The Allen Mouse Brain Common Coordinate Framework: A 3D Reference Atlas.

Recent large-scale collaborations are generating major surveys of cell types and connections in the mouse brain, collecting large amounts of data across modalities, spatial scales, and brain areas. Successful integration of these data requires a standard 3D reference atlas. Here, we present the Allen Mouse Brain Common Coordinate Framework (CCFv3) as such a resource. We constructed an average template brain at 10 µm voxel resolution by interpolating high resolution in-plane serial two-photon tomography images with 100 µm z-sampling from 1,675 young adult C57BL/6J mice. Then, using multimodal reference data, we parcellated the entire brain directly in 3D, labeling every voxel with a brain structure spanning 43 isocortical areas and their layers, 329 subcortical gray matter structures, 81 fiber tracts, and 8 ventricular structures. CCFv3 can be used to analyze, visualize, and integrate multimodal and multiscale datasets in 3D and is openly accessible (https://atlas.brain-map.org/).

The RNA-binding proteins hnRNP H and F regulate splicing of a MYC-dependent HRAS exon in prostate cancer cells.

The MYC proto-oncogene contributes to the pathogenesis of more than half of human cancers. Malignant transformation by MYC transcriptionally up-regulates the core pre-mRNA splicing machinery and causes misregulation of alternative splicing. However, our understanding of how splicing changes are directed by MYC is limited. We performed a signaling pathway-guided splicing analysis to identify MYC-dependent splicing events. These included an HRAS cassette exon repressed by MYC across multiple tumor types. To molecularly dissect the regulation of this HRAS exon, we used antisense oligonucleotide tiling to identify splicing enhancers and silencers in its flanking introns. RNA-binding motif prediction indicated multiple binding sites for hnRNP H and hnRNP F within these cis-regulatory elements. Using siRNA knockdown and cDNA expression, we found that both hnRNP H and F activate the HRAS cassette exon. Mutagenesis and targeted RNA immunoprecipitation implicate two downstream G-rich elements in this splicing activation. Analyses of ENCODE RNA-seq datasets confirmed hnRNP H regulation of HRAS splicing. Analyses of RNA-seq datasets across multiple cancers showed a negative correlation of HNRNPH gene expression with MYC hallmark enrichment, consistent with the effect of hnRNP H on HRAS splicing. Interestingly, HNRNPF expression showed a positive correlation with MYC hallmarks and thus was not consistent with the observed effects of hnRNP F. Loss of hnRNP H/F altered cell cycle progression and induced apoptosis in the PC3 prostate cancer cell line. Collectively, our results reveal mechanisms for MYC-dependent regulation of splicing and point to possible therapeutic targets in prostate cancers.

IRIS: Discovery of cancer immunotherapy targets arising from pre-mRNA alternative splicing.

Alternative splicing (AS) is prevalent in cancer, generating an extensive but largely unexplored repertoire of novel immunotherapy targets. We describe Isoform peptides from RNA splicing for Immunotherapy target Screening (IRIS), a computational platform capable of discovering AS-derived tumor antigens (TAs) for T cell receptor (TCR) and chimeric antigen receptor T cell (CAR-T) therapies. IRIS leverages large-scale tumor and normal transcriptome data and incorporates multiple screening approaches to discover AS-derived TAs with tumor-associated or tumor-specific expression. In a proof-of-concept analysis integrating transcriptomics and immunopeptidomics data, we showed that hundreds of IRIS-predicted TCR targets are presented by human leukocyte antigen (HLA) molecules. We applied IRIS to RNA-seq data of neuroendocrine prostate cancer (NEPC). From 2,939 NEPC-associated AS events, IRIS predicted 1,651 epitopes from 808 events as potential TCR targets for two common HLA types (A*02:01 and A*03:01). A more stringent screening test prioritized 48 epitopes from 20 events with "neoantigen-like" NEPC-specific expression. Predicted epitopes are often encoded by microexons of ≤30 nucleotides. To validate the immunogenicity and T cell recognition of IRIS-predicted TCR epitopes, we performed in vitro T cell priming in combination with single-cell TCR sequencing. Seven TCRs transduced into human peripheral blood mononuclear cells (PBMCs) showed high activity against individual IRIS-predicted epitopes, providing strong evidence of isolated TCRs reactive to AS-derived peptides. One selected TCR showed efficient cytotoxicity against target cells expressing the target peptide. Our study illustrates the contribution of AS to the TA repertoire of cancer cells and demonstrates the utility of IRIS for discovering AS-derived TAs and expanding cancer immunotherapies.

