Analysis of genetic variability and mapping of point mutations in influenza virus by the RNase A mismatch cleavage method

We have applied the RNase A mismatch cleavage method to analyze genetic variability in RNA viruses by using influenza virus as a model system. Uniformly labeled RNA probes synthesized from a cloned hemagglutinin gene of a given viral strain were hybridized to RNA isolated from other strains of characterized or uncharacterized genetic composition. The RNA.RNA heteroduplexes containing a variable number of base mismatches were digested with RNase A, and the resistant products were analyzed by denaturing polyacrylamide gel electrophoresis. We show that many of these single base mismatches are cleaved by RNase A, generating unique and characteristic patterns of resistant RNA fragments specific for each of the different viral strains. Comparative analysis of the cleavage patterns allows a qualitative estimation of the genetic relatedness and evolution of field strains. We also show that cleavage by RNase A at single base mismatches can readily detect and localize point mutations present in monoclonal antibody-resistant variants. This method should have wide applications in the study of RNA viruses, not only for epidemiological analysis but also in some diagnostic problems, such as characterization of phenotypic mutants.This work was supported by National Institutes of Health Grant CA33021 awarded by the Nationa l Cancer Institute to M.P. and by grants from the Comision Asesora de Investigacion Cientificay Tecnica (Grant 608/438) and Fondo de Investigaciones Sanitarias to J.O. and J.A.M.C.L.-G. was a recipient of a NATO short-term post doctoral fellow-ship while on leave from the Centro Nacional de Microbiologia, Majadahonda, Madri

Systems Biology of Tissue-Specific Response to Anaplasma phagocytophilum Reveals Differentiated Apoptosis in the Tick Vector Ixodes scapularis

Anaplasma phagocytophilum is an emerging pathogen that causes human granulocytic anaplasmosis. Infection with this zoonotic pathogen affects cell function in both vertebrate host and the tick vector, Ixodes scapularis. Global tissue-specific response and apoptosis signaling pathways were characterized in I. scapularis nymphs and adult female midguts and salivary glands infected with A. phagocytophilum using a systems biology approach combining transcriptomics and proteomics. Apoptosis was selected for pathway-focused analysis due to its role in bacterial infection of tick cells. The results showed tissue-specific differences in tick response to infection and revealed differentiated regulation of apoptosis pathways. The impact of bacterial infection was more pronounced in tick nymphs and midguts than in salivary glands, probably reflecting bacterial developmental cycle. All apoptosis pathways described in other organisms were identified in I. scapularis, except for the absence of the Perforin ortholog. Functional characterization using RNA interference showed that Porin knockdown significantly increases tick colonization by A. phagocytophilum. Infection with A. phagocytophilum produced complex tissue-specific alterations in transcript and protein levels. In tick nymphs, the results suggested a possible effect of bacterial infection on the inhibition of tick immune response. In tick midguts, the results suggested that A. phagocytophilum infection inhibited cell apoptosis to facilitate and establish infection through up-regulation of the JAK/STAT pathway. Bacterial infection inhibited the intrinsic apoptosis pathway in tick salivary glands by down-regulating Porin expression that resulted in the inhibition of Cytochrome c release as the anti-apoptotic mechanism to facilitate bacterial infection. However, tick salivary glands may promote apoptosis to limit bacterial infection through induction of the extrinsic apoptosis pathway. These dynamic changes in response to A. phagocytophilum in I. scapularis tissue-specific transcriptome and proteome demonstrated the complexity of the tick response to infection and will contribute to characterize gene regulation in ticks.This research was supported by grants BFU2011-23896, the EU FP7 ANTIGONE project number 278976, the Oklahoma Agricultural Experiment Grant 1669 and the Walter R. Sitlington Endowed Chair for Food Animal Research to KMK. NA and RCG were funded by MEC, Spain. RS was supported by the project Postdok\_BIOGLOBE (CZ.1.07/2.3.00/30.0032) and the Grant 13-12816P (GA CR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Impact of a training project for primary health-care providers (FOCO project) in the HIV screening and HIV late diagnosis

Poster [P043] OBJECTIVE Reducing HIV late diagnosis remains an epidemiological challenge . The objective of this project was to promote early HIV diagnosis through the training of primary health - care providers (PHCP) . METHODS HIV specialists conducted training sessions in 108 primary care centers (PCC) from six Spanish regions during 2016 and 2017 , and with 1804 PHCP involved . The intervention was evaluated using a pre - experimental design collecting the dependent variables both in the six months before and after the intervention . Number of requests for HIV tests from the PCC trained and clinical data of new HIV diagnosed patients were collected . Parametric and non - parametric tests were used to assess differences between pre and post - intervention data . RESULTS 3. Differences in clinical variables in pre and post intervention period

Characterization of Patients with Chronic Diseases and Complex Care Needs: A New High-Risk Emergent Population

