An Efficiently Cleaved HIV-1 Clade C Env Selectively Binds to Neutralizing Antibodies

An ideal HIV-1 Env immunogen is expected to mimic the native trimeric conformation for inducing broadly neutralizing antibody responses. The native conformation is dependent on efficient cleavage of HIV-1 Env. The clade B isolate, JRFL Env is efficiently cleaved when expressed on the cell surface. Here, for the first time, we report the identification of a native clade C Env, 4-2.J41 that is naturally and efficiently cleaved on the cell surface as confirmed by its biochemical and antigenic characteristics. In addition to binding to several conformation-dependent neutralizing antibodies, 4-2.J41 Env binds efficiently to the cleavage-dependent antibody PGT151; thus validating its native cleaved conformation. In contrast, 4-2.J41 Env occludes non-neutralizing epitopes. The cytoplasmic-tail of 4-2.J41 Env plays an important role in maintaining its conformation. Furthermore, codon optimization of 4-2.J41 Env sequence significantly increases its expression while retaining its native conformation. Since clade C of HIV-1 is the prevalent subtype, identification and characterization of this efficiently cleaved Env would provide a platform for rational immunogen design.     

Interaction among Non-toxic Phytoplankton,Toxic Phytoplankton and Zooplankton: Inferences from Field Observations

We explore the mutual dependencies and interactions among different groups of species of the plankton population, based on an analysis of the long-term field observations carried out by our group in the North–West coast of the Bay of Bengal. The plankton community is structured into three groups of species, namely, non-toxic phytoplankton (NTP), toxic phytoplankton (TPP) and zooplankton. To find the pair-wise dependencies among the three groups of plankton, Pearson and partial correlation coefficients are calculated. To explore the simultaneous interaction among all the three groups, a time series analysis is performed. Following an Expectation Maximization (E-M) algorithm, those data points which are missing due to irregularities in sampling are estimated, and with the completed data set a Vector Auto-Regressive (VAR) model is analyzed. The overall analysis demonstrates that toxin-producing phytoplankton play two distinct roles: the inhibition on consumption of toxic substances reduces the abundance of zooplankton, and the toxic materials released by TPP significantly compensate for the competitive disadvantages among phytoplankton species. Our study suggests that the presence of TPP might be a possible cause for the generation of a complex interaction among the large number of phytoplankton and zooplankton species that might be responsible for the prolonged coexistence of the plankton species in a fluctuating biomass.     

Safety and Immunogenicity of a Live Oral Recombinant Cholera Vaccine VA1.4: A Randomized,Placebo Controlled Trial in Healthy Adults in a Cholera Endemic Area in Kolkata,India

Background

A live oral cholera vaccine VA 1.4 developed from a non-toxigenic Vibrio cholerae O1 El Tor strain using ctxB gene insertion was further developed into a clinical product following cGMP and was evaluated in a double-blind randomized placebo controlled parallel group two arm trial with allocation ratio of 1∶1 for safety and immunogenicity in men and women aged 18–60 years from Kolkata, India.

Method

A lyophilized dose of 1.9×10⁹ CFU (n = 44) or a placebo (n = 43) reconstituted with a diluent was administered within 5 minutes of drinking 100 ml of a buffer solution made of sodium bicarbonate and ascorbic acid and a second dose on day 14.

Result

The vaccine did not elicit any diarrhea related adverse events. Other adverse events were rare, mild and similar in two groups. One subject in the vaccine group excreted the vaccine strain on the second day after first dose. The proportion of participants who seroconverted (i.e. had 4-folds or higher rise in reciprocal titre) in the vaccine group were 65.9% (95% CI: 50.1%–79.5%) at both 7 days (i.e. after 1^st dose) and 21 days (i.e. after 2^nd dose). None of the placebo recipients seroconverted. Anti-cholera toxin antibody was detected in very few recipients of the vaccine.

Conclusion

This study demonstrates that VA 1.4 at a single dose of 1.9×10⁹ is safe and immunogenic in adults from a cholera endemic region. No additional benefit after two doses was seen.

Trial Registration

Clinical Trials Registry-India, National Institute of Medical Statistics (Indian Council of Medical Research) CTRI/2012/04/002582     

Bacopasaponins with cytotoxic activity against human breast cancer cells in vitro

Molecular Biology Reports - Globally, breast cancer is a serious concern that exhibits a persistent rise in its incidence and related mortality even after significant advancement in the field of...     

