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Tau pathology drives neuronal dysfunction in 4- repeat tauopathies. Here, the authors combine tau-PET, resting-state fMRI and histopathology data, to show that brain connectivity is associated with tau deposition patterns in 4-repeat tauopathies.

Antiskyrmions are topological spin textures with negative vorticity. Like skyrmions, they have considerable technological promise, but have only been stabilised in Heusler compounds. Here, Heigl et al. succeed in stabilising first and second order antiskyrmions in a new class of materials.

Computational decision support systems could provide clinical value in whole-body FDG PET/CT workflows, but labeled data is scarce and PET/CT imaging exams are cumbersome. Here, the authors describe a weak supervision framework that extracts regional abnormality labels from free-text radiology reports.

Tau pathology is the main driver of neuronal dysfunction in 4-repeat tauopathies, including cortico-basal degeneration and progressive supranuclear palsy. Tau is assumed to spread prion-like across connected neurons, but the mechanisms of tau propagation are largely elusive in 4-repeat tauopathies, characterized not only by neuronal but also by astroglial and oligodendroglial tau accumulation. Here, we assess whether connectivity is associated with 4R-tau deposition patterns by combining resting-state fMRI connectomics with both 2nd generation 18F-PI-2620 tau-PET in 46 patients with clinically diagnosed 4-repeat tauopathies and post-mortem cell-type-specific regional tau assessments from two independent progressive supranuclear palsy patient samples (n = 97 and n = 96). We find that inter-regional connectivity is associated with higher inter-regional correlation of both tau-PET and post-mortem tau levels in 4-repeat tauopathies. In regional cell-type specific post-mortem tau assessments, this association is stronger for neuronal than for astroglial or oligodendroglial tau, suggesting that connectivity is primarily associated with neuronal tau accumulation. Using tau-PET we find further that patient-level tau patterns are associated with the connectivity of subcortical tau epicenters. Together, the current study provides combined in vivo tau-PET and histopathological evidence that brain connectivity is associated with tau deposition patterns in 4-repeat tauopathies.

The cellular mechanisms driving cardiac tissue formation remain poorly understood, largely due to the structural and functional complexity of the heart. It is unclear whether newly generated myocytes originate from cardiac stem/progenitor cells or from pre-existing cardiomyocytes that re-enter the cell cycle. Here, we identify the source of new cardiomyocytes during mouse development and after injury. Our findings suggest that cardiac progenitors maintain proliferative potential and are the main source of cardiomyocytes during development; however, the onset of αMHC expression leads to reduced cycling capacity. Single-cell RNA sequencing reveals a proliferative, "progenitor-like" population abundant in early embryonic stages that decreases to minimal levels postnatally. Furthermore, cardiac injury by ligation of the left anterior descending artery was found to activate cardiomyocyte proliferation in neonatal but not adult mice. Our data suggest that clonal dominance of differentiating progenitors mediates cardiac development, while a distinct subpopulation of cardiomyocytes may have the potential for limited proliferation during late embryonic development and shortly after birth.

Abstract A significant challenge for chimeric antigen receptor (CAR) T cell therapy against glioblastoma (GBM) is its immunosuppressive microenvironment, which is densely populated by protumoral glioma-associated microglia and macrophages (GAMs). Myeloid immune checkpoint therapy targeting the CD47-signal regulatory protein alpha (SIRPα) axis induces GAM phagocytic function, but CD47 blockade monotherapy is associated with toxicity and low bioavailability in solid tumors. In this work, we engineer a CAR T cell against epidermal growth factor receptor variant III (EGFRvIII), constitutively secreting a signal regulatory protein gamma-related protein (SGRP) with high affinity to CD47. Anti-EGFRvIII-SGRP CAR T cells eradicate orthotopic EGFRvIII-mosaic GBM in vivo, promoting GAM-mediated tumor cell phagocytosis. In a subcutaneous CD19+ lymphoma mouse model, anti-CD19-SGRP CAR T cell therapy is superior to conventional anti-CD19 CAR T. Thus, combination of CAR and SGRP eliminates bystander tumor cells in a manner that could overcome main mechanisms of CAR T cell therapy resistance, including immune suppression and antigen escape.

Abstract Classical Zn2+-dependent deac(et)ylases play fundamental regulatory roles in life and are well characterized in eukaryotes regarding their structures, substrates and physiological roles. In bacteria, however, classical deacylases are less well understood. We construct a Generalized Profile (GP) and identify thousands of uncharacterized classical deacylases in bacteria, which are grouped into five clusters. Systematic structural and functional characterization of representative enzymes from each cluster reveal high functional diversity, including polyamine deacylases and protein deacylases with various acyl-chain type preferences. These data are supported by multiple crystal structures of enzymes from different clusters. Through this extensive analysis, we define the structural requirements of substrate selectivity, and discovered bacterial de-d-/l-lactylases and long-chain deacylases. Importantly, bacterial deacylases are inhibited by archetypal HDAC inhibitors, as supported by co-crystal structures with the inhibitors SAHA and TSA, and setting the ground for drug repurposing strategies to fight bacterial infections. Thus, we provide a systematic structure-function analysis of classical deacylases in bacteria and reveal the basis of substrate specificity, acyl-chain preference and inhibition.

