ARAMIS: From systematic errors of NGS long reads to accurate assemblies.

NGS long-reads sequencing technologies (or third generation) such as Pacific BioSciences (PacBio) have revolutionized the sequencing field over the last decade improving multiple genomic applications like de novo genome assemblies. However, their error rate, mostly involving insertions and deletions (indels), is currently an important concern that requires special attention to be solved. Multiple algorithms are available to fix these sequencing errors using short reads (such as Illumina), although they require long processing times and some errors may persist. Here, we present Accurate long-Reads Assembly correction Method for Indel errorS (ARAMIS), the first NGS long-reads indels correction pipeline that combines several correction software in just one step using accurate short reads. As a proof OF concept, six organisms were selected based on their different GC content, size and genome complexity, and their PacBio-assembled genomes were corrected thoroughly by this pipeline. We found that the presence of systematic sequencing errors in long-reads PacBio sequences affecting homopolymeric regions, and that the type of indel error introduced during PacBio sequencing are related to the GC content of the organism. The lack of knowledge of this fact leads to the existence of numerous published studies where such errors have been found and should be resolved since they may contain incorrect biological information. ARAMIS yields better results with less computational resources needed than other correction tools and gives the possibility of detecting the nature of the found indel errors found and its distribution along the genome. The source code of ARAMIS is available at https://github.com/genomics-ngsCBMSO/ARAMIS.git.

Human genomics projects and precision medicine.

The completion of the Human Genome Project (HGP) in 2001 opened the floodgates to a deeper understanding of medicine. There are dozens of HGP-like projects which involve from a few tens to several million genomes currently in progress, which vary from having specialized goals or a more general approach. However, data generation, storage, management and analysis in public and private cloud computing platforms have raised concerns about privacy and security. The knowledge gained from further research has changed the field of genomics and is now slowly permeating into clinical medicine. The new precision (personalized) medicine, where genome sequencing and data analysis are essential components, allows tailored diagnosis and treatment according to the information from the patient's own genome and specific environmental factors. P4 (predictive, preventive, personalized and participatory) medicine is introducing new concepts, challenges and opportunities. This review summarizes current sequencing technologies, concentrates on ongoing human genomics projects, and provides some examples in which precision medicine has already demonstrated clinical impact in diagnosis and/or treatment.

Acute stroke care during the COVID-19 pandemic. Ictus Madrid Program recommendations. / Atención al ictus agudo durante la pandemia por COVID-19. Recomendaciones Plan Ictus Madrid.

INTRODUCTION: The COVID-19 pandemic has resulted in complete saturation of healthcare capacities, making it necessary to reorganise healthcare systems. In this context, we must guarantee the provision of acute stroke care and optimise code stroke protocols to reduce the risk of SARS-CoV-2 infection and rationalise the use of hospital resources. The Madrid Stroke multidisciplinary group presents a series of recommendations to achieve these goals. METHODS: We conducted a non-systematic literature search using the keywords "stroke" and "COVID-19" or "coronavirus" or "SARS-CoV-2." Our literature review also included other relevant studies known to the authors. Based on this literature review, a series of consensus recommendations were established by the Madrid Stroke multidisciplinary group and its neurology committee. RESULTS: These recommendations address 5 main objectives: 1) coordination of action protocols to ensure access to hospital care for stroke patients; 2) recognition of potentially COVID-19-positive stroke patients; 3) organisation of patient management to prevent SARS-CoV-2 infection among healthcare professionals; 4) avoidance of unnecessary neuroimaging studies and other procedures that may increase the risk of infection; and 5) safe, early discharge and follow-up to ensure bed availability. This management protocol has been called CORONA (Coordinate, Recognise, Organise, Neuroimaging, At home). CONCLUSIONS: The recommendations presented here may assist in the organisation of acute stroke care and the optimisation of healthcare resources, while ensuring the safety of healthcare professionals.

Multiple myeloma patients in long-term complete response after autologous stem cell transplantation express a particular immune signature with potential prognostic implication.

