bluuebunny/crossref_metadata_2025_split huggingface dataset:
Oxidative stress response mediated by the yeast Rho5 GTPase depends on the proper spatiotemporal distribution of its dimeric GEF
Authors: Bischof Linnet , Heinisch Jürgen J. 
Published: August 2024

AbstractThe small GTPase Rho5 acts as a central hub to mediate the yeast’s response to adverse environmental conditions, including oxidative stress, with the concomitant induction of mitophagy and apoptosis. A proper cellular stress response has been correlated with the rapid translocation of the GTPase to the mitochondria, which depends on its activating dimeric GDP/GTP exchange factor (GEF). Here, the small ALFA tag was attached to Rho5 or the GEF subunits Dck1 and Lmo1 to efficiently trap the functional fusion proteins to specific cellular membranes, i.e. the plasma membrane, the mitochon-drial outer membrane, or the nuclear membrane,viafusions of integral membrane proteins residing in these compartments with an ALFA nanobody. The trapped components were subjected to life-cell fluorescence microscopy in combination with GFP fusions of the GTPase or its GEF subunits to investigate their interactionin vivo. We found that the dimeric GEF tends to auto-assemble and form stable dimers independent of its intracellular localization. On the other hand, GFP-Rho5 does not stably colocalize with the trapped GEF, attributed to its transient interaction. Phenotypic analyses of strains with the misslocalized proteins indicate that for a proper oxidative stress response Lmo1 needs to associate with the plasma membrane. In contrast, Rho5 only exerts its role at the mitochondrial surface when it is there in its active conformation. These data underline the importance of the proper spatio-temporal distribution of Rho5-GTP during oxidative stress response.
Human rod photoreceptor outer segments are supported by accessory inner segment structures
Authors: Lewis Tylor R. , Klementieva Natalia V. , Phan Sebastien , Castillo Carson M. , Kim Keun-Young , Cao Lauren Y. , Ellisman Mark H. , Arshavsky Vadim Y. , Alekseev Oleg 
Published: August 2024

AbstractThe first steps in vision take place in photoreceptor cells, which are highly compartmentalized neurons exhibiting significant structural variation across species. The light-sensitive ciliary compartment, called the outer segment, is located atop of the cell soma, called the inner segment. In this study, we present an ultrastructural analysis of human photoreceptors, which reveals that, in contrast to this classic arrangement, the inner segment of human rods extends alongside the outer segment to form a structure hereby termed the “accessory inner segment”. While reminiscent of the actin-based microvilli known as “calyceal processes” observed in other species, the accessory inner segment is a unique structure: (1) it contains an extensive microtubule-based cytoskeleton, (2) it extends far alongside the outer segment, (3) its diameter is comparable to that of the outer segment, (4) it contains numerous mitochondria, and (5) it forms electron-dense structures that likely mediate adhesion to the outer segment. Given that the spacing of extrafoveal human photoreceptors is more sparse than in non-primate species, with vast amounts of interphotoreceptor matrix present between cells, the closely apposed accessory inner segment likely provides structural support to the outer segment. This discovery expands our understanding of the human retina and directs future studies of human photoreceptor function in health and disease.
Auditory Cellular Cooperativity Probed Via Spontaneous Otoacoustic Emissions
Authors: Bergevin Christopher , Whiley Rebecca , Wit Hero , Manley Geoffrey , van Dijk Pim 
Published: August 2024

