Role of Toll-Like Receptor (TLR) 2 in Experimental Bacillus cereus Endophthalmitis

Bacillus cereus causes a uniquely rapid and blinding intraocular infection, endophthalmitis. B. cereus replicates in the eye, synthesizes numerous toxins, and incites explosive intraocular inflammation. The mechanisms involved in the rapid and explosive intraocular immune response have not been addressed. Because Toll-like receptors (TLRs) are integral to the initial recognition of organisms during infection, we hypothesized that the uniquely explosive immune response observed during B. cereus endophthalmitis is directly influenced by the presence of TLR2, a known Gram-positive pathogen recognition receptor. To address this hypothesis, we compared the courses of experimental B. cereus endophthalmitis in wild type C57BL/6J mice to that of age-matched homozygous TLR2-/- mice. Output parameters included analysis of bacterial growth, inflammatory cell (PMN) infiltration, cytokine/chemokine kinetics, retinal function testing, and histology, with N≥4 eyes/assay/time point/mouse strain. B. cereus grew at similar rates to108 CFU/eye by 12 h, regardless of the mouse strain. Retinal function was preserved to a greater degree in infected TLR2-/- eyes compared to that of infected wild type eyes, but infected eyes of both mouse strains lost significant function. Retinal architecture was preserved in infected TLR2-/- eyes, with limited retinal and vitreal cellular infiltration compared to that of infected wild type eyes. Ocular myeloperoxidase activities corroborated these results. In general, TNFα, IFNγ, IL6, and KC were detected in greater concentrations in infected wild type eyes than in infected TLR2-/- eyes. The absence of TLR2 resulted in decreased intraocular proinflammatory cytokine/chemokine levels and altered recruitment of inflammatory cells into the eye, resulting in less intraocular inflammation and preservation of retinal architecture, and a slightly greater degree of retinal function. These results demonstrate TLR2 is an important component of the initial ocular response to B. cereus endophthalmitis

Ocular Delivery of Compacted DNA-Nanoparticles Does Not Elicit Toxicity in the Mouse Retina

Subretinal delivery of polyethylene glycol-substituted lysine peptide (CK30PEG)-compacted DNA nanoparticles results in efficient gene expression in retinal cells. This work evaluates the ocular safety of compacted DNA nanoparticles. CK30PEG-compacted nanoparticles containing an EGFP expression plasmid were subretinally injected in adult mice (1 µl at 0.3, 1.0 and 3.0 µg/µl). Retinas were examined for signs of inflammation at 1, 2, 4 and 7 days post-injection. Neither infiltration of polymorphonuclear neutrophils or lymphocytes was detected in retinas. In addition, elevation of macrophage marker F4/80 or myeloid marker myeloperoxidase was not detected in the injected eyes. The chemokine KC mRNA increased 3–4 fold in eyes injected with either nanoparticles or saline at 1 day post-injection, but returned to control levels at 2 days post-injection. No elevation of KC protein was observed in these mice. The monocyte chemotactic protein-1, increased 3–4 fold at 1 day post-injection for both nanoparticle and saline injected eyes, but also returned to control levels at 2 days. No elevations of tumor necrosis factor alpha mRNA or protein were detected. These investigations show no signs of local inflammatory responses associated with subretinal injection of compacted DNA nanoparticles, indicating that the retina may be a suitable target for clinical nanoparticle-based interventions

A study on trypsin, Aspergillus flavus and Bacillus sp. protease inhibitory activity in Cassia tora (L.) syn Senna tora (L.) Roxb. seed extract

