-05-21T21:00:00Z

by Emilia Romano, Caterina Michetti, Angela Caruso, Giovanni Laviola, Maria Luisa Scattoni

Reelin is a large secreted extracellular matrix glycoprotein playing an important role in early neurodevelopment. Several genetic studies found an association between RELN gene and increased risk of autism suggesting that reelin deficiency may be a vulnerability factor in its etiology. Moreover, a reduced reelin expression has been observed in several brain regions of subjects with Autism Spectrum Disorders. Since a number of reports have documented presence of vocal and neuromotor abnormalities in patients with autism and suggested that these dysfunctions predate the onset of the syndrome, we performed a fine-grain characterization of the neonatal vocal and motor repertoire in reelin mutant mice to explore the developmental precursors of the disorder. Our findings evidence a general delay in motor and vocal development in heterozygous (50% reduced reelin) and reeler (lacking reelin gene) mutant mice. As a whole, an increased number of calls characterized heterozygous pup's emission. Furthermore, the typical ontogenetic peak in the number of calls characterizing wild-type pups on postnatal day 4 appeared slightly delayed in heterozygous pups (to day 6) and was quite absent in reeler littermates, which exhibited a flat profile during development. We also detected a preferential use of a specific call category (two-components) by heterozygous and reeler mice at postnatal days 6 and 8 as compared to their wild-type littermates. With regard to the analysis of spontaneous movements, a differential profile emerged early in development among the three genotypes. While only slight coordination difficulties are exhibited by heterozygous pups, all indices of motor development appear delayed in reeler mice. Overall, our results evidence a genotype-dependent deviation in ultrasonic vocal repertoire and a general delay in motor development in reelin mutant pups.

Lognormal Infection Times of Online Information Spread

2013-05-21T21:00:00Z

by Christian Doerr, Norbert Blenn, Piet Van Mieghem

The infection times of individuals in online information spread such as the inter-arrival time of Twitter messages or the propagation time of news stories on a social media site can be explained through a convolution of lognormally distributed observation and reaction times of the individual participants. Experimental measurements support the lognormal shape of the individual contributing processes, and have resemblance to previously reported lognormal distributions of human behavior and contagious processes.

Manipulation of BDNF Signaling Modifies the Experience-Dependent Plasticity Induced by Pure Tone Exposure during the Critical Period in the Primary Auditory Cortex

2013-05-21T21:00:00Z

by Renata Anomal, Etienne de Villers-Sidani, Michael M. Merzenich, Rogerio Panizzutti

Sensory experience powerfully shapes cortical sensory representations during an early developmental “critical period” of plasticity. In the rat primary auditory cortex (A1), the experience-dependent plasticity is exemplified by significant, long-lasting distortions in frequency representation after mere exposure to repetitive frequencies during the second week of life. In the visual system, the normal unfolding of critical period plasticity is strongly dependent on the elaboration of brain-derived neurotrophic factor (BDNF), which promotes the establishment of inhibition. Here, we tested the hypothesis that BDNF signaling plays a role in the experience-dependent plasticity induced by pure tone exposure during the critical period in the primary auditory cortex. Elvax resin implants filled with either a blocking antibody against BDNF or the BDNF protein were placed on the A1 of rat pups throughout the critical period window. These pups were then exposed to 7 kHz pure tone for 7 consecutive days and their frequency representations were mapped. BDNF blockade completely prevented the shaping of cortical tuning by experience and resulted in poor overall frequency tuning in A1. By contrast, BDNF infusion on the developing A1 amplified the effect of 7 kHz tone exposure compared to control. These results indicate that BDNF signaling participates in the experience-dependent plasticity induced by pure tone exposure during the critical period in A1.

A Novel Monoclonal Anti-CD81 Antibody Produced by Genetic Immunization Efficiently Inhibits Hepatitis C Virus Cell-Cell Transmission

2013-05-21T21:00:00Z

by Isabel Fofana, Fei Xiao, Christine Thumann, Marine Turek, Laetitia Zona, Rajiv G. Tawar, Fritz Grunert, John Thompson, Mirjam B. Zeisel, Thomas F. Baumert

Background and Aims

Hepatitis C virus (HCV) infection is a challenge to prevent and treat because of the rapid development of drug resistance and escape. Viral entry is required for initiation, spread, and maintenance of infection, making it an attractive target for antiviral strategies.

Methods

Using genetic immunization, we produced four monoclonal antibodies (mAbs) against the HCV host entry factor CD81. The effects of antibodies on inhibition of HCV infection and dissemination were analyzed in HCV permissive human liver cell lines.

Results

The anti-CD81 mAbs efficiently inhibited infection by HCV of different genotypes as well as a HCV escape variant selected during liver transplantation and re-infecting the liver graft. Kinetic studies indicated that anti-CD81 mAbs target a post-binding step during HCV entry. In addition to inhibiting cell-free HCV infection, one antibody was also able to block neutralizing antibody-resistant HCV cell-cell transmission and viral dissemination without displaying any detectable toxicity.

Conclusion

A novel anti-CD81 mAb generated by genetic immunization efficiently blocks HCV spread and dissemination. This antibody will be useful to further unravel the role of virus-host interactions during HCV entry and cell-cell transmission. Furthermore, this antibody may be of interest for the development of antivirals for prevention and treatment of HCV infection.

