The DNA of all eukaryotic organisms is packaged into nucleosomes, the basic repeating units of chromatin. The nucleosome consists of a histone octamer around which a DNA core is wrapped and the linker histone H1, which is associated with linker DNA. By altering the accessibility of DNA sequences, the nucleosome has profound effects on all DNA-dependent processes. Understanding the factors that influence nucleosome positioning is of great importance for the study of genomic control mechanisms. Transcription factors (TFs) have been suggested to play a role in nucleosome positioning in vivo.

Gene expression variation is a phenotypic trait of particular interest as it represents the initial link between genotype and other phenotypes. Analyzing how such variation apportions among and within groups allows for the evaluation of how genetic and environmental factors influence such traits. It also provides opportunities to identify genes and pathways that may have been influenced by non-neutral processes. Here we use a population genetics framework and next generation sequencing to evaluate how gene expression variation is apportioned among four human groups in a natural biological tissue, the placenta.

We reported previously that pre-programming mesenchymal stem cells with the GATA-4 gene increases significantly cell survival in an ischemic environment. In this study, we tested whether regulation of microRNAs and their target proteins was associated with the cytoprotective effects of GATA-4.

It was previously reported that mRNA expression levels in the prefrontal cortex at old age start to resemble pre-adult levels. Such expression reversals could imply loss of cellular identity in the aging brain, and provide a link between aging-related molecular changes and functional decline. Here we analyzed 19 brain transcriptome age-series datasets, comprising 17 diverse brain regions, to investigate the ubiquity and functional properties of expression reversal in the human brain. Across all 19 datasets, 25 genes were consistently up-regulated during postnatal development and down-regulated in aging, displaying an "up-down" pattern that was significant as determined by random permutations. In addition, 113 biological processes, including neuronal and synaptic functions, were consistently associated with genes showing an up-down tendency among all datasets. Genes up-regulated during in vitro neuronal differentiation also displayed a tendency for up-down reversal, although at levels comparable to other genes. We argue that reversals may not represent aging-related neuronal loss. Instead, expression reversals may be associated with aging-related accumulation of stochastic effects that lead to loss of functional and structural identity in neurons.

Lipids are essential to brain functions, yet they remain largely unexplored. Here we investigated the lipidome composition of prefrontal cortex gray matter in 396 cognitively healthy individuals with ages spanning 100 years, as well as 67 adult individuals diagnosed with autism (ASD), schizophrenia (SZ), and Down syndrome (DS). Of the 5024 detected lipids, 95% showed significant age-dependent concentration differences clustering into four temporal stages, and resulting in a gradual increase in membrane fluidity in individuals ranging from newborn to nonagenarian. Aging affects 14% of the brain lipidome with late-life changes starting predominantly at 50-55 years of age-a period of general metabolic transition. All three diseases alter the brain lipidome composition, leading-among other things-to a concentration decrease in glycerophospholipid metabolism and endocannabinoid signaling pathways. Lipid concentration decreases in SZ were further linked to genetic variants associated with disease, indicating the relevance of the lipidome changes to disease progression.

The TREX complex (TREX) plays key roles in nuclear export of mRNAs. However, little is known about its transcriptome-wide binding targets. We used individual cross-linking and immunoprecipitation (iCLIP) to identify the binding sites of ALYREF, an mRNA export adaptor in TREX, in human cells. Consistent with previous in vitro studies, ALYREF binds to a region near the 5' end of the mRNA in a CBP80-dependent manner. Unexpectedly, we identified PABPN1-dependent ALYREF binding near the 3' end of the mRNA. Furthermore, the 3' processing factor CstF64 directly interacts with ALYREF and is required for the overall binding of ALYREF on the mRNA. In addition, we found that numerous middle exons harbor ALYREF binding sites and identified ALYREF-binding motifs that promote nuclear export of intronless mRNAs. Together, our study defines enrichment of ALYREF binding sites at the 5' and the 3' regions of the mRNA in vivo, identifies export-promoting ALYREF-binding motifs, and reveals CstF64- and PABPN1-mediated coupling of mRNA nuclear export to 3' processing.

