bluuebunny/crossref_metadata_2025 huggingface dataset:
Deep Transfer Learning-based COVID-19 prediction using Chest X-rays
Authors: Kumar Saurabh , Mishra Shweta , Singh Sunil Kumar
Published: May 2020

The novel coronavirus disease (COVID-19) is spreading very rapidly across the globe because of its highly contagious nature, and is declared as a pandemic by world health organization (WHO). Scientists are endeavoring to ascertain the drugs for its efficacious treatment. Because, till now, no full-proof drug is available to cure this deadly disease. Therefore, identifying COVID-19 positive people and to quarantine them, can be an effective solution to control its spread. Many machine learning and deep learning techniques are being used quite effectively to classify positive and negative cases. In this work, a deep transfer learning-based model is proposed to classify the COVID-19 cases using chest X-rays or CT scan images of infected persons. The proposed model is based on the ensembling of DenseNet121 and SqueezeNet1.0, which is named as DeQueezeNet. The model can extract the importance of various influential features from the X-ray images, which are effectively used to classify the COVID-19 cases. The performance study of the proposed model depicts its effectiveness in terms of accuracy and precision. A comparative study has also been done with the recently published works and it is observed the performance of the proposed model is significantly better.
Interactions of Upstream and Downstream Promoter Regions with RNA Polymerase are Energetically Coupled and a Target of Regulation in Transcription Initiation
Authors: Sosa Robert P. , Florez-Ariza Alfredo J. , Díaz-Celis César , Onoa Bibiana , Cassago Alexandre , de Oliveira Paulo S. L. , Portugal Rodrigo V. , Guerra Daniel G. , Bustamante Carlos J.
Published: May 2020

During transcription initiation, the RNA polymerase holoenzyme (RNAP) and the promoter form an open complex. For many promoters, this interaction involves upstream DNA wrapping, downstream promoter bending, and associated large-scale protein rearrangements. Although these processes have been reported across the life kingdom, their structure, energetics, and role in transcription remain an area of active research. Using optical tweezers, we find that these processes become energetically and reversibly coupled after the formation of the open promoter complex, providing the main contribution to their stability. Using electron microscopy and single particle analysis, we find that the interaction encompasses from positions −76 to +18 along the template, that it involves an overall DNA bent angle of ~245°, and that the upstream wrapping is enabled by interactions between the C-terminal domains of RNAP’s alpha subunits and proximal and middle upstream promoter regions. The energy associated with upstream wrapping, downstream bending and its coupling to downstream rearrangements does not require specific upstream promoter sequence, and correlate positively with the rate of transcription DNA bubble formation as reported by a real-time fluorescence assay. Our results suggest that the coupling between upstream and downstream events are part of acis-regulatory network established after the opening of the DNA bubble, that could furnish a control mechanism of gene expression by protein factors and regulatory metabolites.

The first step of gene expression involves transcription of DNA into RNA by RNA polymerase (RNAP). RNAP recognizes a promoter sequence forming the transcriptionally active open complex. For several promoters, DNA wraps around the RNAP. We find that upstream wrapping contacts are energetically coupled and occur cooperatively with downstream rearrangements in the open complexes, providing the largest contribution to their stability. We also determined that upstream wrapping is enabled by interactions between non-specific upstream promoter regions and RNAP α subunit C-terminal domains. Significantly, the strength of these contacts correlates with the rate of DNA bubble opening, and is regulated by factors such as the transcriptional regulator ppGpp. We suggest that any modulator altering upstream wrapping and downstream rearrangements could finely tune gene expression in response to the needs of the cell
High temperature inhibits cnidarian-dinoflagellate symbiosis establishment through nitric oxide signaling
Authors: Hill Lilian J. , Salgado Leonardo T. , Salomon Paulo S. , Guse Annika
Published: May 2020

