NMR chemical shift and relaxation measurements provide evidence for the coupled folding and binding of the p53 transactivation domain

The interaction between the acidic transactivation domain of the human tumor suppressor protein p53 (p53TAD) and the 70 kDa subunit of human replication protein A (hRPA70) was investigated using heteronuclear magnetic resonance spectroscopy. A ¹H–¹⁵N heteronuclear single quantum coherence (HSQC) titration experiment was performed on a ¹⁵N-labeled fragment of hRPA70, containing the N-terminal 168 residues (hRPA70_1–168) and p53TAD. HRPA70_1–168 residues important for binding were identified and found to be localized to a prominent basic cleft. This binding site overlapped with a previously identified single-stranded DNA-binding site, suggesting that a competitive binding mechanism may regulate the formation of p53TAD–hRPA70 complex. The amide ¹H and ¹⁵N chemical shifts of an uniformly ¹⁵N-labeled sample of p53TAD were also monitored before and after the addition of unlabeled hRPA70_1–168. In the presence of unlabeled hRPA70_1–168, resonance lineshapes increased and corresponding intensity reductions were observed for specific p53TAD residues. The largest intensity reductions were observed for p53TAD residues 42–56. Minimal binding was observed between p53TAD and a mutant form of hRPA70_1–168, where the basic cleft residue R41 was changed to a glutamic acid (R41E), demonstrating that ionic interactions play an important role in specifying the binding interface. The region of p53TAD most affected by binding hRPA70_1–168 was found to have some residual alpha helical and beta strand structure; however, this structure was not stabilized by binding hRPA70_1–168. ¹⁵N relaxation experiments were performed to monitor changes in backbone dynamics of p53TAD when bound to hRPA70_1–168. Large changes in both the transverse (R₂) and rotating frame (R_1ρ) relaxation rates were observed for a subset of the p53TAD residues that had ¹H–¹⁵N HSQC resonance intensity reductions during the complex formation. The folding of p53TAD upon complex formation is suggested by the pattern of changes observed for both R₂ and R_1ρ. A model that couples the formation of a weak encounter complex between p53TAD and hRPA70_1–168 to the folding of p53TAD is discussed in the context of a functional role for the p53–hRPA70 complex in DNA repair.     

Hepatitis B,C, and D virus and human T-cell leukemia virus types 1 and 2 infections and correlates among men who have sex with men in Ouagadougou,Burkina Faso

Background

Men who have sex with men (MSM) are considered to be at significant risk for sexually transmitted infections (STI) and bloodborne viruses including viral hepatitis types B, C, and D (HBV, HCV, and HDV) and human T-cell leukemia virus types 1 and 2 (HTLV 1&2). This study aimed to assess the seroprevalence and correlates of HBV, HCV, HDV, and HTLV 1&2 antibodies among MSM in Ouagadougou, Burkina Faso.

Methods

We conducted a cross-sectional survey to assess the biological and behavourial characteristics among MSM in Ouagadougou from January to April 2013. Serum specimens obtained were tested for the presence of HBV, HCV, HDV and HTLV-1&2 infections. MSM 18?years and older were recruited using respondent driven sampling (RDS). Population estimates and 95% confidence intervals (CI) adjusted for the RDS design were calculated using RDS Analysis Tool (RDSAT) version 6.0.1 (RDS, Inc., Ithaca, NY). Bivariate and multivariate logistic regression analyses were conducted to assess correlates of these infections using Stata 14.

Results

A total of 329 MSM were tested. Prevalence was 20.4% (95% CI: 16.4–25.1) for HBV, 11.0% (95% CI: 8.0–14.8) for HCV, and 0.0% for HDV. Anti-HTLV 1&2 antibodies were found in 4.0% (95% CI: 2.3–6.8) of MSM. Factors independently associated with HBV infection were lack of condom use during the last anal sex act with a main male sexual partner and experience of condom tearing during anal sex. Presence of anti-HTLV 1&2 antibodies was associated with history of genital or anal lesions and injection drug use. None of the variables included in our study were associated with HCV.

