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Aparna Rao,1,2 Prity Sahay,1,2 Munmun Chakraborty,1,2 Birendra Kumar Prusty,3 Sandhya Srinivasan,4 Gagan deep Jhingan,4 Pragyan Mishra Snr,2 Rahul Modak,2 Mrutyunjay Suar2 1Glaucoma Service, MTC campus, L.V. Prasad Eye Institute, Bhubaneswar, Odisha, 751024, India; 2KIIT School of Biotechnology, Bhubaneswar, Odisha, India; 3Institute of Life Sciences, Bhubaneswar, Odisha, India; 4Vproteomics, Green Park, New Delhi, IndiaCorrespondence: Aparna Rao Email vinodini10375@yahoo.comPurpose: The key differences in cell death mechanisms in the trabecular meshwork (TM) in adult moderate and severe primary glaucoma remain still unanswered. This study explored key differences in cell death mechanisms in the trabecular meshwork (TM) in adult moderate and severe primary glaucoma.Design: In-vitro laboratory study on surgical specimens and primary cell lines.Methods: Select cell death-related proteins differentially expressed on mass spectrometric analysis in ex-vivo dissected TM specimens patients with severe adult primary open-angle (POAG) or angle-closure glaucoma (PACG) compared to controls (cadaver donor cornea) were validated for temporal changes in cell death-related gene expression on in-vitro primary human TM cell culture after 48 hours (moderate) or 72 hours (severe) oxidative stress with H2O2 (400– 1000 uM concentration). These were compared with histone modifications after oxidative stress in human TM (HTM) culture and peripheral blood of patients with moderate and severe glaucoma.Results: Autophagy-related proteins seemed to be the predominant cell-death mechanism over apoptosis in ex-vivo dissected TM specimens in severe glaucoma. Analyzing HTM cell gene expression at 48 hours and 72 hours of oxidative stress, autophagy genes were up-regulated at 48– 72 hours of exposure in contrast to apoptosis-related genes, showing down-regulation at 72 hours. There was associated increased expression of H3K14ac in HTM after 72 hours of oxidative stress and in peripheral blood of severe POAG and PACG.Conclusion: A preference of autophagy over apoptosis may underlie stage transition from moderate to severe glaucoma in the trabecular meshwork or peripheral blood, which may be tightly regulated by epigenetic modulators.Keywords: trabecular meshwork, cell death, autophagy, apoptosis, glaucoma

Nucleoside analogues like 4-thiouridine (4sU) are used to metabolically label newly synthesized RNA. Chemical conversion of 4sU before sequencing induces T-to-C mismatches in reads sequenced from labelled RNA, allowing to obtain total and labelled RNA expression profiles from a single sequencing library. Cytotoxicity due to extended periods of labelling or high 4sU concentrations has been described, but the effects of extensive 4sU labelling on expression estimates from nucleotide conversion RNA-seq have not been studied. Here, we performed nucleotide conversion RNA-seq with escalating doses of 4sU with short-term labelling (1h) and over a progressive time course (up to 2h) in different cell lines. With high concentrations or at later time points, expression estimates were biased in an RNA half-life dependent manner. We show that bias arose by a combination of reduced mappability of reads carrying multiple conversions, and a global, unspecific underrepresentation of labelled RNA emerging during library preparation and potentially global reduction of RNA synthesis. We developed a computational tool to rescue unmappable reads, which performed favourably compared to previous read mappers, and a statistical method, which could fully remove remaining bias. All methods developed here are freely available as part of our GRAND-SLAM pipeline and grandR package., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Abstract: Human cytomegalovirus (HCMV) reactivation poses a substantial risk to patients receiving tranplants. Effective risk stratification and vaccine development is hampered by a lack of HCMV-derived immunogenic peptides in patients with common HLA-A∗03:01 and HLA-B∗15:01 haplotypes. This study aimed to discover novel HCMV immunogenic peptides for these haplotypes by combining ribosome sequencing (Ribo-seq) and mass spectrometry with state-of-the-art computational tools, Peptide-PRISM and Probabilistic Inference of Codon Activities by an EM Algorithm. Furthermore, using machine learning, an algorithm was developed to predict immunogenicity based on translational activity, binding affinity, and peptide localization within small open reading frames to identify the most promising peptides for in vitro validation. Immunogenicity of these peptides was subsequently tested by analyzing peptide-specific T-cell responses of HCMV-seropositive and -seronegative healthy donors as well as patients with transplants. This resulted in the direct identification of 3 canonical and 1 cryptic HLA-A∗03-restricted immunogenic peptides as well as 5 canonical and 1 cryptic HLA-B∗15-restricted immunogenic peptide, with a specific interferon gamma-positive (IFN-γ+)/CD8+ T-cell response of ≥0.02%. High T-cell responses were detected against 2 HLA-A∗03-restricted and 3 HLA-B∗15-restricted canonical peptides with frequencies of up to 8.77% IFN-γ+/CD8+ T cells in patients after allogeneic stem cell transplantation. Therefore, our comprehensive strategy establishes a framework for efficient identification of novel immunogenic peptides from both existing and novel Ribo-seq data sets., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS) is a severe adverse event (mortality of 10%). Its pathophysiology involves herpesviruses, particularly HHV-6, but the exact mechanisms are still poorly understood., Objective: To describe severe cases of DRESS and especially their association with herpesvirus reactivation., Methods: This study was a multicentre case series conducted between 2007 and 2021 at five University Hospital Centres in France. The study included patients who had severe DRESS, which was defined as death, transfer to the intensive care unit (ICU), or severe damage to internal organs. We excluded patients without blood PCR sample, without a drug formally attributed or with RegiSCAR score < 6. We collected data on severity, causative drug, associated visceral damage and results of viral blood PCRs. HHV-6 reactivation was studied in skin biopsies by detection of small non-coding transcripts (HHV-6 miR-aU14) and a late viral protein (GP82/105)., Results: Fifty-two patients were included (29 female, median age 62, interquartile range (IQR) [37;72]). Eight patients (15%) died, 13 (27%) were admitted to ICU. Most patients (n = 34; 65%) had multisystem involvement: most frequent was liver (n = 46; 88%), then renal failure (n = 24; 46%). Forty patients (77%) had at least one blood viral reactivation among HHV-6, EBV or CMV, of which 21 (53%) had at least two. Median time of blood HHV-6 reactivation was 24 days (IQR [20;35]). HHV-6 reactivation was demonstrated in 15 out of 20 skin biopsies, with a median time of 11 days [9;17]., Conclusions: We confirmed the high rate of HHV-6 reactivation in severe DRESS and demonstrated cutaneous HHV-6 reactivation using small non-coding transcripts (HHV-6 miR-aU14), which preceded viral PCR positivity in blood. These results suggest that HHV-6 reactivation during DRESS may start in skin. Furthermore, search for miR-aU14 in skin biopsy could become a useful diagnostic tool for early detection of HHV-6 reactivation., (© 2023 European Academy of Dermatology and Venereology.)

