bluuebunny/crossref_metadata_2025_split huggingface dataset:
Using RNA-targeting CRISPR-Cas13 and engineered U1 systems to reduceABCA4splice variants in Stargardt disease
Authors: Liou Roxanne Hsiang-Chi , Urrutia-Cabrera Daniel , Westin Ida Maria , Golovleva Irina , Liu Guei-Sheung , Kumar Satheesh , McLenachan Samuel , Chen Fred Kuanfu , Hsu Fei-Ting , Edwards Tom , Martin Keith R , Cheng Albert Wu , Wong Raymond C.B. 
Published: March 2024

AbstractDysregulation of the alternative splicing process results in aberrant mRNA transcripts, leading to dysfunctional proteins or nonsense-mediated decay that cause a wide range of mis-splicing diseases. Development of therapeutic strategies to target the alternative splicing process could potentially shift the mRNA splicing from disease isoforms to a normal isoform and restore functional protein. As a proof of concept, we focus on Stargardt disease (STGD1), an autosomal recessive inherited retinal disease caused by biallelic genetic variants in theABCA4gene. The splicing variants c.5461-10T>C and c.4773+3A>G inABCA4cause the skipping of exon 39-40 and exon 33-34 respectively. In this study, we compared the efficacy of different RNA-targeting systems to modulate theseABCA4splicing defects, including four CRISPR-Cas13 systems (CASFx-1, CASFx-3, RBFOX1N-dCas13e-C and RBFOX1N-dPspCas13b-C) as well as an engineered U1 system (ExSpeU1). Using a minigene system containingABCA4variants in the human retinal pigment epithelium ARPE19, our results show that RBFOX1N-dPspCas13b-C is the best performing CRISPR-Cas system, which enabled up to 80% reduction of the mis-splicedABCA4c.5461-10T>C variants and up to 78% reduction of theABCA4c.4773+3A>G variants. In comparison, delivery of a single ExSpeU1 was able to effectively reduce the mis-splicedABCA4c.4773+3A>G variants by up to 84%. We observed that the effectiveness of CRISPR-based and U1 splicing regulation is strongly dependent on the sgRNA/snRNA targeting sequences, highlighting that optimal sgRNA/snRNA designing is crucial for efficient targeting of mis-spliced transcripts. Overall, our study demonstrated the potential of using RNA-targeting CRISPR-Cas technology and engineered U1 to reduce mis-spliced transcripts forABCA4, providing an important step to advance the development of gene therapy to treat STGD1.
Comprehensive analyses of a large human gut Bacteroidales culture collection reveal species and strain level diversity and evolution
Authors: Zhang Zhenrun J. , Cole Cody G. , Coyne Michael J. , Lin Huaiying , Dylla Nicholas , Smith Rita C. , Waligurski Emily , Ramaswamy Ramanujam , Woodson Che , Burgo Victoria , Little Jessica C. , Moran David , Rose Amber , McMillin Mary , McSpadden Emma , Sundararajan Anitha , Sidebottom Ashley M. , Pamer Eric G. , Comstock Laurie E. 
Published: March 2024

SummarySpecies of the Bacteroidales order are among the most abundant and stable bacterial members of the human gut microbiome with diverse impacts on human health. While Bacteroidales strains and species are genomically and functionally diverse, order-wide comparative analyses are lacking. We cultured and sequenced the genomes of 408 Bacteroidales isolates from healthy human donors representing nine genera and 35 species and performed comparative genomic, gene-specific, mobile gene, and metabolomic analyses. Families, genera, and species could be grouped based on many distinctive features. However, we also show extensive DNA transfer between diverse families, allowing for shared traits and strain evolution. Inter- and intra-specific diversity is also apparent in the metabolomic profiling studies. This highly characterized and diverse Bacteroidales culture collection with strain-resolved genomic and metabolomic analyses can serve as a resource to facilitate informed selection of strains for microbiome reconstitution.
Botrytis cinereadetoxifies the sesquiterpenoid phytoalexin rishitin through multiple metabolizing pathways
Authors: Bulasag Abriel Salaria , Ashida Akira , Miura Atsushi , Pring Sreynich , Kuroyanagi Teruhiko , Camagna Maurizio , Tanaka Aiko , Sato Ikuo , Chiba Sotaro , Ojika Makoto , Takemoto Daigo 
Published: March 2024

