Harnessing the regenerative potential of interleukin11 to enhance heart repair.

Balancing between regenerative processes and fibrosis is crucial for heart repair. However, strategies to regulate the balance between these two process are a barrier to the development of effective therapies for heart regeneration. While Interleukin 11 (IL11) is known as a fibrotic factor for the heart, its contribution to heart regeneration remains poorly understood. Here, we uncovered that il11a can initiate robust regenerative programs in the zebrafish heart, including cell cycle reentry of cardiomyocytes (CMs) and coronary expansion, even in the absence of injury. However, the prolonged il11a induction in uninjured hearts causes persistent fibroblast emergence, resulting in cardiac fibrosis. While deciphering the regenerative and fibrotic effects, we found that il11-dependent fibrosis, but not il11-dependent regeneration, is mediated through ERK activity, implying that the dual effects of il11a on regeneration and fibrosis can be uncoupled. To harness the regenerative ability of il11a for injured hearts, we devised a combinatorial treatment through il11a induction with ERK inhibition. Using this approach, we observed enhanced CM proliferation with mitigated fibrosis, achieving a balance between stimulating regenerative processes and curbing fibrotic outcomes. Thus, our findings unveil the mechanistic insights into regenerative roles of il11 signaling, offering the potential therapeutic avenues that utilize a paracrine regenerative factor to foster cardiac repair without exacerbating the fibrotic responses.

Methods for dynamic and whole volume imaging of the zebrafish heart.

Tissue development and regeneration are dynamic processes involving complex cell migration and cell-cell interactions. We have developed a protocol for complementary time-lapse and three-dimensional (3D) imaging of tissue for developmental and regeneration studies which we apply here to the zebrafish cardiac vasculature. 3D imaging of fixed specimens is used to first define the subject at high resolution then live imaging captures how it changes dynamically. Hearts from adult and juvenile zebrafish are extracted and cleaned in preparation for the different imaging modalities. For whole-mount 3D confocal imaging, single or multiple hearts with native fluorescence or immuno-labeling are prepared for stabilization or clearing, and then imaged. For live imaging, hearts are placed in a prefabricated fluidic device and set on a temperature-controlled microscope for culture and imaging over several days. This protocol allows complete visualization of morphogenic processes in a 3D context and provides the ability to follow cell behaviors to complement in vivo and fixed tissue studies. This culture and imaging protocol can be applied to different cell and tissue types. Here, we have used it to observe zebrafish coronary vasculature and the migration of coronary endothelial cells during heart regeneration.

A beta cell subset with enhanced insulin secretion and glucose metabolism is reduced in type 2 diabetes.

The pancreatic islets are composed of discrete hormone-producing cells that orchestrate systemic glucose homeostasis. Here we identify subsets of beta cells using a single-cell transcriptomic approach. One subset of beta cells marked by high CD63 expression is enriched for the expression of mitochondrial metabolism genes and exhibits higher mitochondrial respiration compared with CD63lo beta cells. Human and murine pseudo-islets derived from CD63hi beta cells demonstrate enhanced glucose-stimulated insulin secretion compared with pseudo-islets from CD63lo beta cells. We show that CD63hi beta cells are diminished in mouse models of and in humans with type 2 diabetes. Finally, transplantation of pseudo-islets generated from CD63hi but not CD63lo beta cells into diabetic mice restores glucose homeostasis. These findings suggest that loss of a specific subset of beta cells may lead to diabetes. Strategies to reconstitute or maintain CD63hi beta cells may represent a potential anti-diabetic therapy.

Cross-species single-cell comparison of systemic and cardiac inflammatory responses after cardiac injury.

