APOBEC2 safeguards skeletal muscle cell fate through binding chromatin and regulating transcription of non-muscle genes during myoblast differentiation.

The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide (APOBEC) family is composed of nucleic acid editors with roles ranging from antibody diversification to RNA editing. APOBEC2, a member of this family with an evolutionarily conserved nucleic acid-binding cytidine deaminase domain, has neither an established substrate nor function. Using a cellular model of muscle differentiation where APOBEC2 is inducibly expressed, we confirmed that APOBEC2 does not have the attributed molecular functions of the APOBEC family, such as RNA editing, DNA demethylation, and DNA mutation. Instead, we found that during muscle differentiation APOBEC2 occupied a specific motif within promoter regions; its removal from those regions resulted in transcriptional changes. Mechanistically, these changes reflect the direct interaction of APOBEC2 with histone deacetylase (HDAC) transcriptional corepressor complexes. We also found that APOBEC2 could bind DNA directly, in a sequence-specific fashion, suggesting that it functions as a recruiter of HDAC to specific genes whose promoters it occupies. These genes are normally suppressed during muscle cell differentiation, and their suppression may contribute to the safeguarding of muscle cell fate. Altogether, our results reveal a unique role for APOBEC2 within the APOBEC family.

Integrated multi-omics single cell atlas of the human retina.

Single-cell sequencing has revolutionized the scale and resolution of molecular profiling of tissues and organs. Here, we present an integrated multimodal reference atlas of the most accessible portion of the mammalian central nervous system, the retina. We compiled around 2.4 million cells from 55 donors, including 1.4 million unpublished data points, to create a comprehensive human retina cell atlas (HRCA) of transcriptome and chromatin accessibility, unveiling over 110 types. Engaging the retina community, we annotated each cluster, refined the Cell Ontology for the retina, identified distinct marker genes, and characterized cis-regulatory elements and gene regulatory networks (GRNs) for these cell types. Our analysis uncovered intriguing differences in transcriptome, chromatin, and GRNs across cell types. In addition, we modeled changes in gene expression and chromatin openness across gender and age. This integrated atlas also enabled the fine-mapping of GWAS and eQTL variants. Accessible through interactive browsers, this multimodal cross-donor and cross-lab HRCA, can facilitate a better understanding of retinal function and pathology.

An integrated cell atlas of the lung in health and disease.

Single-cell technologies have transformed our understanding of human tissues. Yet, studies typically capture only a limited number of donors and disagree on cell type definitions. Integrating many single-cell datasets can address these limitations of individual studies and capture the variability present in the population. Here we present the integrated Human Lung Cell Atlas (HLCA), combining 49 datasets of the human respiratory system into a single atlas spanning over 2.4 million cells from 486 individuals. The HLCA presents a consensus cell type re-annotation with matching marker genes, including annotations of rare and previously undescribed cell types. Leveraging the number and diversity of individuals in the HLCA, we identify gene modules that are associated with demographic covariates such as age, sex and body mass index, as well as gene modules changing expression along the proximal-to-distal axis of the bronchial tree. Mapping new data to the HLCA enables rapid data annotation and interpretation. Using the HLCA as a reference for the study of disease, we identify shared cell states across multiple lung diseases, including SPP1+ profibrotic monocyte-derived macrophages in COVID-19, pulmonary fibrosis and lung carcinoma. Overall, the HLCA serves as an example for the development and use of large-scale, cross-dataset organ atlases within the Human Cell Atlas.

Predicting cellular responses to complex perturbations in high-throughput screens.

