Heterogeneity of Mycobacterium tuberculosis Strains Circulating in Panama's Western Region.

Tuberculosis remains a challenge in both rural and urban areas. Although a majority of countries display a higher burden in urban areas compared with rural areas, Panama continues to report the highest mortality rate in Central America. Urban areas, such as Panama City, report a high tuberculosis burden, whereas Panama's western region, including the provinces of Chiriquí, Bocas del Toro (both semiurban) and Ngäbe-Bugle (rural), show a lower burden. We aimed to identify highly transmitted Mycobacterium tuberculosis strains within rural and semiurban settings of Panama's western region during a 3-year period (2017, 2019, 2021). We randomly selected 87 M. tuberculosis isolates from a biobank from Panama's western region and analyzed them using allele-specific oligonucleotide polymerase chain reaction and 24-mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). Our results show only 11.7% (10/85) of M. tuberculosis strains identified as prevalent A-Beijing, B-Haarlem, or C-LAM Strains. We found a low prevalence of A, B, and C M. tuberculosis strains in both rural and semirural settings compared with isolates collected from the Eastern Colon Province. MIRU-VNTR genotyping revealed a high degree of diversity with no clusters with single loci variation of ≥ 2 loci. These results support the notion that tuberculosis prevalence in the rural and semiurban western region of Panama are not due to previously described highly transmitted strains but is influenced instead by other health determinants, including poor health system access and a lack of systematic transmission chain monitoring. For remote rural and semiurban settings, we recommend allocating resources to reinforce efforts to prevent tuberculosis spread.

BRD4 orchestrates genome folding to promote neural crest differentiation.

Higher-order chromatin structure regulates gene expression, and mutations in proteins mediating genome folding underlie developmental disorders known as cohesinopathies. However, the relationship between three-dimensional genome organization and embryonic development remains unclear. Here we define a role for bromodomain-containing protein 4 (BRD4) in genome folding, and leverage it to understand the importance of genome folding in neural crest progenitor differentiation. Brd4 deletion in neural crest results in cohesinopathy-like phenotypes. BRD4 interacts with NIPBL, a cohesin agonist, and BRD4 depletion or loss of the BRD4-NIPBL interaction reduces NIPBL occupancy, suggesting that BRD4 stabilizes NIPBL on chromatin. Chromatin interaction mapping and imaging experiments demonstrate that BRD4 depletion results in compromised genome folding and loop extrusion. Finally, mutation of individual BRD4 amino acids that mediate an interaction with NIPBL impedes neural crest differentiation into smooth muscle. Remarkably, loss of WAPL, a cohesin antagonist, rescues attenuated smooth muscle differentiation resulting from BRD4 loss. Collectively, our data reveal that BRD4 choreographs genome folding and illustrates the relevance of balancing cohesin activity for progenitor differentiation.

Responsiveness to perturbations is a hallmark of transcription factors that maintain cell identity in vitro.

Identifying the particular transcription factors that maintain cell type in vitro is important for manipulating cell type. Identifying such transcription factors by their cell-type-specific expression or their involvement in developmental regulation has had limited success. We hypothesized that because cell type is often resilient to perturbations, the transcriptional response to perturbations would reveal identity-maintaining transcription factors. We developed perturbation panel profiling (P3) as a framework for perturbing cells across many conditions and measuring gene expression responsiveness transcriptome-wide. In human iPSC-derived cardiac myocytes, P3 showed that transcription factors important for cardiac myocyte differentiation and maintenance were among the most frequently upregulated (most responsive). We reasoned that one function of responsive genes may be to maintain cellular identity. We identified responsive transcription factors in fibroblasts using P3 and found that suppressing their expression led to enhanced reprogramming. We propose that responsiveness to perturbations is a property of transcription factors that help maintain cellular identity in vitro. A record of this paper's transparent peer review process is included in the supplemental information.

A transcriptional switch governs fibroblast activation in heart disease.

