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Background: Macrophages are the most common infiltrating immune cells in gliomas and play a wide variety of pro-tumor and anti-tumor roles. However, the different subpopulations of macrophages and their effects on the tumor microenvironment remain poorly understood., Methods: We combined new and previously published single-cell RNA-seq data from 98,015 single cells from a total of 66 gliomas to profile 19,331 individual macrophages., Results: Unsupervised clustering revealed a pro-tumor subpopulation of bone marrow-derived macrophages characterized by the scavenger receptor MARCO, which is almost exclusively found in IDH1-wild-type glioblastomas. Previous studies have implicated MARCO as an unfavorable marker in melanoma and non-small cell lung cancer; here, we find that bulk MARCO expression is associated with worse prognosis and mesenchymal subtype. Furthermore, MARCO expression is significantly altered over the course of treatment with anti-PD1 checkpoint inhibitors in a response-dependent manner, which we validate with immunofluorescence imaging., Conclusions: These findings illustrate a novel macrophage subpopulation that drives tumor progression in glioblastomas and suggest potential therapeutic targets to prevent their recruitment.

Background: Preclinical studies require models that recapitulate the cellular diversity of human tumors and provide insight into the drug sensitivities of specific cellular populations. The ideal platform would enable rapid screening of cell type-specific drug sensitivities directly in patient tumor tissue and reveal strategies to overcome intratumoral heterogeneity., Methods: We combine multiplexed drug perturbation in acute slice culture from freshly resected tumors with single-cell RNA sequencing (scRNA-seq) to profile transcriptome-wide drug responses in individual patients. We applied this approach to drug perturbations on slices derived from six glioblastoma (GBM) resections to identify conserved drug responses and to one additional GBM resection to identify patient-specific responses., Results: We used scRNA-seq to demonstrate that acute slice cultures recapitulate the cellular and molecular features of the originating tumor tissue and the feasibility of drug screening from an individual tumor. Detailed investigation of etoposide, a topoisomerase poison, and the histone deacetylase (HDAC) inhibitor panobinostat in acute slice cultures revealed cell type-specific responses across multiple patients. Etoposide has a conserved impact on proliferating tumor cells, while panobinostat treatment affects both tumor and non-tumor populations, including unexpected effects on the immune microenvironment., Conclusions: Acute slice cultures recapitulate the major cellular and molecular features of GBM at the single-cell level. In combination with scRNA-seq, this approach enables cell type-specific analysis of sensitivity to multiple drugs in individual tumors. We anticipate that this approach will facilitate pre-clinical studies that identify effective therapies for solid tumors.

Immune response dynamics in coronavirus disease 2019 (COVID-19) and their severe manifestations have largely been studied in circulation. Here, we examined the relationship between immune processes in the respiratory tract and circulation through longitudinal phenotypic, transcriptomic, and cytokine profiling of paired airway and blood samples from patients with severe COVID-19 relative to heathy controls. In COVID-19 airways, T cells exhibited activated, tissue-resident, and protective profiles; higher T cell frequencies correlated with survival and younger age. Myeloid cells in COVID-19 airways featured hyperinflammatory signatures, and higher frequencies of these cells correlated with mortality and older age. In COVID-19 blood, aberrant CD163 + monocytes predominated over conventional monocytes, and were found in corresponding airway samples and in damaged alveoli. High levels of myeloid chemoattractants in airways suggest recruitment of these cells through a CCL2-CCR2 chemokine axis. Our findings provide insights into immune processes driving COVID-19 lung pathology with therapeutic implications for targeting inflammation in the respiratory tract., Competing Interests: Declaration of interests J.Z., M.S., and S.M. have competing interests with IsoPlexis. The remaining authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Plasmacytoid dendritic cells (pDCs) can rapidly produce interferons and other soluble factors in response to extracellular viruses or virus mimics such as CpG-containing DNA. pDCs can also recognize live cells infected with certain RNA viruses, but the relevance and functional consequences of such recognition remain unclear. We studied the response of primary DCs to the prototypical persistent DNA virus, human cytomegalovirus (CMV). Human pDCs produced high amounts of type I interferon (IFN-I) when incubated with live CMV-infected fibroblasts but not with free CMV; the response involved integrin-mediated adhesion, transfer of DNA-containing virions to pDCs, and the recognition of DNA through TLR9. Compared with transient polyfunctional responses to CpG or free influenza virus, pDC response to CMV-infected cells was long-lasting, dominated by the production of IFN-I and IFN-III, and lacked diversification into functionally distinct populations. Similarly, pDC activation by influenza-infected lung epithelial cells was highly efficient, prolonged, and dominated by interferon production. Prolonged pDC activation by CMV-infected cells facilitated the activation of natural killer cells critical for CMV control. Last, patients with CMV viremia harbored phenotypically activated pDCs and increased circulating IFN-I and IFN-III. Thus, recognition of live infected cells is a mechanism of virus detection by pDCs that elicits a unique antiviral immune response., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The harsh microenvironment of ductal carcinoma in situ (DCIS) exerts strong evolutionary selection pressures on cancer cells. We hypothesize that the poor metabolic conditions near the ductal center foment the emergence of a Warburg Effect (WE) phenotype, wherein cells rapidly ferment glucose to lactic acid, even in normoxia. To test this hypothesis, we subjected low-glycolytic breast cancer cells to different microenvironmental selection pressures using combinations of hypoxia, acidosis, low glucose, and starvation for many months and isolated single clones for metabolic and transcriptomic profiling. The two harshest conditions selected for constitutively expressed WE phenotypes. RNA sequencing analysis of WE clones identified the transcription factor KLF4 as potential inducer of the WE phenotype. In stained DCIS samples, KLF4 expression was enriched in the area with the harshest microenvironmental conditions. We simulated in vivo DCIS phenotypic evolution using a mathematical model calibrated from the in vitro results. The WE phenotype emerged in the poor metabolic conditions near the necrotic core. We propose that harsh microenvironments within DCIS select for a WE phenotype through constitutive transcriptional reprogramming, thus conferring a survival advantage and facilitating further growth and invasion., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Background: Smooth muscle cells (SMCs) play significant roles in atherosclerosis via phenotypic switching, a pathological process in which SMC dedifferentiation, migration, and transdifferentiation into other cell types. Yet how SMCs contribute to the pathophysiology of atherosclerosis remains elusive., Methods: To reveal the trajectories of SMC transdifferentiation during atherosclerosis and to identify molecular targets for disease therapy, we combined SMC fate mapping and single-cell RNA sequencing of both mouse and human atherosclerotic plaques. We also performed cell biology experiments on isolated SMC-derived cells, conducted integrative human genomics, and used pharmacological studies targeting SMC-derived cells both in vivo and in vitro., Results: We found that SMCs transitioned to an intermediate cell state during atherosclerosis, which was also found in human atherosclerotic plaques of carotid and coronary arteries. SMC-derived intermediate cells, termed "SEM" cells (stem cell, endothelial cell, monocyte), were multipotent and could differentiate into macrophage-like and fibrochondrocyte-like cells, as well as return toward the SMC phenotype. Retinoic acid (RA) signaling was identified as a regulator of SMC to SEM cell transition, and RA signaling was dysregulated in symptomatic human atherosclerosis. Human genomics revealed enrichment of genome-wide association study signals for coronary artery disease in RA signaling target gene loci and correlation between coronary artery disease risk alleles and repressed expression of these genes. Activation of RA signaling by all-trans RA, an anticancer drug for acute promyelocytic leukemia, blocked SMC transition to SEM cells, reduced atherosclerotic burden, and promoted fibrous cap stability., Conclusions: Integration of cell-specific fate mapping, single-cell genomics, and human genetics adds novel insights into the complexity of SMC biology and reveals regulatory pathways for therapeutic targeting of SMC transitions in atherosclerotic cardiovascular disease.

