Search

2.AbstractMethicillin-resistantStaphylococcus aureus(MRSA) is one of the major human pathogens, causing a wide range of infections from food poisoning to necrotizing pneumonia, endocarditis, or septicemia. It could carry numerous resistance genes and virulence factors, some of which are related to the severity of the infection, being also regarded as a potential “Super Bug”. In Paraguay, the prevalence of CC30-ST30-IV clones is the leading cause ofS. aureusinfections both at the regional level and in pediatric population. Here we aimed to deeply analyze the genomic features of MRSA isolates that cause invasive infections in Paraguayan children. An observational, descriptive, cross-sectional study was designed to analyze representative MRSA isolates of the main clones identified between 2009 to 2013 in Paraguayan children. All the genetically linked MRSA isolates were recovered from diverse clinical sources, patients, and hospitals at broad gap periods. Cases were primarily community-acquired, which excludes in-hospital transmission or outbreaks. MRSA isolates were analyzed with short-read paired-end sequencing and assessed for the virulome, resistome, and phylogenetic relationships. The pan-genomic analysis of these clones revealed three major and different clonal complexes (CC8, CC30, and CC5), each composed of clones closely related to each other, despite having different spa types. Furthermore, multiple virulence and resistance genes were identified for the first time in this study, pointing out the complex virulence profiles of MRSA circulating in the country. This study opens a wide range of new possibilities for future projects and trials to improve the existing knowledge on the epidemiology of MRSA circulating in Paraguay.3.Impact statementThe increasing prevalence of methicillin-resistantStaphylococcus aureus(MRSA) is a public health problem worldwide. The most frequent MRSA clones identified in Paraguay in previous studies were (including community and hospital-acquired) the Pediatric (CC5-ST5-IV), the Cordobes-Chilean (CC5-ST5-I), the SouthWest Pacific (CC30-ST30-IV) and the Brazilian (CC8-ST239-III) clones. In this study, the pan-genomic analysis of the most representative MRSA clones circulating in invasive infection in Paraguayan children over ten years (2009-2019), such as the CC30-ST30-IV, CC5-ST5-IV, and CC8-ST8-IV, were carried out to evaluate their genetic diversity, their virulence factors repertoire, and antimicrobial resistance mechanisms revealed multiple virulences and resistance genes pointing out the complex virulence profiles of MRSA circulating in Paraguay. Our work is the first genomic study of MRSA in Paraguay and will contribute to the development of genomic surveillance in the region and our understanding of this pathogen’s global epidemiology.4.Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

The non-recombinant region of the Y chromosome (NRY) contains a great number of polymorphic markers that allows to accurately reconstruct pedigree relationships and retrieve ancestral information from study samples. The analysis of NRY is typically implemented in anthropological, medical, and forensic studies. High-throughput sequencing (HTS) has profoundly increased the identification of genetic markers in the NRY genealogy and has prompted the development of automated NRY haplogroup classification tools. Here, we present a benchmarking study of five command-line tools for NRY haplogroup classification. The evaluation was done using empirical short-read HTS data from 50 unrelated donors using paired data from whole-genome sequencing (WGS) and whole-exome sequencing (WES) experiments. Besides, we evaluate the performance of the top-ranked tool in the classification of data of third generation HTS obtained from a subset of donors. Our findings demonstrate that WES can be an efficient approach to infer the NRY haplogroup, albeit generally providing a lower level of genealogical resolution than that recovered by WGS. Among the tools evaluated, YLeaf offers the best performance for both WGS and WES applications. Finally, we demonstrate that YLeaf is able to correctly classify all samples sequenced with nanopore technology from long noisy reads.

