3,165 research outputs found
A unified compendium of prokaryotic and viral genomes from over 300 anaerobic digestion microbiomes
BackgroundThe anaerobic digestion process degrades organic matter into simpler compounds and occurs in strictly anaerobic and microaerophilic environments. The process is carried out by a diverse community of microorganisms where each species has a unique role and it has relevant biotechnological applications since it is used for biogas production. Some aspects of the microbiome, including its interaction with phages, remains still unclear: a better comprehension of the community composition and role of each species is crucial for a cured understanding of the carbon cycle in anaerobic systems and improving biogas production.ResultsThe primary objective of this study was to expand our understanding on the anaerobic digestion microbiome by jointly analyzing its prokaryotic and viral components. By integrating 192 additional datasets into a previous metagenomic database, the binning process generated 11,831 metagenome-assembled genomes from 314 metagenome samples published between 2014 and 2022, belonging to 4,568 non-redundant species based on ANI calculation and quality verification. CRISPR analysis on these genomes identified 76 archaeal genomes with active phage interactions. Moreover, single-nucleotide variants further pointed to archaea as the most critical members of the community. Among the MAGs, two methanogenic archaea, Methanothrix sp. 43zhSC_152 and Methanoculleus sp. 52maCN_3230, had the highest number of SNVs, with the latter having almost double the density of most other MAGs.ConclusionsThis study offers a more comprehensive understanding of microbial community structures that thrive at different temperatures. The findings revealed that the fraction of archaeal species characterized at the genome level and reported in public databases is higher than that of bacteria, although still quite limited. The identification of shared spacers between phages and microbes implies a history of phage-bacterial interactions, and specifically lysogenic infections. A significant number of SNVs were identified, primarily comprising synonymous and nonsynonymous variants. Together, the findings indicate that methanogenic archaea are subject to intense selective pressure and suggest that genomic variants play a critical role in the anaerobic digestion process. Overall, this study provides a more balanced and diverse representation of the anaerobic digestion microbiota in terms of geographic location, temperature range and feedstock utilization
Ruthenium metallotherapeutics: a targeted approach to combatting multidrug resistant pathogens
The discovery of antibiotics revolutionised healthcare practice. However due to overuse, inappropriate use, widespread prophylaxis therapy and the lack of new developments, the threat of antimicrobial resistance is now a major global threat to health. By 2050, it is estimated that mortality due to antimicrobial resistant infections will exceed 10 million people per annum, superseding cancer as the leading cause of global mortality. The use of drug repurposing to identify potential therapies which combat antimicrobial resistance is one potential solution. Metals have been used as antimicrobial agents throughout the history of medicine for a broad range of applications, including the use of Silver as an antimicrobial agent which dates back to antiquity. More recently, Ruthenium metallotherapeutic complexes have been shown to exhibit highly active antimicrobial properties by targeting a range of bacterial species, and in contrast to traditional antibiotics, these compounds are thought to elicit antibacterial activity at multiple sites within the bacterial cell, which may reduce the possibility of resistance evolution. This study aimed to evaluate the antimicrobial activity of a series of Ruthenium metallotherapeutic complexes against multidrug-resistant bacterial pathogens, with a focus on use within wound care applications.
Antimicrobial susceptibility assays identified two lead candidates, Hexaammineruthenium (III) chloride and [Chlorido(η6-p-cymene)(N-(4-chlorophenyl)pyridine-2-carbothioamide) ruthenium (II)] chloride which demonstrated activity against Pseudomonas aeruginosa and Staphylococcus aureus respectively with MIC values ranging between 4 μg mL-1 and 16 μg mL-1. Furthermore, Hexaammineruthenium (III) chloride demonstrated antibiofilm activity in both a time and concentration-dependent manner. Synergy studies combining lead complexes with antibiotics demonstrated the potential for use as resistance breakers. Subsequent in vitro infection modelling using scratch assays with skin cell lines, coupled with a 3D full thickness skin wound infection model was used to determine potential applied applications of Hexaammineruthenium (III) chloride for use as topical antimicrobial agent against P. aeruginosa infections.