Induced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enable highly resolved descriptions of cell states within these systems; however, approaches are needed to directly measure lineage relationships. Here we establish iTracer, a lineage recorder that combines reporter barcodes with inducible CRISPR-Cas9 scarring and is compatible with single-cell and spatial transcriptomics. We apply iTracer to explore clonality and lineage dynamics during cerebral organoid development and identify a time window of fate restriction as well as variation in neurogenic dynamics between progenitor neuron families. We also establish long-term four-dimensional light-sheet microscopy for spatial lineage recording in cerebral organoids and confirm regional clonality in the developing neuroepithelium. We incorporate gene perturbation (iTracer-perturb) and assess the effect of mosaic TSC2 mutations on cerebral organoid development. Our data shed light on how lineages and fates are established during cerebral organoid formation. More broadly, our techniques can be adapted in any iPSC-derived culture system to dissect lineage alterations during normal or perturbed development.

To investigate Armadillo repeat-containing 5 (ARMC5) mutations in Chinese patients with familial and sporadic primary bilateral macronodular adrenal hyperplasia (PBMAH), we performed clinical data collection and ARMC5 sequencing for three PBMAH families and 23 sporadic PBMAH patients. ARMC5 pathogenic germline mutations were identified in all 3 PBMAH families. Secondary ARMC5 somatic mutations were found in two adrenal nodules from two PBMAH family members with ARMC5 germline mutations. PBMAH family members with ARMC5 pathogenic germline mutations displayed various clinical manifestations. ARMC5 pathogenic germline mutations were identified in 5 sporadic PBMAH patients among whom one patient displayed both hypercortisolism and primary aldosteronism. We detected a total of 10 ARMC5 pathogenic mutations, of which 8 had not been previously reported. Our results suggest that ARMC5 pathogenic germline mutations are common in familial and sporadic Chinese PBMAH patients, and demonstrate the importance of ARMC5 screening in PBMAH family members to detect patients with insidious PBMAH.

Homologous recombination (HR) repair (HRR) has been indicated to be a biomarker for immunotherapy, chemotherapy, and poly-ADP ribose polymerase inhibitors inhibitors (PARPis). Nonetheless, their molecular correlates in upper tract urothelial carcinoma (UTUC) have not been well studied. This study aimed to explore the molecular mechanism and tumor immune profile of HRR genes and the relevance of their prognostic value in patients with UTUC.

Study of drug-target interaction networks is an important topic for drug development. It is both time-consuming and costly to determine compound-protein interactions or potential drug-target interactions by experiments alone. As a complement, the in silico prediction methods can provide us with very useful information in a timely manner.

Amyloid fibrillar aggregates of polypeptides are associated with many neurodegenerative diseases. Short peptide segments in protein sequences may trigger aggregation. Identifying these stretches and examining their behavior in longer protein segments is critical for understanding these diseases and obtaining potential therapies. In this study, we combined machine learning and structure-based energy evaluation to examine and predict amyloidogenic segments. Our feature selection method discovered that windows consisting of long amino acid segments of ~30 residues, instead of the commonly used short hexapeptides, provided the highest accuracy. Weighted contributions of an amino acid at each position in a 27 residue window revealed three cooperative regions of short stretch, resemble the β-strand-turn-β-strand motif in A-βpeptide amyloid and β-solenoid structure of HET-s(218-289) prion (C). Using an in-house energy evaluation algorithm, the interaction energy between two short stretches in long segment is computed and incorporated as an additional feature. The algorithm successfully predicted and classified amyloid segments with an overall accuracy of 75%. Our study revealed that genome-wide amyloid segments are not only dependent on short high propensity stretches, but also on nearby residues.

Upper tract urothelial carcinomas (UTUCs) are rare entities that are usually diagnosed at advanced stages. Research on UTUC pathobiology and clinical management has been hampered by the lack of models accurately reflecting disease nature and diversity. In this study, a modified organoid culture system is used to generate a library of 25 patient-derived UTUC organoid lines retaining the histological architectures, marker gene expressions, genomic landscapes, and gene expression profiles of their parental tumors. The study demonstrates that the responses of UTUC organoids to anticancer drugs can be identified and the model supports the exploration of novel treatment strategies. This work proposes a modified protocol for generating patient-derived UTUC organoid lines that may help elucidate UTUC pathophysiology and assess the responses of these diseases to various drug therapies in personalized medicine.

