Isolation and culturing of zebrafish pluripotent cells

A thesis submitted to the University of Bedfordshire in partial fulfilment of the requirements for the degree of Masters by ResearchZebrafish (Dania rerio) is an important model organIsm for the studies of vertebrate development and gene expression in the field of molecular biology and biomedicine. Its embryonic stem cells provide a unique tool linking in vitra and in vivo genetic manipulations of animal genomes. The aim of the project was to determine the most suitable embryo stage for the isolation and culturing of pluripotent cells of zebrafish embryos. Studies were carried out to investigate the expression of two pluripotency markers i.e. Oct4 and Sa1l4 at certain embryonic stages employing Immunohistochemistry. The protein expression studies indicated maximum expression of Oct4 and Sa1l4 at high stage. Primary cultures were initiated from zebrafish high stage embryos in basal nutrient medium; supplemented with insulin, selenite, epidermal growth factor and Foetal Bovine serum. Experiments were conducted for the determination of optimum concentrations of FBS. The growing cultures identified the signs of differentiation i.e. melanocytes and neurite formation. Basic fibroblast growth factor (bFGF) was found to inhibit the differentiation in cultures. The developed pluripotency markers were tested on cultured embryonic cells. Results indicated that these markers work well for the identification ofphenotype of embryonic cell colonies in zebrafish. The establishment of pluripotent cell line will enable knock out and knock in technologies to be used in this species and have important applications in functional genomics research

Invasion and evolutionary history of a generalist fish parasite.

The introduction of non-native species can lead to the introduction of non-native parasites to their introduced range which can pose significant risk to native biodiversity. The cyprinid fish species, Pseudorasbora parva, is a well-studied example of accidental introduction to a new range; it has been accidentally introduced from China to Europe. Pseudorasbora parva has been hypothesized to have also introduced the generalist fish pathogen Sphaerothecum destruens to Europe which has been identified as a potential threat to European fish biodiversity. Due to the management implications associated with the parasite’s status (native or non-native), this work aimed at determining the S. destruens origin and distribution across its native and non-native P. parva populations, whilst also developing eDNA detection methods in order to assess the efficacy of P. parva eradication as a viable control measure for S. destruens. Due to the unique taxonomical position of S. destruens in tree of life, its mitochondrial DNA evolutionary history was also investigated to better decipher its phylogenetic position. Sphaerothecum destruens presence was confirmed in 90 % of the P. parva sampled populations from China, with a maximum prevalence of 10 %. Furthermore, the phylogenetic and demographic analysis of both the host and the parasite support the hypothesis that S. destruens has been introduced to Europe through the accidental introduction of its reservoir host P. parva. The non-native status of S. destruens in Europe has important management implications for the parasite. Furthermore, S. destruens was detected in 50 % of the P. parva samples from 7 populations in the UK and identified new potential hosts for S. destruens in the wild including chub Squalius cephalus, dace leuciscus, roach Rutilus rutilus and brown trout Salmo trutta. The environmental DNA method detected S. destruens in water samples from a P. parva eradicated site 2 years after its eradication which emphasizes that preventive measures against pathogen expansion should be implemented. The phylogenetic tree based on mitochondrial derived protein sequences revealed an interesting position for S. destruens as a sister group to Filasterea and Choanoflagellate and Metazoa group and it has the most derived mitochondrial genome among Choanozoa

Efficacy of Natural and Synthetic Biofilm Inhibitors Associated with Antibiotics in Eradicating Biofilms Formed by Multidrug-Resistant Bacteria