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a devastating disease affecting millions of people worldwide. Due to the 2019 pandemic of coronavirus disease (COVID-19), we are facing a significant increase of ME/CFS prevalence. On May 11th to 12th, 2023, the second international ME/CFS conference of the Charité Fatigue Center was held in Berlin, Germany, focusing on pathomechanisms, diagnosis, and treatment. During the two-day conference, more than 100 researchers from various research fields met on-site and over 700 attendees participated online to discuss the state of the art and novel findings in this field. Key topics from the conference included: the role of the immune system, dysfunction of endothelial and autonomic nervous system, and viral reactivation. Furthermore, there were presentations on innovative diagnostic measures and assessments for this complex disease, cutting-edge treatment approaches, and clinical studies. Despite the increased public attention due to the COVID-19 pandemic, the subsequent rise of Long COVID-19 cases, and the rise of funding opportunities to unravel the pathomechanisms underlying ME/CFS, this severe disease remains highly underresearched. Future adequately funded research efforts are needed to further explore the disease etiology and to identify diagnostic markers and targeted therapies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The genomes of both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) were first sequenced over 20 years ago. Similar to HCMV, the MCMV genome had initially been proposed to harbor ≈170 open reading frames (ORFs). More recently, omics approaches revealed HCMV gene expression to be substantially more complex comprising several hundred viral ORFs. Here, we provide a state-of-the art reannotation of lytic MCMV gene expression based on integrative analysis of a large set of omics data. Our data reveal 365 viral transcription start sites (TiSS) that give rise to 380 and 454 viral transcripts and ORFs, respectively. The latter include >200 small ORFs, some of which represented the most highly expressed viral gene products. By combining TiSS profiling with metabolic RNA labelling and chemical nucleotide conversion sequencing (dSLAM-seq), we provide a detailed picture of the expression kinetics of viral transcription. This not only resulted in the identification of a novel MCMV immediate early transcript encoding the m166.5 ORF, which we termed ie4, but also revealed a group of well-expressed viral transcripts that are induced later than canonical true late genes and contain an initiator element (Inr) but no TATA- or TATT-box in their core promoters. We show that viral upstream ORFs (uORFs) tune gene expression of longer viral ORFs expressed in cis at translational level. Finally, we identify a truncated isoform of the viral NK-cell immune evasin m145 arising from a viral TiSS downstream of the canonical m145 mRNA. Despite being ≈5-fold more abundantly expressed than the canonical m145 protein it was not required for downregulating the NK cell ligand, MULT-I. In summary, our work will pave the way for future mechanistic studies on previously unknown cytomegalovirus gene products in an important virus animal model., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lodha et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

First exposure to various human herpesviruses (HHVs) including HHV-6, HCMV and EBV does not cause a life-threatening disease. In fact, most individuals are frequently unaware of their first exposure to such pathogens. These herpesviruses acquire lifelong latency in the human body where they show minimal genomic activity required for their survival. We hypothesized that it is not the latency itself but a timely, regionally restricted viral reactivation in a sub-set of host cells that plays a key role in disease development. HHV-6 (HHV-6A and HHV-6B) and HHV-7 are unique HHVs that acquire latency by integration of the viral genome into sub-telomeric region of human chromosomes. HHV-6 reactivation has been linked to Alzheimer's Disease, Chronic Fatigue Syndrome, and many other diseases. However, lack of viral activity in commonly tested biological materials including blood or serum strongly suggests tissue specific localization of active HHV-6 genome. Here in this paper, we attempted to analyze active HHV-6 transcripts in postmortem tissue biopsies from a small cohort of ME/CFS patients and matched controls by fluorescence in situ hybridization using a probe against HHV-6 microRNA (miRNA), miR-aU14. Our results show abundant viral miRNA in various regions of the human brain and associated neuronal tissues including the spinal cord that is only detected in ME/CFS patients and not in controls. Our findings provide evidence of tissue-specific active HHV-6 and EBV infection in ME/CFS, which along with recent work demonstrating a possible relationship between herpesvirus infection and ME/CFS, provide grounds for renewed discussion on the role of herpesviruses in ME/CFS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kasimir, Toomey, Liu, Kaiping, Ariza and Prusty.)

Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation 1,2 . A long appreciated, yet undefined relationship exists between the lytic-latent switch and viral non-coding RNAs 3,4 . Here we identify viral microRNA (miRNA)-mediated inhibition of host miRNA processing as a cellular mechanism that human herpesvirus 6A (HHV-6A) exploits to disrupt mitochondrial architecture, evade intrinsic host defences and drive the switch from latent to lytic virus infection. We demonstrate that virus-encoded miR-aU14 selectively inhibits the processing of multiple miR-30 family members by direct interaction with the respective primary (pri)-miRNA hairpin loops. Subsequent loss of miR-30 and activation of the miR-30-p53-DRP1 axis triggers a profound disruption of mitochondrial architecture. This impairs induction of type I interferons and is necessary for both productive infection and virus reactivation. Ectopic expression of miR-aU14 triggered virus reactivation from latency, identifying viral miR-aU14 as a readily druggable master regulator of the herpesvirus lytic-latent switch. Our results show that miRNA-mediated inhibition of miRNA processing represents a generalized cellular mechanism that can be exploited to selectively target individual members of miRNA families. We anticipate that targeting miR-aU14 will provide new therapeutic options for preventing herpesvirus reactivations in HHV-6-associated disorders., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Viruses play a key role in explaining the pathogenesis of various autoimmune disorders, whose underlying principle is defined by the activation of autoreactive T-cells. In many cases, T-cells escape self-tolerance due to the failure in encountering certain MHC-I self-peptide complexes at substantial levels, whose peptides remain invisible from the immune system. Over the years, contribution of unstable defective ribosomal products (DRiPs) in immunosurveillance has gained prominence. A class of unstable products emerge from non-canonical translation and processing of unannotated mammalian and viral ORFs and their peptides are cryptic in nature. Indeed, high throughput sequencing and proteomics have revealed that a substantial portion of our genomes comprise of non-canonical ORFs, whose generation is significantly modulated during disease. Many of these ORFs comprise short ORFs (sORFs) and upstream ORFs (uORFs) that resemble DRiPs and may hence be preferentially presented. Here, we discuss how such products, normally "hidden" from the immune system, become abundant in viral infections activating autoimmune T-cells, by discussing their emerging role in infection and disease. Finally, we provide a perspective on how these mechanisms can explain several autoimmune disorders in the wake of the COVID-19 pandemic., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lodha, Erhard, Dölken and Prusty.)

