Dimerization and Heme Binding Are Conserved in Amphibian and Starfish Homologues of the microRNA Processing Protein DGCR8

Human DiGeorge Critical Region 8 (DGCR8) is an essential microRNA (miRNA) processing factor that is activated via direct interaction with Fe(III) heme. In order for DGCR8 to bind heme, it must dimerize using a dimerization domain embedded within its heme-binding domain (HBD). We previously reported a crystal structure of the dimerization domain from human DGCR8, which demonstrated how dimerization results in the formation of a surface important for association with heme. Here, in an attempt to crystallize the HBD, we search for DGCR8 homologues and show that DGCR8 from Patiria miniata (bat star) also binds heme. The extinction coefficients (ε) of DGCR8-heme complexes are determined; these values are useful for biochemical analyses and allow us to estimate the heme occupancy of DGCR8 proteins. Additionally, we present the crystal structure of the Xenopus laevis dimerization domain. The structure is very similar to that of human DGCR8. Our results indicate that dimerization and heme binding are evolutionarily conserved properties of DGCR8 homologues not only in vertebrates, but also in at least some invertebrates

Automatic Code Placement Alternatives for Ad-Hoc And Sensor Networks

Developing applications for ad-hoc and sensor networks poses significant challenges. Many interesting applications in these domains entail collaboration between components distributed throughout an ad-hoc network. Defining these components, optimally placing them on nodes in the ad-hoc network and relocating them in response to changes is a fundamental problem faced by such applications. Manual approaches to code and data migration are not only platform-dependent and error-prone, but also needlessly complicate application development. Further, locally optimal decisions made by applications that share the same network can lead to globally unstable and energy inefficient behavior. In this paper we describe the design and implementation of a distributed operating system for ad-hoc and sensor networks whose goal is to enable power-aware, adaptive, and easy-to-develop ad-hoc networking applications. Our system achieves this goal by providing a single system image of a unified Java virtual machine to applications over an ad-hoc collection of heterogeneous nodes. It automatically and transparently partitions applications into components and dynamically finds a placement of these components on nodes within the ad-hoc network to reduce energy consumption and increase system longevity. This paper outlines the design of our system and evaluates two practical, power-aware, online algorithms for object placement that form the core of our system. We demonstrate that our algorithms can increase system longevity by a factor of four to five by effectively distributing energy consumption, and are suitable for use in an energy efficient operating system in which applications are distributed automatically and transparently

A comparison of rapid point-of-care tests for the detection of avian influenza A(H7N9) virus, 2013

Six antigen detection-based rapid influenza point-of-care tests were compared for their ability to detect avian influenza A(H7N9) virus. The sensitivity of at least four tests, standardised by viral infectivity (TCID50) or RNA copy number, was lower for the influenza A(H7N9) virus than for seasonal A(H3N2), A(H1N1)pdm09 or other recent avian A(H7) viruses. Comparing detection limits of A(H7N9) virus with Ct values of A(H7N9) clinical specimens suggests the tests would not have detected most clinical specimens

PqqD is a novel peptide chaperone that forms a ternary complex with the radical S-adenosylmethionine protein PqqE in the pyrroloquinoline quinone biosynthetic pathway

Pyrroloquinoline quinone (PQQ) is a product of a ribosomally synthesized and post-translationally modified pathway consisting of five conserved genes, pqqA-E. PqqE is a radical S-adenosylmethionine (RS) protein with a C-terminal SPASM domain, and is proposed to catalyze the formation of a carbon-carbon bond between the glutamate and tyrosine side chains of the peptide substrate PqqA. PqqD is a 10-kDa protein with an unknown function, but is essential for PQQ production. Recently, in Klebsiella pneumoniae (Kp), PqqD and PqqE were shown to interact; however, the stoichiometry and KD were not obtained. Here, we show that the PqqE and PqqD interaction transcends species, also occurring in Methylobacterium extorquens AM1 (Me). The stoichiometry of the MePqqD and MePqqE interaction is 1:1 and the KD, determined by surface plasmon resonance spectroscopy (SPR), was found to be ∼12 μm. Moreover, using SPR and isothermal calorimetry techniques, we establish for the first time that MePqqD binds MePqqA tightly (KD ∼200 nm). The formation of a ternary MePqqA-D-E complex was captured by native mass spectrometry and the KD for the MePqqAD-MePqqE interaction was found to be ∼5 μm. Finally, using a bioinformatic analysis, we found that PqqD orthologues are associated with the RS-SPASM family of proteins (subtilosin, pyrroloquinoline quinone, anaerobic sulfatase maturating enzyme, and mycofactocin), all of which modify either peptides or proteins. In conclusion, we propose that PqqD is a novel peptide chaperone and that PqqD orthologues may play a similar role in peptide modification pathways that use an RS-SPASM protein

