Inherited and De Novo Genetic Risk for Autism Impacts Shared Networks.

We performed a comprehensive assessment of rare inherited variation in autism spectrum disorder (ASD) by analyzing whole-genome sequences of 2,308 individuals from families with multiple affected children. We implicate 69 genes in ASD risk, including 24 passing genome-wide Bonferroni correction and 16 new ASD risk genes, most supported by rare inherited variants, a substantial extension of previous findings. Biological pathways enriched for genes harboring inherited variants represent cytoskeletal organization and ion transport, which are distinct from pathways implicated in previous studies. Nevertheless, the de novo and inherited genes contribute to a common protein-protein interaction network. We also identified structural variants (SVs) affecting non-coding regions, implicating recurrent deletions in the promoters of DLG2 and NR3C2. Loss of nr3c2 function in zebrafish disrupts sleep and social function, overlapping with human ASD-related phenotypes. These data support the utility of studying multiplex families in ASD and are available through the Hartwell Autism Research and Technology portal.

The contributions of rare inherited and polygenic risk to ASD in multiplex families.

Autism spectrum disorder (ASD) has a complex genetic architecture involving contributions from both de novo and inherited variation. Few studies have been designed to address the role of rare inherited variation or its interaction with common polygenic risk in ASD. Here, we performed whole-genome sequencing of the largest cohort of multiplex families to date, consisting of 4,551 individuals in 1,004 families having two or more autistic children. Using this study design, we identify seven previously unrecognized ASD risk genes supported by a majority of rare inherited variants, finding support for a total of 74 genes in our cohort and a total of 152 genes after combined analysis with other studies. Autistic children from multiplex families demonstrate an increased burden of rare inherited protein-truncating variants in known ASD risk genes. We also find that ASD polygenic score (PGS) is overtransmitted from nonautistic parents to autistic children who also harbor rare inherited variants, consistent with combinatorial effects in the offspring, which may explain the reduced penetrance of these rare variants in parents. We also observe that in addition to social dysfunction, language delay is associated with ASD PGS overtransmission. These results are consistent with an additive complex genetic risk architecture of ASD involving rare and common variation and further suggest that language delay is a core biological feature of ASD.

Deterministic programming of human pluripotent stem cells into microglia facilitates studying their role in health and disease.

Microglia, the resident immune cells of the central nervous system (CNS), are derived from yolk-sac macrophages that populate the developing CNS during early embryonic development. Once established, the microglia population is self-maintained throughout life by local proliferation. As a scalable source of microglia-like cells (MGLs), we here present a forward programming protocol for their generation from human pluripotent stem cells (hPSCs). The transient overexpression of PU.1 and C/EBPß in hPSCs led to a homogenous population of mature microglia within 16 d. MGLs met microglia characteristics on a morphological, transcriptional, and functional level. MGLs facilitated the investigation of a human tauopathy model in cortical neuron-microglia cocultures, revealing a secondary dystrophic microglia phenotype. Single-cell RNA sequencing of microglia integrated into hPSC-derived cortical brain organoids demonstrated a shift of microglia signatures toward a more-developmental in vivo-like phenotype, inducing intercellular interactions promoting neurogenesis and arborization. Taken together, our microglia forward programming platform represents a tool for both reductionist studies in monocultures and complex coculture systems, including 3D brain organoids for the study of cellular interactions in healthy or diseased environments.

Enterovirus D68 disease burden and epidemiology in hospital-admitted influenza-like illness, Valencia region of Spain, 2014-2020 influenza seasons.

Enterovirus D68 (EV-D68) is an emerging agent for which data on the susceptible adult population is scarce. We performed a 6-year analysis of respiratory samples from influenza-like illness (ILI) admitted during 2014-2020 in 4-10 hospitals in the Valencia Region, Spain. EV-D68 was identified in 68 (3.1%) among 2210 Enterovirus (EV)/Rhinovirus (HRV) positive samples. Phylogeny of 59 VP1 sequences showed isolates from 2014 clustering in B2 (6/12), B1 (5/12), and A2/D1 (1/12) subclades; those from 2015 (n = 1) and 2016 (n = 1) in B3 and A2/D1, respectively; and isolates from 2018 in A2/D3 (42/45), and B3 (3/45). B1 and B2 viruses were mainly detected in children (80% and 67%, respectively); B3 were equally distributed between children and adults; whereas A2/D1 and A2/D3 were observed only in adults. B3 viruses showed up to 16 amino acid changes at predicted antigenic sites. In conclusion, two EV-D68 epidemics linked to ILI hospitalized cases occurred in the Valencia Region in 2014 and 2018, with three fatal outcomes and one ICU admission. A2/D3 strains from 2018 were associated with severe respiratory infection in adults. Because of the significant impact of non-polio enteroviruses in ILI and the potential neurotropism, year-round surveillance in respiratory samples should be pursued.

