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Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease arising from impaired regeneration of the alveolar epithelium after injury. During regeneration, type 2 alveolar epithelial cells (AEC2s) assume a transitional state that upregulates multiple keratins and ultimately differentiate into AEC1s. In IPF, transitional AECs accumulate with ineffectual AEC1 differentiation. However, whether and how transitional cells cause fibrosis, whether keratins regulate transitional cell accumulation and fibrosis, and why transitional AECs and fibrosis resolve in mouse models but accumulate in IPF are unclear. Here, we show that human keratin 8 (KRT8) genetic variants were associated with IPF. Krt8-/- mice were protected from fibrosis and accumulation of the transitional state. Keratin 8 (K8) regulated the expression of macrophage chemokines and macrophage recruitment. Profibrotic macrophages and myofibroblasts promoted the accumulation of transitional AECs, establishing a K8-dependent positive feedback loop driving fibrogenesis. Finally, rare murine transitional AECs were highly senescent and basaloid and may not differentiate into AEC1s, recapitulating the aberrant basaloid state in human IPF. We conclude that transitional AECs induced and were maintained by fibrosis in a K8-dependent manner; in mice, most transitional cells and fibrosis resolved, whereas in human IPF, transitional AECs evolved into an aberrant basaloid state that persisted with progressive fibrosis.