Background: Distinctions between HHV-6 primary infection in seronegative patients and HHV-6 reactivation in seropositive patients remains largely undescribed in pediatric liver transplant (LT) recipients., Methods: We implemented pretransplant serology testing of HHV-6 in a large pediatric hospital and retrospectively assessed the incidence, manifestations and outcomes of HHV-6 infections over a 3-year period., Results: Among 101 pediatric LT recipients, 96 had pretransplant HHV-6 serologies; 34 (35.4%) were seronegative and 62 (64.6%) seropositive. Posttransplantation, 8/25 (32%) seronegative patients had HHV-6 DNAemia (primary infection) compared to 2/48 (4%) seropositive patients (p=0.002). Compared to seropositive patients, seronegative patients with HHV-6 DNAemia were younger, and had symptoms of fever and/or elevated aminotransferases in association with higher viral loads, in the first month post-transplant. More than 90% of seronegative patients and 77.8% of seropositive patients had HHV-6 detected by PCR in liver biopsy obtained for concerns of allograft rejection, but most had no detectable concomitant DNAemia. Active replication of virus in the liver was confirmed by in situ hybridization in select cases. While HHV-6 infection occurred among patients on prophylaxis doses of antivirals for CMV, HHV-6 DNAemia and presenting symptoms resolved on treatment doses., Conclusions: HHV-6 DNA-emia occurred more frequently in seronegative pediatric LT recipients, usually in the early posttransplant period, and was subsequently detected in allograft biopsies. HHV-6 cannot be ruled out as a cause of hepatitis in the absence of allograft tissue testing and specialized virological assays, as HHV-6 may disrupt local allograft immune homeostasis while evading traditional screening methods using blood or plasma. The assessment of pre-transplant HHV-6 serological status may be important for risk stratification and post-transplant management of pediatric LT recipients., (© The Author(s) 2021. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Purpose: Pseudoexfoliation (PXF) is a unique form of glaucoma characterized by accumulation of exfoliative material in the eyes. Changes in tear profile in disease stages may give us insights into molecular mechanisms involved in causing glaucoma in the eye., Methods: All patients were categorized into three main categories; pseudoexfoliation (PXF), pseudoexfoliation glaucoma (PXG) and cataract, which served as control. Cytokines, transforming growth factor β1 (TGFβ1), matrix metalloproteases (MMPs) and fibronectin (FN1) were assessed with multiplex bead assay, enzyme-linked immunosorbent assay (ELISA), gelatin zymography, and immunohistochemistry (IHC) respectively in different ocular tissues such as tears, tenon's capsule, aqueous humor (AH) and serum samples of patients with PXF stages., Results: We found that TGFβ1, MMP-9 and FN1 protein expression were upregulated in tears, tenon's capsule and AH samples in PXG compared to PXF, though the MMP-9 protein activity was downregulated in PXG compared with control or PXF. We have also found that in PXG tears sample the fold change of TGF-α (Transforming Growth Factor-α), MDC (Macrophage Derived Chemokine), IL-8 (Interleukin-8), VEGF (Vascular Endothelial Growth Factor) were significantly downregulated and the levels of GM-CSF (Granulocyte Macrophage Colony Stimulating Factor), IP-10 (Interferon- γ produced protein-10) were significant upregulated. While in AH; IL-6 (Interleukin-6), IL-8, VEGF, IFN-a2 (Interferon- α2), GRO (Growth regulated alpha protein) levels were found lower and IL1a (Interleukin-1α) level was higher in PXG compared to PXF. And in serum; IFN-a2, Eotaxin, GM-CSF, Fractalkine, IL-10 (Interleukin-10), IL1Ra (Interleukin-1 receptor antagonist), IL-7 (Interleukin-7), IL-8, MIP1β (Macrophage Inflammatory Protein-1β), MCP-1 (Monocyte Chemoattractant Protein-1) levels were significantly upregulated and PDGF-AA (Platelet Derived Growth Factor-AA) level was downregulated in the patients with PXG compared to PXF., Conclusions: Altered expression of these molecules in tears may therefore be used as a signal for onset of glaucoma or for identifying eyes at risk of developing glaucoma in PXF., Competing Interests: The authors have declared that no competing interests exist.

Despite the international guidelines on the containment of the coronavirus disease 2019 (COVID-19) pandemic, the European scientific community was not sufficiently prepared to coordinate scientific efforts. To improve preparedness for future pandemics, we have initiated a network of nine European-funded Cooperation in Science and Technology (COST) Actions that can help facilitate inter-, multi-, and trans-disciplinary communication and collaboration., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Herpes simplex virus 1 (HSV-1) induces a profound host shutoff during lytic infection. The virion host shutoff ( vhs ) protein plays a key role in this process by efficiently cleaving host and viral mRNAs. Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in host transcriptional activity. To dissect relative contributions of both mechanisms and elucidate gene-specific host transcriptional responses throughout the first 8 h of lytic HSV-1 infection, we used transcriptome sequencing of total, newly transcribed (4sU-labeled) and chromatin-associated RNA in wild-type (WT) and Δ vhs mutant infection of primary human fibroblasts. Following virus entry, vhs activity rapidly plateaued at an elimination rate of around 30% of cellular mRNAs per hour until 8 h postinfection (p.i.). In parallel, host transcriptional activity dropped to 10 to 20%. While the combined effects of both phenomena dominated infection-induced changes in total RNA, extensive gene-specific transcriptional regulation was observable in chromatin-associated RNA and was surprisingly concordant between WT and Δ vhs infections. Both induced strong transcriptional upregulation of a small subset of genes that were poorly expressed prior to infection but already primed by H3K4me3 histone marks at their promoters. Most interestingly, analysis of chromatin-associated RNA revealed vhs -nuclease-activity-dependent transcriptional downregulation of at least 150 cellular genes, in particular of many integrin adhesome and extracellular matrix components. This was accompanied by a vhs -dependent reduction in protein levels by 8 h p.i. for many of these genes. In summary, our study provides a comprehensive picture of the molecular mechanisms that govern cellular RNA metabolism during the first 8 h of lytic HSV-1 infection. IMPORTANCE The HSV-1 virion host shutoff ( vhs ) protein efficiently cleaves both host and viral mRNAs in a translation-dependent manner. In this study, we model and quantify changes in vhs activity, as well as virus-induced global loss of host transcriptional activity, during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA levels were dominated by these two global effects. In contrast, chromatin-associated RNA depicted gene-specific transcriptional changes. This revealed highly concordant transcriptional changes in WT and Δvhs infections, confirmed DUX4 as a key transcriptional regulator in HSV-1 infection, and identified vhs -dependent transcriptional downregulation of the integrin adhesome and extracellular matrix components. The latter explained seemingly gene-specific effects previously attributed to vhs -mediated mRNA degradation and resulted in a concordant loss in protein levels by 8 h p.i. for many of the respective genes., (Copyright © 2021 Friedel et al.)

