An Extracellular Ion Pathway Plays a Central Role in the Cooperative Gating of a K2P K+ Channel by Extracellular pH

Proton-gated TASK-3 K⁺ channel belongs to the K_2P family of proteins that underlie the K⁺ leak setting the membrane potential in all cells. TASK-3 is under cooperative gating control by extracellular [H⁺]. Use of recently solved K_2P structures allows us to explore the molecular mechanism of TASK-3 cooperative pH gating. Tunnel-like side portals define an extracellular ion pathway to the selectivity filter. We use a combination of molecular modeling and functional assays to show that pH-sensing histidine residues and K⁺ ions mutually interact electrostatically in the confines of the extracellular ion pathway. K⁺ ions modulate the pK_a of sensing histidine side chains whose charge states in turn determine the open/closed transition of the channel pore. Cooperativity, and therefore steep dependence of TASK-3 K⁺ channel activity on extracellular pH, is dependent on an effect of the permeant ion on the channel pH_o sensors.     

Proteomic Profiling of the TRAF3 Interactome Network Reveals a New Role for the ER-to-Golgi Transport Compartments in Innate Immunity

Tumor Necrosis Factor receptor-associated factor-3 (TRAF3) is a central mediator important for inducing type I interferon (IFN) production in response to intracellular double-stranded RNA (dsRNA). Here, we report the identification of Sec16A and p115, two proteins of the ER-to-Golgi vesicular transport system, as novel components of the TRAF3 interactome network. Notably, in non-infected cells, TRAF3 was found associated with markers of the ER-Exit-Sites (ERES), ER-to-Golgi intermediate compartment (ERGIC) and the cis-Golgi apparatus. Upon dsRNA and dsDNA sensing however, the Golgi apparatus fragmented into cytoplasmic punctated structures containing TRAF3 allowing its colocalization and interaction with Mitochondrial AntiViral Signaling (MAVS), the essential mitochondria-bound RIG-I-like Helicase (RLH) adaptor. In contrast, retention of TRAF3 at the ER-to-Golgi vesicular transport system blunted the ability of TRAF3 to interact with MAVS upon viral infection and consequently decreased type I IFN response. Moreover, depletion of Sec16A and p115 led to a drastic disorganization of the Golgi paralleled by the relocalization of TRAF3, which under these conditions was unable to associate with MAVS. Consequently, upon dsRNA and dsDNA sensing, ablation of Sec16A and p115 was found to inhibit IRF3 activation and anti-viral gene expression. Reciprocally, mild overexpression of Sec16A or p115 in Hec1B cells increased the activation of IFNβ, ISG56 and NF-κB -dependent promoters following viral infection and ectopic expression of MAVS and Tank-binding kinase-1 (TBK1). In line with these results, TRAF3 was found enriched in immunocomplexes composed of p115, Sec16A and TBK1 upon infection. Hence, we propose a model where dsDNA and dsRNA sensing induces the formation of membrane-bound compartments originating from the Golgi, which mediate the dynamic association of TRAF3 with MAVS leading to an optimal induction of innate immune responses.     

Estrogen therapy and brain muscarinic receptor density in healthy females: a SPET study

Estrogen Therapy (ET) may protect against age-related cognitive decline and neuropsychiatric disorders (e.g. Alzheimer's disease). The biological basis for this putative neuroprotective effect is not fully understood, but may include modulation of cholinergic systems. Cholinergic dysfunction has been implicated in age-related memory impairment and Alzheimer's disease. However, to date no one has investigated the effect of long-term ET on brain cholinergic muscarinic receptor aging, and related this to cognitive function. We used Single Photon Emission Tomography (SPET) and (R,R)[(123)I]-I-QNB, a novel ligand with high affinity for m(1)/m(4) muscarinic receptors, to examine the effect of long-term ET and age on brain m(1)/m(4) receptors in healthy females. We included 10 younger premenopausal subjects and 22 postmenopausal women; 11 long-term ET users (all treated following surgical menopause) and 11 ET never-users (surgical menopause, n=2). Also, verbal memory and executive function was assessed in all postmenopausal subjects. Compared to young women, postmenopausal women (ET users and never-users combined) had significantly lower muscarinic receptor density in all brain regions examined. ET users also had higher muscarinic receptor density than ET never-users in all the brain regions, and this reached statistical significance in left striatum and hippocampus, lateral frontal cortex and thalamus. Moreover, in ET users, (R,R)[(123)I]-I-QNB binding in left hippocampus and temporal cortex was significantly positively correlated with plasma estradiol levels. We also found evidence for improved executive function in ET users as compared to ET never-users. However, there was no significant relationship between receptor binding and cognitive function within any of the groups. In healthy postmenopausal women use of long-term ET is associated with reduced age-related differences in muscarinic receptor binding, and this may be related to serum estradiol levels.     

