RNA-mediated interference and reverse transcription control the persistence of RNA viruses in the insect model Drosophila.

How persistent viral infections are established and maintained is widely debated and remains poorly understood. We found here that the persistence of RNA viruses in Drosophila melanogaster was achieved through the combined action of cellular reverse-transcriptase activity and the RNA-mediated interference (RNAi) pathway. Fragments of diverse RNA viruses were reverse-transcribed early during infection, which resulted in DNA forms embedded in retrotransposon sequences. Those virus-retrotransposon DNA chimeras produced transcripts processed by the RNAi machinery, which in turn inhibited viral replication. Conversely, inhibition of reverse transcription hindered the appearance of chimeric DNA and prevented persistence. Our results identify a cooperative function for retrotransposons and antiviral RNAi in the control of lethal acute infection for the establishment of viral persistence.

proANP Metabolism Provides New Insights Into Sacubitril/Valsartan Mode of Action.

BACKGROUND: Sacubitril/valsartan (S/V) treatment is beneficial in patients with heart failure with reduced ejection fraction (HFrEF), but its mode of action remains elusive, although it involves the increase in ANP (atrial natriuretic peptide). METHODS: Combining mass spectrometry and enzymatic assay in the plasma of 73 HFrEF patients treated with S/V and controls, we deciphered proANP processing that converts proANP into 4 vasoactive peptides. RESULTS: We found that proANP processing is sequential and involved meprin B, ECE (endothelin-converting enzyme) 1, and ANPEP (aminopeptidase N). This processing is limited in HFrEF patients via the downregulation of proANP production, corin, and meprin B activities by miR-425 and miR1-3p. S/V restored or compensated proANP processing by downregulating miR-425 and miR1-3p, hence increasing levels of proANP-derived bioactive peptides. In contrast, S/V directly and indirectly partially inhibited ECE1 and ANPEP. ECE1 partial inhibition resulted in a lower-than-expected increase in ET1 (endothelin 1), tilting the vasoactive balance toward vasodilation, and possibly hypotension. Furthermore, proANP glycosylation interferes with the midregional proANP assay -a clinical surrogate for proANP production, preventing any pathophysiological interpretation of the results. The analysis of S/V dose escalation with respect to baseline treatments suggests S/V-specific effects. CONCLUSIONS: These findings offer mechanistic evidence to the natriuretic peptide -defective state in HFrEF, which is improved by S/V. These data also strongly suggests that S/V increases plasma ANP by multiple mechanisms that involve 2 microRNAs, besides its protection from NEP (neprilysin) cleavage. Altogether, these data provide new insights on HFrEF pathophysiology and the mode of action of S/V.

Deconvolution of BNP and NT-proBNP Immunoreactivities by Mass Spectrometry in Heart Failure and Sacubitril/Valsartan Treatment.

BACKGROUND: Elevated BNP and the N-terminal fragment of the proBNP (NT-proBNP) are hallmarks of heart failure (HF). Generally, both biomarkers parallel each other. In patients receiving sacubitril/valsartan, BNP remained stable while NT-proBNP decreased. As BNP and NT-proBNP assays have limited specificity due to cross-reactivity, we quantified by mass spectrometry (MS) the contributing molecular species. METHODS: We included 356 healthy volunteers, 100 patients with acute dyspnoea (49 acute decompensated HF; 51 dyspnoea of non-cardiac origin), and 73 patients with chronic HF and reduced ejection fraction treated with sacubitril/valsartan. BNP and NT-proBNP immunoreactivities (BNPir and NT-proBNPir) were measured by immunoassays (Abbott ARCHITECT and Roche Diagnostics proBNPII) and proBNP-derived peptides and glycosylation at serine 44 by MS on plasma samples. RESULTS: BNPir corresponded to the sum of proBNP1-108, BNP1-32, BNP3-32, and BNP5-32 (R2 = 0.9995), while NT-proBNPir corresponded to proBNP1-108 and NT-proBNP1-76 not glycosylated at serine 44 (R2 = 0.992). NT-proBNPir was better correlated (R2 = 0.9597) than BNPir (R2 = 0.7643) with proBNP signal peptide (a surrogate of proBNP production). In patients receiving sacubitril/valsartan, non-glycosylated NT-proBNP1-76 remained constant (P = 0.84) despite an increase in NT-proBNP1-76 and its glycosylation (P < 0.0001). ProBNP1-108 remained constant (P = 0.12) while its glycosylation increased (P < 0.0001), resulting in a decrease in non-glycosylated proBNP1-108 (P < 0.0001), and in NT-proBNPir. CONCLUSIONS: Glycosylation interfered with NT-proBNPir measurement, explaining the discrepant evolution of these 2 biomarkers in patients receiving sacubitril/valsartan. Both BNPir and NT-proBNPir are surrogates of proBNP1-108 production, NT-proBNPir being more robust in the clinical contexts studied.

