bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision
Authors: Shembrey Carolyn , Yang Ray , Casan Joshua , Hu Wenxin , Sadras Teresa , Prasad Krishneel , Shortt Jake , Johnstone Ricky W , Trapani Joseph A , Ekert Paul G , Fareh Mohamed 
Published: September 2023

ABSTRACTSingle nucleotide variants (SNVs) are extremely prevalent in human cancers. For instance, KRAS mutations occur in over 90% of pancreatic cancers and ∼40% of colorectal cancers. Virtually all KRAS mutations are SNVs, most of which remain clinically unactionable. The programmable RNA nuclease CRISPR-Cas13 has been deployed to specifically target RNAs such as overexpressed oncogenes and fusion transcripts. However, silencing oncogenic SNVs with single-base precision remains extremely challenging due to the intrinsic mismatch tolerance of Cas13. Here, we developed a comprehensive mutagenesis analysis of target-spacer interactions at single-nucleotide resolution, which revealed key spacer nucleotide positions intolerant to mismatches. We show that introducing synthetic mismatches at these precise positions enablesde novadesign of CRISPR RNA (crRNA) with strong preferential silencing of SNV transcripts. We demonstrate that our top-performing crRNAs possess prominent SNV-selectivity with dose-dependent silencing activity against all KRAS G12 variants at both the RNA and protein levels with minimal off-target silencing of wildtype KRAS. We applied these design principles to effectively silence oncogenic NRAS G12D and BRAF V600E transcripts, underscoring the adaptability of this platform to silence various SNVs. These findings demonstrate that the CRISPR Cas13 system can be reprogrammed to target mutant transcripts with single-base precision, showcasing the tremendous potential of this tool in personalized transcriptome editing.Abstract Figure
Virulence and transmission biology of the widespread, ecologically important pathogen of zooplankton,Spirobacillus cienkowskii
Authors: Wale Nina , Freimark Claire B. , Ramirez Justin , Dziuba Marcin K. , Kafri Ahmad Y. , Bilich Rebecca , Duffy Meghan A. 
Published: September 2023

AbstractSpirobacillus cienkowskii(Spirobacillus,hereafter) is a widely distributed bacterial pathogen that has significant impacts on the population dynamics of zooplankton (Daphnia spp.), particularly in months whenDaphniaare asexually reproducing. Yet little is known aboutSpirobacillus’virulence, transmission mode and dynamics. As a result, we cannot explain the dynamics ofSpirobacillusepidemics in nature or useSpirobacillusas a model pathogen, despiteDaphnia’stractability as a model-host. Here, we work to fill these knowledge gaps experimentally. We found thatSpirobacillusis among the most virulent ofDaphniapathogens, killing its host within a week and reducing host fecundity. We further found thatSpirobacillusdid not transmit horizontally among hosts unless the host died or was destroyed (i.e., it is an “obligate killer”). In experiments aimed at quantifying the dynamics of horizontal transmission among asexually reproducingDaphnia, we demonstrated thatSpirobacillustransmits poorly in the laboratory. In mesocosms,Spirobacillusfailed to generate epidemics; in experiments wherein individualDaphniawere exposed,Spirobacillus’transmission success was low. In the (limited) set of conditions we considered,Spirobacillus’transmission success did not change with host density or pathogen dose and declined following environmental incubation. Lastly, we conducted a field survey ofSpirobacillus’prevalence within egg-cases (ephippia) made by sexually reproducingDaphnia. We foundSpirobacillusDNA in ∼40% of ephippia, suggesting that, in addition to transmitting horizontally among asexually reproducingDaphnia,Spirobacillusmay transmit vertically from sexually reproducingDaphnia. Our work fills critical gaps in the biology ofSpirobacillusand illuminates new hypotheses vis-à-vis its life-history.ImportanceSpirobacillus cienkowskiiis a bacterial pathogen of zooplankton, first described in the 19thCentury and recently placed in a new family of bacteria, theSilvanigrellaceae.Spirobacilluscauses epidemics in lake zooplankton populations and increases the probability that zooplankton will be eaten by predators. However, little is known about howSpirobacillustransmits among hosts, its impact on host survival and reproduction (i.e., how virulent it is) in laboratory conditions and what role virulence plays inSpirobacillus’life cycle. Here, we experimentally quantifiedSpirobacillus’ virulence and showed thatSpirobacillusmust kill its host to transmit horizontally. We also found evidence thatSpirobacillusmay transmit vertically viaDaphnia’s seed-like egg cases. Our work will help scientists to (i) understandSpirobacillusepidemics, (ii) useSpirobacillusas a model pathogen for the study of host-parasite interactions and (iii) better understand the unusual group of bacteria to whichSpirobacillusbelongs.
Oligodendrocyte maturation alters the cell death mechanisms that cause demyelination
Authors: Chapman Timothy W. , Piedra Enrique T. , Hill Robert A. 
Published: September 2023