Abstract Lassa virus (LASV) outbreaks in West Africa pose a significant public health threat. We investigated the infection phenotype and transmission (horizontal and vertical) of LASV strain Ba366 in its natural host, Mastomys natalensis. Here we analyze viral RNA levels in body fluids, virus titers in organs and antibody presence in blood. In adults and 2-week-old animals, LASV causes transient infections with subsequent seroconversion. However, mice younger than two weeks exhibit persistent infections lasting up to 16 months despite antibody presence. LASV can be detected in various body fluids, organs, and cell types, primarily in lung, kidney, and gonadal epithelial cells. Despite the systemic virus presence, no pathological alterations in organs are observed. Infected animals efficiently transmit the virus throughout their lives. Our findings underscore the crucial role of persistently infected individuals, particularly infected females and their progeny, in LASV dissemination within the host population.

Abstract Gonadal hormone activities mediated by androgen and estrogen receptors, along with cell-autonomous mechanisms arising from the absence of sex-chromosome dosage compensation, are key factors in avian sexual development. In this study, we generate androgen receptor (AR) knockout chickens (AR−/−) to explore the role of androgen signaling in avian sexual development. Despite developing sex-typical gonads and gonadal hormone production, AR−/− males and females are infertile. While few somatic sex-specific traits persist (body size, spurs, and tail feathers), crucial sexual attributes such as comb, wattles and sexual behaviors remain underdeveloped in both sexes. Testosterone treatment of young AR−/− males fails to induce crow behavior, comb development, or regression of the bursa of Fabricius, which are testosterone-dependent phenotypes. These findings highlight the significance of androgen receptor mechanisms in fertility and sex-specific traits in chickens, challenging the concept of a default sex in birds and emphasizing the dominance of androgen signaling in avian sexual development.

Abstract Eukaryotic translation initiation factor eIF4B is required for efficient cap-dependent translation, it is overexpressed in cancer cells, and may influence stress granule formation. Due to the high degree of intrinsic disorder, eIF4B is rarely observed in cryo-EM structures of translation complexes and only ever by its single structured RNA recognition motif domain, leaving the molecular details of its large intrinsically disordered region (IDR) unknown. By integrating experiments and simulations we demonstrate that eIF4B IDR orchestrates and fine-tunes an intricate transition from monomers to a condensed phase, in which large-size dynamic oligomers form before mesoscopic phase separation. Single-molecule spectroscopy combined with molecular simulations enabled us to characterize the conformational ensembles and underlying intra- and intermolecular dynamics across the oligomerization transition. The observed sensitivity to ionic strength and molecular crowding in the self-association landscape suggests potential regulation of eIF4B nanoscopic and mesoscopic behaviors such as driven by protein modifications, binding partners or changes to the cellular environment.

Abstract Integrated optical tweezers have the potential to enable highly-compact, low-cost, mass-manufactured, and broadly-accessible optical manipulation when compared to standard bulk-optical tweezers. However, integrated demonstrations to date have been fundamentally limited to micron-scale standoff distances and, often, passive trapping functionality, making them incompatible with many existing applications and significantly limiting their utility, especially for biological studies. In this work, we demonstrate optical trapping and tweezing using an integrated OPA for the first time, increasing the standoff distance of integrated optical tweezers by over two orders of magnitude compared to prior demonstrations. First, we demonstrate trapping of polystyrene microspheres 5 mm above the surface of the chip and calibrate the trap force. Next, we show tweezing of polystyrene microspheres in one dimension by non-mechanically steering the trap by varying the input laser wavelength. Finally, we use the OPA tweezers to demonstrate, to the best of our knowledge, the first cell experiments using single-beam integrated optical tweezers, showing controlled deformation of mouse lymphoblast cells. This work introduces a new modality for integrated optical tweezers, significantly expanding their utility and compatibility with existing applications, especially for biological experiments.

