Integrative analysis of long isoform sequencing and functional data identifies distinct cortical layer neuronal subtypes derived from human iPSCs.

Generation of human induced pluripotent stem cells (iPSCs) through reprogramming was a transformational change in the field of regenerative medicine that led to new possibilities for drug discovery and cell replacement therapy. Several protocols have been established to differentiate hiPSCs into neuronal lineages. However, low differentiation efficiency is one of the major drawbacks of these approaches. Here, we compared the efficiency of two methods of neuronal differentiation from iPSCs cultured in two different culture media, StemFlex Medium (SFM) and Essential 8 Medium (E8M). The results indicated that iPSCs cultured in E8M efficiently generated different types of neurons in a shorter time and without the growth of undifferentiated nonneuronal cells in the culture as compared with those generated from iPSCs in SFM. Furthermore, these neurons were validated as functional units immunocytochemically by confirming the expression of mature neuronal markers (i.e., NeuN, ß tubulin, and Synapsin I) and whole cell patch-clamp recordings. Long-read single-cell RNA sequencing confirms the presence of upper and deep layer cortical layer excitatory and inhibitory neuronal subtypes in addition to small populations of GABAergic neurons in day 30 neuronal cultures. Pathway analysis indicated that our protocol triggers the signaling transcriptional networks important for the process of neuronal differentiation in vivo.NEW & NOTEWORTHY Low differentiation efficiency is one of the major drawbacks of the existing protocols to differentiate iPSCs into neuronal lineages. Here, we present time-efficient and robust approach of neuronal differentiation leading to the generation of functional brain units, cortical layer neurons. We found iPSCs cultured in Essential 8 media (E8M) resulted in neuronal differentiation without the signs of growth of spontaneously differentiated cells in culture at any point in 35 days compared with Stemflex media (SFM).

SNCA and TPPP transcripts increase in oligodendroglial cytoplasmic inclusions in multiple system atrophy.

Multiple system atrophy (MSA) is characterized by glial cytoplasmic inclusions (GCIs) containing aggregated α-synuclein (α-syn) in oligodendrocytes. The origin of α-syn accumulation in GCIs is unclear, in particular whether abnormal α-syn aggregates result from the abnormal elevation of endogenous α-syn expression in MSA or ingested from the neuronal source. Tubulin polymerization promoting protein (TPPP) has been reported to play a crucial role in developing GCI pathology. Here, the total cell body, nucleus, and cytoplasmic area density of SNCA and TPPP transcripts in neurons and oligodendrocytes with and without various α-syn pathologies in the pontine base in autopsy cases of MSA (n = 4) and controls (n = 2) were evaluated using RNAscope with immunofluorescence. Single-nucleus RNA-sequencing data for TPPP was evaluated using control frontal cortex (n = 3). SNCA and TPPP transcripts were present in the nucleus and cytoplasm of oligodendrocytes in both controls and diseased, with higher area density in GCIs and glial nuclear inclusions in MSA. Area densities of SNCA and TPPP transcripts were lower in neurons showing cytoplasmic inclusions in MSA. Indeed, TPPP transcripts were unexpectedly found in neurons, while the anti-TPPP antibody failed to detect immunoreactivity. Single-nucleus RNA-sequencing revealed significant TPPP transcript expression predominantly in oligodendrocytes, but also in excitatory and inhibitory neurons. This study addressed the unclear origin of accumulated α-syn in GCIs, proposing that the elevation of SNCA transcripts may supply templates for misfolded α-syn. In addition, the parallel behavior of TPPP and SNCA transcripts in GCI development highlights their potential synergistic contribution to inclusion formation. In conclusion, this study advances our understanding of MSA pathogenesis, offers insights into the dynamics of SNCA and TPPP transcripts in inclusion formation, and proposes regulating their transcripts for future molecular therapy to MSA.

Genomic Insights from a Deeply Phenotyped Highly Consanguineous Neurodevelopmental Disorders Cohort.