ABSTRACTMyelinating oligodendrocytes die in human disease and early in aging. Despite this, little is known about the mechanisms that govern cell death across the oligodendrocyte lineage. Here we used a combination of intravital imaging, single-cell ablation, and cuprizone intoxication to show that oligodendrocyte maturation dictates the dynamics and mechanisms of death. After single-cell genotoxic damage, oligodendrocyte precursor cells underwent programmed cell death within hours, while mature oligodendrocytes died weeks after the same acute damage. Targeting single cells that were actively undergoing oligodendrocyte generation revealed that a switch in the temporal dynamics and morphological progression of death occurs during differentiation. Consistent with this, cuprizone intoxication initiated a caspase-3-dependent form of rapid cell death in differentiating oligodendrocytes, while mature oligodendrocytes never activated this executioner caspase and exhibited delayed cell death initiation. Thus, oligodendrocyte maturation plays a key role in determining the mechanism of death a cell undergoes in response to the same insult. This means that different strategies are likely necessary to confer protection to the entire oligodendrocyte lineage to enable myelin preservation and facilitate the integration of new oligodendrocytes in aging and disease.
Diet-induced obesity impacts influenza disease severity and transmission dynamics in ferrets
Authors: Meliopoulos Victoria , Honce Rebekah , Livingston Brandi , Hargest Virginia , Freiden Pamela , Lazure Lauren , Brigleb Pamela H. , Karlsson Erik , Tillman Heather , Allen E. Kaity , Boyd David , Thomas Paul G. , Schultz-Cherry Stacey 
Published: September 2023

AbstractObesity, and the associated metabolic syndrome, is a risk factor for increased disease severity with a variety of infectious agents, including influenza virus. Yet the mechanisms are only partially understood. As the number of people, particularly children, living with obesity continues to rise, it is critical to understand the role of host status on disease pathogenesis. In these studies, we use a novel diet-induced obese ferret model and new tools to demonstrate that like humans, obesity resulted in significant changes to the lung microenvironment leading to increased clinical disease and viral spread to the lower respiratory tract. The decreased antiviral responses also resulted in obese animals shedding higher infectious virus for longer making them more likely to transmit to contacts. These data suggest the obese ferret model may be crucial to understanding obesity’s impact on influenza disease severity and community transmission, and a key tool for therapeutic and intervention development for this high-risk population.TeaserA new ferret model and tools to explore obesity’s impact on respiratory virus infection, susceptibility, and community transmission.
The impact of phage and phage resistance on microbial community dynamics
Authors: Alseth Ellinor O , Custodio Rafael , Sundius Sarah A , Kuske Rachel A , Brown Sam P. , Westra Edze R 
Published: September 2023

