bluuebunny/crossref_metadata_2025_split huggingface dataset:
Forward genetic screen in zebrafish identifies new fungal regulators that limit host-protectiveCandida-innate immune interaction
Authors: Blair Bailey A. , Bragdon Emma , Dhillon Gursimran , Baker Nnamdi , Stasiak Lena , Muthig Mya , Miramon Pedro , Lorenz Michael C. , Wheeler Robert T. 
Published: February 2025

AbstractCandidais one of the most frequent causes of bloodstream infections, and our first line of defense against these invasive infections is the innate immune system. The early immune response is critical in controllingC. albicansinfection, butC. albicanshas several strategies to evade host immune attack. Phagocytosis ofC. albicansblocks hyphal growth, limiting host damage and virulence, but howC. albicanslimits early recruitment and phagocytosis in vertebrate infection is poorly understood. To study innate immune evasion by intravital imaging, we utilized the transparent larval zebrafish infection model to screen 131C. albicansmutants for altered virulence and phagocyte response. Infections with each of seven hypovirulent mutants led to altered phagocyte recruitment and/or phagocytosis, falling into four categories. Of particular interest among these isNMD5, a predicted β-importin and newly-identified virulence factor. Thenmd5Δ/Δ mutant fails to limit phagocytosis and its virulence defects are eliminated when phagocyte activity is compromised, suggesting that its role in virulence is limited to immune evasion. These quantitative intravital imaging experiments are the first to document alteredCandida-phagocyte interactions for several additional mutants, and clearly distinguish recruitment from phagocytic uptake, suggesting thatCandidamodulates both events. This initial large-scale screen of individualC. albicansmutants in a vertebrate, coupled with high-resolution imaging ofCandida-phagocyte interactions, provides a more nuanced view of how diverse mutations can lead to more effective phagocytosis, a key immune process which blocks germination and drives anti-fungal immunity.ImportanceCandida albicansis part of the human microbial community and is a dangerous opportunistic pathogen, able to prevent its elimination by the host immune system. AlthoughCandidaavoids immune attack through several strategies, we still understand little about how it regulates when immune phagocytes get recruited to the infection site and when they engulf fungal cells. We tested over 130 selectedCandidamutants for their ability to cause lethal infection and found several avirulent mutants which provoked altered innate immune responses, resulting in lower overall inflammation and greater host survival. Of particular interest isNMD5, which acts to limit fungal phagocytosis and is predicted to regulate the activity of stress-associated transcription factors. Our high-content screening was enabled by modelingCandidainfection in transparent vertebrate zebrafish larva. Our findings help us understand howCandidasurvives immune attack during commensal and pathogenic growth, and may eventually inform new strategies for controlling disease.
Analysis of crosslinking sites suggestsC. elegnasPIWI Argonaute exhibits flexible conformations for target recognition
Authors: Wu Wei-Sheng , Lee Dong-En , Chung Chi-Jung , Lu Shang-Yi , Brown Jordan S. , Zhang Donglei , Lee Heng-Chi 
Published: February 2025

AbstractSmall RNAs play critical roles in gene regulation in diverse processes across organisms. Crosslinking, ligation, and analyses of sequence hybrid (CLASH) experiments have shown PIWI and Argonaute proteins bind to diverse mRNA targets, raising questions about their functional relevance and the degree of flexibility in target recognition.As crosslinking-induced mutations (CIMs) provides nucleotide-resolution of RNA binding sites, we developed MUTACLASH to systematically analyze CIMs in piRNA and miRNA CLASH data inC. elegans. We found CIMs are enriched at the nucleotide positions of mRNA corresponding to the center of targeting piRNAs and miRNAs. Notably, CIMs are also enriched at nucleotides with local pairing mismatches to piRNA. In addition, distinct patterns of CIMs are observed between canonical and non-canonical base pairing interactions, suggesting that the worm PIWI Argonaute PRG-1 adopts distinct conformations for canonical vs. non-canonical interactions. Critically, non-canonical miRNA or piRNA binding sites with CIMs exhibit more regulatory effects than those without CIMs, demonstrating CIM analysis as a valuable approach in assessing functional significance of small RNA targeting sites in CLASH data. Together, our analyses reveal the landscapes of Argonaute crosslinking sites on mRNAs and highlight MUTACLASH as an advanced tool in analyzing CLASH data.
Divergent modulation of dopaminergic neurons by hypocretin/orexin receptors-1 and-2 shapes dopaminergic cell activity and socio-emotional behavior
Authors: Tzanoulinou Stamatina , Astori Simone , Grandi Laura Clara , Gullo Francesca , Kalusivikako Richie , Rai Simran , Tafti Mehdi , Becchetti Andrea , Vassalli Anne 
Published: February 2025

