bluuebunny/crossref_metadata_2025_split huggingface dataset:
Xylazine potentiates the interoceptive effects of fentanyl in male and female rats
Authors: Bender Brooke N. , Carew Joseph M. , Bedard Madigan L. , McElligott Zoe A. , Besheer Joyce 
Published: February 2025

AbstractRationaleXylazine, a sedative typically used in veterinary medicine, has been increasingly detected as an adulterant in the unregulated opioid supply and present in opioid overdose deaths. Therefore, xylazine-adulterated fentanyl is a growing public health concern. People who use drugs have reported that xylazine changes and prolongs the effects of fentanylObjectivesWe used standard operant drug discrimination procedures to better understand how xylazine impacts the discriminative stimulus/interoceptive effects of fentanyl.MethodsMale and female Long-Evans rats (n=23) were trained to discriminate fentanyl (0.032 mg/kg intraperitoneal) such that one lever was reinforced with sucrose on days when fentanyl was administered, and the other lever was reinforced when vehicle was administered. Once rats met testing criteria, we tested a dose range of fentanyl to confirm discriminative stimulus control, then we tested if xylazine alone produced fentanyl-like effects and if the addition of xylazine to fentanyl impacted fentanyl interoceptive effects.ResultsStimulus control was confirmed, as rats showed increased percent responses on the fentanyl-appropriate lever as well as decreased response rates for increasing doses of fentanyl. Xylazine alone did not substitute for the stimulus effects of fentanyl but produced similar response rate reductions as fentanyl alone. Xylazine co-administered with fentanyl potentiated the stimulus effects of lower doses of fentanyl in both males and females and potentiated response rate reductions.ConclusionsThese results indicate that xylazine enhances the interoceptive effects of fentanyl, which may inform clinical research about xylazine-adulterated fentanyl.
A piezoelectric electroporator (Piezopen) for enhanced “naked” RNA vaccine delivery
Authors: Michalaki Eleftheria , Van Zanten Ananya , Najjar Jade , Byagathvalli Gaurav 
Published: February 2025

AbstractDespite the success of COVID-19 mRNA vaccines, they still face challenges with high costs, complex manufacturing, off-target biodistribution, and systemic reactogenicity stemming from their inflammatory carriers: lipid nanoparticles (LNPs). While “naked” RNA delivery could in principle solve these issues, studies have suggested that it is infeasible due to rapid degradation by RNases and poor cellular entry, thereby necessitating formulations that enhance intracellular delivery and RNA stability. Now, we challenge this paradigm by showing that a simple and inexpensive (<$1), lighter-derived electroporator with microneedle electrodes (Piezopen) can augment gene expression and immunogenicity to naked mRNA leading to comparable responses to LNPs at low doses. We achieve robust responses in the absence of systemic inflammation and reactogenicity using skin-targeted delivery, administer diverse construct types (i.e., mRNA, self-amplifying RNA (saRNA), circular RNA (circRNA)), and demonstrate cross-species validation in live human skin to derisk subsequent clinical application. Our results introduce Piezopen as an inexpensive, well-tolerated, and efficacious alternative to LNPs for mRNA vaccine delivery, designed to facilitate routine vaccinations and pandemic response.
Somatic mutations distinguish melanocyte subpopulations in human skin
Authors: Tandukar Bishal , Deivendran Delahny , Chen Limin , Bahrani Neda , Weier Beatrice , Sharma Harsh , Cruz-Pacheco Noel , Hu Min , Marks Kayla , Zitnay Rebecca G , Bandari Aravind K. , Nekoonam Rojina , Yeh Iwei , Judson-Torres Robert , Shain A. Hunter 
Published: February 2025