<p>Abstract</p> <p>Background</p> <p>Proteases play an important role in virulence of many human, plant and insect pathogens. The proteinaceous protease inhibitors of plant origin have been reported widely from many plant species. The inhibitors may potentially be used for multiple therapeutic applications in viral, bacterial, fungal diseases and physiological disorders. In traditional Indian medicine system, <it>Cassia tora </it>(<it>Senna tora</it>) is reportedly effective in treatment of skin and gastrointestinal disorders. The present study explores the protease inhibitory activity of the above plant seeds against trypsin, <it>Aspergillus flavus </it>and <it>Bacillus </it>sp. proteases.</p> <p>Methods</p> <p>The crushed seeds of <it>Cassia tora </it>were washed thoroughly with acetone and hexane for depigmentation and defatting. The proteins were fractionated by ammonium sulphate (0-30, 30-60, 60-90%) followed by dialysis and size exclusion chromatography (SEC). The inhibitory potential of crude seed extract and most active dialyzed fraction against trypsin and proteases was established by spot test using unprocessed x-ray film and casein digestion methods, respectively. Electrophoretic analysis of most active fraction (30-60%) and SEC elutes were carried employing Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Gelatin SDS-PAGE. Inhibition of fungal spore germination was studied in the presence of dialyzed active inhibitor fraction. Standard deviation (SD) and ANOVA were employed as statistical tools.</p> <p>Results</p> <p>The crude seeds' extract displayed strong antitryptic, bacterial and fungal protease inhibitory activity on x-ray film. The seed protein fraction 30-60% was found most active for trypsin inhibition in caseinolytic assay (P < 0.001). The inhibition of caseinolytic activity of the proteases increased with increasing ratio of seed extract. The residual activity of trypsin, <it>Aspergillus flavus </it>and <it>Bacillus </it>sp. proteases remained only 4, 7 and 3.1%, respectively when proteases were incubated with 3 mg ml^-1seed protein extract for 60 min. The inhibitory activity was evident in gelatin SDS-PAGE where a major band (~17-19 kD) of protease inhibitor (PI) was detected in dialyzed and SEC elute. The conidial germination of <it>Aspergillus flavus </it>was moderately inhibited (30%) by the dialyzed seed extract.</p> <p>Conclusions</p> <p><it>Cassia tora </it>seed extract has strong protease inhibitory activity against trypsin, <it>Aspergillus flavus </it>and <it>Bacillus </it>sp. proteases. The inhibitor in <it>Cassia tora </it>may attenuate microbial proteases and also might be used as phytoprotecting agent.</p

Bloodstream-To-Eye Infections Are Facilitated by Outer Blood-Retinal Barrier Dysfunction

This work was funded by National Institutes of Health (NIH; http://www.nih.gov) Grants R01EY024140 and R21EY022466 (to M.C.C.) and R01EY019494 (to M.H.E.). Our research is also funded in part by NIH Core Grant P30EY021725 (to Robert E. Anderson, OUHSC) and an unrestricted grant from Research to Prevent Blindness Inc. (http://www.rpbusa.org) to the Dean A. McGee Eye Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.We thank Bolanle Adebayo (Cameron University, Lawton OK), Craig Land (Oklahoma State University, Stillwater OK), Nathan Jia (Oklahoma Christian University, Edmond OK), Kobbe Wiafe (Oklahoma School of Science and Mathematics, Oklahoma City OK), and Amanda Roehrkasse and Madhu Parkunan (OUHSC) for intellectual discussions and technical assistance. The authors also acknowledge thank Nanette Wheatley, Dr. Feng Li, and Mark Dittmar (OUHSC Live Animal Imaging Core, P30EY021725) for their invaluable technical assistance.This work was presented in part at the 2014 Association for Research in Vision and Ophthalmology Annual Conference in Orlando FL.The blood-retinal barrier (BRB) functions to maintain the immune privilege of the eye, which is necessary for normal vision. The outer BRB is formed by tightly-associated retinal pigment epithelial (RPE) cells which limit transport within the retinal environment, maintaining retinal function and viability. Retinal microvascular complications and RPE dysfunction resulting from diabetes and diabetic retinopathy cause permeability changes in the BRB that compromise barrier function. Diabetes is the major predisposing condition underlying endogenous bacterial endophthalmitis (EBE), a blinding intraocular infection resulting from bacterial invasion of the eye from the bloodstream. However, significant numbers of EBE cases occur in non-diabetics. In this work, we hypothesized that dysfunction of the outer BRB may be associated with EBE development. To disrupt the RPE component of the outer BRB in vivo, sodium iodate (NaIO3) was administered to C57BL/6J mice. NaIO3-treated and untreated mice were intravenously injected with 108 colony forming units (cfu) of Staphylococcus aureus or Klebsiella pneumoniae. At 4 and 6 days postinfection, EBE was observed in NaIO3-treated mice after infection with K. pneumoniae and S. aureus, although the incidence was higher following S. aureus infection. Invasion of the eye was observed in control mice following S. aureus infection, but not in control mice following K. pneumoniae infection. Immunohistochemistry and FITC-dextran conjugate transmigration assays of human RPE barriers after infection with an exoprotein-deficient agr/sar mutant of S. aureus suggested that S. aureus exoproteins may be required for the loss of the tight junction protein, ZO-1, and for permeability of this in vitro barrier. Our results support the clinical findings that for both pathogens, complications which result in BRB permeability increase the likelihood of bacterial transmigration from the bloodstream into the eye. For S. aureus, however, BRB permeability is not required for the development of EBE, but toxin production may facilitate EBE pathogenesis.Yeshttp://www.plosone.org/static/editorial#pee

