A transcription factor atlas of directed differentiation.

Transcription factors (TFs) regulate gene programs, thereby controlling diverse cellular processes and cell states. To comprehensively understand TFs and the programs they control, we created a barcoded library of all annotated human TF splice isoforms (>3,500) and applied it to build a TF Atlas charting expression profiles of human embryonic stem cells (hESCs) overexpressing each TF at single-cell resolution. We mapped TF-induced expression profiles to reference cell types and validated candidate TFs for generation of diverse cell types, spanning all three germ layers and trophoblasts. Targeted screens with subsets of the library allowed us to create a tailored cellular disease model and integrate mRNA expression and chromatin accessibility data to identify downstream regulators. Finally, we characterized the effects of combinatorial TF overexpression by developing and validating a strategy for predicting combinations of TFs that produce target expression profiles matching reference cell types to accelerate cellular engineering efforts.

Structures of fungal and plant acetohydroxyacid synthases.

Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, is a flavin adenine dinucleotide-, thiamine diphosphate- and magnesium-dependent enzyme that catalyses the first step in the biosynthesis of branched-chain amino acids1. It is the target for more than 50 commercial herbicides2. AHAS requires both catalytic and regulatory subunits for maximal activity and functionality. Here we describe structures of the hexadecameric AHAS complexes of Saccharomyces cerevisiae and dodecameric AHAS complexes of Arabidopsis thaliana. We found that the regulatory subunits of these AHAS complexes form a core to which the catalytic subunit dimers are attached, adopting the shape of a Maltese cross. The structures show how the catalytic and regulatory subunits communicate with each other to provide a pathway for activation and for feedback inhibition by branched-chain amino acids. We also show that the AHAS complex of Mycobacterium tuberculosis adopts a similar structure, thus demonstrating that the overall AHAS architecture is conserved across kingdoms.

Distinct subnetworks of the thalamic reticular nucleus.

The thalamic reticular nucleus (TRN), the major source of thalamic inhibition, regulates thalamocortical interactions that are critical for sensory processing, attention and cognition1-5. TRN dysfunction has been linked to sensory abnormality, attention deficit and sleep disturbance across multiple neurodevelopmental disorders6-9. However, little is known about the organizational principles that underlie its divergent functions. Here we performed an integrative study linking single-cell molecular and electrophysiological features of the mouse TRN to connectivity and systems-level function. We found that cellular heterogeneity in the TRN is characterized by a transcriptomic gradient of two negatively correlated gene-expression profiles, each containing hundreds of genes. Neurons in the extremes of this transcriptomic gradient express mutually exclusive markers, exhibit core or shell-like anatomical structure and have distinct electrophysiological properties. The two TRN subpopulations make differential connections with the functionally distinct first-order and higher-order thalamic nuclei to form molecularly defined TRN-thalamus subnetworks. Selective perturbation of the two subnetworks in vivo revealed their differential role in regulating sleep. In sum, our study provides a comprehensive atlas of TRN neurons at single-cell resolution and links molecularly defined subnetworks to the functional organization of thalamocortical circuits.

Long-Term Mortality in Patients With Severe Hypercholesterolemia Phenotype From a Racial and Ethnically Diverse US Cohort.