AbstractTo better understand the homeostatic mechanisms governing melanocytes, we performed deep phenotyping of clonal expansions of single melanocytes from human skin. In total, we interrogated the mutational landscapes, gene expression profiles, and morphological features of 297 melanocytes from 31 donors. To our surprise, a population of melanocytes with low mutation burden was maintained in sun damaged skin. These melanocytes were more stem-like, smaller, less dendritic and displayed distinct gene expression profiles compared to their counterparts with high mutation burdens. We used single-cell spatial transcriptomics (10X Xenium) to reveal the spatial distribution of melanocytes inferred to have low and high mutation burdens (LowMut and HighMut cells), based on their gene expression profiles. LowMut melanocytes were found in hair follicles as well as in the interfollicular epidermis, whereas HighMut melanocytes resided almost exclusively in the interfollicular epidermis. We propose that melanocytes in the hair follicle occupy a privileged niche, protected from UV radiation, but periodically migrate out of the hair follicle to replenish the interfollicular epidermis after waves of photodamage. More broadly, our study illustrates the advantages of a cell atlas that includes mutational information, as cells can change their cellular states and positional coordinates over time, but mutations are like scars, providing a historical record of the homeostatic processes that were operative on each cell.
Optogenetic interrogation of the lateral-line sensory system reveals mechanisms of pattern separation in the zebrafish brain
Authors: Velez-Angel Nicolas , Lu Sihao , Fabella Brian , Reagor Caleb C. , Brown Holland R. , Vázquez Yuriria , Jacobo Adrian , Hudspeth A. J. 
Published: February 2025

AbstractThe ability of animals to interact with their environment hinges on the brain’s capacity to distinguish between patterns of sensory information and accurately attribute them to specific sensory organs. The mechanisms by which neuronal circuits discriminate and encode the source of sensory signals remain elusive. To address this, we utilized as a model the posterior lateral line system of larval zebrafish, which is used to detect water currents. This system comprises a series of mechanosensory organs called neuromasts, which are innervated by neurons from the posterior lateral line ganglion. By combining single-neuromast optogenetic stimulation with whole-brain calcium imaging, we developed a novel approach to investigate how inputs from neuromasts are processed. Upon stimulating individual neuromasts, we observed that neurons in the brain of the zebrafish show diverse selectivity properties despite a lack of topographic organization in second-order circuits. We further demonstrated that complex combinations of neuromast stimulation are represented by sparse ensembles of neurons within the medial octavolateralis nucleus (MON) and found that neuromast input can be integrated nonlinearly. Our approach offers an innovative method for spatiotemporally interrogating the zebrafish lateral line system and presents a valuable model for studying whole-brain sensory encoding.
Hexokinase detachment from mitochondria drives the Warburg effect to support compartmentalized ATP production
Authors: Huggler Kimberly S. , Mellado Fritz Carlos A. , Flickinger Kyle M. , Chang Gavin R. , McGuire Meghan F. , Cantor Jason R. 
Published: February 2025

ABSTRACTHexokinase (HK) catalyzes the synthesis of glucose-6-phosphate, marking the first committed step of glucose metabolism. Most cancer cells express two homologous isoforms (HK1 and HK2) that can each bind to the outer mitochondrial membrane (OMM). CRISPR screens across hundreds of cancer cell lines indicate that both are dispensable for cell growth in traditional culture media. By contrast,HK2deletion impairs cell growth in Human Plasma-Like Medium (HPLM). Here, we find that HK2 is required to maintain sufficient cytosolic (OMM-detached) HK activity under conditions that enhance HK1 binding to the OMM. Notably, OMM-detached rather than OMM-docked HK promotes “aerobic glycolysis” (Warburg effect), an enigmatic phenotype displayed by most proliferating cells. We show that several proposed theories for this phenotype cannot explain theHK2dependence and instead find thatHK2deletion severely impairs glycolytic ATP production with little impact on total ATP yield for cells in HPLM. Our results reveal a basis for conditionalHK2essentiality and suggest that demand for compartmentalized ATP synthesis underlies the Warburg effect.
Overexpression of the NEK8 kinase inhibits homologous recombination
Authors: Turner Joshua L. , Moore Georgia , McCraw Tyler J. , Mason Jennifer M. 
Published: February 2025

ABSTRACTHomologous recombination maintains genome stability by repairing double strand breaks and protecting replication fork stability. Defects in homologous recombination results in cancer predisposition but can be exploited due to increased sensitivity to certain chemotherapeutics such as PARP inhibitors. The NEK8 kinase has roles in the replication response and homologous recombination. NEK8 is overexpressed in breast cancer, but the impact of NEK8 overexpression on homologous recombination has not been determined. Here, we demonstrate NEK8 overexpression inhibits RAD51 focus formation resulting in a defect in homologous recombination and degradation of stalled replication forks. Importantly, NEK8 overexpression sensitizes cells to the PARP inhibitor, Olaparib. Together, our results suggest NEK8 overexpressing tumors may be recombination-deficient and respond to chemotherapeutics that target defects in recombination such as Olaparib.
Sweeps in space: leveraging geographic data to identify beneficial alleles inAnopheles gambiae
Authors: Rehmann Clara T. , Small Scott T. , Ralph Peter L. , Kern Andrew D. 
Published: February 2025