Live cell imaging allows direct observation and monitoring of phenotypes that are difficult to infer from transcriptomics. However, existing methods for linking microscopy and single-cell RNA-seq (scRNA-seq) have limited scalability. Here, we describe an upgraded version of Single Cell Optical Phenotyping and Expression (SCOPE-seq2) for combining single-cell imaging and expression profiling, with substantial improvements in throughput, molecular capture efficiency, linking accuracy, and compatibility with standard microscopy instrumentation. We introduce improved optically decodable mRNA capture beads and implement a more scalable and simplified optical decoding process. We demonstrate the utility of SCOPE-seq2 for fluorescence, morphological, and expression profiling of individual primary cells from a human glioblastoma (GBM) surgical sample, revealing relationships between simple imaging features and cellular identity, particularly among malignantly transformed tumor cells.

Understanding the intricacies of lethal prostate cancer poses specific challenges due to difficulties in accurate modeling of metastasis in vivo. Here we show that NPK EYFP mice (for Nkx3.1 CreERT2/+ ; Pten flox/flox ; Kras LSL-G12D/+ ; R26R-CAG - LSL-EYFP/+ ) develop prostate cancer with a high penetrance of metastasis to bone, thereby enabling detection and tracking of bone metastasis in vivo and ex vivo. Transcriptomic and whole-exome analyses of bone metastasis from these mice revealed distinct molecular profiles conserved between human and mouse and specific patterns of subclonal branching from the primary tumor. Integrating bulk and single-cell transcriptomic data from mouse and human datasets with functional studies in vivo unravels a unique MYC/RAS co-activation signature associated with prostate cancer metastasis. Finally, we identify a gene signature with prognostic value for time to metastasis and predictive of treatment response in human patients undergoing androgen receptor therapy across clinical cohorts, thus uncovering conserved mechanisms of metastasis with potential translational significance.

Background: Single-cell RNA sequencing is the reference technique for characterizing the heterogeneity of the tumor microenvironment. The composition of the various cell types making up the microenvironment can significantly affect the way in which the immune system activates cancer rejection mechanisms. Understanding the cross-talk signals between immune cells and cancer cells is of fundamental importance for the identification of immuno-oncology therapeutic targets., Results: We present a novel method, single-cell Tumor-Host Interaction tool (scTHI), to identify significantly activated ligand-receptor interactions across clusters of cells from single-cell RNA sequencing data. We apply our approach to uncover the ligand-receptor interactions in glioma using 6 publicly available human glioma datasets encompassing 57,060 gene expression profiles from 71 patients. By leveraging this large-scale collection we show that unexpected cross-talk partners are highly conserved across different datasets in the majority of the tumor samples. This suggests that shared cross-talk mechanisms exist in glioma., Conclusions: Our results provide a complete map of the active tumor-host interaction pairs in glioma that can be therapeutically exploited to reduce the immunosuppressive action of the microenvironment in brain tumor., (© The Author(s) 2020. Published by Oxford University Press GigaScience.)

Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides in mammalian cells, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise subcellular translation sites using anti-puromycin immunostaining may be confounded by release of puromycylated nascent polypeptide chains prior to fixation., Competing Interests: BH, LK, EH, RG, PS No competing interests declared, (© 2020, Hobson et al.)

In the adult ventricular-subventricular zone (V-SVZ), neural stem cells (NSCs) generate new olfactory bulb (OB) neurons and glia throughout life. To map adult neuronal lineage progression, we profiled >56,000 V-SVZ and OB cells by single-cell RNA sequencing (scRNA-seq). Our analyses reveal the molecular diversity of OB neurons, including fate-mapped neurons, lineage progression dynamics, and an NSC intermediate enriched for Notum, which encodes a secreted WNT antagonist. SCOPE-seq technology, which links live-cell imaging with scRNA-seq, uncovers cell-size transitions during NSC differentiation and preferential NOTUM binding to proliferating neuronal precursors. Consistently, application of NOTUM protein in slice cultures and pharmacological inhibition of NOTUM in slice cultures and in vivo demonstrated that NOTUM negatively regulates V-SVZ proliferation. Timely, context-dependent neurogenesis demands adaptive signaling among neighboring progenitors. Our findings highlight a critical regulatory state during NSC activation marked by NOTUM, which attenuates WNT-stimulated proliferation in NSC progeny., Competing Interests: Declaration of Interests Columbia University has filed patent applications on SCOPE-seq, and P.A.S. and J.Y. are listed as inventors., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Essential tremor (ET) is one of the most common neurological diseases, with a central feature of an 8-12 Hz kinetic tremor. While previous postmortem studies have identified a cluster of morphological changes in the ET cerebellum centered in/around the Purkinje cell (PC) population, including a loss of PCs in some studies, the underlying molecular mechanisms for these changes are not clear. As genomic studies of ET patients have yet to identify major genetic contributors and animal models that fully recapitulate the human disease do not yet exist, the study of human tissue is currently the most applicable method to gain a mechanistic insight into ET disease pathogenesis. To begin exploration of an underlying molecular source of ET disease pathogenesis, we have performed the first transcriptomic analysis by direct sequencing of RNA from frozen cerebellar cortex tissue in 33 ET patients compared to 21 normal controls. Principal component analysis showed a heterogenous distribution of the expression data in ET patients that only partially overlapped with control patients. Differential expression analysis identified 231 differentially expressed gene transcripts ('top gene hits'), a subset of which has defined expression profiles in the cerebellum across neuronal and glial cell types but a largely unknown relationship to cerebellar function and/or ET pathogenesis. Gene set enrichment analysis (GSEA) identified dysregulated pathways of interest and stratified dysregulation among ET cases. By GSEA and mining curated databases, we compiled major categories of dysregulated processes and clustered string networks of known interacting proteins. Here we demonstrate that these 'top gene hits' contribute to regulation of four main biological processes, which are 1) axon guidance, 2) microtubule motor activity, 3) endoplasmic reticulum (ER) to Golgi transport and 4) calcium signaling/synaptic transmission. The results of our transcriptomic analysis suggest there is a range of different processes involved among ET cases, and draws attention to a particular set of genes and regulatory pathways that provide an initial platform to further explore the underlying biology of ET., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Emerging single-cell technologies, like single-cell RNA sequencing (scRNA-seq), enable the study of heterogeneous biological systems at cellular resolution. By profiling the set of expressed transcripts in each cell, single-cell transcriptomics has allowed for the cataloging of the cellular constituents of multiple organs and tissues, both in health and disease. In addition, these technologies have provided mechanistic insights into cellular function, cell state transitions, developmental trajectories and lineage relationships, as well as helped to dissect complex, population-level responses to environmental perturbations. scRNA-seq is particularly useful for characterizing the intestinal epithelium because it is a dynamic, rapidly self-renewing tissue comprised of more than a dozen specialized cell types. Here we discuss the fundamentals of single-cell transcriptomics of the murine small intestinal epithelium. We review the principles of proper experimental design and provide methods for the dissociation of the small intestinal epithelium into single cells followed by fluorescence-activated cell sorting (FACS) and for scRNA-seq using the 10× Genomics Chromium platform.