BACKGROUND: Tools for mortality prediction in patients with the severe hypercholesterolemia phenotype (low-density lipoprotein cholesterol ≥190 mg/dL) are limited and restricted to specific racial and ethnic cohorts. We sought to evaluate the predictors of long-term mortality in a large racially and ethnically diverse US patient cohort with low-density lipoprotein cholesterol ≥190 mg/dL. METHODS: We conducted a retrospective analysis of all patients with a low-density lipoprotein cholesterol ≥190 mg/dL seeking care at Montefiore from 2010 through 2020. Patients <18 years of age or with previous malignancy were excluded. The primary end point was all-cause mortality. Analyses were stratified by age, sex, and race and ethnicity. Patients were stratified by primary and secondary prevention. Cox regression analyses were used to adjust for demographic, clinical, and treatment variables. RESULTS: A total of 18 740 patients were included (37% non-Hispanic Black, 30% Hispanic, 12% non-Hispanic White, and 2% non-Hispanic Asian patients). The mean age was 53.9 years, and median follow-up was 5.2 years. Both high-density lipoprotein cholesterol and body mass index extremes were associated with higher mortality in univariate analyses. In adjusted models, higher low-density lipoprotein cholesterol and triglyceride levels were associated with an increased 9-year mortality risk (adjusted hazard ratio [HR], 1.08 [95% CI, 1.05-1.11] and 1.04 [95% CI, 1.02-1.06] per 20-mg/dL increase, respectively). Clinical factors associated with higher mortality included male sex (adjusted HR, 1.31 [95% CI, 1.08-1.58]), older age (adjusted HR, 1.19 per 5-year increase [95% CI, 1.15-1.23]), hypertension (adjusted HR, 2.01 [95% CI, 1.57-2.57]), chronic kidney disease (adjusted HR, 1.68 [95% CI, 1.36-2.09]), diabetes (adjusted HR, 1.79 [95% CI, 1.50-2.15]), heart failure (adjusted HR, 1.51 [95% CI, 1.16-1.95]), myocardial infarction (adjusted HR, 1.41 [95% CI, 1.05-1.90]), and body mass index <20 kg/m2 (adjusted HR, 3.36 [95% CI, 2.29-4.93]). A significant survival benefit was conferred by lipid-lowering therapy (adjusted HR, 0.57 [95% CI, 0.42-0.77]). In the primary prevention group, high-density lipoprotein cholesterol <40 mg/dL was independently associated with higher mortality (adjusted HR, 1.49 [95% CI, 1.06-2.09]). Temporal trend analyses showed a reduction in statin use over time (P<0.001). In the most recent time period (2019-2020), 56% of patients on primary prevention and 85% of those on secondary prevention were on statin therapy. CONCLUSIONS: In a large, diverse cohort of US patients with the severe hypercholesterolemia phenotype, we identified several patient characteristics associated with increased 9-year all-cause mortality and observed a decrease in statin use over time, in particular for primary prevention. Our results support efforts geared toward early recognition and consistent treatment for patients with severe hypercholesterolemia.

Exercise-Induced Shear Stress Drives mRNA Translation In Vitro.

The vascular endothelium is the first line of defense to prevent cardiovascular disease. Its optimal functioning and health are maintained by the interaction of the proteins-endothelial nitric oxide synthase (eNOS), sirtuin 1 (SIRT1), and endothelin 1 (ET1)-and the genes that encode them-NOS3, SIRT1, and EDN1, respectively. Aerobic exercise improves endothelial function by allegedly increasing endothelial shear stress (ESS). However, there are no current data exploring the acute effects of specific exercise-induced ESS intensities on these regulatory proteins and genes that are associated with endothelial function. The purpose of this study was to assess the acute changes in endothelial proteins and gene expression after exposure to low-, moderate-, and high-intensity exercise-induced ESS. Human umbilical vein endothelial cells (HUVECs) were exposed to resting ESS (18 dynes/cm2, 60 pulses per minute (PPM)), low ESS (35 dynes/cm2, 100 PPM), moderate ESS (50 dynes/cm2, 120 PPM), and high ESS (70 dynes/cm2, 150 PPM). Protein and gene expression were quantified by fluorescent Western blot and RTqPCR, respectively. All exercise conditions showed an increase in eNOS and SIRT1 expression and a decrease in NOS3 and SIRT1 gene expression when compared to resting conditions. In addition, there was no expression of ET1 and an increase in EDN1 gene expression when compared to resting conditions. These results show that (1) exercise-induced ESS increases the expressions of vascular protective proteins and (2) there is an inverse relationship between the proteins and their encoding genes immediately after exercise-induced ESS, suggesting that exercise has a previously unexplored translational role catalyzing mRNA to proteins.

Combination of computed tomography angiography with coronary artery calcium score for improved diagnosis of coronary artery disease: a collaborative meta-analysis of stable chest pain patients referred for invasive coronary angiography.