Biofilms formed by multidrug resistant (MDR) bacteria like methicillin-resistant Staphylococcus aureus (MRSA) and others are the main causes of infections that represent a serious public health issue. Persistent MDR infections are mostly derived from biofilm formation which in turn leads to resistance to conventional antimicrobial therapy. Inhibition of bacterial surface attachment is the new alternative strategy without affecting the bacterial growth. Thus, the discovery of compounds that interfere with biofilm production, virulence factors release and quorum sensing (QS) detection in pathogens is a promising processus. Among these compounds, natural and synthetic molecules are a compelling alternative to attenuate pathogenicity. The combination of these compounds with antibiotics makes the bacteria more vulnerable to the later, once used alone. This combination can restore antibiotic effectiveness against MDR bacteria. Among these molecules, 3-phenylpropan-1-amine (3-PPA) has been found to inhibit Serratia marcescens biofilm formation, PAβN has been proven to inhibit biofilm prodcution in A. baumannii, while brominated Furanone C-30 has been reported to be a potent inhibitor of the QS system and P. aeruginosa biofilm. Therefore, the combination between biofilm-inhibitors and antibiotics represents a promising strategy to mitigate antibiotic resistance in MDR pathogens, which has become a major threat to public healthcare around the globe

Phylogenetic and environmental DNA insights into emerging aquatic parasites: implications for risk management.

Species translocation leads to disease emergence in native species of considerable economic importance. Generalist parasites are more likely to be transported, become established and infect new hosts, thus their risk needs to be evaluated. Freshwater systems are particularly at risk from parasite introductions due to the frequency of fish movements, lack of international legislative controls for non-listed pathogens and inherent difficulties with monitoring disease introductions in wild fish populations. Here we used one of the world's most invasive freshwater fish, the topmouth gudgeon, Pseudorasbora parva, to demonstrate the risk posed by an emergent generalist parasite, Sphaerothecum destruens. Pseudorasbora parva has spread to 32 countries from its native range in China through the aquaculture trade and has introduced S. destruens to at least five of these. We systematically investigated the spread of S. destruens through Great Britain and its establishment in native fish communities through a combination of phylogenetic studies of the host and parasite and a novel environmental DNA detection assay. Molecular approaches confirmed that S. destruens is present in 50% of the P. parva communities tested and was also detected in resident native fish communities but in the absence of notable histopathological changes. We identified specific P. parva haplotypes associated with S. destruens and evaluated the risk of disease emergence from this cryptic fish parasite. We provide a framework that can be applied to any aquatic pathogen to enhance detection and help mitigate future disease risks in wild fish populations

AfriMTE and AfriCOMET : Empowering COMET to Embrace Under-resourced African Languages

Despite the progress we have recorded in scaling multilingual machine translation (MT) models and evaluation data to several under-resourced African languages, it is difficult to measure accurately the progress we have made on these languages because evaluation is often performed on n-gram matching metrics like BLEU that often have worse correlation with human judgments. Embedding-based metrics such as COMET correlate better; however, lack of evaluation data with human ratings for under-resourced languages, complexity of annotation guidelines like Multidimensional Quality Metrics (MQM), and limited language coverage of multilingual encoders have hampered their applicability to African languages. In this paper, we address these challenges by creating high-quality human evaluation data with a simplified MQM guideline for error-span annotation and direct assessment (DA) scoring for 13 typologically diverse African languages. Furthermore, we develop AfriCOMET, a COMET evaluation metric for African languages by leveraging DA training data from high-resource languages and African-centric multilingual encoder (AfroXLM-Roberta) to create the state-of-the-art evaluation metric for African languages MT with respect to Spearman-rank correlation with human judgments (+0.406)