AbstractAs organisms adapt to environmental changes, natural selection modifies the frequency of non-neutral alleles. For beneficial mutations, the outcome of this process may be a selective sweep, in which an allele rapidly increases in frequency and perhaps reaches fixation within a population. Selective sweeps have well-studied effects on patterns of local genetic variation in panmictic populations, but much less is known about the dynamics of sweeps in continuous space. In particular, because limited movement across a landscape leads to unique patterns of population structure, spatial dynamics may influence the trajectory of selected mutations. Here, we use forward-in-time, individual-based simulations in continuous space to study the impact of space on beneficial mutations as they sweep through a population. In particular, we show that selection changes the joint distribution of allele frequency and geographic range occupied by a focal allele and demonstrate that this signal can be used to identify selective sweeps. We then leverage this signal to identify in-progress selective sweeps within the malaria vectorAnopheles gambiae, a species under strong selection pressure from vector control measures. By considering space, we identify multiple previously undescribed variants with potential phenotypic consequences, including mutations im-pacting known IR-associated genes and altering protein structure and properties. Our results demonstrate a novel signal for detecting selection in spatial population genetic data that may have implications for genomic surveillance and understanding geographic patterns of genetic variation.
Curvature generation and engineering principles fromShewanella oneidensismulti-flagellin flagellum
Authors: Lou Qing , Fan Hongcheng , Liu Yang , Miller Jeff F. , Huang Yu , Zhou Z. Hong 
Published: February 2025

AbstractMotility driven by nanoscale flagella is vital to microbial survival and spread in fluid and structured environments. Absence of native flagellum structures, however, has limited our understanding of the mechanisms of microbial motility, hindering efforts to engineer microbe-based microbots for applications. Here, by cryogenic electron tomography (cryoET) and microscopy (cryoEM), we determined the structural basis of motility driven by the single flagellum anchored to one pole ofShewanella oneidensisMR-1 (S. oneidensis), an electrogenic bacterium commonly used in biotechnology. The structures of the curved flagellum, representing the conformation during motion, are captured, allowing delineation of molecular interactions among the subunits of its three components—filament, hook, and hook-filament junction. The structures of the filament, i.e., the propeller, reveal a varying composition of the flagellin isoforms FlaA and FlaB throughout the filament. Distinct inter-subunit interactions are identified at residues 129 and 134, which are the major determinants of functional differences in motility for the two isoforms. The hook—the universal joint—has a significantly larger curvature than that of the filament, despite both containing 11 curvature-defining conformers of their subunits. Transition between the propeller and universal joint is mediated by hook-filament junction, composed of 11 subunits of FlgK and FlgL, reconciling incompatibility between the filament and hook. Correlating these compositional and structural transitions with varying levels of curvature in flagellar segments reveals molecular mechanism enabling propulsive motility. Mechanistic understandings fromS. oneidensissuggest engineering principles for nanoscale biomimetic systems.Graphic abstract
Pneumolysin nanopores with 20 nm inner diameter to characterize the size and shape of Tau oligomers
Authors: Mukhopadhyay Anasua , Chanakul Wachara , Larpin Yu-Noel , Awasthi Saurabh , Protopopova Anna D. , Köffel René , Ianiro Alessandro , Mayer Michael 
Published: February 2025