Hierarchical organization of cortical and thalamic connectivity.

The mammalian cortex is a laminar structure containing many areas and cell types that are densely interconnected in complex ways, and for which generalizable principles of organization remain mostly unknown. Here we describe a major expansion of the Allen Mouse Brain Connectivity Atlas resource1, involving around a thousand new tracer experiments in the cortex and its main satellite structure, the thalamus. We used Cre driver lines (mice expressing Cre recombinase) to comprehensively and selectively label brain-wide connections by layer and class of projection neuron. Through observations of axon termination patterns, we have derived a set of generalized anatomical rules to describe corticocortical, thalamocortical and corticothalamic projections. We have built a model to assign connection patterns between areas as either feedforward or feedback, and generated testable predictions of hierarchical positions for individual cortical and thalamic areas and for cortical network modules. Our results show that cell-class-specific connections are organized in a shallow hierarchy within the mouse corticothalamic network.

Physical and in silico immunopeptidomic profiling of a cancer antigen prostatic acid phosphatase reveals targets enabling TCR isolation.

Tissue-specific antigens can serve as targets for adoptive T cell transfer-based cancer immunotherapy. Recognition of tumor by T cells is mediated by interaction between peptide-major histocompatibility complexes (pMHCs) and T cell receptors (TCRs). Revealing the identity of peptides bound to MHC is critical in discovering cognate TCRs and predicting potential toxicity. We performed multimodal immunopeptidomic analyses for human prostatic acid phosphatase (PAP), a well-recognized tissue antigen. Three physical methods, including mild acid elution, coimmunoprecipitation, and secreted MHC precipitation, were used to capture a thorough signature of PAP on HLA-A*02:01. Eleven PAP peptides that are potentially A*02:01-restricted were identified, including five predicted strong binders by NetMHCpan 4.0. Peripheral blood mononuclear cells (PBMCs) from more than 20 healthy donors were screened with the PAP peptides. Seven cognate TCRs were isolated which can recognize three distinct epitopes when expressed in PBMCs. One TCR shows reactivity toward cell lines expressing both full-length PAP and HLA-A*02:01. Our results show that a combined multimodal immunopeptidomic approach is productive in revealing target peptides and defining the cloned TCR sequences reactive with prostatic acid phosphatase epitopes.

Droplet-based mRNA sequencing of fixed and permeabilized cells by CLInt-seq allows for antigen-specific TCR cloning.

T cell receptors (TCRs) are generated by somatic recombination of V/D/J segments to produce up to 1015 unique sequences. Highly sensitive and specific techniques are required to isolate and identify the rare TCR sequences that respond to antigens of interest. Here, we describe the use of mRNA sequencing via cross-linker regulated intracellular phenotype (CLInt-Seq) for efficient recovery of antigen-specific TCRs in cells stained for combinations of intracellular proteins such as cytokines or transcription factors. This method enables high-throughput identification and isolation of low-frequency TCRs specific for any antigen. As a proof of principle, intracellular staining for TNFα and IFNγ identified cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-reactive TCRs with efficiencies similar to state-of-the-art peptide-MHC multimer methodology. In a separate experiment, regulatory T cells were profiled based on intracellular FOXP3 staining, demonstrating the ability to examine phenotypes based on transcription factors. We further optimized the intracellular staining conditions to use a chemically cleavable primary amine cross-linker compatible with current single-cell sequencing technology. CLInt-Seq for TNFα and IFNγ performed similarly to isolation with multimer staining for EBV-reactive TCRs. We anticipate CLInt-Seq will enable droplet-based single-cell mRNA analysis from any tissue where minor populations need to be isolated by intracellular markers.

Pathway-guided analysis identifies Myc-dependent alternative pre-mRNA splicing in aggressive prostate cancers.

