Structural–functional insights of single and multi-domain Capsicum annuum protease inhibitors

Pin-II protease inhibitors (PIs) are the focus of research interest because of their large structural–functional diversity and relevance in plant defense. Two representative Capsicum annuum PI genes (CanPI-15 and -7) comprising one and four inhibitory repeat domains, respectively, were expressed and recombinant proteins were characterized. β-Sheet and unordered structure was found predominant in CanPI-15 while -7 also displayed the signatures of polyproline fold, as revealed by circular dichroism studies. Inhibition kinetics against bovine trypsin indicated three times higher potency of CanPI-7 (K_i ～ 57 μM) than -15 (～184 μM). Activity and structural stability of these CanPIs were revealed under various conditions of pH, temperature and denaturing agent. Structure prediction, docking studies with proteases and mass spectroscopy revealed the organization of multiple reactive site loops of multi domain PIs in space as well as the steric hindrances imposed while binding to proteases due to their close proximity.     

Zinc regulates the activity of kinase-phosphatase pair (BasPrkC/BasPrpC) in Bacillus anthracis

Bacillus anthracis Ser/Thr protein kinase PrkC (BasPrkC) is important for virulence of the bacterium within the host. Homologs of PrkC and its cognate phosphatase PrpC (BasPrpC) are the most conserved mediators of signaling events in diverse bacteria. BasPrkC homolog in Bacillus subtilis regulates critical processes like spore germination and BasPrpC modulates the activity of BasPrkC by dephosphorylation. So far, biochemical and genetic studies have provided important insights into the roles of BasPrkC and BasPrpC; however, regulation of their activities is not known. We studied the regulation of BasPrkC/BasPrpC pair and observed that Zn²⁺ metal ions can alter their activities. Zn²⁺ promotes BasPrkC kinase activity while inhibits the BasPrpC phosphatase activity. Concentration of Zn²⁺ in growing B. anthracis cells was found to vary with growth phase. Zn²⁺ was found to be lowest in log phase cells while it was highest in spores. This variation in Zn²⁺ concentration is significant for understanding the antagonistic activities of BasPrkC/BasPrpC pair. Our results also show that BasPrkC activity is modulated by temperature changes and kinase inhibitors. Additionally, we identified Elongation Factor Tu (BasEf-Tu) as a substrate of BasPrkC/BasPrpC pair and assessed the impact of their regulation on BasEf-Tu phosphorylation. Based on these results, we propose Zn²⁺ as an important regulator of BasPrkC/BasPrpC mediated phosphorylation cascades. Thus, this study reveals additional means by which BasPrkC can be activated leading to autophosphorylation and substrate phosphorylation.     

An ontological crisis? A review of large felid conservation in India

The need for a solid knowledge base to inform conservation activity is now universally recognised. We critically scrutinised the scientific knowledge of large felids in India located in peer-reviewed research papers to assess the information available to make landscape-level management decisions that aid conservation, which is a stated goal of both the Indian government and the international community. We found two striking patterns: the biological sciences dominate in the published literature, and nearly all the research has been carried out in protected areas, though a substantial number of large felids also live outside protected areas. We argue that these patterns are not incidental, but the result of the dualistic ontology of science that uses processes of ‘purification’ and ‘translation’ to fit complex realities into disciplinary prerogatives organised around creating dichotomies (like nature–culture). In addition, since this body of scientific knowledge locates large felids in ‘pure’ biological landscapes, there is little or no insight from multi-use landscapes. These findings, we believe, highlight important knowledge gaps in our present research-based knowledge of large felids in India, which urgently need to be addressed if progress is to be made in conservation.     

Analysis of FGF-Dependent and FGF-Independent Pathways in Otic Placode Induction

The inner ear develops from a patch of thickened cranial ectoderm adjacent to the hindbrain called the otic placode. Studies in a number of vertebrate species suggest that the initial steps in induction of the otic placode are regulated by members of the Fibroblast Growth Factor (FGF) family, and that inhibition of FGF signaling can prevent otic placode formation. To better understand the genetic pathways activated by FGF signaling during otic placode induction, we performed microarray experiments to estimate the proportion of chicken otic placode genes that can be up-regulated by the FGF pathway in a simple culture model of otic placode induction. Surprisingly, we find that FGF is only sufficient to induce about 15% of chick otic placode-specific genes in our experimental system. However, pharmacological blockade of the FGF pathway in cultured chick embryos showed that although FGF signaling was not sufficient to induce the majority of otic placode-specific genes, it was still necessary for their expression in vivo. These inhibitor experiments further suggest that the early steps in otic placode induction regulated by FGF signaling occur through the MAP kinase pathway. Although our work suggests that FGF signaling is necessary for otic placode induction, it demonstrates that other unidentified signaling pathways are required to co-operate with FGF signaling to induce the full otic placode program.     

