Multimodal approach to explore the pathogenicity of BARD1, ARG 658 CYS,and ILE 738 VAL mutants

BARD1–BRCA1 complex plays an important role in DNA damage repair, apoptosis, chromatin remodeling, and other important processes required for cell survival. BRCA1 and BARD1 heterodimer possess E3 ligase activity and is involved in genome maintenance, by functioning in surveillance for DNA damage, thereby regulating multiple pathways including tumor suppression. BRCT domains are evolutionary conserved domains present in different proteins such as BRCA1, BARD1, XRCC, and MDC1 regulating damage response and cell-cycle control through protein–protein interactions. Nonetheless, the role of BARD1BRCT in the recruitment of DNA repair mechanism and structural integrity with BRCA1 complex is still implicit. To explicate the role of BARD1BRCT in the DNA repair mechanism, in silico, in vitro, and biophysical approach were applied to characterize BARD1 BRCT wild-type and Arg658Cys and Ile738Val mutants. However, no drastic secondary and tertiary structural changes in the mutant proteins were observed. Thermal and chemical denaturation studies revealed that mutants Arg658Cys and Ile738Val have a decrease in T_m and ?G than the wild type. In silico studies of BARD1 BRCT (568-777) and mutant protein indicate loss in structural compactness on the Ile738Val mutant. Comparative studies of wild-type and mutants will thus be helpful in understanding the basic role of BARD1BRCT in DNA damage repair.     

Reproductive seasonality in the Indian pygmy field mouse,Mus terricolor

The Indian pygmy field mouse, Mus terricolor, is a tiny, yet economically and ecologically important crop pest found throughout South-East Asia. There are no systematic reports exploring its reproductive physiology. We report the presence of distinct periods of annual reproductive activity and quiescence in M. terricolor. Body weight in males and females, relative weights of testis, epididymis and seminal vesicle in males, ovarian and uterine weight in females, gonadal histomorphic changes, testicular and ovarian cholesterol, sialic acid in epididymis, fructose in seminal vesicle, uterine protein content, melatonin in males and females, testosterone in males, estradiol, and progesterone in females were studied over a period of three years in both wild-caught and lab-acclimated mice. The number of Graafian follicles and corpora lutea, and plasma estradiol and progesterone, along with relative weights of ovary and uterus in females exhibited a peak in the months of October–January, compared to June. Based on histomorphic and hormonal status, the major reproductively active season is the winter (short-day breeding). There is a brief period of sub-maximal reproductive activity in April. M. terricolor is reproductively inactive in the summer, monsoons, and autumn. The results establish M. terricolor as a seasonal breeder in the field, with interesting implications for pest management.     

Prediction of stenosis behaviour in artery by neural network and multiple linear regressions

Biomechanics and Modeling in Mechanobiology - Blood flow analysis in the artery is a paramount study in the field of arterial stenosis evaluation. Studies conducted so far have reported the...     

Comparative Evaluation of Bivalent Malaria Rapid Diagnostic Tests versus Traditional Methods in Field with Special Reference to Heat Stability Testing in Central India

Background

Malaria presents a diagnostic challenge in areas where both Plasmodium falciparum and P.vivax are co-endemic. Bivalent Rapid Diagnostic tests (RDTs) showed promise as diagnostic tools for P.falciparum and P.vivax. To assist national malaria control programme in the selection of RDTs, commercially available seven malaria RDTs were evaluated in terms of their performance with special reference to heat stability.

Methodology/Principal Findings

This study was undertaken in four forested districts of central India (July, 2011– March, 2012). All RDTs were tested simultaneously in field along with microscopy as gold standard. These RDTs were stored in their original packing at 25°C before transport to the field or they were stored at 35°C and 45°C upto 100 days for testing the performance of RDTs at high temperature. In all 2841 patients with fever were screened for malaria of which 26% were positive for P.falciparum, and 17% for P.vivax. The highest sensitivity of any RDT for P.falciparum was 98% (95% CI; 95.9–98.8) and lowest sensitivity was 76% (95% CI; 71.7–79.6). For P.vivax highest and lowest sensitivity for any RDT was 80% (95% CI; 94.9 - 83.9) and 20% (95% CI; 15.6–24.5) respectively. Heat stability experiments showed that most RDTs for P.falciparum showed high sensitivity at 45°C upto 90 days. While for P.vivax only two RDTs maintained good sensitivity upto day 90 when compared with RDTs kept at room temperature. Agreement between observers was excellent for positive and negative readings for both P.falciparum and P.vivax (Kappa >0.6–0.9).

Conclusion

This is first field evaluation of RDTs regarding their temperature stability. Although RDTs are useful as diagnostic tool for P.falciparum and P.vivax even at high temperature, the quality of RDTs should be regulated and monitored more closely.     

αADα Hybrids of Cryptococcus neoformans: Evidence of Same-Sex Mating in Nature and Hybrid Fitness

Cryptococcus neoformans is a ubiquitous human fungal pathogen that causes meningoencephalitis in predominantly immunocompromised hosts. The fungus is typically haploid, and sexual reproduction involves two individuals with opposite mating types/sexes, α and a. However, the overwhelming predominance of mating type (MAT) α over a in C. neoformans populations limits α–a mating in nature. Recently it was discovered that C. neoformans can undergo same-sex mating under laboratory conditions, especially between α isolates. Whether same-sex mating occurs in nature and contributes to the current population structure was unknown. In this study, natural αADα hybrids that arose by fusion between two α cells of different serotypes (A and D) were identified and characterized, providing definitive evidence that same-sex mating occurs naturally. A novel truncated allele of the mating-type-specific cell identity determinant SXI1α was also identified as a genetic factor likely involved in this process. In addition, laboratory-constructed αADα strains exhibited hybrid vigor both in vitro and in vivo, providing a plausible explanation for their relative abundance in nature despite the fact that AD hybrids are inefficient in meiosis/sporulation and are trapped in the diploid state. These findings provide insights on the origins, genetic mechanisms, and fitness impact of unisexual hybridization in the Cryptococcus population.     