CMV infection remains a significant cause of morbidity among pediatric HTx recipients We explored the implications of CMV infection on post-transplant outcomes among CMV risk-stratified pediatric HTx recipients receiving VGC prophylaxis. Children who underwent HTx between January 2010 and October 2016 were stratified according to CMV risk at time of transplant and evaluated for evidence of post-transplant CMV infection, rejection, CAV, and graft loss. Among 97 children, 41 (42%) were considered HR or IR risk for CMV infection and received VGC prophylaxis. CMV DNAemia was observed in 34% of children, including 71% HR, 40% IR, and 18% LR individuals. Median time to CMV DNAemia following VGC prophylaxis was 32D among HR vs 277D in IR subjects (P = .042). No difference in overall graft loss was noted among groups, but CMV HR children had decreased rejection-free survival (3.5 years) compared to IR (6 years, P = .015) and LR children (8 years, P = .0003). CMV was noted on EMB in 13% of children but was not associated with increased CAV, rejection or graft loss. High-risk CMV status was associated with decreased time to CMV infection despite VGC prophylaxis, compared to IR, and decreased rejection-free survival times compared to both IR and LR recipients. Detection of CMV on EMB was not associated with increased rejection, CAV or graft loss. Additional studies are needed to explore the impact of CMV infection on rejection-free survival in HTx recipients., (© 2020 Wiley Periodicals LLC.)

Salmonella enterica serovar Enteritidis ( S . Enteritidis; wild type (WT)) is a major cause of foodborne illness globally. The ability of this pathogen to survive stress inside and outside the host, such as encountering antimicrobial peptides and heat stress, determines the efficiency of enteric infection. These stressors concertedly trigger virulence factors encoded on Salmonella pathogenicity islands (SPIs). Although RpoS is a well-known central transcriptional stress and virulence regulator, functional information regarding the genes of the regulon is currently limited. Here, we identified SEN1538 as a conserved RpoS-regulated gene belonging to the KGG protein superfamily. We further assessed its role in pathogenic stress responses and virulence. When SEN1538 was deleted (Δ1538), the pathogen showed reduced survival during antimicrobial peptide introduction and heat stress at 55°C compared to WT. The mutant displayed 70% reduced invasion in the HCT116 colon epithelial cell line, 5-fold attenuated phagocytic survival in RAW264.7 cells, and downregulation of several SPI-1 and SPI-2 genes encoding the three secretion system apparatus and effector proteins. Δ1538 also showed decreased virulence compared to WT, demonstrated by its reduced bacterial counts in the feces, mLN, spleen, and cecum of C57BL/6 mice. Comparative transcriptomic analysis of Δ1538 against WT revealed 111 differentially regulated genes, 103 of which were downregulated (fold change ≤ -1.5, P < 0.05). The majority of these genes were in clusters for metabolism, transporters, and pathogenesis, driving pathogenic stress responses and virulence. SEN1538 is, therefore, an important virulence determinant contributing to the resilience of S . Enteritidis to stress factors during infection.

Objectives: The aim of this study is to evaluate HHV-6 and PVB19 infection using polymerase chain reaction (PCR) and immunofluorescent assay (IFA) in the myocardium of pediatric patients with dilated cardiomyopathy (DCM) and the impact of viral persistence in the cardiac allograft after heart transplantation (HT)., Methods: Multiplex droplet digital PCR was used to analyze the prevalence of viral sequences in myocardial samples from 48 pediatric DCM patients and 10 control subjects. Of the 48 DCM patients, 44 underwent HT. After HT, consecutive endomyocardial biopsy (EMB) samples were analyzed for the presence of PVB19 and HHV-6 antigens using IFA and the patients were evaluated for rejections, coronary vasculopathy, and graft loss., Results: Of the 48 DCM patients, 14 had positive viral PCR results in explanted/autopsy hearts. Among them, PVB19 was found in 8/48, HHV6 in 4/48, both PVB19 and HHV6 in 1/48, and enterovirus in one, but no adenovirus was found. The EMB samples obtained after HT were positive for PVB19 and HHV-6 in 7/44 and 3/44 cases, respectively. Viral presence in both the explanted heart and the cardiac allograft was demonstrated in 4 patients, 3 of whom were positive for PVB19, and one of whom was positive for HHV-6 pretransplant. Coronary vasculopathy and graft loss were more common in patients with PVB19-positive myocardial tissues versus those who were PVB19-negative., Conclusions: There is an association between PVB19 and HHV-6 infection and DCM in children. The study suggests the persistence of PVB19 and HHV-6 in the host can lead to subsequent viral reactivation in the transplanted heart, even in those recipients who do not have active myocarditis. PVB19 in the cardiac allograft tended toward higher adverse post-HT events., Competing Interests: There are no conflict of interest., (Copyright: © 2020 Annals of Pediatric Cardiology.)

Japanese encephalitis virus (JEV) comes under the family Flaviviridae and genus flavivirus. Pigs act as reservoir and amplifying intermediate host for JEV. The current investigation was conducted to understand the prevalence of JEV infection in pigs in three different geographical sites in India (Odisha, Assam and Manipur). Total 857 serum samples were tested by ELISA and RT-PCR, while only RT-PCR was performed in case of 275 tonsils tissues for detection of JEV. It was observed that JEV prevalence was highest in Manipur (positive 39, 25.5% in serum and 10% in tonsil) but lower in Assam (positive 15, 3.8% in serum and 0% in tonsils) and Odisha (positive 7, 1.5% in serum and 3.7% in tonsils). Genotype III (GIII) of JEV was the dominant genotype. Further, analysis of E gene revealed sporadic mutations of S83G, H76P, E78Q, C55S, and S64W along with two consistent mutations V46S and V51I in GIII. Whereas, a single mutation S118N was observed in the GI strain. In conclusion, the high JE virus infection rate of pig in the current locations suggests the need for continuous surveillance of this virus in pigs which will ultimately help to adopt an effective control strategy to prevent the spread of JE infection to human., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multifactorial disorder with many possible triggers. Human herpesvirus (HHV)-6 and HHV-7 are two infectious triggers for which evidence has been growing. To understand possible causative role of HHV-6 in ME/CFS, metabolic and antiviral phenotypes of U2-OS cells were studied with and without chromosomally integrated HHV-6 and with or without virus reactivation using the histone deacetylase inhibitor trichostatin-A. Proteomic analysis was conducted by pulsed stable isotope labeling by amino acids in cell culture analysis. Antiviral properties that were induced by HHV-6 transactivation were studied in virus-naive A549 cells challenged by infection with influenza-A (H1N1) or HSV-1. Mitochondria were fragmented and 1-carbon metabolism, dUTPase, and thymidylate synthase were strongly induced by HHV-6 reactivation, whereas superoxide dismutase 2 and proteins required for mitochondrial oxidation of fatty acid, amino acid, and glucose metabolism, including pyruvate dehydrogenase, were strongly inhibited. Adoptive transfer of U2-OS cell supernatants after reactivation of HHV-6A led to an antiviral state in A549 cells that prevented superinfection with influenza-A and HSV-1. Adoptive transfer of serum from 10 patients with ME/CFS produced a similar fragmentation of mitochondria and the associated antiviral state in the A549 cell assay. In conclusion, HHV-6 reactivation in ME/CFS patients activates a multisystem, proinflammatory, cell danger response that protects against certain RNA and DNA virus infections but comes at the cost of mitochondrial fragmentation and severely compromised energy metabolism., (Copyright © 2020 The Authors.)