Several morphological changes, centered in/around Purkinje cells (PCs), have been identified in the cerebellum of essential tremor (ET) patients. These changes have not been contextualized within a broader degenerative disease spectrum, limiting their interpretability. To address this, we compared the severity and patterning of degenerative changes within the cerebellar cortex in patients with ET, other neurodegenerative disorders of the cerebellum (spinocerebellar ataxias (SCAs), multiple system atrophy (MSA)], and other disorders that may involve the cerebellum [Parkinson's disease (PD), dystonia]. Using a postmortem series of 156 brains [50 ET, 23 SCA (6 SCA3; 17 SCA 1, 2 or 6), 15 MSA, 29 PD, 14 dystonia, 25 controls], we generated data on 37 quantitative morphologic metrics, which were grouped into 8 broad categories: (1) PC loss, (2) heterotopic PCs, (3) PC dendritic changes, (4) PC axonal changes (torpedoes), (5) PC axonal changes (other than torpedoes), (6) PC axonal changes (torpedo-associated), (7) basket cell axonal hypertrophy, (8) climbing fiber-PC synaptic changes. Our analyses used z scored raw data for each metric across all diagnoses (5772 total data items). Principal component analysis revealed that diagnostic groups were not uniform with respect to cerebellar pathology. Dystonia and PD each differed from controls in only 2/37 metrics, whereas ET differed in 21, SCA3 in 8, MSA in 19, and SCA1/2/6 in 26 metrics. Comparing ET with primary disorders of cerebellar degeneration (i.e., SCAs), we observed a spectrum of changes reflecting differences of degree, being generally mild in ET and SCA3 and more severe in SCA1/2/6. Comparative analyses across morphologic categories demonstrated differences in relative expression, defining distinctive patterns of changes in these groups. Thus, the degree of cerebellar degeneration in ET aligns it with a milder end in the spectrum of cerebellar degenerative disorders, and a somewhat distinctive signature of degenerative changes marks each of these disorders.

Human T cells coordinate adaptive immunity in diverse anatomic compartments through production of cytokines and effector molecules, but it is unclear how tissue site influences T cell persistence and function. Here, we use single cell RNA-sequencing (scRNA-seq) to define the heterogeneity of human T cells isolated from lungs, lymph nodes, bone marrow and blood, and their functional responses following stimulation. Through analysis of >50,000 resting and activated T cells, we reveal tissue T cell signatures in mucosal and lymphoid sites, and lineage-specific activation states across all sites including distinct effector states for CD8 + T cells and an interferon-response state for CD4 + T cells. Comparing scRNA-seq profiles of tumor-associated T cells to our dataset reveals predominant activated CD8 + compared to CD4 + T cell states within multiple tumor types. Our results therefore establish a high dimensional reference map of human T cell activation in health for analyzing T cells in disease.

Spatial structuring is important for the maintenance of natural ecological systems 1,2 . Many microbial communities, including the gut microbiome, display intricate spatial organization 3-9 . Mapping the biogeography of bacteria can shed light on interactions that underlie community functions 10-12 , but existing methods cannot accommodate the hundreds of species that are found in natural microbiomes 13-17 . Here we describe metagenomic plot sampling by sequencing (MaPS-seq), a culture-independent method to characterize the spatial organization of a microbiome at micrometer-scale resolution. Intact microbiome samples are immobilized in a gel matrix and cryofractured into particles. Neighboring microbial taxa in the particles are then identified by droplet-based encapsulation, barcoded 16S rRNA amplification and deep sequencing. Analysis of three regions of the mouse intestine revealed heterogeneous microbial distributions with positive and negative co-associations between specific taxa. We identified robust associations between Bacteroidales taxa in all gut compartments and showed that phylogenetically clustered local regions of bacteria were associated with a dietary perturbation. Spatial metagenomics could be used to study microbial biogeography in complex habitats.

Flow cytometry and fluorescence-activated sorting are powerful techniques that hold great promise for studying heterogeneous populations of submicron particles such as synaptosomes, but many technical challenges arise in these experiments. To date, most flow cytometry studies of synaptosomes have relied on particle detection using forward scatter (FSC) measurements and size estimation with polystyrene (PS) bead standards. However, these practices have serious limitations, and special care must be taken to overcome the poor sensitivity of conventional flow cytometers in the analysis of submicron particles. Technical artifacts can confound these experiments, especially the detection of multiple particles as a single event. Here, we compared analysis of P2 crude synaptosomal preparations from murine forebrain on multiple flow cytometers using both FSC-triggered and fluorescence-triggered detection. We implemented multicolor fluorescent dye-based assays to quantify coincident particle detection and aggregation, and we assessed the false colocalization of antigens in immunostaining analyses. Our results demonstrate that fluorescence triggering and proper dilution can control for coincident particle detection, but not particle aggregation. We confirmed previous studies showing that FSC-based size estimation with PS beads underestimates biological particle size, and we identified pervasive aggregation in the FSC range analyzed in most synaptosome flow cytometry studies. We found that analyzing P2 samples in sucrose/EDTA/tris (SET) buffer reduces aggregation compared to PBS, but does not completely eliminate the presence of aggregates, especially in immunostaining experiments. Our study highlights challenges and pitfalls in synaptosome flow cytometry and provides a methodological framework for future studies., (Copyright © 2019 Hobson and Sims.)

Single-cell transcriptome profiling has enabled high-resolution analysis of cellular populations in tissues during development, health, and disease. Recent studies make innovative use of single-cell RNA sequencing (scRNAseq) to investigate mechanisms that allow immune cells to interact with tissue components in the lung during development and fibrotic lung disease.

