Dilution of protein‐surfactant complexes: A fluorescence study

Dilution of protein–surfactant complexes is an integrated step in microfluidic protein sizing, where the contribution of free micelles to the overall fluorescence is reduced by dilution. This process can be further improved by establishing an optimum surfactant concentration and quantifying the amount of protein based on the fluorescence intensity. To this end, we study the interaction of proteins with anionic sodium dodecyl sulfate (SDS) and cationic hexadecyl trimethyl ammonium bromide (CTAB) using a hydrophobic fluorescent dye (sypro orange). We analyze these interactions fluourometrically with bovine serum albumin, carbonic anhydrase, and beta‐galactosidase as model proteins. The fluorescent signature of protein–surfactant complexes at various dilution points shows three distinct regions, surfactant dominant, breakdown, and protein dominant region. Based on the dilution behavior of protein–surfactant complexes, we propose a fluorescence model to explain the contribution of free and bound micelles to the overall fluorescence. Our results show that protein peak is observed at 3 mM SDS as the optimum dilution concentration. Furthermore, we study the effect of protein concentration on fluorescence intensity. In a single protein model with a constant dye quantum yield, the peak height increases with protein concentration. Finally, addition of CTAB to the protein–SDS complex at mole fractions above 0.1 shifts the protein peak from 3 mM to 4 mM SDS. The knowledge of protein–surfactant interactions obtained from these studies provides significant insights for novel detection and quantification techniques in microfluidics.     

Patient navigation pathway and barriers to treatment seeking in cancer in India: A qualitative inquiry

Cancer is a leading cause of mortality worldwide. Early diagnosis and treatment of cancer may curb the growing burden of the disease. Understanding cancer patients’ navigation pathways for seeking treatment is important in order to facilitate early diagnosis and treatment. With this background we conducted a hospital-based cross-sectional study comprising 68 randomly selected cancer inpatients in a tertiary cancer specialty hospital in Odisha, India, to explore the treatment-seeking pathways of the cancer patients and the barriers and enablers in seeking treatment. Financial constraint is one of the major reasons for the delay in accessing treatment, even when patients are suspected of or diagnosed with cancer. Low awareness of the presenting signs and symptoms of cancer and limited knowledge of the availability of cancer diagnosis and treatment facilities are major factors contributing to delay. Family and friends’ support is found to be the major enabling factor toward seeking treatment. Generation of awareness of cancer among the general population and primary-care practitioners – including those in alternative systems of medicine – is important. Information on diagnostic and treatment services appears to be a felt need.     

Mechanism of action of the tuberculosis and Crohn disease risk factor IRGM in autophagy

Polymorphisms in the IRGM gene, associated with Crohn disease (CD) and tuberculosis, are among the earliest identified examples documenting the role of autophagy in human disease. Functional studies have shown that IRGM protects against these diseases by modulating autophagy, yet the exact molecular mechanism of IRGM's activity has remained unknown. We have recently elucidated IRGM's mechanism of action. IRGM functions as a platform for assembling, stabilizing, and activating the core autophagic machinery, while at the same time physically coupling it to conventional innate immunity receptors. Exposure to microbial products or bacterial invasion increases IRGM expression, which leads to stabilization of AMPK. Specific protein-protein interactions and post-translational modifications such as ubiquitination of IRGM, lead to a co-assembly with IRGM of the key autophagy regulators ULK1 and BECN1 in their activated forms. IRGM physically interacts with 2 other CD risk factors, ATG16L1 and NOD2, placing these 3 principal players in CD within the same molecular complex. This explains how polymorphisms altering expression or function of any of the 3 factors individually can affect the same process—autophagy. Furthermore, IRGM's interaction with NOD2, and additional pattern recognition receptors such as NOD1, RIG-I, and select TLRs, transduces microbial signals to the core autophagy apparatus. This work solves the long-standing enigma of how IRGM controls autophagy.     

Prevalence and Association of Mycobacterium avium subspecies paratuberculosis with Disease Course in Patients with Ulcero-Constrictive Ileocolonic Disease

Background

Association of Mycobacterium avium subspecies paratuberculosis (MAP) and Crohn’s disease (CD) has been controversial due to contradictory reports. Therefore, we determined the prevalence of MAP in patients with CD and intestinal tuberculosis (ITB) and its association with clinical course.

Methodology

Blood and intestinal biopsies were taken from 69 CD, 32 ITB patients and 41 patients with haemorrhoidal bleed who served as controls. qPCR targeting of MAP-specific IS900 gene was used to detect the presence of MAP DNA. qPCR results were further validated by sequencing. Immunohistochemistry (IHC) was used to detect the presence of MAP antigen in biopsy specimens. CD and ITB patients were followed-up for disease course and response to therapy.

Principal Findings

The frequency of MAP-specific DNA in biopsies by qPCR was significantly higher in CD patients (23.2%, p = 0.03) as compared to controls (7.3%). No significant difference in intestinal MAP presence was observed between ITB patients (12.5%, p = 0.6) and controls (7.3%). MAP presence in blood of CD patients was 10.1% as compared to 4.9% in controls while no patients with ITB were found to be positive (p = 0.1). Using IHC for detection of MAP antigen, the prevalence of MAP in CD was 2.9%, 12.5% in ITB patients and 2.4% in controls. However, long-term follow-up of the patients revealed no significant associations between clinical characteristics and treatment outcomes with MAP positivity.