Inequalities in COVID19 mortality related to ethnicity and socioeconomic deprivation - pre-print paper

Background: Initial reports suggest that ethnic minorities may be experiencing more severe coronavirus disease 2019 (COVID19) outcomes. We therefore assessed the association between ethnic composition, income deprivation and COVID19 mortality rates in England. Methods: We performed a cross-sectional ecological analysis across England's upper-tier local authorities. We assessed the association between the proportion of the population from Black, Asian and Minority Ethnic (BAME) backgrounds, income deprivation and COVID19 mortality rates using multivariable negative binomial regression, adjusting for population density, proportion of the population aged 50-79 and 80+ years, and the duration of the epidemic in each area. Findings: Local authorities with a greater proportion of residents from ethnic minority backgrounds had statistically significantly higher COVID19 mortality rates, as did local authorities with a greater proportion of residents experiencing deprivation relating to low income. After adjusting for income deprivation and other covariates, each percentage point increase in the proportion of the population from BAME backgrounds was associated with a 1% increase in the COVID19 mortality rate [IRR=1.01, 95%CI 1.01-1.02]. Each percentage point increase in the proportion of the population experiencing income deprivation was associated with a 2% increase in the COVID19 mortality rate [IRR=1.02, 95%CI 1.01-1.04]. Interpretation: This study provides evidence that both income deprivation and ethnicity are associated with greater COVID19 mortality. To reduce these inequalities, Government needs to target effective control and recovery measures at these disadvantaged communities, proportionate to their greater needs and vulnerabilities, during and following the pandemic

Differential Regulation of Formyl Peptide and Platelet-Activating Factor Receptors: Role of Phospholipase Cβ3 Phosphorylation by Protein Kinase A

Formylated peptides (e.g. n-formyl-Met-Leu-Phe (fMLP)) and platelet- activating factor (PAF) mediate chemotactic and cytotoxic responses in leukocytes through receptors coupled to G proteins that activate phospholipase C (PLC). In RBL-2H3 cells, fMLP utilizes a pertussis toxin (ptx)-sensitive G protein to activate PLC, whereas PAF utilizes a ptx- insensitive G protein. Here we demonstrate that fMLP, but not PAF, enhanced intracellular cAMP levels via a ptx-sensitive mechanism. Protein kinase A (PKA) inhibition by H-89 enhanced inositol phosphate formation stimulated by fMLP but not PAF. Furthermore, a membrane-permeable cAMP analog 8-(4- chlorophenylthio)-cAMP (cpt-cAMP) inhibited phosphoinositide hydrolysis and secretion stimulated by fMLP but not PAF. Both cpt-cAMP and fMLP stimulated PLCβ3 phosphorylation in intact RBL cells. The purified catalytic subunit of PKA phosphorylated PLCβ3 immunoprecipitated from RBL cell lysate. Pretreatment of intact cells with cpt-cAMP and fMLP, but not PAF, resulted in an inhibition of subsequent PLCβ3 phosphorylation by PKA in vitro. These data demonstrate that fMLP receptor, which couples to a ptx-sensitive G protein, activates both PLC and cAMP production. The resulting PKA activation phosphorylates PLCβ3 and appears to block the ability of G(βγ) to activate PLC. Thus, both fMLP and PAF generate stimulatory signals for PLCβ3, but only fMLP produces a PKA-dependent inhibitory signal. This suggests a novel mechanism for the bidirectional regulation of receptors which activate PLC by ptx-sensitive G proteins

MDCK-SIAT1 cells show improved isolation rates for recent human influenza viruses compared to conventinal MDCK cells