OBJECTIVES: Coronary computed tomography angiography (CCTA) has higher diagnostic accuracy than coronary artery calcium (CAC) score for detecting obstructive coronary artery disease (CAD) in patients with stable chest pain, while the added diagnostic value of combining CCTA with CAC is unknown. We investigated whether combining coronary CCTA with CAC score can improve the diagnosis of obstructive CAD compared with CCTA alone. METHODS: A total of 2315 patients (858 women, 37%) aged 61.1 ± 10.2 from 29 original studies were included to build two CAD prediction models based on either CCTA alone or CCTA combined with the CAC score. CAD was defined as at least 50% coronary diameter stenosis on invasive coronary angiography. Models were built by using generalized linear mixed-effects models with a random intercept set for the original study. The two CAD prediction models were compared by the likelihood ratio test, while their diagnostic performance was compared using the area under the receiver-operating-characteristic curve (AUC). Net benefit (benefit of true positive versus harm of false positive) was assessed by decision curve analysis. RESULTS: CAD prevalence was 43.5% (1007/2315). Combining CCTA with CAC improved CAD diagnosis compared with CCTA alone (AUC: 87% [95% CI: 86 to 89%] vs. 80% [95% CI: 78 to 82%]; p < 0.001), likelihood ratio test 236.3, df: 1, p < 0.001, showing a higher net benefit across almost all threshold probabilities. CONCLUSION: Adding the CAC score to CCTA findings in patients with stable chest pain improves the diagnostic performance in detecting CAD and the net benefit compared with CCTA alone. CLINICAL RELEVANCE STATEMENT: CAC scoring CT performed before coronary CTA and included in the diagnostic model can improve obstructive CAD diagnosis, especially when CCTA is non-diagnostic. KEY POINTS: • The combination of coronary artery calcium with coronary computed tomography angiography showed significantly higher AUC (87%, 95% confidence interval [CI]: 86 to 89%) for diagnosis of coronary artery disease compared to coronary computed tomography angiography alone (80%, 95% CI: 78 to 82%, p < 0.001). • Diagnostic improvement was mostly seen in patients with non-diagnostic C. • The improvement in diagnostic performance and the net benefit was consistent across age groups, chest pain types, and genders.

Carbon Dioxide Sensing by Immune Cells Occurs through Carbonic Anhydrase 2-Dependent Changes in Intracellular pH.

CO2, the primary gaseous product of respiration, is a major physiologic gas, the biology of which is poorly understood. Elevated CO2 is a feature of the microenvironment in multiple inflammatory diseases that suppresses immune cell activity. However, little is known about the CO2-sensing mechanisms and downstream pathways involved. We found that elevated CO2 correlates with reduced monocyte and macrophage migration in patients undergoing gastrointestinal surgery and that elevated CO2 reduces migration in vitro. Mechanistically, CO2 reduces autocrine inflammatory gene expression, thereby inhibiting macrophage activation in a manner dependent on decreased intracellular pH. Pharmacologic or genetic inhibition of carbonic anhydrases (CAs) uncouples a CO2-elicited intracellular pH response and attenuates CO2 sensitivity in immune cells. Conversely, CRISPR-driven upregulation of the isoenzyme CA2 confers CO2 sensitivity in nonimmune cells. Of interest, we found that patients with chronic lung diseases associated with elevated systemic CO2 (hypercapnia) display a greater risk of developing anastomotic leakage following gastrointestinal surgery, indicating impaired wound healing. Furthermore, low intraoperative pH levels in these patients correlate with reduced intestinal macrophage infiltration. In conclusion, CO2 is an immunomodulatory gas sensed by immune cells through a CA2-coupled change in intracellular pH.

Ellagic acid and its metabolites urolithins A/B ameliorate most common disease phenotypes in cellular and mouse models for lysosomal storage disorders by enhancing extracellular vesicle secretion.

Niemann Pick diseases types A (NPDA) and C (NPDC) are lysosomal storage disorders (LSDs) leading to cognitive impairment, neurodegeneration, and early death. NPDA and NPDC have different genetic origins, being caused by mutations in the acid sphingomyelinase (ASM) or the cholesterol transport protein NPC1, respectively. However, they share a common pathological hallmark in the accumulation of lipids in the endolysosomal compartment. Here, we tested the hypothesis that polyphenols reduce lipid overload in NPD cells by enhancing the secretion of extracellular vesicles (ECVs). We show that among the polyphenols tested, the ellagic acid metabolites, urolithin A and B, were the safest and most efficient in increasing ECV secretion. They reduced levels of accumulating lipids and lysosomal size and permeabilization in cultured bone marrow-derived macrophages and neurons from ASMko and NPC1 mutant mice, which mimic NPDA and NPDC, respectively. Moreover, oral treatment with ellagic acid reduced lipid levels, ameliorated lysosomal alterations, and diminished microglia activation in the brain of NPD mice. These results support the therapeutic value of ECV secretion and polyphenols for NPDs, which may also help treat other LSDs characterized by intracellular lipid overload.