Antimicrobial mechanistic studies demonstrated that Hexaammineruthenium (III) chloride targeted the bacterial cell ultrastructure of P. aeruginosa strain PAO1 as cell perturbations were observed when treated cells were analysed by scanning electron microscopy. Furthermore, exposure of P. aeruginosa PAO1 to Hexaammineruthenium (III) chloride also resulted in a concentration dependent membrane depolarisation, which further supported the antimicrobial mechanistic role.
Finally, global changes in gene expression following exposure of P. aeruginosa strain PAO1 to Hexaammineruthenium (III) chloride were explored by RNA sequencing. Genes involved in ribosome function, cofactor biosynthesis and membrane fusion were downregulated, which provided a further insight into the wider mechanisms of antibacterial activity.
The research conducted in the present study indicated the potential use of Hexaammineruthenium (III) chloride (and derivatives) as a potential treatment option for chronic wounds infected with P. aeruginosa, which could be applied as either a direct treatment or used within antimicrobial wound care applications
Advanced sequencing technologies applied to human cytomegalovirus
The betaherpesvirus human cytomegalovirus (HCMV) is a ubiquitous viral pathogen. It is the most common cause of congenital infection in infants and of opportunistic infections in immunocompromised patients worldwide. The large double-stranded DNA genome of HCMV (236 kb) contains several genes that exhibit a high degree of variation among strains within an otherwise highly conserved sequence. These hypervariable genes encode immune escape, tropism or regulatory factors that may affect virulence. Variation arising from these genes and from an evolutionary history of recombination between strains has been hypothesised to be linked to disease severity. To investigate this, the HCMV genome has been scrutinised in detail over the years using a variety of molecular techniques, most looking only at one or a few of these genes at a time. The advent of high-throughput sequencing (HTS) technology 20 years ago then started to enable more in-depth whole-genome analyses. My study extends this field by using both HTS and the more recently developed long-read nanopore technology to determine HCMV genome sequences directly from clinical samples. Firstly, I used an Illumina HTS pipeline to sequence HCMV strains directly from formalin-fixed, paraffin-embedded (FFPE) tissues. FFPE samples are a valuable repository for the study of relatively rare diseases, such as congenital HCMV (cCMV). However, formalin fixation induces DNA fragmentation and cross-linking, making this a challenging sample type for DNA sequencing. I successfully sequenced five whole HCMV genomes from FFPE tissues. Next, I developed a pipeline utilising the single-molecule, long-read sequencer from Oxford Nanopore Technologies (ONT) to sequence HCMV initially from high-titre cellcultured laboratory strains and then from clinical samples with high HCMV loads. Finally, I utilised a direct RNA sequencing protocol with the ONT sequencer to characterise novel HCMV transcripts produced during infection in cell culture, demonstrating the existence of transcript isoforms with multiple splice sites. Overall, my findings demonstrate how advanced sequencing technologies can be used to characterise the genome and transcriptome of a large DNA virus, and will facilitate future studies on HCMV prognostic factors, novel antiviral targets and vaccine development
Breeding Melons for Resistance to Viral and Fungal Diseases. Exploiting the Multi-Resistant Accession TGR-1551
[ES] Las cucurbitáceas son la segunda familia de hortícolas más importante a nivel mundial, solo por detrás de las solanáceas. Tradicionalmente su cultivo se ha llevado a cabo en las zonas templadas del planeta. Sin embargo, las condiciones de cambio climático, el comercio internacional y los modelos de agricultura intensiva favorecen la aparición de nuevas virosis y enfermedades fúngicas en zonas donde antes no estaban presentes. En este sentido, resulta esencial el monitoreo periódico de las principales zonas productoras, para así poder detectar los virus y hongos emergentes en cada territorio y adaptar los programas de mejora a los objetivos específicos de cada zona. En el caso concreto del melón (Cucumis melo) existe una gran variabilidad intraespecífica que puede servir como fuente de alelos de resistencia frente a estos patógenos. Sin embargo, las fuentes de resistencia suelen encontrarse dentro del germoplasma silvestre, normalmente originario de África o Asia, y en el que el nivel de domesticación es reducido. Para un mejor aprovechamiento de las accesiones resistentes, resulta necesario un estudio del control genético de los caracteres de interés, que permita localizar las regiones asociadas a la resistencia y diseñar marcadores moleculares asociadas a las mismas. Esto facilita los programas de mejora orientados a la introgresión de las resistencias manteniendo el fondo genético de las variedades de interés