The ability to isolate and propagate influenza virus is an essential tool for the yearly surveillance of circulating virus strains and to ensure accurate clinical diagnosis for appropriate treatment. The suitability of MDCK-SIAT1 cells, engineered to express increased levels of alpha-2,6-linked sialic acid receptors, as an alternative to conventional MDCK cells for isolation of circulating influenza virus was assessed. A greater number of influenza A (H1N1 and H3N2) and B viruses from stored human clinical specimens collected between 2005 and 2007 were isolated following inoculation in MDCK-SIAT1 cells than in MDCK cells. In addition, a higher titer of virus was recovered following culture in MDCK-SIAT1 cells. All A(H1N1) viruses recovered from MDCK-SIAT1 cells were able to agglutinate both turkey and guinea pig red blood cells (RBC), while half of the A(H3N2) viruses recovered after passage in MDCK-SIAT1 cells lost the ability to agglutinate turkey RBC. Importantly, the HA-1 domain of the hemagglutinin gene was genetically stable after passaging in MDCK-SIAT1 cells, a feature not always seen following MDCK cell or embryonated chicken egg passage of human influenza virus. These data indicate that the MDCK-SIAT1 cell line is superior to conventional MDCK cells for isolation of human influenza virus from clinical specimens and may be used routinely for the isolation and propagation of current human influenza viruses for surveillance, diagnostic, and research purposes

TaqMan real time RT-PCR assays for detecting ferret innate and adaptive immune responses

AbstractThe ferret is an excellent model for many human infectious diseases including influenza, SARS-CoV, henipavirus and pneumococcal infections. The ferret is also used to study cystic fibrosis and various cancers, as well as reproductive biology and physiology. However, the range of reagents available to measure the ferret immune response is very limited. To address this deficiency, high-throughput real time RT-PCR TaqMan assays were developed to measure the expression of fifteen immune mediators associated with the innate and adaptive immune responses (IFNα, IFNβ, IFNγ, IL1α, IL1β, IL2, IL4, IL6, IL8, IL10, IL12p40, IL17, Granzyme A, MCP1, TNFα), as well as four endogenous housekeeping genes (ATF4, HPRT, GAPDH, L32). These assays have been optimized to maximize reaction efficiency, reduce the amount of sample required (down to 1ng RNA per real time RT-PCR reaction) and to select the most appropriate housekeeping genes. Using these assays, the expression of each of the tested genes could be detected in ferret lymph node cells stimulated with mitogens or infected with influenza virus in vitro. These new tools will allow a more comprehensive analysis of the ferret immune responses following infection or in other disease states

‘A system that is struggling’: understanding health protection resilience in England during the COVID-19 pandemic through the experiences of local health protection responders.

Abstract Background Local health protection systems play a crucial role in infectious disease prevention and control and were critical to COVID-19 pandemic responses. Despite this vital function, few studies have explored the lived experience of health protection responders managing COVID-19. We provide new insights by examining how COVID-19 shaped infectious disease prevention and control in local health protection systems in England. Methods Semi-structured interviews were conducted with twenty local health protection responders from three contrasting local authority areas, and Public Health England (PHE) health protection teams, in England between June 2021 - March 2022. Participants were from: PHE health protection teams (n=6); local authority public health teams (n=5); local authority Public Protection Services (n=7); and local authority commissioned Infection Prevention and Control Teams (n=2). Data were analysed using reflexive thematic analysis. Results First, participants acknowledged the pandemic caused an unprecedented workload and disruption to local health protection service delivery. There was not enough capacity within existing local health protection systems to manage the increased workload. PHE health protection teams therefore transferred some COVID-19 related health protection tasks to other staff, mainly those employed by local authorities. Second, health protection responders highlighted how COVID-19 drew attention to the weaknesses in local health protection systems already stressed by reduced funding in the years leading up to the pandemic. Injecting money into the COVID-19 response did not completely overcome former losses in specialist health protection workforce. Third, health protection responders described how pandemic management raised the profile of public health, especially infectious disease prevention and control. Managing COVID-19 strengthened collaborative working, resulting in enhanced capacity of local health protection systems at the time. Conclusion The COVID-19 pandemic challenged the public health preparedness of all countries. Health protection responders in this study also expressed many challenges. There was insufficient resilience in these local health protection systems and an inability to scale up the specialist health protection workforce, as required in a pandemic situation. The UK needs to learn from the pandemic experience by acknowledging and addressing the challenges faced by local health protection responders so that it can more effectively respond to future threats