Epigenetic regulation of human-specific gene expression in the prefrontal cortex.

BACKGROUND: Changes in gene expression levels during brain development are thought to have played an important role in the evolution of human cognition. With the advent of high-throughput sequencing technologies, changes in brain developmental expression patterns, as well as human-specific brain gene expression, have been characterized. However, interpreting the origin of evolutionarily advanced cognition in human brains requires a deeper understanding of the regulation of gene expression, including the epigenomic context, along the primate genome. Here, we used chromatin immunoprecipitation sequencing (ChIP-seq) to measure the genome-wide profiles of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 acetylation (H3K27ac), both of which are associated with transcriptional activation in the prefrontal cortex of humans, chimpanzees, and rhesus macaques. RESULTS: We found a discrete functional association, in which H3K4me3HP gain was significantly associated with myelination assembly and signaling transmission, while H3K4me3HP loss played a vital role in synaptic activity. Moreover, H3K27acHP gain was enriched in interneuron and oligodendrocyte markers, and H3K27acHP loss was enriched in CA1 pyramidal neuron markers. Using strand-specific RNA sequencing (ssRNA-seq), we first demonstrated that approximately 7 and 2% of human-specific expressed genes were epigenetically marked by H3K4me3HP and H3K27acHP, respectively, providing robust support for causal involvement of histones in gene expression. We also revealed the co-activation role of epigenetic modification and transcription factors in human-specific transcriptome evolution. Mechanistically, histone-modifying enzymes at least partially contribute to an epigenetic disturbance among primates, especially for the H3K27ac epigenomic marker. In line with this, peaks enriched in the macaque lineage were found to be driven by upregulated acetyl enzymes. CONCLUSIONS: Our results comprehensively elucidated a causal species-specific gene-histone-enzyme landscape in the prefrontal cortex and highlighted the regulatory interaction that drove transcriptional activation.

Single-cell RNA-sequencing of zebrafish hair cells reveals novel genes potentially involved in hearing loss.

Hair cells play key roles in hearing and balance, and hair cell loss would result in hearing loss or vestibular dysfunction. Cellular and molecular research in hair cell biology provides us a better understanding of hearing and deafness. Zebrafish, owing to their hair cell-enriched organs, have been widely applied in hair cell-related research worldwide. Similar to mammals, zebrafish have inner ear hair cells. In addition, they also have lateral line neuromast hair cells. These different types of hair cells vary in morphology and function. However, systematic analysis of their molecular characteristics remains lacking. In this study, we analyzed the GFP+ cells isolated from Tg(Brn3c:mGFP) larvae with GFP expression in all hair cells using single-cell RNA-sequencing (scRNA-seq). Three subtypes of hair cells, namely macula hair cell (MHC), crista hair cell (CHC), and neuromast hair cell (NHC), were characterized and validated by whole-mount in situ hybridization analysis of marker genes. The hair cell scRNA-seq data revealed hair cell-specific genes, including hearing loss genes that have been identified in humans and novel genes potentially involved in hair cell formation and function. Two novel genes were discovered to specifically function in NHCs and MHCs, corresponding to their specific expression in NHCs and MHCs. This study allows us to understand the specific genes in hair cell subpopulations of zebrafish, which will shed light on the genetics of both human vestibular and cochlear hair cell function.

The repertoire of testicular extracellular vesicle cargoes and their involvement in inter-compartmental communication associated with spermatogenesis.

BACKGROUND: Spermatogenesis is regulated by a complex network of intercellular communication processes. Extracellular vesicles (EVs) are one of the important mediators in intercellular communication. Previous reports have demonstrated the involvement of EVs from the epididymis and prostate in sperm maturation and function. However, the presence of EVs in the testis and their potential involvement in spermatogenesis has not been explored. Here, we have established a testis dissociation protocol that allows the isolation and characterization of testicular EVs. RESULTS: We show that testicular EVs are specifically and efficiently taken up by somatic cells and germ cells, including the spermatozoa in the interstitial space and the seminiferous tubule compartments. We profiled the proteome of testicular EVs and probed the cell types that release them, revealing the potential contributions from the Leydig cells and testicular macrophages. Moreover, we sequenced the small RNA cargoes of testicular EVs and identified sets of small non-coding RNAs that were overlooked in the testis transcriptome. Selected miRNA candidates in testicular EVs were found in sperm RNA payload and demonstrated specific resistance towards ribonuclease A independent of the vesicle membrane. Small molecule inhibition of EV secretion perturbed spermatogenesis via inter-compartmental communication. CONCLUSIONS: Together, our study provides a valuable resource on the repertoire of cargoes carried by testicular EVs and uncovers a physiological function of testicular EVs in inter-compartmental communication associated to spermatogenesis.