Recent advances in multiplexed single-cell transcriptomics experiments facilitate the high-throughput study of drug and genetic perturbations. However, an exhaustive exploration of the combinatorial perturbation space is experimentally unfeasible. Therefore, computational methods are needed to predict, interpret, and prioritize perturbations. Here, we present the compositional perturbation autoencoder (CPA), which combines the interpretability of linear models with the flexibility of deep-learning approaches for single-cell response modeling. CPA learns to in silico predict transcriptional perturbation response at the single-cell level for unseen dosages, cell types, time points, and species. Using newly generated single-cell drug combination data, we validate that CPA can predict unseen drug combinations while outperforming baseline models. Additionally, the architecture's modularity enables incorporating the chemical representation of the drugs, allowing the prediction of cellular response to completely unseen drugs. Furthermore, CPA is also applicable to genetic combinatorial screens. We demonstrate this by imputing in silico 5,329 missing combinations (97.6% of all possibilities) in a single-cell Perturb-seq experiment with diverse genetic interactions. We envision CPA will facilitate efficient experimental design and hypothesis generation by enabling in silico response prediction at the single-cell level and thus accelerate therapeutic applications using single-cell technologies.

MYT1L haploinsufficiency in human neurons and mice causes autism-associated phenotypes that can be reversed by genetic and pharmacologic intervention.

MYT1L is an autism spectrum disorder (ASD)-associated transcription factor that is expressed in virtually all neurons throughout life. How MYT1L mutations cause neurological phenotypes and whether they can be targeted remains enigmatic. Here, we examine the effects of MYT1L deficiency in human neurons and mice. Mutant mice exhibit neurodevelopmental delays with thinner cortices, behavioural phenotypes, and gene expression changes that resemble those of ASD patients. MYT1L target genes, including WNT and NOTCH, are activated upon MYT1L depletion and their chemical inhibition can rescue delayed neurogenesis in vitro. MYT1L deficiency also causes upregulation of the main cardiac sodium channel, SCN5A, and neuronal hyperactivity, which could be restored by shRNA-mediated knockdown of SCN5A or MYT1L overexpression in postmitotic neurons. Acute application of the sodium channel blocker, lamotrigine, also rescued electrophysiological defects in vitro and behaviour phenotypes in vivo. Hence, MYT1L mutation causes both developmental and postmitotic neurological defects. However, acute intervention can normalise resulting electrophysiological and behavioural phenotypes in adulthood.

Comparative chromatin accessibility upon BDNF stimulation delineates neuronal regulatory elements.

Neuronal stimulation induced by the brain-derived neurotrophic factor (BDNF) triggers gene expression, which is crucial for neuronal survival, differentiation, synaptic plasticity, memory formation, and neurocognitive health. However, its role in chromatin regulation is unclear. Here, using temporal profiling of chromatin accessibility and transcription in mouse primary cortical neurons upon either BDNF stimulation or depolarization (KCl), we identify features that define BDNF-specific chromatin-to-gene expression programs. Enhancer activation is an early event in the regulatory control of BDNF-treated neurons, where the bZIP motif-binding Fos protein pioneered chromatin opening and cooperated with co-regulatory transcription factors (Homeobox, EGRs, and CTCF) to induce transcription. Deleting cis-regulatory sequences affect BDNF-mediated Arc expression, a regulator of synaptic plasticity. BDNF-induced accessible regions are linked to preferential exon usage by neurodevelopmental disorder-related genes and the heritability of neuronal complex traits, which were validated in human iPSC-derived neurons. Thus, we provide a comprehensive view of BDNF-mediated genome regulatory features using comparative genomic approaches to dissect mammalian neuronal stimulation.

Parkinson's disease motor symptoms rescue by CRISPRa-reprogramming astrocytes into GABAergic neurons.

Direct reprogramming based on genetic factors resembles a promising strategy to replace lost cells in degenerative diseases such as Parkinson's disease. For this, we developed a knock-in mouse line carrying a dual dCas9 transactivator system (dCAM) allowing the conditional in vivo activation of endogenous genes. To enable a translational application, we additionally established an AAV-based strategy carrying intein-split-dCas9 in combination with activators (AAV-dCAS). Both approaches were successful in reprogramming striatal astrocytes into induced GABAergic neurons confirmed by single-cell transcriptome analysis of reprogrammed neurons in vivo. These GABAergic neurons functionally integrate into striatal circuits, alleviating voluntary motor behavior aspects in a 6-OHDA Parkinson's disease model. Our results suggest a novel intervention strategy beyond the restoration of dopamine levels. Thus, the AAV-dCAS approach might enable an alternative route for clinical therapies of Parkinson's disease.