AbstractWhere there are bacteria, there will be bacteriophages. These viruses are known to be important players in shaping the wider microbial community in which they are embedded, with potential implications for human health. On the other hand, bacteria possess a range of distinct immune mechanisms that provide protection against bacteriophages, including the mutation or complete loss of the phage receptor, and CRISPR-Cas adaptive immunity. Yet little is known about how interactions between phages and these different phage resistance mechanisms affect the wider microbial community in which they are embedded. Here, we conducted a 10-day, fully factorial evolution experiment to examine how phage impact the structure and dynamics of an artificial four-species bacterial community that includes eitherPseudomonas aeruginosawild type or an isogenic mutant unable to evolve phage resistance through CRISPR-Cas. Our results show that the microbial community structure is drastically altered by the addition of phage, withAcinetobacter baumanniibecoming the dominant species andP. aeruginosabeing driven nearly extinct, whereasP. aeruginosaoutcompetes the other species in the absence of phage. Moreover, we find that aP. aeruginosastrain with the ability to evolve CRISPR-based resistance generally does better when in the presence ofA. baumannii, but that this benefit is largely lost over time as phage is driven extinct. Combined, our data highlight how phage-targeting a dominant species allows for the competitive release of the strongest competitor whilst also contributing to community diversity maintenance and potentially preventing the reinvasion of the target species, and underline the importance of mapping community composition before therapeutically applying phage.
Progressive community, biogeochemical and evolutionary remodeling of the soil microbiome underpins long-term desert ecosystem restoration
Authors: Chen Qiong , Yuan Mengyi , Jiang Liuyiqi , Wei Xin , Liu Zhen , Peng Chen , Huang Zinuo , Tang Dongmei , Wu Xiangrong , Sun Jing , Ye Cunqi , Liu Qing , Zhu Xiaowei , Gao Peng , Huang Laibin , Wang Meng , Jiang Mingkai , Jiang Chao 
Published: September 2023

SummaryEcological restoration of degraded lands is essential to human sustainability. Yet, an in-depth community, functional, and evolutionary microbial perspective of long-term restoration of damaged ecosystems is lacking. Herein, we comprehensively assessed the impact of long-term (up to 17 years) restoration of Tengger Desert, China, by multi-omic profiling of 1,910 topsoil samples. The soil biophysiochemical properties, especially soil hydraulics, microbiome stability, and functional diversity, significantly improved during restoration. The soil microbiome transitioned from an extreme oligotrophic and autotrophic community to a diverse copiotrophic ecosystem. The soil microbiota, including fungi, could mediate the soil physicochemical changes through metabolites. Importantly, the systematic rewiring of nutrient cycles featured the multi-domain preference of an efficient carbon fixation strategy in the extreme desert environment. Finally, the microbiome was evolving via positive selections of genes of biogeochemical cycles, resistance, and motility. In summary, we present a comprehensive community, functional, biogeochemical, and evolutionary landscape of the soil microbiome during the long-term restoration of desert environments. We highlight the crucial microbial role in restoration from soil hydraulic and biogeochemical perspectives, offering promising field applications.HighlightsThe desert soil microbiome transformed from simple oligotrophic to a diverse, stable, and nutrient-rich ecosystem with expanded functional diversity.Restoration led to systematically rewired biogeochemical cycles, which are highly efficient in carbon fixation in the desert environment.The microbiome was evolving via positive selections of genes involved in biogeochemical cycles and environmental adaptations.Microbes and metabolites could facilitate desert restoration from hydraulic and biogeochemical aspects, offering promising field applications.
Peptidoglycan-tethered and free forms of the Braun lipoprotein are in dynamic equilibrium inEscherichia coli
Authors: Liang Yucheng , Hugonnet Jean-Emmanuel , Rusconi Filippo , Arthur Michel 
Published: September 2023

ABSTRACTPeptidoglycan (PG) is a giant macromolecule that completely surrounds bacterial cells and prevents lysis in hypo-osmotic environments. This net-like macromolecule is made of glycan strands linked to each other by two types of transpeptidases that form either 4→3 (PBPs) or 3→3 (LDTs) cross-links. Previously, we devised a heavy isotope-based PG full labeling method coupled to mass spectrometry to determine the mode of insertion of new-subunits into the expanding PG network (Atze et al., 2022). We showed that PG polymerization operates according to different modes for the formation of the septum and of the lateral cell walls, as well as for bacterial growth in the presence or absence of β-lactams in engineered strains that can exclusively rely on LDTs for PG cross-linking when drugs are present. Here, we apply our method to the resolution of the kinetics of the reactions leading to the covalent tethering of the Braun lipoprotein (Lpp) to PG and the subsequent hydrolysis of that same covalent link. We find that Lpp and disaccharide-peptide subunits are independently incorporated into the expanding lateral cell walls. Newly synthesized septum PG appears to contain small amounts of tethered Lpp. LDTs did mediate intense shuffling of Lpp between PG stems leading to a dynamic equilibrium between the PG-tethered and free forms of Lpp.
An enhancer RNA recruits MLL1 to regulate transcription ofMyb
Authors: Kim Juhyun , Diaz Luis F. , Miller Matthew J. , Leadem Benjamin , Krivega Ivan , Dean Ann 
Published: September 2023