Disclaimer Statement

The authors have withdrawn their manuscript after further experiments based on reviewer comments did not support the conclusions of the paper. Therefore, the authors do not wish this work to be cited as reference for the project. The authors thank the anonymous reviewer for a critical assessment. If you have any questions, please contact the corresponding authors.
A novel Ca2+signalling pathway co-ordinates environmental phosphorus sensing and nitrogen metabolism in marine diatoms
Authors: Helliwell K. E. , Harrison E. , Christie-Oleza J. , Rees A. P. , Downe J. , Aguilo-Ferretjans M. M. , Al-Moosawi L. , Brownlee C. , Wheeler G. L.
Published: May 2020

Diatoms are a diverse and globally important phytoplankton group, responsible for an estimated 20% of carbon fixation on Earth. They frequently form spatially extensive phytoplankton blooms, responding rapidly to increased availability of nutrients including phosphorus and nitrogen. Although it is well established that diatoms are common first-responders to nutrient influxes in aquatic ecosystems, little is known of the sensory mechanisms that they employ for nutrient perception. Here we show that diatoms use a novel and highly-sensitive Ca2+-dependent signalling pathway, not previously described in eukaryotes, to sense and respond to the critical macronutrient phosphorus. We demonstrate that phosphorus-Ca2+signalling is essential for regulating diatom recovery from phosphorus limitation, by controlling rapid and substantial increases in nitrogen assimilation. Phosphorus-Ca2+signalling thus mediates fundamental cross-talk between the vital nutrients P and N to maximise resource competition, and likely governs the success of diatoms as major bloom formers in regions of pulsed nutrient supply. Importantly, our study demonstrates that distinct mechanisms for nutrient sensing have evolved in photosynthetic eukaryotes.
iDePP: a genetically encoded system for the inducible depletion of PI(4,5)P2inArabidopsis thaliana
Authors: Doumane Mehdi , Colin Léia , Lebecq Alexis , Fangain Aurélie , Bareille Joseph , Hamant Olivier , Belkhadir Youssef , Jaillais Yvon , Caillaud Marie-Cécile
Published: May 2020

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a low abundant lipid present at the plasma membrane of eukaryotic cells. Extensive studies in animal cells revealed the pleiotropic functions of PI(4,5)P2. In plant cells, PI(4,5)P2is involved in various cellular processes including the regulation of cell polarity and tip growth, clathrin-mediated endocytosis, polar auxin transport, actin dynamics or membrane-contact sites. To date, most studies investigating the role of PI(4,5)P2in plants have relied on mutants lacking enzymes responsible for PI(4,5)P2synthesis and degradation. However, such genetic perturbations only allow steady-state analysis of plants undergoing their life cycle in PI(4,5)P2deficient conditions and the corresponding mutants are likely to induce a range of non-causal (untargeted) effects driven by compensatory mechanisms. In addition, there are no small molecule inhibitors that are available in plants to specifically block the production of this lipid. Thus, there is currently no system to fine tune PI(4,5)P2content in plant cells. Here we report a genetically encoded and inducible synthetic system, iDePP (InducibleDepletion ofPI(4,5)P2inPlants), that efficiently removes PI(4,5)P2from the plasma membrane in different organs ofArabidopsis thaliana, including root meristem, root hair and shoot apical meristem. We show that iDePP allows the inducible depletion of PI(4,5)P2in less than three hours. Using this strategy, we reveal that PI(4,5)P2is critical for cortical microtubule organization. Together, we propose that iDePP is a simple and efficient genetic tool to test the importance of PI(4,5)P2in given cellular or developmental responses but also to evaluate the importance of this lipid in protein localization.

Research Organism

A. thaliana
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

Density 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.

Synopsis

The 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

Root 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

Infections 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.