PURPOSE: The genetic underpinning of neurodevelopmental disorders (NDDs) in diverse ethnic populations, especially those with high rates of consanguinity, remains largely unexplored. Here, we aim to elucidate genomic insight from 576 well phenotyped and highly consanguineous (16%) NDD cohort. METHODS: We employed chromosomal microarray (CMA; N:247), exome sequencing (ES; N:127), combined CMA and ES (N:202) and long-read genome sequencing to identify genetic etiology. Deep clinical multi-variate data was coupled with genomic variants for stratification analysis. RESULTS: Genetic diagnosis rates were 17% with CMA, 29.92% with ES, and 37.13% with combined CMA and ES. Notably, children of consanguineous parents showed a significantly higher diagnostic yield (p<0.01) compared to those from non-consanguineous parents. Among the ES-identified pathogenic variants, 36.19% (38/105) were novel, implicating 35 unique genes. Long-read sequencing of seizure participants unresolved by combined test identified expanded FMR1 trinucleotide repeats. Additionally, we identified two recurrent X-linked variants in the G6PD in 3.65% (12/329) of NDD participants. These variants were absent in large population control cohorts and cohort comprising neurodevelopmental and neuropsychiatric populations of European descendants, indicating a possible associated risk factor potentially resulting from ancient genetic drift. CONCLUSION: This study unveils unique clinical and genomic insights from a consanguinity rich Bangladeshi NDD cohort, highlighting a strong association of G6PD with NDD in this population.

Impact of Respiratory Viruses and SARS-CoV-2 on Febrile Seizures in Saudi Children: Insights into Etiologies, Gender, and Familial Associations.

BACKGROUND Childhood febrile seizures occur between 5 months and 6 years of age in children without a previous history of seizure and are associated with high temperature in the absence of intracranial infection. This retrospective study identified 71 children aged 6 months to 5 years with febrile seizures between 2017 and 2021 at a single center in Saudi Arabia and aimed to identify an association between common respiratory virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. MATERIAL AND METHODS Pediatric nasopharyngeal specimens were tested using a multiplex PCR respiratory panel detecting human coronaviruses (NL63, 229E, OC43, HKU1), influenza A/B, human adenovirus, parainfluenza viruses 1-4, respiratory syncytial virus, human metapneumovirus, rhinovirus/enterovirus, Middle East respiratory syndrome coronavirus, and, as of September 2021, SARS-CoV-2, confirmed using the Cepheid Xpert Xpress SARS-CoV2 RT-PCR kit. RESULTS In a cohort of 71 pediatric patients (median age, 19 months; 54.9% female), dominant pathogens included human rhinovirus/enterovirus (23.9%), influenza A/B (26.8%), and SARS-CoV-2 (14.1%). Concurrent infections were noted in 28.2%. Simple seizures occurred in 69%, and complex seizures in 31%. Females exhibited an 8.18-fold increased risk for complex seizures. Each additional fever day reduced complex seizure risk by 36%. Familial seizure history increased risk 8.76-fold. Human rhinovirus/enterovirus or parainfluenza infections inversely affected complex seizure likelihood compared with adenovirus. CONCLUSIONS In Saudi children with febrile seizures, distinct viral etiologies, sex, and familial links play pivotal roles. Given regional viral variations, region-tailored diagnostic and therapeutic strategies are paramount. A multicenter prospective cohort study is essential for comprehensive understanding.

Single-cell reconstruction and mutation enrichment analysis identifies dysregulated cardiomyocyte and endothelial cells in congenital heart disease.

Congenital heart disease (CHD) is one of the most prevalent neonatal congenital anomalies. To catalog the putative candidate CHD risk genes, we collected 16,349 variants [single-nucleotide variants (SNVs) and Indels] impacting 8,308 genes in 3,166 CHD cases for a comprehensive meta-analysis. Using American College of Medical Genetics (ACMG) guidelines, we excluded the 0.1% of benign/likely benign variants and the resulting dataset consisted of 83% predicted loss of function variants and 17% missense variants. Seventeen percent were de novo variants. A stepwise analysis identified 90 variant-enriched CHD genes, of which six (GPATCH1, NYNRIN, TCLD2, CEP95, MAP3K19, and TTC36) were novel candidate CHD genes. Single-cell transcriptome cluster reconstruction analysis on six CHD tissues and four controls revealed upregulation of the top 10 frequently mutated genes primarily in cardiomyocytes. NOTCH1 (highest number of variants) and MYH6 (highest number of recurrent variants) expression was elevated in endocardial cells and cardiomyocytes, respectively, and 60% of these gene variants were associated with tetralogy of Fallot and coarctation of the aorta, respectively. Pseudobulk analysis using the single-cell transcriptome revealed significant (P < 0.05) upregulation of both NOTCH1 (endocardial cells) and MYH6 (cardiomyocytes) in the control heart data. We observed nine different subpopulations of CHD heart cardiomyocytes of which only four were observed in the control heart. This is the first comprehensive meta-analysis combining genomics and CHD single-cell transcriptomics, identifying the most frequently mutated CHD genes, and demonstrating CHD gene heterogeneity, suggesting that multiple genes contribute to the phenotypic heterogeneity of CHD. Cardiomyocytes and endocardial cells are identified as major CHD-related cell types.NEW & NOTEWORTHY Congential heart disease (CHD) is one of the most prevalent neonatal congenital anomalies. We present a comprehensive analysis combining genomics and CHD single-cell transcriptome. Our study identifies 90 potential candidate CHD risk genes of which 6 are novel. The risk genes have heterogenous expression suggestive of multiple genes contributing to the phenotypic heterogeneity of CHD. Cardiomyocytes and endocardial cells are identified as major CHD-related cell types.

