Novel approaches to target infectious diseases: the utility of Auger electrons and scFvs for imaging and control

With an increasing number of infectious agents resistant to one or more existing antibiotics, there is a global requirement for new therapeutics. One possible method for treatment of infectious diseases proposed in this study is the adaptation of a technique developed for cancer treatment, targeted radionuclide therapy, with particular interest in Auger electron radiation. Auger electrons are short range and low energy particles emitted from a range of radioactive isotopes. As a targeting agent, the antimicrobial peptide fragment UBI 29-41 was used. Antimicrobial peptides have a broad range of target microorganisms, ideal for toxicological studies of Auger electrons. Here, the baseline toxicity of UBI 29-41 was established in a range of model organisms. This enabled the analysis of the toxic effect of Auger electron emitting radio-nuclides using the isotope125I. Although no direct toxic effect was observed with this particular isotope, the labelling allows easy replacement with more potent isotopes. To further develop the targeting agent a lipoarabinomannan (LAM) specific single-chain variable fragment (scFv) was developed. LAM is a glycolipid and virulence factor associated with M. tuberculosis, the primary cause of tuberculosis in humans. By isolating the variable domains of a LAM specific monoclonal antibody, linked via a poly-glycine linker, the scFv was assembled in silico. The nucleotide sequence was optimised for and transfected into a Pichia pink expression system. Expression was successful with a ~27kDa product being secreted. The novel anti-LAM scFv generated in this study combined with the potential toxic effects of Auger electrons could provide a new avenue for the treatment and diagnosis of tuberculosis

Incidence, Reproductive Outcome, and Economic Impact of Reciprocal Translocations in the Domestic Pig

Pigs (Sus scrofa) have vast economic importance, with pork accounting for over 30% of the global meat consumption. Chromosomal abnormalities, and in particular reciprocal translocations (RTs), are an important cause of hypoprolificacy (litter size reduction) in pigs. However, these do not necessarily present with a recognizable phenotype and may cause significant economic losses for breeders when undetected. Here, we present a reappraisal of the incidence of RTs across several European pig herds, using contemporary methodology, as well as an analysis modelling the economic impact of these abnormalities. Molecular cytogenetic investigation was completed by karyotyping and/or multiprobe FISH (fluorescence in situ hybridisation) between 2016–2021, testing 2673 animals. We identified 19 types of chromosome abnormalities, the prevalence of these errors in the database was 9.1%, and the estimated incidence of de novo errors was 0.90%. Financial modelling across different scenarios revealed the potential economic impact of an undetected RT, ranging from £69,802 for an individual affected terminal boar in a commercial farm selling weaned pigs, to £51,215,378 for a genetics company with an undetected RT in a dam line boar used in a nucleus farm. Moreover, the added benefits of screening by FISH instead of karyotyping were estimated, providing a strong case for proactive screening by this approach

A Conserved Requirement for fbxo7 during Male Germ Cell Cytoplasmic Remodelling

Fbxo7 is the substrate-recognition subunit of an SCF-type ubiquitin E3 ligase complex. It has physiologically important functions in regulating mitophagy, proteasome activity and the cell cycle in multiple cell types, like neurons, lymphocytes and erythrocytes. Here, we show that in addition to the previously known Parkinsonian and hematopoietic phenotypes, male mice with reduced Fbxo7 expression are sterile. In these males, despite successful meiosis, nuclear elongation and eviction of histones from chromatin, the developing spermatids are phagocytosed by Sertoli cells during late spermiogenesis, as the spermatids undergo cytoplasmic remodeling. Surprisingly, despite the loss of all germ cells, there was no evidence of the symplast formation and cell sloughing that is typically associated with spermatid death in other mouse sterility models, suggesting that novel cell death and/or cell disposal mechanisms may be engaged in Fbxo7 mutant males. Mutation of the Drosophila Fbxo7 ortholog, nutcracker (ntc) also leads to sterility with germ cell death during cytoplasmic remodeling, indicating that the requirement for Fbxo7 at this stage is conserved. The ntc phenotype was attributed to decreased levels of the proteasome regulator, DmPI31 and reduced proteasome activity. Consistent with the fly model, we observe a reduction in PI31 levels in mutant mice; however, there is no alteration in proteasome activity in whole mouse testes. Our results are consistent with findings that Fbxo7 regulates PI31 protein levels, and indicates that a defect at the late stages of spermiogenesis, possibly due to faulty spatial dynamics of proteasomes during cytoplasmic remodeling, may underlie the fertility phenotype in mice

Automated Nuclear Cartography Reveals Conserved Sperm Chromosome Territory Localization across 2 Million Years of Mouse Evolution.

Measurements of nuclear organization in asymmetric nuclei in 2D images have traditionally been manual. This is exemplified by attempts to measure chromosome position in sperm samples, typically by dividing the nucleus into zones, and manually scoring which zone a fluorescence in-situ hybridisation (FISH) signal lies in. This is time consuming, limiting the number of nuclei that can be analyzed, and prone to subjectivity. We have developed a new approach for automated mapping of FISH signals in asymmetric nuclei, integrated into an existing image analysis tool for nuclear morphology. Automatic landmark detection defines equivalent structural regions in each nucleus, then dynamic warping of the FISH images to a common shape allows us to generate a composite of the signal within the entire cell population. Using this approach, we mapped the positions of the sex chromosomes and two autosomes in three mouse lineages (Mus musculus domesticus, Mus musculus musculus and Mus spretus). We found that in all three, chromosomes 11 and 19 tend to interact with each other, but are shielded from interactions with the sex chromosomes. This organization is conserved across 2 million years of mouse evolution