IMPORTANCE

Trypanosoma 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

We 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

Synaptic 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

Nucleic 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).
ChAdOx1 nCoV-19 vaccination prevents SARS-CoV-2 pneumonia in rhesus macaques
Authors: van Doremalen Neeltje , Lambe Teresa , Spencer Alexandra , Belij-Rammerstorfer Sandra , Purushotham Jyothi N. , Port Julia R. , Avanzato Victoria , Bushmaker Trenton , Flaxman Amy , Ulaszewska Marta , Feldmann Friederike , Allen Elizabeth R. , Sharpe Hannah , Schulz Jonathan , Holbrook Myndi , Okumura Atsushi , Meade-White Kimberly , Pérez-Pérez Lizzette , Bissett Cameron , Gilbride Ciaran , Williamson Brandi N. , Rosenke Rebecca , Long Dan , Ishwarbhai Alka , Kailath Reshma , Rose Louisa , Morris Susan , Powers Claire , Lovaglio Jamie , Hanley Patrick W. , Scott Dana , Saturday Greg , de Wit Emmie , Gilbert Sarah C. , Munster Vincent J.
Published: May 2020

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in December 20191,2and is responsible for the COVID-19 pandemic3. Vaccines are an essential countermeasure urgently needed to control the pandemic4. Here, we show that the adenovirus-vectored vaccine ChAdOx1 nCoV-19, encoding the spike protein of SARS-CoV-2, is immunogenic in mice, eliciting a robust humoral and cell-mediated response. This response was not Th2 dominated, as demonstrated by IgG subclass and cytokine expression profiling. A single vaccination with ChAdOx1 nCoV-19 induced a humoral and cellular immune response in rhesus macaques. We observed a significantly reduced viral load in bronchoalveolar lavage fluid and respiratory tract tissue of vaccinated animals challenged with SARS-CoV-2 compared with control animals, and no pneumonia was observed in vaccinated rhesus macaques. Importantly, no evidence of immune-enhanced disease following viral challenge in vaccinated animals was observed. ChAdOx1 nCoV-19 is currently under investigation in a phase I clinical trial. Safety, immunogenicity and efficacy against symptomatic PCR-positive COVID-19 disease will now be assessed in randomised controlled human clinical trials.
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

The 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.
FGF signaling in development beyond canonical pathways
Authors: Ray Ayan T. , Mazot Pierre , Brewer J. Richard , Catela Catarina , Dinsmore Colin J. , Soriano Philippe
Published: May 2020

FGFs are key developmental regulators which engage a signal transduction cascade through receptor tyrosine kinases, typically involving ERK1/2, PI3K/AKT, and other effectors. However, it remains unknown if all FGF activities depend on kinase activity or these canonical signal transduction cascades. To address these questions, we generated allelic series of knock-inFgfr1andFgfr2mouse strains, carrying point mutations that disrupt binding of signaling effectors to the receptors, alone or in combination. We also produced a kinase dead allele ofFgfr2which broadly phenocopies the null mutant. When interrogated in cranial neural crest cells, point mutations in either receptor revealed discrete functions for signaling pathways in specific craniofacial contexts, but failed to recapitulate the single or double null mutant phenotypes even in their most extensive combination. Furthermore, we found that together these signaling mutations abrogated the established FGF-induced signal transduction pathways, yet certain FGF functions such as cell-matrix and cell-cell adhesion remained unaffected. Our studies establish combinatorial roles of bothFgfr1andFgfr2in development and identify novel kinase-dependent cell adhesion properties of FGF receptors, independent of well-established roles in intracellular signaling.
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

CRISPR-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

New 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

Background

To 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.

Method

In 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.

Results

baQFA 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).

Conclusion

Our 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

Wolbachiaare 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 summary

Wolbachiaare 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

Small-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.
The Effect of a Ketogenic Diet and Synergy with Rapamycin in a Mouse Model of Breast Cancer
Authors: Zou Yiyu , Fineberg Susan , Pearlman Alexander , Feinman Richard D. , Fine Eugene J.
Published: May 2020

Background

The effects of diet in cancer, in general, and breast cancer in particular, are not well understood. Insulin inhibition in ketogenic, high fat diets, modulate downstream signaling molecules and are postulated to have therapeutic benefits. Obesity and diabetes have been associated with higher incidence of breast cancer. Addition of anti-cancer drugs together with diet is also not well studied.