Ablation of atrial fibrillation beyond pulmonary vein isolation: Do additional ablation lesions impact left atrial function?

INTRODUCTION: Electrical isolation of pulmonary veins (PVI) is a cornerstone for atrial fibrillation (AF) ablation. The overall effect of AF ablation, and especially lesions beyond PVI, on left atrial (LA) function is currently poorly understood. Our aim was to determine if LA function is different in patients after extensive LA ablation compared to PVI only. We performed non-inferiority analysis of LA function after PVI with additional nonpulmonary vein ablation lesions in LA (PVI+) and PVI alone. METHODS: We studied 68 patients consecutive patients who underwent AF ablation and who had complete transthoracic echocardiogram (TTE) within 12 months before AF ablation and 1-12 months after the procedure. Patients were stratified into two groups: PVI only and PVI+. Primary outcome was change in LA reservoir strain (LASr). Noninferiority margin was defined at 6%. RESULTS: The PVI only group had a higher proportion of patients with paroxysmal AF (70% vs. 30%). The PVI+ group was observed to have a slightly higher increase in LASr compared to PVI alone (5.0% vs. 4.3%, p < .01 for noninferiority). LASr noninferiority was confirmed when adjusted for age, sex, coronary artery disease, hyperlipidemia, and AF type, rhythm at preprocedure TTE in a multivariable linear regression model, 90% CI (-5.46 to 2.04), p < .01. CONCLUSION: LA functional improvement evaluated by LASr was noninferior after PVI with additional LA ablation lesions compared to PVI alone. These findings were confirmed when adjusted for confounding clinical variables, suggesting that more extensive ablation does not negatively affect LA function.

Does Coronary Plaque Morphology Matter Beyond Plaque Burden?

PURPOSE OF REVIEW: Imaging of adverse coronary plaque features by coronary computed tomography angiography (CCTA) has advanced greatly and at a fast pace. We aim to describe the evolution, present and future in plaque analysis, and its value in comparison to plaque burden. RECENT FINDINGS: Recently, it has been demonstrated that in addition to plaque burden, quantitative and qualitative assessment of coronary plaque by CCTA can improve the prediction of future major adverse cardiovascular events in diverse coronary artery disease scenarios. The detection of high-risk non-obstructive coronary plaque can lead to higher use of preventive medical therapies such as statins and aspirin, help identify culprit plaque, and differentiate between myocardial infarction types. Even more, over traditional plaque burden, plaque analysis including pericoronary inflammation can potentially be useful tools for tracking disease progression and response to medical therapy. The identification of the higher risk phenotypes with plaque burden, plaque characteristics, or ideally both can allow the allocation of targeted therapies and potentially monitor response. Further observational data are now required to investigate these key issues in diverse populations, followed by rigorous randomized controlled trials.

Anatomical considerations and clinical interpretation of the 12-lead ECG in the prone position: a prospective multicentre study.

AIMS: The aim of this study is to provide guidance for the clinical interpretation of electrocardiograms (ECGs) in prone position and to establish the electroanatomic explanations for the possible differences to supine position ECGs that may be observed. Additionally, to determine if prone back ECG can be used as an alternative to standard ECG in patients who may benefit from prone position. METHODS AND RESULTS: The ECG in supine (standard ECG), prone back (precordial leads placed on the patient's back), and prone anterior position (precordial leads placed in the standard position with the subjects in prone position) were prospectively examined on 85 subjects. Comparisons of ECG parameters between these positions were performed. Computed tomography (CT) scans were performed in both positions to determine possible electroanatomic aetiologies for prone-associated ECG changes. There were significant differences in QRS amplitude in Leads V1-V5 between supine and prone positions. Q waves were more frequently observed in prone back position vs. supine position (V1: 74.1 vs. 10.6%, P < 0.0001; V2: 23.5 vs. 0%, P < 0.0001, respectively). Flat and inverted T waves were more common in prone back leads (V1: 98 vs. 66%, P < 0.0001; V2: 96 vs. 8%, P < 0.0001; V3: 45 vs. 7%, P < 0.0001). The 3D-CT reconstructions measurements corroborated the significant inverse correlation between QRS amplitude and the distance from the centre of the heart to the estimated lead positions. CONCLUSION: In prone back position ECG, low QRS amplitude should not be misinterpreted as low voltage conditions, neither should Q waves and abnormal T waves are considered anteroseptal myocardial infarction. These changes can be explained by an increased impedance (due to interposing lung tissue) and by the increased distance between the electrodes to the centre of the heart.

