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Validation of autosomal dominant polycystic kidney disease variants using long-range pcr and third generation sequencing

Thu, 01 Jan 2026 00:00:00 GMT

Title: Validation of autosomal dominant polycystic kidney disease variants using long-range pcr and third generation sequencing Abstract: Autosomal dominant polycystic kidney disease (ADPKD) is a hereditary disorder which is characterized by the formation and progressive enlargement of multiple bilateral kidney cysts with an estimated prevalence of 1 in 1000 to 1 in 2500 individuals. ADPKD is also associated with a high phenotypic variability even between affected members of the same family such that the age of onset and the severity of the disease differ significantly between affected individuals. With molecular testing, it is known that 80-85 % of the affected individuals possess a pathogenic variant in PKD1 and almost all the rest are characterized by a pathogenic variant in PKD2. This study involved 3 objectives. The first objective was to confirm the presence, location and pathogenicity of the PKD1 p.C508R variant. Long range (LR) PCR followed by long-read 3rd generation amplicon sequencing were performed which successfully determined that the p.C508R variant is present on PKD1P3 in both ADPKD patients and in non-PKD individuals and thus should be reclassified as a likely benign polymorphism. The second objective was to confirm previously identified variants which were shortlisted by 2nd generation high-throughput sequencing (HTS) in the Malta NGS Project. LR-PCR and/or nested PCR followed by Sanger sequencing were used, which successfully confirmed 7 out of 11 potential causative variants. These variants were re-evaluated to determine pathogenicity. The third and final objective of this project was to carry out further testing on the patients from the 2 pedigrees (P12 and P13) in which no potential causative variant had been identified. PacBio whole genome sequencing (WGS) was performed on a single Sequel IIe SMRT flowcell however, unsatisfactory results were obtained, with insufficient coverage across several parts of the genome and almost no coverage across PKD1. Additional sequencing runs on other Sequel IIe SMRT flowcells per genome or higher throughput HiFi systems such as Revio or Vega are necessary. This study highlights the differences between short-read and long-read sequencing techniques and encourages the integration of LR-PCR with long-read 3rd generation sequencing especially for complex genomic regions or genes with homologous pseudogenes such as PKD1. Description: M.Sc.(Melit.)

Deciphering the compartmentalized host response in sepsis secondary to community-acquired pneumonia

Thu, 01 Jan 2026 00:00:00 GMT

Title: Deciphering the compartmentalized host response in sepsis secondary to community-acquired pneumonia Abstract: Sepsis, currently defined as a complex condition resulting from a dysregulated host response to infection leading to lethal organ dysfunction, is a major global health concern and a leading cause of both morbidity and mortality worldwide. Emerging evidence suggests that immune compartmentalization, variation in the immune response between the circulation and the tissue microenvironment, plays a vital role in the clinical trajectory of sepsis. In sepsis secondary to community-acquired pneumonia (CAP), distinct immune cell distribution and localization of cytokine production may contribute to divergent inflammatory signatures between bronchoalveolar lavage fluid (BALF) and plasma. This project aims to identify compartment-specific immune responses which would aid in further uncovering the pathophysiology of pneumosepsis. This project was a prospective observational single-center study in the intensive care unit (ICU) within Mater Dei Hospital, Malta, forming part of the Molecular Endotype-Specific Dynamics of Lung Endothelial Barrier Integrity in Sepsis (MENDSEP) study. Peripheral blood and BALF samples were obtained from 29 consenting critically ill patients diagnosed with sepsis secondary to CAP (pneumosepsis) between 2023 and 2025. In addition, 16 age, sex, and co-morbidity-matched controls were recruited from the community and from St Vincent de Paul Residence (SVPR). The levels of various cytokines and markers in blood and BALF of septic samples were quantified using multiplex immunoassays. Cytokine analysis revealed marked immune compartmentalization in pneumosepsis. Pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), anti-inflammatory cytokines (IL-10) and anti-viral interferons (IFN-α, IFN-β, IFN-γ) were all found to be significantly enriched in BALF relative to plasma (p<0.001), indicating localized inflammatory and antiviral activity. Conversely, angiopoietin-2 (Ang-2) and the Ang-2:Ang-1 ratio were elevated in the circulation, representing systemic endothelial dysfunction. These findings underscore the compartmentalized nature of the immune response in sepsis, with localized pulmonary inflammation alongside systemic endothelial dysfunction. This study lays the groundwork to further elucidate the complex pathophysiology of sepsis, in improving early diagnosis, and the development of compartment-specific therapeutic targets. Description: M.Sc.(Melit.)