AbstractAs a sound pressure detector that uses energy to boost both its sensitivity and selectivity, the inner ear is an active non-equilibrium system. The collective processes of the inner ear giving rise to this exquisite functionality remain poorly understood. One manifestation of the active ear across the animal kingdom is the presence of spontaneous otoacoustic emission (SOAE), idiosyncratic arrays of spectral peaks that can be measured using a sensitive microphone in the ear canal.1 Current SOAE models attempt to explain how multiple peaks arise, and generally assume a spatially-distributed tonotopic system. However, the nature of the generators, their coupling, and the role of noise (e.g., Brownian motion) are hotly debated, especially given the inner ear morphological diversity across vertebrates. One means of probing these facets of emission generation is studying fluctuations in SOAE peak properties, which produce amplitude (AM) and frequency modulations (FM). These properties are likely related to the presence of noise affecting active cellular generation elements, and the coupling between generators. To better biophysically constrain models, this study characterizes the fluctuations in filtered SOAE peak waveforms, focusing on interrelations within and across peaks. A systematic approach is taken, examining three species that exhibit disparate inner ear morphologies: humans, barn owls, and green anole lizards. To varying degrees across all three groups, SOAE peaks have intra-(IrP) and interpeak (IPP) correlations indicative of interactions between generative elements. Activity from anole lizards, whose auditory sensory organ is relatively much smaller than that of humans or barn owls, showed a much higher incidence of IPP correlations. Taken together, we propose that these data are indicative of SOAE cellular generators acting cooperatively, allowing the ear to function as an optimized detector.Significance StatementThe inner ear is a complex biomechanical system whose function is not well understood. To further elucidate the role of coupling in emission generation, this study systematically compares fluctuations in sound emitted spontaneously from the ear (spontaneous otoacoustic emission, SOAE) across three vertebrates. Ultimately these data serve to illustrate that the inner ear is a non-equilibrium, active system whose cellular elements work cooperatively. A clearer understanding of SOAE generation and how it manifests across the animal kingdom will significantly advance our understanding of both normal and impaired auditory function.
Arousal threshold reveals novel neural markers of sleep depth independently from the conventional sleep stages
Authors: Picchioni Dante , Yang Fan Nils , de Zwart Jacco A. , Wang Yicun , Mandelkow Hendrik , Özbay Pinar S. , Chen Gang , Taylor Paul A. , Lam Niki , Chappel-Farley Miranda G. , Chang Catie , Liu Jiaen , van Gelderen Peter , Duyn Jeff H. 
Published: August 2024

AbstractReports of sleep-specific brain activity patterns have been constrained by assessing brain function as it related to the conventional polysomnographic sleep stages. This limits the variety of sleep states and underlying activity patterns that one can discover. The current study used all-night functional MRI sleep data and defined sleep behaviorally with auditory arousal threshold (AAT) to characterize sleep depth better by searching for novel neural markers of sleep depth that are neuroanatomically localized and temporally unrelated to the conventional stages.Functional correlation values calculated in a four-min time window immediately before the determination of AAT were entered into a linear mixed effects model, allowing multiple arousals across the night per subject into the analysis, and compared to models with sleep stage to determine the unique relationships with AAT. These unique relationships were for thalamocerebellar correlations, the relationship between the right language network and the right “default-mode network dorsal medial prefrontal cortex subsystem,” and the relationship between thalamus and ventral attention network. These novel neural markers of sleep depth would have remained undiscovered if the data were merely analyzed with the conventional sleep stages.Significance StatementThe original classification of sleep into distinct stages used behavioral characteristics.With the proliferation of new techniques, the first experiments might have been to perform correlations with arousal threshold. These experiments have never been performed, either with functional MRI or with any other modern technique. The amount of communication between brain regions as measured by all-night functional magnetic resonance imaging was correlated with arousal threshold. This revealed novel neural markers of sleep depth that would have remained undiscovered if the data were merely analyzed with the conventional stages. This expands our understanding of sleep and its functions beyond the constraints imposed by the conventional stages.
Drosophila melanogasterpigmentation demonstrates adaptive phenotypic parallelism but genomic unpredictability over multiple timescales
Authors: Berardi Skyler , Rhodes Jessica A. , Berner Mary Catherine , Greenblum Sharon I. , Bitter Mark C. , Behrman Emily L. , Betancourt Nicolas J. , Bergland Alan O. , Petrov Dmitri A. , Rajpurohit Subhash , Schmidt Paul 
Published: August 2024