Background: Ependymoma (EPN) posterior fossa group A (PFA) has the highest rate of recurrence and the worst prognosis of all EPN molecular groups. At relapse, it is typically incurable even with re-resection and re-irradiation. The biology of recurrent PFA remains largely unknown; however, the increasing use of surgery at first recurrence has now provided access to clinical samples to facilitate a better understanding of this., Methods: In this large longitudinal international multicenter study, we examined matched samples of primary and recurrent disease from PFA patients to investigate the biology of recurrence., Results: DNA methylome derived copy number variants (CNVs) revealed large-scale chromosome gains and losses at recurrence in PFA. CNV changes were dominated by chromosome 1q gain and/or 6q loss, both previously identified as high-risk factors in PFA, which were present in 23% at presentation but increased to 61% at first recurrence. Multivariate survival analyses of this cohort showed that cases with 1q gain or 6q loss at first recurrence were significantly more likely to recur again. Predisposition to 1q+/6q- CNV changes at recurrence correlated with hypomethylation of heterochromatin-associated DNA at presentation. Cellular and molecular analyses revealed that 1q+/6q- PFA had significantly higher proportions of proliferative neuroepithelial undifferentiated progenitors and decreased differentiated neoplastic subpopulations., Conclusions: This study provides clinically and preclinically actionable insights into the biology of PFA recurrence. The hypomethylation predisposition signature in PFA is a potential risk-classifier for trial stratification. We show that the cellular heterogeneity of PFAs evolves largely because of genetic evolution of neoplastic cells., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Coordinated mineralization of soft tissue is central to organismal form and function, while dysregulated mineralization underlies several human pathologies. Oral epithelial-derived ameloblasts are polarized, secretory cells responsible for generating enamel, the most mineralized substance in the human body. Defects in ameloblast development result in enamel anomalies, including amelogenesis imperfecta. Identifying proteins critical in ameloblast development can provide insight into specific pathologies associated with enamel-related disorders or, more broadly, mechanisms of mineralization. Previous studies identified a role for MEMO1 in bone mineralization; however, whether MEMO1 functions in the generation of additional mineralized structures remains unknown. Here, we identify a critical role for MEMO1 in enamel mineralization. First, we show that Memo1 is expressed in ameloblasts and, second, that its conditional deletion from ameloblasts results in enamel defects, characterized by a decline in mineral density and tooth integrity. Histology revealed that the mineralization defects in Memo1 mutant ameloblasts correlated with a disruption in ameloblast morphology. Finally, molecular profiling of ameloblasts and their progenitors in Memo1 oral epithelial mutants revealed a disruption to cytoskeletal-associated genes and a reduction in late-stage ameloblast markers, relative to controls. Collectively, our findings integrate MEMO1 into an emerging network of molecules important for ameloblast development and provide a system to further interrogate the relationship of cytoskeletal and amelogenesis-related defects., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Plexiform neurofibroma (PN) is a leading cause of morbidity in children with the genetic condition Neurofibromatosis Type 1 (NF1), often disfiguring or threatening vital structures. During formation of PN, a complex tumor microenvironment (TME) develops, with recruitment of neoplastic and non-neoplastic cell types being critical for growth and progression. Due to the cohesive cellularity of PN, single-cell RNA-sequencing is difficult and may result in a loss of detection of critical cellular subpopulations. To bypass this barrier, we performed single-nuclei RNA-sequencing (snRNA-seq) on 8 frozen PN samples, and integrated this with spatial transcriptomics (ST) in 4 PN samples and immunohistochemistry to provide morphological context to transcriptomic data. SnRNA-seq analysis definitively charted the heterogeneous cellular subpopulations in the PN TME, with the predominant fraction being fibroblast subtypes. PN showed a remarkable amount of inter-sample homogeneity regarding cellular subpopulation proportions despite being resected from a variety of anatomical locations. ST analysis identified distinct cellular subpopulations which were annotated using snRNA-seq data and correlated with histological features. Schwann cell/fibroblast interactions were identified by receptor/ligand interaction analysis demonstrating a high probability of Neurexin 1/Neuroligin 1 (NRXN1/NLGN1) receptor-ligand cross-talk predicted between fibroblasts and non-myelinated Schwann cells (NM-SC) and subtypes, respectively. We observed aberrant expression of NRXN1 and NLGN1 in our PN snRNA-seq data compared to a normal mouse sciatic nerve single-cell RNA-seq dataset. This pathway has never been described in PN and may indicate a clear and direct communication pathway between putative NM-SC cells of origin and surrounding fibroblasts, potentially driving disease progression. SnRNA-seq integrated with spatial transcriptomics advances our understanding of the complex cellular heterogeneity of PN TME and identify potential novel communication pathways that may drive disease progression, a finding that could provide translational therapy options for patients with these devastating tumors of childhood and early adulthood., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Members of the HDAC family are predictive biomarkers and regulate the tumorigenesis in several cancers. However, the role of these genes in the biology of intracranial ependymomas (EPNs) remains unexplored. Here, an analysis of eighteen HDACs genes in an EPN transcriptomic dataset, revealed significantly higher levels of HDAC4 in supratentorial ZFTA fusion (ST-ZFTA) compared with ST-YAP1 fusion and posterior fossa EPNs, while HDAC7 and SIRT2 were downregulated in ST-ZFTA. HDAC4 was also overexpressed in ST-ZFTA as measured by single-cell RNA-Seq, quantitative real time-polymerase chain reaction, and immunohistochemistry. Survival analyses showed a significantly worse outcome for EPNs with higher HDAC4 and SIRT1 mRNA levels. Ontology enrichment analysis showed an HDAC4-high signature consistent with viral processes while collagen-containing extracellular matrix and cell-cell junction were enriched in those with an HDAC4-low signature. Immune gene analysis demonstrated a correlation between HDAC4 expression and low levels of NK resting cells. Several small molecules compounds targeting HDAC4 and ABCG2, were predicted by in silico analysis to be effective against HDAC4-high ZFTA. Our results provide novel insights into the biology of the HDAC family in intracranial ependymomas and reveal HDAC4 as a prognostic marker and potential therapeutic target in ST-ZFTA., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: The diverse cellular constituents of childhood brain tumor ependymoma, recently revealed by single cell RNA-sequencing, may underly therapeutic resistance. Here we use spatial transcriptomics to further advance our understanding of the tumor microenvironment, mapping cellular subpopulations to the tumor architecture of ependymoma posterior fossa subgroup A (PFA), the commonest and most deadly childhood ependymoma variant., Methods: Spatial transcriptomics data from intact PFA sections was deconvoluted to resolve the histological arrangement of neoplastic and non-neoplastic cell types. Key findings were validated using immunohistochemistry, in vitro functional assays and outcome analysis in clinically-annotated PFA bulk transcriptomic data., Results: PFA are comprised of epithelial and mesenchymal histological zones containing a diversity of cellular states, each zone including co-existing and spatially distinct undifferentiated progenitor-like cells; a quiescent mesenchymal zone population, and a second highly mitotic progenitor population that is restricted to hypercellular epithelial zones and that is more abundant in progressive tumors. We show that myeloid cell interaction is the leading cause of mesenchymal transition in PFA, occurring in zones spatially distinct from hypoxia-induced mesenchymal transition, and these distinct EMT-initiating processes were replicated using in vitro models of PFA., Conclusions: These insights demonstrate the utility of spatial transcriptomics to advance our understanding of ependymoma biology, revealing a clearer picture of the cellular constituents of PFA, their interactions and influence on tumor progression., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Acute respiratory distress syndrome (ARDS) due to coronavirus disease 2019 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; recovery requires epithelial regeneration. During physiological regeneration in mice, type 2 AECs (AEC2s) proliferate, exit the cell cycle, transiently assume a transitional state, then differentiate into type 1 AECs (AEC1s); in humans, persistence of the transitional state is associated with pulmonary fibrosis. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiological regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or single-cell RNA sequencing revealed that transitional cells in mouse models of physiological regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis., (Copyright © 2022 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Medulloblastoma (MB) is a heterogeneous disease in which neoplastic cells and associated immune cells contribute to disease progression. We aimed to determine the influence of neoplastic and immune cell diversity on MB biology in patient samples and animal models., Methods: To better characterize cellular heterogeneity in MB we used single-cell RNA sequencing, immunohistochemistry, and deconvolution of transcriptomic data to profile neoplastic and immune populations in patient samples and animal models across childhood MB subgroups., Results: Neoplastic cells cluster primarily according to individual sample of origin which is influenced by chromosomal copy number variance. Harmony alignment reveals novel MB subgroup/subtype-associated subpopulations that recapitulate neurodevelopmental processes, including photoreceptor and glutamatergic neuron-like cells in molecular subgroups GP3 and GP4, and a specific nodule-associated neuronally differentiated subpopulation in the sonic hedgehog subgroup. We definitively chart the spectrum of MB immune cell infiltrates, which include subpopulations that recapitulate developmentally related neuron-pruning and antigen-presenting myeloid cells. MB cellular diversity matching human samples is mirrored in subgroup-specific mouse models of MB., Conclusions: These findings provide a clearer understanding of the diverse neoplastic and immune cell subpopulations that constitute the MB microenvironment., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Stem cell-derived β-like cells (sBC) carry the promise of providing an abundant source of insulin-producing cells for use in cell replacement therapy for patients with diabetes, potentially allowing widespread implementation of a practical cure. To achieve their clinical promise, sBC need to function comparably with mature adult β-cells, but as yet they display varying degrees of maturity. Indeed, detailed knowledge of the events resulting in human β-cell maturation remains obscure. Here we show that sBC spontaneously self-enrich into discreet islet-like cap structures within in vitro cultures, independent of exogenous maturation conditions. Multiple complementary assays demonstrate that this process is accompanied by functional maturation of the self-enriched sBC (seBC); however, the seBC still contain distinct subpopulations displaying different maturation levels. Interestingly, the surface protein ENTPD3 (also known as nucleoside triphosphate diphosphohydrolase-3 [NDPTase3]) is a specific marker of the most mature seBC population and can be used for mature seBC identification and sorting. Our results illuminate critical aspects of in vitro sBC maturation and provide important insights toward developing functionally mature sBC for diabetes cell replacement therapy., (© 2021 by the American Diabetes Association.)