Squidpy: a scalable framework for spatial omics analysis.

Spatial omics data are advancing the study of tissue organization and cellular communication at an unprecedented scale. Flexible tools are required to store, integrate and visualize the large diversity of spatial omics data. Here, we present Squidpy, a Python framework that brings together tools from omics and image analysis to enable scalable description of spatial molecular data, such as transcriptome or multivariate proteins. Squidpy provides efficient infrastructure and numerous analysis methods that allow to efficiently store, manipulate and interactively visualize spatial omics data. Squidpy is extensible and can be interfaced with a variety of already existing libraries for the scalable analysis of spatial omics data.

SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis.

COVID-19-induced "acute respiratory distress syndrome" (ARDS) is associated with prolonged respiratory failure and high mortality, but the mechanistic basis of lung injury remains incompletely understood. Here, we analyze pulmonary immune responses and lung pathology in two cohorts of patients with COVID-19 ARDS using functional single-cell genomics, immunohistology, and electron microscopy. We describe an accumulation of CD163-expressing monocyte-derived macrophages that acquired a profibrotic transcriptional phenotype during COVID-19 ARDS. Gene set enrichment and computational data integration revealed a significant similarity between COVID-19-associated macrophages and profibrotic macrophage populations identified in idiopathic pulmonary fibrosis. COVID-19 ARDS was associated with clinical, radiographic, histopathological, and ultrastructural hallmarks of pulmonary fibrosis. Exposure of human monocytes to SARS-CoV-2, but not influenza A virus or viral RNA analogs, was sufficient to induce a similar profibrotic phenotype in vitro. In conclusion, we demonstrate that SARS-CoV-2 triggers profibrotic macrophage responses and pronounced fibroproliferative ARDS.

Mechanistic insights into transcription factor cooperativity and its impact on protein-phenotype interactions.

Recent high-throughput transcription factor (TF) binding assays revealed that TF cooperativity is a widespread phenomenon. However, a global mechanistic and functional understanding of TF cooperativity is still lacking. To address this, here we introduce a statistical learning framework that provides structural insight into TF cooperativity and its functional consequences based on next generation sequencing data. We identify DNA shape as driver for cooperativity, with a particularly strong effect for Forkhead-Ets pairs. Follow-up experiments reveal a local shape preference at the Ets-DNA-Forkhead interface and decreased cooperativity upon loss of the interaction. Additionally, we discover many functional associations for cooperatively bound TFs. Examination of the link between FOXO1:ETV6 and lymphomas reveals that their joint expression levels improve patient clinical outcome stratification. Altogether, our results demonstrate that inter-family cooperative TF binding is driven by position-specific DNA readout mechanisms, which provides an additional regulatory layer for downstream biological functions.

Efficient and automated large-scale detection of structural relationships in proteins with a flexible aligner.

BACKGROUND: The total number of known three-dimensional protein structures is rapidly increasing. Consequently, the need for fast structural search against complete databases without a significant loss of accuracy is increasingly demanding. Recently, TopSearch, an ultra-fast method for finding rigid structural relationships between a query structure and the complete Protein Data Bank (PDB), at the multi-chain level, has been released. However, comparable accurate flexible structural aligners to perform efficient whole database searches of multi-domain proteins are not yet available. The availability of such a tool is critical for a sustainable boosting of biological discovery. RESULTS: Here we report on the development of a new method for the fast and flexible comparison of protein structure chains. The method relies on the calculation of 2D matrices containing a description of the three-dimensional arrangement of secondary structure elements (angles and distances). The comparison involves the matching of an ensemble of substructures through a nested-two-steps dynamic programming algorithm. The unique features of this new approach are the integration and trade-off balancing of the following: 1) speed, 2) accuracy and 3) global and semiglobal flexible structure alignment by integration of local substructure matching. The comparison, and matching with competitive accuracy, of one medium sized (250-aa) query structure against the complete PDB database (216,322 protein chains) takes about 8 min using an average desktop computer. The method is at least 2-3 orders of magnitude faster than other tested tools with similar accuracy. We validate the performance of the method for fold and superfamily assignment in a large benchmark set of protein structures. We finally provide a series of examples to illustrate the usefulness of this method and its application in biological discovery. CONCLUSIONS: The method is able to detect partial structure matching, rigid body shifts, conformational changes and tolerates substantial structural variation arising from insertions, deletions and sequence divergence, as well as structural convergence of unrelated proteins.