Most causal variants of Mendelian diseases are exonic. Whole-exome sequencing (WES) has become the diagnostic gold standard, but causative variant prioritization constitutes a bottleneck. Here we assessed an in-house sample-to-sequence pipeline and benchmarked free prioritization tools for germline causal variants from WES data. WES of 61 unselected patients with a known genetic disease cause was obtained. Variant prioritizations were performed by diverse tools and recorded to obtain a diagnostic yield when the causal variant was present in the first, fifth, and 10th top rankings. A fraction of causal variants was not captured by WES (8.2%) or did not pass the quality control criteria (13.1%). Most of the applications inspected were unavailable or had technical limitations, leaving nine tools for complete evaluation. Exomiser performed best in the top first rankings, while LIRICAL led in the top fifth rankings. Based on the more conservative top 10th rankings, Xrare had the highest diagnostic yield, followed by a three-way tie among Exomiser, LIRICAL, and PhenIX, then followed by AMELIE, TAPES, Phen-Gen, AIVar, and VarNote-PAT. Xrare, Exomiser, LIRICAL, and PhenIX are the most efficient options for variant prioritization in real patient WES data.

The transactive response DNA-binding protein (TDP-43) is an important regulator of mRNA, being reported to stabilize the anti-HIV factor, histone deacetylase 6 (HDAC6). However, little is known about the role of TDP-43 in HIV infection. In this work, we seek for the TDP-43 function on regulating CD4+ T cell permissibility to HIV infection. We observed that over-expression of wt-TDP-43 in CD4+ T cells stabilized HDAC6, increasing mRNA and the protein levels of this antiviral enzyme. Under this experimental condition, HIV-1 infection was impaired, independently of the viral envelope glycoprotein (Env) complex tropism. The results obtained by using an HIV-1 Env-mediated cell-to-cell fusion model, under the same experimental conditions, suggest that the increase in TDP-43 levels negatively affects the viral Env fusion capacity. Moreover, the specific siRNA silencing of endogenous TDP-43 in target cells lead to a significant decrease in the levels of HDAC6 which consistently induces an increase in the fusogenic and infection activities of the HIV-1 Env. These observations were confirmed by using primary viral Envs from HIV+ individuals with different clinical phenotypes. An increase in the level of expression of wt-TDP-43 strongly reduced the Envs infection activity of viremic non-progressors (VNP) and rapid progressors (RP) HIV+ individuals down to the levels of the inefficient HIV-1 Envs from long-term non-progressor elite controllers (LTNP-EC) individuals. On the contrary, low levels of endogenous TDP-43, obtained after specific siRNA-TDP-43 knocking-down, significantly favors the infection activity of primary HIV-1 Envs of VNP and RP individuals, leading to an increase in the infection ability of the primary HIV-1/LTNP-EC Envs. Based on this evidence, we interpret that TDP-43 conditions cell permissibility to HIV infection by affecting viral Env fusion and infection capacities, at least by altering the cellular levels of the antiviral enzyme HDAC6.

OBJECTIVES Diagnosis of Wilson disease is difficult and, as early detection may prevent all symptoms, it is essential to know the exact prevalence in order to evaluate the cost- efficacy of a screening program. As the number of WD patients was high in our population, we wished to estimate prevalence by determining the carrier frequency for clinically relevant ATP7B mutations. METHODS To estimate prevalence, screening for the most prevalent mutation was performed in 1,661 individuals with ancestry in Gran Canaria, and the frequency of other mutations was estimated from patient records. Alternatively, ATP7B mutations were detected from exomes and genomes from 851 individuals with canarian ancestry, 236 from Gran Canaria, and a public Spanish exome database. RESULTS Estimated carrier frequencies in Gran Canaria ranged from 1 in 20 to 28, depending on the method used, resulting in prevalences of 1 case per 1,547 to 3,140 inhabitants. Alternatively, the estimated affected frequencies were 1 in 5,985 to 7,980 and 1 in 6,278 to 16,510 in the archipelago or mainland Spain respectively. CONCLUSIONS (i) the number of carriers predicts much higher prevalences than reported, suggesting that WD is underdiagnosed; (ii) specific mutations may remain unnoticed due to low penetrance or no signs of disease at all; (iii) regional prevalence rather than national prevalence should be considered in cost-efficacy models in order to approach preventive screening in the asymptomatic population and (iv) genetic screening strategies will have to deal with the genetic heterogeneity of ATP7B in the general population and in patients.

