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Hypoxia-ischemia (HI), which disrupts the oxygen supply-demand balance in the brain by impairing blood oxygen supply and the cerebral metabolic rate of oxygen (CMRO₂), is a leading cause of neonatal brain injury. However, it is unclear how post-HI hypothermia helps to restore the balance, as cooling reduces CMRO₂. Also, how transient HI leads to secondary energy failure (SEF) in neonatal brains remains elusive. Using photoacoustic microscopy, we examined the effects of HI on CMRO₂ in awake 10-day-old mice, supplemented by bioenergetic analysis of purified cortical mitochondria. Our results show that while HI suppresses ipsilateral CMRO₂, it sparks a prolonged CMRO₂-surge post-HI, associated with increased mitochondrial oxygen consumption, superoxide emission, and reduced mitochondrial membrane potential necessary for ATP synthesis-indicating oxidative phosphorylation (OXPHOS) uncoupling. Post-HI hypothermia prevents the CMRO₂-surge by constraining oxygen extraction fraction, reduces mitochondrial oxidative stress, and maintains ATP and N-acetylaspartate levels, resulting in attenuated infarction at 24 hours post-HI. Our findings suggest that OXPHOS-uncoupling induced by the post-HI CMRO₂-surge underlies SEF and blocking the surge is a key mechanism of hypothermia protection. Also, our study highlights the potential of optical CMRO₂-measurements for detecting neonatal HI brain injury and guiding the titration of therapeutic hypothermia at the bedside.

Loss of _m Ca ²⁺ efflux capacity contributes to the pathogenesis and progression of Alzheimer's disease (AD) by promoting mitochondrial Ca ²⁺ ( _m Ca ²⁺ ) overload. Here, we utilized loss-of-function genetic mouse models to causally evaluate the role of _m Ca ²⁺ uptake by conditionally deleting the mitochondrial calcium uniporter channel (mtCU) in a robust mouse model of AD. Loss of neuronal _m Ca ²⁺ uptake reduced Aβ and tau-pathology, synaptic dysfunction, and cognitive decline in 3xTg-AD mice. Knockdown of Mcu in an in vitro model of AD significantly reduced matrix Ca ²⁺ content, redox imbalance, and mitochondrial dysfunction. The preservation of mitochondrial function rescued the AD-dependent decline in autophagic capacity and protected neurons against amyloidosis and cell death. This was corroborated by in vivo data showing improved mitochondrial structure and apposition in AD mice with loss of neuronal Mcu . These results suggest that inhibition of neuronal _m Ca ²⁺ uptake represents a powerful therapeutic target to impede AD progression.

Glioblastoma remains a deadly cancer driven in part by invasion of tumor cells into the brain. Transcriptomic analyses have identified distinct molecular subtypes, but mechanistic differences that account for clinical differences are not clear. Here, we show that, as predicted by the motor-clutch model of cell migration, mesenchymal glioma cells are more spread, generate larger traction forces, and migrate faster in brain tissue compared to proneural cells. Despite their rapid migration and comparable proliferation rates in vitro, mice with mesenchymal tumors survive longer than those with proneural tumors. This improved survival correlated with an immune response in the mesenchymal tumors, including T cell-mediated. Consistently, inducing mesenchymal tumors in immunodeficient mice resulted in shorter survival supporting a protective immune role in mesenchymal tumors. Thus, mesenchymal tumors have aggressive migration, but are immunologically 'hot' which suppresses net proliferation. These two features counteract each other and may explain the lack of a strong survival difference between subtypes clinically, while also opening up new opportunities for subtype-specific therapies.

Automated detection of complex animal behavior remains a challenge in neuroscience. Developments in computer vision have greatly advanced automated behavior detection and allow high-throughput preclinical and mechanistic studies. An integrated hardware and software solution is necessary to facilitate the adoption of these advances in the field of behavioral neurogenetics, particularly for non-computational laboratories. We have published a series of papers using an open field arena to annotate complex behaviors such as grooming, posture, and gait as well as higher-level constructs such as biological age and pain. Here, we present our, integrated rodent phenotyping platform, JAX Animal Behavior System (JABS), to the community for data acquisition, machine learning-based behavior annotation and classification, classifier sharing, and genetic analysis. The JABS Data Acquisition Module (JABS-DA) enables uniform data collection with its combination of 3D hardware designs and software for real-time monitoring and video data collection. JABS-Active Learning Module (JABS-AL) allows behavior annotation, classifier training, and validation. We introduce a novel graph-based framework (ethograph) that enables efficient boutwise comparison of JABS-AL classifiers. JABS-Analysis and Integration Module (JABS-AI), a web application, facilitates users to deploy and share any classifier that has been trained on JABS, reducing the effort required for behavior annotation. It supports the inference and sharing of the trained JABS classifiers and downstream genetic analyses (heritability and genetic correlation) on three curated datasets spanning 168 mouse strains that we are publicly releasing alongside this study. This enables the use of genetics as a guide to proper behavior classifier selection. This open-source tool is an ecosystem that allows the neuroscience and genetics community for shared advanced behavior analysis and reduces the barrier to entry into this new field.

Crossover recombination supports meiotic chromosome inheritance and fertility by establishing chiasmata between homologous chromosomes prior to the first meiotic division. In addition to the physical exchange of DNA mediated by meiotic recombination, chiasma formation also involves restructuring of the underlying chromosome axis, possibly to help with chiasma maturation or to resolve chromosomal interlocks. Here, we identify condensin as an important regulator of axis remodeling in S. cerevisiae. Condensin is recruited near sites of meiotic crossover designation by pro-crossover factors but is largely dispensable for DNA exchange. Instead, condensin helps to create discontinuities in the meiotic chromosome axis by promoting removal of cohesin. In addition, chromosomes of condensin mutants exhibit unusually common parallel chromatin clouds and experience a chromosomal buildup of the conserved axis remodeler Pch2. Consistent with an important role of axis restructuring at crossover sites, the canonical anaphase-bridge phenotype of condensin mutants is partly rescued by redirecting meiotic DNA repair to sister chromatids instead of homologous chromosomes, suggesting that crossover-associated axis reorganization is important for faithful meiotic chromosome segregation.