EGFR, FLT1 and Heparanase as Markers Identifying Patients at Risk of Short Survival in Cholangiocarcinoma

2013-05-21T21:00:00Z

by Andreas-Claudius Hoffmann, Eray Goekkurt, Peter V. Danenberg, Sylvia Lehmann, Gerhard Ehninger, Daniela E. Aust, Jan Stoehlmacher-Williams

Background

Cholangiocarcinoma remains to be a tumor with very few treatment choices and limited prognosis. In this study, we sought to determine the prognostic role of fms-related tyrosine kinase 1/vascular endothelial growth factor receptor 1 (FLT1/VEGFR1), heparanase (HPSE) and epidermal growth factor receptor (EGFR) gene expression in patients with resected CCC.

Methods

47 formalin-fixed paraffin embedded FFPE tumor samples from patients with resected CCC were analyzed. FFPE tissues were dissected using laser-captured microdissection and analyzed for FLT1, FLT4, HPSE, Hif1a, VEGFA/C, HB-EGF, PDGFA, PDGF-RA and EGFR mRNA expression using a quantitative real-time RT-PCR method. Gene expression values (relative mRNA levels) are expressed as ratios between the target gene and internal reference genes (beta-actin, b2mg, rplp2, sdha).

Results

EGFR, FLT1 and HPSE expression levels were significantly associated with overall survival (OS). FLT1 showed the strongest significant independent association with overall survival in a multivariate cox regression analysis when compared to the other genes and clinicopathological factors with a nearly 5 times higher relative risk (4.74) of dying earlier when expressed in low levels (p = 0.04). ROC Curve Analysis revealed that measuring EGFR potentially identifies patients at risk of a worsened outcome with a sensitivity of 80% and a specificity of 75% (p = 0.01).

Conclusions

EGFR and FLT1 seem to be potential markers to identify those patients at high risk of dying from cholangiocarcinoma. Therefore these markers may help to identify patient subgroups in need for a more aggressive approach in a disease that is in desperate need for new approaches.

Targeted Deep Resequencing Identifies Coding Variants in the PEAR1 Gene That Play a Role in Platelet Aggregation

2013-05-21T21:00:00Z

by Yoonhee Kim, Bhoom Suktitipat, Lisa R. Yanek, Nauder Faraday, Alexander F. Wilson, Diane M. Becker, Lewis C. Becker, Rasika A. Mathias

Platelet aggregation is heritable, and genome-wide association studies have detected strong associations with a common intronic variant of the platelet endothelial aggregation receptor1 (PEAR1) gene both in African American and European American individuals. In this study, we used a sequencing approach to identify additional exonic variants in PEAR1 that may also determine variability in platelet aggregation in the GeneSTAR Study. A 0.3 Mb targeted region on chromosome 1q23.1 including the entire PEAR1 gene was Sanger sequenced in 104 subjects (45% male, 49% African American, age = 52±13) selected on the basis of hyper- and hypo- aggregation across three different agonists (collagen, epinephrine, and adenosine diphosphate). Single-variant and multi-variant burden tests for association were performed. Of the 235 variants identified through sequencing, 61 were novel, and three of these were missense variants. More rare variants (MAF<5%) were noted in African Americans compared to European Americans (108 vs. 45). The common intronic GWAS-identified variant (rs12041331) demonstrated the most significant association signal in African Americans (p = 4.020×10⁻⁴); no association was seen for additional exonic variants in this group. In contrast, multi-variant burden tests indicated that exonic variants play a more significant role in European Americans (p = 0.0099 for the collective coding variants compared to p = 0.0565 for intronic variant rs12041331). Imputation of the individual exonic variants in the rest of the GeneSTAR European American cohort (N = 1,965) supports the results noted in the sequenced discovery sample: p = 3.56×10⁻⁴, 2.27×10⁻⁷, 5.20×10⁻⁵ for coding synonymous variant rs56260937 and collagen, epinephrine and adenosine diphosphate induced platelet aggregation, respectively. Sequencing approaches confirm that a common intronic variant has the strongest association with platelet aggregation in African Americans, and show that exonic variants play an additional role in platelet aggregation in European Americans.

In Vitro Activity of Daptomycin against Enterococcus faecalis under Various Conditions of Growth-Phases, Inoculum and pH

2013-05-21T21:00:00Z

by Xavier Argemi, Yves Hansmann, Daniel Christmann, Sophie Lefebvre, Benoit Jaulhac, François Jehl

Enterococcus faecalis (E. faecalis) has become a major leading cause of nosocomial endocarditis. Treatment of such infections remains problematic and new therapeutic options are needed. Nine E. faecalis strains were tested: six obtained from patients presenting endocarditis, one with isolated bacteremia, and two reference strains. Antibiotics included daptomycin, alone or in combination, linezolid, tigecycline, rifampicin, gentamicin, teicoplanin, ceftriaxone and amoxicillin. Time-kill studies included colony counts at 1, 4 and 24 h of incubation. Significant bactericidal activity was defined as a decrease of ≥3log₁₀CFU/ml after 24 h of incubation. Antibiotics were tested at a low (10⁶ CFU/ml) and high (10⁹ CFU/ml) inoculum, against exponential- and stationary-phase bacteria. We also performed time kill studies of chemically growth arrested E. faecalis. Various pH conditions were used during the tests. In exponential growth phase and with a low inoculum, daptomycin alone at 60 µg/ml and the combination amoxicillin-gentamicin both achieved a 4-log₁₀ reduction in one hour on all strains. In exponential growth phase with a high inoculum, daptomycin alone was bactericidal at a concentration of 120 µg/ml. All the combinations tested with this drug were indifferent. In stationary phase with a high inoculum daptomycin remained bactericidal but exhibited a pH dependent activity and slower kill rates. All combinations that did not include daptomycin were not bactericidal in conditions of high inoculum, whatever the growth phase. The results indicate that daptomycin is the only antibiotic that may be able of overcoming the effects of growth phase and high inoculum.