Methods

Two diets, one ketogenic, the other standard mouse chow, were tested in a spontaneous breast cancer model in mice. The diets were implemented either with or without added rapamycin, an mTOR inhibitor and potential anti-cancer drug.

Results

Blood glucose and insulin concentrations in mice ingesting the ketogenic diet (KD) were significantly lower, whereas beta hydroxybutyrate (BHB) levels were significantly higher, respectively, than in mice on the standard diet (SD). Growth of primary breast tumors and lung metastases were inhibited, and lifespans were longer in the KD mice compared to mice on the SD (p<0.005). Rapamycin improved survival in both mouse diet groups, but when combined with the KD was more effective than when combined with the SD.

Conclusions

The study provides proof of principle that a ketogenic diet a) results in serum insulin reduction and ketosis in a spontaneous breast cancer mouse model; b) can serve as a therapeutic anti-cancer agent; and c) can enhance the effects of rapamycin, an anti-cancer drug, permitting dose reduction for comparable effect. Further, the ketogenic diet in this model produces superior cancer control than standard mouse chow whether with or without added rapamycin.
Genome-wide identification ofAedes albopictuslong noncoding RNAs and their association with dengue and zika virus infection
Authors: Azlan Azali , Obeidat Sattam M. , Das Kumitaa Theva , Yunus Muhammad Amir , Azzam Ghows
Published: May 2020

The Asian tiger mosquito,Aedes albopictus(Ae. albopictus), is an important vector that transmits arboviruses such as dengue (DENV), Zika (ZIKV) and Chikungunya virus (CHIKV). On the other hand, long noncoding RNAs (lncRNAs) are known to regulate various biological processes. Knowledge onAe. albopictuslncRNAs and their functional role in virus-host interactions are still limited. Here, we identified and characterized the lncRNAs in the genome of an arbovirus vector,Ae. albopictus, and evaluated their potential involvement in DENV and ZIKV infection. We used 148 public datasets, and identified a total of 10, 867 novel lncRNA transcripts, of which 5,809, 4,139, and 919 were intergenic, intronic and antisense respectively. TheAe. albopictuslncRNAs shared many characteristics with other species such as short length, low GC content, and low sequence conservation. RNA-sequencing ofAe. albopictuscells infected with DENV and ZIKV showed that the expression of lncRNAs was altered upon virus infection. Target prediction analysis revealed thatAe. albopictuslncRNAs may regulate the expression of genes involved in immunity and other metabolic and cellular processes. To verify the role of lncRNAs in virus infection, we generated mutation in lncRNA loci using CRISPR-Cas9, and discovered that two lncRNA loci mutations, namely XLOC_029733 (novel lncRNA transcript id: lncRNA_27639.2) and LOC115270134 (known lncRNA transcript id: XR_003899061.1) resulted in enhancement of DENV and ZIKV replication. The results presented here provide an important foundation for future studies of lncRNAs and their relationship with virus infection inAe. albopictus.

Author summary

Ae. albopictusis an important vector of arboviruses such as dengue and Zika. Studies on virus-host interaction at gene expression and molecular level are crucial especially in devising methods to inhibit virus replication inAedesmosquito. Previous reports showed that, besides protein-coding genes, noncoding RNAs such as lncRNAs are also involved in virus-host interaction. In this study, we report a comprehensive catalog of novel lncRNA transcripts in the genome ofAe. albopictus. We also show that the expression of lncRNAs was altered upon infection with dengue and Zika. Additionally, depletion of certain lncRNAs resulted in increased replication of dengue and Zika; hence, suggesting potential association of lncRNAs in virus infection. Results of this study provide a new avenue to the investigation of mosquito-virus interactions that may potentially pave way to the development of novel methods in vector control.
Comparison of the knockdown resistance locus (kdr) inAnopheles stephensi,An. arabiensis, andCulex pipiens s.l.suggests differing mechanisms of pyrethroid resistance in east Ethiopia
Authors: Carter Tamar E. , Gebresilassie Araya , Hansel Shantoy , Damodaran Lambodhar , Montgomery Callum , Bonnell Victoria , Lopez Karen , Janies Daniel , Yared Solomon
Published: May 2020

