Identification of potential HIV restriction factors by combining evolutionary genomic signatures with functional analyses.

BACKGROUND: Known antiretroviral restriction factors are encoded by genes that are under positive selection pressure, induced during HIV-1 infection, up-regulated by interferons, and/or interact with viral proteins. To identify potential novel restriction factors, we performed genome-wide scans for human genes sharing molecular and evolutionary signatures of known restriction factors and tested the anti-HIV-1 activity of the most promising candidates. RESULTS: Our analyses identified 30 human genes that share characteristics of known restriction factors. Functional analyses of 27 of these candidates showed that over-expression of a strikingly high proportion of them significantly inhibited HIV-1 without causing cytotoxic effects. Five factors (APOL1, APOL6, CD164, TNFRSF10A, TNFRSF10D) suppressed infectious HIV-1 production in transfected 293T cells by >90% and six additional candidates (FCGR3A, CD3E, OAS1, GBP5, SPN, IFI16) achieved this when the virus was lacking intact accessory vpr, vpu and nef genes. Unexpectedly, over-expression of two factors (IL1A, SP110) significantly increased infectious HIV-1 production. Mechanistic studies suggest that the newly identified potential restriction factors act at different steps of the viral replication cycle, including proviral transcription and production of viral proteins. Finally, we confirmed that mRNA expression of most of these candidate restriction factors in primary CD4+ T cells is significantly increased by type I interferons. CONCLUSIONS: A limited number of human genes share multiple characteristics of genes encoding for known restriction factors. Most of them display anti-retroviral activity in transient transfection assays and are expressed in primary CD4+ T cells

Biomarkers to improve functional outcome prediction after ischemic stroke:Results from the SICFAIL, STRAWINSKI, and PREDICT studies

BACKGROUND AND AIMS: Acute ischemic stroke (AIS) outcome prognostication remains challenging despite available prognostic models. We investigated whether a biomarker panel improves the predictive performance of established prognostic scores.METHODS: We investigated the improvement in discrimination, calibration, and overall performance by adding five biomarkers (procalcitonin, copeptin, cortisol, mid-regional pro-atrial natriuretic peptide (MR-proANP), and N-terminal pro-B-type natriuretic peptide (NT-proBNP)) to the Acute Stroke Registry and Analysis of Lausanne (ASTRAL) and age/NIHSS scores using data from two prospective cohort studies (SICFAIL, PREDICT) and one clinical trial (STRAWINSKI). Poor outcome was defined as mRS &gt; 2 at 12 (SICFAIL, derivation dataset) or 3 months (PREDICT/STRAWINSKI, pooled external validation dataset).RESULTS: Among 412 SICFAIL participants (median age 70 years, quartiles 59-78; 63% male; median NIHSS score 3, quartiles 1-5), 29% had a poor outcome. Area under the curve of the ASTRAL and age/NIHSS were 0.76 (95% CI 0.71-0.81) and 0.77 (95% CI 0.73-0.82), respectively. Copeptin (0.79, 95% CI 0.74-0.84), NT-proBNP (0.80, 95% CI 0.76-0.84), and MR-proANP (0.79, 95% CI 0.75-0.84) significantly improved ASTRAL score's discrimination, calibration, and overall performance. Copeptin improved age/NIHSS model's discrimination, copeptin, MR-proANP, and NT-proBNP improved its calibration and overall performance. In the validation dataset (450 patients, median age 73 years, quartiles 66-81; 54% men; median NIHSS score 8, quartiles 3-14), copeptin was independently associated with various definitions of poor outcome and also mortality. Copeptin did not increase model's discrimination but it did improve calibration and overall model performance.DISCUSSION: Copeptin, NT-proBNP, and MR-proANP improved modest but consistently the predictive performance of established prognostic scores in patients with mild AIS. Copeptin was most consistently associated with poor outcome in patients with moderate to severe AIS, although its added prognostic value was less obvious.</p

A Mass Spectrometry-Based Profiling of Interactomes of Viral DDB1- and Cullin Ubiquitin Ligase-Binding Proteins Reveals NF-κB Inhibitory Activity of the HIV-2-Encoded Vpx