AbstractPopulations are capable of responding to environmental change over ecological timescales via adaptive tracking. However, the translation from patterns of allele frequency change to rapid adaptation of complex traits remains unresolved. We used abdominal pigmentation inDrosophila melanogasteras a model phenotype to address the nature, genetic architecture, and repeatability of rapid adaptation in the field. We show thatD. melanogasterpigmentation evolves as a highly parallel and deterministic response to shared environmental gradients across latitude and season in natural North American populations. We then experimentally evolved replicate, genetically diverse fly populations in field mesocosms to remove any confounding effects of demography and/or cryptic structure that may drive patterns in wild populations; we show that pigmentation rapidly responds, in parallel, in fewer than ten generations. Thus, pigmentation evolves concordantly in response to spatial and temporal climatic gradients. We next examined whether phenotypic differentiation was associated with allele frequency change at loci with established links to genetic variance in pigmentation in natural populations. We found that across all spatial and temporal scales, phenotypic patterns were associated with variation at pigmentation-related loci, and the sets of genes we identified in each context were largely nonoverlapping. Therefore, our findings suggest that parallel phenotypic evolution is associated with an unpredictable genomic response, with distinct components of the polygenic architecture shifting across each environmental gradient to produce redundant adaptive patterns.Significance StatementShifts in global climate conditions have heightened our need to understand the dynamics and pace of adaptation in natural populations. In order to anticipate the population-level response to rapidly changing environmental conditions, we need to understand whether trait evolution is predictable over short timescales, and whether the genetic basis of adaptation is shared or distinct across multiple timescales. Here, we explored parallelism in the adaptive response of a complex phenotype,D. melanogasterpigmentation, to shared conditions that varied over multiple spatiotemporal scales. Our results demonstrate that while phenotypic adaptation proceeds as a predictable response to environmental gradients, even over short timescales, the genetic basis of the adaptive response is variable and nuanced across spatial and temporal contexts.
Synthetic mucus biomaterials synergize with antibiofilm agents to combatPseudomonas aeruginosabiofilms
Authors: Yang Sydney , Stern Alexa , Duncan Gregg 
Published: August 2024

AbstractBacterial biofilms are often highly resistant to antimicrobials causing persistent infections which when not effectively managed can significantly worsen clinical outcomes. As such, alternatives to standard antibiotic therapies have been highly sought after to address difficult-to-treat biofilm-associated infections. We hypothesized a biomaterial-based approach using the innate functions of mucins to modulate bacterial surface attachment and virulence could provide a new therapeutic strategy against biofilms. Based on our testing inPseudomonas aeruginosabiofilms, we found synthetic mucus biomaterials can inhibit biofilm formation and significantly reduce the thickness of mature biofilms. In addition, we evaluated if synthetic mucus biomaterials could work synergistically with DNase and/or α-amylase for enhanced biofilm dispersal. Combination treatment with these antibiofilm agents and synthetic mucus biomaterials resulted in up to 3 log reductions in viability of matureP. aeruginosabiofilms. Overall, this work provides a new bio-inspired, combinatorial approach to address biofilms and antibiotic-resistant bacterial infections.
Duplexed CeTEAM drug biosensors reveal determinants of PARP inhibitor selectivity
Authors: Pires Maria J. , Lovric Alen , Alam Seher , Fabbrizi Emanuele , Rotili Dante , Altun Mikael , Valerie Nicholas C.K. 
Published: August 2024

AbstractPARP inhibitors (PARPi) predominantly targeting PARP1 and PARP2 have revolutionized cancer therapy by selectively killing cancer cells with defective DNA repair. However, achieving PARP1 or PARP2-selective inhibitors is difficult due to their close structural homology. Selectivity profiling is typically done with purified proteins, but these lack the complexity of intracellular environments and could therefore be inaccurate. Here, we duplex PARP1 L713F-GFP and PARP2 L269A-mCherry CeTEAM drug biosensors to systematically characterize binding and cell cycle alterations of 27 PARPi at the single cell level. Our results reveal that most PARPi are generally equipotent for both PARPs, including the next-generation drug, senaparib. However, benzimidazole carboxamide (niraparib) derivatives demonstrated PARP1-selective tendencies, while pthalazinones (olaparib) favored PARP2. AZD5305, a reported PARP1-selective inhibitor with characteristics of both series, was the exception and appears ∼1600-fold more potent towards PARP1. In agreement with current understanding, we see that PARP trapping phenotypes positively correlate with PARP1/2 binding potency, while some potent binders, such as veliparib, did not – likely reflecting their allosteric influence on DNA retention. We also assessed the effect of the PARP1/2 active site component, HPF1, on intracellular PARPi binding and see that HPF1 depletion elicits slight deviations in apparent binding potency, while contributing additively to PARP-DNA trapping phenotypes. The PARP1/2 CeTEAM platform thus provides a structural roadmap for the development of selective PARPi and should facilitate the discovery of better targeted therapies. Furthermore, our results highlight that multiplexing CeTEAM biosensors and layered genetic perturbations can systematically profile determinants of intracellular drug selectivity.
Role of U11/U12 minor spliceosome geneZCRB1in Ciliogenesis and WNT Signaling
Authors: Powell-Rodgers Geralle , Pirzada Mujeeb Ur Rehman , Richee Jahmiera , Jungers Courtney F. , Colijn Sarah , Stratman Amber N. , Djuranovic Sergej 
Published: August 2024