Conclusions

This study shows that HBV, HCV and HTLV 1&2 prevalence among MSM in Burkina is high and suggests that comprehensive STI prevention and sexual health education services for this group are needed.

     

<Emphasis Type="Italic">Cryptodiscus muriformis</Emphasis> and <Emphasis Type="Italic">Schizoxylon gilenstamii</Emphasis>, two new species of Stictidaceae (Ascomycota)

Cryptodicus muriformis and Schizoxylon gilenstamii (Stictidaceae, Ostropales, Lecanoromycetes, Ascomycota) are described as new to science from collections made in Europe (Sweden, Austria, Switzerland) and North America (Arizona), based on phylogenetic analyses of three loci. Both species show unique morphological characters that distinguish them from closely related species; Cryptodiscus muriformis, growing on decorticated or sometimes corticated branches of Picea abies and Pseudotsuga menziesii, that resembles C. foveolaris and C. tabularum in the yellowish-orange ascomatal disc, but is clearly distinguished by the muriform ascospores. Schizoxylon gilenstamii grows on dead stems of Globularia vulgaris and has a characteristic unique red-purple pigment in the epithecium and upper part of the excipulum that changes to bluish-green with KOH. We also report for the first time the anamorph of Stictis brunnescens.     

Human Rights Violations among Men Who Have Sex with Men in Southern Africa: Comparisons between Legal Contexts

In 1994, South Africa approved a constitution providing freedom from discrimination based on sexual orientation. Other Southern African countries, including Botswana, Malawi, and Namibia, criminalize same-sex behavior. Men who have sex with men (MSM) have been shown to experience high levels of stigma and discrimination, increasing their vulnerability to negative health and other outcomes. This paper examines the relationship between criminalization of same-sex behavior and experiences of human rights abuses by MSM. It compares the extent to which MSM in peri-urban Cape Town experience human rights abuses with that of MSM in Gaborone, Botswana; Blantyre and Lilongwe, Malawi; and Windhoek, Namibia. In 2008, 737 MSM participated in a cross-sectional study using a structured survey collecting data regarding demographics, human rights, HIV status, and risk behavior. Participants accrued in each site were compared using bivariate and multivariate logistic regression. Encouragingly, the results indicate MSM in Cape Town were more likely to disclose their sexual orientation to family or healthcare workers and less likely to be blackmailed or feel afraid in their communities than MSM in Botswana, Malawi, or Namibia. However, South African MSM were not statistically significantly less likely experience a human rights abuse than their peers in cities in other study countries, showing that while legal protections may reduce experiences of certain abuses, legislative changes alone are insufficient for protecting MSM. A comprehensive approach with interventions at multiple levels in multiple sectors is needed to create the legal and social change necessary to address attitudes, discrimination, and violence affecting MSM.     

Dendritic cell-derived IL-12 is not required for the generation of cytotoxic, IFN-gamma-secreting, CD8(+) CTL in vivo.

By using adoptive transfer of Ag-loaded bone marrow-derived dendritic cells (BMDC), we have established an in vivo model of CTL priming. Activation of CTL in these experiments required both CD4(+) T cells and CD154, demonstrating that this model reflects CD4(+) T cell-dependent dendritic cell (DC) licensing. Because IL-12 has been suggested to play an important role in CTL activation by DC, we examined the ability of BMDC to prime CTL in the complete absence of IL-12 using p40-deficient mice. We observed that the absence of IL-12 does not affect the phenotype or allostimulatory function of BMDC after in vitro maturation. Moreover, there was no difference in the ability of Ag-loaded DC to elicit CTL cytotoxicity, whether the Ag was delivered by virus infection or peptide pulsing. Equal frequencies of Ag-specific, IFN-gamma-secreting CD8(+) T cells developed in both wild-type and IL-12-deficient backgrounds. Finally, CTL generated in the IL-12-deficient environment were capable of protecting immunized mice against tumor challenge, demonstrating that these CTL were fully functional, despite the absence of IL-12 during the maturation process in vivo. These results indicate that IL-12 is not critical for the development of IFN-gamma secreting, CD8(+) T cells and that another mechanism must be used by licensed DC to prime and activate CTL.     

