Finding Your CAR: The Road Ahead for Engineered T Cells.

Adoptive cellular therapy using chimeric antigen receptors (CARs) has transformed immunotherapy by engineering T cells to target specific antigens on tumor cells. As the field continues to advance, pathology laboratories will play increasingly essential roles in the complicated multi-step process of CAR T-cell therapy. These include detection of targetable tumor antigens by flow cytometry or immunohistochemistry at the time of disease diagnosis and the isolation and infusion of CAR T cells. Additional roles include: i) detecting antigen loss or heterogeneity that renders resistance to CAR T cells as well as identifying alternative targetable antigens on tumor cells, ii) monitoring the phenotype, persistence, and tumor infiltration properties of CAR T cells and the tumor microenvironment for factors that predict CAR T-cell therapy success, and iii) evaluating side effects and biomarkers of CAR T-cell cytotoxicity such as cytokine release syndrome. This review highlights existing technologies that are applicable to monitoring CAR T-cell persistence, target antigen identification, and loss. Also discussed are emerging technologies that address new challenges such as how to put a brake on CAR T cells. Although pathology laboratories have already provided companion diagnostic tests important in immunotherapy (eg, programmed death-ligand 1, microsatellite instability, and human epidermal growth factor receptor 2 testing), we draw attention to the exciting new translational research opportunities in adoptive cellular therapy.

ADHD Prevalence Among U.S. Children and Adolescents in 2022: Diagnosis, Severity, Co-Occurring Disorders, and Treatment.

OBJECTIVE: To provide updated national prevalence estimates of diagnosed attention-deficit/hyperactivity disorder (ADHD), ADHD severity, co-occurring disorders, and receipt of ADHD medication and behavioral treatment among U.S. children and adolescents by demographic and clinical subgroups using data from the 2022 National Survey of Children's Health (NSCH). METHOD: This study used 2022 NSCH data to estimate the prevalence of ever diagnosed and current ADHD among U.S. children aged 3-17 years. Among children with current ADHD, ADHD severity, presence of current co-occurring disorders, and receipt of medication and behavioral treatment were estimated. Weighted estimates were calculated overall and for demographic and clinical subgroups (n = 45,169). RESULTS: Approximately 1 in 9 U.S. children have ever received an ADHD diagnosis (11.4%, 7.1 million children) and 10.5% (6.5 million) had current ADHD. Among children with current ADHD, 58.1% had moderate or severe ADHD, 77.9% had at least one co-occurring disorder, approximately half of children with current ADHD (53.6%) received ADHD medication, and 44.4% had received behavioral treatment for ADHD in the past year; nearly one third (30.1%) did not receive any ADHD-specific treatment. CONCLUSIONS: Pediatric ADHD remains an ongoing and expanding public health concern, as approximately 1 million more children had ever received an ADHD diagnosis in 2022 than in 2016. Estimates from the 2022 NSCH provide information on pediatric ADHD during the last full year of the COVID-19 pandemic and can be used by policymakers, government agencies, health care systems, public health practitioners, and other partners to plan for needs of children with ADHD.

Multi-omic profiling reveals early immunological indicators for identifying COVID-19 Progressors.

We sought to better understand the immune response during the immediate post-diagnosis phase of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by identifying molecular associations with longitudinal disease outcomes. Multi-omic analyses identified differences in immune cell composition, cytokine levels, and cell subset-specific transcriptomic and epigenomic signatures between individuals on a more serious disease trajectory (Progressors) as compared to those on a milder course (Non-progressors). Higher levels of multiple cytokines were observed in Progressors, with IL-6 showing the largest difference. Blood monocyte cell subsets were also skewed, showing a comparative decrease in non-classical CD14-CD16+ and intermediate CD14+CD16+ monocytes. In lymphocytes, the CD8+ T effector memory cells displayed a gene expression signature consistent with stronger T cell activation in Progressors. These early stage observations could serve as the basis for the development of prognostic biomarkers of disease risk and interventional strategies to improve the management of severe COVID-19. BACKGROUND: Much of the literature on immune response post-SARS-CoV-2 infection has been in the acute and post-acute phases of infection. TRANSLATIONAL SIGNIFICANCE: We found differences at early time points of infection in approximately 160 participants. We compared multi-omic signatures in immune cells between individuals progressing to needing more significant medical intervention and non-progressors. We observed widespread evidence of a state of increased inflammation associated with progression, supported by a range of epigenomic, transcriptomic, and proteomic signatures. The signatures we identified support other findings at later time points and serve as the basis for prognostic biomarker development or to inform interventional strategies.