SUMMARYTheMybproto-oncogene encodes the transcription factor c-MYB, which is critical for hematopoiesis. Distant enhancers ofMybform a hub of interactions with theMybpromoter. We identified a long non-coding RNA (Myrlin) originating from the −81 kb murineMybenhancer.MyrlinandMybare coordinately regulated during erythroid differentiation.MyrlinTSS deletion using CRISPR/Cas9 reducedMyrlinandMybexpression and LDB1 complex occupancy at theMybenhancers, compromising enhancer contacts and reducing RNA Pol II occupancy in the locus. In contrast, CRISPRi silencing ofMyrlinleft LDB1 and theMybenhancer hub unperturbed, althoughMyrlinandMybexpression were downregulated, decoupling transcription and chromatin looping.Myrlininteracts with the MLL1 complex.MyrlinCRISPRi compromised MLL1 occupancy in theMyblocus, decreasing CDK9 and RNA Pol II binding and resulting in Pol II pausing in theMybfirst exon/intron. Thus,Myrlindirectly participates in activatingMybtranscription by recruiting MLL1.
Mechanism for Vipp1 spiral formation, ring biogenesis and membrane repair
Authors: Naskar Souvik , Merino Andrea , Espadas Javier , Singh Jayanti , Roux Aurelien , Colom Adai , Low Harry H 
Published: September 2023

SummaryESCRT-III family proteins build dynamic filaments that remodel membrane. Although the transition of planar filaments into 3D membrane budding structures is fundamental for their function, the geometric changes in polymer architecture driving the transition remain obscure. Here we show how bacterial Vipp1 polymerises into dynamic planar sheets and spirals on membrane. The spirals converge to form a central ring like those known to bud membrane. To probe how Vipp1 morphs between polymers, we determine the architecture of multiple helical filaments. As well as describing filament constriction and membrane tubulation, the geometric relationship between helical and planar lattices enables Vipp1 sheets and spirals to be modelled. Moreover, the helical structures show filaments twisting – a process needed for Vipp1 to transition between planar and 3D architectures. Given the structural conservation between Vipp1 and ESCRT-III, our results may represent the broad changes in geometry required for some ESCRT-III filaments to switch between 2D and 3D forms.
Replication-dead gammaherpesvirus vaccine protects against acute replication, reactivation from latency, and lethal challenge in mice
Authors: Bland Wesley A. , Owens Shana , McEvoy Kyle , Hogan Chad H. , Boccuzzi Luciarita , Kirillov Varvara , Khairallah Camille , Sheridan Brian S. , Forrest J. Craig , Krug Laurie T. 
Published: September 2023