En la presente tesis doctoral, durante las campañas de verano de 2019 y 2020, se ha llevado a cabo un estudio de la incidencia y diversidad genética de 9 especies virales potencialmente limitantes para el cultivo de cucurbitáceas en el sur este español. Se ha podido observar que los virus transmitidos por pulgones son prevalentes frente a los transmitidos por mosca blanca. Dentro del primer grupo destacó la presencia de watermelon mosaic virus (WMV), cucurbits aphid borne yellows virus (CABYV) y cucumber mosaic virus (CMV), ya que fueron detectados en todas las zonas y cultivos estudiados, apareciendo frecuentemente en infecciones mixtas. Moroccan watermelon mosaic virus (MWMV) y tomato leaf curl New Delhi virus (ToLCNDV) también fueron detectados en algunas zonas, pero con porcentajes de infección más bajos y normalmente en infecciones mixtas con WMV. Los análisis filogenéticos de los distintos aislados encontrados ha permitido la identificación de 7 nuevos perfiles moleculares de WMV y de aislados recombinantes de CMV, lo que es consistente con los resultados obtenidos en otros países y pone de manifiesto la gran variabilidad de estos patógenos.
Las accesiones silvestres de melón recogidas en distintos bancos de germoplasma son un valioso recurso para los programas de mejora genética frente a estreses bióticos. La accesión africana TGR-1551 ha sido descrita previamente como resistente a WMV, CYSDV (cucurbit yellow stunting disorder virus), CABYV y el hongo Podosphaera xanthii (Px, razas 1, 2 y 5) agente causal del oídio en melón. Además, es tolerante a la mosca blanca (Bemisia tabaci) y portadora del gen Vat (virus aphid transmission), el cual limita la transmisión de virus por pulgón. Por lo tanto, esta accesión constituye una buena fuente de alelos de resistencia y, al poder utilizar un único parental donante, su uso acortaría los programas de mejora.
En el marco de la presente tesis doctoral, mediante el desarrollo de poblaciones segregantes de mapeo y el aprovechamiento de las tecnologías de genotipado masivo se han podido cartografiar los QTLs asociados a la resistencia a CYSDV derivados de esta entrada. En el caso de la resistencia a CYSDV, se han detectado dos QTL en el cromosoma 5. El primero de ellos es de efecto mayor y herencia dominante, estando asociado al desarrollo de síntomas. El segundo QTL, de efecto menor y también de herencia dominante, no confiere resistencia por sí mismo y está asociado a la carga viral durante la infección. Siguiendo una estrategia similar se han podido cartografiar y estrecha[CA] Les cucurbitàcies són la segona família d'hortícoles més important a nivell mundial, només per darrere de les solanàcies. Tradicionalment el seu cultiu s'ha dut a terme a les zones temperades del planeta. No obstant això, les condicions de canvi climàtic, el comerç internacional i els models d'agricultura intensiva afavoreixen l'aparició de noves virosis i malalties fúngiques en zones on abans no estaven presents. En aquest sentit, resulta essencial el monitoratge periòdic de les principals zones productores, per a d'aquesta manera, poder detectar els virus i fongs emergents en cada territori i adaptar els programes de millora als objectius específics de cada zona. En el cas concret del meló (Cucumis melo) existeix una gran variabilitat intraespecífica que pot servir com a font d'al·lels de resistència enfront d'aquests patògens. No obstant això, les fonts de resistència solen trobar-se dins del germoplasma silvestre, normalment originari d'Àfrica o Àsia, i en el qual el nivell de domesticació és reduït. Per a un millor aprofitament de les accessions resistents, resulta necessari un estudi del control genètic dels caràcters d'interés, que permeta localitzar les regions associades a la resistència i dissenyar marcadors moleculars associats a aquestes. Això facilita els programes de millora orientats a la introgressió de les resistències mantenint el fons genètic de les varietats d'interés.