We sought to define the landscape of alternative pre-mRNA splicing in prostate cancers and the relationship of exon choice to known cancer driver alterations. To do so, we compiled a metadataset composed of 876 RNA-sequencing (RNA-Seq) samples from five publicly available sources representing a range of prostate phenotypes from normal tissue to drug-resistant metastases. We subjected these samples to exon-level analysis with rMATS-turbo, purpose-built software designed for large-scale analyses of splicing, and identified 13,149 high-confidence cassette exon events with variable incorporation across samples. We then developed a computational framework, pathway enrichment-guided activity study of alternative splicing (PEGASAS), to correlate transcriptional signatures of 50 different cancer driver pathways with these alternative splicing events. We discovered that Myc signaling was correlated with incorporation of a set of 1,039 cassette exons enriched in genes encoding RNA binding proteins. Using a human prostate epithelial transformation assay, we confirmed the Myc regulation of 147 of these exons, many of which introduced frameshifts or encoded premature stop codons. Our results connect changes in alternative pre-mRNA splicing to oncogenic alterations common in prostate and many other cancers. We also establish a role for Myc in regulating RNA splicing by controlling the incorporation of nonsense-mediated decay-determinant exons in genes encoding RNA binding proteins.

A mesoscale connectome of the mouse brain.

Comprehensive knowledge of the brain's wiring diagram is fundamental for understanding how the nervous system processes information at both local and global scales. However, with the singular exception of the C. elegans microscale connectome, there are no complete connectivity data sets in other species. Here we report a brain-wide, cellular-level, mesoscale connectome for the mouse. The Allen Mouse Brain Connectivity Atlas uses enhanced green fluorescent protein (EGFP)-expressing adeno-associated viral vectors to trace axonal projections from defined regions and cell types, and high-throughput serial two-photon tomography to image the EGFP-labelled axons throughout the brain. This systematic and standardized approach allows spatial registration of individual experiments into a common three dimensional (3D) reference space, resulting in a whole-brain connectivity matrix. A computational model yields insights into connectional strength distribution, symmetry and other network properties. Virtual tractography illustrates 3D topography among interconnected regions. Cortico-thalamic pathway analysis demonstrates segregation and integration of parallel pathways. The Allen Mouse Brain Connectivity Atlas is a freely available, foundational resource for structural and functional investigations into the neural circuits that support behavioural and cognitive processes in health and disease.

Prostate epithelial cell of origin determines cancer differentiation state in an organoid transformation assay.

The cell of origin for prostate cancer remains a subject of debate. Genetically engineered mouse models have demonstrated that both basal and luminal cells can serve as cells of origin for prostate cancer. Using a human prostate regeneration and transformation assay, our group previously demonstrated that basal cells can serve as efficient targets for transformation. Recently, a subpopulation of multipotent human luminal cells defined by CD26 expression that retains progenitor activity in a defined organoid culture was identified. We transduced primary human prostate basal and luminal cells with lentiviruses expressing c-Myc and activated AKT1 (myristoylated AKT1 or myrAKT1) to mimic theMYCamplification andPTENloss commonly detected in human prostate cancer. These cells were propagated in organoid culture before being transplanted into immunodeficient mice. We found that c-Myc/myrAKT1-transduced luminal xenografts exhibited histological features of well-differentiated acinar adenocarcinoma, with strong androgen receptor (AR) and prostate-specific antigen (PSA) expression. In contrast, c-Myc/myrAKT1-transduced basal xenografts were histologically more aggressive, with a loss of acinar structures and low/absent AR and PSA expression. Our findings imply that distinct subtypes of prostate cancer may arise from luminal and basal epithelial cell types subjected to the same oncogenic insults. This study provides a platform for the functional evaluation of oncogenes in basal and luminal epithelial populations of the human prostate. Tumors derived in this fashion with defined genetics can be used in the preclinical development of targeted therapeutics.

Activated Prothrombin Complex Concentrate Versus 4-Factor Prothrombin Complex Concentrate for Vitamin K-Antagonist Reversal.