Do plasma neprilysin activity and plasma neprilysin concentration predict cardiac events in chronic kidney disease patients?

Background: Since the introduction of sacubitril/valsartan in clinical cardiology, neprilysin has become a major target for heart failure treatment. Plasma neprilysin concentration has been discussed as a novel biomarker that predicts cardiac events. Natriuretic peptides may inhibit plasma neprilysin. As they accumulate in chronic kidney disease (CKD), we hypothesized that high plasma neprilysin loses its predictive role in CKD patients. Methods: We measured plasma levels of neprilysin concentration, neprilysin activity and brain natriuretic peptide (BNP) in 542 CKD G2-G4 patients within the CARE FOR HOMe study. Patients were followed for predefined endpoints of hospitalization for acute decompensated heart failure and incident atherosclerotic cardiovascular events. Results: During 5.1 ± 2.1 years, 63 patients had acute decompensated heart failure and 125 patients had incident atherosclerotic cardiovascular events. In both Kaplan-Meier and multivariate Cox regression analyses, high plasma BNP and low, rather than elevated, neprilysin activity predicted future hospitalization for acute decompensated heart failure; neprilysin concentration was not predictive. Furthermore, only BNP was an independent predictor of incident atherosclerotic cardiovascular events. Conclusions: In line with experimental studies, high natriuretic peptides may inhibit neprilysin activity in CKD. Therefore, high neprilysin activity and concentrations are not predictors of adverse cardiovascular outcome in CKD patients. Thus neprilysin inhibitors should be implemented with caution in patients with advanced CKD.

The heart regulates the endocrine response to heart failure: cardiac contribution to circulating neprilysin.

Aims: Heart failure (HF) is accompanied by major neuroendocrine changes including the activation of the natriuretic peptide (NP) pathway. Using the unique model of patients undergoing implantation of the CARMAT total artificial heart and investigating regional differences in soluble neprilysin (sNEP) in patients with reduced or preserved systolic function, we studied the regulation of the NP pathway in HF. Methods and results: Venous blood samples from two patients undergoing replacement of the failing ventricles with a total artificial heart were collected before implantation and weekly thereafter until post-operative week 6. The ventricular removal was associated with an immediate drop in circulating NPs, a nearly total disappearance of circulating glycosylated proBNP and furin activity and a marked decrease in sNEP. From post-operative week 1 onwards, NP concentrations remained overall unchanged. In contrast, partial recoveries in glycosylated proBNP, furin activity, and sNEP were observed. Furthermore, while in patients with preserved systolic function (n = 6), sNEP concentrations in the coronary sinus and systemic vessels were similar (all P > 0.05), in patients with reduced left-ventricular systolic function, sNEP concentration, and activity were ∼three-fold higher in coronary sinus compared to systemic vessels (n = 21, all P < 0.0001), while the trans-pulmonary gradient was neutral (n = 5, P = 1.0). Conclusion: The heart plays a pivotal role as a regulator of the endocrine response in systolic dysfunction, not only by directly releasing NPs but also by contributing to circulating sNEP, which in turn determines the bioavailability of other numerous vasoactive peptides.

QSOX1, a novel actor of cardiac protection upon acute stress in mice.

