Immunization with an Autotransporter Protein of Orientia tsutsugamushi Provides Protective Immunity against Scrub Typhus

BackgroundScrub typhus is an acute febrile disease caused by Orientia tsutsugamushi infection. Recently, the rapid increase of scrub typhus incidence in several countries within the endemic region has become a serious public health issue. Despite the wide range of preventative approaches that have been attempted in the past 70 years, all have failed to develop an effective prophylactic vaccine. Currently, the selection of the proper antigens is one of the critical barriers to generating cross-protective immunity against antigenically-variable strains of O. tsutsugamushi.Conclusions/SignificanceImmunization of ScaA proteins provides protective immunity in mice when challenged with the homologous strain and significantly enhanced protective immunity against infection with heterologous strains. To our knowledge, this is the most promising result of scrub typhus vaccination trials against infection of heterologous strains in mouse models thus far.     

Homology and enzymatic requirements of microhomology-dependent alternative end joining

Nonhomologous DNA end joining (NHEJ) is one of the major double-strand break (DSB) repair pathways in higher eukaryotes. Recently, it has been shown that alternative NHEJ (A-NHEJ) occurs in the absence of classical NHEJ and is implicated in chromosomal translocations leading to cancer. In the present study, we have developed a novel biochemical assay system utilizing DSBs flanked by varying lengths of microhomology to study microhomology-mediated alternative end joining (MMEJ). We show that MMEJ can operate in normal cells, when microhomology is present, irrespective of occurrence of robust classical NHEJ. Length of the microhomology determines the efficiency of MMEJ, 5 nt being obligatory. Using this biochemical approach, we show that products obtained are due to MMEJ, which is dependent on MRE11, NBS1, LIGASE III, XRCC1, FEN1 and PARP1. Thus, we define the enzymatic machinery and microhomology requirements of alternative NHEJ using a well-defined biochemical system.DNA double-strand breaks (DSBs) are the most deleterious to the genome among various lesions. Nonhomologous end joining (NHEJ) is one of the major DSB repair pathways in higher eukaryotes.^{1, 2, 3} In the absence of key NHEJ factors, another distinct but error-prone pathway known as alternative NHEJ (A-NHEJ) has been described to have an important role in DSB repair.^{4, 5, 6, 7} It has been shown that majority of A-NHEJ-mediated repair of DSBs utilize distinct microhomology regions, hence termed microhomology-mediated end joining (MMEJ).^{4, 8, 9}A-NHEJ has been proposed as a possible cause for chromosomal translocations. Studies have shown co-amplification of c-MYC and IgH locus from pro-B lymphomas in mice deficient for p53 and NHEJ.¹⁰ A reduced level of class switch recombination (CSR) and increased number of chromosomal rearrangements at IgH locus have been shown in XRCC4- and LIGASE IV-deficient murine B cells.⁸ The occurrence of robust alternative end joining has been reported in the absence of NHEJ proteins, when murine RAG proteins were absent.¹¹Unraveling the enzymatic machinery involved in alternative end joining is currently an active area of research. Recently, it was shown that MRE11-RAD50-NBS1 complex may be involved in a subset of alternative NHEJ,^{5, 12, 13, 14} whereas ATM has a regulatory role.¹⁵ Role of PARP1 in repairing switch regions through a microhomology-mediated pathway leading to IgH/c-MYC translocations during immunoglobulin CSR has been described.¹⁶ Besides, studies have also suggested a role for DNA LIGASE IIIα and WRN in A-NHEJ.¹⁷ Interestingly, XRCC1 was shown to be dispensable in A-NHEJ during CSR, whereas functional relevance of Ligase I, III and Pol λ have been established.^{18, 19, 20} Hence, it can be concluded that canonical NHEJ (C-NHEJ) requires LIGASE IV–XRCC4 complex, while A-NHEJ is predominant in the absence of C-NHEJ proteins and is mainly characterized by joining utilizing microhomology (MMEJ). Further, it has been demonstrated that RPA, when bound to single-stranded DNA can antagonize MMEJ.²¹ Very recently, a genetic system was reported in budding yeast to detect microhomology-mediated repair.²² However, little is known whether alternative NHEJ can be operative when classical NHEJ machinery is intact.²³ A recent study suggested that MMEJ is also functional in normal mammalian cells. Besides, HR and MMEJ share the initial steps of end resection for DSB repair in mammalian cells.²⁴ However, it appears that there is not much consensus among different research groups over its presence and relevance in normal cells.²³ Therefore, several aspects of alternative NHEJ still need to be resolved. For example, its precise mechanism and microhomology length requirements are yet to be fully uncovered. Its occurrence in normal cells needs to be proved beyond doubt. Although there are independent studies showing the role of multiple proteins using gene knockdown or knockout strategies, their involvement needs to be confirmed.In the present study, we have established a cell-free repair assay system using which we show that MMEJ is operative even in the presence of classical NHEJ machinery. Further, our data suggest that MMEJ operates not only in cancer cells but also in normal cells. We show that a minimum of 5 nt microhomology is required for MMEJ and is independent of classical NHEJ proteins such as KU70, KU80 and LIGASE IV. Finally, we show that MRN complex, XRCC1, FEN1, PARP1 and LIGASE III are the factors responsible for joining mediated through microhomology.     