Background: Therapy-related neuroendocrine prostate cancer (NEPC) is a lethal castration-resistant prostate cancer (CRPC) subtype that, at present, lacks well-characterized molecular biomarkers. The clinical diagnosis of this disease is dependent on biopsy and histological assessment: methods that are experience-based and easily misdiagnosed due to tumor heterogeneity. The development of robust diagnostic tools for NEPC may assist clinicians in making medical decisions on the choice of continuing anti-androgen receptor therapy or switching to platinum-based chemotherapy. Methods: Gene expression profiles and clinical characteristics data of 208 samples of metastatic CRPC, including castration-resistant prostate adenocarcinoma (CRPC-adeno) and castration-resistant neuroendocrine prostate adenocarcinoma (CRPC-NE), were obtained from the prad_su2c_2019 dataset. Weighted Gene Co-expression Network Analysis (WGCNA) was subsequently used to construct a free-scale gene co-expression network to study the interrelationship between the potential modules and clinical features of metastatic prostate adenocarcinoma and to identify hub genes in the modules. Furthermore, the least absolute shrinkage and selection operator (LASSO) regression analysis was used to build a model to predict the clinical characteristics of CRPC-NE. The findings were then verified in the nepc_wcm_2016 dataset. Results: A total of 51 co-expression modules were successfully constructed using WGCNA, of which three co-expression modules were found to be significantly associated with the neuroendocrine features and the NEPC score. In total, four novel genes, including NPTX1, PCSK1, ASXL3, and TRIM9, were all significantly upregulated in NEPC compared with the adenocarcinoma samples, and these genes were all associated with the neuroactive ligand receptor interaction pathway. Next, the expression levels of these four genes were used to construct an NEPC diagnosis model, which was successfully able to distinguish CRPC-NE from CRPC-adeno samples in both the training and the validation cohorts. Moreover, the values of the area under the receiver operating characteristic (AUC) were 0.995 and 0.833 for the training and validation cohorts, respectively. Conclusion: The present study identified four specific novel biomarkers for therapy-related NEPC, and these biomarkers may serve as an effective tool for the diagnosis of NEPC, thereby meriting further study.

Self-organizing neural organoids grown from pluripotent stem cells1-3 combined with single-cell genomic technologies provide opportunities to examine gene regulatory networks underlying human brain development. Here we acquire single-cell transcriptome and accessible chromatin data over a dense time course in human organoids covering neuroepithelial formation, patterning, brain regionalization and neurogenesis, and identify temporally dynamic and brain-region-specific regulatory regions. We developed Pando-a flexible framework that incorporates multi-omic data and predictions of transcription-factor-binding sites to infer a global gene regulatory network describing organoid development. We use pooled genetic perturbation with single-cell transcriptome readout to assess transcription factor requirement for cell fate and state regulation in organoids. We find that certain factors regulate the abundance of cell fates, whereas other factors affect neuronal cell states after differentiation. We show that the transcription factor GLI3 is required for cortical fate establishment in humans, recapitulating previous research performed in mammalian model systems. We measure transcriptome and chromatin accessibility in normal or GLI3-perturbed cells and identify two distinct GLI3 regulomes that are central to telencephalic fate decisions: one regulating dorsoventral patterning with HES4/5 as direct GLI3 targets, and one controlling ganglionic eminence diversification later in development. Together, we provide a framework for how human model systems and single-cell technologies can be leveraged to reconstruct human developmental biology.

Human neurons engineered from induced pluripotent stem cells (iPSCs) through neurogenin 2 (NGN2) overexpression are widely used to study neuronal differentiation mechanisms and to model neurological diseases. However, the differentiation paths and heterogeneity of emerged neurons have not been fully explored. Here, we used single-cell transcriptomics to dissect the cell states that emerge during NGN2 overexpression across a time course from pluripotency to neuron functional maturation. We find a substantial molecular heterogeneity in the neuron types generated, with at least two populations that express genes associated with neurons of the peripheral nervous system. Neuron heterogeneity is observed across multiple iPSC clones and lines from different individuals. We find that neuron fate acquisition is sensitive to NGN2 expression level and the duration of NGN2-forced expression. Our data reveal that NGN2 dosage can regulate neuron fate acquisition, and that NGN2-iN heterogeneity can confound results that are sensitive to neuron type.

5-Hydroxymethylcytosine (5hmC) is converted from 5-methylcytosine (5mC) by a group of enzymes termed ten-eleven translocation (TET) family dioxygenases. The loss of 5hmC has been identified as a hallmark of most types of cancer and is related to tumorigenesis and progression. However, the role of 5hmC in bladder cancer is seldom investigated. Vitamin C was recently reported to induce the generation of 5hmC by acting as a cofactor for TET dioxygenases. In this study, we explored the role of 5hmC in bladder cancer and the therapeutic efficacy of vitamin C in increasing the 5hmC pattern.