QSOX1, a sulfhydryl oxidase, was shown to be upregulated in the heart upon acute heart failure (AHF). The aim of the study was to unravel QSOX1 roles during AHF. We generated and characterized mice with QSOX1 gene deletion. The QSOX1-/- mice were viable but adult male exhibited a silent dilated cardiomyopathy. The QSOX1-/- hearts were characterized by low protein SERCA2a levels associated with a calcium homeostasis alteration, high levels of the endoplasmic reticulum (ER) chaperone proteins Grp78/Bip, and of the ER apoptosis sensor CHOP, indicating a chronic unfolded protein response (UPR). Importantly the QSOX1invalidation led to overexpression of two ER oxidases, ERO1-α and PRDX4. Acute stress was induced by isoproterenol injection (ISO, 300 mg/kg/12 h) for 2 days. In both groups, the PERK UPR pathway was transiently activated 6 h after the first ISO injection as indicated by eIF2 phosphorylation. By day-3 after the onset of stress, both WT and QSOX1-/- mice exhibited AHF profile but while high cardiac QSOX1 level was induced in WT hearts, ERO1-α and PRDX4 levels drop down in QSOX1-/-. At that time, QSOX1-/- hearts exhibited an enhanced inflammation (CD68+ cells and Galectin-3 expression) and oxidative stress (DHE staining and oxyblot) when compared to WT ones. In conclusion, the lack of QSOX1 promotes the upregulation of two ER oxidases ERO1α and PRDX4 that likely rescues oxidative protein folding in the hearts. However, signs of chronic ER stress remained present and were associated with a dilated cardiomyopathy. The superimposition of acute stress allowed us to propose that QSOX1 participate to the early response to cardiac stress but not to immediate UPR response. Taken altogether, the data indicated that QSOX1 is required 1) for a proper protein folding in the endo/sarcoplasmic reticulum (ER/SR) and 2) for resolution and protective response during acute stress.

High accuracy of proximity extension assay technology for the quantification of plasma brain natriuretic peptide.

BACKGROUND: Novel multiplex assays allow the simultaneous identification of a large number of plasma proteins. While these new technologies have been shown to be highly sensitive and accurate for the identification of plasma proteins, the use of this technology to quantify those proteins has not been properly investigated. In this pilot study, we tested the accuracy of the proximity extension assay (PEA) for the quantification of the cardiac biomarker brain natriuretic peptide (BNP) compared to a standard clinically approved method. METHODS: Concentrations of BNP were assessed in 120 plasma samples from 30 patients with PEA and compared to chemiluminescent microparticle immunoassay (CMIA). Venous blood samples were collected from in tubes containing ethylenediaminetetraacetic acid, centrifuged within 6 hours at 3,500 rpm for 15 minutes at 4°C, frozen and stored at -80°C until analyzed. Correlation between the CMIA and PEA techniques was tested using the Spearman's rank correlation coefficient (rho) and the agreement was described with a Bland-Altman plot. RESULTS: Brain natriuretic peptide values obtained by CMIA and PEA were highly correlated (Spearman's rho = 0.865, P < .0001). In two patients, PEA consistently overestimated resp. underestimated BNP values compared to CMIA. After removal of those two patients, a very high correlation between the two techniques was shown (rho = 0.966, P < .0001). A high agreement between the two techniques over the whole range of tested concentrations was shown. CONCLUSION: This pilot study showed for the first time an excellent correlation between a clinically approved method and the PEA-based approach for quantification of circulating plasma BNP.

Akt-mediated cardioprotective effects of aldosterone in type 2 diabetic mice.

Studies have shown that aldosterone would have angiogenic effects and therefore would be beneficial in the context of cardiovascular diseases. We thus investigated the potential involvement of aldosterone in triggering a cardiac angiogenic response in the context of type-2 diabetes and the molecular pathways involved. Male 3-wk-old aldosterone synthase (AS)-overexpressing mice and their control wild-type (WT) littermates were fed a standard or high-fat, high-sucrose (HFHS) diet. After 6 mo of diet treatment, mice were euthanized, and cardiac samples were assayed by RT-PCR, immunoblotting, and immunohistology. HFHS diet induced type-2 diabetes in WT (WT-D) and AS (AS-D) mice. VEGFa mRNAs decreased in WT-D (-43%, P<0.05 vs. WT) and increased in AS-D mice (+236%, P< 0.01 vs. WT-D). In WT-D mouse hearts, the proapoptotic p38MAPK was activated (P<0.05 vs. WT and AS-D), whereas Akt activity decreased (-64%, P<0.05 vs. WT). The AS mice, which exhibited a cardiac up-regulation of IGF1-R, showed an increase in Akt phosphorylation when diabetes was induced (P<0.05 vs. WT and AS-D). Contrary to WT-D mice, AS-D mouse hearts did not express inflammatory markers and exhibited a normal capillary density (P<0.05 vs. WT-D). To our knowledge, this is the first study providing new insights into the mechanisms whereby aldosterone prevents diabetes-induced cardiac disorders.

The DNA virus Invertebrate iridescent virus 6 is a target of the Drosophila RNAi machinery.