OBJECTIVES: To compare the international normalized ratio normalization efficacy of activated prothrombin complex concentrates and 4-factor prothrombin complex concentrates and to evaluate the thrombotic complications in patients treated with these products for warfarin-associated hemorrhage. DESIGN: Retrospective, Multicenter Cohort. SETTING: Large, Community, Teaching Hospital. PATIENTS: Patients greater than 18 years old and received either activated prothrombin complex concentrate or 4-factor prothrombin complex concentrate for the treatment of warfarin-associated hemorrhage. We excluded those patients who received either agent for an indication other than warfarin-associated hemorrhage, pregnant, had a baseline international normalized ratio of less than 2, received a massive transfusion as defined by hospital protocol, received plasma for treatment of warfarin-associated hemorrhage, or were treated for an acute warfarin ingestion. INTERVENTIONS: Patients in the activated prothrombin complex concentrate group (enrolled from one hospital) with an international normalized ratio of less than 5 received 500 IU and those with an international normalized ratio greater than 5 received 1,000 IU. Patients in the 4-factor prothrombin complex concentrate (enrolled from a separate hospital) group received the Food and Drug Administration approved dosing algorithm. MEASUREMENTS AND MAIN RESULTS: A total of 158 patients were included in the final analysis (activated prothrombin complex concentrate = 118; 4-factor prothrombin complex concentrate = 40). Those in the 4-factor prothrombin complex concentrate group had a higher pretreatment international normalized ratio (2.7 ± 1.8 vs 3.5 ± 2.9; p = 0.0164). However, the posttreatment international normalized ratio was similar between the groups. In addition, even when controlling for differences in the pretreatment international normalized ratio, there was no difference in the ability to achieve a posttreatment international normalized ratio of less than 1.4 (odds ratio, 0.753 [95% CI, 0.637-0.890]; p = 0.0009). Those in the activated prothrombin complex concentrate group did have higher odds of achieving a posttreatment international normalized ratio of less than 1.2 (odds ratio, 3.23 [95% CI, 1.34-7.81]; p = 0.0088). There was only one posttreatment thrombotic complication reported. CONCLUSIONS: A low, fixed dose of activated prothrombin complex concentrate was as effective as standard dose 4-factor prothrombin complex concentrate for normalization of international normalized ratio. In addition, we did not see an increase in thrombotic events.

Replication stress by Py-Im polyamides induces a non-canonical ATR-dependent checkpoint response.

Pyrrole-imidazole polyamides targeted to the androgen response element were cytotoxic in multiple cell lines, independent of intact androgen receptor signaling. Polyamide treatment induced accumulation of S-phase cells and of PCNA replication/repair foci. Activation of a cell cycle checkpoint response was evidenced by autophosphorylation of ATR, the S-phase checkpoint kinase, and by recruitment of ATR and the ATR activators RPA, 9-1-1, and Rad17 to chromatin. Surprisingly, ATR activation was accompanied by only a slight increase in single-stranded DNA, and the ATR targets RPA2 and Chk1, a cell cycle checkpoint kinase, were not phosphorylated. However, ATR activation resulted in phosphorylation of the replicative helicase subunit MCM2, an ATR effector. Polyamide treatment also induced accumulation of monoubiquitinated FANCD2, which is recruited to stalled replication forks and interacts transiently with phospho-MCM2. This suggests that polyamides induce replication stress that ATR can counteract independently of Chk1 and that the FA/BRCA pathway may also be involved in the response to polyamides. In biochemical assays, polyamides inhibit DNA helicases, providing a plausible mechanism for S-phase inhibition.

Improving reliability and absolute quantification of human brain microarray data by filtering and scaling probes using RNA-Seq.

BACKGROUND: High-throughput sequencing is gradually replacing microarrays as the preferred method for studying mRNA expression levels, providing nucleotide resolution and accurately measuring absolute expression levels of almost any transcript, known or novel. However, existing microarray data from clinical, pharmaceutical, and academic settings represent valuable and often underappreciated resources, and methods for assessing and improving the quality of these data are lacking. RESULTS: To quantitatively assess the quality of microarray probes, we directly compare RNA-Seq to Agilent microarrays by processing 231 unique samples from the Allen Human Brain Atlas using RNA-Seq. Both techniques provide highly consistent, highly reproducible gene expression measurements in adult human brain, with RNA-Seq slightly outperforming microarray results overall. We show that RNA-Seq can be used as ground truth to assess the reliability of most microarray probes, remove probes with off-target effects, and scale probe intensities to match the expression levels identified by RNA-Seq. These sequencing scaled microarray intensities (SSMIs) provide more reliable, quantitative estimates of absolute expression levels for many genes when compared with unscaled intensities. Finally, we validate this result in two human cell lines, showing that linear scaling factors can be applied across experiments using the same microarray platform. CONCLUSIONS: Microarrays provide consistent, reproducible gene expression measurements, which are improved using RNA-Seq as ground truth. We expect that our strategy could be used to improve probe quality for many data sets from major existing repositories.