Ribosome profiling is a valuable methodology for measuring changes in a cell's translational program. The approach can report how efficiently mRNA coding sequences are translated and pinpoint positions along mRNAs where ribosomes slow down or arrest. It can also reveal when translation takes place outside coding regions, often with important regulatory consequences. While many useful software tools have emerged to facilitate analysis of these data, packages can become complex and challenging to adapt to specialized needs. We therefore introduce ribofootPrinter, a suite of Python tools designed to offer an accessible and modifiable set of code for analysis of data from ribosome profiling and related types of small RNA sequencing experiments. Alignments are made to a simplified transcriptome to keep the code intuitive and multiple normalization options help facilitate interpretation of meta analysis, particularly outside coding regions. We demonstrate how mapping of short reads to the transcriptome increases the frequency of matches to multiple sites and we provide multimapper identifier files to highlight these regions. Overall, this tool has the capability to carry out sophisticated analysis while maintaining enough simplicity to make it readily understandable and adaptable.

Most genome-wide association signals for complex disease reside in the noncoding genome, where defining function is nontrivial. MPRAs (massively parallel reporter assays) offer a scalable means to identify functional regulatory elements, but are typically conducted without regard to cell type, pairing cloned fragments with a generic housekeeping promoter. To explore the context-sensitivity of MPRAs, we screened enhancer activity across a panel of nearly 12,000 198-bp fragments spanning over 300 type 2 diabetes- and metabolic trait-associated regions in the 832/13 rat insulinoma beta cell line, a relevant model of pancreatic beta cells. We explored these fragments' context sensitivity by comparing their activities when placed up- or downstream of a reporter gene, and in combination with either a synthetic housekeeping promoter (SCP1) or a more biologically relevant promoter corresponding to the human insulin ( INS ) gene. We identified clear effects of MPRA construct design on enhancer activity. Specifically, a subset of fragments (n = 702/11,656) displayed positional bias, evenly distributed across up- and downstream preference. Promoter choice also influenced MPRA activity (n = 698/11,656), mostly biased towards the cell-specific INS promoter (73.4%). To identify sequence features associated with promoter preference, we used Lasso regression with 562 genomic annotations and discovered that fragments with INS promoter-biased activity are enriched for HNF1 motifs. HNF1 family transcription factors are key regulators of glucose metabolism disrupted in maturity onset diabetes of the young (MODY), suggesting genetic convergence between rare coding variants that cause MODY and common T2D-associated regulatory regions. We designed a follow-up MPRA containing HNF1 motif-enriched fragments and observed several instances where deletion or mutation of HNF1 motifs disrupted the INS promoter-biased enhancer activity, specifically in the beta cell model but not in a skeletal muscle cell line, another diabetes-relevant cell type. Together, our study suggests that cell-specific regulatory activity is partially influenced by enhancer-promoter compatibility and indicates that careful attention should be paid when designing MPRA libraries to capture context-specific regulatory processes at disease-associated genetic signals.

KRAS ^G12C inhibitors (G12Ci) have produced encouraging, albeit modest and transient, clinical benefit in pancreatic ductal adenocarcinoma (PDAC). Identifying and targeting resistance mechanisms to G12Ci treatment is therefore crucial. To better understand the function of KRAS ^G12C and possible G12Ci bypass mechanisms, we developed an autochthonous KRAS ^G12C - driven PDAC model. Compared to the classical KRAS ^G12D PDAC model, the G12C model exhibits slower tumor growth, yet similar histopathological and molecular features. Aligned with clinical experience, G12Ci treatment of KRAS ^G12C tumors produced modest impact despite stimulating a 'hot' tumor immune microenvironment. Immunoprofiling revealed that CD24, a 'don't eat me' signal, is significantly upregulated on cancer cells upon G12Ci treatment. Blocking CD24 enhanced macrophage phagocytosis of cancer cells and significantly sensitized tumors to G12Ci treatment. Similar findings were observed in KRAS ^G12D -driven PDAC. Together, this study reveals common and distinct oncogenic KRAS allele-specific biology and identifies a clinically actionable adaptive mechanism that may improve the efficacy of oncogenic KRAS inhibitor therapy in PDAC.

Significance: Generation of an autochthonous KRAS ^G12C -driven pancreatic cancer model enabled elucidation of specific effects of KRAS ^G12C during tumor development, revealing CD24 as an actionable adaptive mechanism in cancer cells induced upon KRAS ^G12C inhibition.

Touch plays a key role in our perception of our body and shapes our interactions with the world, from the objects we manipulate to the people we touch. While the tactile sensibility of the hand has been extensively characterized, much less is known about touch on other parts of the body. Despite the important role of the breast in lactation as well as in affective and sexual touch, relatively little is known about its sensory properties. To fill this gap, we investigated the spatial acuity of the breast and compared it to that of the hand and back, body regions that span the range of tactile spatial acuity. First, we found that the tactile acuity of the breast was even lower than that of the back, heretofore the paragon of poor acuity. Second, acuity was lower for larger breasts, consistent with the hypothesis that innervation capacity does not scale with body size. Third, touches to different regions of the nipple were largely indistinguishable, suggesting that the nipple is a sensory unit. Fourth, localization errors were systematically biased toward the nipple.