RNA viruses in insects are targets of an RNA interference (RNAi)-based antiviral immune response, in which viral replication intermediates or viral dsRNA genomes are processed by Dicer-2 (Dcr-2) into viral small interfering RNAs (vsiRNAs). Whether dsDNA virus infections are controlled by the RNAi pathway remains to be determined. Here, we analyzed the role of RNAi in DNA virus infection using Drosophila melanogaster infected with Invertebrate iridescent virus 6 (IIV-6) as a model. We show that Dcr-2 and Argonaute-2 mutant flies are more sensitive to virus infection, suggesting that vsiRNAs contribute to the control of DNA virus infection. Indeed, small RNA sequencing of IIV-6-infected WT and RNAi mutant flies identified abundant vsiRNAs that were produced in a Dcr-2-dependent manner. We observed a highly uneven distribution with strong clustering of vsiRNAs to small defined regions (hotspots) and modest coverage at other regions (coldspots). vsiRNAs mapped in similar proportions to both strands of the viral genome, suggesting that long dsRNA derived from convergent overlapping transcripts serves as a substrate for Dcr-2. In agreement, strand-specific RT-PCR and Northern blot analyses indicated that antisense transcripts are produced during infection. Moreover, we show that vsiRNAs are functional in silencing reporter constructs carrying fragments of the IIV-6 genome. Together, our data indicate that RNAi provides antiviral defense against dsDNA viruses in animals. Thus, RNAi is the predominant antiviral defense mechanism in insects that provides protection against all major classes of viruses.

Post-translational modifications enhance NT-proBNP and BNP production in acute decompensated heart failure.

BACKGROUND: Increases in plasma B-type natriuretic peptide (BNP) concentrations in those with acutely decompensated heart failure (ADHF) has been mainly attributed to an increase in NPPB gene transcription. Recently, proBNP glycosylation has emerged as a potential regulatory mechanism in the production of amino-terminal (NT)-proBNP and BNP. The aim of the present study was to investigate proBNP glycosylation, and corin and furin activities in ADHF patients. METHODS AND RESULTS: Plasma levels of proBNP, NT-proBNP, BNP, as well as corin and furin concentration and activity were measured in a large cohort of 683 patients presenting with ADHF (n = 468), non-cardiac dyspnoea (non-ADHF: n = 169) and 46 patients with stable chronic heart failure (CHF); the degree of plasma proBNP glycosylation was assessed in a subset of these patients (ADHF: n = 49, non-ADHF: n = 50, CHF: n = 46). Our results showed a decrease in proBNP glycosylation in ADHF patients that paralleled NT-proBNP overproduction (ρ = -0.62, P < 0.001) but less so to BNP. In addition, we observed an increase in furin activity that is positively related to the plasma levels of proBNP, NT-proBNP and BNP overproduction (all P < 0.001, all ρ > 0.88), and negatively related to the degree of proBNP glycosylation (ρ = -0.62, P < 0.001). CONCLUSION: These comprehensive results provide a paradigm for the post-translational modification of natriuretic peptides in ADHF: as proBNP glycosylation decreases, furin activity increases. This synergistically amplifies the processing of proBNP into BNP and NT-proBNP. CLINICAL TRIAL REGISTRATION: http://clinicaltrials.gov/. Identifier: NCT01374880.

Impact of sacubitril/valsartan on cardiac and systemic hypoxia in chronic heart failure.

In heart failure patients with reduced ejection fraction, Sacubitril/valsartan (S/V) increased proBNP T71 glycosylation, which is regulated negatively by hypoxia via miR-30a in vitro. Using a cohort of 73 HFrEF patients who were transitioned from standard HF medication to S/V, we found that the increase in proBNP T71 glycosylation after S/V was associated with a decrease in cardiac hypoxia. We further found that plasma levels of K709-acteylated HIF1α, HIF-regulated and HIF-independent biomarkers also evolved consistently with a decrease in hypoxia. We further confirmed that biomarker changes were related to hypoxia, in a rat model subjected to isobaric hypoxia. We measured them in rats subjected to isobaric hypoxia. Overall, these data strongly suggest that optimally treated HFrEF patients exhibited subclinical hypoxia that is improved by S/V. The data also posit proBNP T71 glycosylation as a biomarker of cardiac hypoxia.

Patterns of left ventricular remodeling post-myocardial infarction, determinants, and outcome.