Abstract Enhancing thermogenic brown adipose tissue (BAT) function is a promising therapeutic strategy for metabolic disease. However, predominantly thermoneutral modern human living conditions deactivate BAT. We demonstrate that selective adipocyte deficiency of the oxygen-sensor HIF-prolyl hydroxylase (PHD2) gene overcomes BAT dormancy at thermoneutrality. Adipocyte-PHD2-deficient mice maintain higher energy expenditure having greater BAT thermogenic capacity. In human and murine adipocytes, a PHD inhibitor increases Ucp1 levels. In murine brown adipocytes, antagonising the major PHD2 target, hypoxia-inducible factor-(HIF)−2a abolishes Ucp1 that cannot be rescued by PHD inhibition. Mechanistically, PHD2 deficiency leads to HIF2 stabilisation and binding of HIF2 to the Ucp1 promoter, thus enhancing its expression in brown adipocytes. Serum proteomics analysis of 5457 participants in the deeply phenotyped Age, Gene and Environment Study reveal that serum PHD2 associates with increased risk of metabolic disease. Here we show that adipose-PHD2-inhibition is a therapeutic strategy for metabolic disease and identify serum PHD2 as a disease biomarker.

Abstract Apical membrane antigen-1 (AMA1) is a conserved malarial vaccine candidate essential for the formation of tight junctions with the rhoptry neck protein (RON) complex, enabling Plasmodium parasites to invade human erythrocytes, hepatocytes, and mosquito salivary glands. Despite its critical role, extensive surface polymorphisms in AMA1 have led to strain-specific protection, limiting the success of AMA1-based interventions beyond initial clinical trials. Here, we identify an i-body, a humanised single-domain antibody-like molecule that recognises a conserved pan-species conformational epitope in AMA1 with low nanomolar affinity and inhibits the binding of the RON2 ligand to AMA1. Structural characterisation indicates that the WD34 i-body epitope spans the centre of the conserved hydrophobic cleft in AMA1, where interacting residues are highly conserved among all Plasmodium species. Furthermore, we show that WD34 inhibits merozoite invasion of erythrocytes by multiple Plasmodium species and hepatocyte invasion by P. falciparum sporozoites. Despite a short half-life in mouse serum, we demonstrate that WD34 transiently suppressed P. berghei infections in female BALB/c mice. Our work describes the first pan-species AMA1 biologic with inhibitory activity against multiple life-cycle stages of Plasmodium. With improved pharmacokinetic characteristics, WD34 could be a potential immunotherapy against multiple species of Plasmodium.

Abstract Non-covalent interactions play an important role for the framework formation of two-dimensional covalent organic frameworks. Until now, π–π interactions and hydrogen bonding are the main reported forces facilitating the stacking of framework layers. Here, we present a two-dimensional anionic covalent organic framework based on tetratopic borate linkages, where layers are connected by ionic interactions between the linkage site and counter cations. The crystalline covalent organic framework is accessed through the formation of an amorphous borate-based polymer and subsequent solvothermal treatment. The progress of crystallization is investigated, revealing the crystallite growth and morphological change from agglomerated dense particles to hollow crystallite spheres. Due to the pillared nature, the crystallites can be exfoliated into nanosheets by sonication of the material in the presence of methanol. The crystallization and ordered arrangement of the lithium ions in the interlayer space is shown to benefit the conductivity tenfold compared to the amorphous material.

Abstract Multiple myeloma is a hematological malignancy arising from immunoglobulin-secreting plasma cells. It remains poorly understood how chromatin rewiring of regulatory elements contributes to tumorigenesis and therapy resistance in myeloma. Here we generate a high-resolution contact map of myeloma-associated super-enhancers by integrating H3K27ac ChIP-seq and HiChIP from myeloma cell lines, patient-derived myeloma cells and normal plasma cells. Our comprehensive transcriptomic and phenomic analyses prioritize candidate genes with biological and clinical implications in myeloma. We show that myeloma cells frequently acquire SE that transcriptionally activate an oncogene PPP1R15B, which encodes a regulatory subunit of the holophosphatase complex that dephosphorylates translation initiation factor eIF2α. Epigenetic silencing or knockdown of PPP1R15B activates pro-apoptotic eIF2α-ATF4-CHOP pathway, while inhibiting protein synthesis and immunoglobulin production. Pharmacological inhibition of PPP1R15B using Raphin1 potentiates the anti-myeloma effect of bortezomib. Our study reveals that myeloma cells are vulnerable to perturbation of PPP1R15B-dependent protein homeostasis, highlighting a promising therapeutic strategy.

Abstract Activation-induced cytidine deaminase (AID) is a B cell-specific mutator required for antibody diversification. However, it is also implicated in the etiology of several B cell malignancies. Evaluating the AID-induced mutation load in patients at-risk for certain blood cancers is critical in assessing disease severity and treatment options. We have developed a digital PCR (dPCR) assay that allows us to quantify mutations resulting from AID modification or DNA double-strand break (DSB) formation and repair at sites known to be prone to DSBs. Implementation of this assay shows that increased AID levels in immature B cells increase genome instability at loci linked to chromosomal translocation formation. This includes the CRLF2 locus that is often involved in translocations associated with a subtype of acute lymphoblastic leukemia (ALL) that disproportionately affects Hispanics, particularly those with Latin American ancestry. Using dPCR, we characterize the CRLF2 locus in B cell-derived genomic DNA from both Hispanic ALL patients and healthy Hispanic donors and found increased mutations in both, suggesting that vulnerability to DNA damage at CRLF2 may be driving this health disparity. Our ability to detect and quantify these mutations will potentiate future risk identification, early detection of cancers, and reduction of associated cancer health disparities.

