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The syncytial mammalian muscle fiber contains a heterogeneous population of (myo)nuclei. At the neuromuscular junction (NMJ), myonuclei have specialized positioning and gene expression. However, it remains unclear how myonuclei are recruited and what regulates myonuclear output at the NMJ. Here, we identify specific properties of myonuclei located near the Drosophila larval NMJ. These synaptic myonuclei have increased size in relation to their surrounding cytoplasmic domain (size scaling), increased DNA content (ploidy), and increased levels of transcription factor pMad, a readout for BMP signaling activity. Our genetic manipulations show that local BMP signaling affects muscle size, nuclear size, ploidy, and NMJ size and function. In support, RNA sequencing analysis reveals that pMad regulates genes involved in muscle growth, ploidy (i.e., E2f1), and neurotransmission. Our data suggest that muscle BMP signaling instructs synaptic myonuclear output that positively shapes the NMJ synapse. This study deepens our understanding of how myonuclear heterogeneity supports local signaling demands to fine tune cellular function and NMJ activity., (© 2024 von Saucken et al.)

Inherited genetics represents an important contributor to risk of esophageal adenocarcinoma (EAC), and its precursor Barrett's esophagus (BE). Genome-wide association studies have identified ∼30 susceptibility variants for BE/EAC, yet genetic interactions remain unexamined. To address challenges in large-scale G×G scans, we combined knowledge-guided filtering and machine learning approaches, focusing on genes with (A) known/plausible links to BE/EAC pathogenesis (n=493) or (B) prior evidence of biological interactions (n=4,196). ∼75 x 10 6 SNP×SNP interactions were screened via hierarchical group lasso (glinternet) using BEACON GWAS data. The top ∼2000 interactions retained in each scan were prioritized using P values from single logistic models. Identical scans were repeated among males only (78%), with two independent GWAS datasets used for replication. In overall and male-specific primary replications, 11 of 187 and 20 of 191 interactions satisfied P<0.05, respectively. The strongest evidence for secondary replication was for rs17744726×rs3217992 among males, with consistent directionality across all cohorts (P meta =2.19×10 -8 ); rs3217992 "T" was associated with reduced risk only in individuals homozygous for rs17744726 "G". Rs3217992 maps to the CDKN2B 3'UTR and reportedly disrupts microRNA-mediated repression. Rs17744726 maps to an intronic enhancer region in BLK. Through in-silico prioritization and experimental validation, we identified a nearby proxy variant (rs4841556) as a functional modulator of enhancer activity. Enhancer-gene mapping and eQTLs implicated BLK and FAM167A as targets. The first systematic G×G investigation in BE/EAC, this study uncovers differential risk associations for CDKN2B variation by BLK genotype, suggesting novel biological dependency between two risk loci encoding key mediators of tumor suppression and inflammation., (Copyright © 2025. Published by Elsevier Inc.)

Cancer progression involves genetic and epigenetic changes that disrupt chromatin 3D organization, affecting enhancer-promoter interactions and promoting growth. Here, we provide an integrative approach, combining chromatin conformation, accessibility, and transcription analysis, validated by in silico and CRISPR-interference screens, to identify relevant 3D topologies in pediatric T cell leukemia (T-ALL and ETP-ALL). We characterize 3D hubs as regulatory centers for oncogenes and disease markers, linking them to biological processes like cell division, inflammation, and stress response. Single-cell mapping reveals heterogeneous gene activation in discrete epigenetic clones, aiding in patient stratification for relapse risk after chemotherapy. Finally, we identify MYB as a 3D hub regulator in leukemia cells and show that the targeting of key regulators leads to hub dissolution, thereby providing a novel and effective anti-leukemic strategy. Overall, our work demonstrates the relevance of studying oncogenic 3D hubs to better understand cancer biology and tumor heterogeneity and to propose novel therapeutic strategies., Competing Interests: Declaration of interests A.K. is a consultant for Novartis and Rgenta., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The inflammasome plays multifaceted roles in cancer, but less is known about its function during premalignancy upon initial cell transformation. We report a homeostatic function of the inflammasome in suppressing malignant transformation through Ras inhibition. We identified increased hematopoietic stem cell (HSC) proliferation within the bone marrow of inflammasome-deficient mice. HSCs within an inflammasome-deficient stroma expressed a Ras signature associated with increased Ras pathway- and cancer-related transcripts and heightened levels of cytokine, chemokine and growth factor receptors. Stromal inflammasome deficiency established a poised Ras-dependent mitogenic state within HSCs, which fueled progeny B cell lymphomagenesis upon Myc deregulation in a spontaneous model of B cell lymphoma, and shortened its premalignant stage leading to faster onset of malignancy. Thus, the stromal inflammasome preserves tissue balance by restraining Ras to disrupt the most common oncogenic Myc-Ras cooperation and establish a natural defense against transition to malignancy. These findings should inform preventative therapies against hematological malignancies., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Ageing is associated with a decline in the number and fitness of adult stem cells 1,2 . Ageing-associated loss of stemness is posited to suppress tumorigenesis 3,4 , but this hypothesis has not been tested in vivo. Here we use physiologically aged autochthonous genetically engineered 5,6 mouse models and primary cells 5,6 to demonstrate that ageing suppresses lung cancer initiation and progression by degrading the stemness of the alveolar cell of origin. This phenotype is underpinned by the ageing-associated induction of the transcription factor NUPR1 and its downstream target lipocalin-2 in the cell of origin in mice and humans, which leads to functional iron insufficiency in the aged cells. Genetic inactivation of the NUPR1-lipocalin-2 axis or iron supplementation rescues stemness and promotes the tumorigenic potential of aged alveolar cells. Conversely, targeting the NUPR1-lipocalin-2 axis is detrimental to young alveolar cells through ferroptosis induction. Ageing-associated DNA hypomethylation at specific enhancer sites is associated with increased NUPR1 expression, which is recapitulated in young alveolar cells through DNA methylation inhibition. We uncover that ageing drives functional iron insufficiency that leads to loss of stemness and tumorigenesis but promotes resistance to ferroptosis. These findings have implications for the therapeutic modulation of cellular iron homeostasis in regenerative medicine and in cancer prevention. Furthermore, our findings are consistent with a model whereby most human cancers initiate at a young age, thereby highlighting the importance of directing cancer prevention efforts towards young individuals., Competing Interests: Competing interests: T. Tammela is a scientific advisor with equity interests in Lime Therapeutics. His spouse is an employee of and has equity in Recursion Pharmaceuticals. The Tammela Laboratory receives funding from Ono Pharma unrelated to this work. E.S.W. has equity in and her spouse is a co-founder of and equity holder in Gertrude Biomedical Pty. The other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Competing Interests: HH serves without remuneration on an external advisory board of the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, on the International Advisory Board of the University of Vienna, and on the external Executive Advisory Committee of the Sylvester Comprehensive Cancer Center, University of Miami Health System, and as a member of the Standing Advisory Group on Nuclear Applications of the International Atomic Energy Agency. Until Oct 31, 2024, HH also served without remuneration on the Scientific Committee of the German Cancer Research Center (DKFZ) and the Board of Trustees of the DKFZ. She is remunerated for serving on the Board of Directors of Ion Beam Applications and receives stock options for serving on the Board of Directors of iCAD. Her work is partly supported by funding to her institution (Memorial Sloan-Kettering Center support grant/core grant P30 CA008748) from the US National Institutes of Health (NIH)–National Cancer Institute (NCI). JOP reports uncompensated service as President of the Swiss Society of Nuclear Medicine, service to the World Federation of Nuclear Medicine and Biology as the focal point for technical collaboration with WHO, and service as the President of Section and Board of Nuclear Medicine of the European Union of Medical Specialists. AM reports that her work is partly supported by funding to her institution (Memorial Sloan-Kettering Center support grant/core grant P30 CA008748) from NIH–NCI. BA reports support for attending meetings or travel from the International Society of Radiology and the American College of Radiology. AMS reports institutional research funding from the Australian National Health and Medical Research Council, Medical Research Future Fund, and National Imaging Facility; patents planned, issued or pending for his role as an inventor of antibodies to HER2, ADAMs, EGFR, and Ephs; uncompensated participation on data safety monitoring or advisory boards for Telix and ImunOs; uncompensated service as a board member for the Australia and New Zealand Society of Nuclear Medicine and World Federation of Nuclear Medicine and Biology and as theme chair of the National Imaging Facility; stock holdings as founder and Director of Certis Therapeutics; and research and trial funding to his institution from Telix, Clarity, Fusion, Antengene, EMD Serono, ITM, Avid/Lily, Medimmune, and Cyclotek. All other authors declare no competing interests.