ABSTRACTBotrytis cinereais a necrotrophic pathogen that infects across a broad range of plant hosts, including high-impact crop species. Its generalist necrotrophic behavior stems from its ability to detoxify structurally diverse phytoalexins. The current study aims to provide evidence of the ability ofB. cinereato tolerate the sesquiterpenoid phytoalexin rishitin, which is produced by potato and tomato. While the growth of potato pathogensPhytophthora infestans(late blight) andAlternaria solani(early blight) was severely inhibited by rishitin,B. cinereawas tolerant to rishitin. After incubation of rishitin with the mycelia ofB. cinerea, it was metabolized to at least six oxidized forms. Structural analysis of these purified rishitin metabolites revealed a variety of oxidative metabolism including hydroxylation at C7 or C12, ketone formation at C5, and dihydroxylation at the 10,11-olefin. Six rishitin metabolites showed reduced toxicity toP. infestansandA. solani, indicating thatB. cinereahas at least 5 distinct enzymatic reactions to detoxify rishitin. Four host-specialized phytopathogenicBotrytisspecies, namelyB. elliptica, B. allii, B. squamosa,andB. tulipaealso had at least a partial ability to metabolize rishitin asB. cinerea, but their metabolic capacity was significantly weaker than that ofB. cinerea. These results suggest that the ability ofB. cinereato rapidly metabolize rishitin through multiple detoxification mechanisms could be critical for its pathogenicity in potato and tomato.
High-throughput sequencing-based neutralization assay reveals how repeated vaccinations impact titers to recent human H1N1 influenza strains
Authors: Loes Andrea N. , Tarabi Rosario Araceli L. , Huddleston John , Touyon Lisa , Wong Sook San , Cheng Samuel M. S. , Leung Nancy H.L. , Hannon William W. , Bedford Trevor , Cobey Sarah , Cowling Benjamin J. , Bloom Jesse D. 
Published: March 2024

AbstractThe high genetic diversity of influenza viruses means that traditional serological assays have too low throughput to measure serum antibody neutralization titers against all relevant strains. To overcome this challenge, we have developed a sequencing-based neutralization assay that simultaneously measures titers against many viral strains using small serum volumes via a workflow similar to traditional neutralization assays. The key innovation is to incorporate unique nucleotide barcodes into the hemagglutinin (HA) genomic segment, and then pool viruses with numerous different barcoded HA variants and quantify infectivity of all of them simultaneously using next-generation sequencing. With this approach, a single researcher performed the equivalent of 2,880 traditional neutralization assays (80 serum samples against 36 viral strains) in approximately one month. We applied the sequencing-based assay to quantify the impact of influenza vaccination on neutralization titers against recent human H1N1 strains for individuals who had or had not also received a vaccine in the previous year. We found that the viral strain specificities of the neutralizing antibodies elicited by vaccination vary among individuals, and that vaccination induced a smaller increase in titers for individuals who had also received a vaccine the previous year—although the titers six months after vaccination were similar in individuals with and without the previous-year vaccination. We also identified a subset of individuals with low titers to a subclade of recent H1N1 even after vaccination. This study demonstrates the utility of high-throughput sequencing-based neutralization assays that enable titers to be simultaneously measured against many different viral strains. We provide a detailed experimental protocol (DOI:https://dx.doi.org/10.17504/protocols.io.kqdg3xdmpg25/v1) and a computational pipeline (https://github.com/jbloomlab/seqneut-pipeline) for the sequencing-based neutralization assays to facilitate the use of this method by others.
Analyses ofXenorhabdus griffiniaegenomes reveal two distinct sub-species that display intra-species variation due to prophages
Authors: Heppert Jennifer K. , Awori Ryan Musumba , Cao Mengyi , Chen Grischa , McLeish Jemma , Goodrich-Blair Heidi 
Published: March 2024