Viruses and hosts are situated in a molecular arms race. To avoid morbidity and mortality, hosts evolved antiviral restriction factors. These restriction factors exert selection pressure on the viruses and drive viral evolution toward increasingly efficient immune antagonists. Numerous viruses exploit cellular DNA damage-binding protein 1 (DDB1)-containing Cullin RocA ubiquitin ligases (CRLs) to induce the ubiquitination and subsequent proteasomal degradation of antiviral factors expressed by their hosts. To establish a comprehensive understanding of the underlying protein interaction networks, we performed immuno-affinity precipitations for a panel of DDB1-interacting proteins derived from viruses such as mouse cytomegalovirus (MCMV, Murid herpesvirus [MuHV] 1), rat cytomegalovirus Maastricht MuHV2, rat cytomegalovirus English MuHV8, human cytomegalovirus (HCMV), hepatitis B virus (HBV), and human immunodeficiency virus (HIV). Cellular interaction partners were identified and quantified by mass spectrometry (MS) and validated by classical biochemistry. The comparative approach enabled us to separate unspecific interactions from specific binding partners and revealed remarkable differences in the strength of interaction with DDB1. Our analysis confirmed several previously described interactions like the interaction of the MCMV-encoded interferon antagonist pM27 with STAT2. We extended known interactions to paralogous proteins like the interaction of the HBV-encoded HBx with different Spindlin proteins and documented interactions for the first time, which explain functional data like the interaction of the HIV-2-encoded Vpr with Bax. Additionally, several novel interactions were identified, such as the association of the HIV-2-encoded Vpx with the transcription factor RelA (also called p65). For the latter interaction, we documented a functional relevance in antagonizing NF-κB-driven gene expression. The mutation of the DDB1 binding interface of Vpx significantly impaired NF-κB inhibition, indicating that Vpx counteracts NF-κB signaling by a DDB1- and CRL-dependent mechanism. In summary, our findings improve the understanding of how viral pathogens hijack cellular DDB1 and CRLs to ensure efficient replication despite the expression of host restriction factors

Stroke risk associated with balloon based catheter ablation for atrial fibrillation: Rationale and design of the MACPAF Study

<p>Abstract</p> <p>Background</p> <p>Catheter ablation of the pulmonary veins has become accepted as a standard therapeutic approach for symptomatic paroxysmal atrial fibrillation (AF). However, there is some evidence for an ablation associated (silent) stroke risk, lowering the hope to limit the stroke risk by restoration of rhythm over rate control in AF. The purpose of the prospective randomized single-center study "Mesh Ablator versus Cryoballoon Pulmonary Vein Ablation of Symptomatic Paroxysmal Atrial Fibrillation" (MACPAF) is to compare the efficacy and safety of two balloon based pulmonary vein ablation systems in patients with symptomatic paroxysmal AF.</p> <p>Methods/Design</p> <p>Patients are randomized 1:1 for the Arctic Front^®or the HD Mesh Ablator^®catheter for left atrial catheter ablation (LACA). The predefined endpoints will be assessed by brain magnetic resonance imaging (MRI), neuro(psycho)logical tests and a subcutaneously implanted reveal recorder for AF detection. According to statistics 108 patients will be enrolled.</p> <p>Discussion</p> <p>Findings from the MACPAF trial will help to balance the benefits and risks of LACA for symptomatic paroxysmal AF. Using serial brain MRIs might help to identify patients at risk for LACA-associated cerebral thromboembolism. Potential limitations of the study are the single-center design, the existence of a variety of LACA-catheters, the missing placebo-group and the impossibility to assess the primary endpoint in a blinded fashion.</p> <p>Trial registration</p> <p>clinicaltrials.gov NCT01061931</p

HIV-1 Vpu is a potent transcriptional suppressor of NF-κB-elicited antiviral immune responses

Many viral pathogens target innate sensing cascades and/or cellular transcription factors to suppress antiviral immune responses. Here, we show that the accessory viral protein U (Vpu) of HIV-1 exerts broad immunosuppressive effects by inhibiting activation of the transcription factor NF-κB. Global transcriptional profiling of infected CD4 +T cells revealed that vpu-deficient HIV-1 strains induce substantially stronger immune responses than the respective wild type viruses. Gene set enrichment analyses and cytokine arrays showed that Vpu suppresses the expression of NF-κB targets including interferons and restriction factors. Mutational analyses demonstrated that this immunosuppressive activity of Vpu is independent of its ability to counteract the restriction factor and innate sensor tetherin. However, Vpu-mediated inhibition of immune activation required an arginine residue in the cytoplasmic domain that is critical for blocking NF-κB signaling downstream of tetherin. In summary, our findings demonstrate that HIV-1 Vpu potently suppresses NF-κB-elicited antiviral immune responses at the transcriptional level