Phylogenies depicting the evolutionary history of genetically heterogeneous subpopulations of cells from the same cancer, i.e., cancer phylogenies, offer valuable insights about cancer development and guide treatment strategies. Many methods exist that reconstruct cancer phylogenies using point mutations detected with bulk DNA sequencing. However, these methods become inaccurate when reconstructing phylogenies with more than 30 mutations, or, in some cases, fail to recover a phylogeny altogether. Here, we introduce Orchard, a cancer phylogeny reconstruction algorithm that is fast and accurate using up to 1000 mutations. Orchard samples without replacement from a factorized approximation of the posterior distribution over phylogenies, a novel result derived in this paper. Each factor in this approximate posterior corresponds to a conditional distribution for adding a new mutation to a partially built phylogeny. Orchard optimizes each factor sequentially, generating a sequence of incrementally larger phylogenies that ultimately culminate in a complete tree containing all mutations. Our evaluations demonstrate that Orchard outperforms state-of-the-art cancer phylogeny reconstruction methods in reconstructing more plausible phylogenies across 90 simulated cancers and 14 B-progenitor acute lymphoblastic leukemias (B-ALLs). Remarkably, Orchard accurately reconstructs cancer phylogenies using up to 1,000 mutations. Additionally, we demonstrate that the large and accurate phylogenies reconstructed by Orchard are useful for identifying patterns of somatic mutations and genetic variations among distinct cancer cell subpopulations., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kulman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Prime editing (PE) allows for precise genome editing in human pluripotent stem cells (hPSCs), such as introducing single nucleotide modifications, small insertions or deletions at a specific genomic locus. Here, we systematically compare a panel of prime editing conditions in hPSCs and generate a potent prime editor, "PE-Plus", through co-inhibition of mismatch repair and p53-mediated cellular stress responses. We further establish an inducible prime editing platform in hPSCs by incorporating the PE-Plus into a safe-harbor locus and demonstrated temporal control of precise editing in both hPSCs and differentiated cells. By evaluating disease-associated mutations, we show that this platform allows efficient creation of both monoallelic and biallelic disease-relevant mutations in hPSCs. In addition, this platform enables the efficient introduction of single or multiple edits in one step, demonstrating potential for multiplex editing. Our method presents an efficient and controllable multiplex prime editing tool in hPSCs and their differentiated progeny., Competing Interests: Competing interests: L.S. is a scientific co-founder and consultant of BlueRock Therapeutics. The other authors have no competing interests., (© 2024. The Author(s).)

Proteostasis is maintained through regulated protein synthesis and degradation and chaperone-assisted protein folding. However, this is challenging in neuronal projections because of their polarized morphology and constant synaptic proteome remodeling. Using high-resolution fluorescence microscopy, we discover that hippocampal and spinal cord motor neurons of mouse and human origin localize a subset of chaperone mRNAs to their dendrites and use microtubule-based transport to increase this asymmetric localization following proteotoxic stress. The most abundant dendritic chaperone mRNA encodes a constitutive heat shock protein 70 family member (HSPA8). Proteotoxic stress also enhances HSPA8 mRNA translation efficiency in dendrites. Stress-mediated HSPA8 mRNA localization to the dendrites is impaired by depleting fused in sarcoma-an amyotrophic lateral sclerosis-related protein-in cultured spinal cord mouse motor neurons or by expressing a pathogenic variant of heterogenous nuclear ribonucleoprotein A2/B1 in neurons derived from human induced pluripotent stem cells. These results reveal a neuronal stress response in which RNA-binding proteins increase the dendritic localization of HSPA8 mRNA to maintain proteostasis and prevent neurodegeneration., Competing Interests: Competing interests: G.W.Y. is a Scientific Advisory Board member of Jumpcode Genomics and a co-founder, Board of Directors, and Scientific Advisory Board member, equity holder, and paid consultant for Locanabio and Eclipse BioInnovations. G.W.Y. is a visiting professor at the National University of Singapore. G.W.Y.’s interests have been reviewed and approved by the University of California, San Diego in accordance with its conflict-of-interest policies. The remaining authors declare no competing interests., (© 2024. The Author(s).)

