Glutamate-Bound NMDARs Arising from In Vivo-like Network Activity Extend Spatio-temporal Integration in a L5 Cortical Pyramidal Cell Model

In vivo, cortical pyramidal cells are bombarded by asynchronous synaptic input arising from ongoing network activity. However, little is known about how such ‘background’ synaptic input interacts with nonlinear dendritic mechanisms. We have modified an existing model of a layer 5 (L5) pyramidal cell to explore how dendritic integration in the apical dendritic tuft could be altered by the levels of network activity observed in vivo. Here we show that asynchronous background excitatory input increases neuronal gain and extends both temporal and spatial integration of stimulus-evoked synaptic input onto the dendritic tuft. Addition of fast and slow inhibitory synaptic conductances, with properties similar to those from dendritic targeting interneurons, that provided a ‘balanced’ background configuration, partially counteracted these effects, suggesting that inhibition can tune spatio-temporal integration in the tuft. Excitatory background input lowered the threshold for NMDA receptor-mediated dendritic spikes, extended their duration and increased the probability of additional regenerative events occurring in neighbouring branches. These effects were also observed in a passive model where all the non-synaptic voltage-gated conductances were removed. Our results show that glutamate-bound NMDA receptors arising from ongoing network activity can provide a powerful spatially distributed nonlinear dendritic conductance. This may enable L5 pyramidal cells to change their integrative properties as a function of local network activity, potentially allowing both clustered and spatially distributed synaptic inputs to be integrated over extended timescales.     

YPED:An Integrated Bioinformatics Suite and Database for Mass Spectrometry-based Proteomics Research

We report a significantly-enhanced bioinformatics suite and database for proteomics research called Yale Protein Expression Database(YPED) that is used by investigators at more than 300 institutions worldwide. YPED meets the data management, archival, and analysis needs of a high-throughput mass spectrometry-based proteomics research ranging from a singlelaboratory, group of laboratories within and beyond an institution, to the entire proteomics community. The current version is a significant improvement over the first version in that it contains new modules for liquid chromatography–tandem mass spectrometry(LC–MS/MS) database search results, label and label-free quantitative proteomic analysis, and several scoring outputs for phosphopeptide site localization. In addition, we have added both peptide and protein comparative analysis tools to enable pairwise analysis of distinct peptides/proteins in each sample and of overlapping peptides/proteins between all samples in multiple datasets. We have also implemented a targeted proteomics module for automated multiple reaction monitoring(MRM)/selective reaction monitoring(SRM) assay development. We have linked YPED's database search results and both label-based and label-free fold-change analysis to the Skyline Panorama repository for online spectra visualization. In addition, we have built enhanced functionality to curate peptide identifications into an MS/MS peptide spectral library for all of our protein database search identification results.     

Activation of Salmonella Typhi-Specific Regulatory T Cells in Typhoid Disease in a Wild-Type S. Typhi Challenge Model

Salmonella Typhi (S. Typhi), the causative agent of typhoid fever, causes significant morbidity and mortality worldwide. Currently available vaccines are moderately efficacious, and identification of immunological responses associated with protection or disease will facilitate the development of improved vaccines. We investigated S. Typhi-specific modulation of activation and homing potential of circulating regulatory T cells (T_reg) by flow and mass cytometry using specimens obtained from a human challenge study. Peripheral blood mononuclear cells were obtained from volunteers pre- and at multiple time-points post-challenge with wild-type S. Typhi. We identified differing patterns of S. Typhi-specific modulation of the homing potential of circulating T_reg between volunteers diagnosed with typhoid (TD) and those who were not (No TD). TD volunteers demonstrated up-regulation of the gut homing molecule integrin α4ß7 pre-challenge, followed by a significant down-regulation post-challenge consistent with T_reg homing to the gut. Additionally, S. Typhi-specific T_reg from TD volunteers exhibited up-regulation of activation molecules post-challenge (e.g., HLA-DR, LFA-1). We further demonstrate that depletion of T_reg results in increased S. Typhi-specific cytokine production by CD8+ T_EM in vitro. These results suggest that the tissue distribution of activated T_reg, their characteristics and activation status may play a pivotal role in typhoid fever, possibly through suppression of S. Typhi-specific effector T cell responses. These studies provide important novel insights into the regulation of immune responses that are likely to be critical in protection against typhoid and other enteric infectious diseases.     