Hibernators dramatically lower metabolism to save energy while fasting for months. Prolonged fasting challenges metabolic homeostasis, yet small-bodied hibernators emerge each spring ready to resume all aspects of active life, including immediate reproduction. The liver is the body's metabolic hub, processing and detoxifying macromolecules to provide essential fuels to brain, muscle and other organs throughout the body. Here we quantify changes in liver gene expression across several distinct physiological states of hibernation in 13-lined ground squirrels, using RNA-seq to measure the steady-state transcriptome and GRO-seq to measure transcription for the first time in a hibernator. Our data capture key timepoints in both the seasonal and torpor-arousal cycles of hibernation. Strong positive correlation between transcription and the transcriptome indicates that transcriptional control dominates the known seasonal reprogramming of metabolic gene expression in liver for hibernation. During the torpor-arousal cycle, however, discordance develops between transcription and the steady-state transcriptome by at least two mechanisms: 1) although not transcribed during torpor, some transcripts are unusually stable across the torpor bout; and 2) unexpectedly, on some genes, our data suggest continuing, slow elongation with a failure to terminate transcription across the torpor bout. While the steady-state RNAs corresponding to these read through transcripts did not increase during torpor, they did increase shortly after rewarming despite their simultaneously low transcription. Both of these mechanisms would assure the immediate availability of functional transcripts upon rewarming. Integration of transcriptional, post-transcriptional and RNA stability control mechanisms, all demonstrated in these data, likely initiate a serial gene expression program across the short euthermic period that restores the tissue and prepares the animal for the next bout of torpor., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gillen, Fu, Riemondy, Jager, Fang, Lazar and Martin.)