Isolation, structure elucidation, and biological activity of altersolanol P using Staphylococcus aureus fitness test based genome-wide screening.

Bacteria continue to evade existing antibiotics by acquiring resistance by various mechanisms, leading to loss of antibiotic effectiveness. To avoid an epidemic from infections of incurable drug-resistant bacteria, new antibiotics with new modes of action are desperately needed. Using a genome-wide mechanism of action-guided whole cell screening approach based on antisense Staphylococcus aureus fitness test technology, we report herein the discovery of altersolanol P (1), a new tetrahydroanthraquinone from an unknown fungus from the Hypocreales isolated from forest litter collected in Puerto Rico. The structure was elucidated by high-resolution mass spectrometry and 2D NMR spectroscopy. Relative stereochemistry was established by NOESY correlations, and absolute configuration was deduced by the application of MPA ester-based methodology. Observed (1)H and (13)C NMR shifts were well aligned with the corresponding chemical shifts predicted by DFT calculations. Altersolanol P exhibited Gram-positive antibacterial activity (MIC range 1-8 µg/mL) and inhibited the growth of Gram-negative Haemophilus influenzae (MIC 2 µg/mL). The isolation, structure elucidation, and antibacterial activity of altersolanol P are described.

Broadening the spectrum of ß-lactam antibiotics through inhibition of signal peptidase type I.

The resistance of methicillin-resistant Staphylococcus aureus (MRSA) to all ß-lactam classes limits treatment options for serious infections involving this organism. Our goal is to discover new agents that restore the activity of ß-lactams against MRSA, an approach that has led to the discovery of two classes of natural product antibiotics, a cyclic depsipeptide (krisynomycin) and a lipoglycopeptide (actinocarbasin), which potentiate the activity of imipenem against MRSA strain COL. We report here that these imipenem synergists are inhibitors of the bacterial type I signal peptidase SpsB, a serine protease that is required for the secretion of proteins that are exported through the Sec and Tat systems. A synthetic derivative of actinocarbasin, M131, synergized with imipenem both in vitro and in vivo with potent efficacy. The in vitro activity of M131 extends to clinical isolates of MRSA but not to a methicillin-sensitive strain. Synergy is restricted to ß-lactam antibiotics and is not observed with other antibiotic classes. We propose that the SpsB inhibitors synergize with ß-lactams by preventing the signal peptidase-mediated secretion of proteins required for ß-lactam resistance. Combinations of SpsB inhibitors and ß-lactams may expand the utility of these widely prescribed antibiotics to treat MRSA infections, analogous to ß-lactamase inhibitors which restored the utility of this antibiotic class for the treatment of resistant Gram-negative infections.

Kibdelomycin A, a congener of kibdelomycin, derivatives and their antibacterial activities.

Emergence of bacterial resistance has eroded the effectiveness of many life saving antibiotics leading to an urgent need for new chemical classes of antibacterial agents. We have applied a Staphylococcus aureus fitness test strategy to natural products screening to meet this challenge. In this paper we report the discovery of kibdelomycin A, a demethylated congener of kibdelomycin, the representative of a novel class of antibiotics produced by a new strain of Kibdelosporangium. Kibdelomycin A is a potent inhibitor of DNA gyrase and topoisomerase IV, inhibits DNA synthesis and shows whole cell antibiotic activity, albeit, less potently than kibdelomycin. Kibdelomycin C-33 acetate and tetrahydro-bisdechloro derivatives of kibdelomycin were prepared which helped define a basic SAR of the family.

Next-generation brain observatories.

We propose centralized brain observatories for large-scale recordings of neural activity in mice and non-human primates coupled with cloud-based data analysis and sharing. Such observatories will advance reproducible systems neuroscience and democratize access to the most advanced tools and data.

Cellular resolution anatomical and molecular atlases for prenatal human brains.