The proportion of multiple myeloma patients in long-term complete response (LTCR-MM) for more than 6 years after autologous stem cell transplantation (ASCT) is small. To evaluate whether this LTCR is associated with a particular immune signature, peripheral blood samples from 13 LTCR-MM after ASCT and healthy blood donors (HBD) were analysed. Subpopulations of T-cells (naïve, effector, central memory and regulatory), B-cells (naïve, marginal zone-like, class-switched memory, transitional and plasmablasts) and NK-cells expressing inhibitory and activating receptors were quantified by multiparametric flow cytometry (MFC). Heavy/light chains (HLC) were quantified by nephelometry. The percentage of CD4+ T-cells was lower in patients, whereas an increment in the percentage of CD4+ and CD8+ effector memory T-cells was associated with the LTCR. Regulatory T-cells and NK-cells were similar in both groups but a particular redistribution of inhibitory and activating receptors in NK-cells were found in patients. Regarding B-cells, an increase in naïve cells and a corresponding reduction in marginal zone-like and class-switched memory B-cells was observed. The HLC values were normal. Our results suggest that LTCR-MM patients express a particular immune signature, which probably reflects a 'high quality' immune reconstitution that could exert a competent anti-tumor immunological surveillance along with a recovery of the humoral immunity.

Gastric MALT lymphoma: clinical characteristics and prevalence of H. pylori infection in a series of 37 cases.

OBJECTIVE: to perform a retrospective review of the clinical characteristics and prevalence of H. pylori infection in patients with gastric MALT lymphoma diagnosed in our hospital during the last 15 years. METHODS: patients with gastric MALT lymphoma diagnosed in our hospital during the last 15 years were retrospectively included. Demographic, clinic, analytic, endoscopic, and histological variables were reviewed. The extension study, the staging classification, and the presence of H. pylori infection were assessed. RESULTS: thirty-seven patients with gastric MALT lymphoma were identified. Mean age was 61 years, with 62% of males. The most common presentation symptom was dyspepsia (76%), followed by digestive bleeding (11%) and constitutional syndrome (8%). At endoscopy, erosive lesions were identified in 41%, and proliferative or exophytic lesions in 43%. Most lymphomas were classified as low-grade (68%). The stage distribution was EI for 56%, EII for 13%, EIII for 3%, and EIV for 28%. The prevalence of H. pylori infection (histology in all cases, rapid urease test in 19%, and 13C-urea breath test in 24%) was 46%. When only low-grade lymphomas in stage EI were considered, H. pylori prevalence increased to 55%. When H. pylori infection was evaluated by 13C-urea breath testing (in addition to histology), the prevalence of H. pylori infection increased to 78%. CONCLUSIONS: it is probable that the reduced H. pylori prevalence found in some studies, as in ours, could be explained by false-negative results obtained when only one diagnostic method was used. Therefore, at least two (invasive) diagnostic methods should be performed. Furthermore, the performance of a non-invasive diagnostic method (such as a 13C-urea breath test) before the exclusion of H. pylori infection should be considered.

Differential splicing, disease and drug targets.

Genome complexity and diversity can be due to Alternative Splicing (AS), a process by which one gene can generate multiple mRNA isoforms and then several proteins. This is part of a normal process of variation on an individual, and when it is disrupted or modified, may trigger disease. To date, there are many pathologies described due to the effects of altered splicing isoforms, and effort is focused on the description of new ones. The design of drug target has to consider splicing, as in many occasions, a drug might have effect on different isoforms, instead of on the particular one implicated in the pathology. Interestingly, the strategies used to alter splicing can be used to modify a form towards the canonical one, or towards an aberrant one, when the latter one has a beneficial effect on the individual. Here we describe differential splicing, diseases produced by alterations on the mRNA isoforms, and drugs or methods used to restore these alterations.