En la present tesi doctoral, durant les campanyes d'estiu de 2019 i 2020, s'ha dut a terme un estudi de la incidència i diversitat genètica de nou espècies virals potencialment limitants per al cultiu de cucurbitàcies en el sud-est espanyol. S'ha pogut observar que els virus transmesos per pugons són prevalents enfront dels transmesos per mosca blanca. Dins del primer grup va destacar la presència de watermelon mosaic virus (WMV), cucurbits aphid born yellows virus (CABYV) i cucumber mosaic virus (CMV), ja que van ser detectats en totes les zones i cultius estudiats, apareixent sovint en infeccions mixtes. Moroccan watermelon mosaic virus (MWMV) i tomatoleaf curl New Delhi virus (ToLCNDV) també van ser detectats en algunes zones, però amb percentatges d'infecció més baixos i normalment en infeccions mixtes amb WMV. Les anàlisis filogenètiques dels diferents aïllats trobats ha permés la identificació de set nous perfils moleculars de WMV i d'aïllats recombinants de CMV, la qual cosa és consistent amb els resultats obtinguts en altres països i posa de manifest la gran variabilitat d'aquests patògens.
Les accessions silvestres de meló recollides en diferents bancs de germoplasma són un valuós recurs per als programes de millora genètica enfront d'estressos biòtics. L'accessió africana *TGR-1551 ha sigut descrita prèviament com a resistent a WMV, CYSDV (cucurbit yellow stunting disorder virus), CABYV i el fong Podosphaera xanthii (Px, races 1, 2 i 5) agent causal de l'oïdi en meló. A més, és tolerant a la mosca blanca (Bemisia tabaci) i portadora del gen Vat (virus aphid transmission), el qual limita la transmissió de virus per pugó. Per tant, aquesta accessió constitueix una bona font d'al·lels de resistència i, en poder utilitzar un únic parental donant, el seu ús acurtaria els programes de millora.
En el marc de la present tesi doctoral, mitjançant el desenvolupament de poblacions segregants de mapatge i l'aprofitament de les tecnologies de genotipat massiu s'ha pogut cartografiar els QTLs associats a la resistència a CYSDV derivats d'aquesta entrada. En el cas de la resistència a CYSDV, s'han detectat dues QTL en el cromosoma cinc. El primer d'ells és d'efecte major i herència dominant, estant associat al desenvolupament de símptomes. El segon QTL, d'efecte menor i també d'herència dominant, no confereix resistència per si mateix i està associat a la càrrega viral durant la infecció. Seguint una estratègia similar s'han pogut cartografiar i estrényer els *QTLs de resistència enfront de Px. En aquest cas es tracta d'una epistàsia dominant-re[EN] Cucurbits represent the second most important horticultural family worldwide, second only the Solanaceae family. Traditionally, their cultivation has been concentrated in temperate regions across the globe. However, climate change conditions, international trade, and intensive agricultural practices are contributing to the emergence of new viral and fungal diseases in regions where they were previously absent. In this regard, it is crucial to regularly monitor major production areas to detect emerging viruses and fungi specific to each region. This monitoring allows for the adaptation of breeding programs to the unique goals of each area.
In the case of melon (Cucumis melo), it exists significant intraspecific variability that can serve as a source of resistance alleles against these pathogens. However, sources of resistance are often found within wild germplasm, typically originating from Africa or Asia, and characterized by limited domestication. To better utilize these resistant accessions, a study of the genetic control of desirable traits is necessary. This study aims to locate regions associated with resistance and design molecular markers linked to these regions. Such an approach streamlines breeding programs focused on introgressing resistance traits while preserving the genetic background of the desired varieties.
During the summer campaigns of 2019 and 2020, this doctoral thesis conducted a study on the incidence and genetic diversity of nine viral species potentially affecting cucurbit cultivation in southeastern Spain. It was observed that viruses transmitted by aphids were more prevalent than those transmitted by whiteflies. Within the first group, the presence of watermelon mosaic virus (WMV), cucurbits aphid borne yellows virus (CABYV), and cucumber mosaic virus (CMV) stood out, as they were detected in all the studied areas and crops, often in mixed infections. Moroccan watermelon mosaic virus (MWMV) and tomato leaf curl New Delhi virus (ToLCNDV) were also detected in some areas but with lower infection percentages, typically in mixed infections with WMV. Phylogenetic analyses of the found isolates have identified seven new molecular profiles of WMV and recombinant CMV isolates, which is consistent with results from other countries, highlighting the extensive variability of these pathogens.
