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A population of neutrophils recruited into cystic fibrosis (CF) airways is associated with proteolytic lung damage, exhibiting high expression of primary granule exocytosis marker CD63 and reduced phagocytic receptor CD16. Causative factors for this population are unknown, limiting intervention. Here we present a laboratory model to characterize responses of differentiated airway epithelium and neutrophils following respiratory infection. Pediatric primary airway epithelial cells were cultured at the air-liquid interface, challenged individually or in combination with rhinovirus (RV) and Pseudomonas aeruginosa, then apically washed with medical saline to sample epithelial infection milieus. Cytokine multiplex analysis revealed epithelial antiviral signals, including IP-10 and RANTES, increased with exclusive RV infection but were diminished if P. aeruginosa was also present. Proinflammatory signals interleukin-1α and ß were dominant in P. aeruginosa infection milieus. Infection washes were also applied to a published model of neutrophil transmigration into the airways. Neutrophils migrating into bacterial and viral-bacterial co-infection milieus exhibited the in vivo CF phenotype of increased CD63 expression and reduced CD16 expression, while neutrophils migrating into milieus of RV-infected or uninfected cultures did not. Individually, bacterial products lipopolysaccharide and N-formylmethionyl-leucyl-phenylalanine and isolated cytokine signals only partially activated this phenotype, suggesting that additional soluble factors in the infection microenvironment trigger primary granule release. Findings identify P. aeruginosa as a trigger of acute airway inflammation and neutrophil primary granule exocytosis, underscoring potential roles of airway microbes in prompting this neutrophil subset. Further studies are required to characterize microbes implicated in primary granule release, and identify potential therapeutic targets.
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Fibrosis Quística , Infecciones por Pseudomonas , Citocinas/metabolismo , Exocitosis , Humanos , Neutrófilos/metabolismo , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/fisiologíaRESUMEN
Grapevine (Vitis vinifera L.) displays wide plasticity to climate; however, the physiology of dormancy along a seasonal continuum is poorly understood. Here we investigated the apparent disconnect between dormancy and the underlying respiratory physiology and transcriptome of grapevine buds, from bud set in summer to bud burst in spring. The establishment of dormancy in summer was pronounced and reproducible; however, this was coupled with little or no change in physiology, indicated by respiration, hydration, and tissue oxygen tension. The release of dormancy was biphasic; the depth of dormancy declined substantially by mid-autumn, while the subsequent decline towards spring was moderate. Observed changes in physiology failed to explain the first phase of dormancy decline, in particular. Transcriptome data contrasting development from summer through to spring also indicated that dormancy was poorly reflected by metabolic quiescence during summer and autumn. Gene Ontology and enrichment data revealed the prevailing influence of abscisic acid (ABA)-related gene expression during the transition from summer to autumn, and promoter motif analysis suggested that photoperiod may play an important role in regulating ABA functions during the establishment of dormancy. Transcriptomic data from later transitions reinforced the importance of oxidation and hypoxia as physiological cues to regulate the maintenance of quiescence and resumption of growth. Collectively these data reveal a novel disconnect between growth and metabolic quiescence in grapevine following bud set, which requires further experimentation to explain the phenology and dormancy relationships.