Síndrome de pinzamiento isquiofemoral: a propósito de un caso. Diagnóstico diferencial y manejo rehabilitador / Ischiofemoral impingement syndrome: a case report. Differential diagnosis and rehabilitation management

Hay múltiples causas de dolor de cadera. Entre ellas se encuentra el síndrome de pinzamiento isquiofemoral, actualmente de etiología desconocida, aunque inicialmente relacionado con historia previa de traumatismo o cirugía, para el cual no existe un protocolo de actuación unificado (AU)

There are multiple causes of hip pain, including ischiofemoral impingement syndrome. The aetiology of this syndrome is currently unknown, although it is associated with a history of trauma or surgery. Currently, there is no standard of care for this syndrome (AU)

Pulmonary embolism in a patient with severe congenital deficiency for factor V during treatment with fresh frozen plasma.

Thrombosis is a rare complication in patients with congenital clotting factor deficiencies. In most cases, it is related to inherited procoagulant factors, use of central venous catheters or administration of coagulation factor concentrates. There are only a few case reports about thrombotic events during treatment with fresh frozen plasma (FFP). We report the case of a patient with homozygous inherited factor V deficiency, who developed a pulmonary embolism at a time of treatment with methylene blue treated FFP (MBFFP). The patient had only two other factors predisposing to thrombosis and both were acquired: obesity and bed rest. He started anticoagulant treatment with low molecular weight heparin (LMWH) while the deficient factors were replaced with MBFFP. After 8 days of treatment the patient developed a severe respiratory insufficiency. Pulmonary haemorrhage was considered among the differential diagnosis and LMWH was stopped. An inferior vena cava filter was placed without any further thrombotic complications. To our knowledge, there are no reports about patients with clotting factor deficiencies who developed a thrombotic event during treatment with MBFFP.

The novel gene G17, located in the human major histocompatibility complex, encodes PBX2, a homeodomain-containing protein.

Recent characterization of the class III region of the human major histocompatibility complex (MHC), located in chromosome 6p21.3, has revealed that it is very gene dense and contains at least 47 transcriptional units. One of these is the gene G17, which lies 250 kb telomeric of the class II gene DRA. DNA sequence analysis of 5.5 kb of DNA corresponding to the G17 gene has revealed that it encodes PBX2, a homeodomain-containing protein with extensive similarity to PBX1 (which is involved in t(1;19) chromosomal translocations in acute pre-B-cell leukemias). Comparison of the genomic DNA sequence with the published PBX2 cDNA sequence, which are 99.7% identical, indicates that the G17 gene is split into 9 exons, with the intron/exon boundaries conforming to the normal pattern (AG/.../GT) for splice sites. Of the 9 differences observed between the PBX2 cDNA sequence and the G17 genomic sequence, only 1 is contained in the coding sequence and alters the derived amino acid sequence. This results in an Ile (PBX2)-Met (G17) substitution at amino acid 393 near the C-terminus. The PBX2 gene was originally localized only to human chromosome 3q22-q23. However, comparison of genomic and cosmid Southern blots clearly indicates that another copy(ies) of the PBX2 (G17) gene exist(s) in the genome. PCR amplification of exons III and IX of the G17 (PBX2) gene, corresponding to the coding and 3' untranslated regions, respectively, using as template genomic DNA from a panel of monochromosomal somatic human-rodent cell hybrids, gave specific products in hybrids that contain human chromosomes 6, 3, and 1. These results confirm that copies of the PBX2 gene are located on human chromosomes 6 and 3 and indicate that a gene homologous to PBX2 could exist on human chromosome 1. Further PCR analysis of the genes and reverse transcribed mRNA from the hybrid cell lines has revealed that the copies of the PBX2 gene on human chromosomes 6 and 1 are expressed, while the copy on human chromosome 3 may be a processed pseudogene.

African swine fever virus fatty acid acylated proteins.

Labeling experiments with [3H]palmitic and [3H]myristic acids of African swine fever virus-infected Vero cells have shown that 11 proteins induced during infection are covalently bound to myristic acid and that palmitic acid was not attached to viral proteins. The time course of synthesis of the myristylated polypeptides and the requirements of viral DNA replication indicated that the myristylated proteins, with the exception of a 13-kDa protein, belong to the late class of viral proteins. The myristic moiety was not released by hydroxylamine treatment, suggesting that the fatty acid is bound to the polypeptide chain through an amide linkage. The purification of [3H]myristic acid-labeled extracellular virus particles demonstrated that the myristylated 28- and 13-kDa proteins incorporated into the virion.