Characterization of the MUC1-C Cytoplasmic Domain as a Cancer Target

Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly expressed in diverse human carcinomas and certain hematologic malignancies. The oncogenic MUC1 transmembrane C-terminal subunit (MUC1-C) functions in part by transducing growth and survival signals from cell surface receptors. However, little is known about the structure of the MUC1-C cytoplasmic domain as a potential drug target. Using methods for structural predictions, our results indicate that a highly conserved CQCRRK sequence, which is adjacent to the cell membrane, forms a small pocket that exposes the two cysteine residues for forming disulfide bonds. By contrast, the remainder of the MUC1-C cytoplasmic domain has no apparent structure, consistent with an intrinsically disordered protein. Our studies thus focused on targeting the MUC1 CQCRRK region. The results show that L- and D-amino acid CQCRRK-containing peptides bind directly to the CQC motif. We further show that the D-amino acid peptide, designated GO-203, blocks homodimerization of the MUC1-C cytoplasmic domain in vitro and in transfected cells. Moreover, GO-203 binds directly to endogenous MUC1-C in breast and lung cancer cells. Colocalization studies further demonstrate that GO-203 predominantly binds to MUC1-C at the cell membrane. These findings support the further development of agents that target the MUC1-C cytoplasmic domain CQC motif and thereby MUC1-C function in cancer cells.     

A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins

Staphylococcal and streptococcal exotoxins, also known as superantigens, mediate a range of diseases including toxic shock syndrome, and they exacerbate skin, pulmonary and systemic infections caused by these organisms. When present in food sources they can cause enteric effects commonly known as food poisoning. A rapid, sensitive assay for the toxins would enable testing of clinical samples and improve surveillance of food sources. Here we developed a bead-based, two-color flow cytometry assay using single protein domains of the beta chain of T cell receptors engineered for high-affinity for staphylococcal (SEA, SEB and TSST-1) and streptococcal (SpeA and SpeC) toxins. Site-directed biotinylated forms of these high-affinity agents were used together with commercial, polyclonal, anti-toxin reagents to enable specific and sensitive detection with SD₅₀ values of 400 pg/ml (SEA), 3 pg/ml (SEB), 25 pg/ml (TSST-1), 6 ng/ml (SpeA), and 100 pg/ml (SpeC). These sensitivities were in the range of 4- to 80-fold higher than achieved with standard ELISAs using the same reagents. A multiplex format of the assay showed reduced sensitivity due to higher noise associated with the use of multiple polyclonal agents, but the sensitivities were still well within the range necessary for detection in food sources or for rapid detection of toxins in culture supernatants. For example, the assay specifically detected toxins in supernatants derived from cultures of Staphylococcus aureus. Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of Staphylococcus aureus and Streptococcus pyogenes.     

Functional Impairment of Myeloid Dendritic Cells during Advanced Stage of HIV-1 Infection: Role of Factors Regulating Cytokine Signaling

Introduction

Severely immunocompromised state during advanced stage of HIV-1 infection has been linked to functionally defective antigen presentation by dendritic cells (DCs). The molecular mechanisms behind DC impairment are still obscure. We investigated changes in DC function and association of key regulators of cytokine signaling during different stages of HIV-1 infection and following antiretroviral therapy (ART).