The immune system coordinates the response to cardiac injury and is known to control regenerative and fibrotic scar outcomes in the heart and subsequent chronic low-grade inflammation associated with heart failure. Here we profiled the inflammatory response to heart injury using single cell transcriptomics to compare and contrast two experimental models with disparate outcomes. We used adult mice, which like humans lack the ability to fully recover and zebrafish which spontaneously regenerate after heart injury. The extracardiac reaction to cardiomyocyte necrosis was also interrogated to assess the specific peripheral tissue and immune cell reaction to chronic stress. Cardiac macrophages are known to play a critical role in determining tissue homeostasis by healing versus scarring. We identified distinct transcriptional clusters of monocytes/macrophages in each species and found analogous pairs in zebrafish and mice. However, the reaction to myocardial injury was largely disparate between mice and zebrafish. The dichotomous response to heart damage between the mammalian and zebrafish monocytes/macrophages may underlie the impaired regenerative process in mice, representing a future therapeutic target.

leptin b and its regeneration enhancer illustrate the regenerative features of zebrafish hearts.

BACKGROUND: Zebrafish possess a remarkable regenerative capacity, which is mediated by the induction of various genes upon injury. Injury-dependent transcription is governed by the tissue regeneration enhancer elements (TREEs). Here, we utilized leptin b (lepb), an injury-specific factor, and its TREE to dissect heterogeneity of noncardiomyocytes (CMs) in regenerating hearts. RESULTS: Our single-cell RNA sequencing (scRNA-seq) analysis demonstrated that the endothelium/endocardium(EC) is activated to induce distinct subpopulations upon injury. We demonstrated that lepb can be utilized as a regeneration-specific marker to subset injury-activated ECs. lepb+ ECs robustly induce pro-regenerative factors, implicating lepb+ ECs as a signaling center to interact with other cardiac cells. Our scRNA-seq analysis identified that lepb is also produced by subpopulation of epicardium (Epi) and epicardium-derived cells (EPDCs). To determine whether lepb labels injury-emerging non-CM cells, we tested the activity of lepb-linked regeneration enhancer (LEN) with chromatin accessibility profiles and transgenic lines. While nondetectable in uninjured hearts, LEN directs EC and Epi/EPDC expression upon injury. The endogenous LEN activity was assessed using LEN deletion lines, demonstrating that LEN deletion abolished injury-dependent expression of lepb, but not other nearby genes. CONCLUSIONS: Our integrative analyses identify regeneration-emerging cell-types and factors, leading to the discovery of regenerative features of hearts.

Activation of a transient progenitor state in the epicardium is required for zebrafish heart regeneration.

The epicardium, a mesothelial cell tissue that encompasses vertebrate hearts, supports heart regeneration after injury through paracrine effects and as a source of multipotent progenitors. However, the progenitor state in the adult epicardium has yet to be defined. Through single-cell RNA-sequencing of isolated epicardial cells from uninjured and regenerating adult zebrafish hearts, we define the epithelial and mesenchymal subsets of the epicardium. We further identify a transiently activated epicardial progenitor cell (aEPC) subpopulation marked by ptx3a and col12a1b expression. Upon cardiac injury, aEPCs emerge from the epithelial epicardium, migrate to enclose the wound, undergo epithelial-mesenchymal transition (EMT), and differentiate into mural cells and pdgfra+hapln1a+ mesenchymal epicardial cells. These EMT and differentiation processes are regulated by the Tgfß pathway. Conditional ablation of aEPCs blocks heart regeneration through reduced nrg1 expression and mesenchymal cell number. Our findings identify a transient progenitor population of the adult epicardium that is indispensable for heart regeneration and highlight it as a potential target for enhancing cardiac repair.

BRPF1-KAT6A/KAT6B Complex: Molecular Structure, Biological Function and Human Disease.

The bromodomain and PHD finger-containing protein1 (BRPF1) is a member of family IV of the bromodomain-containing proteins that participate in the post-translational modification of histones. It functions in the form of a tetrameric complex with a monocytic leukemia zinc finger protein (MOZ or KAT6A), MOZ-related factor (MORF or KAT6B) or HAT bound to ORC1 (HBO1 or KAT7) and two small non-catalytic proteins, the inhibitor of growth 5 (ING5) or the paralog ING4 and MYST/Esa1-associated factor 6 (MEAF6). Mounting studies have demonstrated that all the four core subunits play crucial roles in different biological processes across diverse species, such as embryonic development, forebrain development, skeletal patterning and hematopoiesis. BRPF1, KAT6A and KAT6B mutations were identified as the cause of neurodevelopmental disorders, leukemia, medulloblastoma and other types of cancer, with germline mutations associated with neurodevelopmental disorders displaying intellectual disability, and somatic variants associated with leukemia, medulloblastoma and other cancers. In this paper, we depict the molecular structures and biological functions of the BRPF1-KAT6A/KAT6B complex, summarize the variants of the complex related to neurodevelopmental disorders and cancers and discuss future research directions and therapeutic potentials.