Multi-omic Profiling Reveals Early Immunological Indicators for Identifying COVID-19 Progressors.

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a rapid response by the scientific community to further understand and combat its associated pathologic etiology. A focal point has been on the immune responses mounted during the acute and post-acute phases of infection, but the immediate post-diagnosis phase remains relatively understudied. We sought to better understand the immediate post-diagnosis phase by collecting blood from study participants soon after a positive test and identifying molecular associations with longitudinal disease outcomes. Multi-omic analyses identified differences in immune cell composition, cytokine levels, and cell subset-specific transcriptomic and epigenomic signatures between individuals on a more serious disease trajectory (Progressors) as compared to those on a milder course (Non-progressors). Higher levels of multiple cytokines were observed in Progressors, with IL-6 showing the largest difference. Blood monocyte cell subsets were also skewed, showing a comparative decrease in non-classical CD14-CD16+ and intermediate CD14+CD16+ monocytes. Additionally, in the lymphocyte compartment, CD8+ T effector memory cells displayed a gene expression signature consistent with stronger T cell activation in Progressors. Importantly, the identification of these cellular and molecular immune changes occurred at the early stages of COVID-19 disease. These observations could serve as the basis for the development of prognostic biomarkers of disease risk and interventional strategies to improve the management of severe COVID-19.

Gaseous polycyclic aromatic hydrocarbons over the South China Sea: Implications for atmospheric transport under monsoon influences.

The seasonal monsoon variations have significant impact on the atmospheric transport of semi-volatile organic pollutants over the South China Sea (SCS). We analyzed polycyclic aromatic hydrocarbons (PAHs) over the basin and island areas (Yongxing Island and Yongshu Island) in 2017. Gaseous PAHs (0.17-1.4 ng m-3) showed spatio-temporal distinctions in their composition and sources among the basin and island areas. Mixed combustion sources of PAHs were identified over the SCS, including a petroleum source near the island areas. The transport routes of PAHs were inferred by the air mass back trajectories and potential source contribution factor analysis, identifying strong biomass burning signals from the Indochina Peninsula and other Southeast Asian countries. Emissions from approximately 90 % of the combustion sources were transported to basin areas by monsoons, whereas the island areas were dominated by local emissions. This study emphasizes the main potential terrestrial source of PAHs over the SCS under monsoon influences.

Preclinical models for prediction of immunotherapy outcomes and immune evasion mechanisms in genetically heterogeneous multiple myeloma.

The historical lack of preclinical models reflecting the genetic heterogeneity of multiple myeloma (MM) hampers the advance of therapeutic discoveries. To circumvent this limitation, we screened mice engineered to carry eight MM lesions (NF-κB, KRAS, MYC, TP53, BCL2, cyclin D1, MMSET/NSD2 and c-MAF) combinatorially activated in B lymphocytes following T cell-driven immunization. Fifteen genetically diverse models developed bone marrow (BM) tumors fulfilling MM pathogenesis. Integrative analyses of ∼500 mice and ∼1,000 patients revealed a common MAPK-MYC genetic pathway that accelerated time to progression from precursor states across genetically heterogeneous MM. MYC-dependent time to progression conditioned immune evasion mechanisms that remodeled the BM microenvironment differently. Rapid MYC-driven progressors exhibited a high number of activated/exhausted CD8+ T cells with reduced immunosuppressive regulatory T (Treg) cells, while late MYC acquisition in slow progressors was associated with lower CD8+ T cell infiltration and more abundant Treg cells. Single-cell transcriptomics and functional assays defined a high ratio of CD8+ T cells versus Treg cells as a predictor of response to immune checkpoint blockade (ICB). In clinical series, high CD8+ T/Treg cell ratios underlie early progression in untreated smoldering MM, and correlated with early relapse in newly diagnosed patients with MM under Len/Dex therapy. In ICB-refractory MM models, increasing CD8+ T cell cytotoxicity or depleting Treg cells reversed immunotherapy resistance and yielded prolonged MM control. Our experimental models enable the correlation of MM genetic and immunological traits with preclinical therapy responses, which may inform the next-generation immunotherapy trials.

Dynamic phosphatase-recruitment controls B-cell selection and oncogenic signaling.