Methods

Phenotypic and functional characteristics of circulating myeloid DCs (mDCs) in 56 ART-naive patients (23 in early and 33 in advanced stage of disease), 36 on ART and 24 healthy controls were evaluated. Sixteen patients were studied longitudinally prior-to and 6 months after the start of ART. For functional studies, monocyte-derived DCs (Mo-DCs) were evaluated for endocytosis, allo-stimulation and cytokine secretion. The expression of suppressor of cytokine signaling (SOCS)-1 and other regulators of cytokine signaling was evaluated by real-time RT-PCR.

Results

The ability to respond to an antigenic stimulation was severely impaired in patients in advanced HIV-1 disease which showed partial recovery in the treated group. Mo-DCs from patients with advanced HIV-disease remained immature with low allo-stimulation and reduced cytokine secretion even after TLR-4 mediated stimulation ex-vivo. The cells had an increased expression of negative regulatory factors like SOCS-1, SOCS-3, SH2-containing phosphatase(SHP)-1 and a reduced expression of positive regulators like Janus kinase(JAK)2 and Nuclear factor kappa-light-chain-enhancer of activated B cells(NF-κB)1. A functional recovery after siRNA mediated silencing of SOCS-1 in these mo-DCs confirms the role of negative regulatory factors in functional impairment of these cells.

Conclusions

Functionally defective DCs in advanced stage of HIV-1 infection seems to be due to imbalanced state of negative and positive regulatory gene expression. Whether this is a cause or effect of increased viral replication at this stage of disease, needs further investigation. The information may be useful in design of novel therapeutic targets for better management of disease.     

Construction of a Genetic Linkage Map and Identification of QTLs for Seed Weight and Seed Size Traits in Lentil (Lens culinaris Medik.)

Seed weight and seed size both are quantitative traits and have been considered as important components of grain yield, thus identification of quantitative trait loci (QTL) for seed traits in lentil (Lens culinaris) would be beneficial for the improvement of grain yield. Hence the main objective of this study was to identify QTLs for seed traits using an intraspecific mapping population derived from a cross between L. culinaris cv. Precoz (seed weight-5.1g, seed size-5.7mm) and L. culinaris cv. L830 (seed weight-2.2g, seed size-4mm) comprising 126 F₈-RILs. For this, two microsatellite genomic libraries enriched for (GA/CT) and (GAA/CTT) motif were constructed which resulted in the development of 501 new genomic SSR markers. Six hundred forty seven SSR markers (including 146 previously published) were screened for parental polymorphism and 219 (33.8%) were found to be polymorphic among the parents. Of these 216 were mapped on seven linkage groups at LOD4.0 spanning 1183.7cM with an average marker density of 5.48cM. Phenotypic data from the RILs was used to identify QTLs for the seed weight and seed size traits by single marker analysis (SMA) followed by composite interval mapping (CIM) which resulted in one QTL each for the 2 traits (qSW and qSS) that were co-localized on LG4 and explained 48.4% and 27.5% of phenotypic variance respectively. The current study would serve as a strong foundation for further validation and fine mapping for utilization in lentil breeding programs.     

A Coarse Grained Model for a Lipid Membrane with Physiological Composition and Leaflet Asymmetry

The resemblance of lipid membrane models to physiological membranes determines how well molecular dynamics (MD) simulations imitate the dynamic behavior of cell membranes and membrane proteins. Physiological lipid membranes are composed of multiple types of phospholipids, and the leaflet compositions are generally asymmetric. Here we describe an approach for self-assembly of a Coarse-Grained (CG) membrane model with physiological composition and leaflet asymmetry using the MARTINI force field. An initial set-up of two boxes with different types of lipids according to the leaflet asymmetry of mammalian cell membranes stacked with 0.5 nm overlap, reliably resulted in the self-assembly of bilayer membranes with leaflet asymmetry resembling that of physiological mammalian cell membranes. Self-assembly in the presence of a fragment of the plasma membrane protein syntaxin 1A led to spontaneous specific positioning of phosphatidylionositol(4,5)bisphosphate at a positively charged stretch of syntaxin consistent with experimental data. An analogous approach choosing an initial set-up with two concentric shells filled with different lipid types results in successful assembly of a spherical vesicle with asymmetric leaflet composition. Self-assembly of the vesicle in the presence of the synaptic vesicle protein synaptobrevin 2 revealed the correct position of the synaptobrevin transmembrane domain. This is the first CG MD method to form a membrane with physiological lipid composition as well as leaflet asymmetry by self-assembly and will enable unbiased studies of the incorporation and dynamics of membrane proteins in more realistic CG membrane models.     