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Latencia en las Plantas , Vitis , Ácido Abscísico/metabolismo , Regulación de la Expresión Génica de las Plantas , Fotoperiodo , Latencia en las Plantas/genética , Estaciones del Año , Vitis/metabolismoRESUMEN
MAIN CONCLUSION: Genome-wide identification, together with gene expression patterns and promoter region analysis of FYVE and PHOX proteins in Physcomitrella patens, emphasized their importance in regulating mainly developmental processes in P. patens. Phosphatidylinositol 3-phosphate (PtdIns3P) is a signaling phospholipid, which regulates several aspects of plant growth and development, as well as responses to biotic and abiotic stresses. The mechanistic insights underlying PtdIns3P mode of action, specifically through effector proteins have been partially explored in plants, with main focus on Arabidopsis thaliana. In this study, we searched for genes coding for PtdIns3P-binding proteins such as FYVE and PHOX domain-containing sequences from different photosynthetic organisms to gather evolutionary insights on these phosphoinositide binding domains, followed by an in silico characterization of the FYVE and PHOX gene families in the moss Physcomitrella patens. Phylogenetic analysis showed that PpFYVE proteins can be grouped in 7 subclasses, with an additional subclass whose FYVE domain was lost during evolution to higher plants. On the other hand, PpPHOX proteins are classified into 5 subclasses. Expression analyses based on RNAseq data together with the analysis of cis-acting regulatory elements and transcription factor (TF) binding sites in promoter regions suggest the importance of these proteins in regulating stress responses but mainly developmental processes in P. patens. The results provide valuable information and robust candidate genes for future functional analysis aiming to further explore the role of this signaling pathway mainly during growth and development of tip growing cells and during the transition from 2 to 3D growth. These studies would identify ancestral regulatory players undertaken during plant evolution.
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Bryopsida/genética , Evolución Molecular , Proteínas de Plantas/genética , Cromosomas de las Plantas/genética , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Genes de Plantas , Anotación de Secuencia Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas/genética , Dominios Proteicos , Estrés Fisiológico/genéticaRESUMEN
Dormant or quiescent buds of woody perennials are often dense and in the case of grapevine (Vitis vinifera L.) have a low tissue oxygen status. The precise timing of the decision to resume growth is difficult to predict, but once committed, the increase in tissue oxygen status is rapid and developmentally regulated. Here, we show that more than a third of the grapevine homologues of widely conserved hypoxia-responsive genes and nearly a fifth of all grapevine genes possessing a plant hypoxia-responsive promoter element were differentially regulated during bud burst, in apparent harmony with resumption of meristem identity and cell-cycle gene regulation. We then investigated the molecular and biochemical properties of the grapevine ERF-VII homologues, which in other species are oxygen labile and function in transcriptional regulation of hypoxia-responsive genes. Each of the 3 VvERF-VIIs were substrates for oxygen-dependent proteolysis in vitro, as a function of the N-terminal cysteine. Collectively, these data support an important developmental function of oxygen-dependent signalling in determining the timing and effective coordination bud burst in grapevine. In addition, novel regulators, including GASA-, TCP-, MYB3R-, PLT-, and WUS-like transcription factors, were identified as hallmarks of the orderly and functional resumption of growth following quiescence in buds.
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Regulación del Desarrollo de la Expresión Génica , Oxígeno/metabolismo , Vitis/fisiología , Meristema/crecimiento & desarrollo , Meristema/fisiología , Latencia en las Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vitis/crecimiento & desarrolloRESUMEN
Vitis vinifera berries are sensitive towards infection by the necrotrophic pathogen Botrytis cinerea, leading to important economic losses worldwide. The combined analysis of the transcriptome and metabolome associated with fungal infection has not been performed previously in grapes or in another fleshy fruit. In an attempt to identify the molecular and metabolic mechanisms associated with the infection, peppercorn-sized fruits were infected in the field. Green and veraison berries were collected following infection for microarray analysis complemented with metabolic profiling of primary and other soluble metabolites and of volatile emissions. The results provided evidence of a reprogramming of carbohydrate and lipid metabolisms towards increased synthesis of secondary metabolites involved in plant defence, such as trans-resveratrol and gallic acid. This response was already activated in infected green berries with the putative involvement of jasmonic acid, ethylene, polyamines, and auxins, whereas salicylic acid did not seem to be involved. Genes encoding WRKY transcription factors, pathogenesis-related proteins, glutathione S-transferase, stilbene synthase, and phenylalanine ammonia-lyase were upregulated in infected berries. However, salicylic acid signalling was activated in healthy ripening berries along with the expression of proteins of the NBS-LRR superfamily and protein kinases, suggesting that the pathogen is able to shut down defences existing in healthy ripening berries. Furthermore, this study provided metabolic biomarkers of infection such as azelaic acid, a substance known to prime plant defence responses, arabitol, ribitol, 4-amino butanoic acid, 1-O-methyl- glucopyranoside, and several fatty acids that alone or in combination can be used to monitor Botrytis infection early in the vineyard.