Antepartum glucose tolerance test results as predictors of type 2 diabetes mellitus in women with a history of gestational diabetes mellitus: A systematic review

Background: Women with a history of gestational diabetes mellitus (GDM) are at high risk for type 2 diabetes mellitus (T2DM).Objective: We reviewed prospective studies of antepartum glucose tolerance test results as risk factors for development of T2DM among women with a history of GDM.Methods: We searched 4 electronic databases and hand-searched 13 journals for literature published through January 2007. The search strategy consisted of medical subject headings and text words for GDM, T2DM, and other relevant terms. Articles were excluded for the following reasons: (1) not written in English; (2) no human data; (3) no original data; (4) <90% of sample was diagnosed with GDM without a separate analysis for women with GDM; (5) case report or series; (6) diagnosis of GDM not based on 3-hour 100-g oral glucose tolerance test (OGTT) or 2-hour 75-g OGTT; (7) T2DM not evaluated as outcome; (8) no relative measure of association or incidence reported; or (9) design did not address antepartum OGTT as a predictor of T2DM. Two investigators independently reviewed citations, performed serial data abstraction on full articles, and assessed the quality of each article. Data were abstracted for study participants and characteristics, T2DM diagnosis, length of follow-up, regression model covariates, and measures of association and variability.Results: Of 11,400 unique citations, we identified 11 articles that evaluated antepartum glucose testing and risk of T2DM in women with a history of GDM. Five studies found that the fasting blood glucose (FBG) on the antepartum diagnostic OGTT was a significant predictor of T2DM (odds ratio [OR] range: 11.1–21.0; relative risk [RR] range: 1.37–1.5; relative hazard [RH] = 2.47). Risk of incident T2DM was predicted by the antepartum 2-hour OGTT plasma glucose in 3 studies (OR range: 1.02–1.03; RR = 1.3) and by the antepartum OGTT glucose AUC in 3 other studies (OR range: 3.64–15; RH = 2.13). Overall, study quality was limited by high losses to follow-up (>20% in 6 studies) and short duration. Few studies adjusted for adiposity, an established diabetes risk factor.Conclusion: FBG, OGTT 2-hour blood glucose, and OGTT glucose AUC appeared to be strong and consistent predictors of subsequent T2DM among women who met diagnostic criteria for GDM using the OGTT.     

Human immunodeficiency virus-specific CD8+ T-cell activity is detectable from birth in the majority of in utero-infected infants

Human immunodeficiency virus (HIV)-infected infants in sub-Saharan Africa typically progress to AIDS or death by 2 years of life in the absence of antiretroviral therapy. This rapid progression to HIV disease has been related to immaturity of the adaptive immune response in infants. We screened 740 infants born to HIV-infected mothers and tracked development and specificity of HIV-specific CD8⁺ T-cell responses in 63 HIV-infected infants identified using gamma interferon enzyme-linked immunospot assays and intracellular cytokine staining. Forty-four in utero-infected and 19 intrapartum-infected infants were compared to 45 chronically infected children >2 years of age. Seventy percent (14 of 20) in utero-infected infants tested within the first week of life demonstrated HIV-specific CD8⁺ T-cell responses. Gag, Pol, and Nef were the principally targeted regions in chronic pediatric infection. However, Env dominated the overall response in one-third (12/36) of the acutely infected infants, compared to only 2/45 (4%) of chronically infected children (P = 0.00083). Gag-specific CD4⁺ T-cell responses were minimal to undetectable in the first 6 months of pediatric infection. These data indicate that failure to control HIV replication in in utero-infected infants is not due to an inability to induce responses but instead suggest secondary failure of adaptive immunity in containing this infection. Moreover, the detection of virus-specific CD8⁺ T-cell responses in the first days of life in most in utero-infected infants is encouraging for HIV vaccine interventions in infants.     

Use of fine needle aspiration biopsy in radiofrequency ablation

OBJECTIVE: To demonstrate the utility of fine needle aspiration biopsy (FNAB) in radiofrequency ablation (RFA) of suspected metastatic tumors at various sites. STUDY DESIGN: Eighteen patients referred for RFA underwent 21 aspirations prior to the procedure. A radiologist performed the FNAB and RFA with radiographic guidance. On-site preliminary evaluation of Diff-Quik-stained smears were followed with Papanicolaou staining. A final diagnosis was rendered and compared to the preliminary diagnosis. RESULTS: Liver was aspirated in 17 cases, lung in 3 cases and pubic bone in 1. Fifteen aspirates were deemed on site as positive or suspicious for malignancy. A preliminary, on-site diagnosis of benign was given in one case and adequate with deferment for review of all slides in four others. One FNAB was unsatisfactory. All but one (patient with benign diagnosis) then immediately proceeded to RFA of the lesion. After review of additional slides, the final diagnosis confirmed metastatic adenocarcinoma in 16, hepatocellular carcinoma in 2 and metastatic squamous cell carcinoma in 1. One FNAB yielded benign hepatocytes, and one was unsatisfactory. CONCLUSION: FNAB is an accurate, safe and rapid method of confirming disease in patients just prior to undergoing RFA.     

Solution structure of the major (Spy0128) and minor (Spy0125 and Spy0130) pili subunits from Streptococcus pyogenes

Adhesion of the serotype M1 Streptococcus pyogenes strain SF370 to human tonsil explants and cultured keratinocytes requires extended polymeric surface structures called pili. In this important human pathogen, pili are assembled from three protein subunits: Spy0125, Spy0128 and Spy0130 through the action of sortase enzymes. For this study, the structural properties of these pili proteins have been investigated in solution. Spy0125 and Spy0128 display characteristics of globular, folded proteins. Circular dichroism suggests a largely β-sheet composition for Spy0128 and Spy0125; Spy0130 appears to contain little secondary structure. Each of the proteins adopts a monodisperse, monomeric state in solution as assessed by analytical ultracentrifugation. Further, small-angle X-ray scattering curves for Spy0125, Spy0128 and Spy0130 suggest each protein adopts an elongated shape, likely comprised of two domains, with similar maximal dimensions. Based on the scattering data, dummy atom models of each of the pili subunits have been reconstructed ab initio. This study provides the first insights into the structure of Streptococcus pyogenes minor pili subunits, and possible implications for protein function are discussed.