Single-cell RNA sequencing (scRNA-seq) provides an unprecedented view of cellular diversity of biological systems. However, across the thousands of publications and datasets generated using this technology, we estimate that only a minority (<25%) of studies provide cell-level metadata information containing identified cell types and related findings of the published dataset. Metadata omission hinders reproduction, exploration, validation, and knowledge transfer and is a common problem across journals, data repositories, and publication dates. We encourage investigators, reviewers, journals, and data repositories to improve their standards and ensure proper documentation of these valuable datasets., Competing Interests: The authors have declared that no competing interests exist.

Hibernation is a physiological and behavioral phenotype that minimizes energy expenditure. Hibernators cycle between profound depression and rapid hyperactivation of multiple physiological processes, challenging our concept of mammalian homeostasis. How the hibernator orchestrates and survives these extremes while maintaining cell to organismal viability is unknown. Here, we enhance the genome integrity and annotation of a model hibernator, the 13-lined ground squirrel. Our new assembly brings this genome to near chromosome-level contiguity and adds thousands of previously unannotated genes. These new genomic resources were used to identify 6,505 hibernation-related, differentially-expressed and processed transcripts using RNA-seq data from three brain regions in animals whose physiological status was precisely defined using body temperature telemetry. A software tool, squirrelBox, was developed to foster further data analyses and visualization. SquirrelBox includes a comprehensive toolset for rapid visualization of gene level and cluster group dynamics, sequence scanning of k -mer and domains, and interactive exploration of gene lists. Using these new tools and data, we deconvolute seasonal from temperature-dependent effects on the brain transcriptome during hibernation for the first time, highlighting the importance of carefully timed samples for studies of differential gene expression in hibernation. The identified genes include a regulatory network of RNA binding proteins that are dynamic in hibernation along with the composition of the RNA pool. In addition to passive effects of temperature, we provide evidence for regulated transcription and RNA turnover during hibernation. Significant alternative splicing, largely temperature dependent, also occurs during hibernation. These findings form a crucial first step and provide a roadmap for future work toward defining novel mechanisms of tissue protection and metabolic depression that may 1 day be applied toward improving human health., Competing Interests: KG is CSO for Fauna Bio and SM and CB serve on its SAB. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fu, Gillen, Grabek, Riemondy, Epperson, Bustamante, Hesselberth and Martin.)