The malaria vector,Anopheles stephensi, which is typically restricted to South Asia and the Middle East, was recently detected in the Horn of Africa. Controlling the spread of this vector could involve integrated vector control that considers the status of insecticide resistance of multiple vector species in the region. Previous reports indicate that the knockdown resistance mutations (kdr) in the voltage-gated sodium channel (vgsc) are absent in both pyrethroid resistant and sensitive variants ofAn. stephensiin east Ethiopia but similar information on other vector species in the same areas is limited. In this study,kdrand the neighboring intron was analyzed inAn. stephensi,An. arabiensis, andCulex pipiens s. l. collected in east Ethiopia between 2016 and 2017. Sequence analysis revealed that all ofCx. pipiens s.l.(n = 42) and 71.6% of theAn. arabiensis(n=67) carriedkdrL1014F known to confer target-site pyrethroid resistance. Intronic variation was only observed inAn. stephensi(segregating sites = 6, haplotypes = 3) previously shown to have no kdr mutations. In addition, no evidence of non-neutral evolutionary processes was detected at theAn. stephensi kdrintron which further supports target-site mechanism not being a major resistance mechanism in thisAn. stephensipopulation. Overall, these results suggest differences in evolved mechanisms of pyrethroid/DDT resistance in populations of vector species from the same region. Variation in insecticide resistance mechanisms in East Ethiopian mosquito vectors highlight possible species or population specific biological factors and distinct environmental exposures that shape their evolution.
SeqEnhDL: sequence-based classification of cell type-specific enhancers using deep learning models
Authors: Wang Yupeng , Jaime-Lara Rosario B. , Roy Abhrarup , Sun Ying , Liu Xinyue , Joseph Paule V.
Published: May 2020

We propose SeqEnhDL, a deep learning framework for classifying cell type-specific enhancers based on sequence features. DNA sequences of “strong enhancer” chromatin states in nine cell types from the ENCODE project were retrieved to build and test enhancer classifiers. For any DNA sequence, sequentialk-mer (k=5, 7, 9 and 11) fold changes relative to randomly selected non-coding sequences were used as features for deep learning models. Three deep learning models were implemented, including multi-layer perceptron (MLP), Convolutional Neural Network (CNN) and Recurrent Neural Network (RNN). All models in SeqEnhDL outperform state-of-the-art enhancer classifiers including gkm-SVM and DanQ, with regard to distinguishing cell type-specific enhancers from randomly selected non-coding sequences. Moreover, SeqEnhDL is able to directly discriminate enhancers from different cell types, which has not been achieved by other enhancer classifiers. Our analysis suggests that both enhancers and their tissue-specificity can be accurately identified according to their sequence features. SeqEnhDL is publicly available athttps://github.com/wyp1125/SeqEnhDL.
Biochemical and Molecular Characterization, and Bioprospecting of Drought Tolerant Actinomycetes from Maize Rhizosphere Soil
Authors: Chukwuneme Chinenyenwa Fortune , Babalola Olubukola Oluranti , Kutu Funso Raphael , Ojuederie Omena Bernard
Published: May 2020

