bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural characterisation of the complete cycle of sliding clamp loading inEscherichia coli
Authors: Xu Zhi-Qiang , Jergic Slobodan , Lo Allen T.Y. , Pradhan Alok C. , Brown Simon H.J. , Bouwer James C. , Ghodke Harshad , Lewis Peter J. , Tolun Gökhan , Oakley Aaron J. , Dixon Nicholas E. 
Published: July 2023

AbstractRing-shaped DNA sliding clamps are essential for DNA replication and genome maintenance. Clamps need to be opened or trapped open and chaperoned onto DNA by clamp loader complexes (CLCs). Detailed understanding of the mechanisms by which CLCs open and place clamps around DNA remains limited. Here, we present a series of six structures of theEscherichia coliCLC bound to an open or closed clamp on and off a primer-template DNA that represent all intermediates in the clamp loading process. We show that the ATP-bound CLC first binds to a clamp, then constricts to hold onto it. The CLC then expands to open the clamp with a gap large enough for double-stranded DNA to enter. Upon binding to DNA, the CLC constricts slightly, allowing ATP hydrolysis and clamp closing around DNA. Although both yeast andE. coliCLCs open clamps by crab claw-like motions, they do it by the CLC expanding in opposite directions. These structures provide critical high-resolution snapshots of clamp loading by theE. coliCLC, revealing how the molecular machine works.
Effect of ‘spent’ nucleotides on nonenzymatic RNA replication
Authors: Patki Gauri M. , Rajamani Sudha 
Published: July 2023

AbstractNonenzymatic template-directed replication would have been affected by co-solutes in a heterogenous prebiotic soup due to lack of enzymatic machinery. Unlike in contemporary biology, these reactions use chemically-activated nucleotides, which undergo rapid hydrolysis forming nucleoside monophosphates (‘spent’ monomers). These co-solutes cannot extend the primer but continue to base pair with the template, thereby interfering with replication. We therefore aimed to understand how a mixture of ‘spent’ ribonucleotides would affect nonenzymatic replication. We observed inhibition of replication in presence of the mixture, wherein predominant contribution came from the cognate Watson-Crick monomer, showing potential sequence dependence. Our study highlights how nonenzymatic RNA replication would have been directly affected by co-solutes, with ramifications for the emergence of functional polymers in an RNA World.
An engineered A549 cell line expressing CD13 and TMPRSS2 is permissive to clinical isolate of human coronavirus 229E
Authors: Lie Laurensius Kevin , Synowiec Aleksandra , Mazur Jedrzej , Pyrć Krzysztof 
Published: July 2023

AbstractThe lack of suitablein vitroculture model has hampered research on wild-type (WT) human coronaviruses. While 3D tissue or organ cultures have been instrumental for this purpose, such models are challenging, time-consuming, expensive and require extensive cell culture adaptation and directed evolution. Consequently, high-throughput applications are beyond reach in most cases. Here we developed a robust A549 cell line permissive to a human coronavirus 229E (HCoV-229E) clinical isolate by transducing CD13 and transmembrane serine protease 2 (TMPRSS2), henceforth referred to as A549++cells. This modification allowed for productive infection, and a more detailed analysis showed that the virus might use the TMPRSS2-dependent pathway but can still bypass this pathway using cathepsin-mediated endocytosis. Overall, our data showed that A549++cells are permissive to HCoV-229E clinical isolate, and applicable for further studies on HCoV-229E infectiology. Moreover, this line constitutes a uniform platform for studies on multiple members of theCoronaviridaefamily.Graphical abstract
Distinct composition of plasma virome in HIV-infected subjects on antiretroviral therapy compared to controls
Authors: Bhagchandani Tannu , Ul Haque Mohd. Maksuf , Malik Md Zubbair , Ray Ashwini Kumar , Kaur S Urvinder , Rai Ankita , Verma Anjali , Sawlani Kamal Kumar , Chaturvedi Rupesh , Himanshu D , Tandon Ravi 
Published: July 2023

