The rockefeller university

Cell movement and division are complex behaviors driven by a dynamic internal cytoskeleton. The molecular components and principles of cytoskeletal assembly are well studied, but less is known about cytoskeletal remodeling events, including how centrioles transition from ciliary base to centrosome. Here, we address this using the chytrid Rhizoclosmatium globosum, a zoosporic fungus that has centrioles and cilia, lost in most fungal lineages. Chytrids undergo reorganization of their microtubule cytoskeleton as they grow from zoospore to multinucleated coenocyte. We use evolutionary comparison, RNA-sequencing, and expansion microscopy to understand this reorganization and further develop this organism as a model for evolutionary cell biology. We find that when motile zoospores transition to sessile sporangia, cilia are retracted into the cytoplasm and degraded, while centrioles detach from the ciliary axoneme yet persist. During the mitotic cycles, short centrioles are associated with a centrosome-like microtubule-organizing center (MTOC) and a dense microtubule array at the spindle pole. After the mitotic cycles, centrioles elongate and form cilia, driven by transcription of genes associated with centriole maturation and ciliogenesis, and microtubule bundles are reorganized. Thus, in chytrids structural remodeling of the centriole is temporally coupled to specific changes in cytoskeletal organization over the coenocytic lifecycle.

Antiretroviral therapy suppresses HIV-1 infection but fails to eliminate a reservoir of intact latent proviruses that reside primarily in CD4+ T cells. The lack of precise understanding of the latent compartment has made it challenging to develop curative strategies for HIV-1 infection. Here we report on the properties of CD4+ T cell clones carrying intact latent proviruses, expanded in vitro from single cells obtained from the reservoir of people living with HIV-1. The latent proviruses in the clones were integrated into ZNF genes, nongenic satellite, and centromeric regions, frequently associated with latency. Despite their descent from single cells, only a fraction of the cells (0.4-14%) expressed relatively low levels of HIV-1 that did not measurably alter host gene transcriptome. Latency-reversing agents (LRAs) variably increased expression, but the effects were modest and clone and LRA specific. The results suggest that pharmacologic and immunologic approaches to clear the reservoir should be optimized to accommodate intra- and inter-clonal diversity.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) systems provide adaptive immunity against phage infection in prokaryotes using an RNA-guided complex that recognizes complementary foreign nucleic acids. Different types of CRISPR-Cas systems have been identified that differ in their mechanism of defense. Upon infection, type III CRISPR-Cas systems employ the Cas10 complex to find phage transcripts and synthesize cyclic oligo-adenylate (cOA) messengers. These ligands bind and activate CARF immune effectors that cause cell toxicity to prevent the completion of the viral lytic cycle. Here, we investigated two proteins containing an N-terminal haloacid dehalogenase (HAD) phosphatase domain followed by four predicted transmembrane helices and a C-terminal CARF domain. We named these proteins Chp for CRISPR-associated HAD phosphatase. We show that, in vivo, Chp localizes to the bacterial membrane and that its activation induces a growth arrest, leads to a depletion of ATP and IMP, and prevents phage propagation during the type III CRISPR-Cas response. In vitro, the CARF domain of Chp binds cyclic tetra-adenylates and the HAD phosphatase domain dephosphorylates dATP, ATP, and IMP. Our findings extend the range of molecular mechanisms employed by CARF effectors to defend prokaryotes against phage infection.

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.

BACKGROUND: The underlying mechanisms of atherosclerosis and strategies for identifying high cardiovascular risk in psoriasis are incompletely understood. Platelet activity is increased in psoriasis and induces vascular dysfunction. We investigated the platelet phenotype and platelet transcriptome as one potential mechanism to explain cardiovascular risk in psoriasis. METHODS: Psoriasis and controls underwent platelet aggregation and activation studies and platelet RNA sequencing to generate a psoriasis platelet transcriptomic score. The relationship between the platelet transcriptomic score and cardiovascular risk was assessed by arterial stiffness, coronary calcium, and longitudinally in an independent cohort of high cardiovascular-risk individuals undergoing lower extremity arterial revascularization. RESULTS: Psoriasis subjects (n=73; median age, 51 years; body surface area of psoriasis, 3%) compared with controls (n=56; median age, 41 years) trended older (P=0.08) and had greater body mass index (P=0.01) and higher hs-CRP (high-sensitivity C-reactive protein) values (P=0.01). Platelet aggregation in response to collagen (P=0.0049) and ADP (P=0.033), and leukocyte-, neutrophil-, and lymphocyte-platelet aggregates (P<0.05 for each comparison) were all higher in psoriasis versus controls. Platelet RNA sequencing comparing 51 patients with psoriasis with 39 controls identified 329 upregulated and 345 downregulated genes (P<0.05). Pathway analysis identified dysregulated platelet activation, apoptosis, VEGF (vascular endothelial growth factor), interferon, senescence, IL (interleukin)-1, and clotting cascade signaling between psoriasis and controls. Using a phenotypic rank-based scoring methodology, a psoriasis platelet transcriptomic score comprised of 142 genes differentiated psoriasis from controls. This score correlated with arterial stiffness (r=0.26; P=0.031) and coronary calcium (r=0.58; P=0.0069). In a separate cohort of high cardiovascular-risk patients undergoing lower extremity arterial revascularization, the psoriasis platelet transcriptomic score associated with incident myocardial infarction (adjusted hazard ratio, 3.7 [95% CI, 1.4-10.1]; P=0.015). CONCLUSIONS: Platelet aggregation and activation are increased in patients with controlled psoriatic disease, with the platelet transcriptome associated with proinflammatory, proatherothrombotic pathways, and cardiovascular risk. Our results warrant further investigation of platelet involvement promoting heightened cardiovascular disease in psoriasis.