Action Prediction in Younger versus Older Adults: Neural Correlates of Motor Familiarity

2013-05-21T21:00:00Z

by Nadine Diersch, Karsten Mueller, Emily S. Cross, Waltraud Stadler, Martina Rieger, Simone Schütz-Bosbach

Generating predictions during action observation is essential for efficient navigation through our social environment. With age, the sensitivity in action prediction declines. In younger adults, the action observation network (AON), consisting of premotor, parietal and occipitotemporal cortices, has been implicated in transforming executed and observed actions into a common code. Much less is known about age-related changes in the neural representation of observed actions. Using fMRI, the present study measured brain activity in younger and older adults during the prediction of temporarily occluded actions (figure skating elements and simple movement exercises). All participants were highly familiar with the movement exercises whereas only some participants were experienced figure skaters. With respect to the AON, the results confirm that this network was preferentially engaged for the more familiar movement exercises. Compared to younger adults, older adults recruited visual regions to perform the task and, additionally, the hippocampus and caudate when the observed actions were familiar to them. Thus, instead of effectively exploiting the sensorimotor matching properties of the AON, older adults seemed to rely predominantly on the visual dynamics of the observed actions to perform the task. Our data further suggest that the caudate played an important role during the prediction of the less familiar figure skating elements in better-performing groups. Together, these findings show that action prediction engages a distributed network in the brain, which is modulated by the content of the observed actions and the age and experience of the observer.

Classifications within Molecular Subtypes Enables Identification of BRCA1/BRCA2 Mutation Carriers by RNA Tumor Profiling

2013-05-21T21:00:00Z

by Martin J. Larsen, Torben A. Kruse, Qihua Tan, Anne-Vibeke Lænkholm, Martin Bak, Anne E. Lykkesfeldt, Kristina P. Sørensen, Thomas v. O. Hansen, Bent Ejlertsen, Anne-Marie Gerdes, Mads Thomassen

Pathogenic germline mutations in BRCA1 or BRCA2 are detected in less than one third of families with a strong history of breast cancer. It is therefore expected that mutations still remain undetected by currently used screening methods. In addition, a growing number of BRCA1/2 sequence variants of unclear pathogen significance are found in the families, constituting an increasing clinical challenge. New methods are therefore needed to improve the detection rate and aid the interpretation of the clinically uncertain variants. In this study we analyzed a series of 33 BRCA1, 22 BRCA2, and 128 sporadic tumors by RNA profiling to investigate the classification potential of RNA profiles to predict BRCA1/2 mutation status. We found that breast tumors from BRCA1 and BRCA2 mutation carriers display characteristic RNA expression patterns, allowing them to be distinguished from sporadic tumors. The majority of BRCA1 tumors were basal-like while BRCA2 tumors were mainly luminal B. Using RNA profiles, we were able to distinguish BRCA1 tumors from sporadic tumors among basal-like tumors with 83% accuracy and BRCA2 from sporadic tumors among luminal B tumors with 89% accuracy. Furthermore, subtype-specific BRCA1/2 gene signatures were successfully validated in two independent data sets with high accuracies. Although additional validation studies are required, indication of BRCA1/2 involvement (“BRCAness”) by RNA profiling could potentially be valuable as a tool for distinguishing pathogenic mutations from benign variants, for identification of undetected mutation carriers, and for selecting patients sensitive to new therapeutics such as PARP inhibitors.

Eosinophils Promote Epithelial to Mesenchymal Transition of Bronchial Epithelial Cells

2013-05-21T21:00:00Z

by Atsushi Yasukawa, Koa Hosoki, Masaaki Toda, Yasushi Miyake, Yuki Matsushima, Takahiro Matsumoto, Daniel Boveda-Ruiz, Paloma Gil-Bernabe, Mizuho Nagao, Mayumi Sugimoto, Yukiko Hiraguchi, Reiko Tokuda, Masahiro Naito, Takehiro Takagi, Corina N. D'Alessandro-Gabazza, Shigeru Suga, Tetsu Kobayashi, Takao Fujisawa, Osamu Taguchi, Esteban C. Gabazza

Eosinophilic inflammation and remodeling of the airways including subepithelial fibrosis and myofibroblast hyperplasia are characteristic pathological findings of bronchial asthma. Epithelial to mesenchymal transition (EMT) plays a critical role in airway remodelling. In this study, we hypothesized that infiltrating eosinophils promote airway remodelling in bronchial asthma. To demonstrate this hypothesis we evaluated the effect of eosinophils on EMT by in vitro and in vivo studies. EMT was assessed in mice that received intra-tracheal instillation of mouse bone marrow derived eosinophils and in human bronchial epithelial cells co-cultured with eosinophils freshly purified from healthy individuals or with eosinophilic leukemia cell lines. Intra-tracheal instillation of eosinophils was associated with enhanced bronchial inflammation and fibrosis and increased lung concentration of growth factors. Mice instilled with eosinophils pre-treated with transforming growth factor(TGF)-β1 siRNA had decreased bronchial wall fibrosis compared to controls. EMT was induced in bronchial epithelial cells co-cultured with human eosinophils and it was associated with increased expression of TGF-β1 and Smad3 phosphorylation in the bronchial epithelial cells. Treatment with anti-TGF-β1 antibody blocked EMT in bronchial epithelial cells. Eosinophils induced EMT in bronchial epithelial cells, suggesting their contribution to the pathogenesis of airway remodelling.