Multimodality Imaging in Aortic Stenosis: Beyond the Valve - Focusing on the Myocardium.

Current guidelines of aortic stenosis (AS) management focus on valve parameters, LV systolic dysfunction, and symptoms; however, emerging data suggest that there may be benefit of aortic valve replacement before it becomes severe by present criteria. Myocardial assessment using novel multimodality imaging techniques exhibits subclinical myocardial injury and remodeling at various stages before guideline-directed interventions, which predicts adverse outcomes. This raises the question of whether implementing serial myocardial assessment should become part of the standard appraisal, thereby identifying high-risk patients aiming to minimize adverse outcomes.

A cross-sectional study evidences regulations of leukocytes in the colostrum of mothers with obesity.

BACKGROUND: Breastmilk is a dynamic fluid whose initial function is to provide the most adapted nutrition to the neonate. Additional attributes have been recently ascribed to breastmilk, with the evidence of a specific microbiota and the presence of various components of the immune system, such as cytokines and leukocytes. The composition of breastmilk varies through time, according to the health status of mother and child, and altogether contributes to the future health of the infant. Obesity is a rising condition worldwide that creates a state of systemic, chronic inflammation including leukocytosis. Here, we asked whether colostrum, the milk produced within the first 48 h post-partum, would contain a distinct leukocyte composition depending on the body mass index (BMI) of the mother. METHODS: We collected peripheral blood and colostrum paired samples from obese (BMI > 30) and lean (BMI < 25) mothers within 48 h post-partum and applied a panel of 6 antibodies plus a viability marker to characterize 10 major leukocyte subpopulations using flow cytometry. RESULTS: The size, internal complexity, and surface expression of CD45 and CD16 of multiple leukocyte subpopulations were selectively regulated between blood and colostrum irrespective of the study groups, suggesting a generalized cell-specific phenotype alteration. In obesity, the colostrum B lymphocyte compartment was significantly reduced, and CD16+ blood monocytes had an increased CD16 expression compared to the lean group. CONCLUSIONS: This is the first characterization of major leukocyte subsets in colostrum of mothers suffering from obesity and the first report of colostrum leukocyte subpopulations in Latin America. We evidence various significant alterations of most leukocyte populations between blood and colostrum and demonstrate a decreased colostrum B lymphocyte fraction in obesity. This pioneering study is a stepping stone to further investigate active immunity in human breastmilk.

Computed tomography angiography versus Agatston score for diagnosis of coronary artery disease in patients with stable chest pain: individual patient data meta-analysis of the international COME-CCT Consortium.

OBJECTIVES: There is conflicting evidence about the comparative diagnostic accuracy of the Agatston score versus computed tomography angiography (CTA) in patients with suspected obstructive coronary artery disease (CAD). PURPOSE: To determine whether CTA is superior to the Agatston score in the diagnosis of CAD. METHODS: In total 2452 patients with stable chest pain and a clinical indication for invasive coronary angiography (ICA) for suspected CAD were included by the Collaborative Meta-analysis of Cardiac CT (COME-CCT) Consortium. An Agatston score of > 400 was considered positive, and obstructive CAD defined as at least 50% coronary diameter stenosis on ICA was used as the reference standard. RESULTS: Obstructive CAD was diagnosed in 44.9% of patients (1100/2452). The median Agatston score was 74. Diagnostic accuracy of CTA for the detection of obstructive CAD (81.1%, 95% confidence interval [CI]: 77.5 to 84.1%) was significantly higher than that of the Agatston score (68.8%, 95% CI: 64.2 to 73.1%, p < 0.001). Among patients with an Agatston score of zero, 17% (101/600) had obstructive CAD. Diagnostic accuracy of CTA was not significantly different in patients with low to intermediate (1 to < 100, 100-400) versus moderate to high Agatston scores (401-1000, > 1000). CONCLUSIONS: Results in our international cohort show CTA to have significantly higher diagnostic accuracy than the Agatston score in patients with stable chest pain, suspected CAD, and a clinical indication for ICA. Diagnostic performance of CTA is not affected by a higher Agatston score while an Agatston score of zero does not reliably exclude obstructive CAD. KEY POINTS: • CTA showed significantly higher diagnostic accuracy (81.1%, 95% confidence interval [CI]: 77.5 to 84.1%) for diagnosis of coronary artery disease when compared to the Agatston score (68.8%, 95% CI: 64.2 to 73.1%, p < 0.001). • Diagnostic performance of CTA was not affected by increased amount of calcium and was not significantly different in patients with low to intermediate (1 to <100, 100-400) versus moderate to high Agatston scores (401-1000, > 1000). • Seventeen percent of patients with an Agatston score of zero showed obstructive coronary artery disease by invasive angiography showing absence of coronary artery calcium cannot reliably exclude coronary artery disease.

