Antibiotic resistance in microorganisms poses a global hazard to human and animal health. To combat antibiotic resistance, a deeper understanding of the mechanisms behind the spread of antibiotic resistance genes (ARGs) in bacteria is crucial. Plasmids are major vehicles for the spreading of ARGs, so their conjugation-mediated transfer is examined in this thesis. Escherichia coli (E. coli) was isolated from a wastewater treatment plant (WWTP) and clean water in Stockholm, Sweden and the isolates were used as donors of mobile plasmids and a lab strain of E. coli (CV601) was used as recipient. Transfer efficiency was analysed by calculating conjugation frequencies (CF), after testing conjugation by fast conjugation and filter mating method. The resulting transconjugants were analysed for antibiotic resistance pattern by the disc diffusion method. Additionally, sequence analysis was used to analyse the transferred plasmid(s) and ARGs. The qPCR was performed to check the expression of transfer genes (TraN and TraJ) during conjugation of IncN plasmids. The results showed that several isolates from WWTP were multi-resistant and successfully transferred plasmids to CV601 and donor strain 18 with a mobile IncN plasmid had the highest CF that depended mainly on the type of pili. The ARGs qnrS1, dfra14, and blaCTX-M-15 were transferred to transconjugants mainly by IncN plasmids and qPCR findings indicated that the level of gene expression of transfer genes in donor strains affected CF. The findings of this study expand our understanding of antibiotic resistance dissemination and knowledge of plasmids found in WWTPs and clean water.

Sclerotinia sclerotiorum, a notorious phytopathogenic fungus, causes sclerotinia stem rot in oilseed rape (Brassica napus), a disease with global consequences for oilseed rape productivity and oil quality. The complexity of factors contributing to sclerotinia stem rot makes prediction and control exceedingly challenging. Early and accurate identification of plant pathogens is crucial for effective disease management. This study aimed to establish a method utilizing MinION nanopore sequencing for identifying S. sclerotiorum and other fungi causing diseases in oilseed rape. Naturally infected leaf, soil and air samples were collected from oilseed rape fields in Sweden, and DNA was extracted. Two primer pairs targeting the ITS region known marker for fungus identification, were amplified by PCR. For nanopore sequencing, six PCR amplicon samples, two from each source were selected based on their purity and stem rot incidences, with one sample spiked-in with gDNA S. sclerotiorum as positive control. Bioinformatic analysis was performed using the CCMetagen tool and EPI2ME. Ascomycota and Basidiomycota constituted 34% and 45%, respectively, of the identified species. S. sclerotiorum was only detected in air field samples, and several other fungal species harmful to oilseed rape production in Sweden, such as Botrytis cinerea, Pyrenopeziza brassicae and many more, were identified. In conclusion, successful identification of plant pathogens, including S. sclerotiorum, was achieved using MinION nanopore sequencer.

The study of viral capsid proteins, such as VP2 of CtenDNAV-II, is essential for understanding viral capsid assembly, infection mechanisms, and host manipulation. Capsid proteins, the protein shells encapsulating viral genetic material, play critical roles in protecting the virus and aiding in its attachment to host cells. By capturing high-resolution images of individual virus particles embedded in vitrified ice, cryo-EM facilitates the reconstruction of three-dimensional (3D) structures of viral capsids. The study of viral capsid structures using cryo-EM provides insights into viral life cycles, host-virus interactions, and evolutionary events. The aim of this research was to gain deeper insights into viral capsid architecture and dynamics by reconstructing the small capsid particle (T=1) and investigating its role in the capsid assembly of CtenDNAV-II. The capsid protein was expressed using Sf9 insect cells that were inoculated with a baculovirus containing the VP2 gene. The capsid protein expression in E. coli was performed by transforming the cells with a ubiquitin-expressing plasmid. An already existing cryo-EM dataset, which has been used to reconstruct the large capsid particle (T=3) was used to reconstruct the small capsid particle and determine whether or not it was hollow inside. The single particle reconstruction of the T=1 capsid particles, realized using cryoSPARC, achieved a fairly high resolution and indicated that the particles are most likely empty inside, containing no genome. It was noticed that many of the small particles were broken, which indicates they were most likely intermediate or incomplete VLP assembly states.

Honeybees are an important pollinator around the world and is used in agriculture, the bees also make products like honey, royal jelly and wax these products make up a market worth 15 billion dollars in the US. Honeybees are on the decline and a problem might be damage to the microbiota in the gut. The MinION is a next generation sequencing tool created by nanopore technologies; it’s often compared to Illumina sequencing. MinION is believed to be relatively inaccurate compared to other sequencing tools, MinION utilizes nanopores to measure electrical signals from the different nucleotides. The aim of this study was to analyze the MinIONs accuracy in identifying bacteria, and analyzing if the MinION is a tool appropriate to use in understanding the gut microbiota of bees. Bacteria was grown on LB plates and the genetic DNA was isolated and amplified using PCR. The 16S gene was sequenced using MinION and analyzed using the Epi2me desktop agent. The large number of analyzing shows that there are about 40% of the materials that never get approved by Epi2me. When identifying bacteria the program showed to be accurate in most cases even the contaminated samples were identified with high accuracy. However, Epi2me had some difficulties correctly identifying specific closely related bacteria. In conclusion the identifying bacterial species using MinION together with the Epi2me software was concluded to be accurate in the majority of cases even though a few blind spots were experienced.