AIM: Left ventricular remodeling (LVR) after myocardial infarction (MI) can lead to heart failure, arrhythmia, and death. We aim to describe adverse LVR patterns at 6 months post-MI and their relationships with subsequent outcomes and to determine baseline. METHODS AND RESULTS: A multicenter cohort of 410 patients (median age 57 years, 87% male) with reperfused MI and at least 3 akinetic LV segments on admission was analyzed. All patients had transthoracic echocardiography performed 4 days and 6 months post-MI, and 214 also had cardiac magnetic resonance imaging performed on day 4. To predict LVR, machine learning methods were employed in order to handle many variables, some of which may have complex interactions. Six months post-MI, echocardiographic increases in LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV), and LV ejection fraction (LVEF) were 14.1% [interquartile range 0.0, 32.0], 5.0% [- 14.0, 25.8], and 8.7% [0.0, 19.4], respectively. At 6 months, ≥ 15% or 20% increases in LVEDV were observed in 49% and 42% of patients, respectively, and 37% had an LVEF < 50%. The rate of death or new-onset HF at the end of 5-year follow-up was 8.8%. Baseline variables associated with adverse LVR were determined best by random forest analysis and included stroke volume, stroke work, necrosis size, LVEDV, LVEF, and LV afterload, the latter assessed by Ea or Ea/Ees. In contrast, baseline clinical and biological characteristics were poorly predictive of LVR. After adjustment for predictive baseline variables, LV dilation > 20% and 6-month LVEF < 50% were significantly associated with the risk of death and/or heart failure: hazard ratio (HR) 2.12 (95% confidence interval (CI) 1.05-4.43; p = 0.04) and HR 2.68 (95% CI 1.20-6.00; p = 0.016) respectively. CONCLUSION: Despite early reperfusion and cardioprotective therapy, adverse LVR remains frequent after acute MI and is associated with a risk of death and HF. A machine learning approach identified and prioritized early variables that are associated with adverse LVR and which were mainly hemodynamic, combining LV volumes, estimates of systolic function, and afterload.

Can Biomarkers Correctly Predict Ventilator-associated Pneumonia in Patients Treated With Targeted Temperature Management After Cardiac Arrest? An Exploratory Study of the Multicenter Randomized Antibiotic (ANTHARTIC) Study.

IMPORTANCE: Ventilator-associated pneumonia (VAP) frequently occurs in patients with cardiac arrest. Diagnosis of VAP after cardiac arrest remains challenging, while the use of current biomarkers such as C-reactive protein (CRP) or procalcitonin (PCT) is debated. OBJECTIVES: To evaluate biomarkers' impact in helping VAP diagnosis after cardiac arrest. DESIGN SETTING AND PARTICIPANTS: This is a prospective ancillary study of the randomized, multicenter, double-blind placebo-controlled ANtibiotherapy during Therapeutic HypothermiA to pRevenT Infectious Complications (ANTHARTIC) trial evaluating the impact of antibiotic prophylaxis to prevent VAP in out-of-hospital patients with cardiac arrest secondary to shockable rhythm and treated with therapeutic hypothermia. An adjudication committee blindly evaluated VAP according to predefined clinical, radiologic, and microbiological criteria. All patients with available biomarker(s), sample(s), and consent approval were included. MAIN OUTCOMES AND MEASURES: The main endpoint was to evaluate the ability of biomarkers to correctly diagnose and predict VAP within 48 hours after sampling. The secondary endpoint was to study the combination of two biomarkers in discriminating VAP. Blood samples were collected at baseline on day 3. Routine and exploratory panel of inflammatory biomarkers measurements were blindly performed. Analyses were adjusted on the randomization group. RESULTS: Among 161 patients of the ANTHARTIC trial with available biological sample(s), patients with VAP (n = 33) had higher body mass index and Acute Physiology and Chronic Health Evaluation II score, more unwitnessed cardiac arrest, more catecholamines, and experienced more prolonged therapeutic hypothermia duration than patients without VAP (n = 121). In univariate analyses, biomarkers significantly associated with VAP and showing an area under the curve (AUC) greater than 0.70 were CRP (AUC = 0.76), interleukin (IL) 17A and 17C (IL17C) (0.74), macrophage colony-stimulating factor 1 (0.73), PCT (0.72), and vascular endothelial growth factor A (VEGF-A) (0.71). Multivariate analysis combining novel biomarkers revealed several pairs with p value of less than 0.001 and odds ratio greater than 1: VEGF-A + IL12 subunit beta (IL12B), Fms-related tyrosine kinase 3 ligands (Flt3L) + C-C chemokine 20 (CCL20), Flt3L + IL17A, Flt3L + IL6, STAM-binding protein (STAMBP) + CCL20, STAMBP + IL6, CCL20 + 4EBP1, CCL20 + caspase-8 (CASP8), IL6 + 4EBP1, and IL6 + CASP8. Best AUCs were observed for CRP + IL6 (0.79), CRP + CCL20 (0.78), CRP + IL17A, and CRP + IL17C. CONCLUSIONS AND RELEVANCE: Our exploratory study shows that specific biomarkers, especially CRP combined with IL6, could help to better diagnose or predict early VAP occurrence in cardiac arrest patients.

