The diversity of Class II transposable elements in mammalian genomes has arisen from ancestral phylogenetic splits during ancient waves of proliferation through the genome

DNA transposons make up three percent of the human genome, roughly the same percentage as genes. However, due to their inactivity, they are often ignored in favour of the more abundant, active, retroelements. Despite this relative ignominy, there are a number of interesting questions to be asked of these transposon families. One particular question relates to the timing of proliferation and inactivation of elements in a family. Does an ongoing process of turnover occur, or is the process more akin to a life cycle for the family, with elements proliferating rapidly before deactivation at a later date? We answer this question by tracing back to the most recent common ancestor of each modern transposon family, using two different methods. The first method identifies the most recent common ancestor of the species in which a family of transposon fossils can still be found, which we assume will have existed soon after the true origin date of the transposon family. The second method uses molecular dating techniques to predict the age of the most recent common ancestor element from which all elements found in a modern genome are descended. Independent data from five pairs of species are used in the molecular dating analysis: Human- Chimpanzee, Human-Orangutan, Dog-Panda, Dog-Cat and Cow-Pig. Orthologous pairs of elements from host species pairs are included, and the divergence dates of these species are used to constrain the analysis. We discover that, in general, the times to element common ancestry, for a given family, are the same for the different species pairs, suggesting that there has been no order-specific process of turnover. Furthermore, for most families, the ages of the common ancestor of the host species and of that of the elements are similar, suggesting a life cycle model for the proliferation of transposons. Where these two ages differ, in families found only in Primates and Rodentia, for example, we find that the host species date is later than that of the common ancestor of the elements, implying that there may be large deletions of elements from host species, examples of which were found in their ancestors

Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana

Background: CRISPR-Cas is a recent and powerful addition to the molecular toolbox which allows programmable genome editing. It has been used to modify genes in a wide variety of organisms, but only two alga to date. Here we present a methodology to edit the genome of Thalassiosira pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas. Results: A single construct was assembled using Golden Gate cloning. Two sgRNAs were used to introduce a precise 37 nt deletion early in the coding region of the urease gene. A high percentage of bi-allelic mutations (≤61.5%) were observed in clones with the CRISPR-Cas construct. Growth of bi-allelic mutants in urea led to a significant reduction in growth rate and cell size compared to growth in nitrate. Conclusions: CRISPR-Cas can precisely and efficiently edit the genome of T. pseudonana. The use of Golden Gate cloning to assemble CRISPR-Cas constructs gives additional flexibility to the CRISPR-Cas method and facilitates modifications to target alternative genes or species

Low prevalence of renal, cardiac, pulmonary, and neurological extra-articular clinical manifestations in spondyloarthritis: analysis of the Brazilian Registry of Spondyloarthritis

OBJECTIVE: To describe the extra-articular manifestations (cardiac, renal, pulmonary, and neurological), usually not related to spondyloarthritis (SpA), in a large cohort of Brazilian patients. MATERIALS AND METHODS: This retrospective study analyzed 1,472 patients diagnosed with SpA and cared for at 29 health care centers distributed in the five major geographic regions in the country, participating in the Brazilian Registry of Spondyloarthritis (BRS). All patients were assessed for the prevalence of major extra-articular manifestations (cardiac, renal, pulmonary, and neurological), classified according to the diagnosis [ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis (ReA), arthritis associated with inflammatory bowel disease (IBD), undifferentiated spondyloarthritis (uSpA), and juvenile SpA], and according to the clinical presentation (axial, peripheral, mixed, and enthesitis). RESULTS: Of the patients with SpA assessed, 963 had AS, 271 PsA, 49 ReA, 48 arthritis associated with IBD, 98 uSpA, and 43 juvenile SpA. Cardiac involvement was reported in 44 patients (3.0%), pulmonary involvement in 19 (1.3%), renal involvement in 17 (1.2%), and neurological involvement in 13 patients (0.9%). Most patients with visceral involvement had AS or PsA, and the mixed (axial + peripheral) and/or predominantly axial clinical form. CONCLUSION: Cardiac, renal, pulmonary, and neurological extra-articular manifestations are quite infrequent in SpA, ranging from 0.9% to 3% in this large Brazilian cohort, and affected predominantly patients with AS and PsA.OBJETIVO: Descrever as manifestações extra-articulares (cardíacas, renais, pulmonares e neurológicas) geralmente não relacionadas às espondiloartrites (EpA) em uma grande coorte de pacientes brasileiros. MÉTODOS: Este estudo retrospectivo analisou 1.472 pacientes com o diagnóstico de EpA atendidos em 29 centros distribuídos pelas cinco principais regiões geográficas do Brasil, integrantes do Registro Brasileiro de Espondiloartrites. Todos os pacientes foram avaliados para a prevalência das principais manifestações extra-articulares (cardíacas, renais, pulmonares e neurológicas), divididas por diagnóstico [espondilite anquilosante (EA), artrite psoriásica (AP), artrite reativa (ARe), artrite associada a doença inflamatória intestinal (DII), EpA indiferenciada (EI) e EpA juvenil] e por forma clínica (axial, periférica, mista e entesítica). RESULTADOS: Dentre os pacientes avaliados com EpA, 963 apresentavam EA, 271 AP, 49 ARe, 48 artrite associada a DII, 98 EI e 43 EpA juvenil. Acometimento cardíaco foi observado em 44 pacientes (3,0%), seguido por acometimento pulmonar em 19 (1,3%), renal em 17 (1,2%) e neurológico em 13 pacientes (0,9%). A maioria dos casos de acometimento visceral ocorreu nos pacientes com EA ou AP e naqueles com forma clínica mista (axial e periférica) e/ou predominantemente axial. CONCLUSÃO: As manifestações extra-articulares cardíacas, renais, pulmonares e neurológicas são muito pouco frequentes nas EpA, variando de 0,9%-3% nesta grande coorte brasileira, estando mais associadas a EA e AP.37938