Characterization of a human MHC class III region gene product with S-thioesterase activity.

Palmitoylated proteins contain a 16-carbon saturated fatty acyl group that is post-translationally attached by a labile thioester bond. These modified proteins are mainly membrane-bound; the lability of the thioester bond allows the process to be reversible, a unique property of this modification. We report here that the gene for G14, located in the class III region of the human MHC, encodes a polypeptide with significant sequence similarity to mammalian palmitoyl protein thioesterase (PPT1), an enzyme that removes palmitate from palmitoylated proteins. The gene for G14, also known as PPT2, is transcribed as at least five different transcripts, which are expressed in different cell lines of the immune system. Immunoprecipitation of these mammalian cells, with an anti-G14 antiserum, showed a specific band of approx. 42 kDa in cell extracts and supernatants. Expression of the G14 cDNA in the baculovirus system revealed that it encoded a secreted glycosylated polypeptide with S-thioesterase activity. The enzymic activity of the recombinant G14 protein was further characterized in quantitative spectrophotometric assays, which revealed that it had the highest S-thioesterase activity for the acyl groups palmitic and myristic acid followed by other long-chain acyl substrates. The S-thioesterase activity of the G14 protein was found to be considerably higher in supernatants than in cell extracts, which was consistent with the protein's being secreted. The G14 polypeptide contains, in addition to an N-terminal lipase domain, a C-terminal domain common to the cytokine receptor superfamily, which might determine the substrate specificity and/or the protein target of the G14 protein.

Characterization of a human lysophosphatidic acid acyltransferase that is encoded by a gene located in the class III region of the human major histocompatibility complex.

Sequence analysis of cDNA clones corresponding to a number of genes located in the class III region of the human major histocompatibility complex (MHC), in the chromosome band 6p21.3, has shown that the G15 gene encodes a 283-amino acid polypeptide with significant homology over the entire polypeptide with the enzyme lysophosphatidic acid acyltransferase (LPAAT) from different yeast, plant, and bacterial species. The amino acid sequence of the MHC-encoded human LPAAT (hLPAATalpha) is 48% identical to the recently described hLPAAT (Eberhardt, C., Gray, P. W., and Tjoelker, L. W. (1997) J. Biol. Chem. 272, 20299-20305), which is encoded by a gene located on chromosome 9p34.3. LPAAT is the enzyme that in lipid metabolism converts lysophosphatidic acid (LPA) into phosphatidic acid (PA). The expression of the hLPAATalpha polypeptide in the baculovirus system and in mammalian cells has shown that it is an intracellular protein that contains LPAAT activity. Cell extracts from insect cells overexpressing hLPAATalpha were analyzed in different LPAAT enzymatic assays using, as substrates, different acyl acceptors and acyl donors. These cell extracts were found to contain up to 5-fold more LPAAT activity compared with control cell extracts, indicating that the hLPAATalpha specifically converts LPA into PA, incorporating different acyl-CoAs with different affinities. The hLPAATalpha polypeptide expressed in the mammalian Chinese hamster ovary cell line was found, by confocal immunofluorescence, to be localized in the endoplasmic reticulum. Due to the known role of LPA and PA in intracellular signaling and inflammation, the hLPAATalpha gene represents a candidate gene for some MHC-associated diseases.

Nucleotide sequence of 21.8 kbp of variola major virus strain Harvey and comparison with vaccinia virus.

