The rockefeller university

The female Aedes aegypti mosquito's remarkable ability to hunt humans and transmit pathogens relies on her unique biology. Here, we present the Aedes aegypti Mosquito Cell Atlas, a comprehensive single-nucleus RNA sequencing dataset of more than 367,000 nuclei from 19 dissected tissues of adult female and male Aedes aegypti, providing cellular-level resolution of mosquito biology. We identify novel cell types and expand our understanding of sensory neuron organization of chemoreceptors across all sensory tissues. Our analysis uncovers male-specific cells and sexually dimorphic gene expression in the antenna and brain. In female mosquitoes, we find that glial cells, rather than neurons, undergo the most extensive transcriptional changes in the brain following blood feeding. Our findings provide insights into the cellular basis of mosquito behavior and sexual dimorphism. The Aedes aegypti Mosquito Cell Atlas resource enables systematic investigation of cell-type-specific expression across all mosquito tissues.

Cancer cells require substantial metabolic adaptations to metastasize to distant organs, but the metabolites essential for successful colonization remain poorly defined. In this study, we used a mitochondrial metabolomics approach to compare primary and metastatic breast cancer cells. This analysis revealed accumulation of mitochondrial glutathione (GSH) during lung metastasis, driven by elevated expression of SLC25A39, a mitochondrial GSH transporter. Loss of SLC25A39 impairs metastatic colonization in genetic screens, cell line models, and patient-derived xenografts, without affecting primary tumor growth. Mitochondrial GSH import is specifically required during early colonization and functions independently of its canonical antioxidant role. CRISPR activation screens identified ATF4, a stress-induced transcription factor, as a bypass mechanism that restores metastatic potential in SLC25A39-deficient cells. Mechanistically, SLC25A39 is required for optimal ATF4 activation during metastasis and under hypoxia, linking mitochondrial GSH availability to integrated stress response signaling. These findings identify mitochondrial GSH as a necessary and limiting metabolite for metastatic progression.Significance: Mitochondrial GSH import via SLC25A39 is essential for early metastatic colonization in breast cancer, linking metabolic adaptation to stress response signaling. Targeting this pathway may uncover a therapeutic vulnerability specific to metastasis without affecting primary tumor growth.

Replicative helicases need loader proteins to assemble at DNA replication origins. Multiple copies of the bacteriophage lambda P (P) loader bind and load the Escherichia coli DnaB (B) replicative helicase onto single-stranded (ss) DNA from the replication origin. We find that the E. coli DnaB center dot lambda P complex exists in two forms: B6P5 and B6P6. In the 2.66 & Aring; cryo-EM structure of B6P5, five lambda P loader copies form a crown-like shape that tightly grips DnaB. In this complex, the closed, planar DnaB is reconfigured into an open spiral with a large enough breach to allow ssDNA to enter an internal chamber. Transition to the open spiral involves lambda P-induced changes to the Docking Helix (DH)-Linker Helix (LH) interface. Unexpectedly, one lambda P chain in B6P5 is positioned across the breach. The disposition of this lambda P chain implies a complex pathway for entry of a replication-origin-derived ssDNA "bubble" ssDNA into the B6P5 complex. We propose that the B6P6 complex is an early intermediate in helicase activation in which neither DnaB nor lambda P has reached its final form. In this complex, DnaB adopts a partially open, ajar planar configuration. lambda P in B6P6 interacts more loosely with DnaB. The ssDNA- and ATP-binding sites in both complexes are not correctly configured for binding or hydrolysis. Our findings detail the distinct conformations of B6P6 and B6P5, allowing us to propose a structural model for the transition from an ajar planar to an open spiral configuration in the helicase loading pathway.

Nucleoside-modified messenger RNA (mRNA) vaccines elicit protective antibodies through their ability to promote T follicular helper (Tfh) cell differentiation. The lipid nanoparticles (LNPs) of mRNA vaccines possess inherent adjuvant activity. However, the extent to which the nucleoside-modified mRNA is sensed and contributes to Tfh cell responses remains undefined. Herein, we deconvolute the signals induced by LNPs and mRNA that instruct dendritic cells (DCs) to promote Tfh cell differentiation. We demonstrate that the mRNA drives the production of type I interferons, which act on DCs to enhance their maturation and Tfh cell differentiation, and favors plasma cells and memory B cell responses. In parallel, LNPs, which allow for mRNA uptake by DCs within the draining lymph node, also modulate Tfh cell responses by shaping the localization of CD25+ DCs. Our work unravels distinct adjuvant features of mRNA and LNPs necessary for the induction of Tfh cells, with implications for rational vaccine design.