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

SummaryBackground Azithromycin has been proposed as a treatment for COVID-19 on the basis of its immunomodulatoryactions. We aimed to evaluate the safety and efficacy of azithromycin in patients admitted to hospital with COVID-19.Methods In this randomised, controlled, open-label, adaptive platform trial (Randomised Evaluation of COVID-19Therapy [RECOVERY]), several possible treatments were compared with usual care in patients admitted to hospitalwith COVID-19 in the UK. The trial is underway at 176 hospitals in the UK. Eligible and consenting patients wererandomly allocated to either usual standard of care alone or usual standard of care plus azithromycin 500 mg once perday by mouth or intravenously for 10 days or until discharge (or allocation to one of the other RECOVERY treatmentgroups). Patients were assigned via web-based simple (unstratified) randomisation with allocation concealment andwere twice as likely to be randomly assigned to usual care than to any of the active treatment groups. Participants andlocal study staff were not masked to the allocated treatment, but all others involved in the trial were masked to theoutcome data during the trial. The primary outcome was 28-day all-cause mortality, assessed in the intention-to-treatpopulation. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936.Findings Between April 7 and Nov 27, 2020, of 16 442 patients enrolled in the RECOVERY trial, 9433 (57%) wereeligible and 7763 were included in the assessment of azithromycin. The mean age of these study participants was65·3 years (SD 15·7) and approximately a third were women (2944 [38%] of 7763). 2582 patients were randomlyallocated to receive azithromycin and 5181 patients were randomly allocated to usual care alone. Overall,561 (22%) patients allocated to azithromycin and 1162 (22%) patients allocated to usual care died within 28 days(rate ratio 0·97, 95% CI 0·87–1·07; p=0·50). No significant difference was seen in duration of hospital stay (median10 days [IQR 5 to >28] vs 11 days [5 to >28]) or the proportion of patients discharged from hospital alive within 28 days(rate ratio 1·04, 95% CI 0·98–1·10; p=0·19). Among those not on invasive mechanical ventilation at baseline, nosignificant difference was seen in the proportion meeting the composite endpoint of invasive mechanical ventilationor death (risk ratio 0·95, 95% CI 0·87–1·03; p=0·24).Interpretation In patients admitted to hospital with COVID-19, azithromycin did not improve survival or otherprespecified clinical outcomes. Azithromycin use in patients admitted to hospital with COVID-19 should be restrictedto patients in whom there is a clear antimicrobial indication

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Screening and Detecting Salmonella in Different Food Matrices in Southern Tunisia Using a Combined Enrichment/Real-Time PCR Method: Correlation with Conventional Culture Method

A combined enrichment/ newly developed invA TaqMan® real-time PCR (qPCR) method as a screening assay to detect Salmonella spp. in 500 naturally food matrices is evaluated. DNA template for qPCR was extracted from an overnight pre-enriched sample in buffered peptone water using lysis–guanidine isothiocyanate method. Heterologous internal amplification control (IAC) was incorporated during qPCR assays and co-amplified with the invA gene of the target pathogen. InvA qPCR exhibited 100% specificity when testing 94 Salmonella strains (inclusivity) and 32 non-Salmonella strains (exclusivity). The qPCR showed a consistent detection of two copies of the invA gene/PCR reaction, a good intra- and inter-run reproducibility with a good PCR efficiency (89.6%). QPCR was sensitive and showed Salmonella detection at 8.5 × 100 CFU mL-1 of artificially spiked poultry meat -BWP solution in less than 40 cycles. When analyzing 500 different food matrices and comparing the results with the ISO 6579:2002 conventional culture method, the sensitivity and specificity were 100 and 76.6%, respectively. QPCR showed Salmonella spp. DNA in raw poultry meat 27/45 (60%), milk 31/93 (33.3%), raw red meat 5/13 (38.5%), and fish 11/46 (23.9%) samples. The prevalence of Salmonella spp. in cakes, dairy, cooked meals, charcuterie products using qPCR was 11/14 (26.8%), 5/22 (22.7%), 32/150 (21.3%), and 5/20 (25%), respectively, compared to 0% as demonstrated by culture. S. Anatum was the most common serovar found associated with red meat compared to S. kentucky isolated from fish and poultry meat. In conclusion, our study is the first to use a combined enrichment/invA qPCR method as a screening assay to detect Salmonella DNA in different types of commercialized food in Southern Tunisia. QPCR results indicate that Salmonella contamination is common in milk and in other types of food samples