FMRP (fragile X mental retardation protein) is a polysome-associated RNA-binding protein encoded by Fmr1 that is lost in fragile X syndrome. Increasing evidence suggests that FMRP regulates both translation initiation and elongation, but the gene specificity of these effects is unclear. To elucidate the impact of Fmr1 loss on translation, we utilize ribosome profiling for genome-wide measurements of ribosomal occupancy and positioning in the cortex of 24-day-old Fmr1 knockout mice. We find a remarkably coherent reduction in ribosome footprint abundance per mRNA for previously identified, high-affinity mRNA binding partners of FMRP and an increase for terminal oligopyrimidine (TOP) motif-containing genes canonically controlled by mammalian target of rapamycin-eIF4E-binding protein-eIF4E binding protein-eukaryotic initiation factor 4E (mTOR-4E-BP-eIF4E) signaling. Amino acid motif- and gene-level analyses both show a widespread reduction of translational pausing in Fmr1 knockout mice. Our findings are consistent with a model of FMRP-mediated regulation of both translation initiation through eIF4E and elongation that is disrupted in fragile X syndrome., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Tissue-resident memory T cells (T RM ) maintain immunity in diverse sites as determined in mouse models, whereas their establishment and role in human tissues have been difficult to assess. Here, we investigated human lung T RM generation, maintenance, and function in airway samples obtained longitudinally from human leukocyte antigen (HLA)-disparate lung transplant recipients, where donor and recipient T cells could be localized and tracked over time. Donor T cells persist specifically in the lungs (and not blood) of transplant recipients and express high levels of T RM signature markers including CD69, CD103, and CD49a, whereas lung-infiltrating recipient T cells gradually acquire T RM phenotypes over months in vivo. Single-cell transcriptome profiling of airway T cells reveals that donor T cells comprise two T RM -like subsets with varying levels of expression of T RM -associated genes, whereas recipient T cells comprised non-T RM and similar T RM -like subpopulations, suggesting de novo T RM generation. Transplant recipients exhibiting higher frequencies of persisting donor T RM experienced fewer adverse clinical events such as primary graft dysfunction and acute cellular rejection compared with recipients with low donor T RM persistence, suggesting that monitoring T RM dynamics could be clinically informative. Together, our results provide spatial and temporal insights into how human T RM develop, function, persist, and affect tissue integrity within the complexities of lung transplantation., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Common approaches to gene signature discovery in single-cell RNA-sequencing (scRNA-seq) depend upon predefined structures like clusters or pseudo-temporal order, require prior normalization, or do not account for the sparsity of single-cell data. We present single-cell hierarchical Poisson factorization (scHPF), a Bayesian factorization method that adapts hierarchical Poisson factorization (Gopalan et al , 2015, Proceedings of the 31st Conference on Uncertainty in Artificial Intelligence , 326) for de novo discovery of both continuous and discrete expression patterns from scRNA-seq. scHPF does not require prior normalization and captures statistical properties of single-cell data better than other methods in benchmark datasets. Applied to scRNA-seq of the core and margin of a high-grade glioma, scHPF uncovers marked differences in the abundance of glioma subpopulations across tumor regions and regionally associated expression biases within glioma subpopulations. scHFP revealed an expression signature that was spatially biased toward the glioma-infiltrated margins and associated with inferior survival in glioblastoma., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

The ventricular-subventricular zone (V-SVZ) harbors adult neural stem cells. V-SVZ neural stem cells exhibit features of astrocytes, have a regional identity, and depending on their location in the lateral or septal wall of the lateral ventricle, generate different types of neuronal and glial progeny. We performed large-scale single-cell RNA sequencing to provide a molecular atlas of cells from the lateral and septal adult V-SVZ of male and female mice. This revealed regional and sex differences among adult V-SVZ cells. We uncovered lineage potency bias at the single-cell level among lateral and septal wall astrocytes toward neurogenesis and oligodendrogenesis, respectively. Finally, we identified transcription factor co-expression modules marking key temporal steps in neurogenic and oligodendrocyte lineage progression. Our data suggest functionally important spatial diversity in neurogenesis and oligodendrogenesis in the adult brain and reveal molecular correlates of adult NSC dormancy and lineage specialization., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Optically decodable beads link the identity of a sample to a measurement through an optical barcode, enabling libraries of biomolecules to be captured on beads in solution and decoded by fluorescence. This approach has been foundational to microarray, sequencing, and flow-based expression profiling technologies. We combine microfluidics with optically decodable beads and show that phenotypic analysis of living cells can be linked to single-cell sequencing. As a proof-of-concept, we demonstrate the accuracy and scalability of our tool called Single Cell Optical Phenotyping and Expression sequencing (SCOPE-Seq) to combine live cell imaging with single-cell RNA sequencing.

Background: Despite extensive molecular characterization, we lack a comprehensive understanding of lineage identity, differentiation, and proliferation in high-grade gliomas (HGGs)., Methods: We sampled the cellular milieu of HGGs by profiling dissociated human surgical specimens with a high-density microwell system for massively parallel single-cell RNA-Seq. We analyzed the resulting profiles to identify subpopulations of both HGG and microenvironmental cells and applied graph-based methods to infer structural features of the malignantly transformed populations., Results: While HGG cells can resemble glia or even immature neurons and form branched lineage structures, mesenchymal transformation results in unstructured populations. Glioma cells in a subset of mesenchymal tumors lose their neural lineage identity, express inflammatory genes, and co-exist with marked myeloid infiltration, reminiscent of molecular interactions between glioma and immune cells established in animal models. Additionally, we discovered a tight coupling between lineage resemblance and proliferation among malignantly transformed cells. Glioma cells that resemble oligodendrocyte progenitors, which proliferate in the brain, are often found in the cell cycle. Conversely, glioma cells that resemble astrocytes, neuroblasts, and oligodendrocytes, which are non-proliferative in the brain, are generally non-cycling in tumors., Conclusions: These studies reveal a relationship between cellular identity and proliferation in HGG and distinct population structures that reflects the extent of neural and non-neural lineage resemblance among malignantly transformed cells.

Conventional differential expression analyses have been successfully employed to identify genes whose levels change across experimental conditions. One limitation of this approach is the inability to discover central regulators that control gene expression networks. In addition, while methods for identifying central nodes in a network are widely implemented, the bioinformatics validation process and the theoretical error estimates that reflect the uncertainty in each step of the analysis are rarely considered. Using the betweenness centrality measure, we identified Etv5 as a potential tissue-level regulator in murine neurofibromatosis type 1 (Nf1) low-grade brain tumors (optic gliomas). As such, the expression of Etv5 and Etv5 target genes were increased in multiple independently-generated mouse optic glioma models relative to non-neoplastic (normal healthy) optic nerves, as well as in the cognate human tumors (pilocytic astrocytoma) relative to normal human brain. Importantly, differential Etv5 and Etv5 network expression was not directly the result of Nf1 gene dysfunction in specific cell types, but rather reflects a property of the tumor as an aggregate tissue. Moreover, this differential Etv5 expression was independently validated at the RNA and protein levels. Taken together, the combined use of network analysis, differential RNA expression findings, and experimental validation highlights the potential of the computational network approach to provide new insights into tumor biology., Competing Interests: The authors have declared that no competing interests exist.

