Cyclic AMP Regulation of Protein Lysine Acetylation in Mycobacterium Tuberculosis

Protein lysine acetylation networks can regulate central processes such as carbon metabolism and gene expression in bacteria. In Escherichia coli, cyclic-AMP (cAMP) regulates protein lysine acetyltransferase (PAT) activity at the transcriptional level, but in Mycobacterium tuberculosis, fusion of a cyclic-nucleotide binding domain to a Gcn5-like PAT domain enables direct cAMP control of protein acetylation. Here we describe the allosteric activation mechanism of M. tuberculosis PAT. The crystal structures of the auto-inhibited and cAMP-activated PAT reveal that cAMP binds to a cryptic site in the regulatory domain over 32 Å from the catalytic site. An extensive conformational rearrangement relieves auto-inhibition by a substrate-mimicking lid that covers the protein-substrate binding surface. A steric double latch couples the domains by harnessing a classic, cAMP-mediated, conformational switch. The structures suggest general features that enable the evolution of long-range communication between linked domains

Complete atrial-specific knockout of sodium-calcium exchange eliminates sinoatrial node pacemaker activity.

The origin of sinoatrial node (SAN) pacemaker activity in the heart is controversial. The leading candidates are diastolic depolarization by "funny" current (If) through HCN4 channels (the "Membrane Clock" hypothesis), depolarization by cardiac Na-Ca exchange (NCX1) in response to intracellular Ca cycling (the "Calcium Clock" hypothesis), and a combination of the two ("Coupled Clock"). To address this controversy, we used Cre/loxP technology to generate atrial-specific NCX1 KO mice. NCX1 protein was undetectable in KO atrial tissue, including the SAN. Surface ECG and intracardiac electrograms showed no atrial depolarization and a slow junctional escape rhythm in KO that responded appropriately to β-adrenergic and muscarinic stimulation. Although KO atria were quiescent they could be stimulated by external pacing suggesting that electrical coupling between cells remained intact. Despite normal electrophysiological properties of If in isolated patch clamped KO SAN cells, pacemaker activity was absent. Recurring Ca sparks were present in all KO SAN cells, suggesting that Ca cycling persists but is uncoupled from the sarcolemma. We conclude that NCX1 is required for normal pacemaker activity in murine SAN

Protein Complexes and Proteolytic Activation of the Cell Wall Hydrolase RipA Regulate Septal Resolution in Mycobacteria

Peptidoglycan hydrolases are a double-edged sword. They are required for normal cell division, but when dysregulated can become autolysins lethal to bacteria. How bacteria ensure that peptidoglycan hydrolases function only in the correct spatial and temporal context remains largely unknown. Here, we demonstrate that dysregulation converts the essential mycobacterial peptidoglycan hydrolase RipA to an autolysin that compromises cellular structural integrity. We find that mycobacteria control RipA activity through two interconnected levels of regulation in vivo—protein interactions coordinate PG hydrolysis, while proteolysis is necessary for RipA enzymatic activity. Dysregulation of RipA protein complexes by treatment with a peptidoglycan synthase inhibitor leads to excessive RipA activity and impairment of correct morphology. Furthermore, expression of a RipA dominant negative mutant or of differentially processed RipA homologues reveals that RipA is produced as a zymogen, requiring proteolytic processing for activity. The amount of RipA processing differs between fast-growing and slow-growing mycobacteria and correlates with the requirement for peptidoglycan hydrolase activity in these species. Together, the complex picture of RipA regulation is a part of a growing paradigm for careful control of cell wall hydrolysis by bacteria during growth, and may represent a novel target for chemotherapy development

Tissue S100/calgranulin expression and blood neutrophil-to-lymphocyte ratio (NLR) in dogs with lower urinary tract urothelial carcinoma.