Slice-based and time-specific hemodynamic measurements discriminate carotid artery vulnerable atherosclerotic plaques.

BACKGROUND AND OBJECTIVE: Hemodynamic patterns play key roles in progression of carotid vulnerable plaques. However, most of previous studies utilized maximum or averaged value of hemodynamic measurements which is not an ideal representative of hemodynamic patterns. This study aimed to investigate the association of slice-based and time-specific hemodynamic measurements with carotid vulnerable plaque using magnetic resonance (MR) vessel wall imaging and histology. METHODS: Thirty-two patients (mean age: 63.9±8.1 years; 25 males) with carotid atherosclerotic stenosis (≥50% stenosis) referred to carotid endarterectomy were recruited and underwent MR vessel wall imaging. Carotid plaque burden was evaluated on MR images and vulnerable plaque features including calcification, lipid-rich necrotic core, and intra-plaque hemorrhage (IPH) were identified by histology. The slice-based and time-specific hemodynamic measurements were extracted from computational fluid dynamics simulation of 3D carotid arterial model. Correlation coefficients between hemodynamic measurements and carotid plaque features were calculated and the logistic regressions with generalized estimating equation (GEE) were conducted. The value in discriminating carotid vulnerable plaque features was determined by receiver-operating-characteristic analysis. RESULTS: Of 102 MR-histology matched slices from 32 patients, time-averaged wall shear stress (TAWSS) (r=0.263, p=0.008), oscillatory shear index (OSI) (r=-0.374, p<0.001), and peakWSS (r=0.232, p=0.019) were significantly associated with carotid IPH. The logistic regression with GEE revealed that peakWSS (OR, 1.206; 95% CI, 1.026-1.418; p, 0.023) and TAWSS (OR, 0.364, 95% CI, 0.138-0.959; p, 0.041) were significantly associated with presence of IPH after adjusting for age and BMI. In discriminating carotid IPH, the AUC of TAWSS, OSI, combined TAWSS with maximum wall thickness (MWT) and combined OSI with MWT was 0.656, 0.722, 0.761, and 0.764, respectively. CONCLUSIONS: Slice-based and time-specific hemodynamic characteristics could effectively discriminate carotid IPH. Combination of hemodynamic measurements with carotid plaque burden might be a stronger indicator for carotid vulnerable plaque features than each measurement alone.

miR-199a Is Upregulated in GDM Targeting the MeCP2-Trpc3 Pathway.

Gestational diabetes mellitus (GDM), the most common medical pregnancy complication, has become a growing problem. More and more studies have shown that microRNAs are closely related to metabolic processes. The purpose of this paper is to investigate the role of up-regulation of miR-199a-5p expression in GDM. We found that miR-199a-5p was significantly up-regulated in the placenta of GDM patients compared with normal pregnant women, and expressed in placental villi. miR-199a-5p can regulate the glucose pathway by inhibiting the expression of methyl CpG-binding protein 2 (MeCP2) and down-regulating canonical transient receptor potential 3 (Trpc3). This suggests that miR-199a-5p may regulate the glucose pathway by regulating methylation levels, leading to the occurrence of GDM.

Enhancer selection dictates gene expression responses in remote organs during tissue regeneration.