Initiation of B-cell receptor (BCR) 1 signaling, and subsequent antigen-encounter in germinal centers 2,3 represent milestones of B-lymphocyte development that are both marked by sharp increases of CD25 surface-expression. Oncogenic signaling in B-cell leukemia (B-ALL) 4 and lymphoma 5 also induced CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells 6-9 , the significance of its expression on B-cells was unclear. Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells assembled an inhibitory complex including PKCδ and SHIP1 and SHP1 phosphatases for feedback control of BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKCδ 10 - 12 , SHIP1 13,14 and SHP1 14, 15,16 , conditional CD25-deletion decimated early B-cell subsets but expanded mature B-cell populations and induced autoimmunity. In B-cell malignancies arising from early (B-ALL) and late (lymphoma) stages of B-cell development, CD25-loss induced cell death in the former and accelerated proliferation in the latter. Clinical outcome annotations mirrored opposite effects of CD25-deletion: high CD25 expression levels predicted poor clinical outcomes for patients with B-ALL, in contrast to favorable outcomes for lymphoma-patients. Biochemical and interactome studies revealed a critical role of CD25 in BCR-feedback regulation: BCR-signaling induced PKCδ-mediated phosphorylation of CD25 on its cytoplasmic tail (S 268 ). Genetic rescue experiments identified CD25-S 268 tail-phosphorylation as central structural requirement to recruit SHIP1 and SHP1 phosphatases to curb BCR-signaling. A single point mutation CD25 S268A abolished recruitment and activation of SHIP1 and SHP1 to limit duration and strength of BCR-signaling. Loss of phosphatase-function, autonomous BCR-signaling and Ca 2+ -oscillations induced anergy and negative selection during early B-cell development, as opposed to excessive proliferation and autoantibody production in mature B-cells. These findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to control oncogenic signaling in B-cell malignancies and negative selection to prevent autoimmune disease.

IRF8 as a Novel Marker to Differentiate Between CD30-Positive Large Cell Lymphomas.

OBJECTIVES: Interferon regulatory factor 8 (IRF8) is a new biomarker shown to be positive in monocytic leukemias as well as in B cells. As a transcription factor, it plays a critical role in pre-B-cell differentiation and induction of tolerance pathways, among other functions. Given the frequent diagnostic dilemma in CD30-positive large cell lymphomas that could resemble both Hodgkin lymphoma and anaplastic large cell lymphoma (ALCL), we sought to determine whether IRF8 can be useful in distinguishing between these neoplasms that require different treatment strategies. METHODS: In this retrospective study, 74 cases of classic Hodgkin lymphoma (CHL) and 7 cases of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) on a tissue microarray (TMA), as well as 15 individual cases of ALK-negative ALCL and 4 cases of ALK-positive ALCL, were stained for IRF8. Paired box 5 (PAX5) immunostaining of the TMA was also performed and compared alongside since that marker is occasionally the only marker to help clinically differentiate between T- and B-cell lymphomas with anaplastic/Hodgkin-like cytology. RESULTS: None (0%) of the ALCLs were positive for IRF8 while all (100%) of the NLPHLs and 85% of the CHLs were positive for IRF8. Six (8%) cases of CHL were PAX5 negative but IRF8 positive. Conversely, seven (10%) cases of CHL were PAX5 positive but IRF8 negative. Four (6%) cases of CHL were negative for both PAX5 and IRF8. CONCLUSIONS: There is significant morphologic and immunophenotypic (CD30 positive and CD45 and CD20 negative) overlap between CHL and ALCL. Since many ALCLs show downregulation of lineage-specific T-cell markers or are "null cell" type, only PAX5 has been a reliable marker to differentiate between borderline cases. This is further confounded by positivity of PAX5 in some ALCLs due to amplification of PAX5. On the basis of recent discoveries of IRF8 function as well as performance as an immunostain, we tested this marker in human lymphoma samples and found that it aids in the discrimination between these tumors.

Changes in Chemical Composition and Copepod Toxicity during Petroleum Photo-oxidation.