The ability of extracellular vesicles (EVs) to regulate a broad range of cellular processes has recently been exploited for the treatment of diseases. For example, EVs secreted by stem cells injected into infarcted hearts can induce recovery through the delivery of stem-cell-specific miRNAs. However, the retention of the EVs and the therapeutic effects are short-lived. Here, we show that an engineered hydrogel patch capable of slowly releasing EVs secreted from cardiomyocytes derived from induced pluripotent stem (iPS) cells reduced arrhythmic burden, promoted ejection-fraction recovery, decreased cardiomyocyte apoptosis 24 hours after infarction, and reduced infarct size and cell hypertrophy 4 weeks post-infarction when implanted onto infarcted rat hearts. We also show that the EVs are enriched with cardiac-specific miRNAs known to modulate cardiomyocyte-specific processes. The extended delivery of EVs secreted from iPS-cell-derived cardiomyocytes into the heart may help understand heart recovery and treat heart injury., Competing Interests: Competing interests statement The authors declare no competing interests.

We and others have shown that transition and maintenance of biological states is controlled by master regulator proteins, which can be inferred by interrogating tissue-specific regulatory models (interactomes) with transcriptional signatures, using the VIPER algorithm. Yet, some tissues may lack molecular profiles necessary for interactome inference (orphan tissues), or, as for single cells isolated from heterogeneous samples, their tissue context may be undetermined. To address this problem, we introduce metaVIPER, an algorithm designed to assess protein activity in tissue-independent fashion by integrative analysis of multiple, non-tissue-matched interactomes. This assumes that transcriptional targets of each protein will be recapitulated by one or more available interactomes. We confirm the algorithm's value in assessing protein dysregulation induced by somatic mutations, as well as in assessing protein activity in orphan tissues and, most critically, in single cells, thus allowing transformation of noisy and potentially biased RNA-Seq signatures into reproducible protein-activity signatures.

Intratumoral heterogeneity is among the greatest challenges in precision cancer therapy. However, developments in high-throughput single-cell RNA sequencing (scRNA-seq) may now provide the statistical power to dissect the diverse cellular populations of tumors. In the future these technologies might inform the selection of targeted combination therapies and enrollment criteria for clinical trials., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Microglia are a major cellular component of gliomas, and abundant in the centre of the tumour and at the infiltrative margins. While glioma is a notoriously infiltrative disease, the dynamics of microglia and glioma migratory patterns have not been well characterized. To investigate the migratory behaviour of microglia and glioma cells at the infiltrative edge, we performed two-colour time-lapse fluorescence microscopy of brain slices generated from a platelet-derived growth factor-B (PDGFB)-driven rat model of glioma, in which glioma cells and microglia were each labelled with one of two different fluorescent markers. We used mathematical techniques to analyse glioma cells and microglia motility with both single cell tracking and particle image velocimetry (PIV). Our results show microglia motility is strongly correlated with the presence of glioma, while the correlation of the speeds of glioma cells and microglia was variable and weak. Additionally, we showed that microglia and glioma cells exhibit different types of diffusive migratory behaviour. Microglia movement fit a simple random walk, while glioma cell movement fits a super diffusion pattern. These results show that glioma cells stimulate microglia motility at the infiltrative margins, creating a correlation between the spatial distribution of glioma cells and the pattern of microglia motility., (© 2018 The Author(s).)

Acute kidney injury (AKI) currently is diagnosed by a temporal trend of a single blood analyte: serum creatinine. This measurement is neither sensitive nor specific to kidney injury or its protean forms. Newer biomarkers, neutrophil gelatinase-associated lipocalin (NGAL, Lipocalin 2, Siderocalin), or kidney injury molecule-1 (KIM-1, Hepatitis A Virus Cellular Receptor 1), accelerate the diagnosis of AKI as well as prospectively distinguish rapidly reversible from prolonged causes of serum creatinine increase. Nonetheless, these biomarkers lack the capacity to subfractionate AKI further (eg, sepsis versus ischemia versus nephrotoxicity from medications, enzymes, or metals) or inform us about the primary and secondary sites of injury. It also is unknown whether all nephrons are injured in AKI, whether all cells in a nephron are affected, and whether injury responses can be stimulus-specific or cell type-specific or both. In this review, we summarize fully agnostic tissue interrogation approaches that may help to redefine AKI in cellular and molecular terms, including single-cell and single-nuclei RNA sequencing technology. These approaches will empower a shift in the current paradigm of AKI diagnosis, classification, and staging, and provide the renal community with a significant advance toward precision medicine in the analysis AKI., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Pharmacological and functional genomic screens play an essential role in the discovery and characterization of therapeutic targets and associated pharmacological inhibitors. Although these screens affect thousands of gene products, the typical readout is based on low complexity rather than genome-wide assays. To address this limitation, we introduce pooled library amplification for transcriptome expression (PLATE-Seq), a low-cost, genome-wide mRNA profiling methodology specifically designed to complement high-throughput screening assays. Introduction of sample-specific barcodes during reverse transcription supports pooled library construction and low-depth sequencing that is 10- to 20-fold less expensive than conventional RNA-Seq. The use of network-based algorithms to infer protein activity from PLATE-Seq data results in comparable reproducibility to 30 M read sequencing. Indeed, PLATE-Seq reproducibility compares favorably to other large-scale perturbational profiling studies such as the connectivity map and library of integrated network-based cellular signatures.Despite the importance of pharmacological and functional genomic screens the readouts are of low complexity. Here the authors introduce PLATE-Seq, a low-cost genome-wide mRNA profiling method to complement high-throughput screening.

Burtch, AR, Ogle, BT, Sims, PA, Harms, CA, Symons, TB, Folz, RJ, and Zavorsky, GS. Controlled frequency breathing reduces inspiratory muscle fatigue. J Strength Cond Res 31(5): 1273-1281, 2017-Controlled frequency breathing (CFB) is a common swim training modality involving holding one's breath for approximately 7-10 strokes before taking another breath. We sought to examine the effects of CFB training on reducing respiratory muscle fatigue. Competitive college swimmers were randomly divided into either the CFB group that breathed every 7-10 strokes or a control group that breathed every 3-4 strokes. Twenty swimmers completed the study. The training intervention included 5-6 weeks (16 sessions) of 12 × 50-m repetitions with breathing 8-10 breaths per 50-m (control group) or 2-3 breaths per 50-m (CFB group). Inspiratory muscle fatigue was defined as the decrease in maximal inspiratory pressure (MIP) between rest and 46 seconds after a 200-yard freestyle swimming race (115 seconds [SD 7]). Aerobic capacity, pulmonary diffusing capacity, and running economy were also measured pre- and posttraining. Pooled results demonstrated a 12% decrease in MIP at 46 seconds post-race (-15 [SD 14] cm H2O, effect size = -0.48, p < 0.01). After 4 weeks of training, only the CFB group prevented a decline in MIP values before to 46 seconds after race (-2 [13] cm H2O, p > 0.05). However, swimming performance, aerobic capacity, pulmonary diffusing capacity, and running economy did not improve (p > 0.05) posttraining in either group. In conclusion, CFB training appears to prevent inspiratory muscle fatigue; yet, no difference was found in performance outcomes.

