A Strain-Promoted Divergent Chemical Steroidation Unveils Potent Anti-Inflammatory Pseudo-Steroidal Glycosides.

The development of novel agents with immunoregulatory effects is a keen way to combat the growing threat of inflammatory storms to global health. To synthesize pseudo-steroidal glycosides tethered by ether bonds with promising immunomodulatory potential, we develop herein a highly effective deoxygenative functionalization of a novel steroidal donor (steroidation) facilitated by strain-release, leveraging cost-effective and readily available Sc(OTf)3 catalysis. This transformation produces a transient steroid-3-yl carbocation which readily reacts with O-, C-, N-, S-, and P-nucleophiles to generate structurally diverse steroid derivatives. DFT calculations were performed to shed light on the mechanistic details of the regioselectivity, underlying an acceptor-dependent steroidation mode. This approach can be readily extended to the etherification of sugar alcohols to enable the achievement of a diversity-oriented, pipeline-like synthesis of pseudo-steroidal glycosides in good to excellent yields with complete stereo- and regiospecific control for anti-inflammatory agent discovery. Immunological studies have demonstrated that a meticulously designed cholesteryl disaccharide can significantly suppress interleukin-6 secretion in macrophages, exhibiting up to 99% inhibition rates compared to the negative control. These findings affirm the potential of pseudo-steroidal glycosides as a prospective category of lead agents for the development of novel anti-inflammatory drugs.

Generation of human excitatory forebrain neurons by cooperative binding of proneural NGN2 and homeobox factor EMX1.

Generation of defined neuronal subtypes from human pluripotent stem cells remains a challenge. The proneural factor NGN2 has been shown to overcome experimental variability observed by morphogen-guided differentiation and directly converts pluripotent stem cells into neurons, but their cellular heterogeneity has not been investigated yet. Here, we found that NGN2 reproducibly produces three different kinds of excitatory neurons characterized by partial coactivation of other neurotransmitter programs. We explored two principle approaches to achieve more precise specification: prepatterning the chromatin landscape that NGN2 is exposed to and combining NGN2 with region-specific transcription factors. Unexpectedly, the chromatin context of regionalized neural progenitors only mildly altered genomic NGN2 binding and its transcriptional response and did not affect neurotransmitter specification. In contrast, coexpression of region-specific homeobox factors such as EMX1 resulted in drastic redistribution of NGN2 including recruitment to homeobox targets and resulted in glutamatergic neurons with silenced nonglutamatergic programs. These results provide the molecular basis for a blueprint for improved strategies for generating a plethora of defined neuronal subpopulations from pluripotent stem cells for therapeutic or disease-modeling purposes.

Evaluating the outcome of screening for glaucoma using colour fundus photography-based referral criteria in a teleophthalmology screening programme for diabetic retinopathy.

AIMS: To evaluate the effectiveness of glaucoma screening using glaucoma suspect (GS) referral criteria assessed on colour fundus photographs in Singapore's Integrated Diabetic Retinopathy Programme (SiDRP). METHODS: A case-control study. This study included diabetic subjects who were referred from SiDRP with and without GS between January 2017 and December 2018 and reviewed at Singapore National Eye Centre. The GS referral criteria were based on the presence of a vertical cup-to-disc ratio (VCDR) of ≥0.65 and other GS features. The final glaucoma diagnosis confirmed from electronic medical records was retrospectively matched with GS status. The sensitivity, specificity and positive predictive value (PPV) of the test were evaluated. RESULTS: Of 5023 patients (2625 with GS and 2398 without GS) reviewed for glaucoma, 451 (9.0%, 95% CI 8.2% to 9.8%) were confirmed as glaucoma. The average follow-up time was 21.5±10.2 months. Using our current GS referral criteria, the sensitivity, specificity and PPV were 81.6% (95% CI 77.7% to 85.1%), 50.6% (95% CI 49.2% to 52.1%) and 14.0% (95% CI 13.4% to 14.7%), respectively, resulting in 2257 false positive cases. Increasing the VCDR cut-off for referral to ≥0.80, the specificity increased to 93.9% (95% CI 93.1% to 94.5%) but the sensitivity decreased to 11.3% (95% CI 8.5% to 14.6%), with a PPV of 15.4% (95% CI 12.0% to 19.4%). CONCLUSIONS: Opportunistic screening for glaucoma in a lower VCDR group could result in a high number of unnecessary referrals. If healthcare infrastructures are limited, targeting case findings on a larger VCDR group with high specificity will still be beneficial.