Ependymoma (EPN) is a brain tumor commonly presenting in childhood that remains fatal in most children. Intra-tumoral cellular heterogeneity in bulk-tumor samples significantly confounds our understanding of EPN biology, impeding development of effective therapy. We, therefore, use single-cell RNA sequencing, histology, and deconvolution to catalog cellular heterogeneity of the major childhood EPN subgroups. Analysis of PFA subgroup EPN reveals evidence of an undifferentiated progenitor subpopulation that either differentiates into subpopulations with ependymal cell characteristics or transitions into a mesenchymal subpopulation. Histological analysis reveals that progenitor and mesenchymal subpopulations co-localize in peri-necrotic zones. In conflict with current classification paradigms, relative PFA subpopulation proportions are shown to determine bulk-tumor-assigned subgroups. We provide an interactive online resource that facilitates exploration of the EPN single-cell dataset. This atlas of EPN cellular heterogeneity increases understanding of EPN biology., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Methods to measure heterogeneity among cells are rapidly transforming our understanding of biology but are currently limited to molecular abundance measurements. We developed an approach to simultaneously measure biochemical activities and mRNA abundance in single cells to understand the heterogeneity of DNA repair activities across thousands of human lymphocytes, identifying known and novel cell-type-specific DNA repair phenotypes., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Assignment of cell types from single-cell RNA sequencing (scRNA-seq) data remains a time-consuming and error-prone process. Current packages for identity assignment use limited types of reference data and often have rigid data structure requirements. We developed the clustifyr R package to leverage several external data types, including gene expression profiles to assign likely cell types using data from scRNA-seq, bulk RNA-seq, microarray expression data, or signature gene lists. We benchmark various parameters of a correlation-based approach and implement gene list enrichment methods. clustifyr is a lightweight and effective cell-type assignment tool developed for compatibility with various scRNA-seq analysis workflows. clustifyr is publicly available at https://github.com/rnabioco/clustifyr., Competing Interests: No competing interests were disclosed., (Copyright: © 2020 Fu R et al.)

Venetoclax-based therapy can induce responses in approximately 70% of older previously untreated patients with acute myeloid leukemia (AML). However, up-front resistance as well as relapse following initial response demonstrates the need for a deeper understanding of resistance mechanisms. In the present study, we report that responses to venetoclax +azacitidine in patients with AML correlate closely with developmental stage, where phenotypically primitive AML is sensitive, but monocytic AML is more resistant. Mechanistically, resistant monocytic AML has a distinct transcriptomic profile, loses expression of venetoclax target BCL2, and relies on MCL1 to mediate oxidative phosphorylation and survival. This differential sensitivity drives a selective process in patients which favors the outgrowth of monocytic subpopulations at relapse. Based on these findings, we conclude that resistance to venetoclax + azacitidine can arise due to biological properties intrinsic to monocytic differentiation. We propose that optimal AML therapies should be designed so as to independently target AML subclones that may arise at differing stages of pathogenesis. SIGNIFICANCE: Identifying characteristics of patients who respond poorly to venetoclax-based therapy and devising alternative therapeutic strategies for such patients are important topics in AML. We show that venetoclax resistance can arise due to intrinsic molecular/metabolic properties of monocytic AML cells and that such properties can potentially be targeted with alternative strategies., (©2020 American Association for Cancer Research.)