Patterns and determinants of elephant attacks on humans in Nepal

Attacks on humans by Asian elephant (Elephas maximus) is an extreme form of human–elephant conflict. It is a serious issue in southern lowland Nepal where elephant‐related human fatalities are higher than other wildlife. Detailed understanding of elephant attacks on humans in Nepal is still lacking, hindering to devising appropriate strategies for human–elephant conflict mitigation. This study documented spatiotemporal pattern of elephant attacks on humans, factors associated with the attacks, and human/elephant behavior contributing to deaths of victims when attacked. We compiled all the documented incidences of elephant attacks on humans in Nepal for last 20 years across Terai and Chure region of Nepal. We also visited and interviewed 412 victim families (274 fatalities and 138 injuries) on elephant attacks. Majority of the victims were males (87.86%) and had low level of education. One fourth of the elephant attacks occurred while chasing the elephants. Solitary bulls or group of subadult males were involved in most of the attack. We found higher number of attacks outside the protected area. People who were drunk and chasing elephants using firecrackers were more vulnerable to the fatalities. In contrast, chasing elephants using fire was negatively associated with the fatalities. Elephant attacks were concentrated in proximity of forests primarily affecting the socioeconomically marginalized communities. Integrated settlement, safe housing for marginalized community, and community grain house in the settlement should be promoted to reduce the confrontation between elephants and humans in entire landscape for their long‐term survival.     

Phosphorylation Status of 72 kDa MMP-2 Determines Its Structure and Activity in Response to Peroxynitrite

Matrix metalloproteinase-2 (MMP-2) is a key intra- and extra-cellular protease which contributes to several oxidative stress related pathologies. A molecular understanding of 72 kDa MMP-2 activity, directly mediated by S-glutathiolation of its cysteine residues in the presence of peroxynitrite (ONOO⁻) and by phosphorylation of its serine and threonine residues, is essential to develop new generation inhibitors of intracellular MMP-2. Within its propeptide and collagen binding domains there is an interesting juxtaposition of predicted phosphorylation sites with nearby cysteine residues which form disulfide bonds. However, the combined effect of these two post-translational modifications on MMP-2 activity has not been studied. The activity of human recombinant 72 kDa MMP-2 (hrMMP-2) following in vitro treatments was measured by troponin I proteolysis assay and a kinetic activity assay using a fluorogenic peptide substrate. ONOO⁻ treatment in the presence of 30 µM glutathione resulted in concentration-dependent changes in MMP-2 activity, with 0.1–1 µM increasing up to twofold and 100 µM attenuating its activity. Dephosphorylation of MMP-2 with alkaline phosphatase markedly increased its activity by sevenfold, either with or without ONOO⁻. Dephosphorylation of MMP-2 also affected the conformational structure of the enzyme as revealed by circular dichroism studies, suggesting an increase in the proportion of α-helices and a decrease in β-strands compared to the phosphorylated form of MMP-2. These results suggest that ONOO⁻ activation (at low µM) and inactivation (at high µM) of 72 kDa MMP-2, in the presence or absence of glutathione, is also influenced by its phosphorylation status. These insights into the role of post-translational modifications in the structure and activity of 72 kDa MMP-2 will aid in the development of inhibitors specifically targeting intracellular MMP-2.     