AbstractProtein nanopores are emerging as versatile tools to fingerprint biomolecules due to their capability to characterize single molecules without the requirement for labeling. A long-standing challenge with biological nanopores is, however, that large biomolecules in their native state are often too large to enter these pores. Here, we report the self-assembly of approximately 35 ± 5 pneumolysin (PLY) toxins to a stable transmembrane pore with a diameter of 20 ± 3 nm, an effective length of 9.5 nm, and excellent low noise characteristics in the context of nanopore-based resistive pulse recordings. The exceptionally large pore diameter enables the characterization of the size and shape of individual proteins and protein complexes ranging in molecular weight from 50 kDa to 0.8 MDa. Moreover, PLY pores make it possible to follow the time course of the formation of oligomers of tau protein in solution by revealing the size, monomer number, approximate shape, and abundance of these oligomers. At least four characteristics make PLY pores well suited for the characterization of heterogeneous amyloid oligomer samples: First, they are not prone to clogging. Second, they provide label-free single particle analysis. Third, their large diameters make it possible to characterize a wide range of amyloid oligomer sizes with high resolution. And fourth, resistive pulse recordings from these pores provide stable open pore current baselines with low electrical noise.
A genetically-diverse mouse model reveals a complex gene-environment regulation of cognitive resilience and susceptibility to Alzheimer’s disease
Authors: Dunn Amy R. , Kaur Harpreet , Dai Miko , Charland Kevin , Ouellette Andrew R. , Hadad Niran , Doyle Patricia H. , Acosta Glen H.G. , Litkowski Elizabeth , Hohman Timothy J. , O’Connell Kristen M.S. , Kaczorowski Catherine C. 
Published: February 2025

AbstractAlzheimer’s disease (AD) has a complex etiology arising from largely unknown interactions between genetic and environmental factors. Even in populations with highly penetrant, disease-causing familial Alzheimer’s disease mutations, there is wide variation in disease onset and progression, suggesting that clinical symptoms are modified by genetics and environment. Identification of such modifiers is critical, as mechanisms that promote resilience to deleterious AD mutations, unhealthy diet, or aging represent promising therapeutic targets for AD and other causes of cognitive decline; global resilience factors that protect against multiple “hits” are among the highest priority for discovery. Both genetic and environmental protective factors in AD have been identified; however, interacting gene-environment (GxE) factors are incredibly difficult to study in human populations given complex genomes, poor self-reporting, data from underrepresented groups, and incompletely documented exposomes. Here, we used a population of mouse strains that model the polygenic nature of human AD to characterize individuals that display cognitive resilience to high-risk genetic and dietary perturbations to define and quantitate roles for genetics, sex, age, diet, and complex interactions that are nearly impossible to elucidate from humans or inbred AD mice. We found that some strains showed improved AD-related outcomes when fed a high-fat high-sugar (HFHS) diet, suggesting the need for personalized recommendations for dietary interventions in AD. We found a locus on Chr 10 that was associated with cognitive resilience to AD in females, and this association was strengthened by HFHS diet, directly pointing to an interaction between specific genetic and environmental factors in AD risk and resilience. In conclusion, this study is the first of its kind to explore characteristics of resilience and GxE interactions in a genetically diverse mouse model. We present a subset of strains that exemplify global cognitive resilience to be leveraged for deep mechanistic studies aimed toward development of resilience-based and personalized therapeutic interventions.HighlightsComprehensive phenotyping of genetically diverse mice (AD-BXDs) revealed cognitively resilient strains to extreme genetic and dietary risk factorsLocus on chromosome 10 harbors resilience factor/s that buffer the impact of familial AD mutations, female sex, and high-fat high-sugar dietHuman cognitive resilience genes are conserved in AD-BXDsAbout 10% of female AD-BXD mouse strains showed an improved AD-related cognitive outcome on a HFHS diet.
Functional relocation of the maize chloroplastatpBgene to the nucleus restores photosynthetic competence to a gene-edited non-photosynthetic mutant
Authors: Mangu Venkata RamanaRao , Jenkins Kelsey , Pratt Anna , Boltz Kara A , Pazouki Leila , Hui Alice Y. , Staub Jeffrey M. 
Published: February 2025