[Capacity of the legal framework of public health institutions in Mexico to support their functional integration]. / Capacidad del marco jurídico de las instituciones públicas de salud de México para apoyar la integración funcional.

OBJECTIVE: Evaluate the capacity of the federal legal framework to govern financing of health institutions in the public sector through innovative schemes--otherwise known as functional integration--, enabling them to purchase and sell health services to and from other public providers as a strategy to improve their performance. MATERIALS AND METHODS: Based on indicators of normative alignment with respect to functional integration across public health provider and governance institutions, content analysis was undertaken of national health programs and relevant laws and guidelines for financial coordination. RESULTS: Significant progress was identified in the implementation of agreements for the coordination of public institutions. While the legal framework provides for a National Health System and a health sector, gaps and contradictions limit their scope. The General Register of Health is also moving forward, yet it lacks the necessary legal foundation to become a comprehensive tool for integration. The medical service exchange agreements are also moving forward based on tariffs and shared guidelines. However, there is a lack of incentives to promote the expansion of these agreements. CONCLUSIONS: It is recommended to update the legal framework for the coordination of the National Health System, ensuring a more harmonious and general focus to provide functional integration with the needed impulse.

Capacidad del marco jurídico de las instituciones públicas de salud de México para apoyar la integración funcional / Capacity of the legal framework of public health institutions in Mexico to support their functional integration

OBJETIVO: Evaluar la capacidad del marco legal federal para gobernar el financiamiento de las instituciones públicas de salud mediante esquemas innovadores que les permiten comprar y vender servicios de salud de y a otros proveedores públicos, como una estrategia para mejorar su desempeño. MATERIAL Y MÉTODOS: Con base en indicadores de alineamiento normativo respecto de la integración funcional entre instituciones proveedoras públicas y de gobernanza, se analizó el contenido de los programas nacionales de salud y de leyes y lineamientos oficiales pertinentes para la coordinación financiera. RESULTADOS: Se identificó un avance considerable en la implementación de convenios para la coordinación de las instituciones públicas. No obstante, el marco legal en apoyo al Sistema Nacional de Salud y al sector salud muestra deficiencias y contradicciones que limitan su alcance, mientras que el Padrón General de Salud carece del sustento legal necesario para convertirse en una herramienta de amplio beneficio para la integración. Si bien los convenios de intercambio de servicios están avanzando con base en tarifas y lineamientos compartidos, se carece de incentivos que promuevan su expansión. CONCLUSIONES: Es recomendable actualizar el marco legal para la coordinación del Sistema Nacional de Salud, procurando un enfoque más armónico y general que impulse la integración funcional.

OBJECTIVE: Evaluate the capacity of the federal legal framework to govern financing of health institutions in the public sector through innovative schemes -otherwise known as functional integration-, enabling them to purchase and sell health services to and from other public providers as a strategy to improve their performance. MATERIALS AND METHODS: Based on indicators of normative alignment with respect to functional integration across public health provider and governance institutions, content analysis was undertaken of national health programs and relevant laws and guidelines for financial coordination. RESULTS: Significant progress was identified in the implementation of agreements for the coordination of public institutions. While the legal framework provides for a National Health System and a health sector, gaps and contradictions limit their scope. The General Register of Health is also moving forward, yet it lacks the necessary legal foundation to become a comprehensive tool for integration. The medical service exchange agreements are also moving forward based on tariffs and shared guidelines. However, there is a lack of incentives to promote the expansion of these agreements. CONCLUSIONS: It is recommended to update the legal framework for the coordination of the National Health System, ensuring a more harmonious and general focus to provide functional integration with the needed impulse.