Wild melon accessions preserved in various germplasm banks represent a valuable resource for breeding programs against biotic stresses. The African accession TGR-1551 has been previously described as resistant to WMV, CYSDV (cucurbit yellow stunting disorder virus), CABYV, and the fungus Podosphaera xanthii (Px, races 1, 2, and 5), which causes powdery mildew in melons. Additionally, it is tolerant to whiteflies (Bemisia tabaci) and carries the Vat gene (Virus Aphid Transmission), limiting virus transmission by aphids. Therefore, this accession constitutes as an excellent source of resistance alleles, and its use, as a single donor parent, can expedite breeding programs.
Within the scope of this doctoral thesis, through the development of segregating mapping populations and the utilization of high-throughput genotyping technologies, the QTLs associated with CYSDV resistance from this accession have been mapped. In the case of CYSDV resistance, two QTLs have been detected on chromosome 5. The first of these, with major effects and dominant inheritance, is associated with symptom development. The second QTL, with minor effects and also dominant inheritance, does not confer resistance by itself and is linked to viral load during infection. A similar strategy was employed to map and narrow down the QTLs for resistance against Px. In this case, it involves a dominant-recessive epistasis, with the recessive gene located on chromosome 12 and the dominant gene on chromosome 5, specifically in the same region where the major CYSDV resistance QTL is located.
Regarding resistance against WMV, previous studies conducted by the researchThis research was funded by the Spanish Ministerio de Ciencia e Innovación
(MCIN/AEI/10.13039/501100011033), grant number PID2020-116055RB (C21 and
C22), and by the Conselleria d’Educació, Investigació, Cultura i Esports de la
Generalitat Valenciana, grant number PROMETEO/2021/072 (to promote
excellence groups, cofinanced with FEDER funds). M.L. is a recipient of a
predoctoral fellowship (PRE2018-083466) of the Spanish Ministerio de Ciencia,
Innovación y Universidades co-financed with FSE funds.López Martín, M. (2023). Breeding Melons for Resistance to Viral and Fungal Diseases. Exploiting the Multi-Resistant Accession TGR-1551 [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/20206
Natural Hosts and Genetic Diversity of the Emerging Tomato Leaf Curl New Delhi Virus in Spain
Knowledge about the host range and genetic structure of emerging plant viruses provides insights into fundamental ecological and evolutionary processes, and from an applied perspective, facilitates the design and implementation of sustainable disease control measures. Tomato leaf curl New Delhi virus (ToLCNDV) is an emerging whitefly transmitted begomovirus that is rapidly spreading and inciting economically important diseases in cucurbit crops of the Mediterranean basin. Genetic characterization of the ToLCNDV Mediterranean populations has shown that they are monophyletic in cucurbit plants. However, the extent to which other alternative (cultivated and wild) hosts may affect ToLCNDV genetic population structure and virus prevalence remains unknown.