Troponin elevation in acute ischemic stroke (TRELAS) - protocol of a prospective observational trial

<p>Abstract</p> <p>Background</p> <p>Levels of the cardiac muscle regulatory protein troponin T (cTnT) are frequently elevated in patients with acute ischemic stroke and elevated cTnT predicts poor outcome and mortality. The pathomechanism of troponin release may relate to co-morbid coronary artery disease and myocardial ischemia or, alternatively, to neurogenic cardiac damage due to autonomic activation after acute ischemic stroke. Therefore, there is uncertainty about how acute ischemic stroke patients with increased cTnT levels should be managed regarding diagnostic and therapeutic workup.</p> <p>Methods/Design</p> <p>The primary objective of the prospective observational trial TRELAS (TRoponin ELevation in Acute ischemic Stroke) is to investigate the frequency and underlying pathomechanism of cTnT elevation in acute ischemic stroke patients in order to give guidance for clinical practice. All consecutive patients with acute ischemic stroke admitted within 72 hours after symptom onset to the Department of Neurology at the Campus Benjamin Franklin of the University Hospital Charité will be screened for cTnT elevations (i.e. >= 0.05 μg/l) on admission and again on the following day. Patients with increased cTnT will undergo coronary angiography within 72 hours. Diagnostic findings of coronary angiograms will be compared with age- and gender-matched patients presenting with Non-ST-Elevation myocardial infarction to the Department of Cardiology. The primary endpoint of the study will be the occurrence of culprit lesions in the coronary angiogram indicating underlying co-morbid obstructive coronary artery disease. Secondary endpoints will be the localization of stroke in the cerebral imaging and left ventriculographic findings of wall motion abnormalities suggestive of stroke-induced global cardiac dysfunction.</p> <p>Discussion</p> <p>TRELAS will prospectively determine the frequency and possible etiology of troponin elevation in a large cohort of ischemic stroke patients. The findings are expected to contribute to clarify pathophysiologic concepts of co-morbid cardiac damage in ischemic stroke patients and also to provide a basis for clinical recommendations for cardiac workup of such patients.</p> <p>Trial registration</p> <p>clinicaltrials.gov <a href="http://www.clinicaltrials.gov/ct2/show/NCT01263964">NCT01263964</a></p

Genetic Basis of Virulence Attenuation Revealed by Comparative Genomic Analysis of Mycobacterium tuberculosis Strain H37Ra versus H37Rv

Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading infectious disease despite the availability of chemotherapy and BCG vaccine. The commonly used avirulent M. tuberculosis strain H37Ra was derived from virulent strain H37 in 1935 but the basis of virulence attenuation has remained obscure despite numerous studies. We determined the complete genomic sequence of H37Ra ATCC25177 and compared that with its virulent counterpart H37Rv and a clinical isolate CDC1551. The H37Ra genome is highly similar to that of H37Rv with respect to gene content and order but is 8,445 bp larger as a result of 53 insertions and 21 deletions in H37Ra relative to H37Rv. Variations in repetitive sequences such as IS6110 and PE/PPE/PE-PGRS family genes are responsible for most of the gross genetic changes. A total of 198 single nucleotide variations (SNVs) that are different between H37Ra and H37Rv were identified, yet 119 of them are identical between H37Ra and CDC1551 and 3 are due to H37Rv strain variation, leaving only 76 H37Ra-specific SNVs that affect only 32 genes. The biological impact of missense mutations in protein coding sequences was analyzed in silico while nucleotide variations in potential promoter regions of several important genes were verified by quantitative RT-PCR. Mutations affecting transcription factors and/or global metabolic regulations related to in vitro survival under aging stress, and mutations affecting cell envelope, primary metabolism, in vivo growth as well as variations in the PE/PPE/PE-PGRS family genes, may underlie the basis of virulence attenuation. These findings have implications not only for improved understanding of pathogenesis of M. tuberculosis but also for development of new vaccines and new therapeutic agents