In diseased organs, stress-activated signalling cascades alter chromatin, thereby triggering maladaptive cell state transitions. Fibroblast activation is a common stress response in tissues that worsens lung, liver, kidney and heart disease, yet its mechanistic basis remains unclear1,2. Pharmacological inhibition of bromodomain and extra-terminal domain (BET) proteins alleviates cardiac dysfunction3-7, providing a tool to interrogate and modulate cardiac cell states as a potential therapeutic approach. Here we use single-cell epigenomic analyses of hearts dynamically exposed to BET inhibitors to reveal a reversible transcriptional switch that underlies the activation of fibroblasts. Resident cardiac fibroblasts demonstrated robust toggling between the quiescent and activated state in a manner directly correlating with BET inhibitor exposure and cardiac function. Single-cell chromatin accessibility revealed previously undescribed DNA elements, the accessibility of which dynamically correlated with cardiac performance. Among the most dynamic elements was an enhancer that regulated the transcription factor MEOX1, which was specifically expressed in activated fibroblasts, occupied putative regulatory elements of a broad fibrotic gene program and was required for TGFß-induced fibroblast activation. Selective CRISPR inhibition of the single most dynamic cis-element within the enhancer blocked TGFß-induced Meox1 activation. We identify MEOX1 as a central regulator of fibroblast activation associated with cardiac dysfunction and demonstrate its upregulation after activation of human lung, liver and kidney fibroblasts. The plasticity and specificity of BET-dependent regulation of MEOX1 in tissue fibroblasts provide previously unknown trans- and cis-targets for treating fibrotic disease.

Pathogenic LMNA variants disrupt cardiac lamina-chromatin interactions and de-repress alternative fate genes.

Pathogenic mutations in LAMIN A/C (LMNA) cause abnormal nuclear structure and laminopathies. These diseases have myriad tissue-specific phenotypes, including dilated cardiomyopathy (DCM), but how LMNA mutations result in tissue-restricted disease phenotypes remains unclear. We introduced LMNA mutations from individuals with DCM into human induced pluripotent stem cells (hiPSCs) and found that hiPSC-derived cardiomyocytes, in contrast to hepatocytes or adipocytes, exhibit aberrant nuclear morphology and specific disruptions in peripheral chromatin. Disrupted regions were enriched for transcriptionally active genes and regions with lower LAMIN B1 contact frequency. The lamina-chromatin interactions disrupted in mutant cardiomyocytes were enriched for genes associated with non-myocyte lineages and correlated with higher expression of those genes. Myocardium from individuals with LMNA variants similarly showed aberrant expression of non-myocyte pathways. We propose that the lamina network safeguards cellular identity and that pathogenic LMNA variants disrupt peripheral chromatin with specific epigenetic and molecular characteristics, causing misexpression of genes normally expressed in other cell types.

BRD4 (Bromodomain-Containing Protein 4) Interacts with GATA4 (GATA Binding Protein 4) to Govern Mitochondrial Homeostasis in Adult Cardiomyocytes.

BACKGROUND: Gene regulatory networks control tissue homeostasis and disease progression in a cell type-specific manner. Ubiquitously expressed chromatin regulators modulate these networks, yet the mechanisms governing how tissue specificity of their function is achieved are poorly understood. BRD4 (bromodomain-containing protein 4), a member of the BET (bromo- and extraterminal domain) family of ubiquitously expressed acetyl-lysine reader proteins, plays a pivotal role as a coactivator of enhancer signaling across diverse tissue types in both health and disease and has been implicated as a pharmacological target in heart failure. However, the cell-specific role of BRD4 in adult cardiomyocytes remains unknown. METHODS: We combined conditional mouse genetics, unbiased transcriptomic and epigenomic analyses, and classic molecular biology and biochemical approaches to understand the mechanism by which BRD4 in adult cardiomyocyte homeostasis. RESULTS: Here, we show that cardiomyocyte-specific deletion of Brd4 in adult mice leads to acute deterioration of cardiac contractile function with mutant animals demonstrating a transcriptomic signature characterized by decreased expression of genes critical for mitochondrial energy production. Genome-wide occupancy data show that BRD4 enriches at many downregulated genes (including the master coactivators Ppargc1a, Ppargc1b, and their downstream targets) and preferentially colocalizes with GATA4 (GATA binding protein 4), a lineage-determining cardiac transcription factor not previously implicated in regulation of adult cardiac metabolism. BRD4 and GATA4 form an endogenous complex in cardiomyocytes and interact in a bromodomain-independent manner, revealing a new functional interaction partner for BRD4 that can direct its locus and tissue specificity. CONCLUSIONS: These results highlight a novel role for a BRD4-GATA4 module in cooperative regulation of a cardiomyocyte-specific gene program governing bioenergetic homeostasis in the adult heart.