BACKGROUND Urothelial carcinoma (UC) is the most common neoplasm of the canine lower urinary tract, affecting approximately 2% of dogs. Elderly female patients of certain breeds are predisposed, and clinical signs of UC can easily be confused with urinary tract infection or urolithiasis. Diagnosis and treatment are challenging given the lack of disease-specific markers and treatments. The S100A8/A9 complex and S100A12 protein are Ca2+-binding proteins expressed by cells of the innate immune system and have shown promise as urinary screening markers for UC. The neutrophil-to-lymphocyte ratio (NLR) can also aid in distinguishing certain neoplastic from inflammatory conditions. Our study aimed to evaluate the tissue expression of S100/calgranulins and the blood NLR in dogs with UC. Urinary bladder and/or urethral tissue samples from dogs with UC (n = 10), non-neoplastic inflammatory lesions (NNUTD; n = 6), and no histologic changes (n = 11) were evaluated using immunohistochemistry. Blood NLRs were analyzed in dogs with UC (n = 22) or NNUTD (n = 26). RESULTS Tissue S100A12-positive cell counts were significantly higher in dogs with lower urinary tract disease than healthy controls (P = 0.0267 for UC, P = 0.0049 for NNUTD), with no significant difference between UC and NNUTD patients. Tissue S100A8/A9-positivity appeared to be higher with NNUTD than UC, but this difference did not reach statistical significance. The S100A8/A9+-to-S100A12+ ratio was significantly decreased in neoplastic and inflamed lower urinary tract tissue compared to histologically normal specimens (P = 0.0062 for UC, P = 0.0030 for NNUTD). NLRs were significantly higher in dogs with UC than in dogs with NNUTD, and a cut-off NLR of ≤ 2.83 distinguished UC from NNUTD with 41% sensitivity and 100% specificity. Higher NLRs were also associated with a poor overall survival time (P = 0.0417). CONCLUSIONS These results confirm that the S100/calgranulins play a role in the immune response to inflammatory and neoplastic lower urinary tract diseases in dogs, but the tissue expression of these proteins appears to differ from their concentrations reported in urine samples. Further investigations of the S100/calgranulin pathways in UC and their potential as diagnostic or prognostic tools and potential therapeutic targets are warranted. The NLR as a routinely available marker might be a useful surrogate to distinguish UC from inflammatory conditions

Inflammatory phenotypes predict changes in arterial stiffness following antiretroviral therapy initiation

Abstract Background Inflammation drives vascular dysfunction in HIV, but in low-income settings causes of inflammation are multiple, and include infectious and environmental factors. We hypothesized that patients with advanced immunosuppression could be stratified into inflammatory phenotypes that predicted changes in vascular dysfunction on ART. Methods We recruited Malawian adults with CD4 <100 cells/μL 2 weeks after starting ART in the REALITY trial (NCT01825031). Carotid femoral pulse-wave velocity (cfPWV) measured arterial stiffness 2, 12, 24, and 42 weeks post–ART initiation. Plasma inflammation markers were measured by electrochemiluminescence at weeks 2 and 42. Hierarchical clustering on principal components identified inflammatory clusters. Results 211 participants with HIV grouped into 3 inflammatory clusters representing 51 (24%; cluster-1), 153 (73%; cluster-2), and 7 (3%; cluster-3) individuals. Cluster-1 showed markedly higher CD4 and CD8 T-cell expression of HLADR and PD-1 versus cluster-2 and cluster-3 (all P < .0001). Although small, cluster-3 had significantly higher levels of cytokines reflecting inflammation (IL-6, IFN-γ, IP-10, IL-1RA, IL-10), chemotaxis (IL-8), systemic and vascular inflammation (CRP, ICAM-1, VCAM-1), and SAA (all P < .001). In mixed-effects models, cfPWV changes over time were similar for cluster-2 versus cluster-1 (relative fold-change, 0.99; 95% CI, .86–1.14; P = .91), but greater in cluster-3 versus cluster-1 (relative fold-change, 1.45; 95% CI, 1.01–2.09; P = .045). Conclusions Two inflammatory clusters were identified: one defined by high T-cell PD-1 expression and another by a hyperinflamed profile and increases in cfPWV on ART. Further clinical characterization of inflammatory phenotypes could help target vascular dysfunction interventions to those at highest risk

BHPR research: qualitative1. Complex reasoning determines patients' perception of outcome following foot surgery in rheumatoid arhtritis