Influence of polyamide composite casings with silver-zinc crystals on the quality of beef and chicken sausages during their storage.

The influence of polyamide 6 composite casings with silver-zinc crystals powder on some of the physicochemicalphysical-chemical, microbiological and sensory indicators of beef and chicken sausages during their storage was evaluated. Beef and chicken sausages were elaborated by using the conventional technology for sausage meat thin pasta; in each case, it was maintained a control batch to compare changes during the storage (4 and 12 °C, 75%-85% RH). To estimate the shelf life was considered sensory evaluation as a criterion for rejection. The results were processed as failure incomplete data via the Weibull distribution and it was admitted 5% of deteriorated units. It did not find a significant effect (p ≤ 0.05) due to the addition of silver-zinc crystals on the values of pH, aw, color, texture and sensory attributes of sausages, but did influence TBARS results, with lower values compared to control products. It reduced the counts of aerobic mesophilic microorganisms and lactic acid bacteria during the storage. The shelf life of chicken sausage was not affected at any storage temperature; while for the beef sausage stored at 4 °C, its shelf life increased in 9 days, although at 12 °C did not exist difference among treatments.

Development of a food colorant from Syzygium cumini L. (Skeels) by spray drying.

The objective of the present work was to develop a powder colorant for food use by spray drying from a hydroalcoholic extract of black cherry (Syzygium cumini [L.] Skeels). The content of total solids significantly affected the contents of anthocyanins and total polyphenols, while the air inlet temperature influenced (p ≤ 0.05) the spray drying performance. The optimal drying conditions were 165 °C as air inlet temperature and 25% of total solids, which allowed obtaining a powder colorant with total anthocyanin contents between 4273 and 5070 mg/1000 g, total polyphenols from 10,142 to 11,184 mg/1000 g, and a drying yield between 67.14 and 67.7%. The colorant presented 5.65% humidity, 25.2% hygroscopicity, poor fluidity, and high cohesiveness, with a dissolution time of 55 s. The degradation of anthocyanins, adjusted to zero-order kinetics, was directly proportional to the increase in temperature and time. The values of the component a* decreased with increasing temperature and time.

FMR1 loss in a human stem cell model reveals early changes to intrinsic membrane excitability.

Fragile X mental retardation 1 (FMR1) encodes the RNA binding protein FMRP. Loss of FMRP drives Fragile X syndrome (FXS), the leading inherited cause of intellectual disability and a leading monogenic cause of autism. While cortical hyperexcitability is a hallmark of FXS, the reported phenotypes and underlying mechanisms, including alterations in synaptic transmission and ion channel properties, are heterogeneous and at times contradictory. Here, we report the generation of new isogenic FMR1y/+ and FMR1y/- human pluripotent stem cell (hPSC) lines using CRISPR-Cas9 to facilitate the study of how complete FMRP loss, independent of genetic background, drives molecular and cellular alterations relevant for FXS. After differentiating these stem cell tools into excitatory neurons, we systematically assessed the impact of FMRP loss on intrinsic membrane and synaptic properties over time. Using whole-cell patch clamp analyses, we found that FMR1y/- neurons overall showed an increased intrinsic membrane excitability compared to age-matched FMR1y/+ controls, with no discernable alternations in synaptic transmission. Surprisingly, longitudinal analyses of cell intrinsic defects revealed that a majority of significant changes emerged early following in vitro differentiation and some were not stable over time. Collectively, this study provides a new isogenic hPSC model which can be further leveraged by the scientific community to investigate basic mechanisms of FMR1 gene function relevant for FXS. Moreover, our results suggest that precocious changes in the intrinsic membrane properties during early developmental could be a critical cellular pathology ultimately contributing to cortical hyperexcitability in FXS.