ABSTRACTMany neuropsychiatric disorders involve dysregulation of the dopaminergic (DA) input to the forebrain. Of particular relevance are DA projections originating from the midbrain ventral tegmental area (VTA). A key neuromodulatory influence onto DAVTAneurons arises from the dense axonal projections emanating from lateral hypothalamic area hypocretin/orexin (OX) neurons. Despite being a major input, the differential action of orexin peptides A and B (OXA and OXB) on orexin receptors 1 and 2 (OX1R and OX2R) in DA cells is poorly characterized. We thus genetically engineered mice whose DA neurons are selectively unresponsive to OX input via OX1R (DAOx1R-KOmice) or OX2R (DAOx2R-KOmice) and compared their behavior and DA cell electrophysiology to genetic controls. We previously showed a profound functional divergence between OX1R-and OX2R-mediated modulation of DA neurons in controlling vigilance states, brain oscillations and cognitive behavior. Inactivation of OX2R, but not OX1R, in DA neurons dramatically increased time spent in EEG theta-rich wakefulness, improved reward-driven learning and attentional skills, while it impaired inhibitory control. Here, we interrogate DAOx1R-KOand DAOx2R-KOmice in further behavioral domains. We show that mice with DA-specific OX1R loss exhibit hyperactivity, or anxiety-like responding, in context-dependent manners. OX2R loss in contrast decreases sociability and aversion-driven learning. We next investigate the underlying electrophysiological substrates and uncover previously unrecognized effects of OX peptides on DAVTAcell responses. In WT and control mice, we show that while OXA enhances, OXB diminishes DAVTAneuronal excitability. OX1R-deficient DA cells lose OXA responding and OX2R-deficient DA cells lose OXB responding. We altogether evidence strikingly distinct functions of OX1vs OX2R signaling in modulating the intrinsic excitability of DAVTAneurons and influencing DA-related behaviors. Our data position OX→DA neurotransmission via OX1or OX2R as relevant to endophenotypes observed in the context of disorders such as obsessive-compulsive, attention-deficit/hyperactivity, and autism spectrum disorders.
Peroxisomal Dysfunction Drives Loss of Dynamic Nutrient Responses to Initiate Metabolic Inflexibility During Aging
Authors: Sharma Arpit , Mistry Meeta , Yao Pallas , Liang Yanshan , Hui Sheng , Mair William B. 
Published: February 2025

ABSTRACTAging results in a loss of metabolic flexibility during fasting, characterized by the inability to acutely switch between metabolic substrates and reduced lipid mobilization. However, the drivers of these effects intracellularly remain unclear. Here, inCaenorhabditis elegans, we show that loss of coordinated inter-organelle dynamics causally initiates metabolic inflexibility with age. In young animals, peroxisomes emerge as the priming orchestrators of the fasting response, simultaneously governing lipid droplets (LDs) utilization and mitochondrial bioenergetics. With age, peroxisomal priming is lost, leading to mitochondrial fragmentation and impaired dynamic nutrient responses during fasting. Notably, dietary restriction (DR) exerts a rejuvenating effect on peroxisomal function, thereby preserving mitochondrial integrity and promoting longevity. Our study uncovers the expansive network of organelles enabling lipid mobilization during youth, providing critical context to the poorly understood role of peroxisomes in actively maintaining organelle homeodynamics and metabolic flexibility throughout the aging process.
Transformation ofGardnerella vaginaliswith aBifidobacterium-Escherichia colishuttle vector plasmid
Authors: Kularatne B. M. D. N. , Hill Janet E. 
Published: February 2025