A Novel Technique of Placing Desfibrillator Leads in Patients with Persistent Left Superior Vena Cava.

Persistent left superior vena cava is the most common thoracic venous anomaly. It is usually asymptomatic, but it can make implanting intracardiac devices difficult.We present a novel technique to facilitate desfibrillator lead implantation in patients with persistent left superior vena cava and the absence of the right superior vena cava. We used a fixed-curve Selectra 3D 65-42 cm sheath (Biotronik), orienting it toward the tricuspid valve (TV) by rotating it counter-clockwise. During follow-up, the electrodes remained stable.Our technique was safe, simple, and feasible for patients with this complex venous anatomy.

Evidence for Strong Mutation Bias toward, and Selection against, U Content in SARS-CoV-2: Implications for Vaccine Design.

Large-scale re-engineering of synonymous sites is a promising strategy to generate vaccines either through synthesis of attenuated viruses or via codon-optimized genes in DNA vaccines. Attenuation typically relies on deoptimization of codon pairs and maximization of CpG dinucleotide frequencies. So as to formulate evolutionarily informed attenuation strategies that aim to force nucleotide usage against the direction favored by selection, here, we examine available whole-genome sequences of SARS-CoV-2 to infer patterns of mutation and selection on synonymous sites. Analysis of mutational profiles indicates a strong mutation bias toward U. In turn, analysis of observed synonymous site composition implicates selection against U. Accounting for dinucleotide effects reinforces this conclusion, observed UU content being a quarter of that expected under neutrality. Possible mechanisms of selection against U mutations include selection for higher expression, for high mRNA stability or lower immunogenicity of viral genes. Consistent with gene-specific selection against CpG dinucleotides, we observe systematic differences of CpG content between SARS-CoV-2 genes. We propose an evolutionarily informed approach to attenuation that, unusually, seeks to increase usage of the already most common synonymous codons. Comparable analysis of H1N1 and Ebola finds that GC3 deviated from neutral equilibrium is not a universal feature, cautioning against generalization of results.

Predicting restriction of life-space mobility: a machine learning analysis of the IMIAS study.

BACKGROUND: Some studies have employed machine learning (ML) methods for mobility prediction modeling in older adults. ML methods could be a helpful tool for life-space mobility (LSM) data analysis. AIM: This study aimed to evaluate the predictive value of ML algorithms for the restriction of life-space mobility (LSM) among elderly people and to identify the most important risk factors for that prediction model. METHODS: A 2-year LSM reduction prediction model was developed using the ML-based algorithms decision tree, random forest, and eXtreme gradient boosting (XGBoost), and tested on an independent validation cohort. The data were collected from the International Mobility in Aging Study (IMIAS) from 2012 to 2014, comprising 372 older patients (≥ 65 years of age). LSM was measured by the Life-Space Assessment questionnaire (LSA) with five levels of living space during the month before assessment. RESULTS: According to the XGBoost algorithm, the best model reached a mean absolute error (MAE) of 10.28 and root-mean-square error (RMSE) of 12.91 in the testing portion. The variables frailty (39.4%), mobility disability (25.4%), depression (21.9%), and female sex (13.3%) had the highest importance. CONCLUSION: The model identified risk factors through ML algorithms that could be used to predict LSM restriction; these risk factors could be used by practitioners to identify older adults with an increased risk of LSM reduction in the future. The XGBoost model offers benefits as a complementary method of traditional statistical approaches to understand the complexity of mobility.

Whole-Genome Analysis Surveillance of Influenza A Virus Resistance to Polymerase Complex Inhibitors in Eastern Spain from 2016 to 2019.