The concept that solid tumors are maintained by a productive interplay between neoplastic and non-neoplastic elements has gained traction with the demonstration that stromal fibroblasts and immune system cells dictate cancer development and progression. While less studied, brain tumor (glioma) biology is likewise influenced by non-neoplastic immune system cells (macrophages and microglia) which interact with neoplastic glioma cells to create a unique physiological state (glioma ecosystem) distinct from that found in the normal tissue. To explore this neoplastic ground state, we leveraged several preclinical mouse models of neurofibromatosis type 1 (NF1) optic glioma, a low-grade astrocytoma whose formation and maintenance requires productive interactions between non-neoplastic and neoplastic cells, and employed whole tumor RNA-sequencing and mathematical deconvolution strategies to characterize this low-grade glioma ecosystem as an aggregate of cellular and acellular elements. Using this approach, we demonstrate that optic gliomas generated by altering the germline Nf1 gene mutation, the glioma cell of origin, or the presence of co-existing genetic alterations represent molecularly-distinct tumors. However, these optic glioma tumors share a 25-gene core signature, not found in normal optic nerve, that is normalized by microglia inhibition (minocycline), but not conventional (carboplatin) or molecularly-targeted (rapamycin) chemotherapy. Lastly, we identify a genetic signature conferred by Pten reduction and corrected by PI3K inhibition. This signature predicts progression-free survival in patients with either low-grade or high-grade glioma. Collectively, these findings support the concept that gliomas are composite ecological systems whose biology and response to therapy may be best defined by examining the tumor as a whole., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Changes in climbing fiber-Purkinje cell (CF-PC) synaptic connections have been found in the essential tremor (ET) cerebellum, and these changes are correlated with tremor severity. Whether these postmortem changes are specific to ET remains to be investigated. We assessed CF-PC synaptic pathology in the postmortem cerebellum across a range of degenerative movement disorders [10 Parkinson's disease (PD) cases, 10 multiple system atrophy (MSA) cases, 10 spinocerebellar ataxia type 1 (SCA1) cases, and 20 ET cases] and 25 controls. We observed differences in terms of CF pathological features across these disorders. Specifically, PD cases and ET cases both had more CFs extending into the parallel fiber (PF) territory, but ET cases had more complex branching and increased length of CFs in the PF territory along with decreased CF synaptic density compared to PD cases. MSA cases and SCA1 cases had the most severely reduced CF synaptic density and a marked paucity of CFs extending into the PF territory. Furthermore, CFs in a subset of MSA cases formed collateral branches parallel to the PC layer, a feature not seen in other diagnostic groups. Using unsupervised cluster analysis, the cases and controls could all be categorized into four clusters based on the CF pathology and features of PC pathology, including counts of PCs and their axonal torpedoes. ET cases and PD cases co-segregated into two clusters, whereas SCA1 cases and MSA cases formed another cluster, separate from the control cluster. Interestingly, the presence of resting tremor seemed to be the clinical feature that separated the cases into the two ET-PD clusters. In conclusion, our study demonstrates that these degenerative movement disorders seem to differ with respect to the pattern of CF synaptic pathology they exhibit. It remains to be determined how these differences contribute to the clinical presentations of these diseases.

Recent developments have enabled rapid, inexpensive RNA sequencing of thousands of individual cells from a single specimen, raising the possibility of unbiased and comprehensive expression profiling from complex tissues. Microwell arrays are a particularly attractive microfluidic platform for single cell analysis due to their scalability, cell capture efficiency, and compatibility with imaging. We report an automated microwell array platform for single cell RNA-Seq with significantly improved performance over previous implementations. We demonstrate cell capture efficiencies of >50%, compatibility with commercially available barcoded mRNA capture beads, and parallel expression profiling from thousands of individual cells. We evaluate the level of cross-contamination in our platform by both tracking fluorescent cell lysate in sealed microwells and with a human-mouse mixed species RNA-Seq experiment. Finally, we apply our system to comprehensively assess heterogeneity in gene expression of patient-derived glioma neurospheres and uncover subpopulations similar to those observed in human glioma tissue., Competing Interests: Columbia University has filed a patent application based on this work.

The number of naive T cells decreases and susceptibility to new microbial infections increases with age. Here we describe a previously unknown subset of phenotypically naive human CD8(+) T cells that rapidly secreted multiple cytokines in response to persistent viral antigens but differed transcriptionally from memory and effector T cells. The frequency of these CD8(+) T cells, called 'memory T cells with a naive phenotype' (TMNP cells), increased with age and after severe acute infection and inversely correlated with the residual capacity of the immune system to respond to new infections with age. CD8(+) TMNP cells represent a potential new target for the immunotherapy of persistent infections and should be accounted for and subtracted from the naive pool if truly naive T cells are needed to respond to antigens.

Ribosome profiling has emerged as a powerful tool for genome-wide measurements of translation, but library construction requires multiple ligation steps and remains cumbersome relative to more conventional deep-sequencing experiments. We report a new, ligation-free approach to ribosome profiling that does not require ligation. Library construction for ligation-free ribosome profiling can be completed in one day with as little as 1 ng of purified RNA footprints. We apply ligation-free ribosome profiling to mouse brain tissue to identify new patterns of cell type-specific translation and test its ability to identify translational targets of mTOR signaling in the brain.

Although immune signaling has emerged as a defining feature of the glioma microenvironment, how the underlying structure of the glioma-infiltrating T-cell population differs from that of the blood from which it originates has been difficult to measure directly in patients. High-throughput sequencing of T-cell receptor (TCR) repertoires (TCRseq) provides a population-wide statistical description of how T cells respond to disease. We have defined immunophenotypes of whole repertoires based on TCRseq of the α- and β-chains from glioma tissue, nonneoplastic brain tissue, and peripheral blood from patients. Using information theory, we partitioned the diversity of these TCR repertoires into that from the distribution of VJ cassette combinations and diversity due to VJ-independent factors, such as selection due to antigen binding. Tumor-infiltrating lymphocytes (TILs) possessed higher VJ-independent diversity than nonneoplastic tissue, stratifying patients according to tumor grade. We found that the VJ-independent components of tumor-associated repertoires diverge more from their corresponding peripheral repertoires than T-cell populations in nonneoplastic brain tissue, particularly for low-grade gliomas. Finally, we identified a "signature" set of TCRs whose use in peripheral blood is associated with patients exhibiting low TIL divergence and is depleted in patients with highly divergent TIL repertoires. This signature is detectable in peripheral blood, and therefore accessible noninvasively. We anticipate that these immunophenotypes will be foundational to monitoring and predicting response to antiglioma vaccines and immunotherapy.