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Botrytis/fisiología , Interacciones Huésped-Patógeno , Metaboloma , Enfermedades de las Plantas/microbiología , Transcriptoma , Vitis/genética , Aciltransferasas/genética , Aciltransferasas/metabolismo , Frutas/genética , Frutas/metabolismo , Ácido Gálico/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenilanina Amoníaco-Liasa/genética , Fenilanina Amoníaco-Liasa/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Poliaminas/metabolismo , Resveratrol , Estilbenos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vitis/metabolismo , Vitis/microbiologíaRESUMEN
Grape and wine production and quality is extremely dependent on the fruit ripening process. Sensory and nutritional characteristics are important aspects for consumers and their development during fruit ripening involves complex hormonal control. In this review, we explored data already published on grape ripening and compared it with the hormonal regulation of ripening of other climacteric and non-climacteric fruits. The roles of abscisic acid, ethylene, and brassinosteroids as promoters of ripening are discussed, as well as the role of auxins, cytokinins, gibberellins, jasmonates, and polyamines as inhibitors of ripening. In particular, the recently described role of polyamine catabolism in grape ripening is discussed, together with its putative interaction with other hormones. Furthermore, other recent examples of cross-talk among the different hormones are presented, revealing a complex interplay of signals during grape development and ripening.
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Frutas/crecimiento & desarrollo , Reguladores del Crecimiento de las Plantas/metabolismo , Vitis/crecimiento & desarrollo , Vino/clasificación , Ácido Abscísico/metabolismo , Brasinoesteroides/metabolismo , Etilenos/metabolismo , Regulación de la Expresión Génica de las Plantas , Metaboloma/fisiología , Transducción de Señal/fisiologíaAsunto(s)
Oxígeno/metabolismo , Hojas de la Planta/fisiología , Brotes de la Planta/fisiología , Vitis/fisiología , Cloroplastos/fisiología , Metabolismo Energético , Regulación de la Expresión Génica de las Plantas , Luz , Meristema/fisiología , Brotes de la Planta/crecimiento & desarrollo , Plastidios/genética , Plastidios/metabolismo , Transducción de SeñalRESUMEN
Introduction: Many survivors of preterm birth (<37 weeks gestation) have lifelong respiratory deficits, the drivers of which remain unknown. Influencers of pathophysiological outcomes are often detectable at the gene level and pinpointing these differences can help guide targeted research and interventions. This study provides the first transcriptomic analysis of primary nasal airway epithelial cells in survivors of preterm birth at approximately 1 year of age. Methods: Nasal airway epithelial brushings were collected, and primary cell cultures established from term (>37 weeks gestation) and very preterm participants (≤32 weeks gestation). Ex vivo RNA was collected from brushings with sufficient cell numbers and in vitro RNA was extracted from cultured cells, with bulk RNA sequencing performed on both the sample types. Differential gene expression was assessed using the limma-trend pipeline and pathway enrichment identified using Reactome and GO analysis. To corroborate gene expression data, cytokine concentrations were measured in cell culture supernatant. Results: Transcriptomic analysis to compare term and preterm cells revealed 2,321 genes differentially expressed in ex vivo samples and 865 genes differentially expressed in cultured basal cell samples. Over one third of differentially expressed genes were related to host immunity, with interferon signalling pathways dominating the pathway enrichment analysis and IRF1 identified as a hub gene. Corroboration of disrupted interferon release showed that concentrations of IFN-α2 were below measurable limits in term samples but elevated in preterm samples [19.4 (76.7) pg/ml/µg protein, p = 0.03]. IFN-γ production was significantly higher in preterm samples [3.3 (1.5) vs. 9.4 (17.7) pg/ml/µg protein; p = 0.01] as was IFN-ß [7.8 (2.5) vs. 13.6 (19.5) pg/ml/µg protein, p = 0.01]. Conclusion: Host immunity may be compromised in the preterm nasal airway epithelium in early life. Altered immune responses may lead to cycles of repeated infections, causing persistent inflammation and tissue damage which can have significant impacts on long-term respiratory function.