Assignment of cell types from single-cell RNA sequencing (scRNA-seq) data remains a time-consuming and error-prone process. Current packages for identity assignment use limited types of reference data and often have rigid data structure requirements. We developed the clustifyr R package to leverage several external data types, including gene expression profiles to assign likely cell types using data from scRNA-seq, bulk RNA-seq, microarray expression data, or signature gene lists. We benchmark various parameters of a correlation-based approach and implement gene list enrichment methods. clustifyr is a lightweight and effective cell-type assignment tool developed for compatibility with various scRNA-seq analysis workflows. clustifyr is publicly available at https://github.com/rnabioco/clustifyr., Competing Interests: No competing interests were disclosed., (Copyright: © 2020 Fu R et al.)

Liver lymphatic vessels support liver function by draining interstitial fluid, cholesterol, fat, and immune cells for surveillance in the liver draining lymph node. Chronic liver disease is associated with increased inflammation and immune cell infiltrate. However, it is currently unknown if or how lymphatic vessels respond to increased inflammation and immune cell infiltrate in the liver during chronic disease. Here we demonstrate that lymphatic vessel abundance increases in patients with chronic liver disease and is associated with areas of fibrosis and immune cell infiltration. Using single-cell mRNA sequencing and multi-spectral immunofluorescence analysis we identified liver lymphatic endothelial cells and found that chronic liver disease results in lymphatic endothelial cells (LECs) that are in active cell cycle with increased expression of CCL21. Additionally, we found that LECs from patients with NASH adopt a transcriptional program associated with increased IL13 signaling. Moreover, we found that oxidized low density lipoprotein, associated with NASH pathogenesis, induced the transcription and protein production of IL13 in LECs both in vitro and in a mouse model. Finally, we show that oxidized low density lipoprotein reduced the transcription of PROX1 and decreased lymphatic stability. Together these data indicate that LECs are active participants in the liver, expanding in an attempt to maintain tissue homeostasis. However, when inflammatory signals, such as oxidized low density lipoprotein are increased, as in NASH, lymphatic function declines and liver homeostasis is impeded.

Many lung diseases result from a failure of efficient regeneration of damaged alveolar epithelial cells (AECs) after lung injury. During regeneration, AEC2s proliferate to replace lost cells, after which proliferation halts and some AEC2s transdifferentiate into AEC1s to restore normal alveolar structure and function. Although the mechanisms underlying AEC2 proliferation have been studied, the mechanisms responsible for halting proliferation and inducing transdifferentiation are poorly understood. To identify candidate signaling pathways responsible for halting proliferation and inducing transdifferentiation, we performed single cell RNA sequencing on AEC2s during regeneration in a murine model of lung injury induced by intratracheal LPS. Unsupervised clustering revealed distinct subpopulations of regenerating AEC2s: proliferating, cell cycle arrest, and transdifferentiating. Gene expression analysis of these transitional subpopulations revealed that TGFβ signaling was highly upregulated in the cell cycle arrest subpopulation and relatively downregulated in transdifferentiating cells. In cultured AEC2s, TGFβ was necessary for cell cycle arrest but impeded transdifferentiation. We conclude that during regeneration after LPS-induced lung injury, TGFβ is a critical signal halting AEC2 proliferation but must be inactivated to allow transdifferentiation. This study provides insight into the molecular mechanisms regulating alveolar regeneration and the pathogenesis of diseases resulting from a failure of regeneration.