Profiling ATM regulated genes in Drosophila at physiological condition and after ionizing radiation.

BACKGROUND: ATM (ataxia-telangiectasia mutated) protein kinase is highly conserved in metazoan, and plays a critical role at DNA damage response, oxidative stress, metabolic stress, immunity, RNA biogenesis etc. Systemic profiling of ATM regulated genes, including protein-coding genes, miRNAs, and long non-coding RNAs, will greatly improve our understanding of ATM functions and its regulation.  RESULTS: 1) differentially expressed protein-coding genes, miRNAs, and long non-coding RNAs in atm mutated flies were identified at physiological condition and after X-ray irradiation. 2) functions of differentially expressed genes in atm mutated flies, regardless of protein-coding genes or non-coding RNAs, are closely related with metabolic process, immune response, DNA damage response or oxidative stress. 3) these phenomena are persistent after irradiation. 4) there is a cross-talk regulation towards miRNAs by ATM, E2f1, and p53 during development and after irradiation. 5) knock-out flies or knock-down flies of most irradiation-induced miRNAs were sensitive to ionizing radiation. CONCLUSIONS: We provide a valuable resource of protein-coding genes, miRNAs, and long non-coding RNAs, for understanding ATM functions and regulations. Our work provides the new evidence of inter-dependence among ATM-E2F1-p53 for the regulation of miRNAs.

Deep Phenotyping on Electronic Health Records Facilitates Genetic Diagnosis by Clinical Exomes.

Integration of detailed phenotype information with genetic data is well established to facilitate accurate diagnosis of hereditary disorders. As a rich source of phenotype information, electronic health records (EHRs) promise to empower diagnostic variant interpretation. However, how to accurately and efficiently extract phenotypes from heterogeneous EHR narratives remains a challenge. Here, we present EHR-Phenolyzer, a high-throughput EHR framework for extracting and analyzing phenotypes. EHR-Phenolyzer extracts and normalizes Human Phenotype Ontology (HPO) concepts from EHR narratives and then prioritizes genes with causal variants on the basis of the HPO-coded phenotype manifestations. We assessed EHR-Phenolyzer on 28 pediatric individuals with confirmed diagnoses of monogenic diseases and found that the genes with causal variants were ranked among the top 100 genes selected by EHR-Phenolyzer for 16/28 individuals (p < 2.2 × 10-16), supporting the value of phenotype-driven gene prioritization in diagnostic sequence interpretation. To assess the generalizability, we replicated this finding on an independent EHR dataset of ten individuals with a positive diagnosis from a different institution. We then assessed the broader utility by examining two additional EHR datasets, including 31 individuals who were suspected of having a Mendelian disease and underwent different types of genetic testing and 20 individuals with positive diagnoses of specific Mendelian etiologies of chronic kidney disease from exome sequencing. Finally, through several retrospective case studies, we demonstrated how combined analyses of genotype data and deep phenotype data from EHRs can expedite genetic diagnoses. In summary, EHR-Phenolyzer leverages EHR narratives to automate phenotype-driven analysis of clinical exomes or genomes, facilitating the broader implementation of genomic medicine.

Systematic profiling of early regulators during tissue regeneration using zebrafish model.