Evaluation of Glucocorticoid Receptor Function in COPD Lung Macrophages Using Beclomethasone-17-Monopropionate

2013-05-21T21:00:00Z

by Jonathan Plumb, Laura Robinson, Simon Lea, Antonia Banyard, John Blaikley, David Ray, Andrea Bizzi, Giorgina Volpi, Fabrizio Facchinetti, Dave Singh

Previous studies of glucocorticoid receptor (GR) function in COPD lung macrophages have used dexamethasone to evaluate inhibition of cytokine production. We have now used the clinically relevant corticosteroid beclomethasone-17-monopropionate (17-BMP) to assess GR function in COPD lung macrophages, and investigated the transactivation of glucocorticoid sensitive genes and GR phosphorylation in addition to cytokine production. Lung macrophages were purified from surgically acquired lung tissue, from patients with COPD, smokers, and non-smokers. The transactivation of glucocorticoid sensitive genes (FKBP51 and GILZ) by 17-BMP were analysed by polymerase chain reaction. 17-BMP suppression of LPS-induced TNFα, IL-6 and CXCL8 was measured by ELISA and GR phosphorylation was measured by immunohistochemistry and Western blot. 17-BMP reduced cytokine release in a concentration dependent manner, with >70% inhibition of all cytokines, and no difference between COPD patients and controls. Similarly, the transactivation of FKBP51 and GILZ, and GR phosphorylation was similar between COPD patients and controls. In this context, GR function in COPD lung macrophages is unaltered. 17-BMP effectively suppresses cytokine production in COPD lung macrophages.

Targeted Sequencing of Cancer-Related Genes in Colorectal Cancer Using Next-Generation Sequencing

2013-05-21T21:00:00Z

by Sae-Won Han, Hwang-Phill Kim, Jong-Yeon Shin, Eun-Goo Jeong, Won-Chul Lee, Kyung-Hun Lee, Jae-Kyung Won, Tae-Yong Kim, Do-Youn Oh, Seock-Ah Im, Yung-Jue Bang, Seung-Yong Jeong, Kyu Joo Park, Jae-Gahb Park, Gyeong Hoon Kang, Jeong-Sun Seo, Jong-Il Kim, Tae-You Kim

Recent advance in sequencing technology has enabled comprehensive profiling of genetic alterations in cancer. We have established a targeted sequencing platform using next-generation sequencing (NGS) technology for clinical use, which can provide mutation and copy number variation data. NGS was performed with paired-end library enriched with exons of 183 cancer-related genes. Normal and tumor tissue pairs of 60 colorectal adenocarcinomas were used to test feasibility. Somatic mutation and copy number alteration were analyzed. A total of 526 somatic non-synonymous sequence variations were found in 113 genes. Among these, 278 single nucleotide variations were 232 different somatic point mutations. 216 SNV were 79 known single nucleotide polymorphisms in the dbSNP. 32 indels were 28 different indel mutations. Median number of mutated gene per tumor was 4 (range 0–23). Copy number gain (>X2 fold) was found in 65 genes in 40 patients, whereas copy number loss (APC in 35 patients (58%), TP53 in 34 (57%), and KRAS in 24 (40%). Altered gene list revealed ErbB signaling pathway as the most commonly involved pathway (25 patients, 42%). Targeted sequencing platform using NGS technology is feasible for clinical use and provides comprehensive genetic alteration data.

Socially-Assigned Race, Healthcare Discrimination and Preventive Healthcare Services

2013-05-21T21:00:00Z

by Tracy MacIntosh, Mayur M. Desai, Tene T. Lewis, Beth A. Jones, Marcella Nunez-Smith

Background

Race and ethnicity, typically defined as how individuals self-identify, are complex social constructs. Self-identified racial/ethnic minorities are less likely to receive preventive care and more likely to report healthcare discrimination than self-identified non-Hispanic whites. However, beyond self-identification, these outcomes may vary depending on whether racial/ethnic minorities are perceived by others as being minority or white; this perception is referred to as socially-assigned race.

Purpose

To examine the associations between socially-assigned race and healthcare discrimination and receipt of selected preventive services.

Methods

Cross-sectional analysis of the 2004 Behavioral Risk Factor Surveillance System “Reactions to Race” module. Respondents from seven states and the District of Columbia were categorized into 3 groups, defined by a composite of self-identified race/socially-assigned race: Minority/Minority (M/M, n = 6,837), Minority/White (M/W, n = 929), and White/White (W/W, n = 25,913). Respondents were 18 years or older, with 61.7% under age 60; 51.8% of respondents were female. Measures included reported healthcare discrimination and receipt of vaccinations and cancer screenings.

Results

Racial/ethnic minorities who reported being socially-assigned as minority (M/M) were more likely to report healthcare discrimination compared with those who reported being socially-assigned as white (M/W) (8.9% vs. 5.0%, p = 0.002). Those reporting being socially-assigned as white (M/W and W/W) had similar rates for past-year influenza (73.1% vs. 74.3%) and pneumococcal (69.3% vs. 58.6%) vaccinations; however, rates were significantly lower among M/M respondents (56.2% and 47.6%, respectively, p-values<0.05). There were no significant differences between the M/M and M/W groups in the receipt of cancer screenings.

Conclusions

Racial/ethnic minorities who reported being socially-assigned as white are more likely to receive preventive vaccinations and less likely to report healthcare discrimination compared with those who are socially-assigned as minority. Socially-assigned race/ethnicity is emerging as an important area for further research in understanding how race/ethnicity influences health outcomes.