AbstractMicrobiome study during HIV infection is widely documented with major emphasis on bacteriome, while virome studies are few. The alteration of plasma virome is reported in HIV patients who are either treatment naïve or have a weak immune system. Less is known about the plasma virome in HIV-infected patients on ART with preserved CD4 counts. In our pilot study, viral DNA isolated from plasma was sequenced on Illumina Nextseq500 platform. With the help of VIROMATCH pipeline, we observed that the plasma virome of HIV patients were significantly distinct from controls on the basis of Bray-Curtis dissimilarity index. The species,Human gammaherpesvirus 4and families,HerpesviridaeandSiphoviridaewere found to be significantly enriched and differentially abundant in HIV patients. Hence, plasma virome is an important component for future study that might influence disease progression among HIV patients during therapy.
Cell division angle regulates the tissue mechanics and tunes the amount of cerebellar folding
Authors: Cook Amber G. , Bishop Taylor V. , Crowe Hannah R. , Stevens Daniel , Reine Lauren , Joyner Alexandra L , Lawton Andrew K 
Published: July 2023

AbstractModeling has proposed that the amount of neural tissue folding is set by the level of differential-expansion between tissue layers and that the wavelength is set by the thickness of the outer layer. Here we used inbred mouse strains with distinct amounts of cerebellar folding to investigate these predictions. We identified a critical period where the folding amount diverges between the strains. In this period, regional changes in the level of differential-expansion between the external granule layer (EGL) and underlying core correlate with the folding amount in each strain. Additionally, the thickness of the EGL is regionally adjusted during the critical period alongside corresponding changes in wavelength. While the number of SHH-expressing Purkinje cells predicts the folding amount, the proliferation rate in the EGL is the same between the strains. However, regional changes in the cell division angle within the EGL predicts both the tangential-expansion and thickness of the EGL. Cell division angle is likely a tunable mechanism whereby both the level of differential-expansion and thickness of the EGL are regionally tuned to set the amount and wavelength of folding.
Autophagy maintains the homeostatic environment in the male reproductive accessory organs playing a key role in fertility
Authors: Jaulim Adil , Cassidy Liam D , Young Andrew RJ , Chan Adelyne SL , Warren Anne Y , Taylor Angela E , Arlt Wiebke , Lan Guochen , Blayney Martyn L , Davidson Olivia , Barratt Christopher LR , Pacey Simon , Narita Masashi 
Published: July 2023

AbstractAutophagy has been implicated in male fertility but its specific role in the post-testicular organs remains unclear. Here, we investigate this in mice expressing a doxycycline-inducible RNAi against Atg5 (Atg5i). Systemic autophagy inhibition in Atg5i mice resulted in the morphological and functional abrogation of the male accessory sex organs, leading to male subfertility. However, the testis was largely protected, likely due to the limited permeability of doxycycline through the blood-testis barrier. Interestingly, restoration of autophagy by doxycycline withdrawal in Atg5i mice led to substantial recovery of the phenotype in the accessory organs. This model offers a unique opportunity to dissect the pre- and post-testicular roles of autophagy, highlighting the non-autonomous impact of autophagy on male fertility.
The Type VI secretion systemof Stenotrophomonas rhizophilaCFBP13503 limits the transmission ofXanthomonas campestrispv. campestris8004 from radish seeds to seedlings
Authors: Garin Tiffany , Brin Chrystelle , Préveaux Anne , Brault Agathe , Briand Martial , Simonin Marie , Barret Matthieu , Journet Laure , Sarniguet Alain 
Published: July 2023

ABSTRACTStenotrophomonas rhizophilaCFBP13503 is a seed-borne commensal bacterial strain, which is efficiently transmitted to seedlings and can outcompete the phytopathogenic bacteriaXanthomonas campestrispv.campestris(Xcc8004). The type VI Secretion System (T6SS), an interference contact-dependent mechanism, is a critical component of interbacterial competition. The involvement of the T6SS ofS. rhizophilaCFBP13503 in the inhibition of Xcc8004 growth and seed-to-seedling transmission was assessed. The T6SS cluster ofS. rhizophilaCFBP13503 and nine putative effectors were identified. Deletion of two T6SS structural genes,hcpandtssB, abolished the competitive advantage ofS. rhizophilaagainst Xcc8004 invitro. The population sizes of these two bacterial species were monitored in seedlings after inoculation of radish seeds with mixtures of Xcc8004 and eitherS. rhizophilawild type (wt) strain or isogenichcpmutant. A significant decrease in the population size of Xcc8004 was observed during confrontation with theS. rhizophilawt in comparison to T6SS- deletion mutants in germinated seeds and seedlings. We found that the T6SS distribution among 835 genomes of theStenotrophomonas genus is scarce. In contrast, in all availableS. rhizophilagenomes, T6SS clusters are widespread and mainly belong to the T6SS group i4. In conclusion, the T6SS of S.rhizophilaCFBP13503 is involved in the antibiosis against Xcc8004 and reduces seedling transmission of Xcc8004 in radish. The distribution of this T6SS cluster in theS. rhizophilacomplex could make it possible to exploit these strains as biocontrol agents againstX. campestrispv.campestris.
ISGylation-independent protection of cell growth by USP18 following interferon stimulation
Authors: Clancy Anne , Rusilowicz-Jones Emma V. , Wallace Iona , Swatek Kirby N. , Urbé Sylvie , Clague Michael J. 
Published: July 2023