Sepsis is a severe systemic inflammatory response to infections that is accompanied by organ dysfunction. Although the ancestral genetic background is a relevant factor for sepsis susceptibility, there is a lack of studies using the genetic singularities of a recently admixed population to identify loci involved in sepsis susceptibility. Here we aimed to discover new sepsis loci by completing the first admixture mapping study of sepsis in Canary Islanders, leveraging their distinctive genetic makeup as a mixture of Europeans and African ancestries. We used a case-control approach and inferred local ancestry blocks from genome-wide data from 113,414 polymorphisms genotyped in 343 patients with sepsis and 410 unrelated controls, all ascertained for grandparental origin in the Canary Islands (Spain). Deviations in local ancestries between cases and controls were tested using logistic regressions, followed by fine-mapping analyses based on imputed genotypes, in silico functional assessments, and gene expression analysis centered on the region of interest. The admixture mapping analysis detected that local European ancestry in a locus spanning 1.2 megabases of chromosome 8p23.1 was associated with sepsis (lowest p = 1.37 × 10−4; Odds Ratio [OR] = 0.51; 95%CI = 0.40–0.66). Fine-mapping studies prioritized the variant rs13249564 within intron 1 of MFHAS1 gene associated with sepsis (p = 9.94 × 10−4; OR = 0.65; 95%CI = 0.50–0.84). Functional and gene expression analyses focused on 8p23.1 allowed us to identify alternative genes with possible biological plausibility such as defensins, which are well-known effector molecules of innate immunity. By completing the first admixture mapping study of sepsis, our results revealed a new genetic locus (8p23.1) harboring a number of genes with plausible implications in sepsis susceptibility.

We describe the case of a 23-year-old immunocompromised male patient with clinically diagnosed X-linked agammaglobulinemia who was admitted to the hospital on the 14th April 2020 due to coronavirus disease of 2019 (COVID-19). Despite COVID-19 test negativizations, the patient was hospitalized most of the time and finally admitted to the intensive care unit where he died from multiorgan failure and shock. Over 149 days, 26 respiratory samples were collected, subjected to viral genome sequencing, and all assigned to the same lineage, supporting a single viral infection event. The accumulation of mutations throughout the course of the infection was accelerated and suggested the presence of compartmentalized viral subpopulations that evolved independently in the upper and lower respiratory airways. These results support that long-term viral shedding in immunocompromised patients is one possible mechanism for the emergence of variants of concern and provide evidence towards the infection control guidelines in these patients.

Starting in December 2020, a sharp increase of COVID-19 cases occurred in Tenerife compared to the rest of the Canary Islands (Spain). Because of the direct touristic connections between Tenerife and the UK, and the rapid transmission and dominance of the SARS-CoV-2 B.1.1.7 variant of concern (VOC-202012/01) by the end of November 2020 in South England, here we measured the proportion of B.1.1.7 cases occurring between the 18th of December 2020 and the 25th of February 2021. Out of the 2,091 COVID-19 positive nasopharyngeal swab samples assessed, 226 showed a spike gene target failure (SGTF). Subsequent viral genome sequencing further confirmed that 93.2% of them corresponded to the B.1.1.7 lineage. Furthermore, a rapid increase in the proportion of SGTF variants was detected in up to 10.7% of positive cases during the Christmas season despite stricter measures for containing the transmission were imposed in Tenerife in the period. These results support the local transmission of SARS-CoV-2 B.1.1.7 lineage in Tenerife since late December 2020 although it is not yet dominant.