Abstract The AMP-forming acetyl-CoA synthetase is regulated by lysine acetylation both in bacteria and eukaryotes. However, the underlying mechanism is poorly understood. The Bacillus subtilis acetyltransferase AcuA and the AMP-forming acetyl-CoA synthetase AcsA form an AcuA•AcsA complex, dissociating upon lysine acetylation of AcsA by AcuA. Crystal structures of AcsA from Chloroflexota bacterium in the apo form and in complex with acetyl-adenosine-5′-monophosphate (acetyl-AMP) support the flexible C-terminal domain adopting different conformations. AlphaFold2 predictions suggest binding of AcuA stabilizes AcsA in an undescribed conformation. We show the AcuA•AcsA complex dissociates upon acetyl-coenzyme A (acetyl-CoA) dependent acetylation of AcsA by AcuA. We discover an intrinsic phosphotransacetylase activity enabling AcuA•AcsA generating acetyl-CoA from acetyl-phosphate (AcP) and coenzyme A (CoA) used by AcuA to acetylate and inactivate AcsA. Here, we provide mechanistic insights into the regulation of AMP-forming acetyl-CoA synthetases by lysine acetylation and discover an intrinsic phosphotransacetylase allowing modulation of its activity based on AcP and CoA levels.

Abstract Plasmodium falciparum is the causative agent of malaria and remains a pathogen of global importance. Asexual blood stage replication, via a process called schizogony, is an important target for the development of new antimalarials. Here we use ultrastructure-expansion microscopy to probe the organisation of the chromosome-capturing kinetochores in relation to the mitotic spindle, the centriolar plaque, the centromeres and the apical organelles during schizont development. Conditional disruption of the kinetochore components, PfNDC80 and PfNuf2, is associated with aberrant mitotic spindle organisation, disruption of the centromere marker, CENH3 and impaired karyokinesis. Surprisingly, kinetochore disruption also leads to disengagement of the centrosome equivalent from the nuclear envelope. Severing the connection between the nucleus and the apical complex leads to the formation of merozoites lacking nuclei. Here, we show that correct assembly of the kinetochore/spindle complex plays a previously unrecognised role in positioning the nascent apical complex in developing P. falciparum merozoites.

Abstract Cancer resistance to immune checkpoint inhibitors motivated investigations into leveraging the immunostimulatory properties of radiotherapy to overcome immune evasion and to improve treatment response. However, clinical benefits of radiotherapy-immunotherapy combinations have been modest. Routine concomitant tumor-draining lymph node irradiation (DLN IR) might be the culprit. As crucial sites for generating anti-tumor immunity, DLNs are indispensable for the in situ vaccination effect of radiotherapy. Simultaneously, DLN sparing is often not feasible due to metastatic spread. Using murine models of metastatic disease in female mice, here we demonstrate that delayed (adjuvant), but not neoadjuvant, DLN IR overcomes the detrimental effect of concomitant DLN IR on the efficacy of radio-immunotherapy. Moreover, we identify IR-induced disruption of the CCR7-CCL19/CCL21 homing axis as a key mechanism for the detrimental effect of DLN IR. Our study proposes delayed DLN IR as a strategy to maximize the efficacy of radio-immunotherapy across different tumor types and disease stages.

Abstract Sickle cell disease is a devastating blood disorder that originates from a single point mutation in the HBB gene coding for hemoglobin. Here, we develop a GMP-compatible TALEN-mediated gene editing process enabling efficient HBB correction via a DNA repair template while minimizing risks associated with HBB inactivation. Comparing viral versus non-viral DNA repair template delivery in hematopoietic stem and progenitor cells in vitro, both strategies achieve comparable HBB correction and result in over 50% expression of normal adult hemoglobin in red blood cells without inducing β-thalassemic phenotype. In an immunodeficient female mouse model, transplanted cells edited with the non-viral strategy exhibit higher engraftment and gene correction levels compared to those edited with the viral strategy. Transcriptomic analysis reveals that non-viral DNA repair template delivery mitigates P53-mediated toxicity and preserves high levels of long-term hematopoietic stem cells. This work paves the way for TALEN-based autologous gene therapy for sickle cell disease.