Overlapping pathogenic de novo CNVs in neurodevelopmental disorders and congenital anomalies impacting constraint genes regulating early development.

Neurodevelopmental disorders (NDDs) and congenital anomalies (CAs) are rare disorders with complex etiology. In this study, we investigated the less understood genomic overlap of copy number variants (CNVs) in two large cohorts of NDD and CA patients to identify de novo CNVs and candidate genes associated with both phenotypes. We analyzed clinical microarray CNV data from 10,620 NDD and 3176 CA cases annotated using Horizon platform of GenomeArc Analytics and applied rigorous downstream analysis to evaluate overlapping genes from NDD and CA CNVs. Out of 13,796 patients, only 195 cases contained 218 validated de novo CNVs. Eighteen percent (31/170) de novo CNVs in NDD cases and 40% (19/48) de novo CNVs in CA cases contained genomic overlaps impacting developmentally constraint genes. Seventy-nine constraint genes (10.1% non-OMIM entries) were found to have significantly enriched genomic overlap within rare de novo pathogenic deletions (P value = 0.01, OR = 1.58) and 45 constraint genes (13.3% non-OMIM entries) within rare de novo pathogenic duplications (P value = 0.01, OR = 1.97). Analysis of spatiotemporal transcriptome demonstrated both pathogenic deletion and duplication genes to be highly expressed during the prenatal stage in human developmental brain (P value = 4.95 X 10-6). From the list of overlapping genes, EHMT1, an interesting known NDD gene encompassed pathogenic deletion CNVs from both NDD and CA patients, whereas FAM189A1, and FSTL5 are new candidate genes from non-OMIM entries. In summary, we have identified constraint overlapping genes from CNVs (including de novo) in NDD and CA patients that have the potential to play a vital role in common disease etiology.

oFlowSeq: a quantitative approach to identify protein coding mutations affecting cell type enrichment using mosaic CRISPR-Cas9 edited cerebral organoids.

Cerebral organoids are comprised of diverse cell types found in the developing human brain, and can be leveraged in the identification of critical cell types perturbed by genetic risk variants in common, neuropsychiatric disorders. There is great interest in developing high-throughput technologies to associate genetic variants with cell types. Here, we describe a high-throughput, quantitative approach (oFlowSeq) by utilizing CRISPR-Cas9, FACS sorting, and next-generation sequencing. Using oFlowSeq, we found that deleterious mutations in autism-associated gene KCTD13 resulted in increased proportions of Nestin+ cells and decreased proportions of TRA-1-60+ cells within mosaic cerebral organoids. We further identified that a locus-wide CRISPR-Cas9 survey of another 18 genes in the 16p11.2 locus resulted in most genes with > 2% maximum editing efficiencies for short and long indels, suggesting a high feasibility for an unbiased, locus-wide experiment using oFlowSeq. Our approach presents a novel method to identify genotype-to-cell type imbalances in an unbiased, high-throughput, quantitative manner.

Separation of CTCs from WBCs using DEP-assisted inertial manipulation: A numerical study.

Isolation and detection of circulating tumor cells (CTCs) hold significant importance for the early diagnosis of cancer and the assessment of therapeutic strategies. However, the scarcity of CTCs among peripheral blood cells presents a major challenge to their detection. Additionally, a similar size range between CTCs and white blood cells (WBCs) makes conventional microfluidic platforms inadequate for the isolation of CTCs. To overcome these challenges, in this study, a novel inertial-dielectrophoretic microfluidic channel for size-independent, single-stage separation of CTCs from WBCs has been presented. The proposed device utilizes a spiral microchannel embedded with interdigitated electrodes. A numerical model is developed and validated to investigate the influence of various parameters related to the channel design, fluid flow, and electrode configuration. It was found that optimal separation of CTCs could be obtained at a relatively low voltage, termed the critical voltage. Furthermore, at the critical voltage of 7.5 V, the hybrid microchannel is demonstrated to be capable of separating CTCs from different WBC subtypes including granulocytes, monocytes, T-, and B-lymphocytes. The unique capabilities of the hybrid spiral microchannel allow for this size-independent isolation of CTCs from a mixture of WBCs. Overall, the proposed technique can be readily utilized for continuous and high-throughput separation of cancer cells.