AbstractGardnerellaspp. significantly influence female reproductive health, and are indicators of bacterial vaginosis, a common gynecological disorder. Lack of genetic tools forGardnerellaspp. is a hindrance in fully understanding their role in the vaginal microbiome and no naturally occurring plasmids have yet been identified in these organisms. The aim of this study was to transformGardnerella vaginalisand characterize transformants carryingBifidobacterium-E. colishuttle vector pKO403-lacZ′-Sp.G. vaginalisATCC 49145 was selected for protocol development based on its high growth rate, lack of restriction activity and susceptibility to spectinomycin. Low efficiency (∼102cfu/µg of plasmid DNA) but reproducible transformation was achieved. The expression of the spectinomycin resistance gene and the β-galactosidase gene of pKO403-lacZ′-Sp inG. vaginalisATCC 49145 resulted in an increase in spectinomycin tolerance from 2 µg/ml (MIC) to >512 µg/ml, and an appreciable increase in β-galactosidase activity compared to the wild type. Plasmid copy number was determined to be ∼3 per genome copy. Plasmid was lost rapidly in the absence of spectinomycin selection, with only ∼13% of colony forming units retaining the resistant phenotype after 24 h of growth without selection. These results demonstrate thatG. vaginaliscan be transformed by electroporation and that pKO403-lacZ′-Sp can be maintained and its genes expressed in this host, offering a starting point for the development of genetic tools for mechanistic studies of this important member of the vaginal microbiome.ImportanceThe healthy human vaginal microbiome is mainly dominated byLactobacillusspp. An imbalance or shift in this population can lead to a gynecological disorder known as bacterial vaginosis (BV). In BV, there is a reduction inLactobacillusspp. and overgrowth of mixed anaerobes and facultative bacteria includingGardnerellaspp. The reason for this increase in theGardnerellapopulation and associated changes in the vaginal microbiota composition is yet not understood, and a lack of genetic tools is one of the major barriers to performing mechanistic research to study the biology of these clinically significant organisms. A first step in developing genetic tools is introducing foreign DNA. In this study, we have developed a protocol for transformation and identified a plasmid that can be maintained inG. vaginalis.
Coro1a promotes the multivesicular body and plasma membrane fusion by facilitating PKM2-mediated SNAP-23 phosphorylation
Authors: Cai Zhenzhai , Li Zhijie , Yan Tingting , Fu Beilei , He Xinyu , Li Nannan , Wang Chenxing , Chen Yujiao , Zhang Dingli , Cai Zhijian , Cai Jing , Zhu Weiguo , Wu Hao 
Published: February 2025

AbstractDuring the formation of endosomal pathway-dependent extracellular vesicles (EVs), intraluminal vesicles in multivesicular bodies (MVBs) fuse with the plasma membrane (PM), releasing intraluminal vesicles to produce exosomes. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, membrane fusion effector, is essential in this process. However, which SNARE complex is involved in MVB and PM fusion and how its assembly is regulated remain elusive. We have demonstrated that neddylation of Coro1a inhibits EV secretion by inducing Rab7-mediated degradation of MVBs. Here, we find that Coro1a increases EV biogenesis by promoting the assembly of SNARE complex STX-12-SNAP-23-VAMP-7 in a neddylation-independent manner. Coro1a activates PKM2 to enhance SNAP-23 phosphorylation in the presence of neddylation inhibitor MLN4924, which drives SNAP-23 to recruit more STX-12 and VAMP7, leading to increased formation of the STX-12-SNAP-23-VAMP-7 complex. Correspondingly, Coro1a-induced EV biogenesis is eliminated by PKM2 inhibitor or SNAP-23 silencing independent of neddylation. Reduced EVs from tumors due to Coro1a knockout cannot be observed in MLN4924-treated tumor mice with PKM2 inhibitor treatment either. Furthermore, Coro1a increases in tumor tissues from lung tumor patients with tumor progression, and high Coro1a indicates unfavorable survival of tumor patients, suggesting that increased Coro1a-mediated enhanced EV production from tumors accelerates tumor progression. Altogether, our data demonstrate that Coro1a facilitates EV biogenesis by promoting the assembly of STX-12-SNAP-23-VAMP-7 complex during MVB and PM fusion independent of neddylation, thereby being involved in the progression of EV-related diseases.
A General Framework for Branch Length Estimation in Ancestral Recombination Graphs
Authors: Deng Yun , Song Yun S. , Nielsen Rasmus 
Published: February 2025