Identification of a molecular basis for the juvenile sleep state.

Across species, sleep in young animals is critical for normal brain maturation. The molecular determinants of early life sleep remain unknown. Through an RNAi-based screen, we identified a gene, pdm3, required for sleep maturation in Drosophila. Pdm3, a transcription factor, coordinates an early developmental program that prepares the brain to later execute high levels of juvenile adult sleep. PDM3 controls the wiring of wake-promoting dopaminergic (DA) neurites to a sleep-promoting region, and loss of PDM3 prematurely increases DA inhibition of the sleep center, abolishing the juvenile sleep state. RNA-Seq/ChIP-Seq and a subsequent modifier screen reveal that pdm3 represses expression of the synaptogenesis gene Msp300 to establish the appropriate window for DA innervation. These studies define the molecular cues governing sleep behavioral and circuit development, and suggest sleep disorders may be of neurodevelopmental origin.

Author Correction: Targeting cardiac fibrosis with engineered T cells.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Targeting cardiac fibrosis with engineered T cells.

Fibrosis is observed in nearly every form of myocardial disease1. Upon injury, cardiac fibroblasts in the heart begin to remodel the myocardium by depositing excess extracellular matrix, resulting in increased stiffness and reduced compliance of the tissue. Excessive cardiac fibrosis is an important factor in the progression of various forms of cardiac disease and heart failure2. However, clinical interventions and therapies that target fibrosis remain limited3. Here we demonstrate the efficacy of redirected T cell immunotherapy to specifically target pathological cardiac fibrosis in mice. We find that cardiac fibroblasts that express a xenogeneic antigen can be effectively targeted and ablated by adoptive transfer of antigen-specific CD8+ T cells. Through expression analysis of the gene signatures of cardiac fibroblasts obtained from healthy and diseased human hearts, we identify an endogenous target of cardiac fibroblasts-fibroblast activation protein. Adoptive transfer of T cells that express a chimeric antigen receptor against fibroblast activation protein results in a significant reduction in cardiac fibrosis and restoration of function after injury in mice. These results provide proof-of-principle for the development of immunotherapeutic drugs for the treatment of cardiac disease.

Early lineage specification defines alveolar epithelial ontogeny in the murine lung.

During the stepwise specification and differentiation of tissue-specific multipotent progenitors, lineage-specific transcriptional networks are activated or repressed to orchestrate cell specification. The gas-exchange niche in the lung contains two major epithelial cell types, alveolar type 1 (AT1) and AT2 cells, and the timing of lineage specification of these cells is critical for the correct formation of this niche and postnatal survival. Integrating cell-specific lineage tracing studies, spatially specific mRNA transcript and protein expression, and single-cell RNA-sequencing analysis, we demonstrate that specification of alveolar epithelial cell fate begins concomitantly with the proximal-distal specification of epithelial progenitors and branching morphogenesis earlier than previously appreciated. By using a newly developed dual-lineage tracing system, we show that bipotent alveolar cells that give rise to AT1 and AT2 cells are a minor contributor to the alveolar epithelial population. Furthermore, single-cell assessment of the transcriptome identifies specified AT1 and AT2 progenitors rather than bipotent cells during sacculation. These data reveal a paradigm of organ formation whereby lineage specification occurs during the nascent stages of development coincident with broad tissue-patterning processes, including axial patterning of the endoderm and branching morphogenesis.

Centromere inheritance through the germline.