AbstractNematodes of the genusSteinernemaand theirXenorhabdusbacterial symbionts are lethal entomopathogens that are useful in the biocontrol of insect pests, as sources of diverse natural products, and as research models for mutualism and parasitism.Xenorhabdusplay a central role in all aspects of theSteinernemalifecycle, and a deeper understanding of their genomes therefore has the potential to spur advances in each of these applications. Here, we report a comparative genomics analysis ofXenorhabdus griffiniae, including the symbiont ofSteinernema hermaphroditumnematodes, for which genetic and genomic tools are being developed. We sequenced and assembled circularized genomes for threeXenorhabdusstrains: HGB2511, ID10 and TH1. We then determined their relationships to otherXenorhabdusand delineated their species via phylogenomic analyses, concluding that HGB2511 and ID10 areXenorhabdus griffiniaewhile TH1 is a novel species. These additions to the existingX. griffiniaelandscape further allowed for the identification of two subspecies within the clade. Consistent with otherXenorhabdus, the analysedX. griffiniaegenomes each encode a wide array of antimicrobials and virulence-related proteins. Comparative genomic analyses, including the creation of a pangenome, revealed that a large amount of the intraspecies variation inX. griffiniaeis contained within the mobilome and attributable to prophage loci. In addition, CRISPR arrays, secondary metabolite potential and toxin genes all varied among strains within theX. griffiniaespecies. Our findings suggest that phage-related genes drive the genomic diversity in closely relatedXenorhabdussymbionts, and that these may underlie some of the traits most associated with the lifestyle and survival of entomopathogenic nematodes and their bacteria: virulence and competition. This study establishes a broad knowledge base for further exploration of not only the relationships betweenX. griffiniaespecies and their nematode hosts but also the molecular mechanisms that underlie their entomopathogenic lifestyle.
Limited evidence of spill over of antimicrobial resistantKlebsiella pneumoniaefrom animal/environmental reservoirs to humans in India
Authors: Jacob Jobin John , V Aravind , Beresford-Jones Benjamin S. , Y Binesh Lal , Shankar Chaitra , M Yesudoss , Abdullah Fiza , Priya T Monisha , Kulkarni Sanika , Baker Stephen , Veeraraghavan Balaji , Walia Kamini 
Published: March 2024

AbstractBackgroundKlebsiella pneumoniaeis a common opportunistic pathogen known for having virulent and antimicrobial resistance (AMR) phenotypes. In addition to accumulating AMR and virulence genes,K. pneumoniaeserves as a vehicle for broadly disseminating these elements into other species. Here, we applied genomic surveillance in a one-health framework to assess the impact of the human-animal-environment interface on AMR transmission.MethodsWe sequenced representative genomes ofKlebsiella pneumoniaeisolated from clinical specimens (n=59), livestock samples (n=71), and hospital sewage samples (n=16) from a two-year surveillance study. We compared the taxonomic and genomic distribution ofK. pneumoniae, the abundance of AMR, virulence genes, and mobile genetic elements between isolates from three sources.ResultsKlebsiellaspp. was the second most commonly isolated species (n=2,569). The clonal distribution ofK. pneumoniaesuggested isolates originating from livestock were clonally distinct from those derived from clinical/hospital effluent isolates. Clinical and hospital sewage isolates typically carried a higher number of resistance/virulence genes. There was limited overlap ofK. pneumoniaeclones, AMR genes, virulence determinants, and plasmids between the different settings.ConclusionCurrently, the spread of XDR or hypervirulent clones ofK. pneumoniaeappears to be confined to humans with no clear linkage with non-clinical sources. Moreover, emerging convergent clones ofK. pneumoniaecarrying both resistance and virulence determinants (ST231, ST2096) are likely to have emerged in hospital settings rather than in animal or natural environments. These data challenge the current view of AMR transmission inK. pneumoniaein a One-Health context.
Tethering NDC80-NUF2 to microparticles is sufficient to enable their biorientation in the spindle
Authors: Asai Kohei , Zhou Yuanzhuo , Kitajima Tomoya S. 
Published: March 2024

SummaryFaithful chromosome segregation requires biorientation, where the pair of kinetochores on the chromosome establish bipolar microtubule attachment. The integrity of the kinetochore, a macromolecular complex built on centromeric DNA, is required for biorientation, but components sufficient for biorientation remain unknown. In this study, we show that tethering the outer kinetochore heterodimer NDC80-NUF2 to the surface of microbeads with no bipolar cue is sufficient for them to establish a biorientation-like state in mouse oocytes. NDC80-NUF2 microbeads efficiently and stably align at the spindle equator, forming bipolar microtubule attachments. Furthermore, they can self-correct alignment errors. They align independently of the outer kinetochore proteins SPC24-SPC25, KNL1, the MIS12 complex, or inner kinetochore proteins. Aurora-mediated NDC80 phosphoregulation promoted microbead alignment under challenging conditions. Interestingly, larger microbeads align more rapidly, suggesting that large platform size enhances NDC80-NUF2-mediated biorientation. This study shows a biohybrid kinetochore design for synthetic biorientation of microscale particles in cells.
Deletion of Nrf1 exacerbates oxidative stress-induced cellular senescence by disrupting the cell homeostasis
Authors: Lyu Da , Wang Meng , Qiu Lu , Hu Shaofan , Zhang Yiguo 
Published: March 2024