Great progresses have been made in comprehension of tissue regeneration process. However, one of the central questions in regeneration research remains to be deciphered is what factors initiate regenerative process. In present study, we focused on systematic profiling of early regulators in tissue regeneration via high-throughput screening on zebrafish caudal fin model. Firstly, 53 GO-annotated regeneration-related genes, which were specifically activated upon fin amputation, were identified according to the transcriptomic analysis. Moreover, qRT-PCR analysis of a couple of randomly selected genes from the aforementioned gene list validated our sequencing results. These studies confirmed the reliability of transcriptome sequencing analysis. Fibroblast growth factor 20a (fgf20a) is a key initial factor in the regeneration of zebrafish. Through a gene expression correlation analysis, we discovered a collection of 70 genes correlating with fgf20a, whose expression increased promptly at 2 days post amputation (dpa) and went down to the basal level until the completion of fin regeneration. In addition, two genes, socs3b and nppc, were chosen to investigate their functions during the fin regeneration. Inhibition of either of those genes significantly delayed the regenerative process. Taken together, we provided a simple and effective time-saving strategy that may serve as a tool for identifying early regulators in regeneration and identified 71 genes as early regulators of fin regeneration.

Deep Learning Methods for Heart Sounds Classification: A Systematic Review.

The automated classification of heart sounds plays a significant role in the diagnosis of cardiovascular diseases (CVDs). With the recent introduction of medical big data and artificial intelligence technology, there has been an increased focus on the development of deep learning approaches for heart sound classification. However, despite significant achievements in this field, there are still limitations due to insufficient data, inefficient training, and the unavailability of effective models. With the aim of improving the accuracy of heart sounds classification, an in-depth systematic review and an analysis of existing deep learning methods were performed in the present study, with an emphasis on the convolutional neural network (CNN) and recurrent neural network (RNN) methods developed over the last five years. This paper also discusses the challenges and expected future trends in the application of deep learning to heart sounds classification with the objective of providing an essential reference for further study.

Primate-specific endogenous retrovirus-driven transcription defines naive-like stem cells.

Naive embryonic stem cells hold great promise for research and therapeutics as they have broad and robust developmental potential. While such cells are readily derived from mouse blastocysts it has not been possible to isolate human equivalents easily, although human naive-like cells have been artificially generated (rather than extracted) by coercion of human primed embryonic stem cells by modifying culture conditions or through transgenic modification. Here we show that a sub-population within cultures of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) manifests key properties of naive state cells. These naive-like cells can be genetically tagged, and are associated with elevated transcription of HERVH, a primate-specific endogenous retrovirus. HERVH elements provide functional binding sites for a combination of naive pluripotency transcription factors, including LBP9, recently recognized as relevant to naivety in mice. LBP9-HERVH drives hESC-specific alternative and chimaeric transcripts, including pluripotency-modulating long non-coding RNAs. Disruption of LBP9, HERVH and HERVH-derived transcripts compromises self-renewal. These observations define HERVH expression as a hallmark of naive-like hESCs, and establish novel primate-specific transcriptional circuitry regulating pluripotency.

A ribosomal DNA-hosted microRNA regulates zebrafish embryonic angiogenesis.

MicroRNAs (miRNAs) are single-stranded small non-coding RNAs, generally 18-25 nucleotides in length, that act as repressors of gene expression. miRNAs are encoded by independent genes or processed from a variety of different RNA species. So far, there is no evidence showing that the ribosomal DNA-hosted microRNA is implicated in vertebrate development. Currently, we found a highly expressed small RNA hosted in ribosomal DNA was predicted as a novel miRNA, named miR-ntu1, in zebrafish endothelial cells by deep sequencing analysis. The miRNA was validated by custom-designed Taqman PCR, Northern Blot, and in silico analysis. Furthermore, we demonstrated that miR-ntu1 played a crucial role in zebrafish angiogenesis via modulation of Notch signaling. Our findings provide a notable case that a miRNA hosted in ribosomal DNA is involved in vertebrate development.

Single-cell RNA-Seq analysis reveals dynamic trajectories during mouse liver development.