Identification of Genes That Promote or Antagonize Somatic Homolog Pairing Using a High-Throughput FISH–Based Screen

The pairing of homologous chromosomes is a fundamental feature of the meiotic cell. In addition, a number of species exhibit homolog pairing in nonmeiotic, somatic cells as well, with evidence for its impact on both gene regulation and double-strand break (DSB) repair. An extreme example of somatic pairing can be observed in Drosophila melanogaster, where homologous chromosomes remain aligned throughout most of development. However, our understanding of the mechanism of somatic homolog pairing remains unclear, as only a few genes have been implicated in this process. In this study, we introduce a novel high-throughput fluorescent in situ hybridization (FISH) technology that enabled us to conduct a genome-wide RNAi screen for factors involved in the robust somatic pairing observed in Drosophila. We identified both candidate “pairing promoting genes” and candidate “anti-pairing genes,” providing evidence that pairing is a dynamic process that can be both enhanced and antagonized. Many of the genes found to be important for promoting pairing are highly enriched for functions associated with mitotic cell division, suggesting a genetic framework for a long-standing link between chromosome dynamics during mitosis and nuclear organization during interphase. In contrast, several of the candidate anti-pairing genes have known interphase functions associated with S-phase progression, DNA replication, and chromatin compaction, including several components of the condensin II complex. In combination with a variety of secondary assays, these results provide insights into the mechanism and dynamics of somatic pairing

Spike-Timing Precision and Neuronal Synchrony Are Enhanced by an Interaction between Synaptic Inhibition and Membrane Oscillations in the Amygdala

The basolateral complex of the amygdala (BLA) is a critical component of the neural circuit regulating fear learning. During fear learning and recall, the amygdala and other brain regions, including the hippocampus and prefrontal cortex, exhibit phase-locked oscillations in the high delta/low theta frequency band (∼2–6 Hz) that have been shown to contribute to the learning process. Network oscillations are commonly generated by inhibitory synaptic input that coordinates action potentials in groups of neurons. In the rat BLA, principal neurons spontaneously receive synchronized, inhibitory input in the form of compound, rhythmic, inhibitory postsynaptic potentials (IPSPs), likely originating from burst-firing parvalbumin interneurons. Here we investigated the role of compound IPSPs in the rat and rhesus macaque BLA in regulating action potential synchrony and spike-timing precision. Furthermore, because principal neurons exhibit intrinsic oscillatory properties and resonance between 4 and 5 Hz, in the same frequency band observed during fear, we investigated whether compound IPSPs and intrinsic oscillations interact to promote rhythmic activity in the BLA at this frequency. Using whole-cell patch clamp in brain slices, we demonstrate that compound IPSPs, which occur spontaneously and are synchronized across principal neurons in both the rat and primate BLA, significantly improve spike-timing precision in BLA principal neurons for a window of ∼300 ms following each IPSP. We also show that compound IPSPs coordinate the firing of pairs of BLA principal neurons, and significantly improve spike synchrony for a window of ∼130 ms. Compound IPSPs enhance a 5 Hz calcium-dependent membrane potential oscillation (MPO) in these neurons, likely contributing to the improvement in spike-timing precision and synchronization of spiking. Activation of the cAMP-PKA signaling cascade enhanced the MPO, and inhibition of this cascade blocked the MPO. We discuss these results in the context of spike-timing dependent plasticity and modulation by neurotransmitters important for fear learning, such as dopamine

The impact of viral mutations on recognition by SARS-CoV-2 specific T cells.