Replicative senescence is a powerful tumor suppressor pathway that curbs proliferation of human cells when a few critically-short telomeres activate the DNA damage response (DDR). We show that ATM is the sole DDR kinase responsible for the induction and maintenance of replicative senescence and that ATM inhibition can induce normal cell divisions in senescent cells. Compared to non-physiological atmospheric (similar to 20%) oxygen, primary fibroblast cells grown at physiological (3%) oxygen were more tolerant to critically short telomeres, explaining their extended replicative lifespan. We show that this tolerance is due to attenuation of the ATM response to double-strand breaks (DSBs) and unprotected telomeres. Our data indicate that the reduced ATM response to DSBs at 3% oxygen is due to increased ROS, which induces disulfide crosslinked ATM dimers that do not respond to DSBs. This regulation of cellular lifespan through attenuation of ATM at physiological oxygen has implications for tumor suppression through telomere shortening.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein continues to evolve, facilitating antibody evasion. It remains unclear whether any conserved RBD epitopes persist across SARS-CoV-2 variants and whether vaccination and/or breakthrough infection (BTI) can elicit antibodies capable of targeting these conserved regions to counter future variants. Here, using a heterogeneous double-bait single B-cell sorting strategy, we identify a subset of antibodies with broad-spectrum RBD binding, including recognition of SARS-CoV-1 and emerging variants such as EG.5.1, BA.2.86, JN.1, and KP.2/3. These broadly binding antibodies (bbAbs) exhibit elevated levels of somatic hypermutation but are infrequently derived from clonally expanded B lymphocytes. Passive transfer of representative bbAbs reduces viral infection in a male hamster model. Structural analyses reveals that these bbAbs primarily target three distinct, highly conserved RBD epitopes, suggesting potential regions of future mutational pressure and highlighting the presence of conserved and immunogenic RBD conformations that may serve as a foundation for the development of broadly protective vaccines.

Membrane protein homo-oligomers named higher-order transient structures (HOTS) are formed through cohesive self-interactions in the range of a few kBT. The small magnitude of these interactions underlies the rapid reversibility of HOTS on the timescale of membrane signaling processes, permitting the dynamic modulation of signals. At the same time, weak interactions present an apparent paradox: HOTS should form only if the concentration of a particular protein is sufficiently high to produce oligomerization by mass action. And yet, HOTS are observed experimentally with membrane proteins present in cell membranes at concentrations of only a few per mu m2. In this study, we employ principles of statistical thermodynamics to explain how cells can alter the configurational entropy of the oligomerization reaction, thereby achieving HOTS formation at low concentrations of the protein in the membrane. We propose that this modification of the configurational entropy, a process we call configurational length scaling, is an important aspect of HOTS formation in cell membranes and possibly other cellular compartments.

Current methods for variant effect prediction do not differentiate between pathogenic variants resulting in different disease outcomes and are restricted in application due to a focus on variants with a single molecular consequence. We have developed Variant-to-Phenotype (V2P), a multi-task, multi-output machine learning model to predict variant pathogenicity conditioned on top-level Human Phenotype Ontology disease phenotypes (n = 23) for single nucleotide variants and insertions/deletions throughout the human genome. V2P leverages a unique approach for the modeling of variant effect that incorporates resultant disease phenotypes as output and during training to improve the quality of variant disease phenotype and effect predictions, simultaneously. We describe the architecture, training strategy, and biological features contributing to V2P's output, revealing initial characteristics underlying the relationship between disease genotype and phenotype. Moreover, we demonstrate the benefit of incorporating disease phenotypes for variant effect predictions by comparing V2P with several variant effect predictors across various high-quality evaluation datasets from manually curated databases and functional assays. Finally, we examine how V2P's predictions result in the successful identification of pathogenic variants in real and simulated patient sequencing data, outperforming other tested methods in initial comparisons. V2P offers a complete mapping of human genetic variants to disease-phenotypes, offering a uniquely conditioned set of variant effect characterizations.

Generation of in vitro human induced pluripotent cell (hiPSC)-derived skeletal muscle progenitor cells (SMPCs) holds great promise for regenerative medicine for skeletal muscle wasting diseases, for example Duchenne muscular dystrophy (DMD). While multiple approaches have been described to obtain SMPCs in vitro, hiPSC-derived SMPCs generated using transgene-free protocols are usually obtained in a low amount and resemble a more embryonal/fetal stage of differentiation. Here, we demonstrate that modulation of the JAK2/STAT3 signaling pathway during an in vitro skeletal muscle differentiation protocol increases the yield of PAX7+and CD54+human SMPCs (hSMPCs) and drives them to a higher maturation stage, in both human embryonic stem (ES) and patient-derived induced pluripotent cells (iPSCs). Importantly, the obtained SMPCs are able to differentiate into multinucleated myotubes in vitro and engraft in vivo. These findings reveal that modulation of the JAK2/STAT3 signaling pathway is a potential therapeutic avenue to generate SMPCs in vitro with potential for cell therapy approaches.