Abstract Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease due to gradual motoneurons (MN) degeneration. Among the processes associated to ALS pathogenesis, there is the formation of cytoplasmic inclusions produced by aggregation of mutant proteins, among which the RNA binding protein FUS. Here we show that, in neuronal cells and in iPSC-derived MN expressing mutant FUS, such inclusions are significantly reduced in number and dissolve faster when the RNA m6A content is diminished. Interestingly, stress granules formed in ALS conditions showed a distinctive transcriptome with respect to control cells, which reverted to similar to control after m6A downregulation. Notably, cells expressing mutant FUS were characterized by higher m6A levels suggesting a possible link between m6A homeostasis and pathological aggregates. Finally, we show that FUS inclusions are reduced also in patient-derived fibroblasts treated with STM-2457, an inhibitor of METTL3 activity, paving the way for its possible use for counteracting aggregate formation in ALS.

Abstract Due to their exceptional solubility and stability, nanobodies have emerged as powerful building blocks for research tools and therapeutics. However, their generation in llamas is cumbersome and costly. Here, by inserting an engineered llama immunoglobulin heavy chain (IgH) locus into IgH-deficient mice, we generate a transgenic mouse line, which we refer to as ‘LamaMouse’. We demonstrate that LamaMice solely express llama IgH molecules without association to Igκ or λ light chains. Immunization of LamaMice with AAV8, the receptor-binding domain of the SARS-CoV-2 spike protein, IgE, IgG2c, and CLEC9A enabled us to readily select respective target-specific nanobodies using classical hybridoma and phage display technologies, single B cell screening, and direct cloning of the nanobody-repertoire into a mammalian expression vector. Our work shows that the LamaMouse represents a flexible and broadly applicable platform for a facilitated selection of target-specific nanobodies.

Abstract The overexpression of the ecotropic viral integration site-1 gene (EVI1/MECOM) marks the most lethal acute myeloid leukemia (AML) subgroup carrying chromosome 3q26 abnormalities. By taking advantage of the intersectionality of high-throughput cell-based and gene expression screens selective and pan-histone deacetylase inhibitors (HDACis) emerge as potent repressors of EVI1. To understand the mechanism driving on-target anti-leukemia activity of this compound class, here we dissect the expression dynamics of the bone marrow leukemia cells of patients treated with HDACi and reconstitute the EVI1 chromatin-associated co-transcriptional complex merging on the role of proliferation-associated 2G4 (PA2G4) protein. PA2G4 overexpression rescues AML cells from the inhibitory effects of HDACis, while genetic and small molecule inhibition of PA2G4 abrogates EVI1 in 3q26 AML cells, including in patient-derived leukemia xenografts. This study positions PA2G4 at the crosstalk of the EVI1 leukemogenic signal for developing new therapeutics and urges the use of HDACis-based combination therapies in patients with 3q26 AML.

Abstract Hedgehog (Hh) signaling relies on the primary cilium, a cell surface organelle that serves as a signaling hub for the cell. Using proximity labeling and quantitative proteomics, we identify Numb as a ciliary protein that positively regulates Hh signaling. Numb localizes to the ciliary pocket and acts as an endocytic adaptor to incorporate Ptch1 into clathrin-coated vesicles, thereby promoting Ptch1 exit from the cilium, a key step in Hh signaling activation. Numb loss impedes Sonic hedgehog (Shh)-induced Ptch1 exit from the cilium, resulting in reduced Hh signaling. Numb loss in spinal neural progenitors reduces Shh-induced differentiation into cell fates reliant on high Hh activity. Genetic ablation of Numb in the developing cerebellum impairs the proliferation of granule cell precursors, a Hh-dependent process, resulting in reduced cerebellar size. This study highlights Numb as a regulator of ciliary Ptch1 levels during Hh signal activation and demonstrates the key role of ciliary pocket-mediated endocytosis in cell signaling.

Abstract 5,10-methylenetetrahydrofolate reductase (MTHFR) commits folate-derived one-carbon units to generate the methyl-donor s-adenosyl-l-methionine (SAM). Eukaryotic MTHFR appends to the well-conserved catalytic domain (CD) a unique regulatory domain (RD) that confers feedback inhibition by SAM. Here we determine the cryo-electron microscopy structures of human MTHFR bound to SAM and its demethylated product s-adenosyl-l-homocysteine (SAH). In the active state, with the RD bound to a single SAH, the CD is flexible and exposes its active site for catalysis. However, in the inhibited state the RD pocket is remodelled, exposing a second SAM-binding site that was previously occluded. Dual-SAM bound MTHFR demonstrates a substantially rearranged inter-domain linker that reorients the CD, inserts a loop into the active site, positions Tyr404 to bind the cofactor FAD, and blocks substrate access. Our data therefore explain the long-distance regulatory mechanism of MTHFR inhibition, underpinned by the transition between dual-SAM and single-SAH binding in response to cellular methylation status.