Genetic Ablation of LAT1 Inhibits Growth of Liver Cancer Cells and Downregulates mTORC1 Signaling.

Through a comprehensive analysis of the gene expression and dependency in HCC patients and cell lines, LAT1 was identified as the top amino acid transporter candidate supporting HCC tumorigenesis. To assess the suitability of LAT1 as a HCC therapeutic target, we used CRISPR/Cas9 to knockout (KO) LAT1 in the epithelial HCC cell line, Huh7. Knockout of LAT1 diminished its branched chain amino acid (BCAA) transport activity and significantly reduced cell proliferation in Huh7. Consistent with in vitro studies, LAT1 ablation led to suppression of tumor growth in a xenograft model. To elucidate the mechanism underlying the observed inhibition of cell proliferation upon LAT1 KO, we performed RNA-sequencing analysis and investigated the changes in the mTORC1 signaling pathway. LAT1 ablation resulted in a notable reduction in phosphorylation of p70S6K, a downstream target of mTORC1, as well as its substrate S6RP. This reduced cell proliferation and mTORC1 activity were rescued when LAT1 was overexpressed. These findings imply an essential role of LAT1 for maintenance of tumor cell growth and additional therapeutic angles against liver cancer.

Danshensu inhibits Aß aggregation and neurotoxicity as one of the main prominent features of Alzheimer's disease.

It has been found that the main cause of neurodegenerative proteinopathies, especially Alzheimer's disease (AD) is the formation of Aß amyloid plaques, which can be regulated by application of potential small molecules. In the present study, we aimed to investigate the inhibitory effect of danshensu on Aß(1-42) aggregation and relevant apoptotic pathway in neurons. A broad range of spectroscopic, theoretical, and cellular assays were done to investigate the anti-amyloidogenic characteristics of danshensu. It was found that danshensu triggers its inhibitory effect against Aß(1-42) aggregation through modulation of hydrophobic patches as well as structural and morphological changes through a stacking interaction. Furthermore, it was observed that incubation of Aß(1-42) samples with danshensu during aggregation process recovered the cell viability and mitigated the expression of caspase-3 mRNA and protein as well caspase-3 activity deregulated by Aß(1-42) amyloid fibrils alone. In general, obtained data showed that danshensu potentially inhibits Aß(1-42) aggregation and associated proteinopathies through regulation of apoptotic pathway in a concentration-dependent manner. Therefore, danshensu may be used as a promising biomolecule against the Aß aggregation and associated proteinopathies, which can be further analyzed in the future studies for the treatment of AD.

Exploring the associations between social support and symptoms of posttraumatic stress disorder among Malaysian and Australian trauma survivors.

Background: Social support is an important feature in understanding posttraumatic stress disorder (PTSD) and its treatment. Non-clinical research has identified distinct profiles of culturally appropriate social support. Despite this, little research has examined cultural influences on social support in the context of PTSD.Objective: This study examined cultural differences in the associations between social support and symptoms of PTSD.Method: The study employed a cross-sectional design. Australian (n = 91) and Malaysian (n = 91) trauma survivors completed an online survey assessing PTSD symptomatology and social support (explicit and implicit social support, perceived helpfulness of support provider, attitudes towards professional help-seeking). A quasi-experimental paradigm assessed the influence of mutual (i.e. the sharing of support between relationship partners) and non-mutual support (i.e. where one person constantly receives support, while the other person constantly provides support) on negative emotion and subjective distress.Results: First, explicit social support was negatively associated with PTSD symptoms for the Australian group but not the Malaysian group. Second, perceived helpfulness of support from family was negatively associated with PTSD symptoms for the Malaysian group but not the Australian group. Third, the Malaysian group reported significantly greater distress for non-mutual support and significantly fewer negative emotions and distress for mutual support than the Australian group. Fourth, the Malaysian group reported that they were significantly more open to acknowledging psychological problems and the possibility of seeking professional help for these problems than the Australian group.Conclusions: As the PTSD social support literature continues to evolve, it is essential that cultural influences are considered given the important theoretical and clinical implications.