Acute trauma stimulates local repair mechanisms but can also impact structures distant from the injury, for example through the activity of circulating factors. To study the responses of remote tissues during tissue regeneration, we profiled transcriptomes of zebrafish brains after experimental cardiac damage. We found that the transcription factor gene cebpd was upregulated remotely in brain ependymal cells as well as kidney tubular cells, in addition to its local induction in epicardial cells. cebpd mutations altered both local and distant cardiac injury responses, altering the cycling of epicardial cells as well as exchange between distant fluid compartments. Genome-wide profiling and transgenesis identified a hormone-responsive enhancer near cebpd that exists in a permissive state, enabling rapid gene expression in heart, brain and kidney after cardiac injury. Deletion of this sequence selectively abolished cebpd induction in remote tissues and disrupted fluid regulation after injury, without affecting its local cardiac expression response. Our findings suggest a model to broaden gene function during regeneration in which enhancer regulatory elements define short- and long-range expression responses to injury.

Identification of enhancer regulatory elements that direct epicardial gene expression during zebrafish heart regeneration.

The epicardium is a mesothelial tissue layer that envelops the heart. Cardiac injury activates dynamic gene expression programs in epicardial tissue, which in zebrafish enables subsequent regeneration through paracrine and vascularizing effects. To identify tissue regeneration enhancer elements (TREEs) that control injury-induced epicardial gene expression during heart regeneration, we profiled transcriptomes and chromatin accessibility in epicardial cells purified from regenerating zebrafish hearts. We identified hundreds of candidate TREEs, which are defined by increased chromatin accessibility of non-coding elements near genes with increased expression during regeneration. Several of these candidate TREEs were incorporated into stable transgenic lines, with five out of six elements directing injury-induced epicardial expression but not ontogenetic epicardial expression in larval hearts. Whereas two independent TREEs linked to the gene gnai3 showed similar functional features of gene regulation in transgenic lines, two independent ncam1a-linked TREEs directed distinct spatiotemporal domains of epicardial gene expression. Thus, multiple TREEs linked to a regeneration gene can possess either matching or complementary regulatory controls. Our study provides a new resource and principles for understanding the regulation of epicardial genetic programs during heart regeneration. This article has an associated 'The people behind the papers' interview.

Diagnostic accuracy of cardiopulmonary ultrasound for pulmonary embolism: A systematic review and meta-analysis.

BACKGROUND: Cardiopulmonary ultrasound has shown varying results in the diagnosis of pulmonary embolism patients around the world. Hence, the current review was done to assess the diagnostic accuracy of cardiopulmonary ultrasound for diagnosis of pulmonary embolism among suspected patients. METHODS: We conducted a systematic search for all studies reporting the diagnostic accuracy of cardiopulmonary ultrasound for pulmonary embolism in the databases of MEDLINE, EMBASE, MEDLINE, SCOPUS, and Cochrane library from inception till May 2021. Meta-analysis was performed using STATA software "midas" package. RESULTS: Ten studies with 4216 patients were included. The pooled sensitivity was 77% (95% CI, 50-92%) and specificity was 99% (95% CI, 97-100%), respectively. The pooled DOR was 382 (95% CI, 77-1883). Pooled LRP was 90 (95% CI, 24-326) and pooled LRN was .23 (.09-.58). There was significant heterogeneity found with the outcome with significant chi-square test and I2 statistic > 75%. CONCLUSION: Cardiopulmonary ultrasound has the ability to be used as an adjunct to CTPA especially in resource constrained settings. Further reviews comparing multiple non-invasive imaging modalities are required to pick the best tool for diagnosis of pulmonary embolism.

Utility of Dual-Layer Spectral Detector CTA to Characterize Carotid Atherosclerotic Plaque Components: An Imaging-Histopathology Comparison in Patients Undergoing Endarterectomy.

