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Genetic bottlenecks in the germline can amplify the impact of mutations, increasing the likelihood that new mutations are transmitted to multiple offspring. Here, we evaluate the timing and consequences of bottlenecks leading to maize pollen. By tracking transposon-induced mutations across tissues, we find that pollen derives from multiple early embryonic cell lineages, maintained by radial symmetries throughout development. In controlled crosses, offspring from bulk pollen or apical branches rarely share mutations, whereas lateral branches sample fewer lineages and produce offspring with a 6.2-fold increase in mutation sharing. Thus, the persistence of multiple, early-diverged cell lineages into the germline reduces mutation recurrence without altering the underlying mutation rate. Similar principles may apply in animals, where diverse mechanisms ensure that multiple cell lineages contribute to the germline.

Acquired resistance to osimertinib remains a major challenge in treating EGFR-mutant (EGFR+) Non-Small-Cell Lung Cancer (NSCLC). Although most patients initially respond to treatment, relapses are universal, even after prolonged remission during which tumor dormancy occurs. Here, we show that osimertinib induces and maintains senescence in EGFR+ NSCLC. Importantly, osimertinib does not kill senescent cells; however, following drug withdrawal, cells escape and resume proliferation. To examine the consequences of recurrent senescence and escape on resistance, we generated four isogenic cell lines clonally expanded through sequential cycles of Osimertinib-Induced Senescence (OsIS). Phylogenetic reconstruction based on de novo somatic variants revealed that these lines form four distinct evolutionary clades with varying degrees of osimertinib resistance. All had elevated tumor mutational burden with distinct single-nucleotide and copy-number variants, and without acquisition of tertiary EGFR mutations or MET amplification. Resistance was predominately associated with chromosomal instability characterized by extensive loss of heterozygosity, high copy-number alteration burden, and mutational signatures consistent with replication-associated DNA damage and repair. A second resistance genotype exhibited extreme focal amplifications with breakage-fusion-bridge-like genome remodeling. Despite profound genomic instability, targeting DNA repair or replication stress pathways was ineffective, whereas sensitivity to platinum-based chemotherapy was retained across clades. Collectively, these findings indicate that recurrent senescence escape drives osimertinib resistance through widespread genomic instability and is most effectively treated by cytotoxic strategies rather than pathway-targeted approaches.

Influenza D virus (IDV), primarily found in livestock species, has demonstrated cross-species transmission potential, yet its threat to humans remains poorly understood. Here, we curated a panel of IDV isolates collected during field surveillance from 2011 to 2020 from swine and cattle to assess their ability to infect human airway cells as a proxy for zoonotic threat assessment. Using lung epithelial cell lines, primary well-differentiated airway epithelial cultures, and precision-cut lung slices, we demonstrated that IDV efficiently propagates in cells and tissues from the human respiratory tract, reaching titers comparable to human influenza A virus (IAV). Infection kinetics in primary porcine airway cultures and respiratory tissues mirrored those from human, suggesting similar infectivity across species. To define host responses to IDV infection, we evaluated innate immune sensing and downstream interferon signaling in human respiratory cells. IDV infection resulted in markedly reduced activation of interferon regulatory factor (IRF) signaling and diminished induction of interferon lambda 1 and interferon-stimulated genes compared to IAV, indicating inefficient activation of innate immune sensing pathways. However, IDV replication was potently restricted in interferon-pretreated cells, demonstrating sensitivity to interferon-mediated antiviral effector mechanisms once an antiviral state was established. Together, these findings show that IDV can efficiently infect the human airway while limiting innate immune sensing, a feature that may facilitate zoonotic spillover. Our study highlights the need for enhanced surveillance of IDV at the animal-human interface and provides a foundation for further investigation into its biology and potential for causing human infection and disease.

RNA-binding proteins (RBPs) are critical regulators of the human transcriptome, but the binding patterns of most RBPs are insufficiently characterized. While sequence context facilitates RBP binding specificity, its precise contribution remains unclear. Existing computational methods to decipher RBP binding patterns are limited by their architecture-dependence, challenging interpretability, and, importantly, lack of focus on context. We present a novel comprehensive approach to address the aforementioned knowledge gaps. We first introduce a natural language-based representation to model RNA sequences using lexical, syntactic, and semantic forms, then devise a sequence decomposition method based on these structures to deconstruct RNA sequences into regions, each containing a target k-mer and its flanking contexts. We leverage this linguistic conceptualization to predict RBP binding under a Multiple Instance Learning (MIL) framework, which we solve using a novel method of significant region extraction termed "iterative relabeling". We demonstrate that our bottom-up approach discovers key regions contributing to RBP binding in an architecture-dependent, accurate, and interpretable manner.

Allosteric regulation enables fine-tuned control of enzyme activity in response to cellular signals, yet its molecular basis often remains unclear. Phosphofructokinase-1 (PFK), the rate-limiting enzyme of glycolysis, is a paradigmatic, well-conserved system whose reaction kinetics conform to the Monod-Wyman-Changeux model of allostery. However, X-ray crystal structures of bacterial PFK orthologs in distinct ligand-bound states do not show the consistent, concerted structural rearrangements expected for classical "relaxed" and "tense" states, revealing a decades-long disconnect between structure and function. We resolve this paradox by integrating biophysical and computational approaches to show that activator and inhibitor binding to the same allosteric pocket differentially reweight the conformational ensemble of Escherichia coli PFK. Activator binding stabilizes conformational substates that preorganize the catalytic site, whereas inhibitor binding upweights apo-like, catalytically incompetent substates. These findings establish an ensemble-based mechanism for PFK regulation and provide an energetic framework for understanding the expanded allosteric architecture of higher PFK orthologs.