Effect of Pretreatment on Biomass Residue Structure and the Application of Pyrolysed and Composted Biomass Residues in Soilless Culture

2013-05-21T21:00:00Z

by Linna Suo, Xiangyang Sun, Weijie Jiang

The changes in the structural characteristics of biomass residues during pyrolysis and composting were investigated. The biomass residues particles were prepared by pyrolysing at temperatures ranging from 350 to 400. For soilless production of the ornamental plant Anthurium andraeanum, pure sphagnum peat moss (P) has traditionally been used as the growing medium. This use of P must be reduced, however, because P is an expensive and nonrenewable resource. The current study investigated the use of biomass residues as substitutes for P in A. andraeanum production. Plants were grown for 15 months in 10 soilless media that contained different proportions of pyrolysed corn cobs (PC), composted corn cobs (C), pyrolysed garden wastes (PG), and P. Although the media altered the plant nutrient content, A. andraeanum growth, development, and yield were similar with media consisting of 50% P+50% PC, 50% P+35% PC+15% PG, and 100% P. This finding indicates that, when pyrolysed, organic wastes, which are otherwise an environmental problem, can be used to reduce the requirement for peat in the soilless culture of A. andraeanum.

Eplerenone Attenuates Pulse Wave Reflection in Chronic Kidney Disease Stage 3–4 - A Randomized Controlled Study

2013-05-21T21:00:00Z

by Lene Boesby, Thomas Elung-Jensen, Svend Strandgaard, Anne-Lise Kamper

Background

Patients with chronic kidney disease (CKD) have high cardiovascular mortality and morbidity associated with increased arterial stiffness. Plasma aldosterone levels are increased in CKD, and aldosterone has been found to increase vascular inflammation and fibrosis. It was hypothesized that aldosterone receptor inhibition with eplerenone could reduce arterial stiffness in CKD stage 3–4.

Study Design

The design was randomized, open, parallel group. Measurements of arterial stiffness markers were undertaken at weeks 1 and 24.

Intervention

24 weeks of add-on treatment with 25–50 mg eplerenone or standard medication.

Outcomes

Primary outcome parameter was carotid-femoral pulse wave velocity (cfPWV). Secondary outcomes were augmentation index (AIx), ambulatory arterial stiffness index (AASI) and urinary albumin excretion.

Results

Fifty-four CKD patients (mean eGFR 36 mL/min/1.73 m², SD 11) were randomized. Forty-six patients completed the trial. The mean difference in cfPWV changes between groups was 0.1 m/s (95%CI: −1.0, 1.3), P = 0.8. The mean difference in AIx changes between groups was 4.4% (0.1, 8.6), P = 0.04. AASI was unchanged in both groups. The ratio of change in urinary albumin excretion in the eplerenone group compared to the control was 0.61 (0.37, 1.01), P = 0.05. Four patients were withdrawn from the eplerenone group including three because of possible side effects; one was withdrawn from the control group. Mild hyperkalemia was seen on three occasions and was easily managed.

Limitations

The full planned number of patients was not attained. The duration of the trial may have been too short to obtain full effect of eplerenone on the arteries.

Conclusions

Add-on treatment with eplerenone in CKD stage 3–4 did not significantly reduce cfPWV. There may be beneficial vascular effects leading to attenuated pulse wave reflection. Treatment was well-tolerated.

Trial Registration

ClinicalTrials.gov NCT01100203

Quantifying the Detrimental Impacts of Land-Use and Management Change on European Forest Bird Populations

2013-05-21T21:00:00Z

by Amy S. I. Wade, Boris Barov, Ian J. Burfield, Richard D. Gregory, Ken Norris, Simon J. Butler

The ecological impacts of changing forest management practices in Europe are poorly understood despite European forests being highly managed. Furthermore, the effects of potential drivers of forest biodiversity decline are rarely considered in concert, thus limiting effective conservation or sustainable forest management. We present a trait-based framework that we use to assess the detrimental impact of multiple land-use and management changes in forests on bird populations across Europe. Major changes to forest habitats occurring in recent decades, and their impact on resource availability for birds were identified. Risk associated with these changes for 52 species of forest birds, defined as the proportion of each species' key resources detrimentally affected through changes in abundance and/or availability, was quantified and compared to their pan-European population growth rates between 1980 and 2009. Relationships between risk and population growth were found to be significantly negative, indicating that resource loss in European forests is an important driver of decline for both resident and migrant birds. Our results demonstrate that coarse quantification of resource use and ecological change can be valuable in understanding causes of biodiversity decline, and thus in informing conservation strategy and policy. Such an approach has good potential to be extended for predictive use in assessing the impact of possible future changes to forest management and to develop more precise indicators of forest health.

Integrative Study of Physiological Changes Associated with Bacterial Infection in Pacific Oyster Larvae

2013-05-21T21:00:00Z

by Bertrand Genard, Philippe Miner, Jean-Louis Nicolas, Dario Moraga, Pierre Boudry, Fabrice Pernet, Réjean Tremblay

Background

Bacterial infections are common in bivalve larvae and can lead to significant mortality, notably in hatcheries. Numerous studies have identified the pathogenic bacteria involved in such mortalities, but physiological changes associated with pathogen exposure at larval stage are still poorly understood. In the present study, we used an integrative approach including physiological, enzymatic, biochemical, and molecular analyses to investigate changes in energy metabolism, lipid remodelling, cellular stress, and immune status of Crassostrea gigas larvae subjected to experimental infection with the pathogenic bacteria Vibrio coralliilyticus.

