bluuebunny/crossref_metadata_2025_split huggingface dataset:
CRISPR-Cas-Docker: Web-basedin silicodocking and machine learning-based classification of crRNAs with Cas proteins
Authors: Park Ho-min , Won Jongbum , Park Yunseol , Anzaku Esla Timothy , Vankerschaver Joris , Van Messem Arnout , De Neve Wesley , Shim Hyunjin 
Published: January 2023

AbstractMotivationCRISPR-Cas-Docker is a web server forin silicodocking experiments with CRISPR RNAs (crRNAs) and Cas proteins. This web server aims at providing experimentalists with the optimal crRNA-Cas pair predicted computationally when prokaryotic genomes have multiple CRISPR arrays and Cas systems, as frequently observed in metagenomic data. CRISPR-Cas-Docker provides two methods to predict the optimal Cas protein given a particular crRN sequence: a structure-based method (in silicodocking) and a sequence-based method (machine learning classification). For the structure-based method, users can either provide experimentally determined 3D structures of these macromolecules or use an integrated pipeline to generate 3D-predicted structures forin silicodocking experiments.ResultsCRISPR-Cas-Docker is an optimized and integrated platform that provides users with 1) 3D-predicted crRNA structures and AlphaFold-predicted Cas protein structures, 2) the top-10 docking models for a particular crRNA-Cas protein pair, and 3) machine learning-based classification of crRNA into its Cas system type.Availability and implementationCRISPR-Cas-Docker is available as an open-source tool under the GNU General Public License v3.0 on GitHub. It is also available as a web server.
Three bacterial DedA subfamilies with distinct functions and phylogenetic distribution
Authors: Todor Horia , Herrera Nadia , Gross Carol 
Published: January 2023

ABSTRACTRecent studies in bacteria suggested that the broadly conserved but enigmatic DedA proteins function as undecaprenyl-phosphate (UndP) flippases, recycling this essential lipid carrier. To determine whether all DedA proteins have UndP flippase activity, we performed a phylogenetic analysis and correlated it to previously published experimental results and predicted structures. We uncovered three major DedA subfamilies: one contains UndP flippases, the second contains putative phospholipid flippases and is associated with aerobic metabolism, and the third is found only in specific Gram-negative phyla.IMPORTANCEDedA-family proteins are highly conserved and nearly ubiquitous integral membrane proteins found in Archaea, Bacteria, and Eukaryotes. Recent work revealed that eukaryotic DedA proteins are phospholipid scramblases and some bacterial DedA proteins are undecaprenyl phosphate flippases. We perform a phylogenetic analysis of this protein family in Bacteria revealing 3 DedA subfamilies with distinct phylogenetic distributions, genomic contexts, and putative functions. Our analysis lays the groundwork for a deeper understanding of DedA proteins and their role in maintaining and modifying the membrane.
ATP is a major determinant of phototrophic bacterial longevity in growth arrest
Authors: Yin Liang , Ma Hongyu , Fones Elizabeth M. , Morris David R. , Harwood Caroline S. 
Published: January 2023

