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YnaI is a member of the family of bacterial MscS (mechanosensitive channel of small conductance)-like channels. Channel gating upon hypoosmotic stress and the role of lipids in this process have been extensively studied for MscS, but are less well understood for YnaI, which features two additional transmembrane helices. Here, we combined cryogenic electron microscopy, molecular dynamics simulations and patch-clamp electrophysiology to advance our understanding of YnaI. The two additional helices move the lipid-filled hydrophobic pockets in YnaI further away from the lipid bilayer and change the function of the pocket lipids from being a critical gating element in MscS to being more of a structural element in YnaI. Unlike MscS, YnaI shows pronounced gating hysteresis and remains open to a substantially lower membrane tension than is needed to initially open the channel. Thus, at near-lytic membrane tension, both MscL and YnaI will open, but while MscL has a large pore and must close quickly to minimize loss of essential metabolites, YnaI only conducts ions and can thus remain open for longer to continue to facilitate pressure equilibration across the membrane.

The Nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain initiates mRNA capping in coronaviruses through a GDP-polyribonucleotidyltransferase reaction, with RNA covalently linked to nsp9. GDP is the preferred substrate for this reaction, but the NiRAN domain can also utilize GTP to produce an authentic 5 ' RNA cap structure, though the GTP-mediated mechanism is unclear. Yan and colleagues claimed to have delineated the reaction mechanism from the analysis of a cryoelectron microscopy (cryo-EM) structure of a trapped catalytic intermediate of the SARS-CoV-2 NiRAN domain with a beta-gamma-non-hydrolyzable GTP analog (GMPPNP) and RNA-nsp9 (PDB: 8GWE). We show that the cryo-EM data used to derive PDB: 8GWE do not support the presence of GMPPNP in the NiRAN active site, and the resulting atomic model is incompatible with fundamental chemical principles. We conclude that Yan and colleagues' conclusions are not experimentally supported and the mechanism for GTP-mediated RNA capping by the SARS-CoV-2 NiRAN domain remains unresolved. This Matters Arising paper is in response to Yan et al. (2022), published in Cell. See also the response by Huang et al. (2025), published in this issue.

Spatial refugia offered by structurally complex habitats mitigate high rates of predation for many aquatic and terrestrial prey species. These refuges are particularly important for small juvenile marine invertebrates, for which predation often represents the greatest cause of mortality. When the availability or quality of habitat landscapes and refugia are diminished by natural or anthropogenic forces, prey populations face further risk. In this study, we examined the utilization of alternative types of submerged aquatic vegetation (SAV) by juvenile bay scallops, Argopecten irradians, in a system where their historical habitat of eelgrass, Zostera marina, has largely disappeared. We found that scallops settled on and remained attached, above the bottom, to 9 species of macroalgae, 6 of which were fine filamentous or fleshy red algae. Macroalgae thus serve as suitable substrates for scallop larval settlement and early juvenile life, clearly important to successful population rebuilding that occurred following commencement of our restoration efforts. However, the much smaller maximum observed size (2-9 mm) and calculated duration of attachment (5-27 days) of scallops in the canopy of red macroalgae were considerably lower than those previously reported for eelgrass and the green macroalgae Codium fragile. With scallops dropping sooner to the bottom from red macroalgae, at smaller sizes, they are accessible to greater numbers of predator species/sizes and higher rates of predation (as shown in supporting laboratory experiments). Furthermore, this transition occurs well before scallops have undergone an ontogenetic shift to evasive swimming or have grown to reach a refuge in larger size. Fine filamentous red macroalgae, in which juvenile scallops demonstrated the highest frequency of attachment in this study and among the shortest duration in the canopy, now predominate in many areas of the Peconic Bays, New York, where eelgrass was formerly widespread. This apparent habitat degradation/replacement is thus acting to compress the length of time scallops are able to utilize a spatial refuge from predation at a critical life history stage, with potential cascading ontogenetic impacts on the use of a subsequent behavioral refuge and possible negative demographic consequences. Few prior studies have revealed such clear impacts of this kind resulting from habitat loss.

The Christchurch mutation (R136S) in the APOE3 (E3S/S) gene is associated with attenuated tau load and cognitive decline despite the presence of a causal PSEN1 mutation and high amyloid burden in the carrier. However, the molecular mechanisms enabling the E3S/S mutation to mitigate tau-induced neurodegeneration remain unclear. Here, we replaced mouse Apoe with wild-type human APOE3 or APOE3S/S on a tauopathy background. The R136S mutation decreased tau load and protected against tau-induced synaptic loss, myelin loss, and reduction in hippocampal theta and gamma power. Additionally, the R136S mutation reduced interferon responses to tau pathology in both mouse and human microglia, suppressing cGAS-STING pathway activation. Treating E3 tauopathy mice with a cGAS inhibitor protected against tau-induced synaptic loss and induced transcriptomic alterations similar to the R136S mutation across brain cell types. Thus, suppression of the microglial cGAS-STING-interferon (IFN) pathway plays a central role in mediating the protective effects of R136S against tauopathy.