BACKGROUND. The composition of noncalcified portions of carotid atherosclerotic plaque is an important marker of plaque vulnerability and ischemia risk. OBJECTIVE. The purpose of this study was to assess the utility of dual-layer spectral detector CTA (DLCTA) parameters for characterization of carotid plaque components with histologic results from carotid endarterectomy as the reference. METHODS. Seven patients (five men, two women; mean age, 61.6 ± 8.5 [SD] years) with carotid plaque awaiting carotid endarterectomy were prospectively enrolled and underwent preoperative supraaortic DLCTA. A neuroradiologist and pathologist performed joint slice-by-slice review of histologic slices of resected plaques and CTA images. With the use of anatomic landmarks, ROIs were placed on noncalcified components (lipid-rich necrotic core [LRNC], intraplaque hemorrhage [IPH], fibrous tissue, loose matrix) on CTA images and compared with corresponding histologic slices. For each ROI, attenuation was recorded for conventional polyenergetic images (CTPI) and virtual monoenergetic images with energy ranging from 40 to 140 keV (CT40-140keV), attenuation spectrum curve slope was calculated, and Z-effective value (representing effective atomic number) was recorded. DLCTA parameters were compared among plaque components. RESULTS. Seven plaques with a total of 65 slices and 364 ROIs (159 fibrous tissue, 96 LRNC, 86 loose matrix, 23 IPH) were analyzed. All parameters (CTPI, CT40-140keV, slope from 40 to 140 keV, Z-effective value) had significant differences between LRNC and the other components (all p < .001). For example, mean CTPI was 37.1 ± 15.1 HU for LRNC, 58.4 ± 21.6 HU for IPH, 69.7 ± 20.5 HU for fibrous tissue, and 69.6 ± 19.6 HU for loose matrix. Mean CT40keV was 28.1 ± 36.7 HU for LRNC, 87.5 ± 48.9 HU for IPH, 106.3 ± 47.5 HU for fibrous tissue, and 102.6 ± 48.0 HU for loose matrix. AUC for differentiating LRNC from other components was highest (0.945) for CT40kev and decreased with higher energy; AUC for CTPI was 0.908. CT40kev also had highest accuracy (90.4%); at a cutoff of 55.7 HU, CT40kev had 88.5% sensitivity and 91.0% specificity. For differentiating IPH from fibrous tissue and loose matrix, AUC was highest at 0.652 for CTPI and 0.645 for CT40kev. CONCLUSION. DLCTA showed strong performance in differentiating LRNC from other noncalcified plaque components; CT40kev had highest accuracy, outperforming CTPI. CLINICAL IMPACT. DLCTA parameters may help characterize carotid plaque composition as a marker of vulnerable plaque and ischemia risk.

Pertussis toxin-induced inflammatory response exacerbates intracerebral haemorrhage and ischaemic stroke in mice.

BACKGROUND: Stroke is a devastating disease, including intracerebral haemorrhage (ICH) and ischaemic stroke. Emerging evidences indicate that systemic inflammatory cascades after stroke contribute to brain damage. However, the direct effects and features of systemic inflammation on brain injury, especially comparing between ischaemic and haemorrhagic stroke, are still obscure. METHODS: Pertussis toxin (PT) was used to build a pro-inflammatory milieu after ICH and ischaemic stroke in mouse model. The neurodeficits, stroke lesion, immune response and blood-brain barrier (BBB) destruction were assessed. RESULTS: In ICH mouse model, PT-induced systemic inflammation exacerbated neurological deficits, and enlarged haemorrhage lesion and perihaematomal oedema. We also found promoted leucocyte infiltration and inflammatory cytokine release into the brain after PT treatment. Moreover, the integrity of the BBB was further disrupted after receiving PT. Furthermore, we demonstrated that PT enhanced brain inflammation and aggravated stroke severity in middle cerebral artery occlusion mouse model. CONCLUSIONS: Our results suggest that PT increases inflammatory response that exacerbates brain injury after ICH or ischaemic stroke in mouse model.

Deficiency of Intellectual Disability-Related Gene Brpf1 Attenuated Hippocampal Excitatory Synaptic Transmission and Impaired Spatial Learning and Memory Ability.