The mitochondrial genome (mtDNA) is of interest for a range of fields including evolutionary, forensic, and medical genetics. Human mitogenomes can be classified into evolutionary related haplogroups that provide ancestral information and pedigree relationships. Because of this and the advent of high-throughput sequencing (HTS) technology, there is a diversity of bioinformatic tools for haplogroup classification. We present a benchmarking of the 11 most salient tools for human mtDNA classification using empirical whole-genome (WGS) and whole-exome (WES) short-read sequencing data from 36 unrelated donors. Besides, because of its relevance, we also assess the best performing tool in third-generation long noisy read WGS data obtained with nanopore technology for a subset of the donors. We found that, for short-read WGS, most of the tools exhibit high accuracy for haplogroup classification irrespective of the input file used for the analysis. However, for short-read WES, Haplocheck and MixEmt were the most accurate tools. Based on the performance shown for WGS and WES, and the accompanying qualitative assessment, Haplocheck stands out as the most complete tool. For third-generation HTS data, we also showed that Haplocheck was able to accurately retrieve mtDNA haplogroups for all samples assessed, although only after following assembly-based approaches (either based on a referenced-based assembly or a hybrid de novo assembly). Taken together, our results provide guidance for researchers to select the most suitable tool to conduct the mtDNA analyses from HTS data.

Whole-exome sequencing has become a popular technique in research and clinical settings, assisting in disease diagnosis and increasing the understanding of disease pathogenesis. In this study, we aimed to compare common enrichment capture solutions available in the market. Peripheral blood-purified DNA samples were enriched with SureSelectQXT V6 (Agilent) and various Illumina solutions: TruSeq DNA Nano, TruSeq DNA Exome, Nextera DNA Exome, and Illumina DNA Prep with Enrichment, and sequenced on a HiSeq 4000. We found that their percentage of duplicate reads was as much as 2 times higher than previously reported values for the previous HiSeq series. SureSelectQXT and Illumina DNA Prep with Enrichment showed the best average on-target coverage, which improved when off-target regions were included. At high coverage levels and in shared bases, these two solutions and TruSeq DNA Exome provided three of the best performances. With respect to the number of small variants detected, SureSelectQXT presented the lowest number of detected variants in target regions. When off-target regions were considered, its ability equalized to other solutions. Our results show SureSelectQXT and Illumina DNA Prep with Enrichment to be the best enrichment capture solutions.

Highlights • Sample pooling level assessment in retrospective samples. • Pooling of five had the highest sensitivity. • Pooling in prospective samples found positives with Ct ranging 16.7-39.4., Objectives Limited testing capacity has characterized the ongoing COVID-19 pandemic in Spain, hampering a timely control of outbreaks and the possibilities to reduce the escalation of community transmissions. Here we investigated the potential of using pooling of samples followed by one-step retrotranscription and quantitative PCR (RT-qPCR) to increase SARS-CoV-2 testing capacity. Methods We first evaluated different sample pooling (1:5, 1:10 and 1:15) prior to RNA extractions followed by standard RT-qPCR for SARS-CoV-2/COVID-19 diagnosis. The pool size achieving reproducible results in independent tests was then used for assessing nasopharyngeal samples in a tertiary hospital during August 2020. Results We found that pool size of five samples achieved the highest sensitivity compared to pool sizes of 10 and 15, showing a mean (± SD) Ct shift of 3.5 ± 2.2 between the pooled test and positive samples in the pool. We then used a pool size of five to test a total of 895 pools (4,475 prospective samples) using two different RT-qPCR kits available at that time. The Real Accurate Quadruplex corona-plus PCR Kit (PathoFinder) reported the lowest mean Ct (± SD) shift (2.2 ± 2.4) among the pool and the individual samples. The strategy allows detecting individual samples in the positive pools with Cts in the range of 16.7-39.4. Conclusions We found that pools of five samples combined with RT-qPCR solutions helped to increase SARS-CoV-2 testing capacity with minimal loss of sensitivity compared to that resulting from testing the samples independently.