Molecular surveillance by whole-genome sequencing was used to monitor the susceptibility of circulating influenza A viruses to three polymerase complex inhibitors. A total of 12 resistance substitutions were found among 285 genomes analyzed, but none were associated with high levels of resistance. Natural resistance to these influenza A antivirals is currently uncommon.

Detection of SARS-CoV-2 in wastewater is influenced by sampling time, concentration method, and target analyzed.

The detection of SARS-CoV in wastewater has been proposed as a tool for monitoring COVID-19 at the community level. Although many reports have been published about detecting viral RNA in wastewater and its presence has been linked to infected people, appropriate analytical methodologies to use this approach have not yet been established. In this study, we compared ultrafiltration, polyethylene glycol precipitation, flocculation using AlCl3, and flocculation with skim milk for the recovery of SARS-CoV-2, using RNA from patients with positive diagnoses for COVID-19 and Pseudomonas phage φ6 as the control. We also evaluated the primers for detecting the E, RdRp, and N genes of the virus, as well as different storage times. Differences in the recovery efficiencies were evident with the different concentration methods, the best being ultrafiltration and precipitation with aluminum, which had recovery rates of 42.0% and 30.0%, respectively, when virus was present at high levels. Significant differences were found between the recoveries using wastewater and deionized water and between different storage times, with better recoveries for 6 and 12 h samplings. The E gene was the only one detected in all the samples analyzed. The results show that although this approach can provide important data for studying the pandemic, clear protocols are necessary for investigations to be comparable.

Validation of electron microscopy initial models via small angle X-ray scattering curves.

MOTIVATION: Cryo electron microscopy (EM) is currently one of the main tools to reveal the structural information of biological macromolecules. The re-construction of three-dimensional (3D) maps is typically carried out following an iterative process that requires an initial estimation of the 3D map to be refined in subsequent steps. Therefore, its determination is key in the quality of the final results, and there are cases in which it is still an open issue in single particle analysis (SPA). Small angle X-ray scattering (SAXS) is a well-known technique applied to structural biology. It is useful from small nanostructures up to macromolecular ensembles for its ability to obtain low resolution information of the biological sample measuring its X-ray scattering curve. These curves, together with further analysis, are able to yield information on the sizes, shapes and structures of the analyzed particles. RESULTS: In this paper, we show how the low resolution structural information revealed by SAXS is very useful for the validation of EM initial 3D models in SPA, helping the following refinement process to obtain more accurate 3D structures. For this purpose, we approximate the initial map by pseudo-atoms and predict the SAXS curve expected for this pseudo-atomic structure. The match between the predicted and experimental SAXS curves is considered as a good sign of the correctness of the EM initial map. AVAILABILITY AND IMPLEMENTATION: The algorithm is freely available as part of the Scipion 1.2 software at http://scipion.i2pc.es/.

Structural and energy determinants in protein-RNA docking.

Deciphering the structural and energetic determinants of protein-RNA interactions harbors the potential to understand key cell processes at molecular level, such as gene expression and regulation. With this purpose, computational methods like docking aim to complement current biophysical and structural biology efforts. However, the few reported docking algorithms for protein-RNA interactions show limited predictive success rates, mainly due to incomplete sampling of the conformational space of both the protein and the RNA molecules, as well as to the difficulties of the scoring function in identifying the correct docking models. Here, we have tested the predictive value of a variety of knowledge-based and energetic scoring functions on a recently published protein-RNA docking benchmark and developed a scoring function able to efficiently discriminate docking decoys. We first performed docking calculations with the bound conformation, which allowed us to analyze the problem in optimal conditions. We found that geometry-based terms and electrostatics were the most important scoring terms, while binding propensities and desolvation were much less relevant for the scoring of protein-RNA models. This is in contrast with what we observed for protein-protein docking. The results also showed an interesting dependence of the predictive rates on the flexibility of the protein molecule, which arises from the observed higher positive charge of flexible interfaces and provides hints for future development of more efficient protein-RNA docking methods.

Influenza vaccine effectiveness in preventing hospitalisation of individuals 60 years of age and over with laboratory-confirmed influenza, Valencia Region, Spain, influenza season 2016/17.