BACKGROUND: The differentiation and maturation trajectories of fetal liver stem/progenitor cells (LSPCs) are not fully understood at single-cell resolution, and a priori knowledge of limited biomarkers could restrict trajectory tracking. RESULTS: We employed marker-free single-cell RNA-Seq to characterize comprehensive transcriptional profiles of 507 cells randomly selected from seven stages between embryonic day 11.5 and postnatal day 2.5 during mouse liver development, and also 52 Epcam-positive cholangiocytes from postnatal day 3.25 mouse livers. LSPCs in developing mouse livers were identified via marker-free transcriptomic profiling. Single-cell resolution dynamic developmental trajectories of LSPCs exhibited contiguous but discrete genetic control through transcription factors and signaling pathways. The gene expression profiles of cholangiocytes were more close to that of embryonic day 11.5 rather than other later staged LSPCs, cuing the fate decision stage of LSPCs. Our marker-free approach also allows systematic assessment and prediction of isolation biomarkers for LSPCs. CONCLUSIONS: Our data provide not only a valuable resource but also novel insights into the fate decision and transcriptional control of self-renewal, differentiation and maturation of LSPCs.

Conserved expression of lincRNA during human and macaque prefrontal cortex development and maturation.

The current annotation of the human genome includes more than 12,000 long intergenic noncoding RNAs (lincRNA). While a handful of lincRNA have been shown to play important regulatory roles, the functionality of most remains unclear. Here, we examined the expression conservation and putative functionality of lincRNA in human and macaque prefrontal cortex (PFC) development and maturation. We analyzed transcriptome sequence (RNA-seq) data from 38 human and 40 macaque individuals covering the entire postnatal development interval. Using the human data set, we detected the expression of 5835 lincRNA annotated in GENCODE and further identified 1888 novel lincRNA. Most of these lincRNA show low DNA sequence conservation, as well as low expression levels. Remarkably, developmental expression patterns of these lincRNA were as conserved between humans and macaques as those of protein-coding genes. Transfection of development-associated lincRNA into human SH-SY5Y cells affected gene expression, indicating their regulatory potential. In brain, expression of these putative target genes correlated with the expression of the corresponding lincRNA during human and macaque PFC development. These results support the potential functionality of lincRNA in primate PFC development.

A germline chromothripsis event stably segregating in 11 individuals through three generations.

PURPOSE: Parentally transmitted germ-line chromothripsis (G-CTH) has been identified in only a few cases. Most of these rearrangements were stably transmitted, in an unbalanced form, from a healthy mother to her child with congenital abnormalities probably caused by de novo copy-number changes of dosage sensitive genes. We describe a G-CTH transmitted through three generations in 11 healthy carriers. METHODS: Conventional cytogenetic analysis, mate-pair sequencing, and polymerase chain reaction (PCR) were used to identify the chromosome rearrangement and characterize the breakpoints in all three generations. RESULTS: We identified an apparently balanced translocation t(3;5), later shown to be a G-CTH, in all individuals of a three-generation family. The G-CTH stably segregated without occurrence of additional rearrangements; however, several spontaneous abortions were reported, possibly due to unbalanced transmission. Although seven protein-coding genes are interrupted, no clinical features can be definitively attributed to the affected genes. However, it can be speculated that truncation of one of these genes, encoding ataxia-telangiectasia and Rad3-related protein kinase (ATR), a key component of the DNA damage response, may be related to G-CTH formation. CONCLUSION: G-CTH rearrangements are not always associated with abnormal phenotypes and may be misinterpreted as balanced two-way translocations, suggesting that G-CTH is an underdiagnosed phenomenon.Genet Med 18 5, 494-500.

MicroRNA-driven developmental remodeling in the brain distinguishes humans from other primates.

While multiple studies have reported the accelerated evolution of brain gene expression in the human lineage, the mechanisms underlying such changes are unknown. Here, we address this issue from a developmental perspective, by analyzing mRNA and microRNA (miRNA) expression in two brain regions within macaques, chimpanzees, and humans throughout their lifespan. We find that constitutive gene expression divergence (species differences independent of age) is comparable between humans and chimpanzees. However, humans display a 3-5 times faster evolutionary rate in divergence of developmental patterns, compared to chimpanzees. Such accelerated evolution of human brain developmental patterns (i) cannot be explained by life-history changes among species, (ii) is twice as pronounced in the prefrontal cortex than the cerebellum, (iii) preferentially affects neuron-related genes, and (iv) unlike constitutive divergence does not depend on cis-regulatory changes, but might be driven by human-specific changes in expression of trans-acting regulators. We show that developmental profiles of miRNAs, as well as their target genes, show the fastest rates of human-specific evolutionary change, and using a combination of computational and experimental methods, we identify miR-92a, miR-454, and miR-320b as possible regulators of human-specific neural development. Our results suggest that different mechanisms underlie adaptive and neutral transcriptome divergence, and that changes in the expression of a few key regulators may have been a major driving force behind rapid evolution of the human brain.