We identify amino acid variants within dominant SARS-CoV-2 T cell epitopes by interrogating global sequence data. Several variants within nucleocapsid and ORF3a epitopes have arisen independently in multiple lineages and result in loss of recognition by epitope-specific T cells assessed by IFN-γ and cytotoxic killing assays. Complete loss of T cell responsiveness was seen due to Q213K in the A∗01:01-restricted CD8+ ORF3a epitope FTSDYYQLY207-215; due to P13L, P13S, and P13T in the B∗27:05-restricted CD8+ nucleocapsid epitope QRNAPRITF9-17; and due to T362I and P365S in the A∗03:01/A∗11:01-restricted CD8+ nucleocapsid epitope KTFPPTEPK361-369. CD8+ T cell lines unable to recognize variant epitopes have diverse T cell receptor repertoires. These data demonstrate the potential for T cell evasion and highlight the need for ongoing surveillance for variants capable of escaping T cell as well as humoral immunity.This work is supported by the UK Medical Research Council (MRC); Chinese Academy of Medical Sciences(CAMS) Innovation Fund for Medical Sciences (CIFMS), China; National Institute for Health Research (NIHR)Oxford Biomedical Research Centre, and UK Researchand Innovation (UKRI)/NIHR through the UK Coro-navirus Immunology Consortium (UK-CIC). Sequencing of SARS-CoV-2 samples and collation of data wasundertaken by the COG-UK CONSORTIUM. COG-UK is supported by funding from the Medical ResearchCouncil (MRC) part of UK Research & Innovation (UKRI),the National Institute of Health Research (NIHR),and Genome Research Limited, operating as the Wellcome Sanger Institute. T.I.d.S. is supported by a Well-come Trust Intermediate Clinical Fellowship (110058/Z/15/Z). L.T. is supported by the Wellcome Trust(grant number 205228/Z/16/Z) and by theUniversity of Liverpool Centre for Excellence in Infectious DiseaseResearch (CEIDR). S.D. is funded by an NIHR GlobalResearch Professorship (NIHR300791). L.T. and S.C.M.are also supported by the U.S. Food and Drug Administration Medical Countermeasures Initiative contract75F40120C00085 and the National Institute for Health Research Health Protection Research Unit (HPRU) inEmerging and Zoonotic Infections (NIHR200907) at University of Liverpool inpartnership with Public HealthEngland (PHE), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford.L.T. is based at the University of Liverpool. M.D.P. is funded by the NIHR Sheffield Biomedical ResearchCentre (BRC – IS-BRC-1215-20017). ISARIC4C is supported by the MRC (grant no MC_PC_19059). J.C.K.is a Wellcome Investigator (WT204969/Z/16/Z) and supported by NIHR Oxford Biomedical Research Centreand CIFMS. The views expressed are those of the authors and not necessarily those of the NIHR or MRC

The burden of bacterial antimicrobial resistance in the WHO African region in 2019: a cross-country systematic analysis

Background A critical and persistent challenge to global health and modern health care is the threat of antimicrobial resistance (AMR). Previous studies have reported a disproportionate burden of AMR in low-income and middle-income countries, but there remains an urgent need for more in-depth analyses across Africa. This study presents one of the most comprehensive sets of regional and country-level estimates of bacterial AMR burden in the WHO African region to date. Methods We estimated deaths and disability-adjusted life-years (DALYs) attributable to and associated with AMR for 23 bacterial pathogens and 88 pathogen–drug combinations for countries in the WHO African region in 2019. Our methodological approach consisted of five broad components: the number of deaths in which infection had a role, the proportion of infectious deaths attributable to a given infectious syndrome, the proportion of infectious syndrome deaths attributable to a given pathogen, the percentage of a given pathogen resistant to an antimicrobial drug of interest, and the excess risk of mortality (or duration of an infection) associated with this resistance. These components were then used to estimate the disease burden by using two counterfactual scenarios: deaths attributable to AMR (considering an alternative scenario where infections with resistant pathogens are replaced with susceptible ones) and deaths associated with AMR (considering an alternative scenario where drug-resistant infections would not occur at all). We obtained data from research hospitals, surveillance networks, and infection databases maintained by private laboratories and medical technology companies. We generated 95% uncertainty intervals (UIs) for final estimates as the 25th and 975th ordered values across 1000 posterior draws, and models were cross-validated for out-of-sample predictive validity. Findings In the WHO African region in 2019, there were an estimated 1·05 million deaths (95% UI 829 000–1 316 000) associated with bacterial AMR and 250 000 deaths (192 000–325 000) attributable to bacterial AMR. The largest fatal AMR burden was attributed to lower respiratory and thorax infections (119 000 deaths [92 000–151 000], or 48% of all estimated bacterial pathogen AMR deaths), bloodstream infections (56 000 deaths [37 000–82 000], or 22%), intra-abdominal infections (26 000 deaths [17 000–39 000], or 10%), and tuberculosis (18 000 deaths [3850–39 000], or 7%). Seven leading pathogens were collectively responsible for 821 000 deaths (636 000–1 051 000) associated with resistance in this region, with four pathogens exceeding 100 000 deaths each: Streptococcus pneumoniae, Klebsiella pneumoniae, Escherichia coli, and Staphylococcus aureus. Third-generation cephalosporin-resistant K pneumoniae and meticillin-resistant S aureus were shown to be the leading pathogen–drug combinations in 25 and 16 countries, respectively (53% and 34% of the whole region, comprising 47 countries) for deaths attributable to AMR. Interpretation This study reveals a high level of AMR burden for several bacterial pathogens and pathogen–drug combinations in the WHO African region. The high mortality rates associated with these pathogens demonstrate an urgent need to address the burden of AMR in Africa. These estimates also show that quality and access to health care and safe water and sanitation are correlated with AMR mortality, with a higher fatal burden found in lower resource settings. Our cross-country analyses within this region can help local governments to leverage domestic and global funding to create stewardship policies that target the leading pathogen–drug combinations. Funding Bill & Melinda Gates Foundation, Wellcome Trust, and Department of Health and Social Care using UK aid funding managed by the Fleming Fund