ABSTRACTDespite the fact that 0.5% of human introns are processed by the U11/U12 minor spliceosome, the latter influences gene expression across multiple cellular processes. The ZCRB1 protein is a recently described core component of the U12 mono-snRNP minor spliceosome, but its functional significance to minor splicing, gene regulation, and biological signaling cascades is poorly understood. Using CRISPR-Cas9 and siRNA targeted knockout and knockdown strategies, we show that human cell lines with a partial reduction in ZCRB1 expression exhibit significant dysregulation of the splicing and expression of U12-type genes, primarily due to dysregulation of U12 mono-snRNA. RNA-Seq and targeted analyses of minor intron-containing genes indicate a downregulation in the expression of genes involved in ciliogenesis, and consequentially an upregulation in WNT signaling. Additionally,zcrb1CRISPR-Cas12a knockdown in zebrafish embryos led to gross developmental and body axis abnormalities, disrupted ciliogenesis, and upregulated WNT signaling, complementing our human cell studies. This work highlights a conserved and essential biological role of the minor spliceosome in general, and the ZCRB1 protein specifically in cellular and developmental processes across species, shedding light on the multifaceted relationship between splicing regulation, ciliogenesis, and WNT signaling.
G protein-Coupled Receptor Associated Sorting Protein GASP1 mediated trafficking of the Glucagon Like Peptide-1 Receptor contributes to the development of tolerance to incretin drugs
Authors: Gaur Anirudh , Keeshen Caroline M , King Madeline , Dirks Noah , Flisher Marcus , Cummings Bethany P , Huising Mark O , Whistler Jennifer L 
Published: August 2024

SummaryIncretin mimetic drugs are in widespread use for the treatment of type 2 diabetes and obesity and more recently have been prescribed for weight loss in otherwise healthy individuals. These drugs are all agonists of the glucagon-like peptide 1 receptor (GLP-1R) and function by supplementing effects produced by the endogenous hormone agonist glucagon-like peptide 1 (GLP-1). The therapeutic benefits of these medications, including improved glucose control and weight loss, require continued usage and wane with time. The molecular mechanisms underlying this loss of effect to incretin drugs remain unknown. Following activation by agonist and signaling to G protein, the GLP-1R engages arrestins and is endocytosed. Here we investigated the role of G protein-coupled receptor associated sorting protein 1 (GASP1), a critical regulator of the post-endocytic trafficking of GLP-1R, on tolerance to GLP-1R agonist drug. We found that tolerance to incretin drug was prevented at the cellular, tissue and whole animal level in mice with a selective disruption of the GASP1 protein in beta cells of the pancreatic islet. These studies implicate post-endocytic sorting of the GLP-1R in the loss of effectiveness of incretin therapeutics with prolonged use. These findings also suggest a novel strategy to prevent tolerance by biasing incretin drugs for G protein and away from arrestin engagement.
Upstream alternative polyadenylation inSCN5Aproduces a short transcript isoform encoding a mitochondria-localized NaV1.5 N-terminal fragment that influences cardiomyocyte respiration
Authors: Witmer Nathan H. , McLendon Jared M. , Stein Colleen S. , Yoon Jin-Young , Berezhnaya Elena , Elrod John W. , London Barry L. , Boudreau Ryan L. 
Published: August 2024