Drought is a major limitation to maize cultivation around the globe. Seven actinomycetes strains were isolated from maize rhizosphere soils in Mahikeng, North-West Province, South Africa. The isolates were biochemically characterized and identified with 16S rRNA gene sequence analysis. Isolates were also screenedin vitrofor abiotic stress tolerance to different concentrations of NaCl, pH, and polyethylene glycol (PEG 8000), as well as for biosynthesis of drought tolerance genes namely Glutathione peroxidase (GPX), Glycine-rich RNA binding protein (GRP), Desiccation protectant protein (DSP), Guanosine triphosphate binding protein (GTP) and plant growth-promoting genes:1-aminocyclopropane-1-carboxylate deaminase (accd) and siderophore biosynthesis (Sid). About 71.43% of isolates were of the genusStreptomyces(99-100% similarity), while 14.29% belong to the genusArthrobacter(R15) and 14.29% to the genusMicrobacterium(S11) respectively (99% similarity). Five isolates had their optimum growth at 35°C.Arthrobacter arilaitensis(R15) grew and tolerated 5%, 10%, and 20% PEG at 120 h. Root length increased by 110.53% in PEG treated maize seeds (−0.30 MPa) inoculated withStreptomyces pseudovenezuelae (S20) compared to the un-inoculated control. Likewise, germination percentage and vigor index increased by 37.53% and 194.81% respectively in PEG treated seeds inoculated with S20 than the un-inoculated PEG treated seeds. ACC deaminase gene was amplified in all the isolates, while the gene for siderophore biosynthesis was amplified in 85.71% of the isolates. Genes for the synthesis of GPX, GRP, DSP and GTP were amplified inArthrobacter arilaitensis(R15) andStreptomyces pseudovenezuelae(S20) which lacked GTP. The amplification of drought-tolerant and plant growth-promoting primers indicates the possible presence of these genes in the isolates. These isolates have the potential for use as bio-inoculants, not only to improve drought tolerance in maize but also to be utilized as biofertilizers and biocontrol agents to facilitate growth promotion.
The anticipated potential nuclear localization sequence of ‘CandidatusPhytoplasma mali’ SAP11-like protein is required for TCP binding but not for transport into the nucleus
Authors: Strohmayer Alisa , Schwarz Timothy , Braun Mario , Krczal Gabi , Boonrod Kajohn
Published: May 2020

Abstract

The plant pathogen ‘CandidatusPhytoplasma mali’ (‘Ca. P. mali’) is the causing agent of apple proliferation that leads to heavy damage in apple production all over Europe. To identify and analyze effector proteins of plant pathogens is an important strategy in plant disease research. Here, we report that the SAP11-like protein of‘Ca. P. mali’ induces crinkled leaves and siliques and witches’ broom symptoms in transgenicArabidopsis thaliana(A. thaliana) plants and binds to 6 members of class I and all members of class II TCP (TEOSINE BRANCHES/ CYCLOIDEA/PROLIFERATING CELL FACTOR) transcription factors ofA. thalianain yeast two-hybrid assays. Moreover, we demonstrate that the protein localizes actively into the plant nucleus without requiring the nuclear leader sequence (NLS). We also identified a 17 amino acid stretch previously predicted to be a nuclear leader sequence that is important for the binding of some of the TCPs and also responsible for the crinkled leaf and silique phenotype in transgenicA. thaliana.
Irrigation water depths and soil covers in carrot crop
Authors: Vieira Joslanny Higino , Aleman Catariny Cabral , de Freitas Elis Marina , Santos Laylton de Albuquerque , da Silva Gustavo Henrique , Franco Fernandes Pedro Henrique
Published: May 2020

The use of soil covers may decrease water consumption and improve the crop sustainability Thus, the objective of this work was to evaluate biophysical parameters and yield of carrot crops (cultivar Brasília) grown under different irrigation water depths and soil cover conditions. The experiment was conducted at the Federal University of Viçosa, MG, Brazil. A randomized block design with four replications was used, in a 5×3 factorial arrangement.The treatments consisted of 5 irrigation water depths, based on the actual soil water capacity (20, 40, 60, 80, and 100% ASWC) and 3 soil covers (white polyethylene, biodegradable semi-kraft paper, and no soil cover - Control). The soil and leaf temperatures, number of leaves, root length, normalized difference vegetation index (NDVI), and fresh root weight were evaluated. The data were subjected to analysis of variance through the F test and the means compared by the Tukey’s test (p≤0.05); regression analysis was carried out using the equation with the highest significant fit. The use of semi-kraft paper was a good option for the carrot crop; and the water depths of up to 60% ASWC did not hinder the crop.