In this study a total of 683 samples from 13 cultivated species, and 203 samples from 24 wild species from three major cucurbit-producing areas of Spain (Murcia, Alicante and Castilla-La Mancha) from five cropping seasons (2012–2016) were analyzed for ToLCNDV infection. Except for watermelon, ToLCNDV was detected in all cultivated cucurbit species as well as in tomato. Among weeds, Ecballium elaterium, Datura stramonium, Sonchus oleraceus, and Solanum nigrum were identified as alternative ToLCNDV plant hosts, which could act as new potential sources of virus inoculum. Furthermore, we performed full-genome deep-sequencing of 80 ToLCNDV isolates from different hosts, location and cropping year. Our phylogenetic analysis supports a Mediterranean virus population that is genetically very homogeneous, with no clustering pattern, and clearly different from Asian virus populations. Additionally, D. stramonium
displayed higher levels of within-host genetic diversity than cultivated plants, and this variability appeared to increase with time. These results suggest that the potential ToLCNDV adaptive evolution occurring in wild plant hosts could serve as a source of virus genetic variability, thereby affecting the genetic structure and spatial-temporal dynamics of the viral population
The role of the oral microbiome in the immunobullous diseases pemphigus vulgaris and mucous membrane pemphigoid and oral lichen planus
Saliva is formed from contributions of salivary glands and the serum exudates principally from gingival margins or damaged mucosa combined with components derived from the environment, including a community of microorganisms - the microbiome. I postulate that changes in microbial diversity and population structure play key roles in the modulation of host- microbial interactions which influence both the hypersensitive autoimmune responses and inflammation seen in these inflammatory mucocutaneous disorders. For my research, a total of 186 participants were recruited: 48 mucous membrane pemphigoid (MMP), 48 pemphigus vulgaris (PV), 50 oral lichen planus (OLP) patients, and 40 healthy controls. Unstimulated whole saliva, subgingival plaque, serum, and plasma samples were collected from 186 participants. In addition, metadata were collected on the following covariates: age, gender, ethnicity, type of the diet, disease history and therapeutic intervention in the preceding six months. Oral disease severity scores (ODSS) were assessed, and periodontal status was examined using a periodontal six pocket chart. To characterise microbiome profiles, saliva and subgingival plaque were processed for sequencing genomic DNA using the NGS Shotgun metagenomics sequencing technique. Inflammatory cytokines and proteases were investigated in saliva and serum using Human Magnetic Luminex Screening Assay (R&D Systems). Selected cytokines were analysed by enzyme-linked immunosorbent assay (ELISA) technique (R&D Systems) to determine host inflammatory responses in saliva and serum samples. Additionally, saliva and plasma samples were analysed for metabolites by nuclear magnetic resonance (NMR). Significant increases in periodontal score (PISA) in all three groups of disease were identified compared to healthy control group with significant positive correlation between oral disease severity (ODSS) and PISA in OLP and PV groups. All three groups of diseases had significantly higher levels of inflammatory Th2/Th17 cytokines (IL-6, IL-13 and IL-17 in saliva samples), as well as higher levels of MMP-3 matrixins in saliva. In addition, there were positive correlations between ODSS and salivary IL-6, IL-13 and MMP-3 in saliva of OLP, salivary and serum levels of IL-6 and MMP-3 in MMP group, and significant association of salivary IL-6, IL-1β and MMP-3 in PV group. Metabolomic data showed that saliva is a better biofluid for correlation of the metabolomic profile with oral disease severity than plasma. Salivary ethanol was corelated with disease severity in the OLP group, whereas in PV was a strong correlation of ODSS with choline. Finally, a unique microbial community was found in each group of diseases. In the MMP group, ODSS was significantly correlated with L. hofstadii, C. sputigena, N. meningitidis, N. cinerea and P. sacchar0lytica. In PV, a positive correlation was found with F. nucleatum, G. morbillorum, and E. corrodens, G. elegans, H. sapiens and T. vincentii. In OLP, the disease tends to worsen when there was reduced abundance of X. cellulosilytica, Actinomyces ICM 47, S. parasanguinis, S. salivarius, L. mirabilis and O. sinus. Lower microbial diversity was correlated with ODSS in saliva and plaque of the OLP group. In conclusion, this study provides strong evidence of the complex interplay between the oral microbiome, immunological factors, and metabolites in the context of immunobullous diseases and OLP. The findings highlight the integral role of oral bacteria in disease progression, the significance of immune dysregulation, and the potential impact of specific microbial species and metabolic pathways. These insights give the way for further research and clinical applications, offering the promise of personalized approaches for diagnosis, and management of OLP, MMP and PV. Future investigations should focus on discovering the mechanistic details underlying these associations and validating the identified biomarkers in larger patient cohorts, ultimately contributing to a deeper understanding of the pathogenesis of these conditions
Development and validation of innovative sequencing tools for the fast and efficient detection of plant virus
Plant viruses are a major cause of crop losses and decreased agricultural productivity worldwide. Rapid and accurate detection of plant viruses is essential for the implementation of effective control measures. Traditional methods of plant virus detection, such as serological and molecular assays, often present very good performance criteria but they are targeted, and they don’t detect new viruses or divergent strains of known viruses.