ABSTRACTWe present a novel approach to photosynthetic gene engineering in maize using a nuclear-encoded, chloroplast-targeted TALE-cytidine deaminase enzyme to create non-photosynthetic knockout mutants of the chloroplastrbcLgene. An off-target mutation in the adjacentatpBgene, encoding the β subunit of ATP synthase, was consistently found in all edited lines, identified as pigment-deficient in tissue culture. These double mutants, carrying mutations in both genes, were purified to homoplasmy using unique leaf-base regeneration techniques. To test mutation complementation and identify the causal gene, nuclear transgenic lines expressing chloroplast-targeted RbcL and AtpB proteins were generated. The results show that nuclear expression of AtpB restores chlorophyll accumulation and supports wild-type growth in tissue culture.
SARS-CoV-2 infection of the brain: the K18-hACE2 mouse model to illustrate the role and response of the vasculature in neurotropic viral infection
Authors: Neck Simon De , Penrice-Randal Rebekah , Hetzel Udo , Seehusen Frauke , Sharma Parul , Bentley Eleanor G. , Helminger Barbara , Kirby Adam , Balistreri Giuseppe , Stewart James P. , Kipar Anja 
Published: February 2025

AbstractSevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) primarily affects the respiratory tract and lungs; however, the associated disease, coronavirus disease 2019 (COVID-19) can also involve the central nervous system. The pathomechanisms underlying neurological impairments in COVID-19 and post COVID-19 condition remain incompletely understood, hence experimental studies under defined conditions, in a suitable animal model, can help to clarify the effect of SARS-CoV-2 infection in the brain. The K18-hACE2 mouse represents such a model, as intranasal challenge with SARS-CoV-2 VOC generally results in widespread neuronal infection with limited tissue response in the brain, consistent with a mild non-suppurative (meningo)encephalitis, with microgliosis, astrogliosis and (peri)vascular leukocyte infiltrates but no evidence of infection of vessel structures. The present study used this model to address the hypothesis that the vascular reaction is secondary to the neuronal infection. It confirmed that in intranasally challenged K18-hACE2 mice the virus targeted the neurons without overt neuropathic effect, whilst sparing the blood vessels, and further characterised the resulting inflammatory response in the brain in situ and through bulk RNA sequencing. The former highlighted the recruitment of leukocytes (neutrophils, monocytes/macrophages, and lymphocytes) via postcapillary venules, with their accumulation in the perivascular space and occasional emigration into the neuroparenchyma, without targeting and/or damage to the vessel wall. At the transcriptome level, this was accompanied by positive enrichment of pathways and the upregulation of genes involved in both the inflammatory reaction and the recruitment (including adhesion and migration) and activity of the leukocytes. We also found morphological and molecular evidence of subtle impairment of blood-brain barrier function, i.e. increased permeability. The results obtained from the model indicate that SARS-CoV-2 infection of the neurons induces neuroinflammation, with leukocyte recruitment and activation as well as blood-brain barrier dysfunction. These data can help to understand more fully the reaction of the CNS in COVID-19 patients, and neurotropic virus infections in general.
Acetylation of histone H2B on lysine 120 regulates BRD4 binding to intergenic enhancers
Authors: Hamilton Gregory A. , Ruiz Penelope D. , Ye Kenny , Gamble Matthew J. 
Published: February 2025

AbstractBRD4 is a bromodomain-containing transcriptional co-regulator that plays important roles in driving transcription by binding to histone acetyl-lysines at enhancers and promoters while recruiting additional transcriptional cofactors. While the mechanisms by which BRD4 regulates transcription have been explored, the critical acetylations primarily responsible for targeting it to chromatin remain unclear. Through a machine learning approach, we determined that distinct sets of histone acetylations dominate the prediction of chromatin accessibility and BRD4 binding in distinct chromatin contexts (e.g. intergenic enhancers, gene body enhancers and promoters). Using human fibroblasts engineered to predominantly express specific histones with lysine-to-arginine mutations, we demonstrate that one such acetylation, H2BK120ac, is required to recruit BRD4 specifically to intergenic enhancers, while not affecting chromatin accessibility. Loss of H2BK120ac did not affect BRD4 binding to either promoters or gene body enhancers, demonstrating that the rules governing BRD4 recruitment to regulatory regions depends on the specific genomic context. Highlighting the importance of H2BK120ac in directing BRD4 recruitment, we found that expression of the H2BK120R mutant significantly reduces the phenotypes driven by BRD4-NUT, an oncogenic fusion protein that drives NUT midline carcinoma. This work demonstrates the critical nature that genomic context plays in BRD4 recruitment to distinct classes of regulatory elements, and suggests that intergenic and gene body enhancers represent classes of functional distinct elements.