Oxidative phosphorylation has emerged as a critical therapeutic vulnerability of M. tuberculosis (Mtb). However, it is unknown how intracellular bacterial pathogens such as Mtb maintain respiration during infection despite the chemical effectors of host immunity. Mtb synthesizes diisonitrile lipopeptides that tightly chelate copper, but the role of these chalkophores in host-pathogen interactions is also unknown. We demonstrate that M. tuberculosis chalkophores maintain the function of the heme-copper bcc:aa 3 respiratory oxidase under copper limitation. Chalkophore deficiency impairs Mtb survival, respiration to oxygen, and ATP production under copper deprivation in culture, effects that are exacerbated by loss of the heme dependent Cytochrome BD respiratory oxidase. Our genetic analyses indicate that maintenance of respiration is the only cellular target of chalkophore mediated copper acquisition. M. tuberculosis lacking chalkophore biosynthesis is attenuated in mice, a phenotype that is also severely exacerbated by loss of the CytBD respiratory oxidase. We find that the host immune pressure that attenuates chalkophore deficient Mtb is independent of adaptive immunity and neutrophils. These data demonstrate that chalkophores counter host inflicted copper deprivation and highlight a multilayered system by which M. tuberculosis maintains respiration during infection., Competing Interests: Conflicts of Interest MSG declares equity and consulting fees from Vedanta biosciences and consulting fees from Fimbrion therapeutics. DST declares no conflicts of interest pertaining to this work.

MYB fusions are recurrently found in select cancers, including blastic plasmacytoid DC neoplasm (BPDCN), an acute leukemia with poor prognosis. They are markedly enriched in BPDCN compared with other blood cancers and, in some patients, are the only obvious somatic mutation detected. This suggests that they may alone be sufficient to drive DC transformation. MYB fusions are hypothesized to alter the normal transcription factor activity of MYB, but, mechanistically, how they promote leukemogenesis is poorly understood. Using CUT&RUN chromatin profiling, we found that, in BPDCN leukemogenesis, MYB switches from being a regulator of DC lineage genes to aberrantly regulating G2/M cell cycle control genes. MYB fusions found in patients with BPDCN increased the magnitude of DNA binding at these locations, and this was linked to BPDCN-associated gene expression changes. Furthermore, expression of MYB fusions in vivo impaired DC differentiation and induced transformation to generate a mouse model of myeloid-dendritic acute leukemia. Therapeutically, we present evidence that all-trans retinoic acid (ATRA) may cause loss of MYB protein and cell death in BPDCN.

Myeloid phagocytes are essential for antifungal immunity against pulmonary Aspergillus fumigatus and systemic Candida albicans infections. However, the molecular mechanisms underlying fungal clearance by phagocytes remain incompletely understood. In this study, we investigated the role of Perforin-2 ( Mpeg1 ) in antifungal immunity. We found that Mpeg1 -/- mice generated on a mixed C57BL/6J-DBA/2 background exhibited enhanced survival, reduced lung fungal burden, and greater neutrophil fungal killing activity compared to wild-type C57BL/6J (B6) mice, suggesting that Perforin-2 may impair antifungal immune responses. However, when we compared Mpeg1 -/- mice with co-housed Mpeg +/+ littermate controls, these differences were no longer observed, indicating that initial findings were likely influenced by differences in the murine genetic background or the microbiota composition. Furthermore, Perforin-2 was dispensable for antifungal immunity during C. albicans bloodstream infection. These results suggest that Perforin-2 is not essential for host defense against fungal infections in otherwise immune-competent mice., Importance: Humans encounter fungal pathogens daily and rely on innate immune cells to clear Aspergillus fumigatus , the leading cause of mold pneumonia worldwide, and Candida albicans , the most common cause of fungal bloodstream infections. The World Health Organization has classified A. fumigatus and C. albicans as critical priority fungal pathogens due to the emergence of drug resistance and the increasing number of susceptible individuals across the globe. The mechanisms by which innate immune cells clear these fungal pathogens remain incompletely defined. In this study, we examined the role of a pore-forming protein called Perforin-2 in host defense against these fungal pathogens, in part because Perforin-2 has been implicated in antibacterial host defense. Our findings reveal that Perforin-2 is dispensable for antifungal immunity against respiratory A. fumigatus and systemic C. albicans infections in mice, suggesting that the antimicrobial activity of Perforin-2 does not extend to these two fungal pathogens.

microRNAs (miRNAs) and small interfering RNAs (siRNAs) are 21- to 22-nucleotide RNAs that guide Argonaute-class effectors to targets for repression. In this work, we uncover 5-aminolevulinic acid synthase 1 (ALAS1), the initiating enzyme for heme biosynthesis, as a general repressor of miRNA accumulation. Although heme is known to be a positive cofactor for the nuclear miRNA processing machinery, ALAS1-but not other heme biosynthesis enzymes-limits the assembly and activity of Argonaute complexes under heme-replete conditions. This involves a cytoplasmic role for ALAS1, previously considered inactive outside of mitochondria. Moreover, conditional depletion of ALAS activity from mouse hepatocytes increases miRNAs and enhances siRNA-mediated knockdown. Notably, because ALAS1 is the target of a Food and Drug Administration-approved siRNA drug, agents that suppress ALAS may serve as adjuvants for siRNA therapies.