Overall, developing and validating innovative sequencing tools for fast and efficient detection of plant viruses gained a lot of leverage. Indeed, high throughput sequencing (HTS) tests followed by bioinformatic analyses can detect several viruses at once (including novel ones) and then characterise their genomes. This very high inclusivity allows better monitoring of agricultural pest presence than traditional methods. In addition, the sensitivity of HTS viral detection is theoretically higher than molecular and serological tests, meaning that low-level infection can be traced more efficiently.
HTS tests have several drawbacks: the price, the high technical requirements and the cross-contamination of sequences between samples nevertheless. The cost of viral detection by sequencing is higher than traditional methods, but the cost gap is reducing over time as HTS is more and more affordable. More technical skills are required for sequencing and analysis of a sample for virus detection, but the laboratory and bioinformatic protocols are becoming simpler and easier to learn and apply. Cross-contamination between samples is a recurrent phenomenon that is challenging the operational activities of laboratories aiming to detect plant pests. The high sensitivity of HTS has a drawback as it means that cross-contamination is an even more pressing issue than with traditional methods.
Cross-contamination is probably one of the main issues when using HTS for viral detection. Indeed, if an unexpected genetic material transfer happens between two samples in the laboratory, one virus can be sequenced in the other sample. Since sequencing sensitivity is high, HTS is more prone to detect this cross-contaminating virus. That may lead to a false positive virus detection (as it is really in the bioinformatic data) while it was not present in the plant. The specificities of HTS technologies (high sensitivity, high inclusivity but with the complexity of laboratory and bioinformatics steps) make their validation difficult compared to traditional tests. Therefore, this thesis describes the side-by-side comparison between traditional tests and HTS technologies for virus indexing of Musa germplasm collection. In addition, an alien control (a specific type of external control) has been used for the first time to
II
monitor cross-contamination in HTS. In addition, a newly described alien-based filter algorithm, called Cont-ID, has been developed and applied to find the most appropriate limit of detection that should be applied for accurate virus detection taking into account the risk of false negatives and false positives. That way, the detection prediction's confidence can be high enough to be considered for its use in plant virus diagnosis.
As written above, HTS technologies can also characterise the genome of the detected viruses. Through variant analysis, the different virus variants can be highlighted. A performance testing was conducted to better understand the difficulties and therefore improve the variants' characterisation.
This thesis has therefore addressed several drawbacks limiting potentially the use of HTS technologies for plant virus detection and genome characterisation. It has delivered several milestones to contribute to these technologies' wider and more reliable applications for plant virus detection. Overall, it has reinforced its high potential for improving the control and management of plant virus diseases.2. Zero hunge
The Host-Microbiota Axis in Chronic Wound Healing
Chronic, non-healing skin wounds represent a substantial area of unmet clinical need, leading to debilitating morbidity and mortality in affected individuals. Due to their high prevalence and recurrence, chronic wounds pose a significant economic burden. Wound infection is a major component of healing pathology, with up to 70% of wound-associated lower limb amputations preceded by infection. Despite this, the wound microbiome remains poorly understood. Studies outlined in this thesis aimed to characterise the wound microbiome and explore the complex interactions that occur in the wound environment. Wound samples were analysed using a novel long-read nanopore sequencing-based approach that delivers quantitative species-level taxonomic identification. Clinical wound specimens were collected at both the point of lower-extremity amputation and via a pilot clinical trial evaluating extracorporeal shockwave therapy (ESWT) for wound healing. Combining microbial community composition, host tissue transcriptional (RNAseq) profiling, with clinical parameters has provided new insight into healing pathology. Specific commensal and pathogenic organisms appear mechanistically linked to healing, eliciting unique host response signatures. Patient- and site-specific shifts in microbial abundance and communitycomposition were observed in individuals with chronic wounds versus healthy skin. Transcriptional profiling (RNAseq) of the wound tissue revealed important insight into functional elements of the host-microbe interaction. Finally, ESWT was shown to confer beneficial effects on both cellular and microbial aspects of healing. High-resolution long-read sequencing offers clinically important genomic insights, including rapid wide-spectrum pathogen identification and antimicrobial resistance profiling, which are not possible using current culture-based diagnostic approaches. Thus, data presented in this thesis provides important new insight into complex host-microbe interactions within the wound microbiome, providing new and exciting future avenues for diagnostic and therapeutic approaches to wound management
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