Oral Wild-Type Salmonella Typhi Challenge Induces Activation of Circulating Monocytes and Dendritic Cells in Individuals Who Develop Typhoid Disease

A new human oral challenge model with wild-type Salmonella Typhi (S. Typhi) was recently developed. In this model, ingestion of 104 CFU of Salmonella resulted in 65% of subjects developing typhoid fever (referred here as typhoid diagnosis -TD-) 5–10 days post-challenge. TD criteria included meeting clinical (oral temperature ≥38°C for ≥12h) and/or microbiological (S. Typhi bacteremia) endpoints. One of the first lines of defense against pathogens are the cells of the innate immune system (e.g., monocytes, dendritic cells -DCs-). Various changes in circulating monocytes and DCs have been described in the murine S. Typhimurium model; however, whether similar changes are present in humans remains to be explored. To address these questions, a subset of volunteers (5 TD and 3 who did not develop typhoid despite oral challenge -NoTD-) were evaluated for changes in circulating monocytes and DCs. Expression of CD38 and CD40 were upregulated in monocytes and DCs in TD volunteers during the disease days (TD-0h to TD-96h). Moreover, integrin α4β7, a gut homing molecule, was upregulated on monocytes but not DCs. CD21 upregulation was only identified in DCs. These changes were not observed among NoTD volunteers despite the same oral challenge. Moreover, monocytes and DCs from NoTD volunteers showed increased binding to S. Typhi one day after challenge. These monocytes showed phosphorylation of p38MAPK, NFkB and Erk1/2 upon stimulation with S. Typhi-LPS-QDot micelles. In contrast, monocytes from TD volunteers showed only a moderate increase in S. Typhi binding 48h and 96h post-TD, and only Erk1/2 phosphorylation. This is the first study to describe different activation and migration profiles, as well as differential signaling patterns, in monocytes and DCs which relate directly to the clinical outcome following oral challenge with wild type S. Typhi.     

Dental and Microbiological Risk Factors for Hospital-Acquired Pneumonia in Non-Ventilated Older Patients

Hospital acquired pneumonia (HAP) is often fatal in older patients. The mouth is the main reservoir of infection and studies have suggested that oral hygiene interventions may prevent HAP. The aim of this study was to investigate associations between HAP and preceding a) heavy dental plaque and b) oral carriage of potential respiratory pathogens in older patients with lower limb fracture to determine the target for intervention studies.

Methods

We obtained a time series of tongue/throat swabs from 90 patients with lower limb fracture, aged 65-101 in a general hospital in the North East of England between April 2009-July 2010. We used novel real-time multiplex PCR assays to detect S. aureus, MRSA, E. coli, P. aeruginosa, S. pneumoniae, H. influenza and Acinetobacter spp. We collected data on dental/denture plaque (modified Quigley-Hein index) and outcomes of clinician-diagnosed HAP.

Results

The crude incidence of HAP was 10% (n = 90), with mortality of 80% at 90 days post discharge. 50% of cases occurred within the first 25 days. HAP was not associated with being dentate, tooth number, or heavy dental/denture plaque. HAP was associated with prior oral carriage with E. coli/S. aureus/P.aeruginosa/MRSA (p = 0.002, OR 9.48 95% CI 2.28-38.78). The incidence of HAP in those with carriage was 35% (4% without), with relative risk 6.44 (95% CI 2.04-20.34, p = 0.002). HAP was associated with increased length of stay (Fishers exact test, p=0.01), with mean 30 excess days (range -11.5-115). Target organisms were first detected within 72 hours of admission in 90% participants, but HAP was significantly associated with S. aureus/MRSA/P. aeruginosa/E. coli being detected at days 5 (OR 4.39, 95%CI1.73-11.16) or 14 (OR 6.69, 95%CI 2.40-18.60).

Conclusions

Patients with lower limb fracture who were colonised orally with E. coli/ S. aureus/MRSA/P. aeruginosa after 5 days in hospital were at significantly greater risk of HAP (p = 0.002).     