Achieving tumor-specific delivery and sustained activation of both cytotoxic and immune-modulating agents remains a critical challenge in chemoimmunotherapy. Here, we present a bacterial platform engineered to combine enzyme/prodrug chemotherapy with immunotherapy, where tumor-homing E. coli Nissle 1917 expresses cytosine deaminase to convert the prodrug 5-fluorocytosine into the cytotoxic drug 5-fluorouracil within tumors. Concurrently, the engineered bacteria produce an IL-15 superagonist and a PD-L1 blocking nanobody to mitigate the immunosuppressive effects of tumor-localized chemotherapy. This platform demonstrated potent antitumor effects in the murine MC38 solid tumor model. Mechanistic studies showed that the combination therapy enhances activation of antigen-presenting cells, T cells and natural killer cells, while reducing immunosuppressive populations. In summary, our approach integrates enzyme/prodrug therapy and immunotherapy into a single bacterial delivery system, overcoming the limitations of conventional therapies and offering a scalable and precision-engineered strategy with an improved safety profile for synergistic cancer treatment.

Detecting a sex difference in response to a treatment or intervention, often reported as a 'sex-specific effect,' requires statistical comparison of the response across sex. Here, we investigated analytical approaches used to test for such effects in the behavioral and brain sciences. Of 200 recent articles containing terms such as 'sex-specific' or 'gender-dependent' in their titles, only 24% presented appropriate evidence supporting the claim: the effect was compared statistically across sex and results consistent with the claim were reported. In most articles (58%), no test was conducted that could have supported the title claim. Only 15% of studies on non-human animals supported the claim with appropriate evidence, which was significantly less frequently than studies on human participants (34%; p = 0.002). The use of appropriate analytical approaches was unrelated to journal rank or the citation impact of the article. We conclude that claims of sex/gender-dependent effects in the behavioral and brain sciences are only infrequently supported by appropriate evidence.

Niemann - Pick Disease Type C1 (NPC1) is a fatal, neurodegenerative disorder, characterized by lysosomal lipid accumulation and dysmyelination. Previous studies have documented some lipid abnormalities in the null mouse focused on the whole brain and liver. However, the specific lipidomic alterations in severely affected brain regions, such as cerebellum and isolated myelin remain understudied. We present a comprehensive LC - MS - based lipidomic analysis of the cerebellum and cortex of Npc1-/- mice during disease progression stages, along with the first comprehensive characterization of the myelin lipidome in NPC1 disease. Our results reveal that the cerebellum accumulates lipid species, including sphingolipids and glycerophospholipids progressively, while the cortex shows an overall decline in lipid levels, indicating region-specific lipid dysregulation. Notably, bis(monoacylglycero)phosphates and their precursors including lysophosphatidylglycerol and hemibismonoacylglycerophosphate exhibit significant accumulation, with a preference for docosahexaenoic acid (DHA) containing species. Despite known cholesterol storage defects in NPC1, we observed reduced free cholesterol levels in both regions, which we attribute to myelin loss. Myelin-specific lipidomics demonstrated extensive dysregulation, particularly in cortical myelin, including severe losses in sulfatides, ether-lipids, and acylcarnitine, alongside striking accumulation of hydroxy-ceramides. These findings identify novel lipid alterations in brain subregions and myelin, offering critical insight into the lipid perturbations under the loss of NPC1, and highlight lipid targets that may be crucial for therapeutic intervention and biomarker development.

The NS3 helicases from the Flaviviridae family of viruses exhibit nucleotide-hydrolysis-dependent, nucleic-acid-unwinding activity. The RNA unwinding activity for NS3 helicases from the Orthoflavivirus genus has not been fully explored and contrasts with NS3 helicase from Hepatitis C virus (HCV) of the Hepacivirus genus, which has thus far served as the prototypical model enzyme from this family of viruses. To begin to understand the functional differences between flavivirus NS3 helicases, we first developed an expression and purification system for full-length untagged NS3 protein from West Nile virus (WNV) and Zika virus (ZIKV). Both enzymes exhibit RNA-stimulated ATPase activity and are dependent on the nucleoside triphosphatase active site of the enzyme. Unlike HCV NS3, orthoflavivirus NS3s do not efficiently pre-assemble on a 3-ssRNA-tailed dsRNA substrate in the absence of ATP-Mg which is a prerequisite for formation of a productive HCV NS3-RNA complex that can exhibit a rapid burst of RNA unwinding. Instead, to observe RNA unwinding by WNV and ZIKV NS3s, low Mg-ATP concentrations are required at a time coincident when NS3 encounters the RNA substrate. In addition, we find that orthoflavivirus NS3s require translocation beyond the displaced strand to completely unwind a dsRNA substrate. Last, we find that orthoflavivirus NS5 stimulates the ability of NS3 to unwind dsRNA. These results suggest that functional differences exist between the flavivirus NS3 helicases and illuminate that orthoflavivirus NS3s require a functional interaction with the NS5 protein for coordination of its activity, as it is believed these two proteins constitute the viral replicase.