RNAi-mediated immunity provides strong protection against the negative-strand RNA vesicular stomatitis virus in Drosophila.

Activation of innate antiviral responses in multicellular organisms relies on the recognition of structural differences between viral and cellular RNAs. Double-stranded (ds)RNA, produced during viral replication, is a well-known activator of antiviral defenses and triggers interferon production in vertebrates and RNAi in invertebrates and plants. Previous work in mammalian cells indicates that negative-strand RNA viruses do not appear to generate dsRNA, and that activation of innate immunity is triggered by the recognition of the uncapped 5' ends of viral RNA. This finding raises the question whether antiviral RNAi, which is triggered by the presence of dsRNA in insects, represents an effective host-defense mechanism against negative-strand RNA viruses. Here, we show that the negative-strand RNA virus vesicular stomatitis virus (VSV) does not produce easily detectable amounts of dsRNA in Drosophila cells. Nevertheless, RNAi represents a potent response to VSV infection, as illustrated by the high susceptibility of RNAi-defective mutant flies to this virus. VSV-derived small RNAs produced in infected cells or flies uniformly cover the viral genome, and equally map the genome and antigenome RNAs, indicating that they derive from dsRNA. Our findings reveal that RNAi is not restricted to the defense against positive-strand or dsRNA viruses but can also be highly efficient against a negative-strand RNA virus. This result is of particular interest in view of the frequent transmission of medically relevant negative-strand RNA viruses to humans by insect vectors.

Impact of IDO activation and alterations in the kynurenine pathway on hyperserotonemia, NAD+ production, and AhR activation in autism spectrum disorder.

Hyperserotonemia is the most replicated biochemical anomaly associated with autism spectrum disorder (ASD) and has been reported in 35-46% of individuals with ASD. Serotonin is synthesised from the essential amino acid tryptophan (TRP). However, the main catabolic route of TRP is the kynurenine pathway (KP), which competes with serotonin synthesis when indoleamine dioxygenase (IDO) is activated. Using the same cohort of individuals with ASD, we used to report extensive studies of the serotonin/melatonin pathway, and found increased kynurenine (KYN), suggesting IDO activation in 58.7% of individuals with ASD (159/271), supported by a strong negative correlation between KYN/TRP ratio and miR-153-3p plasma levels, which negatively regulates IDO. IDO activation was associated with normoserotonemia, suggesting that IDO activation could mask hyperserotonemia which meant that hyperserotonemia, if not masked by IDO activation, could be present in ~94% of individuals with ASD. We also identified several KP alterations, independent of IDO status. We observed a decrease in the activity of 3-hydroxyanthranilate dioxygenase which translated into the accumulation of the aryl hydrocarbon receptor (AhR) selective ligand cinnabarinic acid, itself strongly positively correlated with the AhR target stanniocalcin 2. We also found a deficit in NAD+ production, the end-product of the KP, which was strongly correlated with plasma levels of oxytocin used as a stereotypical neuropeptide, indicating that regulated neuropeptide secretion could be limiting. These results strongly suggest that individuals with ASD exhibit low-grade chronic inflammation that is mediated in most cases by chronic AhR activation that could be associated with the highly prevalent gastrointestinal disorders observed in ASD, and explained IDO activation in ~58% of the cases. Taken together, these results extend biochemical anomalies of TRP catabolism to KP and posit TRP catabolism as a possible major component of ASD pathophysiology.

In silico reconstruction of viral genomes from small RNAs improves virus-derived small interfering RNA profiling.