Background: To analyze the prevalence and main epidemiological, clinical and outcome features of in-Patients with Complex Chronic conditions (PCC) in internal medicine areas, using a pragmatic working definition. Methods: Prospective study in 17 centers from Spain, with 97 in-hospital, monthly prevalence cuts. A PCC was considered when criteria of polypathological patient (two or more major chronic diseases) were met, or when a patient suffered one major chronic disease plus one or more of nine predefined complexity criteria like socio-familial risk, alcoholism or malnutrition among others (PCC without polypathology). A complete set of baseline features as well as 12-months survival were collected. Then, we compared clinical, outcome variables, and PROFUND index accuracy between polypathological patients and PCC without polypathology. Results: The global prevalence of PCC was 61% (40% of them were polypathological patients, and 21% PCC withouth polypathology) out of the 2178 evaluated patients. Their median age was 82 (59.5% men), suffered 2.3 ± 1.1 major diseases (heart diseases (70.5%), neurologic (41.5%), renal (36%), and lung diseases (26%)), 5.5 ± 2.5 other chronic conditions, met 2.5 ± 1.5 complexity criteria, and presented functional decline (Barthel index 55 (25-90)). Compared to polypathological patients, the subgroup of PCC without polypathology were younger, with a different pattern of major diseases and comorbidities, a better functional status, and lower 12-months mortality rates ((36.2% vs 46.8%; p = .003; OR 0.7(0.48-0.86). The PROFUND index obtained adequate calibration and discrimination power (AUC-ROC 0.67 (0.63-0.69)) in predicting 12-month mortality of PCC. Conclusion: Patients with complex chronic conditions are highly prevalent in internal medicine areas; their clinical pattern has changed in parallel to socio-epidemiological modifications, but their death-risk is still adequately predicted by PROFUND index

Further progress in the study of epsilon iron oxide in archaeological baked clays

The occurrence of ε-Fe2O3 in archaeological samples that have been subjected to high temperatures is gradually being detected by the use of micrometric structural characterization techniques. This work provides new information by revealing that the ε-Fe2O3 is formed as a response to temperature, the aggregation state and the position within the baked clay with respect to the nearest heat source. In addition, depending mainly on the atmospheric environment, the temperature reached by the combustion structure, the distance from the heating source and the particle aggregation, other iron oxide magnetic phases are produced. In the baked clay studied here, hematite is found over the whole range of samples studied but its magnetic contribution is negligible. Magnetite is observed at the sample surface, probably due to local atmospheric environment closest to the combustion source. Maghemite is found at all depths up to 6 cm below the sample surface. ε-Fe2O3 has a limited distribution, found within 2–3 cm of the sample surface. Furthermore, the viability of this compound as a palaeofield marker has been evaluated in both archaeological and synthetic samples. The results indicate that ε-Fe2O3 is able to register the direction of the magnetic field. Linear palaeointensity plots have been obtained in synthetic samples, although the value of the palaeofield could be, sometimes, overestimated

The Effect of Pre-Main Sequence Stars on Star Cluster Dynamics

We investigate the effects of the addition of pre-main sequence evolution to star cluster simulations. We allowed stars to follow pre-main sequence tracks that begin at the deuterium burning birthline and end at the zero age main sequence. We compared our simulations to ones in which the stars began their lives at the zero age main sequence, and also investigated the effects of particular choices for initial binary orbital parameters. We find that the inclusion of the pre-main sequence phase results in a slightly higher core concentration, lower binary fraction, and fewer hard binary systems. In general, the global properties of star clusters remain almost unchanged, but the properties of the binary star population in the cluster can be dramatically modified by the correct treatment of the pre-main sequence stage.Comment: 40 pages ApJ preprint style Accepted by Ap

The QUIJOTE experiment: project status and first scientific results

We present the current status of the QUIJOTE (Q-U-I JOint TEnerife) experiment, a new polarimeter with the aim of characterizing the polarization of the Cosmic Microwave Background, and other galactic or extra-galactic physical processes that emit in microwaves in the frequency range 10–42 GHz, and at large angular scales (around 1 degree resolution). The experiment has been designed to reach the required sensitivity to detect a primordial gravitational wave component in the CMB, provided its tensor-to-scalar ratio is larger than r ∼ 0.05. The project consists of two telescopes and three instruments which will survey a large sky area from the Teide Observatory to provide I, Q and U maps of high sensitivity. The first QUIJOTE instrument, known as Multi-Frequency Instrument (MFI), has been surveying the northern sky in four individual frequencies between 10 and 20 GHz since November 2012, providing data with an average sensitivity of 80 µK beam−1 in Q and U in a region of 20, 000 square-degrees. The second instrument, or Thirty-GHz Instrument (TGI), is currently undergoing the commissioning phase, and the third instrument, or Forty-GHz Instrument (FGI), is in the final fabrication phase. Finally, we describe the first scientific results obtained with the MFI. Some specific regions, mainly along the Galactic plane, have been surveyed to a deeper depth, reaching sensitivities of around 40 µK beam−1. We present new upper limits on the polarization of the anomalous dust emission, resulting from these data, in the Perseus molecular complex and in the W43 molecular complex