Findings

Our results showed that V. coralliilyticus exposure induced (1) limited but significant increase of larvae mortality compared with controls, (2) declined feeding activity, which resulted in energy status changes (i.e. reserve consumption, β-oxidation, decline of metabolic rate), (3) fatty acid remodeling of polar lipids (changes in phosphatidylinositol and lysophosphatidylcholine composition`, non-methylene–interrupted fatty acids accumulation, lower content of major C₂₀ polyunsaturated fatty acids as well as activation of desaturases, phospholipase and lipoxygenase), (4) activation of antioxidant defenses (catalase, superoxide dismutase, peroxiredoxin) and cytoprotective processes (heat shock protein 70, pernin), and (5) activation of the immune response (non-self recognition, NF-κκ signaling pathway, haematopoiesis, eiconosoids and lysophosphatidyl acid synthesis, inhibitor of metalloproteinase and antimicrobial peptides).

Conclusion

Overall, our results allowed us to propose an integrative view of changes induced by a bacterial infection in Pacific oyster larvae, opening new perspectives on the response of marine bivalve larvae to infections.

A Novel Epitope from CD22 Regulates Th1 and Th17 Cell Function in Systemic Lupus Erythematosus

2013-05-21T21:00:00Z

by Jing Yuan, Miao Yu, Ai-Lin Cao, Xiao Chen, Li-Hua Zhang, You Song, Xiang Cheng, Zi-Hua Zhou, Min Wang, He-Ping Guo, Rong Du, Yu-Hua Liao

The published antibodies (Abs) against CD22 on B cells including Epratuzumab could inhibit B cell activation mainly through binding to C2-set Ig domain of CD22, but they are rarely reported to modulate the pathogenic CD4⁺ T cell function in systemic lupus erythematosus (SLE). Recently, it was proved that the extracellular amino-terminal V-set Ig domain of CD22 might mediate the interaction of B and T cells, but for now the exact effect of this domain on CD4⁺ T cell biology have not been identified. Thus, in this study, we screened out a peptide termed B2285 from this V-set Ig domain, developed the novel specific anti-B2285 Abs in rabbits, and investigated their effects in MRL/lpr mice with spontaneous SLE. The results showed that anti-B2285 Abs could ameliorate the disease severity obviously in spontaneous SLE mice with the decreased differentiations of Th1 and Th17 cells and no changes of Th2 and Treg cells. In co-cultured B cells and CD4⁺ T cells, this specific anti-CD22 Abs was observed to inhibit the anti-dsDNA Abs production, CD4⁺ T cells proliferation, the protein levels of T-bet and RORγt, and the mRNA levels of TNF-α, IFN-γ, IL-6 and IL-17 in CD4⁺ T cells. Moreover, the expression of CD45RO on CD4⁺ T cells could be also apparently diminished by this novel Abs. The data suggested that anti-B2285 Abs could slow SLE progression significantly by regulating Th1 and Th17 cells function via B-T cell interaction and the cytokine network regulation. The treatment against V-set Ig domain of CD22 would be a valuable therapeutic method for SLE and other autoimmune diseases.

MARCKS Protein Is Phosphorylated and Regulates Calcium Mobilization during Human Acrosomal Exocytosis

2013-05-21T21:00:00Z

by Marcelo J. Rodriguez Peña, Jimena V. Castillo Bennett, Osvaldo M. Soler, Luis S. Mayorga, Marcela A. Michaut

Acrosomal exocytosis is a calcium-regulated exocytosis that can be triggered by PKC activators. The involvement of PKC in acrosomal exocytosis has not been fully elucidated, and it is unknown if MARCKS, the major substrate for PKC, participates in this exocytosis. Here, we report that MARCKS is expressed in human spermatozoa and localizes to the sperm head and the tail. Calcium- and phorbol ester-triggered acrosomal exocytosis in permeabilized sperm was abrogated by different anti-MARCKS antibodies raised against two different domains, indicating that the protein participates in acrosomal exocytosis. Interestingly, an anti-phosphorylated MARCKS antibody was not able to inhibit secretion. Similar results were obtained using recombinant proteins and phospho-mutants of MARCKS effector domain (ED), indicating that phosphorylation regulates MARCKS function in acrosomal exocytosis. It is known that unphosphorylated MARCKS sequesters PIP₂. This phospholipid is the precursor for IP₃, which in turn triggers release of calcium from the acrosome during acrosomal exocytosis. We found that PIP₂ and adenophostin, a potent IP₃-receptor agonist, rescued MARCKS inhibition in permeabilized sperm, suggesting that MARCKS inhibits acrosomal exocytosis by sequestering PIP₂ and, indirectly, MARCKS regulates the intracellular calcium mobilization. In non-permeabilized sperm, a permeable peptide of MARCKS ED also inhibited acrosomal exocytosis when stimulated by a natural agonist such as progesterone, and pharmacological inducers such as calcium ionophore and phorbol ester. The preincubation of human sperm with the permeable MARCKS ED abolished the increase in calcium levels caused by progesterone, demonstrating that MARCKS regulates calcium mobilization. In addition, the phosphorylation of MARCKS increased during acrosomal exocytosis stimulated by the same activators. Altogether, these results show that MARCKS is a negative modulator of the acrosomal exocytosis, probably by sequestering PIP₂, and that it is phosphorylated during acrosomal exocytosis.

Metabolomics Tools for Describing Complex Pesticide Exposure in Pregnant Women in Brittany (France)

2013-05-21T21:00:00Z

by Nathalie Bonvallot, Marie Tremblay-Franco, Cécile Chevrier, Cécile Canlet, Charline Warembourg, Jean-Pierre Cravedi, Sylvaine Cordier

Background

The use of pesticides and the related environmental contaminations can lead to human exposure to various molecules. In early-life, such exposures could be responsible for adverse developmental effects. However, human health risks associated with exposure to complex mixtures are currently under-explored.