Comparison of IRES and F2A-Based Locus-Specific Multicistronic Expression in Stable Mouse Lines

Efficient and stoichiometric expression of genes concatenated by bi- or multi-cistronic vectors has become an invaluable tool not only in basic biology to track and visualize proteins in vivo, but also for vaccine development and in the clinics for gene therapy. To adequately compare, in vivo, the effectiveness of two of the currently popular co-expression strategies - the internal ribosome entry site (IRES) derived from the picornavirus and the 2A peptide from the foot-and-mouth disease virus (FDMV) (F2A), we analyzed two locus-specific knock-in mouse lines co-expressing SRY-box containing gene 9 (Sox9) and enhanced green fluorescent protein (EGFP) linked by the IRES (Sox9IRES-EGFP) or the F2A (Sox9F2A-EGFP) sequence. Both the constructs expressed Sox9 and EGFP proteins in the appropriate Sox9 expression domains, with the IRES construct expressing reduced levels of EGFP compared to that of the F2A. The latter, on the other hand, produced about 42.2% Sox9-EGFP fusion protein, reflecting an inefficient ribosome ‘skipping’ mechanism. To investigate if the discrepancy in the ‘skipping’ process was locus-dependent, we further analyzed the FLAG3-Bapx1F2A-EGFP mouse line and found similar levels of fusion protein being produced. To assess if EGFP was hindering the ‘skipping’ mechanism, we examined another mouse line co-expressing Bagpipe homeobox gene 1 homolog (Bapx1), Cre recombinase and EGFP (Bapx1F2A-Cre-F2A-EGFP). While the ‘skipping’ was highly efficient between Bapx1 and Cre, the ‘skipping’ between Cre and EGFP was highly inefficient. We have thus demonstrated in our comparison study that the efficient and close to equivalent expression of genes linked by F2A is achievable in stable mouse lines, but the EGFP reporter may cause undesirable inhibition of the ‘skipping’ at the F2A sequence. Hence, the use of other reporter genes should be explored when utilizing F2A peptides

Improving educational achievement and anaemia of school children: design of a cluster randomised trial of school-based malaria prevention and enhanced literacy instruction in Kenya

BACKGROUND: Improving the health of school-aged children can yield substantial benefits for cognitive development and educational achievement. However, there is limited experimental evidence on the benefits of school-based malaria prevention or how health interventions interact with other efforts to improve education quality. This study aims to evaluate the impact of school-based malaria prevention and enhanced literacy instruction on the health and educational achievement of school children in Kenya. DESIGN: A factorial, cluster randomised trial is being implemented in 101 government primary schools on the coast of Kenya. The interventions are (i) intermittent screening and treatment of malaria in schools by public health workers and (ii) training workshops and support for teachers to promote explicit and systematic literacy instruction. Schools are randomised to one of four groups: receiving either (i) the malaria intervention alone; (ii) the literacy intervention alone; (iii) both interventions combined; or (iv) control group where neither intervention is implemented. Children from classes 1 and 5 are randomly selected and followed up for 24 months. The primary outcomes are educational achievement and anaemia, the hypothesised mediating variables through which education is affected. Secondary outcomes include malaria parasitaemia, school attendance and school performance. A nested process evaluation, using semi-structured interviews, focus group discussion and a stakeholder analysis will investigate the community acceptability, feasibility and cost-effectiveness of the interventions. DISCUSSION: Across Africa, governments are committed to improve health and education of school-aged children, but seek clear policy and technical guidance as to the optimal approach to address malaria and improved literacy. This evaluation will be one of the first to simultaneously evaluate the impact of health and education interventions in the improvement of educational achievement. Reflection is made on the practical issues encountered in conducting research in schools in Africa. TRIAL REGISTRATION: National Institutes of Health NCT00878007

Refractoriness of hepatitis C virus internal ribosome entry site to processing by Dicer in vivo