Transition of the prion protein from a structured cellular form (PrPC) to the infectious scrapie agent (PrPSc)

Prion diseases in mammals are caused by a conformational transition of the cellular prion protein from its native conformation (PrP^C) to a pathological isoform called “prion protein scrapie” (PrP^Sc). A molecular level of understanding of this conformational transition will be helpful in unveiling the disease etiology. Experimental structural biological techniques (NMR and X‐ray crystallography) have been used to unravel the atomic level structural information for the prion and its binding partners. More than one hundred three‐dimensional structures of the mammalian prions have been deposited in the protein databank. Structural studies on the prion protein and its structural transitions will deepen our understanding of the molecular basis of prion pathogenesis and will provide valuable guidance for future structure‐based drug discovery endeavors.     

Comprehensive SNP Scan of DNA Repair and DNA Damage Response Genes Reveal Multiple Susceptibility Loci Conferring Risk to Tobacco Associated Leukoplakia and Oral Cancer

Polymorphic variants of DNA repair and damage response genes play major role in carcinogenesis. These variants are suspected as predisposition factors to Oral Squamous Cell Carcinoma (OSCC). For identification of susceptible variants affecting OSCC development in Indian population, the “maximally informative” method of SNP selection from HapMap data to non-HapMap populations was applied. Three hundred twenty-five SNPs from 11 key genes involved in double strand break repair, mismatch repair and DNA damage response pathways were genotyped on a total of 373 OSCC, 253 leukoplakia and 535 unrelated control individuals. The significantly associated SNPs were validated in an additional cohort of 144 OSCC patients and 160 controls. The rs12515548 of MSH3 showed significant association with OSCC both in the discovery and validation phases (discovery P-value: 1.43E-05, replication P-value: 4.84E-03). Two SNPs (rs12360870 of MRE11A, P-value: 2.37E-07 and rs7003908 of PRKDC, P-value: 7.99E-05) were found to be significantly associated only with leukoplakia. Stratification of subjects based on amount of tobacco consumption identified SNPs that were associated with either high or low tobacco exposed group. The study reveals a synergism between associated SNPs and lifestyle factors in predisposition to OSCC and leukoplakia.     

Neem Leaf Glycoprotein Activates CD8+ T Cells to Promote Therapeutic Anti-Tumor Immunity Inhibiting the Growth of Mouse Sarcoma

In spite of sufficient data on Neem Leaf Glycoprotein (NLGP) as a prophylactic vaccine, little knowledge currently exists to support the use of NLGP as a therapeutic vaccine. Treatment of mice bearing established sarcomas with NLGP (25 µg/mice/week subcutaneously for 4 weeks) resulted in tumor regression or dormancy (Tumor free/Regressor, 13/24 (NLGP), 4/24 (PBS)). Evaluation of CD8⁺ T cell status in blood, spleen, TDLN, VDLN and tumor revealed increase in cellular number. Elevated expression of CD69, CD44 and Ki67 on CD8⁺ T cells revealed their state of activation and proliferation by NLGP. Depletion of CD8⁺ T cells in mice at the time of NLGP treatment resulted in partial termination of tumor regression. An expansion of CXCR3⁺ and CCR5⁺ T cells was observed in the TDLN and tumor, along with their corresponding ligands. NLGP treatment enhances type 1 polarized T-bet expressing T cells with downregulation of GATA3. Treg cell population was almost unchanged. However, T∶Treg ratios significantly increased with NLGP. Enhanced secretion/expression of IFNγ was noted after NLGP therapy. In vitro culture of T cells with IL-2 and sarcoma antigen resulted in significant enhancement in cytotoxic efficacy. Consistently higher expression of CD107a was also observed in CD8⁺ T cells from tumors. Reinoculation of sarcoma cells in tumor regressed NLGP-treated mice maintained tumor free status in majority. This is correlated with the increment of CD44^hiCD62L^hi central memory T cells. Collectively, these findings support a paradigm in which NLGP dynamically orchestrates the activation, expansion, and recruitment of CD8⁺ T cells into established tumors to operate significant tumor cell lysis.