Objective

This project aims at answering the following questions: What is the influence of exposures to multiple pesticides on the metabolome? What mechanistic pathways could be involved in the metabolic changes observed?

Methods

Based on the PELAGIE cohort (Brittany, France), 83 pregnant women who provided a urine sample in early pregnancy, were classified in 3 groups according to the surface of land dedicated to agricultural cereal activities in their town of residence. Nuclear magnetic resonance-based metabolomics analyses were performed on urine samples. Partial Least Squares Regression-Discriminant Analysis (PLS-DA) and polytomous regressions were used to separate the urinary metabolic profiles from the 3 exposure groups after adjusting for potential confounders.

Results

The 3 groups of exposure were correctly separated with a PLS-DA model after implementing an orthogonal signal correction with pareto standardizations (R2 = 90.7% and Q2 = 0.53). After adjusting for maternal age, parity, body mass index and smoking habits, the most statistically significant changes were observed for glycine, threonine, lactate and glycerophosphocholine (upward trend), and for citrate (downward trend).

Conclusion

This work suggests that an exposure to complex pesticide mixtures induces modifications of metabolic fingerprints. It can be hypothesized from identified discriminating metabolites that the pesticide mixtures could increase oxidative stress and disturb energy metabolism.

Pathophysiological Concepts in Mild Traumatic Brain Injury: Diffusion Tensor Imaging Related to Acute Perfusion CT Imaging

2013-05-21T21:00:00Z

by Zwany Metting, Leonardo Cerliani, Lars A. Rödiger, Joukje van der Naalt

Background

A subgroup of patients with mild traumatic brain injury (TBI) experiences residual symptoms interfering with their return to work. The pathophysiological substrate of the suboptimal outcome in these patients is a source of debate.

Objective

To provide greater insight into the pathophysiological mechanisms of mild TBI.

Methods

Diffusion tensor imaging (DTI) was performed during follow-up of 18 patients with mild TBI and compared with healthy control subjects. DTI data of the patient group were also compared with perfusion CT imaging in the acute phase of injury.

Results

In patients with mild TBI, a trend was observed for a decreased fractional anisotropy (FA) in widespread bilateral frontal white matter areas with increased mean diffusivity (MD) in the parieto-temporal regions, compared to healthy control subjects. Cerebral blood volume (CBV) correlated significantly with FA in several white matter tracts including the corpus callosum, the internal capsule, the inferior fronto-occipital fascicle, the corticospinal tract, the superior and the inferior longitudinal fascicle.

Conclusion

In mild TBI with normal conventional imaging significant associations between cerebral perfusion in the acute phase of injury and DTI analyses in the chronic phase of injury were discerned. The pathophysiological concept of these findings is being outlined.

Molecular Mechanisms of Environmental Enrichment: Impairments in Akt/GSK3β, Neurotrophin-3 and CREB Signaling

2013-05-21T21:00:00Z

by Yuan-Shih Hu, Nancy Long, Gustavo Pigino, Scott T. Brady, Orly Lazarov

Experience of mice in a complex environment enhances neurogenesis and synaptic plasticity in the hippocampus of wild type and transgenic mice harboring familial Alzheimer's disease (FAD)-linked APPswe/PS1ΔE9. In FAD mice, this experience also reduces levels of tau hyperphosphorylation and oligomeric β-amyloid. Although environmental enrichment has significant effects on brain plasticity and neuropathology, the molecular mechanisms underlying these effects are unknown. Here we show that environmental enrichment upregulates the Akt pathway, leading to the downregulation of glycogen synthase kinase 3β (GSK3β), in wild type but not FAD mice. Several neurotrophic signaling pathways are activated in the hippocampus of both wild type and FAD mice, including brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and this increase is accompanied by the upregulation of the BDNF receptor, tyrosine kinase B (TrkB). Interestingly, neurotrophin-3 (NT-3) is upregulated in the brains of wild type mice but not FAD mice, while insulin growth factor-1 (IGF-1) is upregulated exclusively in the brains of FAD mice. Upregulation of neurotrophins is accompanied by the increase of N-Methyl-D-aspartic acid (NMDA) receptors in the hippocampus following environmental enrichment. Most importantly, we observed a significant increase in levels of cAMP response element- binding (CREB) transcripts in the hippocampus of wild type and FAD mice following environmental enrichment. However, CREB phosphorylation, a critical step for the initiation of learning and memory-required gene transcription, takes place in the hippocampus of wild type but not of FAD mice. These results suggest that experience of wild type mice in a complex environmental upregulates critical signaling that play a major role in learning and memory in the hippocampus. However, in FAD mice, some of these pathways are impaired and cannot be rescued by environmental enrichment.