Abstract Homeodomains (HDs) are the second largest class of DNA binding domains (DBDs) among eukaryotic sequence-specific transcription factors (TFs) and are the TF structural class with the largest number of disease-associated mutations in the Human Gene Mutation Database (HGMD). Despite numerous structural studies and large-scale analyses of HD DNA binding specificity, HD-DNA recognition is still not fully understood. Here, we analyze 92 human HD mutants, including disease-associated variants and variants of uncertain significance (VUS), for their effects on DNA binding activity. Many of the variants alter DNA binding affinity and/or specificity. Detailed biochemical analysis and structural modeling identifies 14 previously unknown specificity-determining positions, 5 of which do not contact DNA. The same missense substitution at analogous positions within different HDs often exhibits different effects on DNA binding activity. Variant effect prediction tools perform moderately well in distinguishing variants with altered DNA binding affinity, but poorly in identifying those with altered binding specificity. Our results highlight the need for biochemical assays of TF coding variants and prioritize dozens of variants for further investigations into their pathogenicity and the development of clinical diagnostics and precision therapies.

Abstract Myelodysplastic syndromes (MDS) with mutated SF3B1 gene present features including a favourable outcome distinct from MDS with mutations in other splicing factor genes SRSF2 or U2AF1. Molecular bases of these divergences are poorly understood. Here we find that SF3B1-mutated MDS show reduced R-loop formation predominating in gene bodies associated with intron retention reduction, not found in U2AF1- or SRSF2-mutated MDS. Compared to erythroblasts from SRSF2- or U2AF1-mutated patients, SF3B1-mutated erythroblasts exhibit augmented DNA synthesis, accelerated replication forks, and single-stranded DNA exposure upon differentiation. Importantly, histone deacetylase inhibition using vorinostat restores R-loop formation, slows down DNA replication forks and improves SF3B1-mutated erythroblast differentiation. In conclusion, loss of R-loops with associated DNA replication stress represents a hallmark of SF3B1-mutated MDS ineffective erythropoiesis, which could be used as a therapeutic target.

Abstract Neural stem and progenitor cell (NSPC) maintenance is essential for ensuring that organisms are born with proper brain volumes and head sizes. Microcephaly is a disorder in which babies are born with significantly smaller head sizes and cortical volumes. Mutations in subunits of the DNA organizing complex condensin have been identified in microcephaly patients. However, the molecular mechanisms by which condensin insufficiency causes microcephaly remain elusive. We previously identified conserved roles for condensins in repression of retrotransposable elements (RTEs). Here, we show that condensin subunit knockdown in NSPCs of the Drosophila larval central brain increases RTE expression and mobility which causes cell death, and significantly decreases adult head sizes and brain volumes. These findings suggest that unrestricted RTE expression and activity may lead to improper brain development in condensin insufficient organisms, and lay the foundation for future exploration of causative roles for RTEs in other microcephaly models.

Abstract The significance of transient neuropeptide expression during postnatal brain development is unknown. Here, we show that galanin expression in the ventrobasal thalamus of infant mice coincides with whisker map development and modulates subcortical circuit wiring. Time-resolved neuroanatomy and single-nucleus RNA-seq identified complementary galanin (Gal) and galanin receptor 1 (Galr1) expression in the ventrobasal thalamus and the principal sensory nucleus of the trigeminal nerve (Pr5), respectively. Somatodendritic galanin release from the ventrobasal thalamus was time-locked to the first postnatal week, when Gal1R+ Pr5 afferents form glutamatergic (Slc17a6 +) synapses for the topographical whisker map to emerge. RNAi-mediated silencing of galanin expression disrupted glutamatergic synaptogenesis, which manifested as impaired whisker-dependent exploratory behaviors in infant mice, with behavioral abnormalities enduring into adulthood. Pharmacological probing of receptor selectivity in vivo corroborated that target recognition and synaptogenesis in the thalamus, at least in part, are reliant on agonist-induced Gal1R activation in inbound excitatory axons. Overall, we suggest a neuropeptide-dependent developmental mechanism to contribute to the topographical specification of a fundamental sensory neurocircuit in mice.

Abstract A foundational assumption of quantum error correction theory is that quantum gates can be scaled to large processors without exceeding the error-threshold for fault tolerance. Two major challenges that could become fundamental roadblocks are manufacturing high-performance quantum hardware and engineering a control system that can reach its performance limits. The control challenge of scaling quantum gates from small to large processors without degrading performance often maps to non-convex, high-constraint, and time-dynamic control optimization over an exponentially expanding configuration space. Here we report on a control optimization strategy that can scalably overcome the complexity of such problems. We demonstrate it by choreographing the frequency trajectories of 68 frequency-tunable superconducting qubits to execute single- and two-qubit gates while mitigating computational errors. When combined with a comprehensive model of physical errors across our processor, the strategy suppresses physical error rates by ~3.7× compared with the case of no optimization. Furthermore, it is projected to achieve a similar performance advantage on a distance-23 surface code logical qubit with 1057 physical qubits. Our control optimization strategy solves a generic scaling challenge in a way that can be adapted to a variety of quantum operations, algorithms, and computing architectures.