Photoproducts can be formed rapidly in the initial phase of a marine oil spill. However, their toxicity is not well understood. In this study, oil was irradiated, chemically characterized, and tested for toxicity in three copepod species (Acartia tonsa, Temora longicornis, and Calanus finmarchicus). Irradiation led to a depletion of polycyclic aromatic hydrocarbons (PAHs) and n-alkanes in oil residues, along with an enrichment in aromatic and aliphatic oil photoproducts. Target lipid model-based calculations of PAH toxicity units predicted that PAH toxicities were lower in water-accommodated fractions (WAFs) of irradiated oil residues ("irradiated WAFs") than in WAFs of dark-control samples ("dark WAFs"). In contrast, biomimetic extraction (BE) measurements showed increased bioaccumulation potential of dissolved constituents of irradiated WAFs compared to dark WAFs, mainly driven by photoproducts present in irradiated oil. In line with the BE results, copepod mortality increased in irradiated WAFs compared to dark WAFs. However, low copepod toxicities were observed for WAFs produced with photo-oxidized oil slicks collected during the Deepwater Horizon oil spill. The results of this study suggest that while oil photoproducts have the potential to be a significant source of copepod toxicity, dilution and dispersion of these higher solubility products appear to help mitigate their toxicity at sea.

Global assessment of IRF8 as a novel cancer biomarker.

Interferon regulatory factor 8 (IRF8) is a member of the IRF family that is specific to the hematopoietic cell and is involved in regulating the development of human monocytic and dendritic-lineage cells, as well as B-cells. Because its utility as a sensitive and specific monoblast marker in the context of acute monocytic leukemias has been recently demonstrated, we hypothesized that it may also be useful as a novel immunohistochemical marker in myeloid sarcomas and blastic plasmacytoid dendritic cell neoplasms (BPDCNs) with respect to their differential diagnoses. In this retrospective study, we analyzed the IHC expression pattern of IRF8 in 385 patient samples across 30 types of cancers, referenced to their mRNA expression data available through The Cancer Genome Atlas. In addition, we assessed IRF8 in 35 myeloid sarcomas and 15 BPDCNs. Twenty-four of 35 cases of myeloid sarcomas (68.5%) showed positivity for IRF8, with six cases (17.1%) demonstrating IRF8 expression in the absence of CD34 and MPO. All 15 of 15 BPDCNs (100%) showed strong uniform expression of IRF8 and were occasionally more definitive than CD123. IRF8 was negative in all desmoplastic small round cell tumors, Ewing sarcomas, synovial sarcomas, and undifferentiated pleomorphic sarcomas, as well as all epithelial malignancies tested except for 2 triple negative breast cancers that showed subset weak staining. In conclusion, IRF8 is a novel marker helpful in identifying extranodal hematopoietic tumors that can otherwise be difficult to diagnose given the broad differential diagnoses and frequent loss of more common lineage-defining markers.

Histopathologic and Machine Deep Learning Criteria to Predict Lymphoma Transformation in Bone Marrow Biopsies.

CONTEXT.­: Large cell transformation (LCT) of indolent B-cell lymphomas, such as follicular lymphoma (FL) and chronic lymphocytic leukemia (CLL), signals a worse prognosis, at which point aggressive chemotherapy is initiated. Although LCT is relatively straightforward to diagnose in lymph nodes, a marrow biopsy is often obtained first given its ease of procedure, low cost, and low morbidity. However, consensus criteria for LCT in bone marrow have not been established. OBJECTIVE.­: To study the accuracy and reproducibility of a trained convolutional neural network in identifying LCT, in light of promising machine learning tools that may introduce greater objectivity to morphologic analysis. DESIGN.­: We retrospectively identified patients who had a diagnosis of FL or CLL who had undergone bone marrow biopsy for the clinical question of LCT. We scored morphologic criteria and correlated results with clinical disease progression. In addition, whole slide scans were annotated into patches to train convolutional neural networks to discriminate between small and large tumor cells and to predict the patient's probability of transformation. RESULTS.­: Using morphologic examination, the proportion of large lymphoma cells (≥10% in FL and ≥30% in CLL), chromatin pattern, distinct nucleoli, and proliferation index were significantly correlated with LCT in FL and CLL. Compared to pathologist-derived estimates, machine-generated quantification demonstrated better reproducibility and stronger correlation with final outcome data. CONCLUSIONS.­: These histologic findings may serve as indications of LCT in bone marrow biopsies. The pathologist-augmented with machine system appeared to be the most predictive, arguing for greater efforts to validate and implement these tools to further enhance physician practice.

Balancing life and death: BCL-2 family members at diverse ER-mitochondrial contact sites.