Two distinct lineages, pluripotent epiblast (EPI) and primitive (extra-embryonic) endoderm (PrE), arise from common inner cell mass (ICM) progenitors in mammalian embryos. To study how these sister identities are forged, we leveraged mouse embryonic stem (ES) cells and extra-embryonic endoderm (XEN) stem cells-in vitro counterparts of the EPI and PrE. Bidirectional reprogramming between ES and XEN coupled with single-cell RNA and ATAC-seq analyses showed distinct rates, efficiencies, and trajectories of state conversions, identifying drivers and roadblocks of reciprocal conversions. While GATA4-mediated ES-to-iXEN conversion was rapid and nearly deterministic, OCT4-, KLF4-, and SOX2-induced XEN-to-induced pluripotent stem (iPS) reprogramming progressed with diminished efficiency and kinetics. A dominant PrE transcriptional program, safeguarded by GATA4, alongside elevated chromatin accessibility and reduced DNA methylation of the EPI underscored the differential plasticities of the two states. Mapping in vitro to embryo trajectories tracked reprogramming cells in either direction along EPI and PrE in vivo states, without transitioning through the ICM., Competing Interests: Declaration of interests A.-K.H. is a member of the advisory board of Developmental Cell., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Development of antigen-specific memory upon pathogen exposure is a hallmark of the adaptive immune system. While natural killer (NK) cells are considered part of the innate immune system, humans exposed to the chronic viral pathogen cytomegalovirus (CMV) often possess a distinct NK cell population lacking in individuals who have not been exposed, termed "adaptive" NK cells. To identify the "naïve" population from which this "memory" population derives, we performed phenotypic, transcriptional, and functional profiling of NK cell subsets. We identified immature precursors to the Adaptive NK cells that are equally present in both CMV+ and CMV- individuals, resolved an Adaptive transcriptional state distinct from most mature NK cells and sharing a common gene program with the immature CD56 bright population, and demonstrated retention of proliferative capacity and acquisition of superior IFNγ production in the Adaptive population. Furthermore, we distinguish the CD56 bright and Adaptive NK populations by expression of the transcription factor CXXC5, positioning these memory NK cells at the inflection point between innate and adaptive lymphocytes., Competing Interests: MKP and KCH are inventors on a patent application for the design and use of HLA-E:peptide chimeric molecules. KCH is a scientific advisory board member for Wugen, Inc., and a consultant for Exelixis. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Panjwani, Grassmann, Sottile, Le Luduec, Kontopoulos, van der Ploeg, Sun and Hsu.)

More than 40 years ago, studies of the Drosophila engrailed and Hox genes led to major discoveries that shaped the history of developmental biology. We learned that these genes define the state of determination of cells that populate particular spatially defined regions: the identity of segmental domains by Hox genes, and the identity of posterior developmental compartments by engrailed. Hence, the boundaries that delimit spatial domains depend on engrailed. Here, we review the engrailed field, which now includes orthologs in Drosophila and mouse, as well as many other animals. We focus on fly and mouse and highlight additional functions that span early stages of embryogenesis and neural development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Noncanonical inflammasomes are instrumental in defense against Gram-negative bacteria, activated primarily by bacterial lipopolysaccharide. This review examines commonalities and distinctions in noncanonical inflammasome activation either by virulence factor activity indicating cellular invasion or by detection of bacterial mRNA signaling the undesired presence of live bacteria in sterile tissue. These inflammasome triggers, alongside other examples discussed, reflect properties exclusive to live bacteria. The emerging picture underscores noncanonical inflammasome activation hinging on detection of indicators of bacterial viability such as the presence of certain molecules or activity of specific processes. The complex interpretation of combinatorial signals is essential for inflammasome activation according to the specific facet of infection confronting the host. Decoding these signals and their convergence on inflammasome activation will inform interventions and therapies for infectious diseases., Competing Interests: Declaration of Competing Interest The authors declare no financial conflict., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution has resulted in viral escape from clinically authorized monoclonal antibodies (mAbs), creating a need for mAbs that are resilient to epitope diversification. Broadly neutralizing coronavirus mAbs that are sufficiently potent for clinical development and retain activity despite viral evolution remain elusive. We identified a human mAb, designated VIR-7229, which targets the viral receptor-binding motif (RBM) with unprecedented cross-reactivity to all sarbecovirus clades, including non-ACE2-utilizing bat sarbecoviruses, while potently neutralizing SARS-CoV-2 variants since 2019, including the recent EG.5, BA.2.86, and JN.1. VIR-7229 tolerates extraordinary epitope variability, partly attributed to its high binding affinity, receptor molecular mimicry, and interactions with RBM backbone atoms. Consequently, VIR-7229 features a high barrier for selection of escape mutants, which are rare and associated with reduced viral fitness, underscoring its potential to be resilient to future viral evolution. VIR-7229 is a strong candidate to become a next-generation medicine., Competing Interests: Declaration of interests L.E.R., A.D.M., D.P., D.B., T.R., J.M.E., K.H., H.V.D., M.G., G.C., C.S., F.Z., E.D., A.R., J.N., Q.T., Y.D., B.T., R.A., S.S., B.G., M.A.S., G. Schnell, J.L.M., F.A.L., N.C., E.C., L.A.P., F.B., J.d.I., M.S.P., A.L., A.T., G. Snell, and D.C. are current or previous employees of Vir Biotechnology and may hold shares in Vir Biotechnology. L.E.R., A.D.M., D.P., E.C., F.B., M.S.P., G. Snell, and D.C. are currently listed as inventors on multiple patent applications that disclose the subject matter described in this paper. J.W.C. is an employee and shareholder of ProtaBody. J.W.C. and ProtaBody have received funding from Vir Biotechnology related to the work described in this paper. I.Z., Z.L., S.P.J.W., G.D.d.M., L.K., H.B., and T.N.S. have received funding through sponsored research awards to their respective institutions from Vir Biotechnology related to the work described in this paper. I.Z. is a current employee of Bristol Myers Squibb. L.A.P. is a former employee and shareholder of Regeneron Pharmaceuticals and is a member of the Scientific Advisory Board AI-driven structure-enabled antiviral platform (ASAP). Regeneron provided no funding for this work. L.A.P. is a current employee of Third Rock Ventures. D.V. is named as inventor on patents for coronavirus vaccines filed by the University of Washington. The lab of T.N.S. has received sponsored research agreements unrelated to the present work from Aerium Therapeutics, Inc. and Invivyd, Inc., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the face of rapidly accumulating genomic data, our ability to accurately predict key mature RNA properties that underlie transcript function and regulation remains limited. Pre-trained genomic foundation models offer an avenue to adapt learned RNA representations to biological prediction tasks. However, existing genomic foundation models are trained using strategies borrowed from textual or visual domains that do not leverage biological domain knowledge. Here, we introduce Orthrus, a Mamba-based mature RNA foundation model pre-trained using a novel self-supervised contrastive learning objective with biological augmentations. Orthrus is trained by maximizing embedding similarity between curated pairs of RNA transcripts, where pairs are formed from splice isoforms of 10 model organisms and transcripts from orthologous genes in 400+ mammalian species from the Zoonomia Project. This training objective results in a latent representation that clusters RNA sequences with functional and evolutionary similarities. We find that the generalized mature RNA isoform representations learned by Orthrus significantly outperform existing genomic foundation models on five mRNA property prediction tasks, and requires only a fraction of fine-tuning data to do so. Finally, we show that Orthrus is capable of capturing divergent biological function of individual transcript isoforms.