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BACKGROUND: Respiratory viruses significantly impact global morbidity and mortality, causing more disease in humans than any other infectious agent. Beyond pathogens, various viruses and bacteria colonize the respiratory tract without causing disease, potentially influencing respiratory diseases' pathogenesis. Nevertheless, our understanding of respiratory microbiota is limited by technical constraints, predominantly focusing on bacteria and neglecting crucial populations like viruses. Despite recent efforts to improve our understanding of viral diversity in the human body, our knowledge of viral diversity associated with the human respiratory tract remains limited. METHODS: Following a comprehensive search in bibliographic and sequencing data repositories using keyword terms, we retrieved shotgun metagenomic data from public repositories (n = 85). After manual curation, sequencing data files from 43 studies were analyzed using EVEREST (pipEline for Viral assEmbly and chaRactEriSaTion). Complete and high-quality contigs were further assessed for genomic and taxonomic characterization. RESULTS: Viral contigs were obtained from 194 out of the 868 FASTQ files processed through EVEREST. Of the 1842 contigs that were quality assessed, 8% (n = 146) were classified as complete/high-quality genomes. Most of the identified viral contigs were taxonomically classified as bacteriophages, with taxonomic resolution ranging from the superkingdom level down to the species level. Captured contigs were spread across 25 putative families and varied between RNA and DNA viruses, including previously uncharacterized viral genomes. Of note, airway samples also contained virus(es) characteristic of the human gastrointestinal tract, which have not been previously described as part of the lung virome. Additionally, by performing a meta-analysis of the integrated datasets, ecological trends within viral populations linked to human disease states and their biogeographical distribution along the respiratory tract were observed. CONCLUSION: By leveraging publicly available repositories of shotgun metagenomic data, the present study provides new insights into viral genomes associated with specimens from the human respiratory tract across different disease spectra. Further studies are required to validate our findings and evaluate the potential impact of these viral communities on respiratory tract physiology.
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Genoma Viral , Metagenómica , Sistema Respiratorio , Viroma , Virus , Humanos , Metagenómica/métodos , Sistema Respiratorio/virología , Virus/genética , Virus/clasificación , Virus/aislamiento & purificación , Metagenoma , Simulación por Computador , Filogenia , Biología Computacional/métodos , Microbiota , Bacteriófagos/genética , Bacteriófagos/clasificación , Bacteriófagos/aislamiento & purificaciónRESUMEN
Introduction: Recurrent wheezing disorders including asthma are complex and heterogeneous diseases that affect up to 30% of all children, contributing to a major burden on children, their families, and global healthcare systems. It is now recognized that a dysfunctional airway epithelium plays a central role in the pathogenesis of recurrent wheeze, although the underlying mechanisms are still not fully understood. This prospective birth cohort aims to bridge this knowledge gap by investigating the influence of intrinsic epithelial dysfunction on the risk for developing respiratory disorders and the modulation of this risk by maternal morbidities, in utero exposures, and respiratory exposures in the first year of life. Methods: The Airway Epithelium Respiratory Illnesses and Allergy (AERIAL) study is nested within the ORIGINS Project and will monitor 400 infants from birth to 5 years. The primary outcome of the AERIAL study will be the identification of epithelial endotypes and exposure variables that influence the development of recurrent wheezing, asthma, and allergic sensitisation. Nasal respiratory epithelium at birth to 6 weeks, 1, 3, and 5 years will be analysed by bulk RNA-seq and DNA methylation sequencing. Maternal morbidities and in utero exposures will be identified on maternal history and their effects measured through transcriptomic and epigenetic analyses of the amnion and newborn epithelium. Exposures within the first year of life will be identified based on infant medical history as well as on background and symptomatic nasal sampling for viral PCR and microbiome analysis. Daily temperatures and symptoms recorded in a study-specific Smartphone App will be used to identify symptomatic respiratory illnesses. Discussion: The AERIAL study will provide a comprehensive longitudinal assessment of factors influencing the association between epithelial dysfunction and respiratory morbidity in early life, and hopefully identify novel targets for diagnosis and early intervention.