The outer mitochondrial membrane is a busy place. One essential activity for cellular survival is the regulation of membrane integrity by the BCL-2 family of proteins. Another critical facet of the outer mitochondrial membrane is its close approximation with the endoplasmic reticulum. These mitochondrial-associated membranes (MAMs) occupy a significant fraction of the mitochondrial surface and serve as key signaling hubs for multiple cellular processes. Each of these pathways may be considered as forming their own specialized MAM subtype. Interestingly, like membrane permeabilization, most of these pathways play critical roles in regulating cellular survival and death. Recently, the pro-apoptotic BCL-2 family member BOK has been found within MAMs where it plays important roles in their structure and function. This has led to a greater appreciation that multiple BCL-2 family proteins, which are known to participate in numerous functions throughout the cell, also have roles within MAMs. In this review, we evaluate several MAM subsets, their role in cellular homeostasis, and the contribution of BCL-2 family members to their functions.

Yes, MAM!

Regulation of cell life and death by members of the BCL-2 family of proteins occurs at the mitochondria. Large portions of the mitochondria's outer membrane are found in tight approximation with the endoplasmic reticulum (ER), known as mitochondria-associated membranes (MAMs) or mitochondria-ER contact sites (MERCs). We recently reported that BOK is present within MAMs where it regulates Ca2+ transfer from the ER to the mitochondria, appropriate MAM components and MERC structure, and apoptosis.

A genome-wide screen uncovers multiple roles for mitochondrial nucleoside diphosphate kinase D in inflammasome activation.

Noncanonical inflammasome activation by cytosolic lipopolysaccharide (LPS) is a critical component of the host response to Gram-negative bacteria. Cytosolic LPS recognition in macrophages is preceded by a Toll-like receptor (TLR) priming signal required to induce transcription of inflammasome components and facilitate the metabolic reprograming that fuels the inflammatory response. Using a genome-scale arrayed siRNA screen to find inflammasome regulators in mouse macrophages, we identified the mitochondrial enzyme nucleoside diphosphate kinase D (NDPK-D) as a regulator of both noncanonical and canonical inflammasomes. NDPK-D was required for both mitochondrial DNA synthesis and cardiolipin exposure on the mitochondrial surface in response to inflammasome priming signals mediated by TLRs, and macrophages deficient in NDPK-D had multiple defects in LPS-induced inflammasome activation. In addition, NDPK-D was required for the recruitment of TNF receptor-associated factor 6 (TRAF6) to mitochondria, which was critical for reactive oxygen species (ROS) production and the metabolic reprogramming that supported the TLR-induced gene program. NDPK-D knockout mice were protected from LPS-induced shock, consistent with decreased ROS production and attenuated glycolytic commitment during priming. Our findings suggest that, in response to microbial challenge, NDPK-D-dependent TRAF6 mitochondrial recruitment triggers an energetic fitness checkpoint required to engage and maintain the transcriptional program necessary for inflammasome activation.

Spatial transcriptome profiling by MERFISH reveals fetal liver hematopoietic stem cell niche architecture.

The hematopoietic stem cell (HSC) niche has been extensively studied in bone marrow, yet a more systematic investigation into the microenvironment regulation of hematopoiesis in fetal liver is necessary. Here we investigate the spatial organization and transcriptional profile of individual cells in both wild type (WT) and Tet2-/- fetal livers, by multiplexed error robust fluorescence in situ hybridization. We find that specific pairs of fetal liver cell types are preferentially positioned next to each other. Ligand-receptor signaling molecule pairs such as Kitl and Kit are enriched in neighboring cell types. The majority of HSCs are in direct contact with endothelial cells (ECs) in both WT and Tet2-/- fetal livers. Loss of Tet2 increases the number of HSCs, and upregulates Wnt and Notch signaling genes in the HSC niche. Two subtypes of ECs, arterial ECs and sinusoidal ECs, and other cell types contribute distinct signaling molecules to the HSC niche. Collectively, this study provides a comprehensive picture and bioinformatic foundation for HSC spatial regulation in fetal liver.

IRF8 is a Reliable Monoblast Marker for Acute Monocytic Leukemias.