The molecular mechanism underpinning the specification of the ectoderm, a transient germ-layer tissue, during mouse gastrulation was examined here in a stem cell-based model. We captured a self-renewing cell population with enhanced ectoderm potency from mouse epiblast stem cells (EpiSCs) by suppressing Nodal signaling activity. The transcriptome of the Nodal-inhibited EpiSCs resembles that of the anterior epiblast of embryonic day (E)7.0 and E7.5 mouse embryo, which is accompanied by chromatin modifications that reflect the priming of ectoderm lineage-related genes for expression. Nodal-inhibited EpiSCs show enhanced ectoderm differentiation in vitro and contribute to the neuroectoderm and the surface ectoderm in postimplantation chimeras but lose the propensity for mesendoderm differentiation in vitro and in chimeras. Our findings show that specification of the ectoderm progenitors is enhanced by the repression of Nodal signaling activity, and the ectoderm-like stem cells provide an experimental model to investigate the molecular characters of the epiblast-derived ectoderm.

Healing of full-thickness skin wounds remains a major challenge. Recently, human umbilical cord mesenchymal stem cells (hUC-MSCs) were shown to possess an extraordinary potential to promote skin repair in clinical settings. However, their low survival rate after transplantation limits their therapeutic efficiency in treating full-thickness skin wounds. Hydrogels are considered an ideal cell transplantation vector owing to their three-dimensional mesh structure, good biosafety, and biodegradation. The objective of this study was to investigate the skin wound healing effect of a fibrin hydrogel scaffold loaded with hUC-MSCs. We found that the fibrin hydrogel had a three-dimensional mesh structure and low cytotoxicity and could prolong the time of cell survival in the peri-wound area. The number of green fluorescent protein (GFP)-labeled hUC-MSCs was higher in the full-thickness skin wound of mice treated with hydrogel-hUC-MSCs than those of mice treated with cell monotherapy. In addition, the combination therapy between the hydrogel and hUC-MSCs speed up wound closure, its wound healing rate was significantly higher than those of phosphate-buffered saline (PBS) therapy, hydrogel monotherapy, and hUC-MSCs monotherapy. Furthermore, the results showed that the combination therapy between hydrogel and hUC-MSCs increased keratin 10 and keratin 14 immunofluorescence staining, and upregulated the relative gene expressions of epidermal growth factor (EGF), transforming growth factor-β1 (TGF-β1), and vascular endothelial growth factor A (VEGFA), promoting epithelial regeneration and angiogenesis. In conclusion, the fibrin hydrogel scaffold provides a relatively stable sterile environment for cell adhesion, proliferation, and migration, and prolongs cell survival at the wound site. The hydrogel-hUC-MSCs combination therapy promotes wound closure, re-epithelialization, and neovascularization. It exhibits a remarkable therapeutic effect, being more effective than the monotherapy with hUC-MSCs or hydrogel.

Organoids generated from human pluripotent stem cells provide experimental systems to study development and disease, but quantitative measurements across different spatial scales and molecular modalities are lacking. In this study, we generated multiplexed protein maps over a retinal organoid time course and primary adult human retinal tissue. We developed a toolkit to visualize progenitor and neuron location, the spatial arrangements of extracellular and subcellular components and global patterning in each organoid and primary tissue. In addition, we generated a single-cell transcriptome and chromatin accessibility timecourse dataset and inferred a gene regulatory network underlying organoid development. We integrated genomic data with spatially segmented nuclei into a multimodal atlas to explore organoid patterning and retinal ganglion cell (RGC) spatial neighborhoods, highlighting pathways involved in RGC cell death and showing that mosaic genetic perturbations in retinal organoids provide insight into cell fate regulation.

Human neocortex expansion likely contributed to the remarkable cognitive abilities of humans. This expansion is thought to primarily reflect differences in proliferation versus differentiation of neural progenitors during cortical development. Here, we have searched for such differences by analysing cerebral organoids from human and chimpanzees using immunohistofluorescence, live imaging, and single-cell transcriptomics. We find that the cytoarchitecture, cell type composition, and neurogenic gene expression programs of humans and chimpanzees are remarkably similar. Notably, however, live imaging of apical progenitor mitosis uncovered a lengthening of prometaphase-metaphase in humans compared to chimpanzees that is specific to proliferating progenitors and not observed in non-neural cells. Consistent with this, the small set of genes more highly expressed in human apical progenitors points to increased proliferative capacity, and the proportion of neurogenic basal progenitors is lower in humans. These subtle differences in cortical progenitors between humans and chimpanzees may have consequences for human neocortex evolution.