Single-cell transcriptome profiling highlights the importance of telocyte, kallikrein genes, and alternative splicing in mouse testes aging.

Advancing healthcare for elderly men requires a deeper understanding of testicular aging processes. In this study, we conducted transcriptomic profiling of 43,323 testicular single cells from young and old mice, shedding light on 1032 telocytes-an underexplored testicular cell type in previous research. Our study unveiled 916 age-related differentially expressed genes (age-DEGs), with telocytes emerging as the cell type harboring the highest count of age-DEGs. Of particular interest, four genes (Klk1b21, Klk1b22, Klk1b24, Klk1b27) from the Kallikrein family, specifically expressed in Leydig cells, displayed down-regulation in aged testes. Moreover, cell-type-level splicing analyses unveiled 1838 age-related alternative splicing (AS) events. While we confirmed the presence of more age-DEGs in somatic cells compared to germ cells, unexpectedly, more age-related AS events were identified in germ cells. Further experimental validation highlighted 4930555F03Rik, a non-coding RNA gene exhibiting significant age-related AS changes. Our study represents the first age-related single-cell transcriptomic investigation of testicular telocytes and Kallikrein genes in Leydig cells, as well as the first delineation of cell-type-level AS dynamics during testicular aging in mice.

Classifying Heart-Sound Signals Based on CNN Trained on MelSpectrum and Log-MelSpectrum Features.

The intelligent classification of heart-sound signals can assist clinicians in the rapid diagnosis of cardiovascular diseases. Mel-frequency cepstral coefficients (MelSpectrums) and log Mel-frequency cepstral coefficients (Log-MelSpectrums) based on a short-time Fourier transform (STFT) can represent the temporal and spectral structures of original heart-sound signals. Recently, various systems based on convolutional neural networks (CNNs) trained on the MelSpectrum and Log-MelSpectrum of segmental heart-sound frames that outperform systems using handcrafted features have been presented and classified heart-sound signals accurately. However, there is no a priori evidence of the best input representation for classifying heart sounds when using CNN models. Therefore, in this study, the MelSpectrum and Log-MelSpectrum features of heart-sound signals combined with a mathematical model of cardiac-sound acquisition were analysed theoretically. Both the experimental results and theoretical analysis demonstrated that the Log-MelSpectrum features can reduce the classification difference between domains and improve the performance of CNNs for heart-sound classification.

Epididymis cell atlas in a patient with a sex development disorder and a novel NR5A1 gene mutation.

This study aims to characterize the cell atlas of the epididymis derived from a 46,XY disorders of sex development (DSD) patient with a novel heterozygous mutation of the nuclear receptor subfamily 5 group A member 1 (NR5A1) gene. Next-generation sequencing found a heterozygous c.124C>G mutation in NR5A1 that resulted in a p.Q42E missense mutation in the conserved DNA-binding domain of NR5A1. The patient demonstrated feminization of external genitalia and Tanner stage 1 breast development. The surgical procedure revealed a morphologically normal epididymis and vas deferens but a dysplastic testis. Microfluidic-based single-cell RNA sequencing (scRNA-seq) analysis found that the fibroblast cells were significantly increased (approximately 46.5%), whereas the number of main epididymal epithelial cells (approximately 9.2%), such as principal cells and basal cells, was dramatically decreased. Bioinformatics analysis of cell-cell communications and gene regulatory networks at the single-cell level inferred that epididymal epithelial cell loss and fibroblast occupation are associated with the epithelial-to-mesenchymal transition (EMT) process. The present study provides a cell atlas of the epididymis of a patient with 46,XY DSD and serves as an important resource for understanding the pathophysiology of DSD.