RNA interference (RNAi) is the essential component of antiviral immunity in invertebrates and plants. One of the landmarks of the antiviral RNAi response is the production of virus-derived small interfering RNA (vsiRNA) from viral double-stranded RNA (dsRNA). vsiRNAs constitute a fragmented image of the viral genome sequence that results from Dicer cleavage. vsiRNA sequence profiling is used extensively as a surrogate to study the antiviral RNAi response by determining the nature of the viral dsRNA molecules exposed to and processed by the RNAi machinery. The accuracy of these profiles depends on the actual viral genome sequence used as a reference to align vsiRNA reads, and the interpretation of inaccurate profiles can be misleading. Using Flock house virus and Drosophila melanogaster as a model RNAi-competent organism, we show accurate reconstruction of full-length virus reference sequence from vsiRNAs and prediction of the structure of defective interfering particles (DIs). We developed a Perl script, named Paparazzi, that reconstitutes viral genomes through an iterative alignment/consensus call procedure using a related reference sequence as scaffold. As prevalent DI-derived reads introduce artifacts during reconstruction, Paparazzi eliminates DI-specific reads to improve the quality of the reconstructed genome. Paparazzi constitutes a promising alternative to Sanger sequencing in this context and an effective tool to study antiviral RNAi mechanisms by accurately quantifying vsiRNA along the replicating viral genome. We further discuss Paparazzi as a companion tool for virus discovery as it provides full-length genome sequences and corrects for potential artifacts of assembly.

Impact of natriuretic peptide polymorphisms on diastolic and metabolic function in a populational cohort: insights from the STANISLAS cohort.

AIMS: Elevated brain natriuretic peptide (BNP) and the N-terminal fragment of its pro-hormone (NT-proBNP) have become established biomarkers for heart failure and are associated with cardiovascular morbidity and mortality. Investigating sources of inter-individual heterogeneity, particularly genetic factors, could help better identify patients at risk of future cardiovascular disease. The aim of this study was to estimate the heritability of circulating NT-proBNP levels, to perform a genome-wide association study (GWAS) and gene-candidate analysis focused on NPPB-NPPA genes on these levels, and to examine their association with cardiovascular or metabolic outcomes. METHODS AND RESULTS: A total of 1555 individuals from the STANISLAS study were included. The heritability of circulating NT-proBNP levels was estimated at 15%, with seven single nucleotide polymorphisms (SNPs) reaching the significant threshold in the GWAS. All above SNPs were located on the same gene cluster constituted of MTHFR, CLCN6, NPPA, NPPB, and C1orf167. NPPA gene expression was also associated with NT-proBNP levels. Moreover, six other SNPs from NPPA-NPPB genes were associated with diastolic function (lateral e' on echocardiography) and metabolic features (glycated haemoglobin). CONCLUSIONS: The heritability of natriuretic peptides appears relatively low (15%) and mainly based on the same gene cluster constituted of MTHFR, CLCN6, NPPA, NPPB, and C1orf167. Natriuretic peptide polymorphisms are associated with natriuretic peptide levels and diastolic function. These results suggest that natriuretic peptide polymorphisms may have an impact in the early stages of cardiovascular and metabolic disease.

A proof of concept for targeting the PrPC - Amyloid ß peptide interaction in basal prostate cancer and mesenchymal colon cancer.

The cellular prion protein PrPC partners with caveolin-1 (CAV1) in neurodegenerative diseases but whether this interplay occurs in cancer has never been investigated. By leveraging patient and cell line datasets, we uncover a molecular link between PrPC and CAV1 across cancer. Using cell-based assays, we show that PrPC regulates the expression of and interacts with CAV1. PrPC additionally controls the expression of the amyloid precursor protein APP and of the Aß generating enzyme BACE1, and regulates the levels of Aß, whose accumulation is a central event in Alzheimer's disease. We further identify DKK1 and DKK3, involved in both Alzheimer's disease and cancer progression, as targets of the PrPC-dependent axis. Finally, we establish that antibody-mediated blocking of the Aß-PrPC interaction delays the growth of prostate cancer cell line-derived xenografts and prevents the development of metastases. Our data additionally support an enrichment of the Aß-PrPC-dependent pathway in the basal subtype of prostate cancer, associated with anti-hormonal therapy resistance, and in mesenchymal colon cancer, associated with poor prognosis. Thus, based on a parallel with neurodegenerative diseases, our results bring to light an Aß-PrPC axis and support the potential of targeting this pathway in patients with selected subtypes of prostate and colon cancer.