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BACKGROUND: Atherosclerotic cardiovascular disease (ASCVD) is a leading cause of global mortality, often associated with high blood levels of LDL cholesterol (LDL-c). Medications like statins and PCSK9 inhibitors, are used to manage LDL-c levels and reduce ASCVD risk. Recent findings connect the gut microbiota and its metabolites to ASCVD development. We showed that statins modulate the gut microbiota including the production of microbial metabolites involved in the regulation of cholesterol metabolism such as short chain fatty acids (SCFAs) and bile acids (BAs). Whether this pleiotropic effect of statins is associated with their antimicrobial properties or it is secondary to the modulation of cholesterol metabolism in the host is unknown. In this observational study, we evaluated whether alirocumab, a PCSK9 inhibitor administered subcutaneously, alters the stool-associated microbiota and the profiles of SCFAs and BAs. METHODS: We used stool and plasma collected from patients enrolled in a single-sequence study using alirocumab. Microbial DNA was extracted from stool, and the bacterial component of the gut microbiota profiled following an amplicon sequencing strategy targeting the V3-V4 region of the 16S rRNA gene. Bile acids and SCFAs were profiled and quantified in stool and plasma using mass spectrometry. RESULTS: Treatment with alirocumab did not alter bacterial alpha (Shannon index, p = 0.74) or beta diversity (PERMANOVA, p = 0.89) in feces. Similarly, circulating levels of SCFAs (mean difference (95% confidence interval (CI)), 8.12 [-7.15-23.36] µM, p = 0.25) and BAs (mean difference (95% CI), 0.04 [-0.11-0.19] log10(nmol mg-1 feces), p = 0.56) were equivalent regardless of PCSK9 inhibition. Alirocumab therapy was associated with increased concentration of BAs in feces (mean difference (95% CI), 0.20 [0.05-0.34] log10(nmol mg-1 feces), p = 0.01). CONCLUSION: In statin-treated patients, the use of alirocumab to inhibit PCSK9 leads to elevated levels of fecal BAs without altering the bacterial population of the gut microbiota. The association of alirocumab with increased fecal BA concentration suggests an additional mechanism for the cholesterol-lowering effect of PCSK9 inhibition.
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Introduction: Recurrent wheezing disorders including asthma are complex and heterogeneous diseases that affect up to 30% of all children, contributing to a major burden on children, their families, and global healthcare systems. It is now recognized that a dysfunctional airway epithelium plays a central role in the pathogenesis of recurrent wheeze, although the underlying mechanisms are still not fully understood. This prospective birth cohort aims to bridge this knowledge gap by investigating the influence of intrinsic epithelial dysfunction on the risk for developing respiratory disorders and the modulation of this risk by maternal morbidities, in utero exposures, and respiratory exposures in the first year of life. Methods and Analysis: The Airway Epithelium Respiratory Illnesses and Allergy (AERIAL) study is nested within the ORIGINS Project and will monitor 400 infants from birth to five years. The primary outcome of the AERIAL study will be the identification of epithelial endotypes and exposure variables that influence the development of recurrent wheezing, asthma, and allergic sensitisation. Nasal respiratory epithelium at birth to six weeks, one, three, and five years will be analysed by bulk RNA-seq and DNA methylation sequencing. Maternal morbidities and in utero exposures will be identified on maternal history and their effects measured through transcriptomic and epigenetic analyses of the amnion and newborn epithelium. Exposures within the first year of life will be identified based on infant medical history as well as on background and symptomatic nasal sampling for viral PCR and microbiome analysis. Daily temperatures and symptoms recorded in a study-specific Smartphone App will be used to identify symptomatic respiratory illnesses. Ethics and Dissemination: Ethical approval has been obtained from Ramsey Health Care HREC WA-SA (#1908). Results will be disseminated through open-access peer-reviewed manuscripts, conference presentations, and through different media channels to consumers, ORIGINS families, and the wider community.