In recent decades, the study of the gut microbiota has gained attention as a crucial element in human health and the onset of diseases. Technological advancements, such as high-throughput sequencing, have enabled a deeper study of intestinal microbiota composition and functions. Among the environmental factors modulating the gut microbial configuration, the impact of nutrients is of particular significance. Nonetheless, research on dietary lipids, particularly omega-3 fatty acids, remains underexplored. This study aimed to contribute to the metagenomic analysis of the 16 subunit of the ribosomal RNA gene in faecal samples. A nutritional intervention was carried out focused on fish consumption rich in omega-3. Successful DNA extraction from faecal samples yielded notable, with defined genomic DNA bands in the gel electrophoresis and an adequate concentration. Polymerase chain reactions, essential for library preparation, revealed distinct products in the gel electrophoresis, confirming the successful amplification of the V3-V4 16 ribosomal RNA region with proper adapter addition for DNA sequencing. Comprehensive quality control assessment of sequenced samples confirmed the effectiveness of the refined laboratory procedures. The steps undertaken in this project were critical for subsequent bioinformatics and biostatistics analyses, providing insights into the impact of omega-3-rich fish consumption on the relative abundance and diversity of studied microbial taxa. Integrating metagenomics analysis with other omics and non-omics disciplines, alongside standardising methods across different laboratories, will pave the way for the future application and democratisation of precision nutrition to enhance overall wellbeing.

Registered nurses entering a Nurse Prescriber programme participated in a mixed methods case study to explore the extent of their bioscience knowledge and the confidence with which that knowledge was held. Forty two Nurse Prescriber students, aged 26 – 55 years, from a range of job roles were recruited. Using questionnaires and interviews, both quantitative and qualitative data were obtained. An examination of the Nurse Prescribers’ views of pre-registration nursing demonstrated that the knowledge gained had been related to practice but had been both superficial and lacking in breadth. The bioscience in pre-registration programmes had not sufficiently prepared the participants for their roles as registered nurses. The importance of experiences gained as a registered nurse in the practice setting in the learning of bioscience was strongly emphasised. Participants reported greater learning of bioscience by informal methods such as work experience, use of books and the Internet and discussion with colleagues than from experiences in the classroom. Interviewees placed particularly strong emphasis on the importance of learning from medical colleagues. The role of post-registration programmes emerged as important in learning bioscience because it related to the job role. Post-registration courses also emerged as significant in giving confidence to the registered nurse. Confidence increased not just in terms of the knowledge held, but also in terms of nurses’ ability to communicate with patients, relatives, and doctors, their ability to understand nursing skills, and their willingness to admit when something was not known.

Iron plays a crucial role in various essential functions within the human body, participating in processes vital for overall health and well-being. To address iron deficiency, a wide array of iron supplements are commonly employed. However, it is important to recognize that certain types of iron supplements can have adverse effects on the body, including the induction of oxidative stress and inflammation. Therefore, extensive research is imperative to investigate the inflammatory potential of different sources of iron supplements in order to ensure their safety and effectiveness. In the pursuit of evaluating the inflammatory effects of various iron supplements, researchers frequently employ the Caco2 cell model. In this study, the focus was placed on examining the pro-inflammatory potential of different iron supplements by measuring the levels of a specific inflammatory biomarker, the cytokine IL-6, in the Caco2 cells. To mimic the physiological conditions, the supplements were subjected to a simulated gastrointestinal digestion protocol, ensuring that the Caco2 cells were exposed to digested forms of the supplement after which the levels of IL-6 were determined using ELISA. Surprisingly, the results of the study unveiled intriguing findings. Specifically, the two iron supplements derived from bovine sources exhibited no significant effect on IL-6 levels, indicating a lack of pro-inflammatory activity. However, it was the iron supplement derived from Spirulina, a marine-originated source that captured attention. This particular supplement showcased the ability to decrease the levels of IL-6, suggesting a potentially anti-inflammatory effect on intestinal cells. / <p>Utbytesstudent</p>

Uropathogenic Escherichia coli is a leading cause of urinary tract infections and bacterial prostatitis, the most common UTI complication in men. The initial stages of a successful infection involve bacterial adhesion to host cells through specialized adhesins. FimH, a protein located at the tip of type 1 pili, plays a crucial role as the main mediator for UPEC binding to bladder cells. While the host partners of FimH in the bladder are well-established, the interactions between FimH and prostate cells remain elusive. Consequently, the overarching goal is to enhance comprehension of the initial steps in prostate infection by investigating the interaction of FimH with prostate proteins. To achieve this, a recombinant FimH was constructed and expressed in an inducible expression vector, and an immunofluorescence staining assay was performed which demonstrated distinctive binding patterns in prostate cells compared to the bladder cell line. A Far Western overlay assay, revealed six distinct protein bands in human prostate cells and two in mouse prostate cells, indicating different potential protein partners. These interactions were examined under native conditions by establishing and optimizing a co-immunoprecipitation assay with cell proteins derived from both human and mouse prostates, with the 5637 cell line serving as a positive control. In summary, this study reveals striking differences between FimH binding to prostate and bladder cells, emphasizing the importance of FimH in adhesion and the need for further exploration of FimH interaction with prostate cells.

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Yes / Since the 1980's, changes in nursing education have inadvertently led to diminishing anatomy and physiology content in curricula (Taylor et al., 2015). The need for nurses to have a thorough grounding in these subjects is undisputed; however, the pedagogical principles for anatomy and physiology education have been under scrutiny (Perkins, 2019). Anatomy and physiology are typically incorporated as part of bioscience, which also encompasses genetics, microbiology, pharmacology, and pathophysiology (Horiuchi-Hirose et al., 2023). Registered nurses and nursing students often express anxiety about studying bioscience and its perceived difficulty, largely due to difficulties in applying theory to practice (Craft et al., 2013, Craft et al., 2017, Meedya et al., 2019). Despite this, there remains a recognition that bioscience knowledge is important for effective nursing practice (Danielson and Berntsson, 2007, Horiuchi-Hirose et al., 2023). / The full-text of this article will be released for public view at the end of the publisher embargo on 15 Dec 2024.