Social support is an important feature in understanding posttraumatic stress disorder (PTSD). While non-clinical research has identified distinct profiles of culturally appropriate social support, little research has examined cultural influences on social support in the context of posttraumatic stress disorder.Disclosing the trauma to others and explicating requesting assistance was negatively associated with PTSD symptoms for the Australian group but not the Malaysian group. Support from family was negatively associated with PTSD symptoms for the Malaysian group but not the Australian group. The Malaysian group reported significantly greater distress for non-mutual support and significantly less negative affect and distress for mutual support than the Australian group.As the psychotraumatology literature continues to evolve, it is essential that cultural influences on social support are considered given the important theoretical and clinical implications.

The autism risk factor CHD8 is a chromatin activator in human neurons and functionally dependent on the ERK-MAPK pathway effector ELK1.

The chromodomain helicase DNA-binding protein CHD8 is the most frequently mutated gene in autism spectrum disorder. Despite its prominent disease involvement, little is known about its molecular function in the human brain. CHD8 is a chromatin regulator which binds to the promoters of actively transcribed genes through genomic targeting mechanisms which have yet to be fully defined. By generating a conditional loss-of-function and an endogenously tagged allele in human pluripotent stem cells, we investigated the molecular function and the interaction of CHD8 with chromatin in human neurons. Chromatin accessibility analysis and transcriptional profiling revealed that CHD8 functions as a transcriptional activator at its target genes in human neurons. Furthermore, we found that CHD8 chromatin targeting is cell context-dependent. In human neurons, CHD8 preferentially binds at ETS motif-enriched promoters. This enrichment is particularly prominent on the promoters of genes whose expression significantly changes upon the loss of CHD8. Indeed, among the ETS transcription factors, we identified ELK1 as being most highly correlated with CHD8 expression in primary human fetal and adult cortical neurons and most highly expressed in our stem cell-derived neurons. Remarkably, ELK1 was necessary to recruit CHD8 specifically to ETS motif-containing sites. These findings imply that ELK1 and CHD8 functionally cooperate to regulate gene expression and chromatin states at MAPK/ERK target genes in human neurons. Our results suggest that the MAPK/ERK/ELK1 axis potentially contributes to the pathogenesis caused by CHD8 mutations in human neurodevelopmental disorders.

Scalable Production of Extracellular Vesicles and Its Therapeutic Values: A Review.

Extracellular vesicles (EVs) are minute vesicles with lipid bilayer membranes. EVs are secreted by cells for intercellular communication. Recently, EVs have received much attention, as they are rich in biological components such as nucleic acids, lipids, and proteins that play essential roles in tissue regeneration and disease modification. In addition, EVs can be developed as vaccines against cancer and infectious diseases, as the vesicle membrane has an abundance of antigenic determinants and virulent factors. EVs for therapeutic applications are typically collected from conditioned media of cultured cells. However, the number of EVs secreted by the cells is limited. Thus, it is critical to devise new strategies for the large-scale production of EVs. Here, we discussed the strategies utilized by researchers for the scalable production of EVs. Techniques such as bioreactors, mechanical stimulation, electrical stimulation, thermal stimulation, magnetic field stimulation, topographic clue, hypoxia, serum deprivation, pH modification, exposure to small molecules, exposure to nanoparticles, increasing the intracellular calcium concentration, and genetic modification have been used to improve the secretion of EVs by cultured cells. In addition, nitrogen cavitation, porous membrane extrusion, and sonication have been utilized to prepare EV-mimetic nanovesicles that share many characteristics with naturally secreted EVs. Apart from inducing EV production, these upscaling interventions have also been reported to modify the EVs' cargo and thus their functionality and therapeutic potential. In summary, it is imperative to identify a reliable upscaling technique that can produce large quantities of EVs consistently. Ideally, the produced EVs should also possess cargo with improved therapeutic potential.

Detecting the Knowledge Domains of Compound Semiconductors.

The development of compound semiconductors (CS) has received extensive attention worldwide. This study aimed to detect and visualize CS knowledge domains for quantifying CS research patterns and emerging trends through a scientometric review based on the literature between 2011 and 2020 by using CiteSpace. The combined dataset of 24,622 bibliographic records were collected through topic searches and citation expansion to ensure adequate coverage of the field. While research in "solar cell" and "perovskite tandem" appears to be the two most distinctive knowledge domains in the CS field, research related to thermoelectric materials has grown at a respectable pace. Most notably, the deep connections between "thermoelectric material" and "III-Sb nanowire (NW)" research have been demonstrated. A rapid adaptation of black phosphorus (BP) field-effect transistors (FETs) and gallium nitride (GaN) transistors in the CS field is also apparent. Innovative strategies have focused on the opto-electronics with engineered functionalities, the design, synthesis and fabrication of perovskite tandem solar cells, the growing techniques of Sb-based III-V NWs, and the thermal conductivity of boron arsenide (BAs). This study revealed how the development trends and research areas in the CS field advance over time, which greatly help us to realize its knowledge domains.