Background Hereditary angioedema is a rare genetic condition caused by C1 esterase inhibitor deficiency, dysfunction, or kinin cascade dysregulation, leading to an increased bradykinin plasma concentration. Hereditary angioedema is a poorly recognized clinical entity and is very often misdiagnosed as a histaminergic angioedema. Despite its genetic nature, first-line genetic screening is not integrated in routine diagnosis. Consequently, a delay in the diagnosis, and inaccurate or incomplete diagnosis and treatment of hereditary angioedema are common. Objective In agreement with recent recommendations from the International Consensus on the Use of Genetics in the Management of Hereditary Angioedema, to facilitate the clinical diagnosis and adapt it to the paradigm of precision medicine and next-generation sequencing–based genetic tests, we aimed to develop a genetic annotation tool, termed Hereditary Angioedema Database Annotation (HADA). Methods HADA is built on top of a database of known variants affecting function, including precomputed pathogenic assessment of each variant and a ranked classification according to the current guidelines from the American College of Medical Genetics and Genomics. Results HADA is provided as a freely accessible, user-friendly web-based interface with versatility for the entry of genetic information. The underlying database can also be incorporated into automated command-line stand-alone annotation tools. Conclusions HADA can achieve the rapid detection of variants affecting function for different hereditary angioedema types, and further integrates useful information to reduce the diagnosis odyssey and improve its delay.

Highlights • High-throughput screening of SARS-CoV-2 by RT-qPCR without previous RNA extraction. • Heated nasopharyngeal swab transport medium as template in the RT-qPCR test. • Fast and safety management of SARS-CoV-2 samples in the diagnostic process., Objectives The gold-standard COVID-19 diagnosis relies on detecting SARS-CoV-2 using RNA purification and one-step retrotranscription and quantitative PCR (RT-qPCR). Based on the urgent need for high-throughput screening, we tested the performance of three alternative, simple and affordable protocols to rapidly detect SARS-CoV-2, bypassing the long and tedious RNA extraction step and reducing the time to viral detection. Methods We evaluated three methods based on direct nasopharyngeal swab viral transmission medium (VTM) heating before the RT-qPCR: a) direct without additives; b) in a formamide-EDTA (FAE) buffer, c) in a RNAsnapTM buffer. Results Although with a delay in cycle threshold compared to the gold-standard, we found consistent results in nasopharyngeal swab samples that were subject to a direct 70°C incubation for 10 min. Conclusions Our findings provide valuable options to overcome any supply chain issue and help to increase the throughput of diagnostic tests, thereby complementing standard diagnosis.

BACKGROUND Hereditary angioedema is a rare genetic condition caused by C1 esterase inhibitor deficiency, dysfunction, or kinin cascade dysregulation, leading to an increased bradykinin plasma concentration. Hereditary angioedema is a poorly recognized clinical entity and is very often misdiagnosed as a histaminergic angioedema. Despite its genetic nature, first-line genetic screening is not integrated in routine diagnosis. Consequently, a delay in the diagnosis, and inaccurate or incomplete diagnosis and treatment of hereditary angioedema are common. OBJECTIVE In agreement with recent recommendations from the International Consensus on the Use of Genetics in the Management of Hereditary Angioedema, to facilitate the clinical diagnosis and adapt it to the paradigm of precision medicine and next-generation sequencing–based genetic tests, we aimed to develop a genetic annotation tool, termed Hereditary Angioedema Database Annotation (HADA). METHODS HADA is built on top of a database of known variants affecting function, including precomputed pathogenic assessment of each variant and a ranked classification according to the current guidelines from the American College of Medical Genetics and Genomics. RESULTS HADA is provided as a freely accessible, user-friendly web-based interface with versatility for the entry of genetic information. The underlying database can also be incorporated into automated command-line stand-alone annotation tools. CONCLUSIONS HADA can achieve the rapid detection of variants affecting function for different hereditary angioedema types, and further integrates useful information to reduce the diagnosis odyssey and improve its delay.