Single-cell RNA sequencing (scRNA-seq) methods generate sparse gene expression profiles for thousands of single cells in a single experiment. The information in these profiles is sufficient to classify cell types by distinct expression patterns but the high complexity of scRNA-seq libraries often prevents full characterization of transcriptomes from individual cells. To extract more focused gene expression information from scRNA-seq libraries, we developed a strategy to physically recover the DNA molecules comprising transcriptome subsets, enabling deeper interrogation of the isolated molecules by another round of DNA sequencing. We applied the method in cell-centric and gene-centric modes to isolate cDNA fragments from scRNA-seq libraries. First, we resampled the transcriptomes of rare, single megakaryocytes from a complex mixture of lymphocytes and analyzed them in a second round of DNA sequencing, yielding up to 20-fold greater sequencing depth per cell and increasing the number of genes detected per cell from a median of 1313 to 2002. We similarly isolated mRNAs from targeted T cells to improve the reconstruction of their VDJ-rearranged immune receptor mRNAs. Second, we isolated CD3D mRNA fragments expressed across cells in a scRNA-seq library prepared from a clonal T cell line, increasing the number of cells with detected CD3D expression from 59.7% to 100%. Transcriptome resampling is a general approach to recover targeted gene expression information from single-cell RNA sequencing libraries that enhances the utility of these costly experiments, and may be applicable to the targeted recovery of molecules from other single-cell assays., (© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Leukemia stem cells (LSCs) drive the initiation and perpetuation of AML, are quantifiably associated with worse clinical outcomes, and often persist after conventional chemotherapy resulting in relapse 1-5 . In this report, we show that treatment of older patients with AML with the B cell lymphoma 2 (BCL-2) inhibitor venetoclax in combination with azacitidine results in deep and durable remissions and is superior to conventional treatments. We hypothesized that these promising clinical results were due to targeting LSCs. Analysis of LSCs from patients undergoing treatment with venetoclax + azacitidine showed disruption of the tricarboxylic acid (TCA) cycle manifested by decreased α-ketoglutarate and increased succinate levels, suggesting inhibition of electron transport chain complex II. In vitro modeling confirmed inhibition of complex II via reduced glutathionylation of succinate dehydrogenase. These metabolic perturbations suppress oxidative phosphorylation (OXPHOS), which efficiently and selectively targets LSCs. Our findings show for the first time that a therapeutic intervention can eradicate LSCs in patients with AML by disrupting the metabolic machinery driving energy metabolism, resulting in promising clinical activity in a patient population with historically poor outcomes.

RNA editing diversifies genomically encoded information to expand the complexity of the transcriptome. In ectothermic organisms, including Drosophila and Cephalopoda , where body temperature mirrors ambient temperature, decreases in environmental temperature lead to increases in A-to-I RNA editing and cause amino acid recoding events that are thought to be adaptive responses to temperature fluctuations. In contrast, endothermic mammals, including humans and mice, typically maintain a constant body temperature despite environmental changes. Here, A-to-I editing primarily targets repeat elements, rarely results in the recoding of amino acids, and plays a critical role in innate immune tolerance. Hibernating ground squirrels provide a unique opportunity to examine RNA editing in a heterothermic mammal whose body temperature varies over 30°C and can be maintained at 5°C for many days during torpor. We profiled the transcriptome in three brain regions at six physiological states to quantify RNA editing and determine whether cold-induced RNA editing modifies the transcriptome as a potential mechanism for neuroprotection at low temperature during hibernation. We identified 5165 A-to-I editing sites in 1205 genes with dynamically increased editing after prolonged cold exposure. The majority (99.6%) of the cold-increased editing sites are outside of previously annotated coding regions, 82.7% lie in SINE-derived repeats, and 12 sites are predicted to recode amino acids. Additionally, A-to-I editing frequencies increase with increasing cold-exposure, demonstrating that ADAR remains active during torpor. Our findings suggest that dynamic A-to-I editing at low body temperature may provide a neuroprotective mechanism to limit aberrant dsRNA accumulation during torpor in the mammalian hibernator., (© 2018 Riemondy et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

New tools for reproducible exploratory data analysis of large datasets are important to address the rising size and complexity of genomic data. We developed the valr R package to enable flexible and efficient genomic interval analysis. valr leverages new tools available in the "tidyverse", including dplyr. Benchmarks of valr show it performs similar to BEDtools and can be used for interactive analyses and incorporated into existing analysis pipelines., Competing Interests: Competing interests: No competing interests were disclosed.