Miringoplastias en niños: nuestros resultados / Myringoplasties in children: Our results

Introducción y objetivos: La miringoplastia es una de las intervenciones más frecuentes en otología. Consiste en la reparación de la membrana timpánica cuando la única lesión existente es una perforación y la cadena de huesecillos está íntegra. El objetivo principal de nuestro trabajo es estudiar el resultado de la miringoplastia en pacientes menores de 15 años. También analizar la existencia de factores pronósticos asociados, como la funcionalidad de la trompa de Eustaquio, la técnica quirúrgica empleada y el resultado auditivo tras la cirugía. Métodos: Realizamos un estudio retrospectivo desde el año 1994 hasta el 2010 de un total de 81 niños menores de 15 años intervenidos de miringoplastia. Analizamos los siguientes datos: edad, sexo, técnica empleada, abordaje y tipo de injerto, tipo de perforación, tipo de anestesia, ganancia auditiva y cierre de la perforación. Correlacionamos dichas variables con el éxito de la cirugía. Resultados: El porcentaje de cierre de la perforación fue de 84% (n=68). Las técnicas utilizadas fueron medial en el 79,01% (n=64), lateral 11,11% (n=9) y «sándwich» 9,87% (n=8). El porcentaje de casos que presentan ganancia auditiva tras la intervención fue de 88,40% (n= 61). Conclusión: La miringoplastia es una técnica quirúrgica que ofrece buenos resultados anatómicos y funcionales en niños. No encontramos en nuestro estudio ningún factor pronóstico asociado. Encontramos mejoría auditiva postoperatoria aunque no significativa (AU)

Introduction and objectives: Myringoplasty, one of the most frequent surgical techniques in otology, is the repair of tympanic membrane when the ear has only a perforation without any ossicular damage. The main objective of our work was to study the outcome of myringoplasty in patients less than 15 years of age. We also reviewed the existence of prognostic factors, such as Eustachian tube functionality, surgical technique and the hearing outcome after surgery. Method: We present a retrospective study (1994-2010) with a total of 81 children (under 15 years of age) who had undergone myringoplasty during that period of time. For these children, we analysed age, sex, technique, approach, type of graft, type of perforation, anaesthesia, hearing gain and perforation closure. We correlated these variables with the success of the surgery. Results: The percentage of closure was 84% (n=68). The techniques used were underlay in 79.01% (n=64), overlay in 11.11% (n=9) and sandwich in 9.87% (n=8). The percentage of patients with hearing improvement was 88.40% (n=61). Conclusion: Myringoplasty is a surgical technique that offers good anatomical and functional results in children. We did not find prognostic factors in our study. We found postoperative improvement of hearing but it was not statistically significant (AU)

Myringoplasties in children: our results.

INTRODUCTION AND OBJECTIVES: Myringoplasty, one of the most frequent surgical techniques in otology, is the repair of tympanic membrane when the ear has only a perforation without any ossicular damage. The main objective of our work was to study the outcome of myringoplasty in patients less than 15 years of age. We also reviewed the existence of prognostic factors, such as Eustachian tube functionality, surgical technique and the hearing outcome after surgery. METHOD: We present a retrospective study (1994-2010) with a total of 81 children (under 15 years of age) who had undergone myringoplasty during that period of time. For these children, we analysed age, sex, technique, approach, type of graft, type of perforation, anaesthesia, hearing gain and perforation closure. We correlated these variables with the success of the surgery. RESULTS: The percentage of closure was 84% (n=68). The techniques used were underlay in 79.01% (n=64), overlay in 11.11% (n=9) and sandwich in 9.87% (n=8). The percentage of patients with hearing improvement was 88.40% (n=61). CONCLUSION: Myringoplasty is a surgical technique that offers good anatomical and functional results in children. We did not find prognostic factors in our study. We found postoperative improvement of hearing but it was not statistically significant.

Ventricular assist device as a bridge to transplant, and extracorporeal membrane oxygenation for primary graft failure in a child with hemophilia A.