Conventional optical microscopy imaging of obligate intracellular bacteria is hampered by the small size of bacterial cells, tight clustering exhibited by some bacterial species and challenges relating to labelling such as background from host cells, a lack of validated reagents, and a lack of tools for genetic manipulation. In this study, we imaged intracellular bacteria from the species Orientia tsutsugamushi (Ot) using five different fluorescence microscopy techniques: standard confocal, Airyscan confocal, instant Structured Illumination Microscopy (iSIM), three-dimensional Structured Illumination Microscopy (3D-SIM) and Stimulated Emission Depletion Microscopy (STED). We compared the ability of each to resolve bacterial cells in intracellular clumps in the lateral (xy) axis, using full width half-maximum (FWHM) measurements of a labelled outer membrane protein (ScaA) and the ability to detect small, outer membrane vesicles external to the cells. Comparing the techniques readily available to us (above), 3D-SIM microscopy, in combination with the shortest-wavelength dyes, was found overall to give the best lateral resolution. We next compared the ability of each technique to sufficiently resolve bacteria in the axial (z) direction and found 3D-STED to be the most successful method for this. We then combined this 3D-STED approach with a custom 3D cell segmentation and analysis pipeline using the open-source, deep learning software, Cellpose to segment the cells and subsequently the commercial software Imaris to analyse their 3D shape and size. Using this combination, we demonstrated differences in bacterial shape, but not their size, when grown in different mammalian cell lines. Overall, we compare the advantages and disadvantages of different super-resolution microscopy techniques for imaging this cytoplasmic obligate intracellular bacterium based on the specific research question being addressed., (© 2024 The Author(s). Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

Background: The efficacy of Vesical Imaging-Reporting and Data System (VI-RADS) for the second transurethral resection (TUR) has not been adequately validated. This study aimed to evaluate the utility of the VI-RADS for high-risk patients with non-muscle-invasive bladder cancer (NMIBC) who are candidates for a second TUR., Methods: We retrospectively analyzed 116 patients who received magnetic resonance imaging (MRI) prior to an initial TUR and underwent a second TUR for a diagnosis of high-risk NMIBC at the initial TUR. MRI images were retrospectively classified according to VI-RADS. Second TUR outcomes and recurrence-free and progression-free survival rates were compared with VI-RADS scores., Results: Ninety-nine (91%) patients were diagnosed with T1 bladder cancer at the initial TUR. At the second TUR, residual cancer was found in 53 (49%) cases, including five (4.6%) cases of muscle invasion. With a median follow-up of 41 months, the 2-year bladder recurrence-free survival rate was 71% and the 2-year progression-free rate was 85%. By two radiologists' consensus, 30 (28%)/49 (45%)/16 (15%)/10 (9.2%)/4 (3.7%) cases were classified as VI-RADS 1/2/3/4/5, respectively. Of five pT2 upstage cases, three were VI-RADS 1, one was VI-RADS 2, and one was VI-RADS 3. There was no significant association between VI-RADS and cancer residual rate and pT2 upstage rate in second TUR outcomes, and recurrence-free and progression-free survival rates., Conclusion: In high-risk NMIBCs, a certain number of residual cancers and pT2 upstage cases exist after the initial TUR, and a second TUR should be performed regardless of VI-RADS scores., (© 2024 The Japanese Urological Association.)

As the field of neural organoids and assembloids rapidly expands, there is an emergent need for guidance and advice on designing, conducting and reporting experiments to increase the reproducibility and utility of these models. Here, our consortium- representing specialized laboratories from around the world- presents a framework for the experimental process that ranges from ensuring the quality and integrity of human pluripotent stem cells to characterizing and manipulating neural cells in vitro, and from transplantation techniques to considerations for modeling human development, evolution, and disease. As with all scientific endeavors, we advocate for rigorous experimental designs tailored to explicit scientific questions, and transparent methodologies and data sharing, to provide useful knowledge for both current research practices and for developing regulatory standards., (© 2024. Springer Nature Limited.)