AbstractType 1 interferon stimulation highly up-regulates all elements of a ubiquitin-like conjugation system that leads to ISGylation of target proteins. An ISG15-specific member of the deubiquitylase family, USP18, is up-regulated in a co-ordinated manner. USP18 can also provide a negative feedback by inhibiting JAK-STAT signaling through protein interactions independently of DUB activity. Here, we provide an acute example of this phenomenon, whereby the early expression of USP18, post-interferon treatment of HCT116 colon cancer cells is sufficient to fully suppress the expression of the ISG15 E1 enzyme, UBA7. Stimulation of lung adenocarcinoma A549 cells with interferon reduces their growth rate but they remain viable. In contrast, A549 USP18 knock-out cells show similar growth characteristics under basal conditions, but upon interferon stimulation a profound inhibition of cell growth is observed. We show that this contingency on USP18 is independent of ISGylation, suggesting non catalytic functions are required for viability. We also demonstrate that global deISGylation kinetics are very slow compared with deubiquitylation. This is not influenced by USP18 expression, suggesting that enhanced ISGylation in USP18 KO cells reflects increased conjugating activity.
Non-invasive Analysis of Peptidoglycan from Living Animals
Authors: Ocius Karl L. , Kolli Sree H. , Ahmad Saadman S. , Dressler Jules M. , Chordia Mahendra D. , Jutras Brandon L. , Rutkowski Melanie R. , Pires Marcos M. 
Published: July 2023

AbstractThe role of the intestinal microbiota in host health is increasingly revealed in its contributions to disease states. The host-microbiome interaction is multifactorial and dynamic. One of the factors that has recently been strongly associated with host physiological responses is peptidoglycan from bacterial cell walls. Peptidoglycan from gut commensal bacteria activate peptidoglycan sensors in human cells, including the Nucleotide-binding oligomerization domain containing protein 2 (NOD2). When present in the gastrointestinal tract, both the polymeric form (sacculi) and de-polymerized fragments can modulate host physiology, including checkpoint anticancer therapy efficacy, body temperature and appetite, and postnatal growth. To leverage this growing area of biology towards therapeutic prescriptions, it will be critical to directly analyze a key feature of the host-microbiome interaction from living hosts in a reproducible and non-invasive way. Here we show that metabolically labeled peptidoglycan/sacculi can be readily isolated from fecal samples collected from both mice and humans. Analysis of fecal samples provided a non-invasive route to probe the gut commensal community including the metabolic synchronicity with the host circadian clock. Together, these results pave the way for non-invasive diagnostic tools to interrogate the causal nature of peptidoglycan in host health and disease.
Transcription factor-mediated direct cellular reprogramming yields cell-type specific DNA methylation signature
Authors: Horisawa Kenichi , Miura Shizuka , Araki Hiromitsu , Miura Fumihito , Ito Takashi , Suzuki Atsushi 
Published: July 2023