Assessment of DNA sequencing, antibiotic sensitivity, and resistance in yeast cells and human skin microbiome isolates post-space travel and cosmic radiation exposure

Wed, 01 Jan 2025 00:00:00 GMT

Title: Assessment of DNA sequencing, antibiotic sensitivity, and resistance in yeast cells and human skin microbiome isolates post-space travel and cosmic radiation exposure Abstract: Background: The Maleth research program involved three consecutive spaceflight missions in which tissue samples from diabetic foot ulcers (DFUs), containing both polymicrobial bacterial populations and the model eukaryote Saccharomyces cerevisiae, were sent to the International Space Station (ISS). Each mission exposed these bacterial cells to low Earth orbit conditions—including microgravity and heightened radiation for approximately 30 days. Method: Upon return to Earth, the bacterial communities were carefully recovered, cultured and subsequently identified using MALDI-TOF©. To assess potential alterations in antibiotic resistance profiles, all recovered bacterial isolates were cultured and then were subjected to antimicrobial susceptibility testing via the VITEK® 2 automated system. In parallel, Saccharomyces cerevisiae cells were evaluated for genomic changes through targeted sequencing and comparative genetic analysis against Earth-based controls at Singleron Germany. Results: The results showed that Proteus mirabilis was among the most significantly impacted bacterial species, with its post-spaceflight counts showing statistically significant differences compared to ground controls. Moreover, the isolates of Proteus mirabilis exhibited increased resistance to antibiotics, suggesting that space conditions may exert selective pressure conducive to resistance development. In contrast, analysis of Saccharomyces cerevisiae demonstrated no detectable genomic changes across any of the space-exposed samples, under the tested spaceflight conditions although there is the possibility that the duration in space was short and not sufficient to bring changes in the genome. Conclusion: These findings suggest that exposure to space can induce antimicrobial resistance characteristics of bacterial pathogens, such as in Proteus mirabilis. The implications of this study highlight the potential risks posed by altered microbial behaviour during long-term space and their effects on AMR in patients. Description: M.Sc. Biomed. Sc.(Melit.)

Development of monoclonal ScFv antibodies targeting the t-cell co-inhibitory molecules VISTA and PD-L1

Wed, 01 Jan 2025 00:00:00 GMT

Title: Development of monoclonal ScFv antibodies targeting the t-cell co-inhibitory molecules VISTA and PD-L1 Abstract: Tumour development is a multistep process characterized by aberrant monoclonal cell proliferation. Immune cells exert selective pressure on tumour populations, promoting the survival of low-immunogenic variants that can evade immune detection. One such mechanism involves the recruitment of immunosuppressive cells expressing inhibitory receptors and ligands that suppress T-cell activation. Currently, inhibitory receptors and ligands are being extensively studied as therapeutic targets. The food and drug administration (FDA) has approved several immune checkpoint inhibitors (ICIs) targeting Cytotoxic T-Lymphocyte-Associated Protein 4 (CTLA-4), Programmed Cell Death Protein 1 (PD-1), and its ligand Programmed Cell Death-Ligand 1 (PD-L1). These treatments have shown efficacy in limiting tumour progression and extending patientsurvival.However, due to dynamic intrinsic and extrinsic changes in the tumour microenvironment (TME), a high rate of acquired resistance to ICIs remains a major challenge. To address this, alternative co-inhibitory molecules are being explored for therapeutic targeting, either as standalone agents or in combination therapies. In this study, recombinant single-chain variable fragment (scFv) antibodies with high affinity for the co-inhibitory molecules V-domain Immunoglobulin Suppressor of T-cell Activation (VISTA) and PD-L1 were initially targeted. These co-inhibitory molecules play a critical role in immune regulation by inhibiting T-cell proliferation and cytokine production when interacting with their respective ligands. Both VISTA and PD-L1 have been shown to be overexpressed in various solid tumours including melanoma, non-small cell lung carcinoma (NSCLC) and ovarian cancer. The generation of recombinant scFv antibodies against these co-inhibitory molecules were pursued using the phage display biopanning technique. Due to technical challenges encountered during the biopanning phage display process, a statistically significant increase in binding towards either bait protein was not achieved after four rounds. As a result the project’s focus shifted toward characterizing 60 monoclonal antibodies derived from a previously successful VISTA-directed biopanning experiment conducted in a separate study. These clones were evaluated for specificity against VISTA and control bait proteins, and their binding affinities were analysed using the Enzyme-Linked Immunosorbent Assay (ELISA). ELISA analysis revealed that most of the clones exhibited strong binding affinity to VISTA with minimal cross-reactivity to control proteins. Notably, most clones demonstrated statistically significant preferential binding to their target bait, with p-values <0.000001. To assess sequence diversity, Sanger sequencing was performed on all clones. The results of Sanger sequencing showed that full-length sequencing was completed only on half of the, most of which had an identical sequence, suggesting potential amplification bias during phage display. Thus, in this project, 60 monoclonal scFvs with a high binding affinity towards VISTA and minimal cross-reactivity towards control bait proteins was achieved. Sanger sequencing of these clones showed minimal diversity indicating an amplification bias during biopanning phage display. Further confirmatory and validation techniques are required to validate the functional activity of the scFv binders. Future efforts should focus on optimizing the biopanning phage display protocol to enhance target-specific library generation and minimize bias. Description: M.Sc. Biomed. Sc.(Melit.)