Photocatalytic Degradation of Plastic Waste: A Mini Review.

Plastic waste becomes an immediate threat to our society with ever-increasing negative impacts on our environment and health by entering our food chain. Sunlight is known to be the natural energy source that degrades plastic waste at a very slow rate. Mimicking the role of sunlight, the photocatalytic degradation process could significantly accelerate the degradation rate thanks to the photocatalyst that drastically facilitates the photochemical reactions involved in the degradation process. This mini review begins with an introduction to the chemical compositions of the common plastic waste. The mechanisms of photodegradation of polymers in general were then revisited. Afterwards, a few photocatalysts were introduced with an emphasis on titanium dioxide (TiO2), which is the most frequently used photocatalyst. The roles of TiO2 photocatalyst in the photodegradation process were then elaborated, followed by the recent advances of photocatalytic degradation of various plastic waste. Lastly, our perspectives on the future research directions of photocatalytic plastic degradation are present. Herein, the importance of catalytic photodegradation is emphasized to inspire research on developing new photocatalysts and new processes for decomposition of plastic waste, and then to increase its recycling rate particularly in the current pandemic with the ever-increasing generation of plastic waste.

A Hybrid Fuzzy Decision Model for Evaluating MEMS and IC Integration Technologies.

Integrated devices incorporating MEMS (microelectromechanical systems) with IC (integrated circuit) components have been becoming increasingly important in the era of IoT (Internet of Things). In this study, a hybrid fuzzy MCDM (multi-criteria decision making) model was proposed to effectively evaluate alternative technologies that incorporate MEMS with IC components. This model, composed of the fuzzy AHP (analytic hierarchy process) and fuzzy VIKOR (VIseKriterijumska Optimizacija I Kompromisno Resenje) methods, solves the decision problem of how best to rank MEMS and IC integration technologies in a fuzzy environment. The six important criteria and the major five alternative technologies associated with our research themes were explored through literature review and expert investigations. The priority weights of criteria were derived using fuzzy AHP. After that, fuzzy VIKOR was deployed to rank alternatives. The empirical results show that development schedule and manufacturing capability are the two most important criteria and 3D (three-dimensional) SiP (system-in-package) and monolithic SoC (system-on-chip) are the top two favored technologies. The proposed fuzzy decision model could serve as a reference for the future strategic evaluation and selection of MEMS and IC integration technologies.

Reprogramming of mitochondrial proline metabolism promotes liver tumorigenesis.

Dysregulated cellular energetics has recently been recognized as a hallmark of cancer and garnered attention as a potential targeting strategy for cancer therapeutics. Cancer cells reprogram metabolic activities to meet bio-energetic, biosynthetic and redox requirements needed to sustain indefinite proliferation. In many cases, metabolic reprogramming is the result of complex interactions between genetic alterations in well-known oncogenes and tumor suppressors and epigenetic changes. While the metabolism of the two most abundant nutrients, glucose and glutamine, is reprogrammed in a wide range of cancers, accumulating evidence demonstrates that additional metabolic pathways are also critical for cell survival and growth. Proline metabolism is one such metabolic pathway that promotes tumorigenesis in multiple cancer types, including liver cancer, which is the fourth main cause of cancer mortality in the world. Despite the recent spate of approved treatments, including targeted therapy and combined immunotherapies, there has been no significant gain in clinical benefits in the majority of liver cancer patients. Thus, exploring novel therapeutic strategies and identifying new molecular targets remains a top priority for liver cancer. Two of the enzymes in the proline biosynthetic pathway, pyrroline-5-carboxylate reductase (PYCR1) and Aldehyde Dehydrogenase 18 Family Member A1 (ALDH18A1), are upregulated in liver cancer of both human and animal models, while proline catabolic enzymes, such as proline dehydrogenase (PRODH) are downregulated. Here we review the latest evidence linking proline metabolism to liver and other cancers and potential mechanisms of action for the proline pathway in cancer development.

Pro-neuronal activity of Myod1 due to promiscuous binding to neuronal genes.