The prevalence of asthma symptoms in Canary Islanders, a southwestern European population from Spain, is almost three times higher than the country average. Because the genetic risks identified so far explain −4; odds ratio (OR) = 2.05; 95% confidence interval (CI) = 1.41–3.00]. Fine-mapping studies identified a variant associated with asthma, and results were replicated in independent samples (rs3852738, OR = 1.34; 95% CI = 1.13–1.59, P = 7.58 × 10−4). Whole exome sequencing data from a subset of individuals revealed an enrichment of likely deleterious variants among asthma cases in 16q23.3, particularly in the phospholipase Cγ2 ( PLCG2) gene ( P = 3.67 × 10−4). By completing the first mapping study of asthma in admixed populations from Europe, our results revealed a new plausible asthma locus.

Glucocorticoids are widely used to treat acute and chronic diseases. Unfortunately, their therapeutic use is associated with severe side effects. Glucocorticoids are known to regulate several ion channels in cardiac myocytes, including voltage-dependent Ca2+ channels. Low-voltage-activated T-type Ca2+ channels are expressed in ventricular myocytes during the fetal and perinatal period, but are practically absent in the adult. However, these channels can be re-expressed in adult cardiomyocytes under some pathological conditions. We have investigated the glucocorticoid regulation of T-type Ca2+ channels in rat cardiomyocytes. Molecular studies revealed that dexamethasone induces the upregulation of CaV3.2 mRNA in neonatal rat ventricular myocytes, whereas CaV3.1 mRNA is only slightly affected. Patch-clamp recordings confirmed that T-type Ca2+ channel currents were upregulated in dexamethasone treated cardiomyocytes, and the addition of 50 μmol/L NiCl2 demonstrated that the CaV3.2 channel is responsible for this upregulation. The effect of dexamethasone on CaV3.2 is mediated by the activation and translocation to the cell nucleus of the glucocorticoid receptor (GR). We have isolated the upstream promoter of the Cacna1h gene and tested its activity in transfected ventricular myocytes. The initial in silico analysis of Cacna1h promoter revealed putative glucocorticoid response elements (GREs). Transcriptional activity assays combined with deletion analyses and chromatin immunoprecipitation assays demonstrated that GR binds to a region a GRE located in -1006/-985 bp of Cacna1h promoter. Importantly, upregulation of the CaV3.2 channel is also observed in vitro in adult rat ventricular myocytes, and in vivo in a rat model of excess of glucocorticoids.

The epithelial sodium channel (ENaC) constitutes the rate-limiting step for Na(+) transport across electrically tight epithelia. Regulation of ENaC activity is critical for electrolyte and extracellular volume homeostasis, as well as for lung liquid clearance and colon Na(+) handling. ENaC activity is tightly controlled by a combination of mechanisms involving changes in open probability and plasma membrane abundance. The latter reflects a combination in channel biosynthesis and trafficking to and from the membrane. Studying ENaC trafficking with different techniques in a variety of expression systems has yielded inconsistent results, indicating either fast or slow rates of insertion and retrieval, which range from the order of minutes to several hours. Here, we use Xenopus oocytes as ENaC expression system to study channel insertion rate in the membrane using two different techniques under comparable conditions: (1) confocal microscopy coupled to fluorescence recovery after photobleaching (FRAP) measurements; and (2) fluorescent bungarotoxin (BTX) binding to ENaC subunits modified to include BTX binding sites (BBSs) in their extracellular domain, a technique that has not been previously used to study ENaC trafficking. Our confocal-FRAP data indicate a fast rate of ENaC incorporation to the membrane in a process conditioned by channel subunit composition. On the other hand, BTX binding experiments indicate much slower channel insertion rates, with matching slow ENaC retrieval rates. The data support a model that includes fast recycling of endocytosed ENaC with parallel incorporation of newly synthesized channels at a slower rate.