The original 100.3 Mb reference genome for Caenorhabditis elegans, generated from the wild-type laboratory strain N2, has been crucial for analysis of C. elegans since 1998 and has been considered complete since 2005. Unexpectedly, this long-standing reference was shown to be incomplete in 2019 by a genome assembly from the N2-derived strain VC2010. Moreover, genetically divergent versions of N2 have arisen over decades of research and hindered reproducibility of C. elegans genetics and genomics. Here we provide a 106.4 Mb gap-free, telomere-to-telomere genome assembly of C. elegans, generated from CGC1, an isogenic derivative of the N2 strain. We use improved long-read sequencing and manual assembly of 43 recalcitrant genomic regions to overcome deficiencies of prior N2 and VC2010 assemblies and to assemble tandem repeat loci, including a 772 kb sequence for the 45S rRNA genes. Although many differences from earlier assemblies come from repeat regions, unique additions to the genome are also found. Of 19,972 protein-coding genes in the N2 assembly, 19,790 (99.1%) encode products that are unchanged in the CGC1 assembly. The CGC1 assembly also may encode 183 new protein-coding and 163 new ncRNA genes. CGC1 thus provides both a completely defined reference genome and corresponding isogenic wild-type strain for C. elegans, allowing unique opportunities for model and systems biology.

Background Recent advances in machine learning have revolutionized medical imaging. Currently, identifying brown adipose tissue (BAT) relies on manual identification and segmentation on Fluorine-(18) fluorodeoxyglucose positron emission tomography/computed tomography (F-18-FDG PET/CT) scans. However, the process is time-consuming, especially for studies involving a large number of cases, and is subject to bias due to observer dependency. The introduction of machine learning algorithms, such as the PET/CT algorithm implemented in the TriDFusion (3DF) Image Viewer, represents a significant advancement in BAT detection. In the context of cancer care, artificial intelligence (AI)-driven BAT detection holds immense promise for rapid and automatic differentiation between malignant lesions and non-malignant BAT confounds. By leveraging machine learning to discern intricate patterns in imaging data, this study aims to advance the automation of BAT recognition and provide precise quantitative assessment of radiographic features. Results We used a semi-automatic, threshold-based 3DF workflow to segment 317 PET/CT scans containing BAT. To minimize manual edits, we defined exclusion zones via machine-learning-based CT organ segmentation and used those organ masks to assign each volume of interest (VOI) to its anatomical site. Three physicians then reviewed and corrected all segmentations using the 3DF contour panel. The final, edited masks were used to train an nnU-Net V2 model, which we subsequently applied to 118 independent PET/CT scans. Across all anatomical sites, physicians reviewed the network's automated segmentations to be approximately 90% accurate. Conclusion Although nnU-Net V2 effectively identified BAT from PET/CT scans, training an AI model capable of perfect BAT segmentation remains a challenge due to factors such as PET/CT misregistration and the absence of visible BAT activity across contiguous slices.

Pregnancy brings about profound changes in the mammary gland to prepare for lactation, yet immunocyte changes that accompany this rapid remodeling are incompletely understood. We comprehensively analyzed mammary T cells, revealing a marked increase in CD4+ and CD8+ T effector cells, including an expansion of T cell receptor (TCR)alpha beta+CD8 alpha alpha+ cells, in pregnancy and lactation. T cells were localized in the mammary epithelium, resembling intraepithelial lymphocytes (IELs) typically found in mucosal tissues. Similarity to mucosal tissues was substantiated by demonstrating partial dependence on microbial cues, T cell migration from the intestine to the mammary gland in late pregnancy and shared TCR clonotypes between intestinal and mammary tissues, including intriguing public TCR families. Putative counterparts of mammary IELs were found in human breast and milk. Mammary IELs are thus poised to manage the transition from a nonmucosal tissue to a mucosal barrier during lactogenesis.

The Rev response element (RRE) forms an oligomeric complex with the viral protein Rev to facilitate the nuclear export of intron-retaining viral RNAs during the late phase of HIV-1 (human immunodeficiency virus type 1) infection. However, the structures and mechanisms underlying this process remain largely unknown. Here, we determined the crystal structure of the HIV-1 RRE stem-loop II (SLII), revealing a unique three-way junction architecture in which the base stem (IIa) bifurcates into the stem-loops (IIb and IIc) to compose Rev binding sites. The crystal structures of various SLII mutants demonstrated that while some mutants retain the same "compact" fold as the wild type, other single-nucleotide mutants induce drastic conformational changes, forming an "extended" SLII structure. Through in vitro Rev binding assays and Rev activity measurements in HIV-1-infected cells using structure-guided SLII mutants designed to favor specific conformers, we showed that while the compact fold represents a functional SLII, the alternative extended conformation inhibits Rev binding and oligomerization and consequently stimulates HIV-1 RNA nuclear export dysfunction. The propensity of SLII to adopt multiple conformations as captured in crystal structures and their influence on Rev oligomerization illuminate emerging perspectives on RRE structural plasticity-based regulation of HIV-1 nuclear export and provide opportunities for developing anti-HIV drugs targeting specific RRE conformations.

Mismatch repair deficient (MMRd) tumors harbor thousands of somatic mutations enriched for insertion-deletion (indels) conferring high sensitivity to immunotherapy. We sought to reproduce this phenotype using mutagenic agents to engineer an MMRd genotype in immunoresistant cells. The combination of temozolomide (TMZ) and cisplatin led to a rapid accumulation of a high mutational load enriched for indels in murine cell lines resulting from the epigenetic loss of Msh2. Pretreated cells showed sensitivity to PD-1 blockade. Systemic treatment with TMZ, cisplatin, and anti-PD-1 bearing immunoresistant tumor cells led to increased survival, intratumoral T cell infiltration, and downregulation of Msh2 expression without affecting healthy tissues. In a clinical trial with 18 patients with refractory mismatch repair proficient colorectal cancer, no responses were seen, but MMRd signatures emerged in cell-free DNA. These findings show that recapitulating an MMRd genotype through chemical mutagenesis can generate an immunogenic phenotype.