Effect of Physical Violence on Sexually Transmitted Infections and Treatment Seeking Behaviour among Female Sex Workers in Thane District,Maharashtra, India

Background

Violence against sex workers can heighten their vulnerability to HIV and other sexually transmitted infections (STIs). Evidence suggests the risk of acquiring STI/HIV infections among female sex workers (FSWs) who have experienced violence to be almost three-times higher than FSWs, who have not experienced violence. Moreover, an experience of physical and sexual violence makes it difficult for them to negotiate safer sex with their partners and often act as a barrier to utilization of prevention services.

Methods

This study utilizes data from 2785 FSWs aged 18 years and above who participated in a cross-sectional behavioural study conducted during 2013–14 in Thane district, Maharashtra. A probability-based two-stage cluster sampling method was used for data collection. This study assesses the effect of physical violence on self-reported STI symptoms (any STI and multiple STIs) and treatment seeking for the last STI symptom using propensity score matching method.

Results

About 18% of sampled FSWs reported physical violence at the time of the survey. The likelihood of experiencing such violence was significantly higher among FSWs who solicited clients at public places, engaged in other economic activities apart from sex work, had savings, and reported high client volume per week. FSWs experiencing violence were also inconsistent condom users while engaging in sex with regular partners and clients. The average adjusted effect of violence clearly depicted an increase in the risk of any STI (11%, p<0.05) and multiple STIs (8%, p<0.10) and reduction in treatment seeking (10%, p<0.05).

Conclusions

This study demonstrates a significant effect of physical violence on reporting of any STI symptom and treatment seeking. Findings call for the immediate inclusion of strategies aimed to address violence related challenges in HIV prevention program currently being provided at Thane district. Such strategies would further help in enhancing the access to tailored STI prevention and care services among FSWs in the district.     

Induction of IL-10 and TGFβ from CD4+CD25+FoxP3+ T Cells Correlates with Parasite Load in Indian Kala-azar Patients Infected with Leishmania donovani

Background

Visceral leishmaniasis (VL) is distinguished by a complex interplay of immune response and parasite multiplication inside host cells. However, the direct association between different immunological correlates and parasite numbers remains largely unknown.

Methodology/Principal Findings

We examined the plasma levels of different disease promoting/protective as well as Th17 cytokines and found IL-10, TGFβ and IL-17 to be significantly correlated with parasite load in VL patients (r = 0.52, 0.53 and 0.51 for IL-10, TGFβ and IL-17, respectively). We then extended our investigation to a more antigen-specific response and found leishmanial antigen stimulated levels of both IL-10 and TGFβ to be significantly associated with parasite load (r = 0.71 and 0.72 for IL-10 and TGFβ respectively). In addition to cytokines we also looked for different cellular subtypes that could contribute to cytokine secretion and parasite persistence. Our observations manifested an association between different Treg cell markers and disease progression as absolute numbers of CD4⁺CD25⁺ (r = 0.55), CD4⁺CD25^hi (r = 0.61) as well as percentages of CD4⁺CD25⁺FoxP3⁺ T cells (r = 0.68) all correlated with parasite load. Encouraged by these results, we investigated a link between these immunological components and interestingly found both CD4⁺CD25⁺ and CD4⁺CD25⁺FoxP3⁺ Treg cells to secrete significantly (p<0.05) higher amounts of not only IL-10 but also TGFβ in comparison to corresponding CD25^- T cells.

Conclusions/Significance

Our findings shed some light on source(s) of TGFβ and suggest an association between these disease promoting cytokines and Treg cells with parasite load during active disease. Moreover, the direct evidence of CD4⁺CD25⁺FoxP3⁺ Treg cells as a source of IL-10 and TGFβ during active VL could open new avenues for immunotherapy towards cure of this potentially fatal disease.