IntroductionSeasonal influenza vaccination is widely recommended for people with risk factors, especially for people who are elderly. However, influenza vaccine effectiveness (IVE) varies year after year because of the variable antigenic composition of the circulating viruses and the vaccine composition. Methods: We summarise the results of IVE and the impact of previous vaccination among subjects 60 years of age and over in a multicentre prospective study in the Valencia Hospital Surveillance Network for the Study of Influenza and Respiratory Viruses Disease (VAHNSI) in Spain. We applied the test-negative design taking laboratory-confirmed influenza as outcome and vaccination status as exposure. Information about potential confounders was obtained from clinical registries or directly from patients. Results: Adjusted IVE was 19% (95% confidence interval (CI): -15 to 43). For patients vaccinated in the current season but not in the two previous seasons, effectiveness was 49% (95% CI: -20 to 78) and for patients vaccinated in the current and any of two previous seasons, effectiveness was 29% (95% CI: -3 to 52). For those patients not vaccinated in the current season but vaccinated in any of the two previous seasons, effectiveness was 53% (95% CI: 8 to 76). Conclusions: Our data show a low vaccine effectiveness for the 2016/17 influenza season.

Isolation and Antifouling Activity of Azulene Derivatives from the Antarctic Gorgonian Acanthogorgia laxa.

Three azulenoid sesquiterpenes (1 - 3) were isolated from the Antarctic gorgonian Acanthogorgia laxa collected by bottom trawls at -343 m. Besides linderazulene (1), and the known ketolactone 2, a new brominated C16 linderazulene derivative (3) was also identified. This compound has an extra carbon atom at C(7) of the linderazulene framework. The antifouling activity of compounds 1 and 2 was assayed in the laboratory with Artemia salina larvae, and also in field tests, by incorporation in soluble-matrix experimental antifouling paints. The results obtained after a 45 days field trial of the paints, showed that compounds 1 and 2 displayed good antifouling potencies against a wide array of organisms. Compound 3, a benzylic bromide, was unstable and for this reason was not submitted to bioassays. Two known cembranolides: pukalide and epoxypukalide, were also identified as minor components of the extract.

ScipionCloud: An integrative and interactive gateway for large scale cryo electron microscopy image processing on commercial and academic clouds.

New instrumentation for cryo electron microscopy (cryoEM) has significantly increased data collection rate as well as data quality, creating bottlenecks at the image processing level. Current image processing model of moving the acquired images from the data source (electron microscope) to desktops or local clusters for processing is encountering many practical limitations. However, computing may also take place in distributed and decentralized environments. In this way, cloud is a new form of accessing computing and storage resources on demand. Here, we evaluate on how this new computational paradigm can be effectively used by extending our current integrative framework for image processing, creating ScipionCloud. This new development has resulted in a full installation of Scipion both in public and private clouds, accessible as public "images", with all the required preinstalled cryoEM software, just requiring a Web browser to access all Graphical User Interfaces. We have profiled the performance of different configurations on Amazon Web Services and the European Federated Cloud, always on architectures incorporating GPU's, and compared them with a local facility. We have also analyzed the economical convenience of different scenarios, so cryoEM scientists have a clearer picture of the setup that is best suited for their needs and budgets.

Prox1 Is Required for Oligodendrocyte Cell Identity in Adult Neural Stem Cells of the Subventricular Zone.

Adult neural stem cells with the ability to generate neurons and glia cells are active throughout life in both the dentate gyrus (DG) and the subventricular zone (SVZ). Differentiation of adult neural stem cells is induced by cell fate determinants like the transcription factor Prox1. Evidence has been provided for a function of Prox1 as an inducer of neuronal differentiation within the DG. We now show that within the SVZ Prox1 induces differentiation into oligodendrocytes. Moreover, we find that loss of Prox1 expression in vivo reduces cell migration into the corpus callosum, where the few Prox1 deficient SVZ-derived remaining cells fail to differentiate into oligodendrocytes. Thus, our work uncovers a novel function of Prox1 as a fate determinant for oligodendrocytes in the adult mammalian brain. These data indicate that the neurogenic and oligodendrogliogenic lineages in the two adult neurogenic niches exhibit a distinct requirement for Prox1, being important for neurogenesis in the DG but being indispensable for oligodendrogliogenesis in the SVZ. Stem Cells 2016;34:2115-2129.

The RNA helicase DDX6 regulates cell-fate specification in neural stem cells via miRNAs.