Background: Active infections of human herpesvirus 6B (HHV-6B) are frequent in immunocompromised recipients after transplantation. Nevertheless, they need to be distinguished from latent inherited chromosomally integrated genomes (iciHHV-6) present in about 1% of the population to avoid unnecessary administration of toxic antivirals., Methods: A 5-year-old child presented with acute liver allograft rejection associated with HHV-6 DNA in plasma, which led to an unfavorable outcome. We investigated the possibility of HHV-6 infection derived from an iciHHV-6 present in the donor's liver using molecular and histopathology studies in various tissues, including quantification of HHV-6 DNA, genotyping, sequencing for antiviral resistance genes, relative quantification of viral transcripts, and detection of gB and gH viral proteins., Results: The presence of iciHHV-6B was evidenced in the donor with signs of reactivation in the gallbladder and transplanted liver (detection of HHV-6B mRNA and late proteins). This localized expression could have played a role in liver rejection. Low viral loads in the recipient's plasma, with identical partial U39 sequences, were in favor of viral DNA released from the transplanted liver rather than a systemic infection., Conclusions: Determination of iciHHV-6 status before transplantation should be considered to guide clinical decisions, such as antiviral prophylaxis, viral load monitoring, and antiviral therapy., (© 2019 Wiley Periodicals, Inc.)

Combining integrative genomics and systems biology approaches has revealed new and conserved features in the genome of human herpesvirus 6., Competing Interests: BP, AW No competing interests declared, (© 2020, Prusty and Whisnant.)

Infection by viruses, including herpes simplex virus-1 (HSV-1), and cellular stresses cause widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes. However, the underlying mechanisms remain unclear. Here, we demonstrate that the HSV-1 immediate early protein ICP27 induces DoTT by directly binding to the essential mRNA 3' processing factor CPSF. It thereby induces the assembly of a dead-end 3' processing complex, blocking mRNA 3' cleavage. Remarkably, ICP27 also acts as a sequence-dependent activator of mRNA 3' processing for viral and a subset of host transcripts. Our results unravel a bimodal activity of ICP27 that plays a key role in HSV-1-induced host shutoff and identify CPSF as an important factor that mediates regulation of transcription termination. These findings have broad implications for understanding the regulation of transcription termination by other viruses, cellular stress and cancer.

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine associated with autoimmune and infectious diseases. Importance of TNF-α in P. falciparum malaria and systemic lupus erythematosus (SLE) have been demonstrated. However, association of functional promoter variants with SLE and malaria is lacking in malaria endemic population. A total of 204 female SLE patients and 224 age and sex matched healthy controls were enrolled in the study. Three hundred fourteen P. falciparum infected patients with different clinical phenotypes were included. TNF-α polymorphisms (G-238A & G-308A) were genotyped by PCR-RFLP. Plasma levels of TNF-α was quantified by ELISA. Heterozygous mutants and minor alleles of TNF-α (G-238A and G-308A) polymorphisms were significantly higher in SLE patients compared to healthy controls and associated with development of lupus nephritis. In addition, both promoter variants were associated with severe P. falciparum malaria. SLE patients demonstrated higher levels of plasma TNF-α compared to healthy controls. TNF-α (G-238A and G-308A) variants were associated with higher plasma TNF-α. In conclusion, TNF-α (G-238A & G-308A) variants are associated with higher plasma TNF-α levels in SLE patients residing in malaria endemic areas and could be a contributing factor in the development of SLE and susceptibility to severe P. falciparum malaria.

Soil-transmitted helminth (STH) infections and malaria are parasitic diseases with enormous global health burdens. Research has demonstrated a relationship between each of these parasites and the gut microbiome, suggesting that the gut microbiota may be implicated in governing host susceptibility to diverse pathogens, and perhaps even coinfection by different pathogens, through similar microbiome-influenced pathways. Here, we have derived a first microbiome community profile associated with STH infections in Odisha, India, and tested the hypothesis that the gut microbiome can modulate host susceptibility to multiple parasite infections through the same pathways. This study revealed several bacterial taxa negatively associated with specific STH infections, including Lactobacillus and Lachnospiracaea. Our results also suggest that relative abundance of Lactobacillus is driven by the STH infection status more so than by the Plasmodium infection status. This study contributes to efforts to understand the effects of the microbiome on host susceptibility to parasitic infections in endemic communities.

Background/Objectives: With mucocutaneous candidiasis being highly prevalent in HIV patients, the emergence of fluconazole-resistant Candida species forms a major challenge in treating and eradicating these infections. The objective of this study was to establish the antifungal activity of K21, a membrane-rupturing antimicrobial compound derived from a silica quaternary ammonium compound (SiQAC) with tetraethoxysilane (TEOS). Methods: The study sample included 81 Candida species of which 9 were type strains and 72 were clinical isolates. Minimum inhibitory concentrations, synergy, fractional inhibitory concentration index (FICI), and time kill assays were determined by broth microdilution. Electron microscopy (EM) was used to determine the qualitative changes brought about after treatment with K21. Results: K21 inhibited the growth of all fluconazole-resistant and susceptible Candida strains with only 2 h of exposure required to effectively kill 99.9% of the inoculum, and a definite synergistic effect was observed with a combination of K21 and fluconazole. EM demonstrated the presence of two forms of extracellular vesicles indicative of biofilm formation and cell lysis. Conclusion: The study established the efficacy of K21 as an antifungal agent and with fluconazole-resistant candidiasis on the increase, the development of K21 can provide a promising alternative to combat acquired drug resistance.

Chlamydia trachomatis infections have been associated with ovarian cancer by several epidemiological studies. Here, we show that C. trachomatis-infected primary human ovarian epithelial cells display elevated oxidative DNA damage. Base excision repair, an important cellular mechanism to repair oxidative DNA lesions, was impaired in infected primary ovarian and in several other types of cells. Polymerase β was downregulated in infected cells associated with upregulation of microRNA-499a (miR-499a). Stabilising polymerase β by inhibiting miR-499a significantly improved repair. Moreover, downregulation of tumour suppressor p53 also resulted in attenuated repair in these cells. Thus, our data show that downregulation of polymerase β by direct inhibition through miR-499a and downregulation of p53 debilitate the host-cell base excision repair during C. trachomatis infection., (© 2018 John Wiley & Sons Ltd.)