Conclusion

We report significantly high prevalence of MAP in intestinal biopsies of CD patients. However, the presence of MAP does not affect the disease course and treatment outcomes in either CD or ITB patients.     

Is MTHFR 677 C>T Polymorphism Clinically Important in Polycystic Ovarian Syndrome (PCOS)? A Case-Control Study,Meta-Analysis and Trial Sequential Analysis

Background

Optimum efficiency of the folate pathway is considered essential for adequate ovarian function. 677 C>T substitution in the 5, 10-methylene tertrahydrofolatereductase (MTHFR) gene compromises activity of the MTHFR enzyme by about 50%. The significance of correlation between 677C>T substitution and PCOS remains dubious due to the low power of published studies.

Methods and Results

We analyzed MTHFR 677 C>T site in ethnically two different PCOS case-control groups (total 261 cases and 256 controls) from India. The data analysis revealed a lack of association between this polymorphism and PCOS [OR = 1.11 (95%CI = 0.71–1.72), P = 0.66]. Group-wise analysis on the basis of ethnicity also revealed no association in any of the ethnic groups [Indo-Europeans, P = 1; Dravidians, P = 0.70]. Homocysteine levels did not differ significantly between cases (15.51 μmol/L, SD = 2.89) and controls (15.89 μmol/L, SD = 2.23). We also undertook a meta-analysis on 960 cases and 1028 controls, which suggested a significant association of the substitution with PCOS in the dominant model of analysis (OR = 1.47 (95%CI = 1.04–2.09), P = 0.032]. Trial sequential analysis corroborated findings of the traditional meta-analysis. However, we found that the conclusions of meta-analysis were strongly influenced by studies that deviated from the Hardy Weinberg equilibrium. A careful investigation of each study and a trial sequential analysis suggested that 677 C>T substitution holds no clinical significance in PCOS in most of the populations.

Conclusion

In conclusion, MTHFR 677 C>T polymorphism does not affect PCOS risk in India. The association seen in the meta-analysis is due to an outlier study and studies showing deviation from the Hardy Weinberg equilibrium.     

Generation and Characterization of an Immortalized Human Esophageal Myofibroblast Line

Stromal cells with a myofibroblast phenotype present in the normal human esophagus are increased in individuals with gastro-esophageal reflux disease (GERD). We have previously demonstrated that myofibroblasts stimulated with acid and TLR4 agonists increase IL-6 and IL-8 secretion using primary cultures of myofibroblasts established from normal human esophagus. While primary cultures have the advantage of reflecting the in vivo environment, a short life span and unavoidable heterogeneity limits the usefulness of this model in larger scale in vitro cellular signaling studies. The major aim of this paper therefore was to generate a human esophageal myofibroblast line with an extended lifespan. In the work presented here we have generated and characterized an immortalized human esophageal myofibroblast line by transfection with a commercially available GFP-hTERT lentivirus. Immortalized human esophageal myofibroblasts demonstrate phenotypic, genotypic and functional similarity to primary cultures of esophageal myofibroblasts we have previously described. We found that immortalized esophageal myofibroblasts retain myofibroblast spindle-shaped morphology at low and high confluence beyond passage 80, and express α-SMA, vimentin, and CD90 myofibroblast markers. Immortalized human esophageal myofibroblasts also express the putative acid receptor TRPV1 and TLR4 and retain the functional capacity to respond to stimuli encountered in GERD with secretion of IL-6. Finally, immortalized human esophageal myofibroblasts also support the stratified growth of squamous esophageal epithelial cells in 3D organotypic cultures. This newly characterized immortalized human esophageal myofibroblast cell line can be used in future cellular signaling and co-culture studies.     

Identification,validation, and expression of ABC transporters in Podophyllum hexandrum and their role in podophyllotoxin biosynthesis

Podophyllum hexandrum Royle is an important medicinal herb of North-Western Himalayas, and podophyllotoxin, being its major metabolite, has been used extensively in the preparation of several anticancer drugs. Podophyllotoxin accumulates in rhizomes; however, no information exists on the role of ATP-binding cassette (ABC) transporters vis-à-vis podophyllotoxin content. The present study reports identification, validation, and expression analysis of ABC transporter genes from P. hexandrum. Total 252 ABC transporter genes were identified as unigenes out of which 22 were further validated using real time qPCR in different tissues of varying podophyllotoxin content. Differential expression analysis and Pearson’s correlation coefficient revealed two candidate genes PhABC6 and PhABCIII having a positive correlation with the podophyllotoxin content. PhABCIV showed the highest expression in rhizomes (20.53-folds compared to shoots) suggesting its possible role in transport and accumulation of podophyllotoxin.     

Metallic Slit–Loaded Ring Resonator–Based Plasmonic Demultiplexer with Large Crosstalk

Plasmonics - Photonics provides a key solution to limit the myriads of challenges offered by existing silicon technology. Here, we propose a simple 2-channel Plasmonic demultiplexer with...