AbstractDirect reprogramming is a technique for inducing the conversion of one type of somatic cell into another by the forced expression of defined transcription factors. Cell differentiation is generally determined by specific gene expression profiles based on distinct genome-wide epigenetic signatures. Although the CpG methylation of genomic DNA is an essential epigenetic factor that affects the transcriptional state of genes, little is known about how DNA methylation changes and what roles it plays in direct reprogramming. Here, we performed comparative genome-wide DNA methylation analyses of mouse embryonic fibroblasts (MEFs) and cells composing organoids formed by intestinal stem cells (ISCs) or induced ISCs (iISCs) that were directly induced from MEFs to investigate the impact of DNA methylation dynamics on direct reprogramming. We found that the methylation state of CpG was similar between cells forming ISC organoids and iISC organoids, while they differed widely from those in MEFs. Moreover, genomic regions that were differentially methylated between ISC organoid- and iISC organoid-forming cells did not significantly affect gene expression. These results demonstrate the accuracy and safety of iISC induction, as they show that the DNA methylation state transitions to a state close to that of ISCs during direct reprogramming from MEFs to iISCs.
Spatiotemporal and single-cell atlases to dissect cell lineage differentiation and regional specific cell types in mouse ovary morphogenesis
Authors: Zhao Zheng-Hui , Meng Tie-Gang , Gao Fei , Schatten Heide , Sun Qing-Yuan 
Published: July 2023

AbstractCharacterization of cell heterogeneity and molecular diversity using single-cell RNA sequencing has greatly enhanced our understanding of the ovary’s dynamic differentiation processes. However, regional specification of ovarian cells for certain physiological functions remains largely unexplored in the physical space. Here, we combine spatial transcriptomics with single-cell RNA sequencing technologies to build a spatiotemporal and single-cell atlas of ovaries from fetal to adult stages. We construct the pseudotime trajectories of female germ cells and bipotential pregranulosa cells and define key regulatory transcription factors responsible for their differentiation processes. Specifically, we dissect the relationships between two waves of meiosis initiation, oogenesis processes and folliculogenesis. Moreover, we characterize the region-specific subtypes of granulosa cells and luteal cells and construct pseudo-space-time trajectories from granulosa cells to luteal cells. Notably, we identify small luteal cells, a novel cell type, which highly expressOnecut2and exclusively locate at the corpus luteum. Altogether, this study comprehensively delineates ovary development and regional specific ovarian cell types.
Cas9-directed long-read sequencing to resolve optical genome mapping findings in leukemia diagnostics
Authors: de Boer Eddy N. , Vroom Vincent , Scheper Arjen J. , Johansson Lennart F. , Bosscher Laura , Rietema Nettie , Commandeur Sabrina Z. , Knoers Nine V.A.M. , Sikkema-Raddatz Birgit , van den Berg Eva , van Diemen Cleo C. 
Published: July 2023

AbstractLeukemias are genetically heterogeneous, and knowing the type of genetic aberration is clinically important for progression prognosis and choice of treatment. The standard-of-care (SOC) methods in leukemia diagnostics are karyotyping, SNP-array and FISH. Optical genome mapping (OGM) has emerged as their replacement as it can detect different types of structural aberrations simultaneously. However, although OGM can additionally detect much smaller aberrations (500 bp vs. 5–10 Mb with karyotyping), its resolution is too low to define the location of aberrations between two labels and the breakpoints are disputable when labels are not distinct enough. Here, we test whether Cas9-directed long-read sequencing (LRS) can fill this gap.From an internal Bionano implementation study we selected ten OGM calls either located in low resolution areas or with disputable breakpoints that could not be validated with SOC methods. Per variant we designed crRNAs for Cas9 enrichment, prepared libraries and sequenced them on a MinION / GridION device.We could confirm all the OGM aberrations with Cas9-directed LRS and the actual breakpoints of the OGM calls were located between 0.2–5.5 kb of the OGM-estimated breakpoints, confirming the high reliability of OGM. Furthermore, we show examples of redefinition of aberrations between labels that enable judgment of clinical relevance.We show that Cas9-directed LRS can fill the gap of low resolution OGM areas thereby improving the prediction of clinical significance. We envisage that this technique can be an relevant secondary technique in diagnostic workflows including OGM.
Expanding the Substrate Scope ofN- andO-Methyltransferases from Plants for Chemoselective Alkylation
Authors: Jockmann Emely , Subrizi Fabiana , Mohr Michael K. F. , Carter Eve M. , Hebecker Pia M. , Popadić Désirée , Hailes Helen C. , Andexer Jennifer N. 
Published: July 2023