AbstractInference of Ancestral Recombination Graphs (ARGs) is of central interest in the analysis of genomic variation. ARGs can be specified in terms of topologies and coalescence times. The coalescence times are usually estimated using an informative prior derived from coalescent theory, but this may generate biased estimates and can also complicate downstream inferences based on ARGs. Here we introduce, POLEGON, a novel approach for estimating branch lengths for ARGs which uses an uninformative prior. Using extensive simulations, we show that this method provides improved estimates of coalescence times and lead to more accurate inferences of effective population sizes under a wide range of demographic assumptions. It also improves other downstream inferences including estimates of mutation rates. We apply the method to data from the 1000 Genomes Project to investigate population size histories and differential mutation signatures across populations. We also estimate coalescence times in the HLA region, and show that they exceed 30 million years in multiple segments.
Identification of PKN2 and MOB4 as Coordinators of Collective Cell Migration
Authors: Fokin Artem I. , Lin Yueying , Guschin Dmitry Y. , Chen Hsiang-Ying , James John , Yan Jie , Silberzan Pascal , Gautreau Alexis M. 
Published: February 2025

ABSTRACTIn animals, collective cell migration is critical during development and adult life for repairing organs. It remains, however, poorly understood compared with single cell migration. Polymerization of branched actin by the RAC1-WAVE-Arp2/3 pathway is well established to power membrane protrusions at the front of migrating cells, but also to maintain cell junctions in epithelial monolayers. Here we performed a screen for novel regulators of collective cell migration by identifying genes associated with the RAC1-WAVE-Arp2/3 pathway at cell junctions using dependency maps and by inactivating these candidates using CRISPR/Cas9. In wound healing, MCF10A epithelial cells collectively migrate towards the free space in a coordinated manner. PKN2 knockout (KO) cells display decreased collective migration due to destabilization of adherens junctions, whereas MOB4 KO cells display increased collective migration with a swirling behavior. Upon wound healing, PKN2 relocalizes to lateral junctions and maintains coordinated migration in the monolayer, whereas MOB4 relocalizes to the front edge of cells migrating towards the wound. The role of MOB4 in controlling collective migration requires YAP1, since MOB4 KO cells fail to activate YAP1 and their phenotype is rescued by constitutively active YAP1. Together, our data reveal two complementary activities required for coordinating cells in collective migration.
Nested parasitism in hypersaline environments: viruses and virus satellites of haloarchaea and their nanosized cellular symbionts
Authors: Zhou Yifan , Gutiérrez-Preciado Ana , Liu Ying , Moreira David , Yakimov Michail M. , López-García Purificación , Krupovic Mart 
Published: February 2025

AbstractIt is increasingly recognized that hyperparasitism, whereby a parasite exploits a host which itself is a parasite, is a common phenomenon across ecosystems and domains of life. Here, to explore hyperparasitism in Archaea, we focused on ultra-small archaea of the phylum Nanohaloarchaeota, a distinct lineage within the DPANN superphylum, which establish obligate symbiotic interactions with extreme halophiles of the class Halobacteria. We assembled five metagenomes originating from geothermally influenced salt lakes in the Danakil Depression, Ethiopia, and reconstructed the viromes associated with both haloarchaea and nanohaloarchaea. Both archaeal lineages were associated with viruses from four distinct groups, including head-tailed viruses (classCaudoviricetes), tailless icosahedral viruses, pleomorphic viruses and spindle-shaped viruses, which represent 12 previously undescribed families. The haloarchaeal viruses (HVs) and nanohaloarchaeal viruses (NHVs) are only distantly related, suggesting that viruses from the four groups co-evolved with their respective hosts for an extended period, likely since the divergence of the two archaeal lineages from their last common ancestor. Consistently, our results show that HVs and NHVs are well-adapted to replicate in their respective hosts and to thrive in hypersaline environments. No evidence of host switching between haloarchaea and nanohaloarchaea was obtained, but multiple horizontal transfers of genes implicated in virion structure and morphogenesis between HVs and NHVs were detected. We also identified several NHVs-encoded auxiliary metabolic genes implicated in nucleotide and amino acid metabolisms, which could enhance the metabolic capabilities of the nanohaloarchaeal hosts that have highly reduced genomes. Finally, in addition to HVs and NHVs, we describe plasmid-derived virus satellites that appear to have originated convergently to parasitize spindle-shaped viruses of both haloarchaea and nanohaloarchaea, uncovering an additional layer of parasitism. Collectively, our findings fill the knowledge gap on the diversity of HVs and NHVs, highlight the complexity of virus-host and virus-virus interactions in hypersaline environments, and open doors for further mining of the virosphere of the globally distributed DPANN archaea.
Simultaneous in vitro expression of minimal 21 transfer RNAs by tRNA array method
Authors: Miyachi Ryota , Shimizu Yoshihiro , Ichihashi Norikazu 
Published: February 2025