Inherited and acquired disorders that enhance the activity of transporters mediating renal tubular Na(+) reabsorption are well established causes of hypertension. It is unclear, however, whether primary activation of an Na(+)-independent chloride transporter in the kidney can also play a pathogenic role in this disease. Here, mice overexpressing the chloride transporter pendrin in intercalated cells of the distal nephron (Tg(B1-hPDS) mice) displayed increased renal absorption of chloride. Compared with normal mice, these transgenic mice exhibited a delayed increase in urinary NaCl and ultimately, developed hypertension when exposed to a high-salt diet. Administering the same sodium intake as NaHCO3 instead of NaCl did not significantly alter BP, indicating that the hypertension in the transgenic mice was chloride-sensitive. Moreover, excessive chloride absorption by pendrin drove parallel absorption of sodium through the epithelial sodium channel ENaC and the sodium-driven chloride/bicarbonate exchanger (Ndcbe), despite an appropriate downregulation of these sodium transporters in response to the expanded vascular volume and hypertension. In summary, chloride transport in the distal nephron can play a primary role in driving NaCl transport in this part of the kidney, and a primary abnormality in renal chloride transport can provoke arterial hypertension. Thus, we conclude that the chloride/bicarbonate exchanger pendrin plays a major role in controlling net NaCl absorption, thereby influencing BP under conditions of high salt intake.

Regulation of sodium balance is a critical factor in the maintenance of euvolemia, and dysregulation of renal sodium excretion results in disorders of altered intravascular volume, such as hypertension. The amiloride-sensitive epithelial sodium channel (ENaC) is thought to be the only mechanism for sodium transport in the cortical collecting duct (CCD) of the kidney. However, it has been found that much of the sodium absorption in the CCD is actually amiloride insensitive and sensitive to thiazide diuretics, which also block the Na-Cl cotransporter (NCC) located in the distal convoluted tubule. In this study, we have demonstrated the presence of electroneutral, amiloride-resistant, thiazide-sensitive, transepithelial NaCl absorption in mouse CCDs, which persists even with genetic disruption of ENaC. Furthermore, hydrochlorothiazide (HCTZ) increased excretion of Na+ and Cl- in mice devoid of the thiazide target NCC, suggesting that an additional mechanism might account for this effect. Studies on isolated CCDs suggested that the parallel action of the Na+-driven Cl-/HCO3- exchanger (NDCBE/SLC4A8) and the Na+-independent Cl-/HCO3- exchanger (pendrin/SLC26A4) accounted for the electroneutral thiazide-sensitive sodium transport. Furthermore, genetic ablation of SLC4A8 abolished thiazide-sensitive NaCl transport in the CCD. These studies establish what we believe to be a novel role for NDCBE in mediating substantial Na+ reabsorption in the CCD and suggest a role for this transporter in the regulation of fluid homeostasis in mice.

The collecting duct of the kidney is composed of two morphologically and physiologically distinct cell types, principal and intercalated cells. To better understand intercalated cell function we generated a transgenic mouse expressing Cre recombinase under the control of a cell type- specific promoter. We used 7 kb of the ATP6V1B1 5' untranslated region (B1 promoter), a gene found in the intercalated cells of the kidney and the male reproductive tract. We first crossed these B1-Cre transgenic mice with the ROSA26-loxP-stop-loxP-yellow fluorescent protein reporter mice to assess the specificity of Cre expression. Immunohistochemistry and confocal fluorescence microscopy showed that Cre is selectively active in all intercalated cells (type A, type B, and non-A/B cells) within the collecting duct and most cells of the connecting segment. About half of the principal cells of the connecting segment also expressed Cre, a pattern also seen in B1-driven enhanced green fluorescent protein transgenic mice. Cre was found to be active in the male reproductive tract and at a low level in limited non-ATP6V1B1 expressing tissues. The B1-Cre transgenic mice are healthy, breed normally, produce regular sized litters, and transmit the transgene in Mendelian fashion. This new cell-specific Cre expressing mouse should prove useful for the study of intercalated cell physiology and development.