The Canary Islands’ indigenous people have been the subject of substantial archaeological, anthropological, linguistic and genetic research pointing to a most probable North African Berber source. However, neither agreement about the exact point of origin nor a model for the indigenous colonization of the islands has been established. To shed light on these questions, we analyzed 48 ancient mitogenomes from 25 archaeological sites from the seven main islands. Most lineages observed in the ancient samples have a Mediterranean distribution, and belong to lineages associated with the Neolithic expansion in the Near East and Europe (T2c, J2a, X3a…). This phylogeographic analysis of Canarian indigenous mitogenomes, the first of its kind, shows that some lineages are restricted to Central North Africa (H1cf, J2a2d and T2c1d3), while others have a wider distribution, including both West and Central North Africa, and, in some cases, Europe and the Near East (U6a1a1, U6a7a1, U6b, X3a, U6c1). In addition, we identify four new Canarian-specific lineages (H1e1a9, H4a1e, J2a2d1a and L3b1a12) whose coalescence dates correlate with the estimated time for the colonization of the islands (1stmillennia CE). Additionally, we observe an asymmetrical distribution of mtDNA haplogroups in the ancient population, with certain haplogroups appearing more frequently in the islands closer to the continent. This reinforces results based on modern mtDNA and Y-chromosome data, and archaeological evidence suggesting the existence of two distinct migrations. Comparisons between insular populations show that some populations had high genetic diversity, while others were probably affected by genetic drift and/or bottlenecks. In spite of observing interinsular differences in the survival of indigenous lineages, modern populations, with the sole exception of La Gomera, are homogenous across the islands, supporting the theory of extensive human mobility after the European conquest.

Despite the reduced read length, the so-called Next-Generation Sequencing (NGS) of second-generation has allowed rapid and complete genome characterization of many species. MinION (Oxford Nanopore Technologies), a portable third-generation NGS device, enables sequencing of long DNA fragments at low cost. Here we used a low-coverage MinION sequencing in combination with short-read NGS to improve genome assembly. We tested this possibility by using MinION R9.0 with Rapid 1D kit and MiSeq with >300X paired-end 300 bp reads (Illumina, Inc.) for the genome assembly of a Streptococcus agalactiae clinical isolate (2.2 Mb). With as few as 1,171 MinION reads that covered the genome at 2.4X (the longest read being 186 Kb long), the hybrid assembly combining MinION and Illumina reads increased the N50 by 4.9-fold compared to the assembly using Illumina data alone. Almost 50% of the genome was represented into a single contig (1.02 Mb). Besides, this allowed the full reconstruction of mobile elements, including a plasmid, and improved gene annotation. Taken together, our results support that shallow MinION sequencing combined with high-throughput second-generation NGS constitutes a cost-efficient strategy for the assembly of whole genomes.

Despite the genetic resemblance of Canary Islanders to other southern European populations, their geographical isolation and the historical admixture of aborigines (from North Africa) with sub-Saharan Africans and Europeans have shaped a distinctive genetic makeup that likely affects disease susceptibility and health disparities. Based on single nucleotide polymorphism array data and whole genome sequencing (30×), we inferred that the last African admixture took place ∼14 generations ago and estimated that up to 34% of the Canary Islander genome is of recent African descent. The length of regions in homozygosis and the ancestry-related mosaic organization of the Canary Islander genome support the view that isolation has been strongest on the two smallest islands. Furthermore, several genomic regions showed significant and large deviations in African or European ancestry and were significantly enriched in genes involved in prevalent diseases in this community, such as diabetes, asthma, and allergy. The most prominent of these regions were located near LCT and the HLA, two well-known targets of selection, at which 40‒50% of the Canarian genome is of recent African descent according to our estimates. Putative selective signals were also identified in these regions near the SLC6A11-SLC6A1, KCNMB2, and PCDH20-PCDH9 genes. Taken together, our findings provide solid evidence of a significant recent African admixture, population isolation, and adaptation in this part of Europe, with the favoring of African alleles in some chromosome regions. These findings may have medical implications for populations of recent African ancestry.