ABSTRACTHow bacteria transition into growth arrest as part of stationary phase has been well-studied, but our knowledge of features that help cells to stay alive in the following days and weeks is incomplete. Most studies have used heterotrophic bacteria that are growth-arrested by depletion of substrates used for both biosynthesis and energy generation, making is difficult to disentangle the effects of the two. In contrast, when grown anaerobically in light, the phototrophic bacteriumRhodopseudomonas palustrisgenerates ATP from light via cyclic photophosphorylation and builds biomolecules from organic substrates such as acetate. As such, energy generation and carbon utilization are independent from one another. Here we compared the physiological and molecular responses ofR. palustristo growth arrest caused by carbon source depletion in light (energy-replete) and dark (energy-depleted) conditions. Both sets of cells remained viable for six to ten days, at which point dark-incubated cells lost viability whereas light-incubated cells remained fully viable for 60 days. Dark-incubated cells were depleted in intracellular ATP prior to losing viability, suggesting that ATP depletion is a cause of cell death. Dark-incubated cells also shut down measurable protein synthesis, whereas light-incubated cells continued to synthesize proteins at low levels. Cells incubated in both conditions continued to transcribe genes. We suggest thatR. palustrismay completely shut down protein synthesis in dark, energy-depleted, conditions as a strategy to survive the nighttime hours of day/night cycles it experiences in nature, where there is a predictable source of energy in the form of sunlight during days.IMPORTANCEThe molecular and physiological basis of bacterial longevity in growth arrest is important to investigate for several reasons. Such investigations could improve treatment of chronic infections, advance use of non-growing bacteria as biocatalysts to make high yields of value-added products, and improve estimates of microbial activities in natural habitats, where cells are often growing slowly or not at all. Here we compared survival of the phototrophic bacteriumRhodopseudomonas palustrisunder conditions where it generates ATP (incubation in light) and where it does not generate ATP (incubation in dark) to directly assess effects of energy depletion on long-term viability. We found that ATP is important for long-term survival over weeks. However,R. palustrissurvives 12h periods of ATP depletion without loss of viability, apparently in anticipation of sunrise and restoration of its ability to generate ATP. Our work suggests that cells respond to ATP depletion by shutting down protein synthesis.
Highly pathogenic avian influenza A H5 virus outbreaks in broiler farms in the Netherlands – Clinical signs, transmission and identification of reporting thresholds
Authors: Gonzales Jose L. , Hobbelen Peter H.F. , van der Spek Arco N. , Vries Edwin P. , Elbers Armin R.W. 
Published: January 2023

AbstractBackgroundFor a successful control of highly pathogenic avian influenza virus (HPAIV) epidemics in poultry, early detection is key and it is mostly dependent on the farmer’s prompt identification of disease and reporting suspicions. The latter could be further improved by providing references to farmers for triggering suspicions.MethodsHere we report observations on clinical signs of HPAIV H5N1 and H5N8 infected broiler farms in the Netherlands and analyze their daily mortality and feed and water intake data in order to identify thresholds for reporting suspicions. In addition, mortality data was used to characterize the transmissibility of these viruses, which could help estimate how fast infection spreads within the flock and when infection was likely introduced.ResultsThe most frequently observed clinical signs in affected flocks were sudden increase in mortality, cyanosis of wattles comb and/or legs and hemorrhagic conjunctiva. Analysis of mortality data indicate that daily mortality higher than 0.17% is an effective threshold for reporting a HPAIV-suspicion. Reliable thresholds for food or water intake could not be stablished. The estimated within-flock transmission rates ranged from 1.1 to 2.0 infections caused by one infectious broiler chicken per day.ConclusionsWe identified effective mortality thresholds for reporting suspicions of HPAIV infections. The estimated transmission rates appear to indicate a slow progression of a H5 HPAIV outbreak in affected broiler flocks. The information here provided can be used to improve syndromic surveillance and guide outbreak response.
Intermediate molecular phenotypes to identify genetic markers of anthracycline-induced cardiotoxicity risk
Authors: Gómez-Vecino Aurora , Corchado-Cobos Roberto , Blanco-Gómez Adrián , García-Sancha Natalia , Castillo-Lluva Sonia , Martín-García Ana , Mendiburu-Eliçabe Marina , Prieto Carlos , Ruiz-Pinto Sara , Pita Guillermo , Velasco-Ruiz Alejandro , Patino-Alonso Carmen , Galindo-Villardón Purificación , Vera-Pedrosa María Linarejos , Jalife José , Mao Jian-Hua , de Plasencia Guillermo Macías , Castellanos-Martín Andrés , del Mar Sáez Freire María , Fraile-Martín Susana , Rodrigues-Teixeira Telmo , García-Macías Carmen , Galvis-Jiménez Julie Milena , García-Sánchez Asunción , Isidoro-García María , Fuentes Manuel , García-Cenador María Begoña , García-Criado Francisco Javier , García Juan Luis , Hernández-García María Ángeles , Cruz Hernández Juan Jesús , Rodríguez-Sánchez César Augusto , Martín-Ruiz Alejandro , Pérez-López Estefanía , Pérez-Martínez Antonio , Gutiérrez-Larraya Federico , Cartón Antonio J. , García-Sáenz José Ángel , Patiño-García Ana , Martín Miguel , Gordoa Teresa Alonso , Vulsteke Christof , Croes Lieselot , Hatse Sigrid , Van Brussel Thomas , Lambrechts Diether , Wildiers Hans , Hang Chang , Holgado-Madruga Marina , González-Neira Anna , Sánchez Pedro L , Pérez Losada Jesús 
Published: January 2023

AbstractCardiotoxicity due to anthracyclines (CDA) affects cancer patients, but we cannot predict who may suffer from this complication. CDA is a complex disease whose polygenic component is mainly unidentified. We propose that levels of intermediate molecular phenotypes in the myocardium associated with histopathological damage could explain CDA susceptibility; so that variants of genes encoding these intermediate molecular phenotypes could identify patients susceptible to this complication. A genetically heterogeneous cohort of mice generated by backcrossing (N = 165) was treated with doxorubicin and docetaxel. Cardiac histopathological damage was measured by fibrosis and cardiomyocyte size by an Ariol slide scanner. We determine intramyocardial levels of intermediate molecular phenotypes of CDA associated with histopathological damage and quantitative trait loci (ipQTLs) linked to them. These ipQTLs seem to contribute to the missing heritability of CDA because they improve the heritability explained by QTL directly linked to CDA (cda-QTLs) through genetic models. Genes encoding these molecular subphenotypes were evaluated as genetic markers of CDA in three cancer patient cohorts (N = 517) whose cardiac damage was quantified by echocardiography or Cardiac Magnetic Resonance. Many SNPs associated with CDA were found using genetic models. LASSO multivariate regression identified two risk score models, one for pediatric cancer patients and the other for women with breast cancer. Molecular intermediate phenotypes associated with heart damage can identify genetic markers of CDA risk, thereby allowing a more personalized patient management. A similar strategy could be applied to identify genetic markers of other complex trait diseases.
TheFIBRILLINmultigene family in tomato, their roles in plastoglobuli structure and metabolism
Authors: Almeida Juliana , Perez-Fons Laura , Drapal Margit , Liew Kit , Fraser Paul D. 
Published: January 2023

AbstractPlastoglobuli (PG) are plant lipoprotein compartments, present in plastid organelles. They are involved in the formation and/or storage of lipophilic metabolites. FIBRILLINs (FBN) are one of the main PG-associated proteins and are particularly abundant in carotenoid-enriched chromoplasts found in ripe fruits and flowers. To address the contribution of different FBNs to isoprenoid sequestration and PG function, a multiplex gene editing approach was undertaken. Analysis of single and high-orderfbnmutants for the major PG-related FBNs in tomato, namelySlFBN1,SlFBN2a,SlFBN4,SlFBN7a, revealed functional redundancy. High orderfbnmutants displayed phenotypes associated with abnormal isoprenoid accumulation, and aberrant PG formation and morphology. Lipidomic analysis highlighted broader changes in lipid metabolism. Paralog-specific roles were also observed and included the regulation of specific isoprenoids (e.g., plastochromanol) and control of plastidial esterification capability by SlFBN7a. Collectively, the results support both structural and regulatory roles of SlFBNs in PGs. Our findings expose fundamental aspects of metabolic compartmentalisation in plant cells and the importance of lipoprotein particles for their plastid metabolism/physiology.Significance statementIn the chromoplast of ripe tomato fruit and flower, plastoglobuli (PGs) are associated with several important biotechnological traits, due to their functional involvement in metabolism, developmental transitions, and environmental adaption. FIBRILLINS (FBN) are a multigene family of proteins that are collectively major components of the PG. Using a multiplex CRISPR-Cas9 approach single and high-orderfbnmutants have been developed. Functional redundancy amongst the members of theFBNmultigene family was evident, but also paralog specific functions/influence. Aberrant plastoglobuli formation and altered lipid metabolism are evident amongfbnmutants. Characterisation of this resource has shed light on the functional role of FBN and their role in PG formation. This strategy offers new potential for the development of nutritional enhanced and climate resilient crops.
Cell-free expression and characterization of multivalent rhamnose-binding lectins using biolayer interferometry
Authors: Warfel Katherine F. , Laigre Eugénie , Sobol Sarah E. , Gillon Emilie , Varrot Annabelle , Renaudet Olivier , Dejeu Jerome , Jewett Michael C. , Imberty Anne 
Published: January 2023

AbstractLectins are important biological tools for binding glycans, but recombinant protein expression poses challenges for some lectin classes, limiting the pace of discovery and characterization. To discover and engineer lectins with new functions, workflows amenable to rapid expression and subsequent characterization are needed. Here, we present bacterial cell-free protein synthesis as a means for efficient, small-scale expression of multivalent, disulfide bond-rich, rhamnose-binding lectins. Furthermore, we demonstrate that the cell-free expressed lectins can be directly coupled with BLI analysis, either in solution or immobilized on the sensor, to measure interaction with carbohydrate ligands without purification. This workflow enables determination of lectin substrate specificity and estimation of binding affinity. Overall, we believe that this method will enable high-throughput expression, screening, and characterization of new and engineered multivalent lectins for applications in synthetic glycobiology.
A genomic test of subspecies in theEunota togataspecies group (Coleoptera: Cicindelidae): Morphology masks evolutionary relationships and taxonomy
Authors: Laroche Robert A. , Duran Daniel P. , Lee Cin-Ty A. , Godwin William , Roman Stephen J. , Herrmann David P. , Egan Scott P. 
Published: January 2023

AbstractMost of the world’s biodiversity is described primarily or exclusively using morphological traits that may not always reflect the true evolutionary units. Accurate taxonomy is critical for conservation efforts and re-evaluation of traditional taxonomy may often be warranted since species and subspecies are frequently the focus of conservation and faunistic studies. Here, we test comprehensive taxonomic hypotheses of morphologically defined subspecies in the tiger beetle,Eunota togata(LaFerté-Sénectère, 1841). The four recognized subspecies were delineated based mainly on the dorsal coloration and extent of white markings termed maculations. We combine inferences from mtDNA genealogies and genome-wide multilocus data to elucidate the evolutionary relationships within the group and assess the taxonomic implications. Three of the four subspecific taxa delineated by morphology were not supported by the genomic or mtDNA data. In fact, the species-level diversity in this group was underestimated, asE. togatawas found to represent three well-supported distinct species in all genetic analyses. Emerging from these analyses, we also document an intriguing example of convergent evolution in lighter coloredE. togataadapting to similar white sand backgrounds. Our collective work underscores the importance of integrating molecular methods with morphology for species and subspecies delimitation and conservation.
Competition co-immunoprecipitation reveals interactors of the chloroplast CPN60 chaperonin machinery
Authors: Ries Fabian , Weil Heinrich Lukas , Herkt Claudia , Mühlhaus Timo , Sommer Frederik , Schroda Michael , Willmund Felix 
Published: January 2023

SUMMARYThe functionality of essential metabolic processes in chloroplasts depends on a balanced integration of nuclear-and chloroplast-encoded polypeptides into the plastid’s proteome. The chloroplast chaperonin machinery is an essential player in chloroplast protein folding with a more intricate structure and subunit composition compared to the orthologous GroEL/ES chaperonin ofEscherichia coli. However, its exact role in chloroplasts remains obscure, mainly because of a very limited knowledge about the folded substrates. We employed the competition immunoprecipitation method for the identification of the chaperonin’s substrates inChlamydomonas reinhardtii. Co-immunoprecipitation of the target complex in the presence of increasing amounts of isotope-labelled competitor epitope and subsequent mass spectrometry analysis specifically allowed to distinguish true interactors from unspecifically co-precipitated proteins. Besides known substrates such as RbcL, we revealed numerous new substrates with high confidence. Identified substrate proteins differ from bulk chloroplast proteins by a higher content of beta-sheets, lower alpha-helical content and increased aggregation propensity. Immunoprecipitations performed with a subunit of the co-chaperonin lid revealed the ClpP protease as a specific partner complex, with altered interactions during heat stress, pointing to a close collaboration of these machineries to maintain protein homeostasis in the chloroplast.
Global gene expression of human malaria parasite liver stages throughout intrahepatocytic development
Authors: Zanghi Gigliola , Patel Hardik , Camargo Nelly , Smith Jenny L. , Bae Yeji , Flannery Erika L. , Chuenchob Vorada , Fishbaugher Matthew E. , Mikolajczak Sebastian A , Roobsoong Wanlapa , Sattabongkot Jetsumon , Hayes Kiera , Vaughan Ashley M. , Kappe Stefan H. I. 
Published: January 2023

ABSTRACTPlasmodium falciparum(Pf) is causing the greatest malaria burden, yet the liver stages (LS) of this most important parasite species have remained poorly studied. Here, we used a human liver-chimeric mouse model in combination with a novel fluorescentPfNF54 parasite line (PfNF54cspGFP) to isolatePfLS-infected hepatocytes and generate transcriptomes that cover the major LS developmental phases in human hepatocytes. RNA-seq analysis of earlyPfLS trophozoites two days after infection, revealed a central role of translational regulation in the transformation of the extracellular invasive sporozoite into intracellular LS. The developmental time course gene expression analysis indicated that fatty acid biosynthesis, isoprenoid biosynthesis and iron metabolism are sustaining LS development along with amino acid metabolism and biosynthesis. Countering oxidative stress appears to play an important role during intrahepatic LS development. Furthermore, we observed expression of the variant PfEMP1 antigen-encodingvargenes, and we confirmed expression of PfEMP1 protein during LS development. Transcriptome comparison of the latePfliver stage schizonts withP. vivax(Pv) late liver stages revealed highly conserved gene expression profiles among orthologous genes. A notable difference however was the expression of genes regulating sexual stage commitment. WhilePvschizonts expressed markers of sexual commitment, thePfLS parasites were not sexually committed and showed expression of gametocytogenesis repression factors. Our results provide the first comprehensive gene expression profile of the human malaria parasitePfLS isolated duringin vivointrahepatocytic development. This data will inform biological studies and the search for effective intervention strategies that can prevent infection.
Comparison of SARS-CoV-2 entry inhibitors based on ACE2 receptor or engineered Spike-binding peptides
Authors: Llewellyn George N. , Chen Hsu-Yu , Rogers Geoffrey L. , Huang Xiaoli , Sell Philip J. , Henley Jill E. , Cannon Paula M. 
Published: January 2023

AbstractWith increasing resistance of SARS-CoV-2 variants to antibodies, there is interest in developing entry inhibitors that target essential receptor binding regions of the viral Spike protein and thereby present a high bar for viral resistance. Such inhibitors can be derivatives of the viral receptor, ACE2, or peptides engineered to interact specifically with the receptor-binding pocket. We compared the efficacy of a series of both types of entry inhibitors, constructed as fusions to an antibody Fc domain. Such a design can increase protein stability and act to both neutralize free virus and recruit effector functions to clear infected cells. We tested the reagents against prototype variants of SARS-CoV-2, using both Spike pseudotyped VSV vectors and viral plaque assays. These analyses revealed that an optimized ACE2 derivative could neutralize all variants we tested with high efficacy. In contrast, the Spike-binding peptides had varying activities against different variants, with resistance observed for the Spike proteins from Beta, Gamma and Omicron. The resistance mapped to mutations at Spike residues K417 and N501 and could be overcome for one of the peptides by linking two copies in tandem, effectively creating a tetrameric reagent in the Fc fusion. Finally, both the optimized ACE2 and tetrameric peptide inhibitors provided some protection to human ACE2 transgenic mice challenged with the SARS-CoV-2 Delta variant, which typically causes death in this model within 7-9 days.ImportanceThe increasing resistance of SARS-CoV-2 variants to therapeutic antibodies has highlighted the need for new treatment options, especially in individuals who do not respond to vaccination. Receptor decoys that block viral entry are an attractive approach because of the presumed high bar to developing viral resistance. Here, we compare two entry inhibitors based on derivatives of the ACE2 receptor or engineered peptides that bind to the receptor binding pocket of the SARS-CoV-2 Spike protein. In each case, the inhibitors were fused to immunoglobulin Fc domains, which can further enhance therapeutic properties, and compared for activity against different SARS-CoV-2 variants. Potent inhibition against multiple SARS-CoV-2 variants was demonstratedin vitro, and even relatively low single doses of optimized reagents provided some protection in mouse models, confirming their potential as an alternative to antibody therapies.