Treatment-Induced Changes in Plasma Adiponectin Do Not Reduce Urinary Albumin Excretion in the Diabetes Prevention Program Cohort

Background and Objectives

Molecular data suggests that adiponectin may directly regulate urinary albumin excretion. In the Diabetes Prevention Program (DPP) we measured adiponectin and albuminuria before and after intervention, and we previously reported increases in adiponectin with interventions. Here we have used the DPP dataset to test the hypothesis that treatment-related increases in adiponectin may reduce albuminuria in obesity.

Design, Setting, Participants and Methods

We evaluated cross-sectional correlations between plasma adiponectin and urinary albumin excretion at baseline, and the relationship of treatment-related changes in adiponectin and albuminuria. Baseline and follow-up urine albumin to creatinine ratios (ACR (albumin to creatinine ratio)) and plasma adiponectin concentration were available in 2553 subjects.

Results

Adjusting for age, sex and race/ethnicity, we observed a statistically significant but weak inverse relationship between adiponectin and ACR at baseline (conditional Spearman’s rho = (-) 0.04, p = 0.04). Although DPP treatments significantly increased plasma adiponectin, there were no treatment effects on ACR and no differences in ACR across treatment groups. There was a weak direct (not inverse) association between change in adiponectin and change in albuminuria (adjusted Spearman’s rho = (+) 0.04, p = 0.03).

Conclusions

In a large, well-characterized cohort of obese dysglycemic subjects we observed a weak inverse association between circulating adiponectin concentrations and urinary albumin excretion at baseline. Contrary to the hypothesized effect, treatment-related increases in plasma adiponectin were not associated with a reduction in ACR. The association of change in adiponectin with change in ACR should be assessed in populations with overt albuminuria before excluding a beneficial effect of increasing adiponectin to reduce ACR in obesity.     

Staff Perception on Biomedical or Health Care Waste Management: A Qualitative Study in a Rural Tertiary Care Hospital in India

Background

Health care or biomedical waste, if not managed properly, can be of high risk to the hospital staff, the patients, the community, public health and the environment, especially in low and middle income settings where proper disposal norms are often not followed. Our aim was to explore perceptions of staff of an Indian rural tertiary care teaching hospital on hospital waste management.

Method

A qualitative study was conducted using 10 focus group discussions (FGDs), with different professional groups, cleaning staff, nurses, medical students, doctors and administrators. The FGD guide included the following topics: (i) role of Health Care Waste Management (HCWM) in prevention of health care associated infections, (ii) awareness of and views about HCWM-related guidelines/legislation, (iii) current HCWM practices, (iv) perception and preparedness related to improvements of the current practices, and (v) proper implementation of the available guidelines/legislation. The FGDs were recorded, transcribed verbatim, translated to English (when conducted in Hindi) and analysed using content analysis.

Results

Two themes were identified: Theme (A), ‘Challenges in integration of HCWM in organizational practice,’ with the categories (I) Awareness and views about HCWM, (II) Organizational practices regarding HCWM, and (III) Challenges in Implementation of HCWM; and Theme (B), ‘Interventions to improve HCWM,’ with three categories, (I) Educational and motivational interventions, (II) Organizational culture change, and (III) Policy-related interventions.

Conclusion

A gap between knowledge and actual practice regarding HCWM was highlighted in the perception of the hospital staff. The participants suggested organizational changes, training and monitoring to address this. The information generated is relevant not merely to the microsystem studied but to other institutions in similar settings.