Background: Foot surgery is common in patients with RA but research into surgical outcomes is limited and conceptually flawed as current outcome measures lack face validity: to date no one has asked patients what is important to them. This study aimed to determine which factors are important to patients when evaluating the success of foot surgery in RA Methods: Semi structured interviews of RA patients who had undergone foot surgery were conducted and transcribed verbatim. Thematic analysis of interviews was conducted to explore issues that were important to patients. Results: 11 RA patients (9 ♂, mean age 59, dis dur = 22yrs, mean of 3 yrs post op) with mixed experiences of foot surgery were interviewed. Patients interpreted outcome in respect to a multitude of factors, frequently positive change in one aspect contrasted with negative opinions about another. Overall, four major themes emerged. Function: Functional ability & participation in valued activities were very important to patients. Walking ability was a key concern but patients interpreted levels of activity in light of other aspects of their disease, reflecting on change in functional ability more than overall level. Positive feelings of improved mobility were often moderated by negative self perception ("I mean, I still walk like a waddling duck”). Appearance: Appearance was important to almost all patients but perhaps the most complex theme of all. Physical appearance, foot shape, and footwear were closely interlinked, yet patients saw these as distinct separate concepts. Patients need to legitimize these feelings was clear and they frequently entered into a defensive repertoire ("it's not cosmetic surgery; it's something that's more important than that, you know?”). Clinician opinion: Surgeons' post operative evaluation of the procedure was very influential. The impact of this appraisal continued to affect patients' lasting impression irrespective of how the outcome compared to their initial goals ("when he'd done it ... he said that hasn't worked as good as he'd wanted to ... but the pain has gone”). Pain: Whilst pain was important to almost all patients, it appeared to be less important than the other themes. Pain was predominately raised when it influenced other themes, such as function; many still felt the need to legitimize their foot pain in order for health professionals to take it seriously ("in the end I went to my GP because it had happened a few times and I went to an orthopaedic surgeon who was quite dismissive of it, it was like what are you complaining about”). Conclusions: Patients interpret the outcome of foot surgery using a multitude of interrelated factors, particularly functional ability, appearance and surgeons' appraisal of the procedure. While pain was often noted, this appeared less important than other factors in the overall outcome of the surgery. Future research into foot surgery should incorporate the complexity of how patients determine their outcome Disclosure statement: All authors have declared no conflicts of interes

Photo-Induced Protonation And Conductivity Of Polyaniline/Poly(Ethylene Glycol) And Polyaniline/[Poly(Ethylene Glycol)-Grafted Polyaniline] Composites

Composites of polyaniline in its emeraldine base form (PANI-EB) and photo-acid generators (PAG) show an increase in conductivity upon photo-irradiation due to the protonation of PANI-EB. Such materials may be utilized to fabricate conducting patterns by photo-irradiation. However, the conductivity obtained by direct irradiation of PANI-EB/PAG composites was normally quite low (\u3c10-3 S/cm) due to aggregation of highly loaded PAG. In this work, poly(ethylene glycol) (PEG), which is a proton transfer polymer, was added to PANI-EB/PAG. Results showed that addition of low Mw (550) PEG significantly enhance the photo-induced conductivity. Conductivities as high as 10-1-100 S/cm were observed after photo-irradiation. This conductivity is comparable to that of PANI-salt synthesized by oxidizing aniline in the presence of an acid. High Mw (8000) PEG is much less effective than PEG 550, which is attributed to its lower compatibility with PANI. PEG-grafted PANI (N-PEG-PANI) was also studied as an additive. Composites of PANI-EB and N-PEG-PANI showed conductivity as high as 102 S/cm after treatment with HCl vapor. The photo-induced conductivity of the N-PEG-PANI/PANI-EB/PAG composite reached 10 -2-10-1 S/cm. Copyright © 2013 Wiley Periodicals, Inc

Differential effects of press vs. pulse seawater intrusion on microbial communities of a tidal freshwater marsh

Abstract Tidal freshwater marshes (TFMs) are threatened by seawater intrusion, which can affect microbial communities and alter biogeochemical processes. Here, we report on a long‐term, large‐scale manipulative field experiment that investigated continuous (press) and episodic (pulse, 2 months/yr) inputs of brackish water on microbial communities in a TFM. After 2.5 yr, microbial diversity was lower in press treatments than in control (untreated) plots whereas diversity in pulse plots was unaffected by brackish water additions. Sulfate reducer abundance increased in response to both press and pulse treatments whereas methanogens did not differ among treatments. Our results, along with other lab and field measurements that show reduced soil respiration and extracellular enzyme activity suggest that continuous seawater intrusion will decrease macrophyte C inputs that reduce bacterial diversity in ways that also diminish ecosystem carbon cycling