Background and Purpose— Rho-kinase, an effector of RhoA, is associated with various cardiovascular diseases in circulating blood cells. However, the role of RhoA/Rho-kinase in peripheral blood mononuclear cells from patients with spontaneous aneurysmal subarachnoid hemorrhage (aSAH) has not yet been studied in relation to the severity of this disease. Therefore, we analyzed the expression and activity of RhoA as a possible biomarker in aSAH. Methods— Twenty-four patients with aSAH and 15 healthy subjects were examined. Peripheral blood mononuclear cells were collected, and RhoA activity and expression were determined by RhoA activation assay kit (G-LISA) and enzyme-linked immunosorbent assay tests, respectively. The severity of aSAH was determined from the World Federation of Neurological Surgeon scale, and vasospasm was evaluated using clinical symptoms, arteriography, and sonography. Results— RhoA expression was significantly increased in peripheral blood mononuclear cells from patients on days 0, 2, and 4 after aSAH versus healthy subjects ( P =0.036, 0.010, and 0.018, respectively, by U Mann–Whitney analysis). There was a significant correlation between RhoA expression and injury severity on days 2 and 4 (Spearman test, day 2: r =0.682, n=14, P =0.007; day 4: r =0.721, n=14, P =0.004). No significant correlation was observed on day 0 (day 0: r =0.131, n=6, P =0.805). Active RhoA was not significantly different in patients and healthy subjects on days 0, 2, and 4 ( P =0.243, 0.222, and 0.600, respectively) nor did it increase significantly on days 0 and 2 in patients with vasospasm versus patients without vasospasm ( P =0.064 and 0.519, respectively). In contrast, active RhoA was significantly higher on day 4 in patients who developed vasospasm versus patients without vasospasm ( P =0.028). Conclusions— Our preliminary results indicate that RhoA expression and activity in peripheral blood mononuclear cells might be related with aSAH severity and cerebral vasospasm. RhoA is a potential biomarker of the risks associated with aSAH.

[Background/Aims] Protein kinase C (PKC)- and RhoA/Rho-associated kinase (ROCK) play important roles in arterial sustained contraction. Although depolarization-elicited RhoA/ROCK activation is accepted, the role of PKC in depolarized vascular smooth muscle cells (VSMCs) is a subject of controversy. Our aim was to study the role of PKC in arterial contraction and its interaction with RhoA/ROCK., [Methods] Mass spectrometry was used to identify the PKC isoenzymes. PKCα levels and RhoA activity were analyzed by western blot and G-LISA, respectively, and isometric force was measured in arterial rings., [Results] In depolarized VSMCs RhoA and PKCα were translocated to the plasma membrane, where they colocalize and coimmunoprecipitate. Interestingly, depolarization-induced RhoA activation was downregulated by PKCα, effect reverted by PKCα inhibition. Phorbol 12,13-dibutyrate (PDBu) induced the translocation of PKCα to the plasma membrane, increased the level of RhoA in the cytosol and reduced RhoA/ROCK activity. These effects were reverted when PKC was inhibited. Pharmacological or siRNA inhibition of PKCα synergistically potentiated the vasorelaxant effect of RhoA/ROCK inhibition., [Conclusion] The present study provides the first evidence that RhoA activity is downregulated by PKCα in depolarized and PDBu treated freshly isolated VSMCs and arteries, with an important physiological role on arterial contractility., This work was supported by the "Red de Investigación Cardiovascular, RIC, RD12/0042/0041" of the Instituto de Salud Carlos III and by Ministerio de Economía y Competitividad and FEDER (SAF2013-46806-R and SAF2017-89474-R). W

Several perennial legumes endemic to the Canary Islands have been recognized as forage resources for semi-arid zones. In the period 2008–2010, seed collecting missions focused on the genera Adenocarpus DC., Bituminaria Heist. ex Fabr., Chamaecytisus Link, Cicer L., Genista L., Teline Medik. and Vicia L. were carried out in the archipelago. Sixty-nine samples were gathered from 31 endemic taxa, eight of which are threatened. These accessions are conserved at the gene bank of the Instituto Canario de Investigaciones Agrarias for further characterization and evaluation. Collection and ex situ conservation contribute to preserve endemic legumes genetic resources for utilization in the Canary Islands and in other semi-arid regions of the world.