In neural stem cells (NSCs), the balance between stem cell maintenance and neuronal differentiation depends on cell-fate determinants such as TRIM32. Previously, we have shown that TRIM32 associates with the RNA-induced silencing complex and increases the activity of microRNAs such as Let-7a. However, the exact mechanism of microRNA regulation by TRIM32 during neuronal differentiation has yet to be elucidated. Here, we used a mass spectrometry approach to identify novel protein-protein interaction partners of TRIM32 during neuronal differentiation. We found that TRIM32 associates with proteins involved in neurogenesis and RNA-related processes, such as the RNA helicase DDX6, which has been implicated in microRNA regulation. We demonstrate, that DDX6 colocalizes with TRIM32 in NSCs and neurons and that it increases the activity of Let-7a. Furthermore, we provide evidence that DDX6 is necessary and sufficient for neuronal differentiation and that it functions in cooperation with TRIM32.

Structural bases for the interaction and stabilization of the human amino acid transporter LAT2 with its ancillary protein 4F2hc.

Heteromeric amino acid transporters (HATs) are the unique example, known in all kingdoms of life, of solute transporters composed of two subunits linked by a conserved disulfide bridge. In metazoans, the heavy subunit is responsible for the trafficking of the heterodimer to the plasma membrane, and the light subunit is the transporter. HATs are involved in human pathologies such as amino acidurias, tumor growth and invasion, viral infection and cocaine addiction. However structural information about interactions between the heavy and light subunits of HATs is scarce. In this work, transmission electron microscopy and single-particle analysis of purified human 4F2hc/L-type amino acid transporter 2 (LAT2) heterodimers overexpressed in the yeast Pichia pastoris, together with docking analysis and crosslinking experiments, reveal that the extracellular domain of 4F2hc interacts with LAT2, almost completely covering the extracellular face of the transporter. 4F2hc increases the stability of the light subunit LAT2 in detergent-solubilized Pichia membranes, allowing functional reconstitution of the heterodimer into proteoliposomes. Moreover, the extracellular domain of 4F2hc suffices to stabilize solubilized LAT2. The interaction of 4F2hc with LAT2 gives insights into the structural bases for light subunit recognition and the stabilizing role of the ancillary protein in HATs.

Assembly and channel opening in a bacterial drug efflux machine.

Drugs and certain proteins are transported across the membranes of Gram-negative bacteria by energy-activated pumps. The outer membrane component of these pumps is a channel that opens from a sealed resting state during the transport process. We describe two crystal structures of the Escherichia coli outer membrane protein TolC in its partially open state. Opening is accompanied by the exposure of three shallow intraprotomer grooves in the TolC trimer, where our mutagenesis data identify a contact point with the periplasmic component of a drug efflux pump, AcrA. We suggest that the assembly of multidrug efflux pumps is accompanied by induced fit of TolC driven mainly by accommodation of the periplasmic component.

Neural stem cells in Parkinson's disease: a role for neurogenesis defects in onset and progression.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, leading to a variety of motor and non-motor symptoms. Interestingly, non-motor symptoms often appear a decade or more before the first signs of motor symptoms. Some of these non-motor symptoms are remarkably similar to those observed in cases of impaired neurogenesis and several PD-related genes have been shown to play a role in embryonic or adult neurogenesis. Indeed, animal models deficient in Nurr1, Pitx3, SNCA and PINK1 display deregulated embryonic neurogenesis and LRRK2 and VPS35 have been implicated in neuronal development-related processes such as Wnt/ß-catenin signaling and neurite outgrowth. Moreover, adult neurogenesis is affected in both PD patients and PD animal models and is regulated by dopamine and dopaminergic (DA) receptors, by chronic neuroinflammation, such as that observed in PD, and by differential expression of wild-type or mutant forms of PD-related genes. Indeed, an increasing number of in vivo studies demonstrate a role for SNCA and LRRK2 in adult neurogenesis and in the generation and maintenance of DA neurons. Finally, the roles of PD-related genes, SNCA, LRRK2, VPS35, Parkin, PINK1 and DJ-1 have been studied in NSCs, progenitor cells and induced pluripotent stem cells, demonstrating a role for some of these genes in stem/progenitor cell proliferation and maintenance. Together, these studies strongly suggest a link between deregulated neurogenesis and the onset and progression of PD and present strong evidence that, in addition to a neurodegenerative disorder, PD can also be regarded as a developmental disorder.