Dengue is the most rapidly spreading viral disease transmitted by the bite of infected Aedes mosquitos. The pathogenesis of dengue is still unclear; although host immune responses and virus serotypes have been proposed to contribute to disease severity. In this study, we examined the circulating dengue virus (DENV) and measured plasma levels of inflammatory mediators. Ninety-eight patients during a dengue outbreak in eastern India in 2016 were included in the study. The presence of DENV was demonstrated by detecting NS1 antigen; IgM capture ELISA and serotypes were discriminated by type-specific RT-PCR and/or sequencing. Plasma samples were assayed for 41-plex cytokine/chemokines using multiplex Luminex assay. Eighty-five (87%) samples were positive by NS1/IgM capture ELISA/RT-PCR. All four serotypes of DENV were detected in this outbreak, with DENV-2 as the predominant type, seen in 55% of cases. Mixed infections were seen in 39% of subjects. Among the host inflammatory biomarkers, GM-CSF, IFN-γ, IL-10, IL-15, IL-8, MCP-1, IL-6, MIP-1β, and TNF-α levels were significantly increased in dengue with and without warning signs, in severe dengue patients in comparison to healthy controls. Four cytokines IFN-γ, GM-CSF, IL-10, and MIP-1β correlated significantly with disease severity and could serve as potential predictor for disease severity. Information on the host biomarkers and the dengue serotype may help guide in optimizing effective intervention strategies.

Background and Main Text: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex and controversial clinical condition without having established causative factors. Increasing numbers of cases during past decade have created awareness among patients as well as healthcare professionals. Chronic viral infection as a cause of ME/CFS has long been debated. However, lack of large studies involving well-designed patient groups and validated experimental set ups have hindered our knowledge about this disease. Moreover, recent developments regarding molecular mechanism of pathogenesis of various infectious agents cast doubts over validity of several of the past studies., Conclusions: This review aims to compile all the studies done so far to investigate various viral agents that could be associated with ME/CFS. Furthermore, we suggest strategies to better design future studies on the role of viral infections in ME/CFS.

Human herpesvirus 6A and 6B frequently acquires latency. HHV-6 activation has been associated with various human diseases. Germ line inheritance of chromosomally integrated HHV-6 makes viral DNA-based analysis difficult for determination of early stages of viral activation. We characterized early stages of HHV-6 activation using high throughput transcriptomics studies and applied the results to understand virus activation under clinical conditions. Using a latent HHV-6A cell culture model in U2OS cells, we identified an early stage of viral reactivation, which we define as transactivation that is marked by transcription of several viral small non-coding RNAs (sncRNAs) in the absence of detectable increase in viral replication and proteome. Using deep sequencing approaches, we detected previously known as well as a new viral sncRNAs that characterized viral transactivation and differentiated it from latency. Here we show changes in human transcriptome upon viral transactivation that reflect multiple alterations in mitochondria-associated pathways, which was supported by observation of increased mitochondrial fragmentation in virus reactivated cells. Furthermore, we present here a unique clinical case of DIHS/DRESS associated death where HHV-6 sncRNA-U14 was abundantly detected throughout the body of the patient in the presence of low viral DNA. In this study, we have identified a unique and early stage of viral activation that is characterized by abundant transcription of viral sncRNAs, which can serve as an ideal biomarker under clinical conditions., Competing Interests: The authors declare no competing interests.

Early-life infections and associated neuroinflammation is incriminated in the pathogenesis of various mood disorders. Infection with human roseoloviruses, HHV-6A and HHV-6B, allows viral latency in the central nervous system and other tissues, which can later be activated causing cognitive and behavioral disturbances. Hence, this study was designed to evaluate possible association of HHV-6A and HHV-6B activation with three different groups of psychiatric patients. DNA qPCR, immunofluorescence and FISH studies were carried out in post-mortem posterior cerebellum from 50 cases each of bipolar disorder (BPD), schizophrenia, 15 major depressive disorder (MDD) and 50 appropriate control samples obtained from two well-known brain collections (Stanley Medical Research Institute). HHV-6A and HHV-6B late proteins (indicating active infection) and viral DNA were detected more frequently ( p < 0.001 for each virus) in human cerebellum in MDD and BPD relative to controls. These roseolovirus proteins and DNA were found less frequently in schizophrenia cases. Active HHV-6A and HHV-6B infection in cerebellar Purkinje cells were detected frequently in BPD and MDD cases. Furthermore, we found a significant association of HHV-6A infection with reduced Purkinje cell size, suggesting virus-mediated abnormal Purkinje cell function in these disorders. Finally, gene expression analysis of cerebellar tissue revealed changes in pathways reflecting an inflammatory response possibly to HHV-6A infection. Our results provide molecular evidence to support a role for active HHV-6A and HHV-6B infection in BPD and MDD.

Chlamydia trachomatis, an obligate intracellular human pathogen, is a major cause of sexually transmitted diseases. Infections often occur without symptoms, a feature that has been attributed to the ability of the pathogen to evade the host immune response. We show here that C. trachomatis paralyses the host immune system by preventing the activation of polymorphic nuclear leukocytes (PMNs). PMNs infected with Chlamydia fail to produce neutrophil extracellular traps and the bacteria are able to survive in PMNs for extended periods of time. We have identified the secreted chlamydial protease-like activating factor (CPAF) as an effector mediating the evasion of the innate immune response since CPAF-deficient Chlamydia activate PMNs and are subsequently efficiently killed. CPAF suppresses the oxidative burst and interferes with chemical-mediated activation of neutrophils. We identified formyl peptide receptor 2 (FPR2) as a target of CPAF. FPR2 is cleaved by CPAF and released from the surface of PMNs. In contrast to previously described subversion mechanisms that mainly act on already activated PMNs, we describe here details of how Chlamydia actively paralyses PMNs, including the formation of neutrophil extracellular traps, to evade the host's innate immune response.

Obligate intracellular pathogenic Chlamydia trachomatis express several serine proteases whose roles in chlamydial development and pathogenicity are not completely understood. The chlamydial protease CPAF is expressed during the replicative phase of the chlamydial developmental cycle and is secreted into the lumen of the Chlamydia -containing vacuole called inclusion. How the secreted protease is activated in the inclusion lumen is currently not fully understood. We have identified human serine peptidase inhibitor PI15 as a potential host factor involved in the regulation of CPAF activation. Silencing expression as well as over expression of PI15 affected normal development of Chlamydia . PI15 was transported into the chlamydial inclusion lumen where it co-localized with CPAF aggregates. We show that PI15 binds to the CPAF zymogen and potentially induces CPAF protease activity at low concentrations. However, at high concentrations PI15 inhibits CPAF activity possibly by blocking its protease domain. Our findings shed light on a new aspect of chlamydial host co-evolution which involves the recruitment of host cell proteins into the inclusion to control the activation of bacterial proteases like CPAF that are important for the normal development of Chlamydia .