Investigation of hospital discharge cases and SARS-CoV-2 introduction into Lothian care homes

Summary Background The first epidemic wave of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Scotland resulted in high case numbers and mortality in care homes. In Lothian, over one-third of care homes reported an outbreak, while there was limited testing of hospital patients discharged to care homes. Aim To investigate patients discharged from hospitals as a source of SARS-CoV-2 introduction into care homes during the first epidemic wave. Methods A clinical review was performed for all patients discharges from hospitals to care homes from 1st March 2020 to 31st May 2020. Episodes were ruled out based on coronavirus disease 2019 (COVID-19) test history, clinical assessment at discharge, whole-genome sequencing (WGS) data and an infectious period of 14 days. Clinical samples were processed for WGS, and consensus genomes generated were used for analysis using Cluster Investigation and Virus Epidemiological Tool software. Patient timelines were obtained using electronic hospital records. Findings In total, 787 patients discharged from hospitals to care homes were identified. Of these, 776 (99%) were ruled out for subsequent introduction of SARS-CoV-2 into care homes. However, for 10 episodes, the results were inconclusive as there was low genomic diversity in consensus genomes or no sequencing data were available. Only one discharge episode had a genomic, time and location link to positive cases during hospital admission, leading to 10 positive cases in their care home. Conclusion The majority of patients discharged from hospitals were ruled out for introduction of SARS-CoV-2 into care homes, highlighting the importance of screening all new admissions when faced with a novel emerging virus and no available vaccine

Tracking SARS-CoV-2 mutations and variants through the COG-UK-Mutation Explorer

COG-UK Mutation Explorer (COG-UK-ME, https://sars2.cvr.gla.ac.uk/cog-uk/—last accessed date 16 March 2022) is a web resource that displays knowledge and analyses on SARS-CoV-2 virus genome mutations and variants circulating in the UK, with a focus on the observed amino acid replacements that have an antigenic role in the context of the human humoral and cellular immune response. This analysis is based on more than 2 million genome sequences (as of March 2022) for UK SARS-CoV-2 data held in the CLIMB-COVID centralised data environment. COG-UK-ME curates these data and displays analyses that are cross-referenced to experimental data collated from the primary literature. The aim is to track mutations of immunological importance that are accumulating in current variants of concern and variants of interest that could alter the neutralising activity of monoclonal antibodies (mAbs), convalescent sera, and vaccines. Changes in epitopes recognised by T cells, including those where reduced T cell binding has been demonstrated, are reported. Mutations that have been shown to confer SARS-CoV-2 resistance to antiviral drugs are also included. Using visualisation tools, COG-UK-ME also allows users to identify the emergence of variants carrying mutations that could decrease the neutralising activity of both mAbs present in therapeutic cocktails, e.g. Ronapreve. COG-UK-ME tracks changes in the frequency of combinations of mutations and brings together the curated literature on the impact of those mutations on various functional aspects of the virus and therapeutics. Given the unpredictable nature of SARS-CoV-2 as exemplified by yet another variant of concern, Omicron, continued surveillance of SARS-CoV-2 remains imperative to monitor virus evolution linked to the efficacy of therapeutics