In recent years, machine learning has transformed many aspects of the drug discovery process, including small molecule design, for which the prediction of bioactivity is an integral part. Leveraging structural information about the interactions between a small molecule and its protein target has great potential for downstream machine learning scoring approaches but is fundamentally limited by the accuracy with which protein-ligand complex structures can be predicted in a reliable and automated fashion. With the goal of finding practical approaches to generating useful kinase-inhibitor complex geometries for downstream machine learning scoring approaches, we present a kinase-centric docking benchmark assessing the performance of different classes of docking and pose selection strategies to assess how well experimentally observed binding modes are recapitulated in a realistic cross-docking scenario. The assembled benchmark data set focuses on the well-studied protein kinase family and comprises a subset of 589 protein structures cocrystallized with 423 ATP-competitive ligands. We find that the docking methods biased by the cocrystallized ligand, utilizing shape overlap with or without maximum common substructure matching, are more successful in recovering binding poses than standard physics-based docking alone. Also, docking into multiple structures significantly increases the chance of generating a low root-mean-square deviation (RMSD) docking pose. Docking utilizing an approach that combines all three methods (Posit) into structures with the most similar cocrystallized ligands according to the maximum common substructure (MCS) proved to be the most efficient way to reproduce binding poses, achieving a success rate of 70.4% across all included systems. The studied docking and pose selection strategies, which utilize the OpenEye Toolkits, were implemented into pipelines of the KinoML framework, allowing automated and reliable protein-ligand complex generation for future downstream machine learning tasks. Although focused on protein kinases, we believe that the general findings can also be transferred to other protein families.

Alternative splicing is essential for plants, enabling a single gene to produce multiple transcript variants to boost functional diversity and fine-tune responses to environmental and developmental cues. At-RS31, a plant-specific splicing factor in the Serine/Arginine (SR)-rich protein family, responds to light and the Target of Rapamycin (TOR) signaling pathway, yet its downstream targets and regulatory impact remain unknown.To identify At-RS31 targets, we applied individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) and RNAcompete assays. Transcriptomic analyses of At-RS31 mutant and overexpressing plants further revealed its effects on alternative splicing.iCLIP identified 4,034 At-RS31 binding sites across 1,421 genes, enriched in CU-rich and CAGA RNA motifs. Comparative iCLIP and RNAcompete data indicate that the RS domain of At-RS31 may influence its binding specificity in planta , underscoring the value of combining in vivo and in vitro approaches. Transcriptomic analysis showed that At-RS31 modulates diverse splicing events, particularly intron retention and exitron splicing, and influences other splicing modulators, acting as a hierarchical regulator.By regulating stress-response genes and genes in both TOR and abscisic acid (ABA) signaling pathways, At-RS31 may help integrate these signals, balancing plant growth with environmental adaptability through alternative splicing., Competing Interests: COMPETING INTERESTS None declared.

MicroRNAs (miRNAs) play a crucial role in regulating gene expression and influence many biological processes. Despite their importance, understanding of how genetic variation affects miRNA expression in the brain and how this relates to brain disorders remains limited. Here we investigated these questions by identifying microRNA expression quantitative trait loci (miR-QTLs), or genetic variants associated with brain miRNA levels, using genome-wide small RNA sequencing profiles from dorsolateral prefrontal cortex samples of 604 older adult donors of European ancestry. Here we show that nearly half (224 of 470) of the analyzed miRNAs have associated miR-QTLs, many of which fall in regulatory regions such as brain promoters and enhancers. We also demonstrate that intragenic miRNAs often have genetic regulation independent from their host genes. Furthermore, by integrating our findings with 16 genome-wide association studies of psychiatric and neurodegenerative disorders, we identified miRNAs that likely contribute to bipolar disorder, depression, schizophrenia and Parkinson's disease. These findings advance understanding of the genetic regulation of miRNAs and their role in brain health and disease., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Abstract: There are only a few options for patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL), thus, this is a major area of unmet medical need. In this study, we reveal that the inclusion of a poison exon in RBM39, which could be induced by both CDK9 or CDK9 independent cyclin-dependent kinases, mitogen-activated protein kinases, glycogen synthase kinases, CDC-like kinases (CMGC) kinase inhibition, is recognized by the nonsense-mediated messenger RNA decay pathway for degradation. Targeting this poison exon in RBM39 with CMGC inhibitors led to protein downregulation and the inhibition of ALL growth, particularly in relapsed/refractory B-ALL. Mechanistically, disruption of cotranscriptional splicing by the inhibition of CMGC kinases, including DYRK1A, or inhibition of CDK9, which phosphorylate the C-terminal domain of RNA polymerase II (Pol II), led to alteration in the SF3B1 and Pol II association. Disruption of SF3B1 and the transcriptional elongation complex altered Pol II pausing, which promoted the inclusion of a poison exon in RBM39. Moreover, RBM39 ablation suppressed the growth of human B-ALL, and targeting RBM39 with sulfonamides, which degrade RBM39 protein, showed strong antitumor activity in preclinical models. Our data reveal that relapsed/refractory B-ALL is susceptible to pharmacologic and genetic inhibition of RBM39 and provide 2 potential strategies to target this axis., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

In recent years, large-scale sequencing efforts have identified targetable driver mutations in haematopoietic stem cells. These efforts have led to the development and approval of nine novel agents for relapsed or refractory acute myelogenous leukaemia (R/R AML). However, despite an expansion in targeted therapies, achieving a durable remission in AML and high-risk myelodysplastic syndrome (HR-MDS) remains a significant challenge, and there is an urgent need for new effective treatments. Modulation of aberrant RNA splicing has emerged as a novel therapeutic approach in myeloid diseases. Aberrant splicing drives dysregulated gene expression that promotes tumourigenesis through increased proliferation and metastatic potential, immune evasion, decreased apoptosis, and chemotherapy resistance. Mutations in spliceosomal components have been identified in numerous cancer subtypes, with mutations in RNA binding proteins SF3B1, SRSF2, U2AF1, and ZRSR2 occurring frequently in AML and in up to 60% of patients with MDS, as well as in chronic myelomonocytic leukaemia and in 10% of patients with chronic lymphocytic leukaemia. In this review, we explore therapeutic strategies targeting aberrant splicing and the potential of these approaches to drive clinical responses., (© 2024 British Society for Haematology and John Wiley & Sons Ltd.)