Radioimmunotherapy using Ac-225, a highly cytotoxic alpha-particle emitter, has potential for treating advanced breast cancer, especially human epidermal growth factor receptor 2 (HER2)-positive cases. We use a pretargeted radioimmunotherapy (PRIT) approach consisting of a 3-step intravenous regimen (step 1: bispecific anti-HER2/anti-DOTA antibody; step 2: clearing agent; step 3: Ac-225-radiolabeled Proteus DOTA, or [Ac-225]Ac-Pr). Our goal was to establish curative Ac-225-PRIT with high therapeutic indices. Methods: The impact of [Ac-225]Ac-Pr specific activity was evaluated in the BT-474 breast xenograft model. We tested the effects of [Ac-225]Ac-Pr dosing during PRIT on tumor-targeting efficiency and tissue biodistribution. Using a Ac-225-PRIT regimen consisting of a ratio of 1.19 nmol of bispecific antibody to 0.60-0.66 nmol of [Ac-225]Ac-Pr, we evaluated therapy in the BT-474 model and a patient-derived xenograft model. BT-474-tumor-bearing mice were treated with 1 or 2 cycles of Ac-225-PRIT (37 kBq/cycle) separated by 1 wk. A dose escalation study was performed on the BT-474 model to establish an absorbed radiation dose of approximately 40 Gy (relative biological effectiveness [RBE], 5) as a nephrotoxic dose, as no such histologic findings were observed in prior studies at the 20.7-Gy (RBE, 5) renal dose level. Results: In the BT-474 model, 100% (20/20) achieved complete responses and histologic cure in 17 of 20 (85%) of the treated animals. One-cycle and 2-cycle treatments were equally effective. Treatments were well tolerated, with no chronic radiation toxicity documented during necropsy at 175 d. Dosimetry estimates (RBE, 5) per 37 kBq administered for tumors and kidneys were 210 and 3.5 Gy, respectively. In the patient-derived xenograft model, a single Ac-225-PRIT treatment led to 60% (3/5) complete response and prolonged survival (>93 d) versus no treatment (30 d; P = 0.0185). Lastly, a Ac-225-PRIT regimen was identified that induces severe chronic nephrotoxicity (41.4 Gy/592 kBq; RBE, 5). Conclusion: Safe and effective Ac-225-PRIT regimens were developed in 2 preclinical models of advanced HER2-positive human breast cancer with tumor cure without dose-limiting nephrotoxicity. This study establishes crucial preclinical dosimetry benchmarks for Ac-225-PRIT and provides a compelling rationale for its advancement into the clinic.

The synaptic vesicle glycoprotein 2A (SV2A), a member of the major facilitator superfamily (MFS), is a key target for antiseizure medications and a biomarker for synaptic density imaging. Despite its clinical importance, the mechanisms underlying SV2A ligand binding and modulation remain poorly understood. Here, we report sub-3 & Aring; resolution cryo-electron microscopy (cryo-EM) structures of human SV2A in its apo form and in complex with FDA-approved antiseizure medication levetiracetam; PET imaging tracer UCB-J; experimental antiseizure drug padsevonil; and allosteric modulator UCB1244283. We find that levetiracetam and UCB-J induce vestibule occlusion, a hallmark conformational transition of MFS transporters that had not been observed in previous SV2A structures. UCB1244283 binds to an allosteric site and enhances orthosteric ligand engagement by stabilizing the occluded state and slowing ligand dissociation. Notably, padsevonil occupies both orthosteric and allosteric sites, functionally precluding modulation. These findings uncover an allosteric mechanism of regulation and provide a structural framework for the development of modulators targeting SV2A and related MFS transporters.

The ability to precisely control gene expression using small-molecule drugs is a valuable tool in research and has important therapeutic potential. However, existing systems are often limited by the toxicity of the drugs and the need to alter gene sequences or endogenous regulatory elements. Here, we introduce Cyclone (acyclovir-controlled poison exon), an acyclovir-controlled poison exon cassette that can be used for small-molecule control of both transgene and endogenous gene expression. Cyclone is a portable 'intron-poison exon-intron' element that can be inserted into nearly any gene and is completely removed upon acyclovir treatment, leaving the native transcript intact. Cyclone offers tunable, reversible gene expression with nearly undetectable background and a similar to 295-fold activation. We also present Pac-Cyclone, a cassette that simplifies the generation of cell lines with acyclovir-controlled endogenous gene expression. Finally, we demonstrate the programmability of Cyclone, underscoring its potential for developing diverse genetic circuits controlled by various ligands.

Taylor's power law of fluctuation scaling has been well approximated empirically in many fields, including physics, meteorology, computer science, finance and ecology. In ecology, it describes well the abundances of many species, humans and non-humans. Taylor's law asserts that, in a collection of probability distributions, the variance of each distribution is directly proportional to a power of the mean of each distribution, exactly for population moments and, whether or not population moments exist, approximately for sample moments. Linear regression of log variance as a function of log mean is widely used to estimate the parameters of Taylor's power law. We provide large-sample asymptotics for this kind of inference under general conditions, and we derive confidence intervals for the parameters. In many ecological applications, the means and variances are estimated over time or across space from arrays of abundance data collected at different locations and time points. Our results depend on the asymptotic relation between the time-series length and the number of spatial points. When their ratio converges to a constant as both become large, the usual normalized statistics are asymptotically biased. We provide a bias correction to get correct confidence intervals. Taylor's law, widely studied in multiple sciences, is a source of challenging new statistical problems in a non-stationary spatio-temporal framework. We illustrate our results with both simulated and real data.