Activation of parvalbumin-expressing neurons reconfigures neuronal ensembles in murine striatal microcircuits.

The striatum is the largest entrance to the basal ganglia. Diverse neuron classes make up striatal microcircuit activity, consisting in the sequential activation of neuronal ensembles. How different neuron classes participate in generating ensemble sequences is unknown. In control mus musculus brain slices in vitro, providing excitatory drive generates ensemble sequences. In Parkinsonian microcircuits captured by a highly recurrent ensemble, a cortical stimulus causes a transitory reconfiguration of neuronal groups alleviating Parkinsonism. Alternation between neuronal ensembles needs interconnectivity, in part due to interneurons, preferentially innervated by incoming afferents. One main class of interneuron expresses parvalbumin (PV+ neurons) and mediates feed-forward inhibition. However, its more global actions within the microcircuit are unknown. Using calcium imaging in ex vivo brain slices simultaneously recording dozens of neurons, we aimed to observe the actions of PV+ neurons within the striatal microcircuit. PV+ neurons in active microcircuits are 5%-11% of the active neurons even if, anatomically, they are <1% of the total neuronal population. In resting microcircuits, optogenetic activation of PV+ neurons turns on circuit activity by activating or disinhibiting, more neurons than those actually inhibited, showing that feed-forward inhibition is not their only function. Optostimulation of PV+ neurons in active microcircuits inhibits and activates different neuron sets, resulting in the reconfiguration of neuronal ensembles by changing their functional connections and ensemble membership, showing that neurons may belong to different ensembles at different situations. Our results show that PV+ neurons participate in the mechanisms that generate alternation of neuronal ensembles, therefore provoking ensemble sequences.

Anti-tuberculosis chemotherapy alters TNFR2 expression on CD4+ lymphocytes in both drug-sensitive and -resistant tuberculosis: however, only drug-resistant tuberculosis maintains a pro-inflammatory profile after a long time.

BACKGROUND: Tuberculosis (TB) is an infectious disease. During TB, regulatory T cells (Treg) are related to poor prognosis. However, information about conventional and unconventional Treg (cTreg and uTreg, respectively) is limited. The tumour necrosis factor (TNF) and its receptors (TNFR1 and TNFR2) are necessary for mycobacterial infection, and TNFR2 signalling is required to maintain Treg. METHODS: A blood sample of drug-susceptible (DS-TB) and drug-resistant tuberculosis (DR-TB) patients was obtained before (basal) and after 2 and 6 months of anti-TB therapy. Expression of TNF, TNFR1, and TNFR2 (transmembrane form, tm) on cTreg, uTreg, activated CD4+ (actCD4+), and CD4+ CD25- (CD4+) T cell subpopulations were evaluated. The main objective was to identify immunological changes associated with sensitive/resistant Mtb strains and with the use of anti-TB therapy. RESULTS: We found that after 6 months of anti-TB therapy, both DS- and DR-TB patients have decreased the frequency of cTreg tmTNF+, CD4+ tmTNFR1+ and CD4+ tmTNFR2+. Nevertheless, after 6 months of therapy, only DR-TB patients decreased the frequency of actCD4+ tmTNF+ and actCD4+ tmTNFR2+, exhibited a systemic inflammatory status (high levels of TNF, IFN-γ and IL-12), and their purified CD4+ T cells showed that TNF and TNFR2 are up-regulated at the transcriptional level. Moreover, DS- and DR-TB down-regulated TNFR1 and other proteins associated with Treg (FOXP3 and TGFß1) in response to the anti-TB therapy. CONCLUSION: These results partially explain the differences in the immune response of DS-TB vs DR-TB. The frequency of actCD4+ tmTNFR2+ cells and inflammatory status should be considered in the follow-up of therapy in DR-TB patients.