New antivirals are required to prevent rising antimicrobial resistance from replication inhibitors. The aim of this study was to analyse the range of emerging mutations in herpesvirus by whole genome deep sequencing. We tested human herpesvirus 6 treatment with novel antiviral K21, where evidence indicated distinct effects on virus envelope proteins. We treated BACmid cloned virus in order to analyse mechanisms and candidate targets for resistance. Illumina based next generation sequencing technology enabled analyses of mutations in 85 genes to depths of 10,000 per base detecting low prevalent minority variants (<1%). After four passages in tissue culture the untreated virus accumulated mutations in infected cells giving an emerging mixed population (45-73%) of non-synonymous SNPs in six genes including two envelope glycoproteins. Strikingly, treatment with K21 did not accumulate the passage mutations; instead a high frequency mutation was selected in envelope protein gQ2, part of the gH/gL complex essential for herpesvirus infection. This introduced a stop codon encoding a truncation mutation previously observed in increased virion production. There was reduced detection of the glycoprotein complex in infected cells. This supports a novel pathway for K21 targeting virion envelopes distinct from replication inhibition., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Specialized assembly factors facilitate the formation of many macromolecular complexes in vivo. The formation of Sm core structures of spliceosomal U-rich small nuclear ribonucleoprotein particles (UsnRNPs) requires assembly factors united in protein arginine methyltransferase 5 (PRMT5) and survival motor neuron (SMN) complexes. We demonstrate that perturbations of this assembly machinery trigger complex cellular responses that prevent aggregation of unassembled Sm proteins. Inactivation of the SMN complex results in the initial tailback of Sm proteins on the PRMT5 complex, followed by down-regulation of their encoding mRNAs. In contrast, reduction of pICln, a PRMT5 complex subunit, leads to the retention of newly synthesized Sm proteins on ribosomes and their subsequent lysosomal degradation. Overexpression of Sm proteins under these conditions results in a surplus of Sm proteins over pICln, promoting their aggregation. Our studies identify an elaborate safeguarding system that prevents individual Sm proteins from aggregating, contributing to cellular UsnRNP homeostasis., (© 2017 Prusty et al.)

Obligate intracellular bacteria such as Chlamydia trachomatis depend on metabolites of the host cell and thus protect their sole replication niche by interfering with the host cells' stress response. Here, we investigated the involvement of host microRNAs (miRNAs) in maintaining the viability of C. trachomatis -infected primary human cells. We identified miR-30c-5p as a prominently up-regulated miRNA required for the stable down-regulation of p53, a major suppressor of metabolite supply in C. trachomatis- infected cells. Loss of miR-30c-5p led to the up-regulation of Drp1, a mitochondrial fission regulator and a target gene of p53, which, in turn, severely affected chlamydial growth and had a marked effect on the mitochondrial network. Drp1-induced mitochondrial fragmentation prevented replication of C. trachomatis even in p53-deficient cells. Additionally, Chlamydia maintain mitochondrial integrity during reactive oxygen species-induced stress that occurs naturally during infection. We show that C. trachomatis require mitochondrial ATP for normal development and hence postulate that they preserve mitochondrial integrity through a miR-30c-5p-dependent inhibition of Drp1-mediated mitochondrial fission., (© 2017 Chowdhury et al.)

Human herpesvirus 6A (HHV-6A) and 6B (HHV-6B) are two different species of betaherpesviruses that integrate into sub-telomeric ends of human chromosomes, for which different prevalence rates of integration have been reported. It has been demonstrated that integrated viral genome is stable and is fully retained. However, study of chromosomally integrated viral genome in individuals carrying inherited HHV-6 (iciHHV-6) showed unexpected number of viral DR copies. Hence, we created an in vitro infection model and studied retention of full or partial viral genome over a period of time. We observed an exceptional event where cells retained viral direct repeats (DRs) alone in the absence of the full viral genome. Finally, we found evidence for non-telomeric integration of HHV-6A DR in both cultured cells and in an iciHHV-6 individual. Our results shed light on several novel features of HHV-6A chromosomal integration and provide valuable information for future screening techniques.

Obligate intracellular Chlamydia trachomatis replicate in a membrane-bound vacuole called inclusion, which serves as a signaling interface with the host cell. Here, we show that the chlamydial deubiquitinating enzyme (Cdu) 1 localizes in the inclusion membrane and faces the cytosol with the active deubiquitinating enzyme domain. The structure of this domain revealed high similarity to mammalian deubiquitinases with a unique α-helix close to the substrate-binding pocket. We identified the apoptosis regulator Mcl-1 as a target that interacts with Cdu1 and is stabilized by deubiquitination at the chlamydial inclusion. A chlamydial transposon insertion mutant in the Cdu1-encoding gene exhibited increased Mcl-1 and inclusion ubiquitination and reduced Mcl-1 stabilization. Additionally, inactivation of Cdu1 led to increased sensitivity of C. trachomatis for IFNγ and impaired infection in mice. Thus, the chlamydial inclusion serves as an enriched site for a deubiquitinating activity exerting a function in selective stabilization of host proteins and protection from host defense.

Human herpesvirus 7 (HHV-7) is a betaherpesvirus, and is phylogenetically related to both HHV-6A and HHV-6B. The presence of telomeric repeat sequences at both ends of its genome should make it equally likely to integrate into the human telomere as HHV-6. However, numerous studies have failed to detect germline integration of HHV-7, suggesting an important difference between the HHV-6A/-6B and HHV-7 genomes. In search of possible germline integrated HHV-7, we developed a sensitive and quantitative real-time PCR assay and discovered that primers designed against some parts of the HHV-7 genome can frequently miss HHV-7 positive clinical samples even though they work efficiently in cell-culture-derived HHV-7 positive materials. Using a primer pair against the U90 ORF of HHV-7, we identified a possible case of germline integration of HHV-7 with one copy of viral genome per cell in both peripheral blood cells and hair follicles. Chromosomal integration of HHV-7 in these individuals was confirmed by fluorescence in situ hybridization analysis. Germline integration of HHV-7 was further confirmed by detection of ~2.6 copies of HHV-7 in the hair follicles of one of the parents. Our results shed light on the complex nature of the HHV-7 genome in human-derived materials in comparison to cell-culture-derived materials and show the need for stringent criteria in the selection of primers for epidemiological HHV-7 studies.