A 21.8 kbp region of the genome of variola major virus (strain Harvey), a virus that caused haemorrhagic-type smallpox, has been sequenced and shown to possess 96% nucleotide identity to the corresponding region of vaccinia virus, the smallpox vaccine. Overall the gene arrangement in the two viruses is highly similar and individual open reading frames (ORFs) display a high degree of amino acid identity, for instance 26 of the 32 variola virus ORFs have > or = 90% identity with their vaccinia virus counterparts. A remarkable difference is the disruption of seven vaccinia virus ORFs into small fragments in variola virus. These include the variola virus homologue of vaccinia virus SalF2R, which encodes a protein related to C-type animal lectins, and SalF7L, which encodes an active 3 beta-hydroxysteroid dehydrogenase enzyme that contributes to vaccinia virus virulence. Upstream of the variola virus haemagglutinin gene there is a deletion of 1910 bp so that the equivalent of vaccinia virus gene SalF17R is truncated, and SalF16R, which shows amino acid similarity to the tumour necrosis factor receptor, is absent. The region sequenced includes the genes for thymidylate kinase and DNA ligase both of which are active in vaccinia virus and are highly conserved in variola virus. Other conserved ORFs with interesting homologies are those encoding profilin, superoxide dismutase and part of guanylate kinase. Two vaccinia virus genes encoding glycoproteins of the outer envelope of extracellular enveloped virus are also conserved in variola virus and this homology is likely to have contributed to the immunological protection which vaccinia virus evoked against smallpox. Lastly, there are multiple instances in which short oligonucleotide direct repeats flank a region absent from either variola or vaccinia virus.

G6b, a novel immunoglobulin superfamily member encoded in the human major histocompatibility complex, interacts with SHP-1 and SHP-2.

The G6b gene, located in the class III region of the human major histocompatibility complex, has been suggested to encode a putative receptor of the immunoglobulin superfamily. Genomic sequence information was used as a starting point to clone the corresponding cDNA. Reverse transcriptase polymerase chain reaction showed that expression of the gene is restricted to certain hematopoietic cell lines including K562, Molt 4, and Jurkat. Several splice variants were detected, varying only in their C-terminal parts. One of the potential membrane-bound isoforms contained two immunoreceptor tyrosine-based inhibitory motifs in its cytoplasmic tail. Four of the isoforms were expressed as epitope-tagged proteins in the cell lines K562 and COS-7. The two splice isoforms lacking the hydrophobic transmembrane segment were secreted from the cell. Glycosidase treatment of the four recombinant proteins provided evidence for N- and O-glycosylation. Immunofluorescence studies indicated that the spliced isoforms having a transmembrane segment were directed to the cell membrane. The G6b isoform containing two immunoreceptor tyrosine-based inhibitory motifs in its cytoplasmic tail was found to be phosphorylated on tyrosine residues after pervanadate treatment of cells and, subsequently, interacts with the SH2-containing protein-tyrosine phosphatases SHP-1 and SHP-2. Mutagenesis studies showed that phosphorylation of tyrosine 211 is critical for the interaction of G6b with SHP-1 and SHP-2.

Linfoma MALT gástrico: características clínicas y prevalencia de la infección por H. pylori en una serie de 37 casos / Gastric MALT lymphoma: clinical characteristics and prevalence of H. pylori infection in a series of 37 cases

Objetivo: revisar retrospectivamente las características clínicasy la prevalencia de infección por H. pylori en los pacientescon linfoma MALT gástrico diagnosticados en nuestro hospitaldurante los últimos 15 años.Métodos: se identificaron retrospectivamente todos los pacientesdiagnosticados de linfoma MALT gástrico en nuestro centroen los últimos 15 años. Se revisaron las variables demográficas,clínicas, analíticas, endoscópicas, histológicas, el estudio deextensión, la estadificación y la infección por H. pylori.Resultados: se identificaron 37 pacientes con linfoma MALTgástrico, con una edad media de 61 años, el 62% varones. El síntomade presentación más frecuente fue la dispepsia (76%), seguidode la hemorragia digestiva alta (11%) y el síndrome constitucional(8%). Durante la endoscopia se identificaron lesiones erosivasen el 41% y proliferativas/exofíticas en el 43%. La mayoría de loslinfomas fueron de bajo grado (68%). Estadificación: EI (56%), EII(13%), EIII (3%) y EIV (28%). La prevalencia de H. pylori (histologíaen todos los casos, test rápido de la ureasa en el 19% y pruebadel aliento con 13C-urea en el 24%) fue del 46%. Cuando únicamentese consideraron los linfomas de bajo grado en estadio EI, laprevalencia de H. pylori ascendió al 55%. Cuando la presencia deH. pylori se valoró mediante la prueba del aliento (además de lahistología), la prevalencia de infección ascendió al 78%.Conclusiones: es probable que la reducida prevalencia de infecciónpor H. pylori encontrada en algunos estudios, como elnuestro, sea debida a resultados falsos negativos obtenidos al utilizarun sólo test diagnóstico, por lo que se sugiere el empleo de almenos dos, e incluso añadir un método diagnóstico “no invasivo”como la prueba del aliento con 13C-urea antes de excluir definitivamentela infección por H. pylori