AbstractMethylation reactions are of significant interest when generating pharmaceutically active molecules and building blocks for other applications. Synthetic methylating reagents are often toxic and unselective due to their high reactivity.S-Adenosyl-L-methionine (SAM)-dependent methyltransferases (MTs) present a chemoselective and environmentally friendly alternative. The anthranilateN-MT fromRuta graveolens(RgANMT) is involved in acridone alkaloid biosynthesis, methylating anthranilate. Although it is known to methylate substrates only at theN-position, the closest relatives with respect to amino acid sequence similarities of over 60% areO-MTs catalysing the methylation reaction of caffeate and derivatives containing only hydroxyl groups (CaOMTs). In this study, we investigated the substrate range ofRgANMT and a CaOMT fromPrunus persica(PpCaOMT) using compounds with both, an amino- and hydroxyl group (aminophenols) as possible methyl group acceptors. For both enzymes, the reaction was highly chemoselective. Furthermore, generating cofactor derivatives in situ enabled the transfer of other alkyl chains onto the aminophenols, leading to an enlarged pool of products. Selected MT reactions were performed at a preparative biocatalytic scale in in vitro and in vivo experiments resulting in yields of up to 62%.
Parallel evolution and enhanced virulence uponin vivopassage of an RNA virus inDrosophila melanogaster
Authors: Lezcano Oscar M. , Fuhrmann Lara , Ramakrishnan Gayatri , Beerenwinkel Niko , Huynen Martijn A. , van Rij Ronald P. 
Published: July 2023

AbstractVirus evolution is strongly affected by antagonistic co-evolution of virus and host. Host immunity positively selects for viruses that evade the immune response, which in turn may drive counter-adaptations in host immune genes. We investigated how host immune pressure shapes virus populations, using the fruit flyDrosophila melanogasterand its natural pathogen Drosophila C virus (DCV), as a model. We performed an experimental evolution study in which DCV was serially passaged for ten generations in three fly genotypes differing in their antiviral RNAi response: wild-type flies and flies in which the endonuclease geneDicer-2was either overexpressed or inactivated. All evolved virus populations replicated more efficientlyin vivoand were more virulent than the parental stock. The number of polymorphisms increased in all three host genotypes with passage number, which was most pronounced inDicer-2knockout flies. Mutational analysis showed strong parallel evolution, as mutations accumulated in a specific region of the VP3 capsid protein in every lineage in a host genotype-independent manner. The parental tyrosine at position 95 of VP3 was substituted with either one of five different amino acids in 14 out of 15 lineages. However, no consistent amino acid changes were observed in the viral RNAi suppressor gene 1A, nor elsewhere in the genome in any of the host backgrounds. Our study indicates that the RNAi response restricts the sequence space that can be explored by viral populations. Moreover, our study illustrates how evolution towards higher virulence can be a highly reproducible, yet unpredictable process.
Autophagy restricts fungal accommodation in the roots ofArabidopsis thaliana
Authors: Zecua-Ramirez Patricia , Llamas Ernesto , Charura Nyasha M. , Dunken Nick , De Quattro Concetta , Mandel Alexander , Langen Gregor , Dagdas Yasin , Zuccaro Alga 
Published: July 2023

AbstractEndophytic colonization ofArabidopsis thalianaby the beneficial root endophyteSerendipita indicais characterized by an initial biotrophic phase followed by a restricted host cell death-associated phase. This latter phase involves regulated cell death (RCD) for fungal accommodation. However, the host molecular pathways that limitS. indicacolonization and govern symbiosis remain largely unknown. Our study demonstrates that autophagy, a major cellular degradation pathway, is activated duringS. indicacolonization and is required to restrict fungal colonization in Arabidopsis. Independent Arabidopsis knockout (KO) mutants deficient in autophagosome formation are more susceptible to deoxyadenosine (dAdo), a cell death inducer produced by two secretedS. indicaeffectors at the onset of the cell death-associated phase. In theatg5autophagy mutant background, impaired dAdo uptake prevents dAdo-induced and symbiosis-mediated cell death. Based on our data, we propose that autophagy-mediated pro-survival responses in the host are crucial for maintaining a balanced symbiotic interaction betweenS. indicaand Arabidopsis.In a NutshellOur study reveals that during colonization ofArabidopsis thalianaroots by the beneficial root endophyteSerendipita indica, autophagy, a key cellular degradation pathway, is activated to limit fungal colonization. Autophagy-deficient Arabidopsis mutants are more susceptible to deoxyadenosine (dAdo), a cell death inducer produced byS. indica. We propose that autophagy-mediated pro-survival responses are essential for maintaining a balanced symbiotic interaction betweenS. indicaand Arabidopsis.