Abstract Clinical translation of AAV-mediated gene therapy requires preclinical development across different experimental models, often confounded by variable transduction efficiency. Here, we describe a human liver chimeric transgene-free Il2rg −/− /Rag2 −/− /Fah −/− /Aavr −/− (TIRFA) mouse model overcoming this translational roadblock, by combining liver humanization with AAV receptor (AAVR) ablation, rendering murine cells impermissive to AAV transduction. Using human liver chimeric TIRFA mice, we demonstrate increased transduction of clinically used AAV serotypes in primary human hepatocytes compared to humanized mice with wild-type AAVR. Further, we demonstrate AAV transduction in human teratoma-derived primary cells and liver cancer tissue, displaying the versatility of the humanized TIRFA mouse. From a mechanistic perspective, our results support the notion that AAVR functions as both an entry receptor and an intracellular receptor essential for transduction. The TIRFA mouse should allow prediction of AAV gene transfer efficiency and the study of AAV vector biology in a preclinical human setting.

Abstract In the context of continuous emergence of SARS-CoV-2 variants of concern (VOCs), one strategy to prevent the severe outcomes of COVID-19 is developing safe and effective broad-spectrum vaccines. Here, we present preclinical studies of a RBD vaccine derived from the Gamma SARS-CoV-2 variant adjuvanted with Alum. The Gamma-adapted RBD vaccine is more immunogenic than the Ancestral RBD vaccine in terms of inducing broader neutralizing antibodies. The Gamma RBD presents more immunogenic B-cell restricted epitopes and induces a higher proportion of specific-B cells and plasmablasts than the Ancestral RBD version. The Gamma-adapted vaccine induces antigen specific T cell immune responses and confers protection against Ancestral and Omicron BA.5 SARS-CoV-2 challenge in mice. Moreover, the Gamma RBD vaccine induces higher and broader neutralizing antibody activity than homologous booster vaccination in mice previously primed with different SARS-CoV-2 vaccine platforms. Our study indicates that the adjuvanted Gamma RBD vaccine is highly immunogenic and a broad-spectrum vaccine candidate.

Abstract Walking slowly after stroke reduces health and quality of life. This multi-site, prospective, interventional, 2-arm randomized controlled trial (NCT04121754) evaluated the safety and efficacy of an autonomous neurorehabilitation system (InTandemTM) designed to use auditory-motor entrainment to improve post-stroke walking. 87 individuals were randomized to 5-week walking interventions with InTandem or Active Control (i.e., walking without InTandem). The primary endpoints were change in walking speed, measured by the 10-meter walk test pre-vs-post each 5-week intervention, and safety, measured as the frequency of adverse events (AEs). Clinical responder rates were also compared. The trial met its primary endpoints. InTandem was associated with a 2x larger increase in speed (Δ: 0.14 ± 0.03 m/s versus Δ: 0.06 ± 0.02 m/s, F(1,49) = 6.58, p = 0.013), 3x more responders (40% versus 13%, χ2(1) ≥ 6.47, p = 0.01), and similar safety (both groups experienced the same number of AEs). The auditory-motor intervention autonomously delivered by InTandem is safe and effective in improving walking in the chronic phase of stroke.

Abstract The Escherichia coli TetR-related transcriptional regulator RutR is involved in the coordination of pyrimidine and purine metabolism. Here we report that lysine acetylation modulates RutR function. Applying the genetic code expansion concept, we produced site-specifically lysine-acetylated RutR proteins. The crystal structure of lysine-acetylated RutR reveals how acetylation switches off RutR-DNA-binding. We apply the genetic code expansion concept in E. coli in vivo revealing the consequences of RutR acetylation on the transcriptional level. We propose a model in which RutR acetylation follows different kinetic profiles either reacting non-enzymatically with acetyl-phosphate or enzymatically catalysed by the lysine acetyltransferases PatZ/YfiQ and YiaC. The NAD+-dependent sirtuin deacetylase CobB reverses enzymatic and non-enzymatic acetylation of RutR playing a dual regulatory and detoxifying role. By detecting cellular acetyl-CoA, NAD+ and acetyl-phosphate, bacteria apply lysine acetylation of transcriptional regulators to sense the cellular metabolic state directly adjusting gene expression to changing environmental conditions.

Abstract Near continuous phase transitions, universal power-law scaling, characterized by critical exponents, emerges. This behavior reflects the singular responses of physical systems to continuous control parameters like temperature or external fields. Universal scaling extends to non-equilibrium dynamics in isolated quantum systems after a quench, where time takes the role of the control parameter. Our research unveils critical scaling in time also during the relaxation dynamics of an open quantum system. Here we experimentally realize such a system by the spin of individual Cesium atoms dissipatively coupled through spin-exchange processes to a bath of ultracold Rubidium atoms. Through a finite-size scaling analysis of the entropy dynamics via numerical simulations, we identify a critical point in time in the thermodynamic limit. This critical point is accompanied by the divergence of a characteristic length, which is described by critical exponents that turn out to be unaffected by system specifics.