AbstractCellular senescence has been accepted as a fundamental contributor to ageing and a variety of age-related diseases, in which oxidative stress has been further recognized to play a critical initiation role. However, the anti-senescence potential of antioxidant nuclear factor erythroid-derived 2-like 1 (Nrf1, encoded byNfe2l1) remains elusive to date, even though the hitherto accumulating evidence demonstrates that it is an indispensable redox-determining transcription factor for maintaining cellular homeostasis and organ integrity. Herein, we discovered that deletion of Nrf1 resulted in markedly elevated senescence characteristics inNrf1α−/−cells, as characterized by two distinct experimental models induced by oxidative stress, which are evinced by typically heightened activity of senescence-associated β-galactosidase and progressive senescence-associated secretory phenotype (SASP), along with decreased cell vitality and intensified cell cycle arrest. Further experimental investigation also uncovered that such acceleration of oxidative stress-induced senescence resulted from heightened disturbance in the cellular homeostasis, because deficiency of Nrf1α leads to the STAG2- and SMC3-dependent chromosomal stability disruption and autophagy dysfunction, though as accompanied by excessive accumulation of Nrf2 (encoded byNfe2l2). The aberrant hyperactive Nrf2 cannot effectively counteract the escalating disturbance of cellular homeostasis caused byNrf1α−/−. Overall, this study has provided a series of evidence supporting that Nrf1 indeed exerts an essential protective function against oxidative stress-induced cellular senescence, thereby, highlighting its primary indispensable contribution to maintaining robust cell homeostasis.
Sex-specific viability effects of mutations inDrosophila melanogaster
Authors: Melde Robert H. , Abraham JoHanna M. , Ugolini Maryn R. , Castle Madison P. , Fjalstad Molly M. , Blumstein Daniela M. , Sharp Nathaniel P. 
Published: March 2024

AbstractIn populations with separate sexes, genetic load due to deleterious mutations may be expressed differently in males and females. Evidence from insect models suggests that selection against mutations is stronger in males, with a positive intersexual correlation for fitness. This pattern will reduce deleterious allele frequencies at the expense of males, such that female mean fitness is greater than expected, preserving population persistence in the face of high mutation rates. While previous studies focus on reproductive success, mutation load depends on total selection in each sex, including selection for viability. In fruit flies, we might expect minimal sex differences in viability effects, since male and female larvae behave similarly, but many genes show sex-biased expression in larvae. We measured the sex-specific viability effects of nine “marker” mutations and 33 mutagenized chromosomes. We find that both types of mutations generally reduce viability in both sexes. Among marker mutations we detect instances of sex biased selection in both directions, but mutagenized chromosomes show little sign of sex-specific mutational variance. We conclude that some mutations can indeed affect viability in a sex-specific manner, but that the pattern of male-biased mutational effects observed previously for reproductive success is not apparent at the pre-reproductive stage.
Evaluating the feasibility of study methods for a future trial-based economic evaluation of a multistage shared decision-making program for type 2 diabetes mellitus: protocol for a cluster-randomized controlled pilot study
Authors: Tichler A. , Hertroijs D.F.L. , van Mastrigt G.A.P.G. , Brouwers M.C.G.J. , Ruwaard D. , Elissen A.M.J. 
Published: March 2024