SUMMARYSCN5Aencodes the cardiac voltage-gated Na+ channel, NaV1.5, that initiates action potentials.SCN5Agene variants cause arrhythmias and increased heart failure risk. Mechanisms controlling NaV1.5 expression and activity are not fully understood. We recently found a well-conserved alternative polyadenylation (APA) signal downstream of the firstSCN5Acoding exon. This yields aSCN5A-shorttranscript isoform expressed in several species (e.g. human, pig, and cat), though rodents lack this upstream APA. Reanalysis of transcriptome-wide cardiac APA-seq and mRNA-seq data shows reductions in both upstream APA usage and short/full-lengthSCN5AmRNA ratios in failing hearts. Knock-in of the humanSCN5AAPA sequence into mice is sufficient to enable expression ofSCN5A-short transcript, while significantly decreasing expression of full-lengthSCN5AmRNA. Notably,SCN5A-short transcript encodes a novel protein (NaV1.5-NT), composed of an N-terminus identical to NaV1.5 and a unique C-terminus derived from intronic sequence. AAV9 constructs were able to achieve stable NaV1.5-NT expression in mouse hearts, and western blot of human heart tissues showed bands co-migrating with NaV1.5-NT transgene-derived bands. NaV1.5-NT is predicted to contain a mitochondrial targeting sequence and localizes to mitochondria in cultured cardiomyocytes and in mouse hearts. NaV1.5-NT expression in cardiomyocytes led to elevations in basal oxygen consumption rate, ATP production, and mitochondrial ROS, while depleting NADH supply. Native PAGE analyses of mitochondria lysates revealed that NaV1.5-NT expression resulted in increased levels of disassembled complex V subunits and accumulation of complex I-containing supercomplexes. Overall, we discovered that APA-mediated regulation ofSCN5Aproduces a short transcript encoding NaV1.5-NT. Our data support that NaV1.5-NT plays a multifaceted role in influencing mitochondrial physiology: 1) by increasing basal respiration likely through promoting complex V conformations that enhance proton leak, and 2) by increasing overall respiratory efficiency and NADH consumption by enhancing formation and/or stability of complex I-containing respiratory supercomplexes, though the specific molecular mechanisms underlying each of these remain unresolved.
Pleiotropic cellular responses underlying antibiotic tolerance inCampylobacter jejuni
Authors: Cho Eunshin , Kim Jinshil , Hur Jeong In , Ryu Sangryeol , Jeon Byeonghwa 
Published: August 2024

AbstractAntibiotic tolerance enables antibiotic-susceptible bacteria to withstand prolonged exposure to high concentrations of antibiotics. Although antibiotic tolerance presents a major challenge for public health, its underlying molecular mechanisms remain to be elucidated. Previously, we have demonstrated thatCampylobacter jejunidevelops tolerance to clinically important antibiotics, including ciprofloxacin and tetracycline. To identify cellular responses associated with antibiotic tolerance, we conducted RNA-sequencing analysis onC. jejunifollowing the induction of antibiotic tolerance through exposure to elevated levels of ciprofloxacin or tetracycline. Additionally, we constructed knockout mutants for genes that showed significant changes in expression levels during antibiotic tolerance. We observed a significant upregulation of genes involved in protein chaperones, bacterial motility, DNA repair system, drug efflux pump, and iron homeostasis during antibiotic tolerance. Furthermore, the viability of these knockout mutants was significantly reduced compared to the wild-type strain, indicating the critical role of these cellular responses in sustaining antibiotic tolerance. Notably, we also found that the protein chaperone mutants ΔdnaK, ΔclpB, and ΔgroESLexhibited increased protein aggregates under antibiotic treatment, suggesting protein chaperones play a critical role in managing protein aggregation and facilitating the survival ofC. jejuniduring antibiotic tolerance. In conclusion, our findings demonstrate that various cellular defense mechanisms collectively contribute to sustaining antibiotic tolerance inC. jejuni, providing novel insights into the molecular mechanisms of antibiotic tolerance that enableC. jejunito withstand the lethal effects of antibiotics.