Objective: to perform a retrospective review of the clinicalcharacteristics and prevalence of H. pylori infection in patientswith gastric MALT lymphoma diagnosed in our hospital duringthe last 15 years.Methods: patients with gastric MALT lymphoma diagnosed inour hospital during the last 15 years were retrospectively included.Demographic, clinic, analytic, endoscopic, and histological variableswere reviewed. The extension study, the staging classification,and the presence of H. pylori infection were assessed.Results: thirty-seven patients with gastric MALT lymphomawere identified. Mean age was 61 years, with 62% of males. Themost common presentation symptom was dyspepsia (76%), followedby digestive bleeding (11%) and constitutional syndrome(8%). At endoscopy, erosive lesions were identified in 41%, andproliferative or exophytic lesions in 43%. Most lymphomas wereclassified as low-grade (68%). The stage distribution was EI for56%, EII for 13%, EIII for 3%, and EIV for 28%. The prevalenceof H. pylori infection (histology in all cases, rapid urease test in19%, and 13C-urea breath test in 24%) was 46%. When only lowgradelymphomas in stage EI were considered, H. pylori prevalenceincreased to 55%. When H. pylori infection was evaluatedby 13C-urea breath testing (in addition to histology), the prevalenceof H. pylori infection increased to 78%.Conclusions: it is probable that the reduced H. pylori prevalencefound in some studies, as in ours, could be explained byfalse-negative results obtained when only one diagnostic methodwas used. Therefore, at least two (invasive) diagnostic methodsshould be performed. Furthermore, the performance of a non-invasivediagnostic method (such as a 13C-urea breath test) beforethe exclusion of H. pylori infection should be considered

El dolor lumbar en el año 2009 / Lumbar pain in 2009

Todo el mundo reconoce la frecuencia tan elevada de las lumbalgias y su importante repercusión socioprofesional. El dolor lumbar es una de las patologías más frecuentes en las consultas de medicina general y de los especialistas del aparato locomotor (traumatólogos, reumatólogos y rehabilitadores). Tiene características de epidemia en las sociedades más desarrolladas y ha sido denominado por algunos autores como la “enfermedad del siglo”. Entre las consultas médicas, después de los síntomas del resfriado le siguen inmediatamente los dolores de espalda.El mantenimiento en el empleo de los trabajadores que sufren de la espalda y la prevención de la lumbalgia crónica constituyen preocupaciones crecientes de los responsables de la salud pública en razón de los costes elevados que esta problemática de salud genera en la colectividad. Para hacer frente a este problema mayor de salud pública, la literatura científica, apoyándose principalmente en estudios escandinavos y norteamericanos, propone y considera muy importante la actuación médica precoz y adecuada de la lumbalgia en la fase aguda (AU)

Almost everyone will experience low back pain at some point in their lives and recognizes the very high frequency of lower back pain and its significant social and occupational impact. Low back pain is one of the most common conditions in general practice consultations and musculoskeletal specialists (orthopedists, rheumatologists and rehabilitation). It has characteristics of an epidemic in most developed societies, and has been dubbed by some as "disease of the century". Between doctor visits, after cold symptoms followed immediately back pain.The job retention of workers suffering from back and preventing chronic back pain are growing concerns of those responsible for public health because of the high costs this creates health problems in the community. To address this major public health problem, the scientific literature, relying mainly on studies Scandinavian and North American, proposes and attaches great importance to early and appropriate medical intervention in low back pain in the acute phase (AU)