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

2025-01-01T00:00:00Z

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

2025-01-01T00:00:00Z

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.)

Identification of single chain variable fragment (ScFv) antibodies specific to immune checkpoints by phage display

2025-01-01T00:00:00Z

Title: Identification of single chain variable fragment (ScFv) antibodies specific to immune checkpoints by phage display Abstract: The inhibition of immune checkpoints is a significant emerging strategy in immunotherapy for the treatment of several malignancies, with much potential for further applications. However, currently there are many pitfalls and drawbacks to the widespread use of this therapy in a clinical setting. One such drawback is the lack of diverse options in the choice for immune checkpoints targeted. This study therefore aimed to identify, isolate, and validate high affinity single chain variable fragment (scFv) antibodies specific to three known immune checkpoints (CD47, TIGIT, and GITR), in an effort to further drive the research and development of new immunotherapeutic strategies for the treatment and management of various cancers. This was accomplished in collaboration with the International Centre for Cancer Vaccine Science in Gdansk, as well as the Roslin Institute, University of Edinburgh, using the phage display method. Numerous rounds of biopanning were performed, beginning from a naïve canine scFv bacteriophage library to create enriched libraries of phages carrying scFv with high affinity to the target proteins. The affinity of the generated scFv antibodies was verified using ELISA, after which, specific phage clones were selected from the biopanning round which showed highest affinity to the target, which were then further amplified and validated with ELISA. The protein sequences of these high affinity phage clones were determined using Sanger sequencing and manually compared. Of the three targets, high affinity scFv clones were only successfully produced against GITR His-tag, isolating 60 scFv phage clones, 43 of which showed high affinity and specificity against GITR protein. All but three clones were successfully sequenced, showing a relatively high amount of diversity of scFv sequences. Further research is required to fully explore the drawbacks of this methodology, as well as successfully identify high affinity scFvs against CD47 and TIGIT. Description: M.Sc. Biomed. Sc.(Melit.)

Sampling barber shops and hair salons for anthropophilic dermatophytes potentially causing tinea capitis in adults

2025-01-01T00:00:00Z

Title: Sampling barber shops and hair salons for anthropophilic dermatophytes potentially causing tinea capitis in adults Abstract: Tinea capitis has emerged as an increasingly common scalp infection in adults in Malta, despite previously being rare and mostly affecting children. Researchers observed a notable rise in cases, particularly those caused by Trichophyton tonsurans, often linked to poor hygiene practices in hairdressing environments. This study investigates the factors contributing to the growing prevalence of adult tinea capitis in Malta. It explores transmission routes, diagnostic approaches, and public health concerns, aiming to highlight the importance of effective hygiene protocols and early detection in controlling the spread of infection. This cross-sectional study used questionnaires and microbiological analysis of hair samples collected from barber shops across Malta and Gozo. Researchers cultured samples on Sabouraud Dextrose Agar with Chloramphenicol and Cycloheximide (SDCC) medium and identified fungi using microscopy and MALDI-TOF MS. Historical T. tonsurans data was retrieved from the Laboratory Information System for comparative analysis. The study identified Maltese males aged between 30-39 as the majority respondents, mainly barber shop owners. It recorded a wide regional distribution of shops and detected two anthropophilic dermatophyte cases in the South. The study highlighted varied hygiene practices and found hair trims, fades, and usage of blades to be popular trends. The study reveals a sharp rise in Trichophyton tonsurans cases in Malta since 2022, peaking in 2024. Poor hygiene in barber shops, like low-temperature laundering, tool reuse, and infrequent cleaning, likely drives transmission. Findings highlight the need for public health interventions, stricter sanitation protocols, and further research to curb dermatophyte spread in high-contact grooming environments. Description: B.Sc. (Hons)(Melit.)