Successful pregnancy relies directly on the placenta's complex, dynamic, gene-regulatory networks. Disruption of this vast collection of intercellular and intracellular programs leads to pregnancy complications and developmental defects. In the present study, we generated a comprehensive, spatially resolved, multimodal cell census elucidating the molecular architecture of the first trimester human placenta. We utilized paired single-nucleus (sn)ATAC (assay for transposase accessible chromatin) sequencing and RNA sequencing (RNA-seq), spatial snATAC-seq and RNA-seq, and in situ sequencing and hybridization mapping of transcriptomes at molecular resolution to spatially reconstruct the joint epigenomic and transcriptomic regulatory landscape. Paired analyses unraveled intricate tumor-like gene expression and transcription factor motif programs potentially sustaining the placenta in a hostile uterine environment; further investigation of gene-linked cis-regulatory elements revealed heightened regulatory complexity that may govern trophoblast differentiation and placental disease risk. Complementary spatial mapping techniques decoded these programs within the placental villous core and extravillous trophoblast cell column architecture while simultaneously revealing niche-establishing transcriptional elements and cell-cell communication. Finally, we computationally imputed genome-wide, multiomic single-cell profiles and spatially characterized the placental chromatin accessibility landscape. This spatially resolved, single-cell multiomic framework of the first trimester human placenta serves as a blueprint for future studies on early placental development and pregnancy., Competing Interests: Competing interests: A patent application related to this work about discovering novel immune modulators has been filed by the Massachusetts General Hospital. J.S. is a scientific advisor for Johnson & Johnson. F.C. is an academic co-founder of Curio Bioscience and Doppler Bio, and an advisor to Amber Bio. F.C., A.J.C.R. and N.M.N. are listed as inventors on a patent application related to Slide-tags. The other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Purpose: Although germline genetic testing (GT) is recommended for all patients with ovarian cancer (OC) and some patients with endometrial cancer (EC), uptake remains low with multiple barriers. Our center performs GT in parallel with somatic testing via a targeted sequencing assay (MSK-IMPACT) and initiates testing in oncology clinics (mainstreaming). We sought to optimize our GT processes for OC/EC., Methods: We performed a quality improvement study to evaluate our GT processes within gynecologic surgery/medical oncology clinics. All eligible patients with newly diagnosed OC/EC were identified for GT and tracked in a REDCap database. Clinical data and GT rates were collected by the study team, who reviewed data for qualitative themes., Results: From February 2023 to April 2023, we identified 116 patients with newly diagnosed OC (n = 57) and EC (n = 59). Patients were mostly White (62%); English was the preferred language for 90%. GT was performed in 52 (91%) patients with OC (seven external, 45 MSK-IMPACT) and in 44 (75%) patients with EC (three external, 41 MSK-IMPACT). GT results were available within 3 months for 100% and 95% of patients with OC and EC, respectively. Reasons for not undergoing GT included being missed by the clinical team where there was no record that GT was recommended, feeling overwhelmed, financial and privacy concerns, and language barriers. In qualitative review, we found that resources were concentrated in the initial visit with little follow-up to encourage GT at subsequent points of care., Conclusion: A mainstreaming approach that couples somatic and germline GT resulted in high testing rates in OC/EC; however, barriers were identified. Processes that encourage GT at multiple care points and allow self-directed, multilingual digital consenting should be piloted.