Zinc stabilizes adenomatous polyposis coli (APC) protein levels and induces cell cycle arrest in colon cancer cells

In the present study, we investigated the mechanisms by which zinc causes growth arrest in colon cancer cells. The results suggest that zinc treatment stabilizes the levels of the wild-type adenomatous polyposis coli (APC) protein at the post-translational level since the APC mRNA levels and the promoter activity of the APC gene were decreased in HCT-116 cells (which express the wild-type APC gene) after treatment with ZnCl2. Increased levels of wild-type but not truncated APC proteins were required for the ZnCl2-mediated G2/M phase arrest in different colon cancer cell lines. We further tested whether serum-stimulation, which induces cell cycle arrest in the S phase, can relieve ZnCl2-induced G2/M phase arrest of HCT-116 cells. Results showed that in the HCT-116 cells pretreated with ZnCl2, the serum-stimulation neither changed the distribution of G2/M phase arrested cells nor the increased levels of APC protein. The G2/M phase arrest correlated with retarded growth of HCT-116 cells. To further establish that wild-type APC protein plays a role in ZnCl2-induced G2/M arrest, we treated SW480 colon cancer cells that express truncated APC protein. We found that ZnCl2 treatment did not induce G2/M phase arrest in SW480 cells; however, the cell growth was retarded due to the loss of E-cadherin and alpha-tubulin levels. These results suggest that ZnCl2 inhibits the proliferation of colon cancer cells (which carry the wild-type APC gene) through stabilization of the APC protein and cell cycle arrest in the G2/M phase. On the other hand, ZnCl2 inhibits the proliferation of colon cancer cells (which carry the mutant APC gene) by disrupting cellular attachment and microtubule stability.     

Changes in endogenous indole-3-acetic acid and some biochemical parameters during seed development in <Emphasis Type="Italic">Camellia sinensis</Emphasis> (L.) O. Kuntze

The role of endogenous indole-3-acetic acid (IAA), soluble proteins and RNA in the development of tea (Camellia sinensis (L). O. Kuntze) seeds was investigated in the present study. The state of continuum even at full maturity and lack of a clear end point to seed development as indicated by the persistence of appreciable contents of proteins at full maturity in all the seed parts further confirmed the ‘recalcitrant nature’ of the tea seeds. Unlike the orthodox seeds, the level of free IAA in tea embryos also remained high even at full maturity. The total RNA content remained high in the stages with high moisture content but declined with progressive decline in moisture content.     

Increased band sharing in DNA fingerprints of an inbred human population

Summary We have compared band sharing between the DNA fingerprints of members of an inbred human population with band sharing between members of an outbred population. It had not previously been determined whether the high rate of mutation at minisatellite loci is sufficient to prevent an increase in band sharing in moderately inbred populations. We have found that there is an increase in band sharing in the 2-kb to 9-kb size range, but not in the >9-kb size range, in the inbred population. The difference was consistently observed using four different multi-locus probes, viz. 33.6, 33.15, (CAC)₅ and M13. Thus, we have demonstrated that moderate but prolonged inbreeding can lead to increased similarity in human DNA fingerprints. This should be considered when analysing DNA fingerprints in forensic or paternity cases involving members of an inbred community.     

Genetic variability of Cotton leaf curl betasatellite in Northern India

Cotton is an important crop and its production is affected by various disease pathogens. Monopartite begomovirus associated betasatellites cause Cotton leaf curl disease (CLCuD) in Northern India. In order to access the occurrence and genetic variability of Cotton leaf curl betasatellites, an extensive field survey was conducted in states of Rajasthan, Punjab and Haryana. We selected the betasatellite sequence for analysis as they are reported as important for disease severity and sequence variability. Based on the field observations, the disease incidence ranged from 30% to 80% during the survey. Full genome and DNA β were amplified from various samples while no amplicon was obtained in some samples. The nucleotide sequence homology ranged from 90.0% to 98.7% with Cotton leaf curl virus (CLCuV), 55.2–55.5% with Bhendi yellow vein mosaic virus, 55.8% with Okra leaf curl virus and 51.70% with Tomato leaf curl virus isolates. The lowest similarity (47.8%) was found in CLCuV-Sudan isolate. Phylogenetic analysis showed that analyzed isolates formed a close cluster with various CLCuV isolates reported earlier. The analysis results show sequence variation in Cotton leaf curl betasatellite which could be the result of recombination. The results obtained by genome amplification and sequence variability indicate that some new variants are circulating and causing leaf curl disease in Rajasthan, Punjab and Haryana.Abbreviations: CLCuD, Cotton leaf curl disease; CLCuV, Cotton leaf curl virus; PCR, polymerase chain reaction; SCR, satellite conserved region     

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.