Glioblastoma (GBM) is the most common malignant primary brain cancer. With a median survival of about a year, new approaches to treating this disease are necessary. To identify signaling molecules regulating GBM progression in a genetically engineered murine model of proneural GBM, we quantified phosphotyrosine-mediated signaling using mass spectrometry. Oncogenic signals, including phosphorylated ERK MAPK, PI3K, and PDGFR, were found to be increased in the murine tumors relative to brain. Phosphorylation of CDK1 pY15, associated with the G2 arrest checkpoint, was identified as the most differentially phosphorylated site, with a 14-fold increase in phosphorylation in the tumors. To assess the role of this checkpoint as a potential therapeutic target, syngeneic primary cell lines derived from these tumors were treated with MK-1775, an inhibitor of Wee1, the kinase responsible for CDK1 Y15 phosphorylation. MK-1775 treatment led to mitotic catastrophe, as defined by increased DNA damage and cell death by apoptosis. To assess the extensibility of targeting Wee1/CDK1 in GBM, patient-derived xenograft (PDX) cell lines were also treated with MK-1775. Although the response was more heterogeneous, on-target Wee1 inhibition led to decreased CDK1 Y15 phosphorylation and increased DNA damage and apoptosis in each line. These results were also validated in vivo, where single-agent MK-1775 demonstrated an antitumor effect on a flank PDX tumor model, increasing mouse survival by 1.74-fold. This study highlights the ability of unbiased quantitative phosphoproteomics to reveal therapeutic targets in tumor models, and the potential for Wee1 inhibition as a treatment approach in preclinical models of GBM. Mol Cancer Ther; 15(6); 1332-43. ©2016 AACR., (©2016 American Association for Cancer Research.)

Background & Aims: Activin, a member of the transforming growth factor-β (TGFB) family, might be involved in pancreatic tumorigenesis, similar to other members of the TGFB family. Human pancreatic ductal adenocarcinomas contain somatic mutations in the activin A receptor type IB (ACVR1B) gene, indicating that ACVR1B could be a suppressor of pancreatic tumorigenesis., Methods: We disrupted Acvr1b specifically in pancreata of mice (Acvr1b(flox/flox);Pdx1-Cre mice) and crossed them with LSL-KRAS(G12D) mice, which express an activated form of KRAS and develop spontaneous pancreatic tumors. The resulting Acvr1b(flox/flox);LSL-KRAS(G12D);Pdx1-Cre mice were monitored; pancreatic tissues were collected and analyzed by histology and immunohistochemical analyses. We also analyzed p16(flox/flox);LSL-Kras(G12D);Pdx1-Cre mice and Cre-negative littermates (controls). Genomic DNA, total RNA, and protein were isolated from mouse tissues and primary pancreatic tumor cell lines and analyzed by reverse-transcription polymerase chain reaction, sequencing, and immunoblot analyses. Human intraductal papillary mucinous neoplasm (IPMN) specimens were analyzed by immunohistochemistry., Results: Loss of ACVR1B from pancreata of mice increased the proliferation of pancreatic epithelial cells, led to formation of acinar to ductal metaplasia, and induced focal inflammatory changes compared with control mice. Disruption of Acvr1b in LSL-KRAS(G12D);Pdx1-Cre mice accelerated the growth of pancreatic IPMNs compared with LSL-KRAS(G12D);Pdx1-Cre mice, but did not alter growth of pancreatic intraepithelial neoplasias. We associated perinuclear localization of the activated NOTCH4 intracellular domain to the apical cytoplasm of neoplastic cells with the expansion of IPMN lesions in Acvr1b(flox/flox);LSL-KRAS(G12D);Pdx1-Cre mice. Loss of the gene that encodes p16 (Cdkn2a) was required for progression of IPMNs to pancreatic ductal adenocarcinomas in Acvr1b(flox/flox);LSL-Kras(G12D);Pdx1-Cre mice. We also observed progressive loss of p16 in human IPMNs of increasing grades., Conclusions: Loss of ACVR1B accelerates growth of mutant KRAS-induced pancreatic IPMNs in mice; this process appears to involve NOTCH4 and loss of p16. ACVR1B suppresses early stages of pancreatic tumorigenesis; the activin signaling pathway therefore might be a therapeutic target for pancreatic cancer., (Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The proliferative and invasive nature of malignant cancers drives lethality. In glioblastoma, these two processes are presumed mutually exclusive and hence termed "go or grow." We identified a molecular target that shuttles between these disparate cellular processes-the molecular motor KIF11. Inhibition of KIF11 with a highly specific small-molecule inhibitor stopped the growth of the more treatment-resistant glioblastoma tumor-initiating cells (TICs, or cancer stem cells) as well as non-TICs and impeded tumor initiation and self-renewal of the TIC population. Targeting KIF11 also hit the other arm of the "go or grow" cell fate decision by reducing glioma cell invasion. Administration of a KIF11 inhibitor to mice bearing orthotopic glioblastoma prolonged their survival. In its role as a shared molecular regulator of cell growth and motility across intratumoral heterogeneity, KIF11 is a compelling therapeutic target for glioblastoma., (Copyright © 2015, American Association for the Advancement of Science.)

Many important biological questions demand single-cell transcriptomics on a large scale. Hence, new tools are urgently needed for efficient, inexpensive manipulation of RNA from individual cells. We report a simple platform for trapping single-cell lysates in sealed, picoliter microwells capable of printing RNA on glass or capturing RNA on beads. We then develop a scalable technology for genome-wide, single-cell RNA-Seq. Our device generates pooled libraries from hundreds of individual cells with consumable costs of $0.10-$0.20 per cell and includes five lanes for simultaneous experiments. We anticipate that this system will serve as a general platform for single-cell imaging and sequencing.