The on-target pioneer factors Ascl1 and Myod1 are sequence-related but induce two developmentally unrelated lineages-that is, neuronal and muscle identities, respectively. It is unclear how these two basic helix-loop-helix (bHLH) factors mediate such fundamentally different outcomes. The chromatin binding of Ascl1 and Myod1 was surprisingly similar in fibroblasts, yet their transcriptional outputs were drastically different. We found that quantitative binding differences explained differential chromatin remodelling and gene activation. Although strong Ascl1 binding was exclusively associated with bHLH motifs, strong Myod1-binding sites were co-enriched with non-bHLH motifs, possibly explaining why Ascl1 is less context dependent. Finally, we observed that promiscuous binding of Myod1 to neuronal targets results in neuronal reprogramming when the muscle program is inhibited by Myt1l. Our findings suggest that chromatin access of on-target pioneer factors is primarily driven by the protein-DNA interaction, unlike ordinary context-dependent transcription factors, and that promiscuous transcription factor binding requires specific silencing mechanisms to ensure lineage fidelity.

Metabolic pathway analyses identify proline biosynthesis pathway as a promoter of liver tumorigenesis.

BACKGROUND & AIM: Under the regulation of various oncogenic pathways, cancer cells undergo adaptive metabolic programming to maintain specific metabolic states that support their uncontrolled proliferation. As it has been difficult to directly and effectively inhibit oncogenic signaling cascades with pharmaceutical compounds, focusing on the downstream metabolic pathways that enable indefinite growth may provide therapeutic opportunities. Thus, we sought to characterize metabolic changes in hepatocellular carcinoma (HCC) development and identify metabolic targets required for tumorigenesis. METHODS: We compared gene expression profiles of Morris Hepatoma (MH3924a) and DEN (diethylnitrosamine)-induced HCC models to those of liver tissues from normal and rapidly regenerating liver models, and performed gain- and loss-of-function studies of the identified gene targets for their roles in cancer cell proliferation in vitro and in vivo. RESULTS: The proline biosynthetic enzyme PYCR1 (pyrroline-5-carboxylate reductase 1) was identified as one of the most upregulated genes in the HCC models. Knockdown of PYCR1 potently reduced cell proliferation of multiple HCC cell lines in vitro and tumor growth in vivo. Conversely, overexpression of PYCR1 enhanced the proliferation of the HCC cell lines. Importantly, PYCR1 expression was not elevated in the regenerating liver, and KD or overexpression of PYCR1 had no effect on proliferation of non-cancerous cells. Besides PYCR1, we found that additional proline biosynthetic enzymes, such as ALDH18A1, were upregulated in HCC models and also regulated HCC cell proliferation. Clinical data demonstrated that PYCR1 expression was increased in HCC, correlated with tumor grade, and was an independent predictor of clinical outcome. CONCLUSION: Enhanced expression of proline biosynthetic enzymes promotes HCC cell proliferation. Inhibition of PYCR1 or ALDH18A1 may be a novel therapeutic strategy to target HCC. LAY SUMMARY: Even with the recently approved immunotherapies against liver cancer, currently available medications show limited clinical benefits or efficacy in the majority of patients. As such, it remains a top priority to discover new targets for effective liver cancer treatment. Here, we identify a critical role for the proline biosynthetic pathway in liver cancer development, and demonstrate that targeting key proteins in the pathway, namely PYCR1 and ALDH18A1, may be a novel therapeutic strategy for liver cancer.

Direct Reprogramming of Human Neurons Identifies MARCKSL1 as a Pathogenic Mediator of Valproic Acid-Induced Teratogenicity.

Human pluripotent stem cells can be rapidly converted into functional neurons by ectopic expression of proneural transcription factors. Here we show that directly reprogrammed neurons, despite their rapid maturation kinetics, can model teratogenic mechanisms that specifically affect early neurodevelopment. We delineated distinct phases of in vitro maturation during reprogramming of human neurons and assessed the cellular phenotypes of valproic acid (VPA), a teratogenic drug. VPA exposure caused chronic impairment of dendritic morphology and functional properties of developing neurons, but not those of mature neurons. These pathogenic effects were associated with VPA-mediated inhibition of the histone deacetylase (HDAC) and glycogen synthase kinase-3 (GSK-3) pathways, which caused transcriptional downregulation of many genes, including MARCKSL1, an actin-stabilizing protein essential for dendritic morphogenesis and synapse maturation during early neurodevelopment. Our findings identify a developmentally restricted pathogenic mechanism of VPA and establish the use of reprogrammed neurons as an effective platform for modeling teratogenic pathways.