Cell-specific MAPT gene expression is preserved in neuronal and glial tau cytopathologies in progressive supranuclear palsy.

Microtubule-associated protein tau (MAPT) aggregates in neurons, astrocytes and oligodendrocytes in a number of neurodegenerative diseases, including progressive supranuclear palsy (PSP). Tau is a target of therapy and the strategy includes either the elimination of pathological tau aggregates or reducing MAPT expression, and thus the amount of tau protein made to prevent its aggregation. Disease-associated tau affects brain regions in a sequential manner that includes cell-to-cell spreading. Involvement of glial cells that show tau aggregates is interpreted as glial cells taking up misfolded tau assuming that glial cells do not express enough MAPT. Although studies have evaluated MAPT expression in human brain tissue homogenates, it is not clear whether MAPT expression is compromised in cells accumulating pathological tau. To address these perplexing aspects of disease pathogenesis, this study used RNAscope combined with immunofluorescence (AT8), and single-nuclear(sn) RNAseq to systematically map and quantify MAPT expression dynamics across different cell types and brain regions in controls (n = 3) and evaluated whether tau cytopathology affects MAPT expression in PSP (n = 3). MAPT transcripts were detected in neurons, astrocytes and oligodendrocytes, and varied between brain regions and within each cell type, and were preserved in all cell types with tau aggregates in PSP. These results propose a complex scenario in all cell types, where, in addition to the ingested misfolded tau, the preserved cellular MAPT expression provides a pool for local protein production that can (1) be phosphorylated and aggregated, or (2) feed the seeding of ingested misfolded tau by providing physiological tau, both accentuating the pathological process. Since tau cytopathology does not compromise MAPT gene expression in PSP, a complete loss of tau protein expression as an early pathogenic component is less likely. These observations provide rationale for a dual approach to therapy by decreasing cellular MAPT expression and targeting removal of misfolded tau.

Left atrial appendage closure device outcomes among cirrhosis patients with atrial fibrillation: a United States National Cohort Study.

AIMS: Literature regarding outcomes associated with atrial fibrillation among cirrhosis patients who had left atrial appendage occlusion (LAAO) device procedure is limited. We aim to evaluate the in-hospital clinical outcomes and 30-day readmissions among LAAO with and without cirrhosis. METHODS AND RESULTS: We performed a retrospective study of all hospitalizations associated with the LAAO procedure, using the Nationwide Readmissions Database for the years 2016-19. Primary outcomes were in-hospital clinical outcomes and 30-day readmissions. A total of 54 897 index hospitalizations for LAAO (female 41.8%) were reported. Of these, 905(1.65%) had cirrhosis. Gastrointestinal (GI) bleeding was reported in 44 (4.9%) vs. 1606 (2.97%) and coagulopathy in 21 (2.3%) vs. 521 (0.96%) in cirrhosis and without-cirrhosis groups, respectively. A total of 872 (1.59%) patients needed blood transfusion, 24 (2.7%) vs. 848(1.57%) in cirrhosis vs. without-cirrhosis groups (P = 0.047). Fresh frozen plasma (FFP) transfusion was reported among 888 (1.62%), with cirrhosis 26 (3%) vs. without cirrhosis 862 (1.6%) (P = 0.05). On adjusted multivariate logistic regression analysis, acute kidney injury, coagulopathy, FFP transfusion, and blood transfusion were strongly associated with cirrhosis, and GI bleeding, ischaemic stroke, and intracranial haemorrhage were not associated with cirrhosis. Readmissions in 30 days were 5028 (9.18%), 167 (18.5%) in the cirrhosis group and 4861 (9%) without-cirrhosis group (P = 0.01). On multivariate Cox regression, CHA2DS2-Vasc score of six was significantly associated with 30-day readmission compared with other scores [hazard ratio 2.24; 95% confidence interval (1.58-3.16); P < 0.001]. CONCLUSION: Left atrial appendage occlusion procedure in patients with cirrhosis had relatively similar GI bleeding and stroke rates, however, had higher rates of 30-day readmission. A higher CHA2DS2-Vasc score was more likely to be associated with 30-day readmissions and hence would help in discharge planning. The long-term safety and efficacy of LAAO in the cirrhosis population need to be demonstrated.