We have created a novel quaternary ammonium silane, K21 through sol-gel chemistry, using an ethoxylated version of an organosilane quaternary ammonium compound and TetraEthyl Ortho Silicate (TEOS) as precursors. Previous studies using the precursor molecule quaternary ammonium compounds (QACs) and a methacryloxy version of K21, primarily designed for use in dental healthcare, have shown inhibited growth properties against several types of gram-positive and gram-negative bacteria including Escherichia coli, Streptococcus mutans, Actinomyces naeslundii and Candida albicans etc. Here we tested the effect of K21 on HSV-1, HHV-6A and HHV-7 in in vitro cell culture infection models. Our results show growth inhibitory effect of K21 on HSV-1, HHV-6A and HHV-7 infection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Acetaminophen (N-acetyl-para-aminophenol (APAP)) toxicity causes acute liver failure by inducing centrilobular hepatic damage as a consequence of mitochondrial oxidative stress. Sterile inflammation, triggered by hepatic damage, facilitates gut bacterial translocation leading to systemic inflammation; TLR4-mediated activation by LPS has been shown to have a critical role in APAP-mediated hepatotoxicity. In this study, we demonstrate significant protection mediated by chitohexaose (Chtx) in mice challenged with a lethal dose of APAP (400 mg/kg b.w.). Decreased mortality by Chtx was associated with reduced hepatic damage, increased peritoneal migration of neutrophils, decreased mRNA expression of IL-1β as well as inhibition of inflammasome activation in liver. Further, an alternate mouse model of co-administration of a sublethal doses of APAP (200 mg/kg b.w.) and LPS (5 mg/kg b.w.) operating synergistically and mediating complete mortality was developed. Overwhelming inflammation, characterized by increased inflammatory cytokines (TNF-α, IL-1β and so on) in liver as well as in circulation and mortality was demonstrable in this model. Also, Chtx administration mediated significant reversal of mortality in APAP+LPS co-administered mice, which was associated with reduced IL-1β in liver and plasma cytokines in this model. In conclusion, Chtx being a small molecular weight linear carbohydrate offers promise for clinical management of liver failure associated with APAP overdose.

Fatal myocarditis is a rare complication in immunosuppressed children. Recent reports have linked human herpesvirus 6 (HHV-6) infection, typically a benign infection in childhood, with myocarditis. HHV-6 can reactivate during periods of immunosuppression. Here, we report 2 cases in which children were immunosuppressed, one for treatment of Evans syndrome and the other post hematopoietic stem cell transplantation, who developed rapid and fatal HHV-6-associated myocarditis. These cases suggest that HHV-6 infection should be considered as an etiology of myocarditis in immunosuppressed patients regardless of correlating blood levels. Early treatment of HHV-6 in patients with myocarditis could improve morbidity and mortality., (Copyright © 2016 by the American Academy of Pediatrics.)

Certain pathogenic bacteria adopt an intracellular lifestyle and proliferate in eukaryotic host cells. The intracellular niche protects the bacteria from cellular and humoral components of the mammalian immune system, and at the same time, allows the bacteria to gain access to otherwise restricted nutrient sources. Yet, intracellular protection and access to nutrients comes with a price, i.e., the bacteria need to overcome cell-autonomous defense mechanisms, such as the bactericidal endocytic pathway. While a few bacteria rupture the early phagosome and escape into the host cytoplasm, most intracellular pathogens form a distinct, degradation-resistant and replication-permissive membranous compartment. Intracellular bacteria that form unique pathogen vacuoles include Legionella, Mycobacterium, Chlamydia, Simkania, and Salmonella species. In order to understand the formation of these pathogen niches on a global scale and in a comprehensive and quantitative manner, an inventory of compartment-associated host factors is required. To this end, the intact pathogen compartments need to be isolated, purified and biochemically characterized. Here, we review recent progress on the isolation and purification of pathogen-modified vacuoles and membranes, as well as their proteomic characterization by mass spectrometry and different validation approaches. These studies provide the basis for further investigations on the specific mechanisms of pathogen-driven compartment formation.

CD46 and CD134 mediate attachment of Human Herpesvirus 6A (HHV-6A) and HHV-6B to host cell, respectively. But many cell types interfere with viral infection through rapid degradation of viral DNA. Hence, not all cells expressing these receptors are permissive to HHV-6 DNA replication and production of infective virions suggesting the involvement of additional factors that influence HHV-6 propagation. Here, we used a proteomics approach to identify other host cell proteins necessary for HHV-6 binding and entry. We found host cell chaperone protein GP96 to interact with HHV-6A and HHV-6B and to interfere with virus propagation within the host cell. In human peripheral blood mononuclear cells (PBMCs), GP96 is transported to the cell surface upon infection with HHV-6 and interacts with HHV-6A and -6B through its C-terminal end. Suppression of GP96 expression decreased initial viral binding but increased viral DNA replication. Transient expression of human GP96 allowed HHV-6 entry into CHO-K1 cells even in the absence of CD46. Thus, our results suggest an important role for GP96 during HHV-6 infection, which possibly supports the cellular degradation of the virus.

Obligate intracellular bacteria depend entirely on nutrients from the host cell for their reproduction. Here, we show that obligate intracellular Chlamydia downregulate the central tumor suppressor p53 in human cells. This reduction of p53 levels is mediated by the PI3K-Akt signaling pathway, activation of HDM2, and subsequent proteasomal degradation of p53. The stabilization of p53 in human cells severely impaired chlamydial development and caused the loss of infectious particle formation. DNA-damage-induced p53 interfered with chlamydial development through downregulation of the pentose phosphate pathway (PPP). Increased expression of the PPP key enzyme glucose-6-phosphate dehydrogenase rescued the inhibition of chlamydial growth induced by DNA damage or stabilized p53. Thus, downregulation of p53 is a key event in the chlamydial life cycle that reprograms the host cell to create a metabolic environment supportive of chlamydial growth.

Background: Apoptosis of several host cells induced by parasites/parasite products has been investigated in human filariasis to understand immune hyporesponsiveness. However, apoptosis of monocytes-one of the major antigen presenting cells in peripheral circulation, which are chronically exposed to filarial antigens in infected subjects-is yet to be understood., Methods: Apoptosis of human monocytes with Brugia pahangi antigen (BpA) was demonstrated by scoring several apoptotic markers using flow cytometry. Ability of BpA and plasma of infected subjects to suppress lymphocyte proliferation was demonstrated by (3)H thymidine incorporation assay and carboxyfluorescein succinimidyl ester dilution assay., Results: BpA induced significant apoptosis of normal human monocytes, primarily through Toll-like receptor 4 (TLR4), and suppressed phytohemagglutinin (PHA)-mediated proliferation of normal human T lymphocytes. However, monocytes of Wuchereria bancrofti-infected subjects were resistant to BpA-induced apoptosis. Plasma of infected subjects also mediated apoptosis of normal monocytes, presumably due to circulating filarial antigens, and resulted in inhibition of PHA-induced proliferation., Conclusion: Normal human monocytes were found to be qualitatively different from those of filariasis-infected subjects; whereas filarial antigens mediate apoptosis of normal human monocytes through TLR4, those of infected subjects were found to be resistant., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)