Glioblastomas (GBMs) diffusely infiltrate the brain, making complete removal by surgical resection impossible. The mixture of neoplastic and nonneoplastic cells that remain after surgery form the biological context for adjuvant therapeutic intervention and recurrence. We performed RNA-sequencing (RNA-seq) and histological analysis on radiographically guided biopsies taken from different regions of GBM and showed that the tissue contained within the contrast-enhancing (CE) core of tumors have different cellular and molecular compositions compared with tissue from the nonenhancing (NE) margins of tumors. Comparisons with the The Cancer Genome Atlas dataset showed that the samples from CE regions resembled the proneural, classical, or mesenchymal subtypes of GBM, whereas the samples from the NE regions predominantly resembled the neural subtype. Computational deconvolution of the RNA-seq data revealed that contributions from nonneoplastic brain cells significantly influence the expression pattern in the NE samples. Gene ontology analysis showed that the cell type-specific expression patterns were functionally distinct and highly enriched in genes associated with the corresponding cell phenotypes. Comparing the RNA-seq data from the GBM samples to that of nonneoplastic brain revealed that the differentially expressed genes are distributed across multiple cell types. Notably, the patterns of cell type-specific alterations varied between the different GBM subtypes: the NE regions of proneural tumors were enriched in oligodendrocyte progenitor genes, whereas the NE regions of mesenchymal GBM were enriched in astrocytic and microglial genes. These subtype-specific patterns provide new insights into molecular and cellular composition of the infiltrative margins of GBM.

Glioma growth is driven by signaling that ultimately regulates protein synthesis. Gliomas are also complex at the cellular level and involve multiple cell types, including transformed and reactive cells in the brain tumor microenvironment. The distinct functions of the various cell types likely lead to different requirements and regulatory paradigms for protein synthesis. Proneural gliomas can arise from transformation of glial progenitors that are driven to proliferate via mitogenic signaling that affects translation. To investigate translational regulation in this system, we developed a RiboTag glioma mouse model that enables cell-type-specific, genome-wide ribosome profiling of tumor tissue. Infecting glial progenitors with Cre-recombinant retrovirus simultaneously activates expression of tagged ribosomes and delivers a tumor-initiating mutation. Remarkably, we find that although genes specific to transformed cells are highly translated, their translation efficiencies are low compared with normal brain. Ribosome positioning reveals sequence-dependent regulation of ribosomal activity in 5'-leaders upstream of annotated start codons, leading to differential translation in glioma compared with normal brain. Additionally, although transformed cells express a proneural signature, untransformed tumor-associated cells, including reactive astrocytes and microglia, express a mesenchymal signature. Finally, we observe the same phenomena in human disease by combining ribosome profiling of human proneural tumor and non-neoplastic brain tissue with computational deconvolution to assess cell-type-specific translational regulation., (Copyright © 2014 the authors 0270-6474/14/3410924-13$15.00/0.)

Proneural glioblastoma is defined by an expression pattern resembling that of oligodendrocyte progenitor cells and carries a distinctive set of genetic alterations. Whether there is a functional relationship between the proneural phenotype and the associated genetic alterations is unknown. To evaluate this possible relationship, we performed a longitudinal molecular characterization of tumor progression in a mouse model of proneural glioma. In this setting, the tumors acquired remarkably consistent genetic deletions at late stages of progression, similar to those deleted in human proneural glioblastoma. Further investigations revealed that p53 is a master regulator of the transcriptional network underlying the proneural phenotype. This p53-centric transcriptional network and its associated phenotype were observed at both the early and late stages of progression, and preceded the proneural-specific deletions. Remarkably, deletion of p53 at the time of tumor initiation obviated the acquisition of later deletions, establishing a link between the proneural transcriptional network and the subtype-specific deletions selected during glioma progression., (©2014 AACR)

The crystal structure of AdhP, a recombinantly expressed alcohol dehydrogenase from Escherichia coli K-12 (substrain MG1655), was determined to 2.01 Å resolution. The structure, which was solved using molecular replacement, also included the structural and catalytic zinc ions and the cofactor nicotinamide adenine dinucleotide (NAD). The crystals belonged to space group P21, with unit-cell parameters a = 68.18, b = 118.92, c = 97.87 Å, β = 106.41°. The final R factor and Rfree were 0.138 and 0.184, respectively. The structure of the active site of AdhP suggested a number of residues that may participate in a proton relay, and the overall structure of AdhP, including the coordination to structural and active-site zinc ions, is similar to those of other tetrameric alcohol dehydrogenase enzymes.

Molecular subtypes of glioblastoma (GBM) with distinct alterations have been identified. There is need for reproducible, versatile preclinical models that resemble specific GBM phenotypes to facilitate preclinical testing of novel therapies. We present a cell line-based murine proneural GBM model and characterize its response to radiation therapy. Proneural gliomas were generated by injecting PDGF-IRES-Cre retrovirus into the subcortical white matter of adult mice that harbor floxed tumor suppressors (Pten and p53) and stop-floxed reporters. Primary cell cultures were generated from the retrovirus induced tumors and maintained in vitro for multiple passages. RNA sequencing-based expression profiling of the resulting cell lines was performed. The tumorigenic potential of the cells was assessed by intracranial injection into adult naïve mice from different strains. Tumor growth was assessed by bioluminescence imaging (BLI). BLI for tumor cells and brain slices were obtained and compared to in vivo BLI. Response to whole-brain radiation was assessed in glioma-bearing animals. Intracranial injection of Pdgf(+)Pten(-/-)p53(-/-)luciferase(+) glioma cells led to formation of GBM-like tumors with 100 % efficiency (n = 48) and tumorigenesis was retained for more than 3 generations. The cell lines specifically resembled proneural GBM based on expression profiling by RNA-Seq. Pdgf(+)Pten(-/-)p53(-/-)luciferase(+) cell number correlated with BLI signal. Serial BLI measured tumor growth and correlated with size and location by ex vivo imaging. Moreover, BLI predicted tumor-related mortality with a 93 % risk of death within 5 days following a BLI signal between 1 × 10(8) and 5 × 10(8) photons/s cm(2). BLI signal had transient but significant response following radiotherapy, which corresponded to a modest survival benefit for radiated mice (p < 0.05). Intracranial injection of Pdgf(+)Pten(-/-)p53(-/-)luciferase(+) cells constitutes a novel and highly reproducible model, recapitulating key features of human proneural GBM, and can be used to evaluate tumor-growth and response to therapy.

Real-time on-site monitoring of analytes is currently in high demand for food contamination, water, medicines, and ingestible household products that were never tested appropriately. Here we introduce chemical methods for the rapid quantification of a wide range of chemical and microbial contaminations using a simple instrument. Within the testing procedure, we used a multichannel, multisample, UV-vis spectrophotometer/fluorometer that employs two frequencies of light simultaneously to interrogate the sample. We present new enzyme- and dye-based methods to detect (di)ethylene glycol in consumables above 0.1 wt % without interference and alcohols above 1 ppb. Using DNA intercalating dyes, we can detect a range of pathogens ( E. coli , Salmonella , V. Cholera, and a model for Malaria) in water, foods, and blood without background signal. We achieved universal scaling independent of pathogen size above 10(4) CFU/mL by taking advantage of the simultaneous measurement at multiple wavelengths. We can detect contaminants directly, without separation, purification, concentration, or incubation. Our chemistry is stable to ± 1% for >3 weeks without refrigeration, and measurements require <5 min.