Millions of skeletal remains from South Asia were exported in red markets (the underground economy of human tissues/organs) to educational institutions globally for over a century. It is time to recognize the personhood of the people who were systematically made into anatomical objects and acknowledge the scientific racism in creating and continuing to use them.

Abstract The resilience of cellular proteostasis declines with age, which drives protein aggregation and compromises viability. The nucleus has emerged as a key quality control compartment that handles misfolded proteins produced by the cytosolic protein biosynthesis system. Here, we find that age-associated metabolic cues target the yeast protein disaggregase Hsp104 to the nucleus to maintain a functional nuclear proteome during quiescence. The switch to respiratory metabolism and the accompanying decrease in translation rates direct cytosolic Hsp104 to the nucleus to interact with latent translation initiation factor eIF2 and to suppress protein aggregation. Hindering Hsp104 from entering the nucleus in quiescent cells results in delayed re-entry into the cell cycle due to compromised resumption of protein synthesis. In sum, we report that cytosolic-nuclear partitioning of the Hsp104 disaggregase is a critical mechanism to protect the latent protein synthesis machinery during quiescence in yeast, ensuring the rapid restart of translation once nutrients are replenished.

Abstract Medulloblastomas with extensive nodularity are cerebellar tumors characterized by two distinct compartments and variable disease progression. The mechanisms governing the balance between proliferation and differentiation in MBEN remain poorly understood. Here, we employ a multi-modal single cell transcriptome analysis to dissect this process. In the internodular compartment, we identify proliferating cerebellar granular neuronal precursor-like malignant cells, along with stromal, vascular, and immune cells. In contrast, the nodular compartment comprises postmitotic, neuronally differentiated malignant cells. Both compartments are connected through an intermediate cell stage resembling actively migrating CGNPs. Notably, we also discover astrocytic-like malignant cells, found in proximity to migrating and differentiated cells at the transition zone between the two compartments. Our study sheds light on the spatial tissue organization and its link to the developmental trajectory, resulting in a more benign tumor phenotype. This integrative approach holds promise to explore intercompartmental interactions in other cancers with varying histology.

Abstract Biomolecular condensates serve as membrane-less compartments within cells, concentrating proteins and nucleic acids to facilitate precise spatial and temporal orchestration of various biological processes. The diversity of these processes and the substantial variability in condensate characteristics present a formidable challenge for quantifying their molecular dynamics, surpassing the capabilities of conventional microscopy. Here, we show that our single-photon microscope provides a comprehensive live-cell spectroscopy and imaging framework for investigating biomolecular condensation. Leveraging a single-photon detector array, single-photon microscopy enhances the potential of quantitative confocal microscopy by providing access to fluorescence signals at the single-photon level. Our platform incorporates photon spatiotemporal tagging, which allowed us to perform time-lapse super-resolved imaging for molecular sub-diffraction environment organization with simultaneous monitoring of molecular mobility, interactions, and nano-environment properties through fluorescence lifetime fluctuation spectroscopy. This integrated correlative study reveals the dynamics and interactions of RNA-binding proteins involved in forming stress granules, a specific type of biomolecular condensates, across a wide range of spatial and temporal scales. Our versatile framework opens up avenues for exploring a broad spectrum of biomolecular processes beyond the formation of membrane-less organelles.

Abstract Epigenetic dysregulation may influence disease progression. Here we explore whether epigenetic alterations in human pancreatic islets impact insulin secretion and type 2 diabetes (T2D). In islets, 5,584 DNA methylation sites exhibit alterations in T2D cases versus controls and are associated with HbA1c in individuals not diagnosed with T2D. T2D-associated methylation changes are found in enhancers and regions bound by β-cell-specific transcription factors and associated with reduced expression of e.g. CABLES1, FOXP1, GABRA2, GLR1A, RHOT1, and TBC1D4. We find RHOT1 (MIRO1) to be a key regulator of insulin secretion in human islets. Rhot1-deficiency in β-cells leads to reduced insulin secretion, ATP/ADP ratio, mitochondrial mass, Ca2+, and respiration. Regulators of mitochondrial dynamics and metabolites, including L-proline, glycine, GABA, and carnitines, are altered in Rhot1-deficient β-cells. Islets from diabetic GK rats present Rhot1-deficiency. Finally, RHOT1methylation in blood is associated with future T2D. Together, individuals with T2D exhibit epigenetic alterations linked to mitochondrial dysfunction in pancreatic islets.