A Model for How Signal Duration Can Determine Distinct Outcomes of Gene Transcription Programs

The reason why IL-6 induces a pro-inflammatory response, while IL-10 induces an anti-inflammatory response, despite both cytokines activating the same transcription factor, STAT3, is not well understood. It is known that IL-6 induces a transient STAT3 signal and that IL-10 induces a sustained STAT3 signal due to the STAT3-induced inhibitor SOCS3's ability to bind to the IL-6R and not the IL-10R. We sought to develop a general transcriptional network that is capable of translating sustained signals into one response, while translating transient signals into a second response. The general structure of such a network is that the transcription factor STAT3 can induce both an inflammatory response and an anti-inflammatory response by inducing two different genes. The anti-inflammatory gene can bind to and inhibit the inflammatory gene's production and the inflammatory gene can bind to its own promoter and induce its own transcription in the absence of the signal. One prediction that can be made from such a network is that in SOCS3−/− mice, where IL-6 induces a sustained STAT3 signal, that IL-6 would act as an anti-inflammatory cytokine, which has indeed been observed experimentally in the literature

Mycobacterial Acid Tolerance Enables Phagolysosomal Survival and Establishment of Tuberculous Infection In Vivo.

The blockade of phagolysosomal fusion is considered a critical mycobacterial strategy to survive in macrophages. However, viable mycobacteria have been observed in phagolysosomes during infection of cultured macrophages, and mycobacteria have the virulence determinant MarP, which confers acid resistance in vitro. Here we show in mice and zebrafish that innate macrophages overcome mycobacterial lysosomal avoidance strategies to rapidly deliver a substantial proportion of infecting bacteria to phagolysosomes. Exploiting the optical transparency of the zebrafish, we tracked the fates of individual mycobacteria delivered to phagosomes versus phagolysosomes and discovered that bacteria survive and grow in phagolysosomes, though growth is slower. MarP is required specifically for phagolysosomal survival, making it an important determinant for the establishment of mycobacterial infection in their hosts. Our work suggests that if pathogenic mycobacteria fail to prevent lysosomal trafficking, they tolerate the resulting acidic environment of the phagolysosome to establish infection.National Institutes of Health (Grant IDs: R37AI054503, R01 AI076327, 5T32HD007233, 5F30HL110455), Wellcome Trust, National Institute of Health Research Cambridge Biomedical Research CentreThis is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.chom.2016.07.00

Jean Briggs's Never in Anger as an Ethnography of Experience

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66542/2/10.1177_0308275X9301300406.pd

Isolation of a Rickettsial Pathogen from a Non-Hematophagous Arthropod

Rickettsial diversity is intriguing in that some species are transmissible to vertebrates, while others appear exclusive to invertebrate hosts. Of particular interest is Rickettsia felis, identifiable in both stored product insect pests and hematophagous disease vectors. To understand rickettsial survival tactics in, and probable movement between, both insect systems will explicate the determinants of rickettsial pathogenicity. Towards this objective, a population of Liposcelis bostrychophila, common booklice, was successfully used for rickettsial isolation in ISE6 (tick-derived cells). Rickettsiae were also observed in L. bostrychophila by electron microscopy and in paraffin sections of booklice by immunofluorescence assay using anti-R. felis polyclonal antibody. The isolate, designated R. felis strain LSU-Lb, resembles typical rickettsiae when examined by microscopy. Sequence analysis of portions of the Rickettsia specific 17-kDa antigen gene, citrate synthase (gltA) gene, rickettsial outer membrane protein A (ompA) gene, and the presence of the R. felis plasmid in the cell culture isolate confirmed the isolate as R. felis. Variable nucleotide sequences from the isolate were obtained for R. felis-specific pRF-associated putative tldD/pmbA. Expression of rickettsial outer membrane protein B (OmpB) was verified in R. felis (LSU-Lb) using a monoclonal antibody. Additionally, a quantitative real-time PCR assay was used to identify a significantly greater median rickettsial load in the booklice, compared to cat flea hosts. With the potential to manipulate arthropod host biology and infect vertebrate hosts, the dual nature of R. felis provides an excellent model for the study of rickettsial pathogenesis and transmission. In addition, this study is the first isolation of a rickettsial pathogen from a non-hematophagous arthropod

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment

Evaluation of cerebral acetate transport and metabolic rates in the rat brain in vivo using 1H-[13C]-NMR

Acetate is a well-known astrocyte-specific substrate that has been used extensively to probe astrocytic function in vitro and in vivo. Analysis of amino acid turnover curves from 13C-acetate has been limited mainly for estimation of first-order rate constants from exponential fitting or calculation of relative rates from steady-state 13C enrichments. In this study, we used 1H-[13C]-Nuclear Magnetic Resonance spectroscopy with intravenous infusion of [2-13C]acetate-Na+ in vivo to measure the cerebral kinetics of acetate transport and utilization in anesthetized rats. Kinetics were assessed using a two-compartment (neuron/astrocyte) analysis of the 13C turnover curves of glutamate-C4 and glutamine-C4 from [2-13C]acetate-Na+, brain acetate levels, and the dependence of steady-state glutamine-C4 enrichment on blood acetate levels. The steady-state enrichment of glutamine-C4 increased with blood acetate concentration until 90% of plateau for plasma acetate of 4 to 5 mmol/L. Analysis assuming reversible, symmetric Michaelis–Menten kinetics for transport yielded 27±2mmol/L and 1.3±0.3 μmol/g/min for Kt and Tmax, respectively, and for utilization, 0.17±0.24 mmol/L and 0.14±0.02 μmol/g/min for KM_util and Vmax_util, respectively. The distribution space for acetate was only 0.32±0.12 mL/g, indicative of a large excluded volume. The astrocytic and neuronal tricarboxylic acid cycle fluxes were 0.37±0.03 μmol/g/min and 1.41±0.11 μmol/g/min, respectively; astrocytes thus comprised ∼21%±3% of total oxidative metabolism

Quantification of High-Resolution ¹H‑[¹³C] NMR Spectra from Rat Brain Extracts

NMR spectroscopy in combination with ¹³C-labeled substrate infusion is a unique technique to obtain information about dynamic metabolic fluxes noninvasively in vivo. In many cases, the in vivo information content obtained during dynamic ¹³C studies in rodents can be enhanced by high-resolution ¹H-[¹³C] NMR spectroscopy on brain extracts. Previously, it has been shown that ¹H NMR spectra from rat brain extracts can be accurately quantified with a spectral fitting routine utilizing simulated basis sets using complete prior knowledge of chemical shifts and scalar couplings. The introduction of ¹³C label into the various metabolites presents complications that demand modifications of the spectral fitting routine. As different multiplets within a given molecule accumulate various amounts of ¹³C label, the fixed amplitude relationship between multiplets typical for ¹H NMR spectra must be abandoned. In addition, ¹³C isotope effects lead to spectral multiplet patterns that become dependent on the amount of ¹³C label accumulation, thereby preventing the use of a common basis set. Here a modified spectral fitting routine is presented that accommodates variable ¹³C label accumulation and ¹³C isotope effects. Spectral fitting results are quantitatively compared to manual integration on column-separated samples in which spectral overlap is minimized