Coverage of the 2011 Q fever vaccination campaign in the Netherlands, using retrospective population-based prevalence estimation of cardiovascular risk-conditions for chronic Q fever

Background: In 2011, a unique Q fever vaccination campaign targeted people at risk for chronic Q fever in the southeast of the Netherlands. General practitioners referred patients with defined cardiovascular risk-conditions (age >15 years). Prevalence rates of those risk-conditions were lacking, standing in the way of adequate planning and coverage estimation. We aimed to obtain prevalence rates retrospectively in order to estimate coverage of the Q fever vaccination campaign. Methods: With broad search terms for these predefined risk-conditions, we extracted patient-records from a large longitudinal general-practice research-database in the Netherlands (IPCI-database). Afte

Coverage of the 2011 Q fever vaccination campaign in the Netherlands, using retrospective population-based prevalence estimation of cardiovascular risk-conditions for chronic Q fever

Background: In 2011, a unique Q fever vaccination campaign targeted people at risk for chronic Q fever in the southeast of the Netherlands. General practitioners referred patients with defined cardiovascular risk-conditions (age >15 years). Prevalence rates of those risk-conditions were lacking, standing in the way of adequate planning and coverage estimation. We aimed to obtain prevalence rates retrospectively in order to estimate coverage of the Q fever vaccination campaign. Methods: With broad search terms for these predefined risk-conditions, we extracted patient-records from a large longitudinal general-practice research-database in the Netherlands (IPCI-database). Afte

Targeted knock-down of miR21 primary transcripts using snoMEN vectors induces apoptosis in human cancer cell lines

We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2

Solving integral equations in η→3π\eta\to 3\pi

A dispersive analysis of $\eta\to 3\pi$ decays has been performed in the past by many authors. The numerical analysis of the pertinent integral equations is hampered by two technical difficulties: i) The angular averages of the amplitudes need to be performed along a complicated path in the complex plane. ii) The averaged amplitudes develop singularities along the path of integration in the dispersive representation of the full amplitudes. It is a delicate affair to handle these singularities properly, and independent checks of the obtained solutions are demanding and time consuming. In the present article, we propose a solution method that avoids these difficulties. It is based on a simple deformation of the path of integration in the dispersive representation (not in the angular average). Numerical solutions are then obtained rather straightforwardly. We expect that the method also works for $\omega\to 3\pi$.Comment: 11 pages, 10 Figures. Version accepted for publication in EPJC. The ancillary files contain an updated set of fundamental solutions. The numerical differences to the former set are tiny, see the READMEv2 file for detail

NF90 Binds the Dengue Virus RNA 3′ Terminus and is a Positive Regulator of Dengue Virus Replication

Background Viral RNA translation and replication are regulated by sequence and structural elements in the 5′ and 3′ untranslated regions (UTR) and by host cell and/or viral proteins that bind them. Dengue virus has a single-stranded RNA genome with positive polarity, a 5′ m7GpppG cap, and a conserved 3′-terminal stem loop (SL) that is linked to proposed functions in viral RNA transcription and translation. Mechanisms explaining the contributions of host proteins to viral RNA translation and replication are poorly defined, yet understanding host protein-viral RNA interactions may identify new targets for therapeutic intervention. This study was directed at identifying functionally significant host proteins that bind the conserved dengue virus RNA 3′ terminus. Methodology/Principal Findings Proteins eluted from a dengue 3′ SL RNA affinity column at increasing ionic strength included two with double-strand RNA binding motifs (NF90/DRBP76 and DEAH box polypeptide 9/RNA helicase A (RHA)), in addition to NF45, which forms a heterodimer with NF90. Although detectable NF90 and RHA proteins localized to the nucleus of uninfected cells, immunofluorescence revealed cytoplasmic NF90 in dengue virus-infected cells, leading us to hypothesize that NF90 has a functional role(s) in dengue infections. Cells depleted of NF90 were used to quantify viral RNA transcript levels and production of infectious dengue virus. NF90 depletion was accompanied by a 50%-70% decrease in dengue RNA levels and in production of infectious viral progeny. Conclusions/Significance The results indicate that NF90 interacts with the 3′ SL structure of the dengue RNA and is a positive regulator of dengue virus replication. NF90 depletion diminished the production of infectious dengue virus by more than 50%, which may have important significance for identifying therapeutic targets to limit a virus that threatens more than a billion people worldwide.Ruth L. Kirschstein National Research Service Award (NIH-NRSA GM64985)UNCF-Merck Postdoctoral FellowshipNational Institute of Allergy and Infectious Diseases (U.S.)Ellison Medical Foundatio

Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines

The snoMEN (snoRNA Modulator of gene ExpressioN) vector technology was developed from a human box C/D snoRNA, HBII-180C, which contains an internal sequence that can be manipulated to make it complementary to RNA targets, allowing knock-down of targeted genes. Here we have screened additional human nucleolar snoRNAs and assessed their application for gene specific knock-downs to improve the efficiency of snoMEN vectors. We identify and characterise a new snoMEN vector, termed 47snoMEN, that is derived from box C/D snoRNA U47, demonstrating its use for knock-down of both endogenous cellular proteins and G/YFP-fusion proteins. Using multiplex 47snoMEM vectors that co-express multiple 47snoMEN in a single transcript, each of which can target different sites in the same mRNA, we document >3-fold increase in knock-down efficiency when compared with the original HBII-180C based snoMEN. The multiplex 47snoMEM vector allowed the construction of human protein replacement cell lines with improved efficiency, including the establishment of novel GFP–HIF-1α replacement cells. Quantitative mass spectrometry analysis confirmed the enhanced efficiency and specificity of protein replacement using the 47snoMEN-PR vectors. The 47snoMEN vectors expand the potential applications for snoMEN technology in gene expression studies, target validation and gene therapy