Global DNA methylation remodeling during direct reprogramming of fibroblasts to neurons.

Direct reprogramming of fibroblasts to neurons induces widespread cellular and transcriptional reconfiguration. Here, we characterized global epigenomic changes during the direct reprogramming of mouse fibroblasts to neurons using whole-genome base-resolution DNA methylation (mC) sequencing. We found that the pioneer transcription factor Ascl1 alone is sufficient for inducing the uniquely neuronal feature of non-CG methylation (mCH), but co-expression of Brn2 and Mytl1 was required to establish a global mCH pattern reminiscent of mature cortical neurons. Ascl1 alone induced promoter CG methylation (mCG) of fibroblast specific genes, while BAM overexpression additionally targets a competing myogenic program and directs a more faithful conversion to neuronal cells. Ascl1 induces local demethylation at its binding sites. Surprisingly, co-expression with Brn2 and Mytl1 inhibited the ability of Ascl1 to induce demethylation, suggesting a contextual regulation of transcription factor - epigenome interaction. Finally, we found that de novo methylation by DNMT3A is required for efficient neuronal reprogramming.

The novel lncRNA lnc-NR2F1 is pro-neurogenic and mutated in human neurodevelopmental disorders.

Long noncoding RNAs (lncRNAs) have been shown to act as important cell biological regulators including cell fate decisions but are often ignored in human genetics. Combining differential lncRNA expression during neuronal lineage induction with copy number variation morbidity maps of a cohort of children with autism spectrum disorder/intellectual disability versus healthy controls revealed focal genomic mutations affecting several lncRNA candidate loci. Here we find that a t(5:12) chromosomal translocation in a family manifesting neurodevelopmental symptoms disrupts specifically lnc-NR2F1. We further show that lnc-NR2F1 is an evolutionarily conserved lncRNA functionally enhances induced neuronal cell maturation and directly occupies and regulates transcription of neuronal genes including autism-associated genes. Thus, integrating human genetics and functional testing in neuronal lineage induction is a promising approach for discovering candidate lncRNAs involved in neurodevelopmental diseases.

Rapid Chromatin Switch in the Direct Reprogramming of Fibroblasts to Neurons.

How transcription factors (TFs) reprogram one cell lineage to another remains unclear. Here, we define chromatin accessibility changes induced by the proneural TF Ascl1 throughout conversion of fibroblasts into induced neuronal (iN) cells. Thousands of genomic loci are affected as early as 12 hr after Ascl1 induction. Surprisingly, over 80% of the accessibility changes occur between days 2 and 5 of the 3-week reprogramming process. This chromatin switch coincides with robust activation of endogenous neuronal TFs and nucleosome phasing of neuronal promoters and enhancers. Subsequent morphological and functional maturation of iN cells is accomplished with relatively little chromatin reconfiguration. By integrating chromatin accessibility and transcriptome changes, we built a network model of dynamic TF regulation during iN cell reprogramming and identified Zfp238, Sox8, and Dlx3 as key TFs downstream of Ascl1. These results reveal a singular, coordinated epigenomic switch during direct reprogramming, in contrast to stepwise cell fate transitions in development.

Redox Homeostasis Plays Important Roles in the Maintenance of the Drosophila Testis Germline Stem Cells.

Oxidative stress influences stem cell behavior by promoting the differentiation, proliferation, or apoptosis of stem cells. Thus, characterizing the effects of reactive oxygen species (ROS) on stem cell behavior provides insights into the significance of redox homeostasis in stem cell-associated diseases and efficient stem cell expansion for cellular therapies. We utilized the Drosophila testis as an in vivo model to examine the effects of ROS on germline stem cell (GSC) maintenance. High levels of ROS induced by alteration in Keap1/Nrf2 activity decreased GSC number by promoting precocious GSC differentiation. Notably, high ROS enhanced the transcription of the EGFR ligand spitz and the expression of phospho-Erk1/2, suggesting that high ROS-mediated GSC differentiation is through EGFR signaling. By contrast, testes with low ROS caused by Keap1 inhibition or antioxidant treatment showed an overgrowth of GSC-like cells. These findings suggest that redox homeostasis regulated by Keap1/Nrf2 signaling plays important roles in GSC maintenance.

Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq.

Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states. However, the intermediate stages through which individual cells progress during reprogramming are largely undefined. Here we use single-cell RNA sequencing at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts to induced neuronal cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts, suggesting that the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.