Resistance to chimaeric antigen receptor (CAR) T cell therapy develops through multiple mechanisms, most notably antigen loss and tumour-induced immune suppression. It has been suggested that T cells expressing multiple CARs may overcome the resistance of tumours and that T cells expressing receptors that switch inhibitory immune-checkpoint signals into costimulatory signals may enhance the activity of the T cells in the tumour microenvironment. However, engineering multiple features into a single T cell product is difficult because of the transgene-packaging constraints of current gene-delivery vectors. Here we describe a cell-sorting method that leverages leucine zippers for the selective single-step immunomagnetic purification of cells co-transduced with two vectors. Such 'Zip sorting' facilitated the generation of T cells simultaneously expressing up to four CARs and coexpressing up to three 'switch' receptors. In syngeneic mouse models, T cells with multiple CARs and multiple switch receptors eliminated antigenically heterogeneous populations of leukaemia cells coexpressing multiple inhibitory ligands. By combining diverse therapeutic strategies, Zip-sorted multi-CAR multi-switch-receptor T cells can overcome multiple mechanisms of CAR T cell resistance., Competing Interests: Competing interests: S.E.J., L.J., and M.R.M.v.d.B. are co-inventors on patent applications related to this manuscript (‘Leucine zipper-based compositions and methods of use’, nos. US20210171601A1 (USA), EP3836944A4 (Europe), WO2020037178A1 (WIPO), CA3109630A1 (Canada) and CN112930186A (China); ‘Cell sorting systems and methods of use’, nos. US20210179686A1 (USA), EP3837287A4 (Europe), WO2020037181A2 (WIPO), CA3109635A1 (Canada) and CN112996819A (China)). A.P.B. has consulted for Bristol Myers Squibb and Cancer Study Group, LLC, and has received honoraria from OncLive. F.D.B. is a founder of Biocept and has intellectual property licensed to Novartis. J.U.P. reports research funding, intellectual property fees and travel reimbursement from Seres Therapeutics as well as consulting fees from Da Volterra, CSL Behring and MaaT Pharma. He serves on an Advisory board of, and holds equity in, Postbiotics Plus Research. He has filed intellectual property applications related to the microbiome. S.A.V. has received funding from Bristol-Meyers Squibb and has received consulting fees from Koch Disruptive Technologies and Generate Biomedicine. C.A.K. has previously filed intellectual property applications related to the FasDNR featured in this manuscript. C.A.K. is a scientific co-founder and holds equity in Affini-T Therapeutics. C.A.K. has previously consulted for or is on the scientific and/or clinical advisory boards of: Achilles Therapeutics, Affini-T Therapeutics, Aleta BioTherapeutics, Bellicum Pharmaceuticals, Bristol Myers Squibb, Catamaran Bio, Cell Design Labs, Decheng Capital, G1 Therapeutics, Klus Pharma, Obsidian Therapeutics, PACT Pharma, Roche/Genentech, Royalty Pharma and T-knife. M.R.M.v.d.B. has received research support and stock options from Seres Therapeutics and stock options from Notch Therapeutics and Pluto Therapeutics; has received royalties from Wolters Kluwer; has consulted, received honoraria from or participated in advisory boards for Seres Therapeutics, Vor Biopharma, Rheos Medicines, Frazier Healthcare Partners, Nektar Therapeutics, Notch Therapeutics, Ceramedix, Lygenesis, Pluto Therapeutics, GlaxoSmithKline, Da Volterra, Thymofox, Garuda, Novartis (spouse), Synthekine (spouse), Beigene (spouse), Kite (spouse); has intellectual property licensing with Seres Therapeutics and Juno Therapeutics; and holds a fiduciary role on the Foundation Board of DKMS (a non-profit organization). MSKCC has institutional financial interests relative to Seres Therapeutics., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Selective vulnerability of neuronal populations occurs in both Down syndrome (DS) and Alzheimer's disease (AD), resulting in disproportional degeneration of pyramidal neurons (PNs) affecting memory and executive function. Elucidating the cellular mechanisms underlying the selective vulnerability of these populations will provide pivotal insights for disease progression in DS and AD. Single population RNA-sequencing analysis was performed on neurons critical for executive function, prefrontal cortex Brodmann area 9 (BA9) layer III (L3) and layer V (L5) excitatory PNs in postmortem human DS and age- and sex-matched control (CTR) brains. Data mining was performed on differentially expressed genes (DEGs) from PNs in each lamina with DEGs divergent between lamina identified and interrogated. Bioinformatic inquiry of L3 PNs revealed more unique/differentially expressed DEGs (uDEGs) than in L5 PNs in DS compared to CTR subjects, indicating gene dysregulation shows both spatial and cortical laminar projection neuron dependent dysregulation. DS triplicated human chromosome 21 (HSA21) comprised a subset of DEGs only dysregulated in L3 or L5 neurons, demonstrating partial cellular specificity in HSA21 expression. These HSA21 uDEGs had a disproportionally high number of noncoding RNAs, suggesting lamina specific dysfunctional gene regulation. L3 uDEGs revealed overall more dysregulation of cellular pathways and processes, many relevant to early AD pathogenesis, while L5 revealed processes suggestive of frank AD pathology. These findings indicate that trisomy differentially affects a subpopulation of uDEGs in L3 and L5 BA9 projection neurons in aged individuals with DS, which may inform circuit specific pathogenesis underlying DS and AD., Competing Interests: Declarations. Ethics approval: De-identified postmortem brain tissues were employed. Ethics approval was waived at the Nathan Kline Institute and New York University Grossman School of Medicine. Animal tissue use is not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Bis(monoacylglycero)phosphate (BMP) is an abundant lysosomal phospholipid required for degradation of lipids, particularly gangliosides. Alterations in BMP levels are associated with neurodegenerative diseases. Unlike typical glycerophospholipids, lysosomal BMP has two chiral glycerol carbons in the S (rather than the R) stereo-conformation, protecting it from lysosomal degradation. How this unusual and yet crucial S,S-stereochemistry is achieved is unknown. Here, we report that phospholipases D3 and D4 (PLD3 and PLD4) synthesize lysosomal S,S-BMP, with either enzyme catalyzing the critical glycerol stereo-inversion reaction in vitro. Deletion of PLD3 or PLD4 markedly reduced BMP levels in cells or in murine tissues where either enzyme is highly expressed (brain for PLD3; spleen for PLD4), leading to gangliosidosis and lysosomal abnormalities. PLD3 mutants associated with neurodegenerative diseases, including risk of Alzheimer's disease, diminished PLD3 catalytic activity. We conclude that PLD3/4 enzymes synthesize lysosomal S,S-BMP, a crucial lipid for maintaining brain health., Competing Interests: Declaration of interests R.V.F. serves gratis as a board member of the Bluefield Project to Cure FTD., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Alchemical free energy campaigns can be planned using graph theory by building networks that contain nodes representing molecules that are connected by possible transformations as edges. We introduce Konnektor, an open-source Python package, for systematically planning, modifying, and analyzing free energy calculation networks. Konnektor is designed to aid in the drug discovery process by enabling users to easily setup free energy campaigns using complex graph manipulation methods. The package contains functions for network operations including concatenation of networks, deletion of transformations, and clustering of molecules along with a framework for combining these tools with existing network generation algorithms to enable the development of more complex methods for network generation. A comparison of the various network layout features offered is carried out using toy data sets. Additionally, Konnektor contains visualization and analysis tools, making the investigation of network features much simpler. Besides the content of the package, the paper also offers application examples, demonstrating how Konnektor can be used and how the different networks perform from a graph theory perspective. Konnektor is freely available via GitHub at https://github.com/OpenFreeEnergy/konnektor under the permissive MIT License.

Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated stereoisomeric acrylamide EV96 and its analogues lead to a selective T cell state-dependent loss of interleukin 2-inducible T cell kinase (ITK) by targeting one of the core splicing factors SF3B1. Mechanistic investigations suggest that the state-dependency stems from a combination of differential protein turnover rates and extensive ITK mRNA alternative splicing. We further introduce the most comprehensive list to date of proteins involved in splicing and leverage cysteine- and protein-directed activity-based protein profiling with electrophilic scout fragments to demonstrate covalent ligandability for many classes of splicing factors and splicing regulators in T cells. Taken together, our findings show how chemical perturbation of splicing can lead to immune state-dependent changes in protein expression and provide evidence for the broad potential to target splicing factors with covalent chemistry., Competing Interests: Declaration of interests B.F.C. is a founder and advisor to Vividion Therapeutics. E.V.V. is a co-inventor on patents with Vividion Therapeutics. O.A.-W. is a founder and advisor to Codify Therapeutics. S.J.H. is an employee of AbbVie., (Published by Elsevier Ltd.)