OBJECTIVE: To report the management of hemophilia in a patient with dilated cardiomyopathy during application of the Berlin-Heart biventricular assist. DESIGN: Case report. INTERVENTIONS AND RESULTS: The Berlin-Heart biventricular assist was successfully used as a bridge to heart transplantation (178 days) in a child with hemophilia A; post-transplant extracorporeal membrane oxygenation was implanted until the patient recovered from primary graft failure. Anticoagulant therapy varied as a function of patient status: 1) postoperative bleeding was treated by partial replacement of deficient factors (VII and VIII) and hemoderivatives in order to maintain factor VIII at 50% of normal levels; 2) once the bleeding had stopped, the effect of the hemophilia itself maintained the degree of anticoagulation required by the ventricular assist device; and 3) transplant surgery was followed by complete replacement of factor VIII and intravenous heparinization (a simple way of preventing clot formation in the device and to ensure proper scarring of surgical wounds). CONCLUSIONS: Selection of anticoagulant therapy as a function of patient status in terms of bleeding and surgical-wound scarring progress is vital for the proper functioning of support techniques (Berlin-Heart biventricular assist and extracorporeal membrane oxygenation) in hemophiliac patients. Collagen dressings placed on surgical wounds achieved good functional and aesthetic results, as well as mechanically isolating the scars from the exterior.

Interactive software tool to comprehend the calculation of optimal sequence alignments with dynamic programming.

SUMMARY: Dynamic programming (DP) is a general optimization strategy that is successfully used across various disciplines of science. In bioinformatics, it is widely applied in calculating the optimal alignment between pairs of protein or DNA sequences. These alignments form the basis of new, verifiable biological hypothesis. Despite its importance, there are no interactive tools available for training and education on understanding the DP algorithm. Here, we introduce an interactive computer application with a graphical interface, for the purpose of educating students about DP. The program displays the DP scoring matrix and the resulting optimal alignment(s), while allowing the user to modify key parameters such as the values in the similarity matrix, the sequence alignment algorithm version and the gap opening/extension penalties. We hope that this software will be useful to teachers and students of bioinformatics courses, as well as researchers who implement the DP algorithm for diverse applications. AVAILABILITY AND IMPLEMENTATION: The software is freely available at: http:/melolab.org/sat. The software is written in the Java computer language, thus it runs on all major platforms and operating systems including Windows, Mac OS X and LINUX. CONTACT: All inquiries or comments about this software should be directed to Francisco Melo at fmelo@bio.puc.cl.

Carcinoma de vesícula biliar y litiasis vesicular en material de autopsia: estudio de 25 años / Gallbladder carcinoma and lithiasis in autopsy material: a 25 years study

Se estudiaron 13,545 autopsias, de las cuales 7,045 correspondieron a masculinos y 6,500 a femeninos. De estos 13,545, el carcinoma de vesícula biliar ocurrió en 82 casos (0.6 por ciento); 16 masculinos y 66 femeninos (M/F 1/4.1); la edad de presentación fue de los 30 y 100 años, con una media de 65 años. La frecuencia de carcinoma de vesícula en los casos con litiasis vesicular resultó 3.1 veces mayo en femeninos (7 por ciento) que en masculinos (2.2 por ciento). En los casos sin litiasis, la frecuencia de cáncer de vesícula es igualmente 3 veces más alta en femeninos que en masculinos (0.09 y 0.03 por ciento respectivamente), independientemente de la presencia de colelitiasis (p<0.05). Hubo 1,261 casos con litiasis vesicular (9.1 por ciento), 483 fueron masculinos y 778 femeninos, con edades entre 20 y 100 años (x 60 años). De 1261 casos con litiasis vesicular, el carcinoma de vesícula se presentó en 66 (5.2 por ciento) y de 12,284 casos sin litiasis, su frecuencia fue de 0.13 por ciento. Es así que la frecuencia de carcinoma de vesícula fue 40 veces más alta en los casos con litiasis que sin litiasis (p<0.01).