The centromere directs chromosome segregation and genetic inheritance but is not itself heritable in a canonical, DNA-based manner. In most species, centromeres are epigenetically defined by the presence of a histone H3 variant centromere protein A (CENP-A), independent of underlying DNA sequence. Therefore, centromere inheritance depends on maintaining the CENP-A nucleosome mark across generations. Experiments in cycling somatic cells have led to a model in which centromere identity is maintained by a cell cycle-coupled CENP-A chromatin assembly pathway. However, the processes of animal gametogenesis pose unique challenges to centromere inheritance because of the extended cell cycle arrest and the massive genome reorganization in the female and male germline, respectively. Here, we review our current understanding of germline centromere inheritance and highlight outstanding questions.

Prognostic Abilities and Quality Assessment of Models for the Prediction of 90-Day Mortality in Liver Transplant Waiting List Patients.

BACKGROUND: Model of end-stage liver disease (MELD)-score and diverse variants are widely used for prognosis on liver transplant waiting-lists. METHODS: 818 consecutive patients on the liver transplant waiting-list included to calculate the MELD, MESO Index, MELD-Na, UKELD, iMELD, refitMELD, refitMELD-Na, upMELD and PELD-scores. Prognostic abilities for 90-day mortality were investigated applying Receiver-operating-characteristic-curve analysis. Independent risk factors for 90-day mortality were identified with multivariable binary logistic regression modelling. Methodological quality of the underlying development studies was assessed with a systematic assessment tool. RESULTS: 74 patients (9%) died on the liver transplant waiting list within 90 days after listing. All but one scores, refitMELD-Na, had acceptable prognostic performance with areas under the ROC-curves (AUROCs)>0.700. The iMELD performed best (AUROC = 0.798). In pediatric cases, the PELD-score just failed to reach the acceptable threshold with an AUROC = 0.699. All scores reached a mean quality score of 72.3%. Highest quality scores could be achieved by the UKELD and PELD-scores. Studies specifically lack statistical validity and model evaluation. CONCLUSIONS: Inferior quality assessment of prognostic models does not necessarily imply inferior prognostic abilities. The iMELD might be a more reliable tool representing urgency of transplantation than the MELD-score. PELD-score is assumedly not accurate enough to allow graft allocation decision in pediatric liver transplantation.

What Is the Added Benefit of Oropharyngeal Swabs Compared to Nasal Swabs Alone for Respiratory Virus Detection in Hospitalized Children Aged <10 Years?

We evaluated the added value of collecting both nasal and oropharyngeal swabs, compared with collection of nasal swabs alone, for detection of common respiratory viruses by reverse transcription-polymerase chain reaction in hospitalized children aged <10 years. Nasal swabs had equal or greater sensitivity than oropharyngeal swabs for detection of respiratory syncytial virus, adenovirus, human metapneumovirus, rhinovirus, and influenza virus but not parainfluenza virus. The addition of an oropharyngeal swab, compared with use of a nasal swab alone, increased the frequency of detection of each respiratory virus by no more than 10% in children aged <10 years.

Impacto del serotipo 5 en la enfermedad invasora por Streptococcus pneumoniae en la población pediátrica ingresada en el Hospital Materno Infantil José Domingo de Obaldía