Higher Trophic Levels Overwhelm Climate Change Impacts on Terrestrial Ecosystem Functioning

Forest floor food webs play pivotal roles in carbon cycling, but they are rarely considered in models of carbon fluxes, including soil carbon dioxide emissions (respiration), under climatic warming. The indirect effects of invertebrates on heterotrophic (microbial and invertebrate) respiration through interactions with microbial communities are significant and will be altered by warming. However, the interactive effects of invertebrates and warming on heterotrophic respiration in the field are poorly understood. In this study we combined field and common garden laboratory approaches to examine relationships between warming, forest floor food web structure, and heterotrophic respiration. We found that soil animals can overwhelm the effects of warming (to 5 degrees Celsius above ambient) on heterotrophic respiration. In particular, the presence of higher trophic levels and burrowing detritivores strongly determined heterotrophic respiration rates in temperate forest soils. These effects were, however, context-dependent, with greater effects in a lower-latitude site. Without isolating and including the significant impact of invertebrates, climate models will be incomplete, hindering well-informed policy decisions.     

Developmental timing of mRNA translation—integration of distinct regulatory elements

Targeted mRNA translation is emerging as a critical mechanism to control gene expression during developmental processes. Exciting new findings have revealed a critical role for regulatory elements within the mRNA untranslated regions to direct the timing of mRNA translation. Regulatory elements can be targeted by sequence‐specific binding proteins to direct either repression or activation of mRNA translation in response to developmental signals. As new regulatory elements continue to be identified it has become clear that targeted mRNAs can contain multiple regulatory elements, directing apparently contradictory translational patterns. How is this complex regulatory input integrated? In this review, we focus on a new challenge area—how sequence‐specific RNA binding proteins respond to developmental signals and functionally integrate to regulate the extent and timing of target mRNA translation. We discuss current understanding with a particular emphasis on the control of cell cycle progression that is mediated through a complex interplay of distinct mRNA regulatory elements during Xenopus oocyte maturation. Mol. Reprod. Dev. 77: 662–669, 2010. © 2010 Wiley‐Liss, Inc.     

RRP1B targets PP1 to mammalian cell nucleoli and is associated with Pre-60S ribosomal subunits

A pool of protein phosphatase 1 (PP1) accumulates within nucleoli and accounts for a large fraction of the serine/threonine protein phosphatase activity in this subnuclear structure. Using a combination of fluorescence imaging with quantitative proteomics, we mapped the subnuclear localization of the three mammalian PP1 isoforms stably expressed as GFP-fusions in live cells and identified RRP1B as a novel nucleolar targeting subunit that shows a specificity for PP1β and PP1γ. RRP1B, one of two mammalian orthologues of the yeast Rrp1p protein, shows an RNAse-dependent localization to the granular component of the nucleolus and distributes in a similar manner throughout the cell cycle to proteins involved in later steps of rRNA processing. Quantitative proteomic analysis of complexes containing both RRP1B and PP1γ revealed enrichment of an overlapping subset of large (60S) ribosomal subunit proteins and pre-60S nonribosomal proteins involved in mid-late processing. Targeting of PP1 to this complex by RRP1B in mammalian cells is likely to contribute to modulation of ribosome biogenesis by mechanisms involving reversible phosphorylation events, thus playing a role in the rapid transduction of cellular signals that call for regulation of ribosome production in response to cellular stress and/or changes in growth conditions.     

Marginal zinc deficiency increases oxidative DNA damage in the prostate after chronic exercise

Approximately 12% of Americans do not consume the recommended level of zinc and could be at risk for marginal zinc deficiency. Zinc functions in antioxidant defense and DNA repair and could be important for prostate health. We hypothesized that marginal zinc deficiency sensitizes the prostate to oxidative stress and DNA damage. Rats were fed a zinc-adequate (ZA; 30 mg Zn/kg) or marginally zinc-deficient (MZD; 5–6 mg Zn/kg) diet for 6 weeks. MZD increased p53 and PARP expression but no change in 8-hydroxy-2′-deoxyguanosine levels was detected. To examine the susceptibility to exogenous oxidative stress, rats fed a ZA or MZD diet were assigned to exercising (EXE) or sedentary (SED) groups for 9 weeks. MZD or EXE alone did not affect oxidative DNA damage in the prostate; however, combined MZD + EXE increased DNA damage in the dorsolateral lobe. PARP and p53 expression was not further induced with MZD + EXE, suggesting that MZD interferes with DNA repair responses to stress. Finally, the addition of phytase to the MZD diet successfully restored zinc levels in the prostate and decreased DNA damage back to ZA levels. Overall, this study suggests that marginal zinc deficiency sensitizes the prostate to oxidative stress and demonstrates the importance of maintaining optimal zinc nutrition in physically active populations.