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Pseudomonas aeruginosa is an opportunistic pathogen. Here, we report the isolation of four bacteriophages from wastewater. All four bacteriophages belong to the Myoviridae family. Kara-mokiny 8, 13, and 16 are of the Pbunavirus genus and have genomes between 65,527 and 66,420-bp. Boorn-mokiny 1 is of the Phikzvirus genus and has a 278,796-bp genome.
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Here, we present the complete genome sequence of Pseudomonas aeruginosa phages Kara-mokiny 1, Kara-mokiny 2, and Kara-mokiny 3. These phages have lytic capabilities against P. aeruginosa and belong to the myovirus morphotype. The genomes of Kara-mokiny 1 and Kara-mokiny 2 are 67,075 bp while that of Kara-mokiny 3 is 66,019 bp long.
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We identified a double-stranded DNA (dsDNA) bacteriophage appearing to belong to Herelleviridae, genus Kayvirus. The bacteriophage, Biyabeda-mokiny 1, was isolated from breast milk using a clinical isolate of Staphylococcus aureus. The genome is 141,091 bp in length and encodes 230 putative coding sequences.
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Two lytic double-stranded DNA (dsDNA) bacteriophages, belonging to the family Herelleviridae, were isolated from wastewater in Western Australia. Biyabeda-mokiny 2 appears to belong to the genus Kayvirus, and Koomba-kaat 1 to Silviavirus.
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Physcomitrium patens apical growing protonemal cells have the singularity that they continue to undergo cell divisions as the plant develops. This feature provides a valuable tool to study autophagy in the context of a multicellular apical growing tissue coupled to development. Herein, we showed that the core autophagy machinery is present in the moss P. patens, and characterized the 2D and 3D growth and development of atg5 and atg7 loss-of-function mutants under optimal and nutrient-deprived conditions. Our results showed that 2D growth of the different morphological and functional protonemata apical growing cells, chloronema and caulonema, is differentially modulated by this process. These differences depend on the protonema cell type and position along the protonemal filament, and growth condition. As a global plant response, the absence of autophagy favors the spread of the colony through protonemata growth at the expense of a reduction of the 3D growth, such as the buds and gametophore development, and thus the adult gametophytic and reproductive phases. Altogether this study provides valuable information suggesting that autophagy has roles during apical growth with differential responses within the cell types of the same tissue and contributes to life cycle progression and thus the growth and development of the 2D and 3D tissues of P. patens.
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Viruses are the cause of a considerable burden to human, animal and plant health, while on the other hand playing an important role in regulating entire ecosystems. The power of new sequencing technologies combined with new tools for processing "Big Data" offers unprecedented opportunities to answer fundamental questions in virology. Virologists have an urgent need for virus-specific bioinformatics tools. These developments have led to the formation of the European Virus Bioinformatics Center, a network of experts in virology and bioinformatics who are joining forces to enable extensive exchange and collaboration between these research areas. The EVBC strives to provide talented researchers with a supportive environment free of gender bias, but the gender gap in science, especially in math-intensive fields such as computer science, persists. To bring more talented women into research and keep them there, we need to highlight role models to spark their interest, and we need to ensure that female scientists are not kept at lower levels but are given the opportunity to lead the field. Here we showcase the work of the EVBC and highlight the achievements of some outstanding women experts in virology and viral bioinformatics.