Immunization with Brugia malayi Myosin as Heterologous DNA Prime Protein Boost Induces Protective Immunity against B. malayi Infection in Mastomys coucha

The current control strategies employing chemotherapy with diethylcarbamazine, ivermectin and albendazole have reduced transmission in some filaria-endemic areas, there is growing interest for complementary approaches, such as vaccines especially in light of threat of parasite developing resistance to mainstay drugs. We earlier demonstrated recombinant heavy chain myosin of B. malayi (Bm-Myo) as a potent vaccine candidate whose efficacy was enhanced by heterologous DNA prime/protein boost (Myo-pcD+Bm-Myo) vaccination in BALB/c mice. BALB/c mouse though does not support the full developmental cycle of B. malayi, however, the degree of protection may be studied in terms of transformation of challenged infective larvae (L3) to next stage (L4) with an ease of delineating the generated immunological response of host. In the current investigation, DNA vaccination with Bm-Myo was therefore undertaken in susceptible rodent host, Mastomys coucha (M. coucha) which sustains the challenged L3 and facilitates their further development to sexually mature adult parasites with patent microfilaraemia. Immunization schedule consisted of Myo-pcD and Myo-pcD+Bm-Myo followed by B. malayi L3 challenge and the degree of protection was evaluated by observing microfilaraemia as well as adult worm establishment. Myo-pcD+Bm-Myo immunized animals not only developed 78.5% reduced blood microfilarial density but also decreased adult worm establishment by 75.3%. In addition, 75.4% of the recovered live females revealed sterilization over those of respective control animals. Myo-pcD+Bm-Myo triggered higher production of specific IgG and its isotypes which induced marked cellular adhesion and cytotoxicity (ADCC) to microfilariae (mf) and L3 in vitro. Both Th1 and Th2 cytokines were significantly up-regulated displaying a mixed immune response conferring considerable protection against B. malayi establishment by engendering a long-lasting effective immune response and therefore emerges as a potential vaccination method against LF.     

Comparative Membrane Channel Size and Activity of Botulinum Neurotoxins A and E

In an effort to compare the molecular basis of differential toxic activity of botulinum neurotoxin A (BoNT/A) and BoNT/E, we have analyzed their membrane channel activity by measuring calcein release from liposomes. Both BoNT/A and /E showed a same level of membrane channel activity that was specifically blocked by IgG specific to the neurotoxins. With the use of fluorescein-labeled dextran, we determined that the size of the channel is at least 24.2 A which is appropriate for the translocation of a protein of 50 kDa (the light chain of BoNT). These findings would suggest that the difference in the toxicity level of the two BoNT serotypes might reflect differences in either endopeptidase activity or their binding to receptor(s).     

A novel inhibitor protein of N-myristoyltransferase from Escherichia coli

Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristate to the N-terminal of the glycine residue of various eukaryotic and viral proteins of diverse functions. Earlier, we have demonstrated that NMT activity is elevated in colon and gall bladder cancer. Attenuation of NMT activity may prove a novel therapeutic protocol for cancer. We report here a novel inhibitor protein of NMT being expressed in Escherichia coli cells containing the human NMT gene on increasing the incubation period from 5 to 24h. The inhibitor protein was purified by SP-Sepharose column chromatography, heat treatment, ammonium sulfate precipitation, and Superose 12 HR/30 FPLC column chromatography. The inhibitor protein had an apparent molecular mass of 10kDa by gel filtration. It inhibited human NMT in a concentration-dependent manner with 50% inhibition at 640+/-4.68nM. The inhibitor protein showed no direct interaction with myristoyl-CoA and demonstrated no demyristoylase or protease activity. Therefore, we conclude that the inhibitor protein acts directly on NMT.     

Identifying highly connected counties compensates for resource limitations when evaluating national spread of an invasive pathogen

Surveying invasive species can be highly resource intensive, yet near-real-time evaluations of invasion progress are important resources for management planning. In the case of the soybean rust invasion of the United States, a linked monitoring, prediction, and communication network saved U.S. soybean growers approximately $200 M/yr. Modeling of future movement of the pathogen (Phakopsora pachyrhizi) was based on data about current disease locations from an extensive network of sentinel plots. We developed a dynamic network model for U.S. soybean rust epidemics, with counties as nodes and link weights a function of host hectarage and wind speed and direction. We used the network model to compare four strategies for selecting an optimal subset of sentinel plots, listed here in order of increasing performance: random selection, zonal selection (based on more heavily weighting regions nearer the south, where the pathogen overwinters), frequency-based selection (based on how frequently the county had been infected in the past), and frequency-based selection weighted by the node strength of the sentinel plot in the network model. When dynamic network properties such as node strength are characterized for invasive species, this information can be used to reduce the resources necessary to survey and predict invasion progress.