Patients with monoallelic bromodomain and PHD finger-containing protein 1 (BRPF1) mutations showed intellectual disability. The hippocampus has essential roles in learning and memory. Our previous work indicated that Brpf1 was specifically and strongly expressed in the hippocampus from the perinatal period to adulthood. We hypothesized that mouse Brpf1 plays critical roles in the morphology and function of hippocampal neurons, and its deficiency leads to learning and memory deficits. To test this, we performed immunofluorescence, whole-cell patch clamp, and mRNA-Seq on shBrpf1-infected primary cultured hippocampal neurons to study the effect of Brpf1 knockdown on neuronal morphology, electrophysiological characteristics, and gene regulation. In addition, we performed stereotactic injection into adult mouse hippocampus to knock down Brpf1 in vivo and examined the learning and memory ability by Morris water maze. We found that mild knockdown of Brpf1 reduced mEPSC frequency of cultured hippocampal neurons, before any significant changes of dendritic morphology showed. We also found that Brpf1 mild knockdown in the hippocampus showed a decreasing trend on the spatial learning and memory ability of mice. Finally, mRNA-Seq analyses showed that genes related to learning, memory, and synaptic transmission (such as C1ql1, Gpr17, Htr1d, Glra1, Cxcl10, and Grin2a) were dysregulated upon Brpf1 knockdown. Our results showed that Brpf1 mild knockdown attenuated hippocampal excitatory synaptic transmission and reduced spatial learning and memory ability, which helps explain the symptoms of patients with BRPF1 mutations.

SCG3 Protein Expression in Glioma Associates With less Malignancy and Favorable Clinical Outcomes.

Introduction: Secretogranin III (SCG3) physiologically participates in neurotransmitter storage/transport and is widely expressed in neuroendocrine tumors. However, there is no report on SCG3 protein expression in gliomas. Methods: The method of immunohistochemical staining on a glioma tissue microarray was utilized to detect SCG3 protein expression and investigate the correlations of its expression with clinicopathological and genetic features in gliomas. The RNA-seq data of SCG3 in The Cancer Genome Atlas database was exploited to explore these correlations at the transcriptional level. Results: There were 57.5% (130/226) glioma cases having SCG3 cytoplasmic staining in the tissue microarray. SCG3 expression inversely correlated with malignancy grade at both transcriptional and protein levels. The highest level was observed in oligodendroglial tumors, especially in oligodendrogliomas (ODs) with IDH-mutation/1p19q-codeletion. The lowest SCG3 expression was observed in glioblastomas (GBMs), especially in the mesenchymal subtype. Nearly a half of GBM cases (44.4%, 64/144) had any discernible SCG3 staining, and were defined as SCG3-positive by the microarray study. SCG3-positive GBM cases exhibited improved overall survival as compared with the SCG3-negative cases (29.3 vs. 14.5 months; Hazard ratio, 0.364; 95% CI, 0.216-0.612; p < 0.001). A multivariate Cox regression analysis also revealed SCG3 positivity as an independent favorable prognosticator in GBM patients. Conclusion: SCG3 protein expression inversely correlates with glioma malignancy and predicts favorable outcomes in GBM patients.

The effects of CPET-guided cardiac rehabilitation on the cardiopulmonary function, the exercise endurance, and the NT-proBNP and hscTnT levels in CHF patients.

OBJECTIVE: To explore the effects of cardiopulmonary exercise testing (CPET) on the cardiopulmonary function, the exercise endurance, and the NT-proBNP and hscTnT levels in chronic heart failure (CHF) patients. METHODS: Altogether 98 patients with CHF were randomly divided into a control group and a CPET group, with 49 cases in each group. The control group was administered routine treatment, and the CPET group was administered CPET cardiac rehabilitation training in addition to the routine treatment. Heart and lung function, exercise endurance, and the peripheral blood NTproBNP, hscTnT, and CRP levels were observed. The patients' quality of life, anxiety, and depression were observed using the scale. RESULTS: After the treatment, the left ventricular end systolic diameters (LVESD) and the left ventricular end diastolic diameters (LVEDD) were significantly decreased, the left ventricular ejection fractions (LVEF), the stroke volumes (SV), and the CI levels were significantly increased, and there were significant differences in these indexes between the CPET group and the control group (all P<0.05). After the treatment, the carbon dioxide ventilation equivalent slope (VE/VCO2slop) decreased significantly, the peak oxygen consumption (peakVO2) and anaerobic threshold oxygen consumption (VO2AT) levels increased significantly, and there were significant differences in these indicators between the CPET group and the control group (all P<0.05). Compared with the control group, the exercise endurance, the maximum oxygen uptake capacity (VO2max), the maximum power, the exhaustion times, and the six-minute walking test (6MWT) levels in the CPET group increased significantly (all P<0.05). After the treatment, the N-terminal precursor brain natriuretic peptide (NTproBNP), the high sensitivity cardiac troponin (hscTnT), and the C-reactive protein (CRP) levels in the two groups were decreased compared with their pre-treatment levels, and there were significant differences in these indexes between the CPET group and the control group (all P<0.05). After the treatment, the Minnesota living with heart failure questionnaire (MLHFQ), the self-rating anxiety scale (SAS), and the self-rating depression scale (SDS) scores in the two groups were significantly lower than they were before the treatment, and there were significant differences in the two scores between the CPET group and the control group (all P<0.05). CONCLUSION: CPET for patients with CHF helps increase heart and lung function, improves exercise endurance, reduces the NT-proBNP and hscTnT levels, and improves patients' quality of life.