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Biología Computacional , Investigadores , Virus , Europa (Continente) , Femenino , Humanos , Investigadores/estadística & datos numéricos , Virus/genéticaRESUMEN
BACKGROUND: Grapes (Vitis vinifera L.) are economically the most important fruit crop worldwide. However, the complexity of molecular and biochemical events that lead to the onset of ripening of nonclimacteric fruits is not fully understood which is further complicated in grapes due to seasonal and cultivar specific variation. The Portuguese wine variety Trincadeira gives rise to high quality wines but presents extremely irregular berry ripening among seasons probably due to high susceptibility to abiotic and biotic stresses. RESULTS: Ripening of Trincadeira grapes was studied taking into account the transcriptional and metabolic profilings complemented with biochemical data. The mRNA expression profiles of four time points spanning developmental stages from pea size green berries, through véraison and mature berries (EL 32, EL 34, EL 35 and EL 36) and in two seasons (2007 and 2008) were compared using the Affymetrix GrapeGen® genome array containing 23096 probesets corresponding to 18726 unique sequences. Over 50% of these probesets were significantly differentially expressed (1.5 fold) between at least two developmental stages. A common set of modulated transcripts corresponding to 5877 unigenes indicates the activation of common pathways between years despite the irregular development of Trincadeira grapes. These unigenes were assigned to the functional categories of "metabolism", "development", "cellular process", "diverse/miscellanenous functions", "regulation overview", "response to stimulus, stress", "signaling", "transport overview", "xenoprotein, transposable element" and "unknown". Quantitative RT-PCR validated microarrays results being carried out for eight selected genes and five developmental stages (EL 32, EL 34, EL 35, EL 36 and EL 38). Metabolic profiling using 1H NMR spectroscopy associated to two-dimensional techniques showed the importance of metabolites related to oxidative stress response, amino acid and sugar metabolism as well as secondary metabolism. These results were integrated with transcriptional profiling obtained using genome array to provide new information regarding the network of events leading to grape ripening. CONCLUSIONS: Altogether the data obtained provides the most extensive survey obtained so far for gene expression and metabolites accumulated during grape ripening. Moreover, it highlighted information obtained in a poorly known variety exhibiting particular characteristics that may be cultivar specific or dependent upon climatic conditions. Several genes were identified that had not been previously reported in the context of grape ripening namely genes involved in carbohydrate and amino acid metabolisms as well as in growth regulators; metabolism, epigenetic factors and signaling pathways. Some of these genes were annotated as receptors, transcription factors, and kinases and constitute good candidates for functional analysis in order to establish a model for ripening control of a non-climacteric fruit.
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Frutas/crecimiento & desarrollo , Metaboloma , Transcriptoma , Vitis/genética , Aminoácidos/metabolismo , Metabolismo de los Hidratos de Carbono , Análisis por Conglomerados , Frutas/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Análisis de Secuencia por Matrices de Oligonucleótidos , Reguladores del Crecimiento de las Plantas/metabolismo , ARN Mensajero/genética , ARN de Planta/genética , Transducción de Señal , Vitis/crecimiento & desarrollo , Vitis/metabolismoRESUMEN
Antimicrobial resistance is a current global health crisis, and the increasing emergence of multidrug resistant infections has led to the resurgent interest in bacteriophages as an alternative treatment. Prior to clinical application, phage suitability is assessed, via susceptibility testing and breadth of host range to bacteriophage, however, these are both large-scale manual processes and labor-intensive. The aim of the study was to establish and validate a scaled down methodology for high-throughput screening to reduce procedural footprint. In this paper, we describe a scaled-down adapted methodology that can successfully screen bacteriophages, isolated and purified from wastewater samples. Furthermore, we describe a miniaturized host range assay against clinical Pseudomonas aeruginosa isolates using a spot test (2 µL/ drop) that was found to be both sensitive (94.6%) and specific (94.7%). It also demonstrated a positive predictive value (PPV) of 86.4% and negative predictive value (NPV) of 98%. The breadth of host range of bacteriophages that exhibited lytic activity on P. aeruginosa isolates was corroborated using the scaled down assay. The high correlation achieved in this study confirms miniaturization as the first step in future automation that could test phage diversity and efficacy as antimicrobials.