A Yeast Model of FUS/TLS-Dependent Cytotoxicity

FUS/TLS is a nucleic acid binding protein that, when mutated, can cause a subset of familial amyotrophic lateral sclerosis (fALS). Although FUS/TLS is normally located predominantly in the nucleus, the pathogenic mutant forms of FUS/TLS traffic to, and form inclusions in, the cytoplasm of affected spinal motor neurons or glia. Here we report a yeast model of human FUS/TLS expression that recapitulates multiple salient features of the pathology of the disease-causing mutant proteins, including nuclear to cytoplasmic translocation, inclusion formation, and cytotoxicity. Protein domain analysis indicates that the carboxyl-terminus of FUS/TLS, where most of the ALS-associated mutations are clustered, is required but not sufficient for the toxicity of the protein. A genome-wide genetic screen using a yeast over-expression library identified five yeast DNA/RNA binding proteins, encoded by the yeast genes ECM32, NAM8, SBP1, SKO1, and VHR1, that rescue the toxicity of human FUS/TLS without changing its expression level, cytoplasmic translocation, or inclusion formation. Furthermore, hUPF1, a human homologue of ECM32, also rescues the toxicity of FUS/TLS in this model, validating the yeast model and implicating a possible insufficiency in RNA processing or the RNA quality control machinery in the mechanism of FUS/TLS mediated toxicity. Examination of the effect of FUS/TLS expression on the decay of selected mRNAs in yeast indicates that the nonsense-mediated decay pathway is probably not the major determinant of either toxicity or suppression.Fidelity Biosciences (Firm)Fidelity Biosciences (Firm) (Research Inititative)ALS Therapy AllianceNational Institutes of Health (U.S.) (NIH 1RC1NS06839)National Institutes of Health (U.S.) (NIH U01NS05225-03)National Institutes of Health (U.S.) (NIH R01NS050557-05)National Institutes of Health (U.S.) (NIH 1RC2NS070342-01)Pierre L. de Bourgknecht ALS Research FoundationNational Science Foundation (U.S.) (NS614192

Host Factors interacting with the Pestivirus N terminal protease, Npro are Components of the Ribonucleoprotein Complex

The viral N-terminal protease N(pro) of pestiviruses counteracts cellular antiviral defenses through inhibition of IRF3. Here we used mass spectrometry to identify a new role for N(pro) through its interaction with over 55 associated proteins, mainly ribosomal proteins and ribonucleoproteins, including RNA helicase A (DHX9), Y-box binding protein (YBX1), DDX3, DDX5, eIF3, IGF2BP1, multiple myeloma tumor protein 2, interleukin enhancer binding factor 3 (IEBP3), guanine nucleotide binding protein 3, and polyadenylate-binding protein 1 (PABP-1). These are components of the translation machinery, ribonucleoprotein particles (RNPs), and stress granules. Significantly, we found that stress granule formation was inhibited in MDBK cells infected with a noncytopathic bovine viral diarrhea virus (BVDV) strain, Kyle. However, ribonucleoproteins binding to N(pro) did not inhibit these proteins from aggregating into stress granules. N(pro) interacted with YBX1 though its TRASH domain, since the mutant C112R protein with an inactive TRASH domain no longer redistributed to stress granules. Interestingly, RNA helicase A and La autoantigen relocated from a nuclear location to form cytoplasmic granules with N(pro). To address a proviral role for N(pro) in RNP granules, we investigated whether N(pro) affected RNA interference (RNAi), since interacting proteins are involved in RISC function during RNA silencing. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) silencing with small interfering RNAs (siRNAs) followed by Northern blotting of GAPDH, expression of N(pro) had no effect on RNAi silencing activity, contrasting with other viral suppressors of interferon. We propose that N(pro) is involved with virus RNA translation in the cytoplasm for virus particle production, and when translation is inhibited following stress, it redistributes to the replication complex. IMPORTANCE Although the pestivirus N-terminal protease, N(pro), has been shown to have an important role in degrading IRF3 to prevent apoptosis and interferon production during infection, the function of this unique viral protease in the pestivirus life cycle remains to be elucidated. We used proteomic mass spectrometry to identify novel interacting proteins and have shown that N(pro) is present in ribosomal and ribonucleoprotein particles (RNPs), indicating a translational role in virus particle production. The virus itself can prevent stress granule assembly from these complexes, but this inhibition is not due to N(pro). A proviral role to subvert RNA silencing through binding of these host RNP proteins was not identified for this viral suppressor of interferon

Drainage of a deep magma reservoir near Mayotte inferred from seismicity and deformation

The dynamics of magma deep in the Earth’s crust are difficult to capture by geophysical monitoring. Since May 2018, a seismically quiet area offshore of Mayotte in the western Indian Ocean has been affected by complex seismic activity, including long-duration, very-long-period signals detected globally. Global Navigation Satellite System stations on Mayotte have also recorded a large surface deflation offshore. Here we analyse regional and global seismic and deformation data to provide a one-year-long detailed picture of a deep, rare magmatic process. We identify about 7,000 volcano-tectonic earthquakes and 407 very-long-period seismic signals. Early earthquakes migrated upward in response to a magmatic dyke propagating from Moho depth to the surface, whereas later events marked the progressive failure of the roof of a magma reservoir, triggering its resonance. An analysis of the very-long-period seismicity and deformation suggests that at least 1.3 km3 of magma drained from a reservoir of 10 to 15 km diameter at 25 to 35 km depth. We demonstrate that such deep offshore magmatic activity can be captured without any on-site monitoring