Deficiency of intellectual disability-related gene Brpf1 reduced inhibitory neurotransmission in MGE-derived GABAergic interneurons.

Intellectual disability is closely related to impaired GABA neurotransmission. Brpf1 was specifically expressed in medial ganglionic eminence (MGE), a developmental niche of GABAergic interneurons, and patients with BRPF1 mutations showed intellectual disability. To test its role in the development and function of MGE-derived GABAergic interneurons, we performed immunofluorescence staining, whole-cell patch-clamp, MGE transplantation, and mRNA-Seq to understand its effect on neuronal differentiation, dendritic morphology, electrophysiology, migration, and gene regulation, using mouse MGE-derived GABAergic interneurons infected with AAV-shBrpf1. The results showed that Brpf1 knockdown had a decreasing trend, although not significant, on the differentiation of GABAergic interneurons into parvalbumin+ interneurons. Moreover, increased firing threshold, decreased number of evoked action potentials, and a reduced amplitude of miniature inhibitory postsynaptic currents were observed before any significant change of MAP2+ dendritic morphology and in vivo migration ability appeared. Finally, mRNA-Seq analysis revealed that genes related to neurodevelopment and synaptic transmission such as Map2k7 were dysregulated. Our results demonstrated a key role of Brpf1 in inhibitory neurotransmission and related gene expression of GABAergic interneurons.

Microfluidic Devices for Monitoring the Root Morphology of Arabidopsis Thaliana in situ.

Plant roots play critical roles in absorbing nutrients for the growth and development of plants as well as adapting different environments. Currently, there is no satisfactory way to track dynamic information when studying roots at the high temporal and spatial resolution. Herein, a simple microfluidic device with crossed microchannels was utilized for a microscopic investigation of Arabidopsis thaliana roots in situ. Our experimental results showed that the microfluidic system combined with a microscope could be conveniently utilized for the quantification of primary roots and root hairs with a change of micrometers within a time of minutes. Using the same approach, the influences of high salinity stress could also be investigated on different parts of roots, including the root cap, meristematic zone, elongation zone, mature zone, and root hairs. More importantly, the growth of roots and root hairs could be quantified and compared in a solution of abscisic acid and indole-3-acetic acid, respectively. Our study suggested that the microfluidic system could become a powerful tool for the quantitative investigation of Arabidopsis thaliana roots.

Ex Vivo Techniques to Study Heart Regeneration in Zebrafish.

Due to its pronounced regenerative capacity, the zebrafish heart represents an advantageous model system for exploring the cellular and molecular mechanisms of cardiac regeneration. Upon injury, the epicardium, the outermost mesothelial tissue layer of vertebrate hearts, serves dual purposes in the regenerating heart as both a signaling center and a source for crucial cell types. Traditional in vivo genetic approaches to study heart regeneration can be time consuming and are not applicable to large-scale approaches and live surveillance of cellular behaviors. Here, we demonstrate ex vivo methods to culture, maintain, and study the regenerative responses of epicardial tissue in excised zebrafish hearts. Epicardial cell proliferation and migration are monitored in real time after uninjured or injured hearts are excised, washed, and cultured for up to 30 days. In addition to these techniques, we describe ex vivo genetic ablation of the epicardium, cell proliferation assays, partial ventricular explant culturing, and chemical screening.