AbstractIntroductionWe developed a multistage shared decision-making program for type 2 diabetes that aims to support person-centered type 2 diabetes management in primary care. The program consists of an online patient decision aid, a preparatory consult for patients, and interprofessional training for healthcare professionals. The short– and long-term effectiveness of the multistage shared decision-making program needs to be researched in a trial-based economic evaluation. To evaluate the feasibility of study methods for future economic evaluation, we will conduct a pilot study that focuses on sample recruitment and retention, study management, and feasibility of outcome and cost measurements.Methods and analysisThe multistage shared decision-making program will be pilot-tested in a cluster-randomized controlled trial in four primary care practices (located in the region of Gorinchem, the Netherlands) using a mixed-methods approach. The intervention practices will adopt the program, whereas the control practices provide usual care. Data collection will include recruitment, retention, and consent rates, patients’ sociodemographic and clinical characteristics, and the assessment of primary and secondary outcomes of the future trial-based economic evaluation. We will also collect data on the usage behavior of patients when completing questionnaires of the primary and secondary outcomes (i.e. time needed to complete questionnaires). Semi-structured interviews with patients will be conducted to obtain insights into the understandability and usability of measurement tools. Moreover, focus groups with healthcare professionals from participating practices will be organized to complement the quantitative data on sample representativeness and to assess the study management challenges of participating practices.DiscussionThe pilot will address uncertainties around the feasibility of a future trial-based economic evaluation, focusing on sample recruitment and retention, study management, and the feasibility of outcome and cost measurements. The results will guide the improvement of study procedures for the economic evaluation of our multistage shared decision-making program for type 2 diabetes.
The association between prolonged SARS-CoV-2 symptoms and work outcomes
Authors: Venkatesh Arjun , Yu Huihui , Malicki Caitlin , Gottlieb Michael , Elmore Joann G. , Hill Mandy J. , Idris Ahamed H. , Montoy Juan Carlos C. , O’Laughlin Kelli N. , Rising Kristin L. , Stephens Kari A. , Spatz Erica S. , Weinstein Robert A. ,   the INSPIRE Group
Published: March 2024

AbstractWhile the early effects of the COVID-19 pandemic on the United States labor market are well-established, less is known about the long-term impact of SARS-CoV-2 infection and Long COVID on employment. To address this gap, we analyzed self-reported data from a prospective, national cohort study to estimate the effects of SARS-CoV-2 symptoms at three months post-infection on missed workdays and return to work. The analysis included 2,939 adults in the Innovative Support for Patients with SARS-CoV-2 Infections Registry (INSPIRE) study who tested positive for their initial SARS-CoV-2 infection at the time of enrollment, were employed before the pandemic, and completed a baseline and three-month electronic survey. At three months post-infection, 40.8% of participants reported at least one SARS-CoV-2 symptom and 9.6% of participants reported five or more SARS-CoV-2 symptoms. When asked about missed work due to their SARS-CoV-2 infection at three months, 7.1% of participants reported missing ≥10 workdays and 13.9% of participants reported not returning to work since their infection. At three months, participants with ≥5 symptoms had a higher adjusted odds ratio (aOR) of missing ≥10 workdays (2.96, 95% CI 1.81-4.83) and not returning to work (2.44, 95% CI 1.58-3.76) compared to those with no symptoms. Prolonged SARS-CoV-2 symptoms were common, affecting 4-in-10 participants at three-months post-infection, and were associated with increased odds of work loss, most pronounced among adults with ≥5 symptoms at three months. Despite the end of the Federal COVID-19 Public Health Emergency and efforts to “return to normal”, policymakers must consider the clinical and economic implications of the COVID-19 pandemic on people’s employment status and work absenteeism, particularly as data characterizing the numerous health and well-being impacts of Long COVID continue to emerge. Improved understanding of risk factors for lost work time may guide efforts to support people in returning to work.
A fungal transcription factorBOT6facilitates the transition of a beneficial root fungus into an adapted anthracnose pathogen
Authors: Ujimatsu Ren , Takino Junya , Nakamura Masami , Haba Hiromi , Minami Atsushi , Hiruma Kei 
Published: March 2024

AbstractThe infection strategies employed by plant endophytes are attributed to their ability to overcome durable nonhost resistance and adapt to the host environment. However, the regulatory genetic background underlying how they adapt to the host and determine their lifestyles remains enigmatic. Here, we show that theCtBOT6, a cluster-residing transcription factor in the root-associated fungusColletotrichum tofieldiae(Ct), plays a pivotal role in regulating virulence-related gene expression and in producing metabolites both not only within and but unexpectedly outside of the cluster. Genetic manipulation ofCtBOT6toward activation alone is sufficient to transition a root beneficial Ct along the mutualist-pathogen continuum even toward a leaf pathogen capable of overcoming nonhost resistance, partly dependent on the host abscisic acid and ethylene pathways. Our findings indicate that the status of CtBOT6 serves as a critical determinant for the endophytic fungus to adapt to the plant different environments and manifest diverse infection strategies.