Blast evaluation in patients with acute monocytic leukemias (AMoL) is notoriously difficult due to the lack of reliable surface markers and cytologic subtleties on the aspirate smears. While blasts of most nonmonocytic acute leukemias express CD34, available immunohistochemical antibodies to monocytic blasts also mark normal background mature monocytes. We searched for a potential biomarker candidate by surveying specific gene expression profiles of monocyte progenitors. Our investigations led us to IRF8, which is a lineage-specific transcription factor critical for the production of monocytic and dendritic cell progenitors. In this study, we tested and validated a monoclonal antibody to IRF8 as a novel immunohistochemical stain for trephine core biopsies of human bone marrow. We assessed the expression of IRF8 in 90 cases of AMoL, including posttherapy staging bone marrows, 23 cases of chronic myelomonocytic leukemia, 26 cases of other acute myeloid leukemia subtypes, and 18 normal control marrows. In AMoL, there was high correlation of IRF8-positive cells to aspirate blast count (R=0.95). Comparison of IRF8 staining to aspirate blast percentage in chronic myelomonocytic leukemia also showed good correlation (R=0.86). In contrast, IRF8-positive cells did not correlate with blast count in other subtypes of acute myeloid leukemia (R=0.56) and staining was <5% in all normal control marrows, even those with reactive monocytosis. We found that IRF8 was also weakly reactive in B cells and hematogones, with the latter accounting for rare cases of discrepancies. When IRF8 was used to categorize cases as AMoL, positive for residual leukemia or negative, the sensitivity was 98%, specificity was 82%, positive predictive value was 86%, and negative predictive value was 98%. These results demonstrate that IRF8 may serve as a clinically useful immunostain to diagnose and track AMoLs on bone marrow core biopsies. This can be particularly impactful in the setting of poor aspiration and focal blast increase. In the era of new targeted therapies that have been reported to induce monocytic outgrowths of leukemia, a marker for malignant monoblasts may prove even more critical.

SIGNAL: A web-based iterative analysis platform integrating pathway and network approaches optimizes hit selection from genome-scale assays.

Hit selection from high-throughput assays remains a critical bottleneck in realizing the potential of omic-scale studies in biology. Widely used methods such as setting of cutoffs, prioritizing pathway enrichments, or incorporating predicted network interactions offer divergent solutions yet are associated with critical analytical trade-offs. The specific limitations of these individual approaches and the lack of a systematic way by which to integrate their rankings have contributed to limited overlap in the reported results from comparable genome-wide studies and costly inefficiencies in secondary validation efforts. Using comparative analysis of parallel independent studies as a benchmark, we characterize the specific complementary contributions of each approach and demonstrate an optimal framework to integrate these methods. We describe selection by iterative pathway group and network analysis looping (SIGNAL), an integrated, iterative approach that uses both pathway and network methods to optimize gene prioritization. SIGNAL is accessible as a rapid user-friendly web-based application (https://signal.niaid.nih.gov). A record of this paper's transparent peer review is included in the Supplemental information.

BOK controls apoptosis by Ca2+ transfer through ER-mitochondrial contact sites.

Calcium transfer from the endoplasmic reticulum (ER) to mitochondria is a critical contributor to apoptosis. B cell lymphoma 2 (BCL-2) ovarian killer (BOK) localizes to the ER and binds the inositol 1,4,5-trisphosophate receptor (IP3R). Here, we show that BOK is necessary for baseline mitochondrial calcium levels and stimulus-induced calcium transfer from the ER to the mitochondria. Murine embryonic fibroblasts deficient for BOK have decreased proximity of the ER to the mitochondria and altered protein composition of mitochondria-associated membranes (MAMs), which form essential calcium microdomains. Rescue of the ER-mitochondrial juxtaposition with drug-inducible interorganelle linkers reveals a kinetic disruption, which when overcome in Bok-/- cells is still insufficient to rescue thapsigargin-induced calcium transfer and apoptosis. Likewise, a BOK mutant unable to interact with IP3R restores ER-mitochondrial proximity, but not ER-mitochondrial calcium transfer, MAM protein composition, or apoptosis. This work identifies the dynamic coordination of ER-mitochondrial contact by BOK as an important control point for apoptosis.

Multiplexed imaging of nucleome architectures in single cells of mammalian tissue.

The three-dimensional architecture of the genome affects genomic functions. Multiple genome architectures at different length scales, including chromatin loops, domains, compartments, and lamina- and nucleolus-associated regions, have been discovered. However, how these structures are arranged in the same cell and how they are mutually correlated in different cell types in mammalian tissue are largely unknown. Here, we develop Multiplexed Imaging of Nucleome Architectures that measures multiscale chromatin folding, copy numbers of numerous RNA species, and associations of numerous genomic regions with nuclear lamina, nucleoli and surface of chromosomes in the same, single cells. We apply this method in mouse fetal liver, and identify de novo cell-type-specific chromatin architectures associated with gene expression, as well as cell-type-independent principles of chromatin organization. Polymer simulation shows that both intra-chromosomal self-associating interactions and extra-chromosomal interactions are necessary to establish the observed organization. Our results illustrate a multi-faceted picture and physical principles of chromatin organization.