PUF60 promotes cell cycle and lung cancer progression by regulating alternative splicing of CDC25C.

Alternative splicing (AS) has been implicated in cell cycle regulation and cancer, but the underlying mechanisms are poorly understood. The poly(U)-binding splicing factor 60 (PUF60) is essential for embryonic development and is overexpressed in multiple types of cancer. Here, we report that PUF60 promotes mitotic cell cycle and lung cancer progression by controlling AS of the cell division cycle 25C (CDC25C). Systematic analysis of splicing factors deregulated in lung adenocarcinoma (LUAD) identifies that elevated copy number and expression of PUF60 correlate with poor prognosis. PUF60 depletion inhibits LUAD cell-cycle G2/M transition, cell proliferation, and tumor development. Mechanistically, PUF60 knockdown leads to exon skipping enriched in mitotic cell cycle genes, including CDC25C. Exon 3 skipping in the full-length CDC25C results in nonsense-mediated mRNA decay and a decrease of CDC25C protein, thereby inhibiting cell proliferation. This study establishes PUF60 as a cell cycle regulator and an oncogenic splicing factor in lung cancer.

The unique interplay of mitochondrial oxidative phosphorylation (OXPHOS) and immunity and its potential implication for the sex- and age-related morbidity of severe COVID-19 patients.

Aged male patients are more vulnerable to severe or critical symptoms of COVID-19, but the underlying mechanism remains elusive. In this study, we analyzed previously published scRNA-seq data from a large cohort of COVID-19 patients, castrated and regenerated mice, and bulk RNA-seq of a RNAi library of 400 genes, and revealed that both immunity and OXPHOS displayed cell-type-, sex-, and age-related variation in the severe or critical COVID-19 patients during disease progression, with a more prominent increase in immunity and decrease in OXPHOS in myeloid cells in the males relative to the females (60-69 years old). Male severe or critical patients above 70 years old were an exception in that the compromised negative correlation between OXPHOS and immunity in these patients was associated with its disordered transcriptional regulation. Finally, the expression levels of OXPHOS and androgens were revealed to be positively correlated, and the responses of macrophages to android fluctuation were more striking than other types of detected immune cells in the castrated mice model. Therefore, the interplay of OXPHOS and immunity displayed a cell-type-specific, age-related, and sex-biased pattern, and the underlying potential regulatory role of the hormonal milieu should not be neglected.

Single Cell Transcriptome Sequencing of Zebrafish Testis Revealed Novel Spermatogenesis Marker Genes and Stronger Leydig-Germ Cell Paracrine Interactions.

Spermatogenesis in testis is an important process for sexual reproduction, and worldwide about 10-15 percent of couples suffer from infertility. It is of importance to study spermatogenesis at single cell level in both of human and model organisms. Currently, single-cell RNA sequencing technologies (scRNA-seq) had been extensively applied to the study of cellular components and its gene regulations in the testes of different species, including human, monkey, mouse, and fly, but not in zebrafish. Zebrafish was a widely used model organism in biology and had been extensively used for the study of spermatogenesis in the previous studies. Therefore, it is also important to profile the transcriptome of zebrafish testis at single cell level. In this study, the transcriptomes of 14, 315 single cells from adult male zebrafish testes were profiled by scRNA-seq, and 10 cell populations were revealed, including Leydig cell, Sertoli cell, spermatogonia cell (SPG), spermatocyte, and spermatids. Notably, thousands of cell-type specific novel marker genes were identified, including sumo3b for SPG, krt18a.1 for Sertoli cells, larp1b and edrf1 for spermatids, which were also validated by RNA in situ hybridization experiments. Interestingly, through Ligand-Receptor (LR) analyses, zebrafish Leydig cells demonstrated stronger paracrine influence on germ cells than Sertoli cells. Overall, this study could be an important resource for the study of spermatogenesis in zebrafish and might also facilitate the study of the genes associated with human infertility through using zebrafish as a model organism.