AbstractGammaherpesviruses (GHVs) are oncogenic viruses that establish lifelong infections and are significant causes of human morbidity and mortality. While several vaccine strategies to limit GHV infection and disease are in development, there are no FDA-approved vaccines for human GHVs. As a new approach to gammaherpesvirus vaccination, we developed and tested a replication-dead virus (RDV) platform, using murine gammaherpesvirus 68 (MHV68), a well-established mouse model for gammaherpesvirus pathogenesis studies and preclinical therapeutic evaluations. We employed codon-shuffling-based complementation to generate revertant-free RDV lacking expression of the essential replication and transactivator protein (RTA) encoded byORF50to arrest viral gene expression early afterde novoinfection. Inoculation with RDV-50.stop exposes the host to intact virion particles and leads to limited lytic gene expression in infected cells. Prime-boost vaccination of mice with RDV-50.stop elicited virus-specific neutralizing antibody and effector T cell responses in the lung and spleen. Vaccination with RDV-50.stop resulted in a near complete abolishment of virus replication in the lung 7 days post-challenge and virus reactivation from spleen 16 days post-challenge with WT MHV68.Ifnar1-/-mice, which lack the type I interferon receptor, exhibit severe disease upon infection with WT MHV68. RDV-50.stop vaccination ofIfnar1-/-mice prevented wasting and mortality upon challenge with WT MHV68. These results demonstrate that prime-boost vaccination with a GHV that is unable to undergo lytic replication offers protection against acute replication, reactivation, and severe disease upon WT virus challenge.ImportanceGammaherpesviruses establish chronic infections that place a host at life-long risk for the development of lymphoproliferative disorders. While these viruses are endemic within the adult human population, there are currently no FDA-approved vaccines available to prevent acute infection or the later onset of lymphoproliferative disorders. We report here the use of a prime-boost vaccination strategy utilizing replication-dead virus strains to protect mice from acute disease following challenge with WT virus. We demonstrate that vaccination with replication-dead viruses is both safe and effective within immunocompetent and immunodeficient mice, and protects from viral replication, reactivation, and onset of severe disease.
Unveiling multi-drug resistant (MDR) gram negative pathogenic bacteria from poultry chickens in the Noakhali region of Bangladesh
Authors: Munim Md. Adnan , Das Shuvo Chandra , Hossain Md. Murad , Hami Ithmam , Topu Mridul Gope , Gupta Shipan Das 
Published: September 2023

AbstractRapidly increasing antibiotic-resistant bacterial strains in Bangladesh’s food and farm animals stem from the excessive and inappropriate use of antibiotics. To assess the prevalence of multi drug resistant (MDR) gram-negative bacteria in poultry chicks, we sought to isolate and identify strains carrying antimicrobial resistance genes. Isolation and identification involved biochemical tests,16SrRNA sequencing, and PCR screening of species-specific genes. MDR patterns were evaluated using CLSI guidelines with seventeen antibiotics across twelve classes. Targeted gene sequences were amplified for detection of ESBL, carbapenem, tetracycline, sulfonamide, and colistin resistance genes. Common isolates, such asEscherichia coli,Klebsiella pneumoniae, Proteus penneri,andEnterobacter hormaechei, exhibited average Multiple Antimicrobial Resistance (MAR) indices of 0.66, 0.76, 0.8, 0.84, and 0.81, 0.76, 0.84, 0.41 for broiler and layer chicken, respectively.Providencia stuartiiandSalmonella enterica, exclusive to broiler samples, had MAR indices of 0.82 and 0.84, respectively. Additional isolatesMorganella morganii,Aeromonasspp., andWohlfahrtiimonas chitiniclasticawere found in layers (Average MAR indices: 0.73, 0.71, and 0.91). Notably,M. morganii, E. hormaechei and W. chitiniclasticawere identified for the first time in Bangladeshi poultry chicken, although their evolution is yet to understand. In this study, Pan-drug resistance was observed in oneP. stuartii(broiler) and oneAeromonasspp. (layer) with a MAR index 1, while all isolates exhibited MAR indices >0.2, indicating MDR. Antimicrobial resistance (AMR) gene screening identifiedblaTEM,blaSHV,tetA, andsul1in a majority of the MDR strains. Interestingly,E. coli(lactose positive and negative) andE. hormaecheiwere exclusively found to possess thetetBgene. In addition,E. coli(lactose negative),Klebsiella pneumoniae,Enterobacter hormaechei,M. morganii, andP. stuartiiwere observed to carry the colistin-resistantmcr1gene, whereassul2was detected inE. coli(lactose positive and negative),E. hormaechei,P. stuartii, andP. penneri. These findings emphasize the health risk of our consumers of both broiler and layer chickens as they have turned to a potent reservoir of various AMR gene carrying MDR and Pan-drug resistant bacteria.