Antiviral Lectins from Red and Blue-Green Algae Show Potent In Vitro and In Vivo Activity against Hepatitis C Virus

2013-05-21T21:00:00Z

by Yutaka Takebe, Carrie J. Saucedo, Garry Lund, Rie Uenishi, Saiki Hase, Takayo Tsuchiura, Norman Kneteman, Koreen Ramessar, D. Lorne J. Tyrrell, Masayuki Shirakura, Takaji Wakita, James B. McMahon, Barry R. O'Keefe

Hepatitis C virus (HCV) infection is a significant public health problem with over 170,000,000 chronic carriers and infection rates increasing worldwide. Chronic HCV infection is one of the leading causes of hepatocellular carcinoma which was estimated to result in ∼10,000 deaths in the United States in the year 2011. Current treatment options for HCV infection are limited to PEG-ylated interferon alpha (IFN-α), the nucleoside ribavirin and the recently approved HCV protease inhibitors telaprevir and boceprevir. Although showing significantly improved efficacy over the previous therapies, treatment with protease inhibitors has been shown to result in the rapid emergence of drug-resistant virus. Here we report the activity of two proteins, originally isolated from natural product extracts, which demonstrate low or sub-nanomolar in vitro activity against both genotype I and genotype II HCV. These proteins inhibit viral infectivity, binding to the HCV envelope glycoproteins E1 and E2 and block viral entry into human hepatocytes. In addition, we demonstrate that the most potent of these agents, the protein griffithsin, is readily bioavailable after subcutaneous injection and shows significant in vivo efficacy in reducing HCV viral titers in a mouse model system with engrafted human hepatocytes. These results indicate that HCV viral entry inhibitors can be an effective component of anti-HCV therapy and that these proteins should be studied further for their therapeutic potential.

Diffusion Weighted Imaging Evaluated the Early Therapy Effect of Tamoxifen in an MNU-Induced Mammary Cancer Rat Model

2013-05-21T21:00:00Z

by Guihua Zhai, Clinton J. Grubbs, Cecil R. Stockard, Heidi R. Umphrey, T. Mark Beasley, Hyunki Kim

Purpose

To assess the optimal time point of diffusion-weighted imaging (DWI) for early prognosis of breast cancer following tamoxifen therapy using a methylnitrosourea (MNU)-induced ER-positive breast-cancer model.

Methods

Two groups of Sprague-Dawley rats (n = 15 for group 1; n = 10 for group 2) were used. All animals (50 days old) were intravenously injected with MNU (50 mg/kg body weight) to induce ER-positive mammary tumors. When tumors were approximately 2 cm in diameter, DWI was performed on days 0, 3, and 7, and intratumoral apparent diffusion coefficient (ADC) values were measured. Therapy started on day 0 with tamoxifen (10 mg/kg diet) and continued for 4 weeks for group 1, but only 1 week for group 2, while tumor volume was measured by caliper twice weekly. All animals of group 2 were euthanized on day 7 after imaging, and Ki-67, TUNEL, ERα, and ERβ staining were performed on tumor tissue.

Results

DW images of MNU-induced mammary tumors were successfully obtained with minimal motion artifact. For group 1, ADC change for 3 days after therapy initiation (ADC_3D) was significantly correlated with tumor-volume change until day 11, but the significant correlation between ADC change for 7 days (ADC_7D) and the tumor-volume change was observed until day 18. Similarly, for group 2, either ADC_7D or ADC_3D was significantly correlated with the tumor-volume change, but the higher significance was observed for ADC_7D. Furthermore, ADC_7D was significantly correlated with apoptotic (TUNEL stained), proliferative (Ki-67 stained), and ERβ-positive cell densities, but ADC_3D was not significantly correlated with any of those.

Conclusions

ADC_7D might be a more reliable surrogate imaging biomarker than ADC_3D to assess effectiveness of tamoxifen therapy for ER-positive breast cancer, which may enable personalized treatment. The significant correlation between ADC_7D and ERβ-positive cell density suggests that ERβ may play an important role as a therapeutic indicator of tamoxifen.

Recurrent Pregnancy Loss in Polycystic Ovary Syndrome: Role of Hyperhomocysteinemia and Insulin Resistance

2013-05-21T21:00:00Z

by Pratip Chakraborty, S. K. Goswami, Shweta Rajani, Sunita Sharma, Syed N. Kabir, Baidyanath Chakravarty, Kuladip Jana

Recurrent pregnancy loss (RPL) in polycystic ovary syndrome (PCOS), which occurs in ∼50% of total pregnancies is a frequent obstetric complication. Among the several hypotheses, insulin resistance (IR), obesity and hyperhomocysteinemia (HHcy) play significant role/s in RPL. This study was conducted to assess the link between elevated levels of homocysteine and IR in PCOS-associated women with RPL in Kolkata, India. A retrospective study was conducted of one hundred and twenty six PCOS women (<30 years) who experienced two or more spontaneous abortions during the first trimester presenting to Institute of Reproductive Medicine (IRM) in Kolkata during the period of March 2008 through February 2011. One hundred and seventeen non-PCOS subjects with matching age range were randomly chosen as controls. Incidence of HHcy and IR was 70.63% (n = 89) and 56.34% (n = 71), respectively, in RPL-affected PCOS population which was significantly higher (p<0.04; p<0.0001) when compared to the non-PCOS set (HHcy: 57.26%; IR: 6.83%). Rates of miscarriage were significantly higher (p<0.008; p<0.03) in hyperhomocysteinemia-induced miscarriage when compared to the normohomocysteinemic segment (PCOS: 70.63% vs.29.36% & non-PCOS: 57.26% vs. 42.73%) along with the insulin resistant (p<0.04; p<0.0001) population (PCOS: 70.63% vs. 56.34% & non-PCOS: 57.26% vs. 6.83%) in both groups. A probabilistic causal model evaluated HHcy as the strongest plausible factor for diagnosis of RPL. A probability percentage of 43.32% in the cases of HHcy- mediated RPL suggests its increased tendency when compared to IR mediated miscarriage (37.29%), further supported by ROC-AUC (HHcy: 0.778vs. IR: 0.601) values. Greater susceptibility towards HHcy may increase the incidence for miscarriage in women in India and highlights the need to combat the condition in RPL control programs in the subcontinent.