<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus (HCV) is a positive-strand RNA virus harboring a highly structured internal ribosome entry site (IRES) in the 5' nontranslated region of its genome. Important for initiating translation of viral RNAs into proteins, the HCV IRES is composed of RNA structures reminiscent of microRNA precursors that may be targeted by the host RNA silencing machinery.</p> <p>Results</p> <p>We report that HCV IRES can be recognized and processed into small RNAs by the human ribonuclease Dicer in vitro. Furthermore, we identify domains II, III and VI of HCV IRES as potential substrates for Dicer in vitro. However, maintenance of the functional integrity of the HCV IRES in response to Dicer overexpression suggests that the structure of the HCV IRES abrogates its processing by Dicer in vivo.</p> <p>Conclusion</p> <p>Our results suggest that the HCV IRES may have evolved to adopt a structure or a cellular context that is refractory to Dicer processing, which may contribute to viral escape of the host RNA silencing machinery.</p

MEF2C Enhances Dopaminergic Neuron Differentiation of Human Embryonic Stem Cells in a Parkinsonian Rat Model

Human embryonic stem cells (hESCs) can potentially differentiate into any cell type, including dopaminergic neurons to treat Parkinson's disease (PD), but hyperproliferation and tumor formation must be avoided. Accordingly, we use myocyte enhancer factor 2C (MEF2C) as a neurogenic and anti-apoptotic transcription factor to generate neurons from hESC-derived neural stem/progenitor cells (NPCs), thus avoiding hyperproliferation. Here, we report that forced expression of constitutively active MEF2C (MEF2CA) generates significantly greater numbers of neurons with dopaminergic properties in vitro. Conversely, RNAi knockdown of MEF2C in NPCs decreases neuronal differentiation and dendritic length. When we inject MEF2CA-programmed NPCs into 6-hydroxydopamine—lesioned Parkinsonian rats in vivo, the transplanted cells survive well, differentiate into tyrosine hydroxylase-positive neurons, and improve behavioral deficits to a significantly greater degree than non-programmed cells. The enriched generation of dopaminergic neuronal lineages from hESCs by forced expression of MEF2CA in the proper context may prove valuable in cell-based therapy for CNS disorders such as PD

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2–4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Quality of life in spondyloarthritis : analysis of a large Brazilian cohort

Objetivo: analisar as variáveis demográficas e clínicas associadas à diminuição da qualidade de vida em uma grande coorte brasileira de pacientes com espondiloartrite (EpA). Métodos: Foi aplicado um protocolo de pesquisa único a 1.465 pacientes brasileiros classificados como tendo EpA de acordo com os critérios do European Spondyloarthropaties Study Group (ESSG), atendidos em 29 centros de referência em reumatologia do Brasil. Foram registradas as variáveis clínicas e demográficas. A qualidade de vida foi analisada por meio do questionário Ankylosing Spondylitis Quality of Life (ASQoL). Resultados: A pontuação média do ASQoL foi de 7,74 (+ 5,39). Ao analisar doenças específicas no grupo de EpA, as pontuações do ASQoL não apresentaram diferença estatisticamente significativa. Os dados demográficos mostraram piores escores de ASQoL associados ao gênero feminino (p = 0,014) e etnia negra (p < 0,001). Quanto aos sintomas clínicos, a dor na região glútea (p = 0,032), a dor cervical (p < 0,001) e a dor no quadril (p = 0,001), estiveram estatisticamente associadas a piores escores no ASQoL. O uso contínuo de fármacos anti-inflamatórios não esteroides (p < 0,001) e agentes biológicos (p = 0,044) esteve associado a escores mais elevados de ASQoL, enquanto outros medicamentos não interferiram nos escores do ASQoL. Conclusão: Nesta grande série de pacientes com EpA, o sexo feminino e a etnia negra, bem como sintomas predominantemente axiais, estiveram associados a uma qualidade de vida reduzida.Objective: to analyze quality of life and demographic and clinical variables associated to its impairment in a large Brazilian cohort of patients with spondyloarthritis (SpA). Methods: A common protocol of investigation was applied to 1465 Brazilian patients classified as SpA according to the European Spondyloarthropaties Study Group (ESSG) criteria, attended at 29 reference centers for Rheumatology in Brazil. Clinical and demographic variables were recorded. Quality of life was analyzed through the Ankylosing Spondylitis Quality of Life (ASQoL) questionnaire. Results: The mean ASQoL score was 7.74 (± 5.39). When analyzing the specific diseases in the SpA group, the ASQoL scores did not present statistical significance. Demographic data showed worse scores of ASQoL associated with female gender (p = 0.014) and African-Brazilian ethnicity (p < 0.001). Regarding clinical symptoms, buttock pain (p = 0.032), cervical pain (p < 0.001) and hip pain (p = 0.001), were statistically associated with worse scores of ASQoL. Continuous use of nonsteroidal anti-inflammatory drugs (p < 0.001) and biologic agents (p = 0.044) were associated with higher scores of ASQoL, while the other medications did not interfere with the ASQoL scores. Conclusion: In this large series of patients with SpA, female gender and African-Brazilian ethnicity, as well as predominant axial symptoms, were associated with impaired quality of life

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe