bluuebunny/crossref_metadata_2025_split huggingface dataset:
Density modification of cryo-EM maps
Authors: Terwilliger Thomas C. , Sobolev Oleg V. , Afonine Pavel V. , Adams Paul D. , Read Randy J. 
Published: May 2020

AbstractDensity modification uses expectations about features of a map such as a flat solvent and expected distributions of density in the region of the macromolecule to improve individual Fourier terms representing the map. This process transfers information from one part of a map to another and can improve the accuracy of a map. Here the assumptions behind density modification for maps from electron cryomicroscopy are examined and a procedure is presented that allows incorporation of model-based information. Density modification works best in cases where unfiltered, unmasked maps with clear boundaries between macromolecule and solvent are visible and where there is substantial noise in the map, both in the region of the macromolecule and the solvent. It also is most effective if the characteristics of the map are relatively constant within regions of the macromolecule and the solvent. Model-based information can be used to improve density modification, but model bias can in principle occur. Here model bias is reduced by using ensemble models that allow estimation of model uncertainty. A test of model bias is presented suggesting that even if the expected density in a region of a map is specified incorrectly by using an incorrect model, the incorrect expectations do not strongly affect the final map.SynopsisThe prerequisites for density modification of maps from electron cryomicroscopy are examined and a procedure for incorporating model-based information is presented.
VST Family Proteins are Regulators of Root System Architecture in Rice andArabidopsis
Authors: Shao Yanlin , Lehner Kevin R. , Zhou Hongzhu , Taylor Isaiah , Mao Chuanzao , Benfey Philip N. 
Published: May 2020

AbstractRoot System Architecture (RSA) is a key factor in the efficiency of nutrient capture and water uptake in plants. Understanding the genetic control of RSA will be useful in minimizing fertilizer and water usage in agricultural cropping systems. Using a hydroponic screen and a gel-based imaging system we identified a rice gene,OsVST1, which plays a key role in controlling RSA. This gene encodes a homolog of theArabidopsisVAP-RELATED SUPPRESSORS OF TMM (VSTs), a class of proteins that promote signaling in stomata by mediating plasma membrane-endoplasmic reticulum contacts.OsVST1mutants have shorter primary roots, decreased root meristem size, and a more compact root system architecture. We show that theArabidopsisVST triple mutants have similar phenotypes, with reduced primary root growth and smaller root meristems. Expression ofOsVST1largely complements the short root length and reduced plant height in theArabidopsistriple mutant, supporting conservation of function between rice andArabidopsisVST proteins. In a field trial, mutations inOsVST1do not adversely affect grain yield, suggesting that modulation of this gene could be used as a way to optimize RSA without an inherent yield penalty.
In vivoanalysis ofTrypanosoma cruzipersistence foci at single cell resolution
Authors: Ward Alexander I. , Lewis Michael D. , Khan Archie , McCann Conor J. , Francisco Amanda F. , Jayawardhana Shiromani , Taylor Martin C. , Kelly John M. 
Published: May 2020

ABSTRACTInfections withTrypanosoma cruziare usually life-long despite generating a strong adaptive immune response. Identifying the sites of parasite persistence is therefore crucial to understand howT. cruziavoids immune-mediated destruction. However, this is a major technical challenge because the parasite burden during chronic infections is extremely low. Here, we describe an integrated approach involving comprehensive tissue processing,ex vivoimaging, and confocal microscopy, which has allowed us to visualise infected host cells in murine tissue, with exquisite sensitivity. Using bioluminescence-guided tissue sampling, with a detection level of <20 parasites, we show that in the colon, smooth muscle myocytes in the circular muscle layer are the most common infected host cell type. Typically, during chronic infections, the entire colon of a mouse contains only a few hundred parasites, often concentrated in a small number of cells containing >200 parasites, that we term mega-nests. In contrast, during the acute stage, when the total parasite burden is considerably higher and many cells are infected, nests containing >50 parasites are rarely found. In C3H/HeN mice, but not BALB/c, we identified skeletal muscle as a major site of persistence during the chronic stage, with most parasites found in large mega-nests within the muscle fibres. Finally, we report that parasites are also frequently found in the skin during chronic murine infections, often in multiple infection foci. In addition to being a site of parasite persistence, this anatomical reservoir could play an important role in insect-mediated transmission, and have implications for drug development.IMPORTANCETrypanosoma cruzicauses Chagas disease, the most important parasitic infection in Latin America. Major pathologies include severe damage to the heart and digestive tract, although symptoms do not usually appear until decades after infection. Research has been hampered by the complex nature of the disease and technical difficulties in locating the extremely low number of parasites. Here, using highly sensitive imaging technology, we reveal the sites of parasite persistence in experimental mice at single-cell resolution. We show that parasites are frequently located in smooth muscle cells in the circular muscle layer of the colon, and that skeletal muscle cells and the skin can also be important reservoirs. This information provides a framework for investigating how the parasite is able to survive as a life-long infection, despite a vigorous immune response. It also informs drug-development strategies by identifying tissue sites that must be accessed to achieve a curative outcome.
Gain of function mutagenesis through activation tagging identifiesXPB2andSEN1helicase genes as potential targets for drought stress tolerance in rice
Authors: Dutta Mouboni , Moin Mazahar , Saha Anusree , Bakshi Achala , Kirti P.B. 
Published: May 2020

AbstractWe have earlier reported on the development of an activation tagged gain-of-function mutant population in anindicarice variety, BPT-5204 (Moin et al. 2016). Screening of these gain of function mutants for water-use efficiency (WUE) followed by physiological analyses revealed the activation of two helicases, ATP-dependent RNA (SEN1) and DNA (XPB2) encoding unwinding proteins in two different mutant lines. In the current study, we examined the roles of these genes in stable activation tagged mutants of rice for drought stress responses. Transcript profiling ofSEN1andXPB2showed their significant up-regulation under various stresses (particularly ABA and PEG). TheSEN1andXPB2tagged mutants exhibited reduced leaf wilting, improved revival efficiency, high chlorophyll and proline contents, profuse tillering, high quantum efficiency and yield-related traits in response to simulated drought (PEG) and hormone (ABA) treatments with respect to their controls. These observations were further validated under greenhouse conditions by periodic withdrawal of water. Germination of the seeds of these mutant lines indicates their ABA insensitivity under high ABA concentration. Also, the associated high up-regulation of stress-specific genes suggests that their drought tolerance might have been because of the coordinated expression of several stress responsive genes in these two mutants. Altogether, our results provided a firm basis forSEN1andXPB2as potential candidates for manipulation of drought tolerance and improving rice performance and yield under limited water conditions.
Insulin receptor activation by proinsulin preserves synapses and vision in retinitis pigmentosa
Authors: Sánchez-Cruz Alonso , Hernández-Pinto Alberto , Lillo Concepción , Isiegas Carolina , Marchena Miguel , Lizasoain Ignacio , Bosch Fátima , de la Villa Pedro , Hernández-Sánchez Catalina , de la Rosa Enrique J. 
Published: May 2020

ABSTRACTSynaptic loss, neuronal death, and circuit remodeling are common features of central nervous system neurodegenerative disorders. Retinitis pigmentosa (RP), the leading cause of inherited blindness, is a group of retinal dystrophies characterized by photoreceptor dysfunction and death. The insulin receptor, a key controller of metabolism, also regulates neuronal survival and synaptic formation, maintenance, and activity. Indeed, deficient insulin receptor signaling has been implicated in several brain neurodegenerative pathologies. We present evidence linking impaired insulin receptor signaling with RP. We describe a selective decrease in the levels of the insulin receptor and its downstream effector phospho-S6 in retinal horizontal cell axons in the rd10 mouse model of RP, as well as aberrant synapses between rod photoreceptors and the postsynaptic terminals of horizontal and bipolar cells. A gene therapy strategy to induce sustained proinsulin production restored retinal insulin receptor signaling, by increasing S6 phosphorylation, without peripheral metabolic consequences. Moreover, proinsulin preserved photoreceptor synaptic connectivity and prolonged visual function in electroretinogram and optomotor tests. These findings support the therapeutic potential of proinsulin in RP.
CRISPR-Assisted DNA Detection, a novel dCas9-based DNA detection technique
Authors: Xu Xinhui , Luo Tao , Gao Jinliang , Lin Na , Li Weiwei , Xia Xinyi , Wang Jinke 
Published: May 2020

AbstractNucleic acid detection techniques are always critical to diagnosis, especially in the background of the present COVID-19 pandemic. The simple and rapid detection techniques with high sensitivity and specificity are always urgently needed. However, the current nucleic acid detection techniques are still limited the traditional amplification and hybridization. To overcome the limitation, we here develop a CRISPR/Cas9-assisted DNA detection (CADD). In this detection, DNA sample is incubated with a pair of capture sgRNAs (sgRNAa and sgRNAb) specific to a target DNA, dCas9, a signal readout-related probe, and an oligo-coated solid support beads or microplate at room temperature for 15 min. During this incubation, the dCas9-sgRNA-DNA complex is formed and captured on solid support by the capture sequence of sgRNAa and the signal readout-related probe is captured by the capture sequence of sgRNAb. Finally the detection result is reported by a fluorescent or colorimetric signal readout. This detection was verified by detecting DNA of bacteria, cancer cell and virus. Especially, by designing a set of sgRNAs specific to 15 high-risk human papillomaviruses (HPVs), the HPV infection in 64 clinical cervical samples were successfully detected by the method. All detections can be finished in 30 minutes at room temperature. This detection holds promise for rapid on-the-spot detection or point-of-care testing (POCT).
Repeated global migrations on different plant hosts by the tropical pathogenPhytophthora palmivora
Authors: Wang Jianan , Coffey Michael D. , De Maio Nicola , Goss Erica M. 
Published: May 2020

AbstractThe genetic structure and diversity of plant pathogen populations are the outcomes of evolutionary interactions with hosts and local environments, and migration at different scales, including human-enabled long-distance dispersal events. As a result, patterns of genetic variation in present populations may elucidate the history of pathogens.Phytophthora palmivorais a devastating oomycete that causes disease in a broad range of plant hosts in the tropics and subtropics worldwide. The center of diversity ofP. palmivorais in Southeast Asia, but it is a destructive pathogen of hosts native to South America. Our objective was to use multilocus sequence analysis to resolve the origin and historical migration pathways ofP. palmivora. Our analysis supports Southeast Asia as a center of diversity ofP. palmivoraand indicates that a single colonization event was responsible for the global pandemic of black pod disease of cacao. Analysis using the structured coalescent indicated thatP. palmivoraemerged on cacao and that cacao has been the major source of migrants to populations in Asia, Africa, and Pacific Islands. To explain these results, we hypothesize widespread introgression between the pandemic cacao lineage and populations native to Asia and the Pacific Islands. The complex evolutionary history ofP. palmivorais a consequence of geographic isolation followed by long-distance movement and host jumps that allowed global expansion with cacao, coconut and other hosts.
Efficient Gene Targeting in Maize using Inducible CRISPR-Cas9 and Marker-Free Donor Template
Authors: Barone Pierluigi , Wu Emily , Lenderts Brian , Anand Ajith , Gordon-Kamm William , Svitashev Sergei , Kumar Sandeep 
Published: May 2020

AbstractCRISPR-Cas9 is a powerful tool for generating targeted mutations and genomic deletions. However, precise gene insertion or sequence replacement remains a major hurdle before application of CRISPR-Cas9 technology is fully realized in plant breeding. Here we report high frequency, selectable marker-free intra-genomic gene targeting (GT) in maize. Heat shock-inducible Cas9 was used for generating targeted double-strand breaks (DSBs) and simultaneous mobilization of the donor template from pre-integrated T-DNA. The construct was designed such that release of the donor template and subsequent DNA repair activated expression of the selectable marker gene within the donor locus. This approach generated up to 4.7% targeted insertion of the donor sequence into the target locus in T0 plants, with up to 86% detected donor template release and 99% mutation rate were observed at the donor loci and the genomic target site, respectively. Unlike previousin plantaor intra-genomic homologous recombination reports, that required multiple generations and extensive screening, our method provides non-chimeric, heritable GT in the T0 generation.
Functional prediction and comparative population analysis of variants in genes for proteases and innate immunity related to SARS-CoV-2 infection
Authors: Klaassen Kristel , Stankovic Biljana , Zukic Branka , Kotur Nikola , Gasic Vladimir , Pavlovic Sonja , Stojiljkovic Maja 
Published: May 2020

AbstractNew coronavirus SARS-CoV-2 is capable to infect humans and cause a novel disease COVID-19. Aiming to understand a host genetic component of COVID-19, we focused on variants in genes encoding proteases and genes involved in innate immunity that could be important for susceptibility and resistance to SARS-CoV-2 infection.Analysis of sequence data of coding regions ofFURIN, PLG, PRSS1, TMPRSS11a, MBL2andOAS1genes in 143 unrelated individuals from Serbian population identified 22 variants with potential functional effect.In silicoanalyses (PolyPhen-2, SIFT, MutPred2 and Swiss-Pdb Viewer) predicted that 10 variants could impact the structure and/or function of proteins. These protein-altering variants (p.Gly146Ser inFURIN; p.Arg261His and p.Ala494Val inPLG; p.Asn54Lys inPRSS1; p.Arg52Cys, p.Gly54Asp and p.Gly57Glu inMBL2; p.Arg47Gln, p.Ile99Val and p.Arg130His inOAS1) may have predictive value for inter-individual differences in the response to the SARS-CoV-2 infection.Next, we performed comparative population analysis for the same variants using extracted data from the 1000 genomes project. Population genetic variability was assessed using delta MAF and Fst statistics. Our study pointed to 7 variants inPLG, TMPRSS11a, MBL2andOAS1genes with noticeable divergence in allelic frequencies between populations worldwide. Three of them, all inMBL2gene, were predicted to be damaging, making them the most promising population-specific markers related to SARS-CoV-2 infection.Comparing allelic frequencies between Serbian and other populations, we found that the highest level of genetic divergence related to selected loci was observed with African, followed by East Asian, Central and South American and South Asian populations. When compared with European populations, the highest divergence was observed with Italian population.In conclusion, we identified 4 variants in genes encoding proteases (FURIN, PLGandPRSS1) and 6 in genes involved in the innate immunity (MBL2andOAS1) that might be relevant for the host response to SARS-CoV-2 infection.
Quantifying variation in bacterial reproductive fitness with and without antimicrobial resistance: a high-throughput method
Authors: Frey Pascal M. , Baer Julian , Bergadà Pijuan Judith , Lawless Conor , Bühler Philipp K. , Kouyos Roger D. , Lemon Katherine P. , Zinkernagel Annelies S. , Brugger Silvio D. 
Published: May 2020

ABSTRACTBackgroundTo evaluate changes in reproductive fitness of bacteria, e.g., after acquisition of antimicrobial resistance, a low-cost high-throughput method to analyse bacterial growth on agar is desirable for broad usability, including in low-resource settings.MethodIn our bacterial quantitative fitness analysis (baQFA), cultures are spotted in a predefined array on agar plates and photographed sequentially while growing. These time-lapse images are analysed using a purpose-built open source software to derive normalised image intensity values for each culture spot. Subsequently, a Gompertz growth model is fitted to these optical intensity values of each culture spot and fitness is calculated from parameters of the model. For image segmentation validation, we investigated the association between normalised intensity values and colony-forming unit (CFU) counts. To represent a range of clinically important pathogenic bacteria, we used different strains ofEnterococcus faecium, Escherichia coliandStaphylococcus aureus, with and without antimicrobial resistance. Relative competitive fitness (RCF) was defined as the mean fitness ratio of two strains growing competitively on one plate.ResultsbaQFA permitted the accurate construction of growth curves from bacteria grown on semisolid agar plates and fitting of Gompertz models: Normalised image intensity values showed a strong association with the total CFU/ml count per spotted culture (p<0.001) for all strains of the three species. Bacterial QFA showed relevant reproductive fitness differences between individual strains, suggesting substantial higher fitness of methicillin-resistantS. aureusJE2 than Cowan (RCF 1.60, p<0.001). Similarly, the vancomycin-resistantE. faeciumST172b showed higher competitive fitness than susceptibleE. faeciumST172 (RCF 1.72, p<0.001).ConclusionOur baQFA adaptation allows detection of fitness differences between our bacterial strains, and is likely to be applicable to other bacteria. In the future, baQFA may help to estimate epidemiological antimicrobial persistence or contribute to the prediction of clinical outcomes in severe infections at a low cost.
Evolution-guided mutagenesis of the cytoplasmic incompatibility proteins: Identifying CifA’s complex functional repertoire and new essential regions in CifB
Authors: Shropshire J. Dylan , Kalra Mahip , Bordenstein Seth R. 
Published: May 2020

AbstractWolbachiaare the world’s most common, maternally-inherited, arthropod endosymbionts. Their worldwide distribution is due in part to a selfish drive system termed cytoplasmic incompatibility (CI) that confers a relative fitness advantage to females that transmitWolbachiato their offspring. CI results in embryonic death when infected males mate with uninfected females but not infected females. Under the Two-by-One genetic model of CI, males expressing the two phage WO proteins CifA and CifB cause CI, and females expressing CifA rescue CI. While each protein is predicted to harbor three functional domains, there is no knowledge on how sites across these Cif domains, rather than in any one particular domain, contribute to CI and rescue. Here, we use evolution-guided, substitution mutagenesis of conserved amino acids across the Cif proteins, coupled with transgenic expression in uninfectedDrosophila melanogaster, to determine the functional impacts of conserved residues evolving mostly under purifying selection. We report that amino acids in CifA’s N-terminal unannotated region and annotated catalase-related domain are important for both complete CI and rescue, whereas C-terminal residues in CifA’s putative domain of unknown function are solely important for CI. Moreover, conserved CifB amino acids in the predicted nucleases, peptidase, and unannotated regions are essential for CI. Taken together, these findings indicate that (i) all CifA amino acids determined to be involved in rescue are correspondingly involved in CI, (ii) an additional set of CifA amino acids are uniquely important in CI, and (iii) CifB amino acids across the protein, rather than in one particular domain, are all essential for CI. We discuss how these findings advance an expanded view of Cif protein evolution and function, inform the mechanistic and biochemical bases of Cif-induced CI/rescue, and continue to substantiate the Two-by-One genetic model of CI.Article summaryWolbachiaare maternally-transmitted, intracellular bacteria that occur in approximately half of arthropod species worldwide. They can spread rapidly though host populations via the cytoplasmic incompatibility (CI) drive system. CI causes embryonic death when infected males mate with infected females, but offspring of infected females are rescued. Two proteins, CifA and CifB, underlie the genetic basis of CI and rescue, but how amino acid sites across these proteins contribute to CI and/or rescue remain unknown. Here, we employed evolution-guided, combinatorial mutagenesis on conserved amino acids to understand their relative contributions to CI and rescue. The results of this study reveal a phenotypic complexity underlying the expression of these proteins and provide relevance to the biochemical and mechanistic bases of CI and rescue.
Assessment of a small-scale fishery: Lane Snapper (Lutjanus synagris) using a length metric method
Authors: Castillo Liliana Sierra , Fujiwara Masami 
Published: May 2020

ABSTRACTSmall-scale fisheries are hard to assess because of the limited availability of data. Therefore, a method requiring easy-to-obtain catch-data is important for the assessment and management of small-scale fisheries. The objectives of this study were to assess the effect of fishing gear selectivity on a length-based metric method proposed by Froese by estimating three indicators using catch-data from Lane Snapper (Lutjanus synagris) collected in Honduras. These indicators are (1) the percentage of mature individuals in the catch, (2) the percentage of fish within the range of estimated optimal lengths to be captured and (3) the percentage of fish larger than the optimal length. These indicators determine the level of overfishing. The indicators were estimated separately for catchdata corresponding to gillnets, and each indicator was estimated with and without selectivity correction. Selectivity and mesh sizes of the fishing gear had a major impact in the estimation of indicators 1 and 2. As for indicator 3, it consistently showed a high level of exploitation. The three estimated indicators suggested that the Lane Snapper fishery in Honduras, is experiencing overfishing. Overall, the method proposed by Froese appears to be promising for the assessment of small-scale fisheries, but it should be used cautiously.