La vigilancia de la enfermedad invasora por Streptococcus pneumoniae permite detectar los cambios geográficos y cronológicos en los serotipos circulantes y en la sensibilidad antimicrobiana, monitorizando además el impacto de las vacunas sobre la enfermedad. Objetivos: Describir los serotipos aislados y la sensibilidad antimicrobiana en los pacientes pediatrics hospitalizados con enfermedad invasora por Streptococcus pneumoniae. Describir la enfermedad invasora por Streptococcus pneumoniae y sus complicaciones clínicas asociadas. Metodología: Estudio retrospectivo, longitudinal, incluye pacientes pediátricos hospitalizados en el Hospital Materno Infantil José Domingo de Obaldía en el periodo de enero de 2010 a junio de 2011; los cuales se cursaron con enfermedad invasora por Streptococcus pneumoniae como etiología en este periodo. El diagnóstico microbiológico se realizó por coaglutinación en líquidos corporales y cultivo del microorganismo. La cepa se aisló en el laboratorio de microbiología y se tipificó en el Laboratorio Central. La enfermedad invasora fue definida por el cuadro clínico y el aislamiento de Streptococcus pneumoniae de un sitio previamente estéril. Variables analizadas: edad, sexo, procedencia, sitio de infección, complicaciones, tiempo hospitalario, mortalidad, ingreso a UCI, anemia, VIH , letalidad. Resultados: Se revisaron 25 expedientes de pacientes con enfermedad invasora y aislamiento de Streptococcus pneumoniae. Dos fueron excluidos. 20 pacientes fueron menores de 5 años y 3 mayores de 5 años. 11 varones y 12 niñas. El 91% fueron indigenas. El 60.8% procedía de la Comarca Ngöbe Buglé. Promedio hospitalario 25.3 días, rango 1-93 días. 60.8% ingresó con enfermedad grave a la UCIP; 92.8% ameritó ventilación mecánica y 78.5% presentó choque. 34%(8) cursó con compromiso de SNC y el 30% (7) bacteriemia. Se tipificaron 14 cepas , el (78.5%) fue serotipo 5. Todos los aislados fueron sensibles a penicilina. 31% (5) resistentes a TMP/SMX. El 74% de los casos tenia hemoglobina menor de 10 g y solamente 3 habían completado su vacunación con PCV-7. Todo fueron VIH negativo. 30% de los pacientes fallecieron. Conclusiones: En los caso evaluados encontramos un predominio del Streptococcus pneumoniae tipo 5 en el 78.5% de los casos. Los serotipos 24F, 18C y 4 fueron aislados en 1 caso cada uno; el 100% fue sensible a penicilina. La población más afectada fue la raza indígena y los menores de 5 años. El SNC y respiratorio fueron los sitios primarios de infección. El 61% ingreso con enfermedad grave y el 30% falleció.

The monitoring of the invading disease by S. pneumoniae allows detecting geographic and temporary changes in antimicrobial sensitivity, besides monitoring the impact of the vaccines on the disease. Objectives: To describe to the isolate serotypes and antimicrobial sensitivy of Streptococcus pneumoniae to associate invasive disease in the pediatric patients hospitalized. Besides clinical manifestations and complications. Methods: Retrospective, longitudinal study. Pediatrics patients hospitalized in the Maternal Hospital in January 2010 to June 2011; in which Streptococcus pneumoniae was isolated as etiology of invasive disease. The microbiological diagnosis for Streptococcus pneumoniae was realized with coaglutination and culture of body fluids. The strain was isolated in the microbiology laboratory and it was typified in the Central Laboratory in Panama City. An invasive disease was defined by clinical manifestations and the isolation of Streptococcus pneumoniae of a previously sterile site. Analyzes variables: age, sex, origins, site of infection, complications, hospital time, death, admission to UCI, anemia , HIV, lethality. Results: 25 files with Streptococcus pneumoniae isolation and invasive disease were reviewed. Two were excluded. 20 patients were younger than 5 years and 3 oldest of 5 years. 11 were boys and 12 girls. 91% were indigenous. 60.8% came from Comarca Ngöbe Bugle. Hospital average 25, 3 days with range 1-93 days. 60.8% had serious disease and went to the UCIP; 92.8% need mechanical ventilation and 78.5% suffering shock. 34% (8) presented with commitment respiratory and SNC simultaneously. 70% (20) patients with pneumonia presented pleural effusion. 21,7% pericardial effusion and 30% (7) bacteremia. 14 isolate were typified, serotype 5 (78.5%). All were sensible to penicillin; TMP/SMX resistant 31% (5). 74% had hemoglobin less than 10 g and only 3 had completed their vaccination with PCV-7. All were HIV negative. 30% lethality. Conclusions: Streptococcus pneumoniae serotype 5 were strains predominance in the 78.5%. Others as 24 F, 18C and 4, 1 case of each serotypes; 100% were sensible to penicillin. The people more affected was indigenous race and the younger than 5 years. The respiratory and SNC were the primary infections sites. 61% come with serious illness and 30% died.