AbstractTransfer RNA (tRNA) plays a central role in translation. The simultaneousin vitrosynthesis of minimal yet sufficient tRNA species (at least 21) poses a challenge for constructing a self-reproducible artificial cell. A key obstacle is the processing of the 5’ and 3’ ends, which requires a multi-step reaction in natural cells. In this study, we developed a simplified processing method that allows simultaneous expression of all 21 tRNAs in a reconstituted transcription/translation system (PURE system). We tested three available methods (leader, 5’-G variants, and HDVR attachment methods) and one new method (direct tRNA linkage method). Using these methods, we succeeded in simultaneous expression of six non-G-start tRNA from monocistronic six DNA templates in the PURE system. Furthermore, we developed a method that combines the direct tRNA linkage and HDVR attachment methods (termed tRNA array method). Using this method, we succeeded in simultaneous expression of all 21 tRNAs from a single polycistronic DNA template in the PURE system. The tRNA mixture produced by the tRNA array method supported a similar level of translation to the individually synthesized tRNA mixture. Additionally, we demonstrated that the minimal tRNA sets prepared by the tRNA array method can be used for genetic code engineering. This study represents a step toward the realization of self-reproducible artificial cells and also provides an easy method for preparing all tRNAs useful for genetic code engineering.
Reference-based chemical-genetic interaction profiling to elucidate small molecule mechanism of action inMycobacterium tuberculosis
Authors: Bond Austin N. , Orzechowski Marek , Zhang Shuting , Ben-Zion Ishay , Lemmer Allison , Garry Nathaniel , Lee Katie , Chen Michael , Delano Kayla , Gath Emily , Golas Aaron , Nietupski Raymond , Fitzgerald Michael , Ehrt Sabine , Rubin Eric J. , Sassetti Christopher M. , Schnappinger Dirk , Shoresh Noam , Hunt Diana K. , Gomez James E. , Hung Deborah T. 
Published: February 2025

AbstractIn an era of increasing resistance, new and effective strategies are needed for antibiotic discovery. Whole-cell active screens yield candidate compounds lacking mechanism-of-action (MOA) information and thus do not provide biological insight for prioritization. We previously reported PROSPECT (PRimary screeningOfStrains toPrioritizeExpandedChemistry andTargets), an antimicrobial discovery strategy that measures chemical-genetic interactions between small molecules and a pool ofMycobacterium tuberculosismutants, each depleted of a different essential protein target. PROSPECT facilitates efficient hit prioritization by simultaneously identifying whole-cell active compounds with high sensitivity and providing early insights into their MOA. Here, we report a reference-based approach to infer MOA from often complex PROSPECT data. For this aim, we curated a reference set of 437 compounds with published, annotated MOA and known or suspected antitubercular activity, and applied PROSPECT to it. We then developedPerturbagenCLass (PCL) analysis, a computational method that predicts MOA by comparing chemical-genetic interaction profiles of unknown compounds to those of this reference set. In leave-one-out cross-validation, PCL analysis correctly predicted MOA with 70% sensitivity and 75% precision. When applied to 75 antitubercular leads with known MOA previously reported by GlaxoSmithKline (GSK), PCL analysis similarly achieved 69% sensitivity and 87% precision. We also analyzed 98 GSK compounds lacking MOA information, predicting 60 of them to act via a reference MOA, and followed up with functional validation of 29 compounds predicted to target respiration-related MOAs. Finally, we applied PROSPECT and PCL analysis to ∼5,000 compounds from larger unbiased libraries that had not been preselected for antitubercular activity. PCL analysis identified a novel scaffold lacking wild-type activity but predicted to inhibit respiration via QcrB, and we confirmed this prediction while chemically optimizing this scaffold to achieve wild-type activity. PCL analysis of PROSPECT data thus enables rapid MOA assignment and hit prioritization, advancing the discovery of new, potent antitubercular compounds.