Increasing interest in studies of prenatal human brain development, particularly using new single-cell genomics and anatomical technologies to create cell atlases, creates a strong need for accurate and detailed anatomical reference atlases. In this study, we present two cellular-resolution digital anatomical atlases for prenatal human brain at postconceptional weeks (PCW) 15 and 21. Both atlases were annotated on sequential Nissl-stained sections covering brain-wide structures on the basis of combined analysis of cytoarchitecture, acetylcholinesterase staining, and an extensive marker gene expression dataset. This high information content dataset allowed reliable and accurate demarcation of developing cortical and subcortical structures and their subdivisions. Furthermore, using the anatomical atlases as a guide, spatial expression of 37 and 5 genes from the brains, respectively, at PCW 15 and 21 was annotated, illustrating reliable marker genes for many developing brain structures. Finally, the present study uncovered several novel developmental features, such as the lack of an outer subventricular zone in the hippocampal formation and entorhinal cortex, and the apparent extension of both cortical (excitatory) and subcortical (inhibitory) progenitors into the prenatal olfactory bulb. These comprehensive atlases provide useful tools for visualization, segmentation, targeting, imaging, and interpretation of brain structures of prenatal human brain, and for guiding and interpreting the next generation of cell census and connectome studies.

HLA-A∗02:01 restricted T cell receptors against the highly conserved SARS-CoV-2 polymerase cross-react with human coronaviruses.

Cross-reactivity and direct killing of target cells remain underexplored for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific CD8+ T cells. Isolation of T cell receptors (TCRs) and overexpression in allogeneic cells allows for extensive T cell reactivity profiling. We identify SARS-CoV-2 RNA-dependent RNA polymerase (RdRp/NSP12) as highly conserved, likely due to its critical role in the virus life cycle. We perform single-cell TCRαß sequencing in human leukocyte antigen (HLA)-A∗02:01-restricted, RdRp-specific T cells from SARS-CoV-2-unexposed individuals. Human T cells expressing these TCRαß constructs kill target cell lines engineered to express full-length RdRp. Three TCR constructs recognize homologous epitopes from common cold coronaviruses, indicating CD8+ T cells can recognize evolutionarily diverse coronaviruses. Analysis of individual TCR clones may help define vaccine epitopes that can induce long-term immunity against SARS-CoV-2 and other coronaviruses.

Distinct Transcriptomic Cell Types and Neural Circuits of the Subiculum and Prosubiculum along the Dorsal-Ventral Axis.

Subicular regions play important roles in spatial processing and many cognitive functions, and these are mainly attributed to the subiculum (Sub) rather than the prosubiculum (PS). Using single-cell RNA sequencing, we identify 27 transcriptomic cell types residing in sub-domains of the Sub and PS. Based on in situ expression of reliable transcriptomic markers, the precise boundaries of the Sub and PS are consistently defined along the dorsoventral axis. Using these borders to evaluate Cre-line specificity and tracer injections, we find bona fide Sub projections topographically to structures important for spatial processing and navigation. In contrast, the PS sends its outputs to widespread brain regions crucial for motivation, emotion, reward, stress, anxiety, and fear. The Sub and PS, respectively, dominate dorsal and ventral subicular regions and receive different afferents. These results reveal two molecularly and anatomically distinct circuits centered in the Sub and PS, respectively, providing a consistent explanation for historical data and a clearer foundation for future studies.

Transcriptomic evidence that von Economo neurons are regionally specialized extratelencephalic-projecting excitatory neurons.

von Economo neurons (VENs) are bipolar, spindle-shaped neurons restricted to layer 5 of human frontoinsula and anterior cingulate cortex that appear to be selectively vulnerable to neuropsychiatric and neurodegenerative diseases, although little is known about other VEN cellular phenotypes. Single nucleus RNA-sequencing of frontoinsula layer 5 identifies a transcriptomically-defined cell cluster that contained VENs, but also fork cells and a subset of pyramidal neurons. Cross-species alignment of this cell cluster with a well-annotated mouse classification shows strong homology to extratelencephalic (ET) excitatory neurons that project to subcerebral targets. This cluster also shows strong homology to a putative ET cluster in human temporal cortex, but with a strikingly specific regional signature. Together these results suggest that VENs are a regionally distinctive type of ET neuron. Additionally, we describe the first patch clamp recordings of VENs from neurosurgically-resected tissue that show distinctive intrinsic membrane properties relative to neighboring pyramidal neurons.