Charles River Laboratories (Netherlands)

Microbial identification and strain typing are critical in pharmaceutical microbiology for ensuring product safety, controlling contaminants, and meeting regulatory requirements. Traditional strain typing methods like pulsed-field gel electrophoresis and multi-locus sequence typing have their limitations in scalability and resolution. Increased use of next-generation sequencing (NGS) techniques for microbial identification and characterization, coupled with the availability of bacterial whole genomes, advanced sequence analysis approaches such as single nucleotide polymorphisms (SNPs), core and pan-genome analysis, is opening the door for more modern approaches to microbial strain typing. However, several of these approaches are species-specific and require optimization per species or project. In this study, we analyzed conserved genes from several bacterial species to analyze the possibility of using these genes as a set of universal genes for prokaryotic strain typing. We compared the strain typing results using these universal genes to results from species-specific housekeeping genes and with species with no established strain typing methods. Our data demonstrated that these conserved genes successfully distinguished bacterial strains when compared to the results from established species-specific strain typing schemes, providing a path forward in the development of a rapid, universal strain typing solution using bacterial genomes that is not limited to a single species.

We demonstrate a hybrid system combining Raman spectroscopy and partial wave spectroscopy, improving tissue classification accuracy and detecting variation in tissues from an intestinal tumorigenesis mouse model, showcasing the system’s potential for field cancerization studies.

Infections with antimicrobial resistant pathogens are a major threat to human health. Inhibitors of the replicative polymerase PolC are a promising novel class of antimicrobials against Gram-positive pathogens, but the structural basis for their activity remains unknown. The first-in-class PolC-targeting antimicrobial, ibezapolstat, is a guanine analogue in late-stage clinical development for the treatment of Clostridioides difficile infections, and related inhibitors are being developed for systemic treatment of infections with methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Here, we present the cryo-electron microscopy structures of Enterococcus faecium PolC bound to DNA and in complex with ibezapolstat or the previously-undescribed inhibitor ACX-801. Both inhibitors form base-pairing interactions with the DNA in the active site, thereby competing with incoming dGTP nucleotides. We identify a crucial susceptibility determinant in PolC that is conserved in other organisms, such as C. difficile. This is explained by an unusual non-planar conformation of the inhibitors that induce a binding pocket in PolC. By combining structural, biochemical, bioinformatic and genetic analyses, this work lays the foundation for the rational development of an innovative class of antimicrobials against Gram-positive priority pathogens.

Estrogen receptor beta (ERβ) is a favorable therapeutic target for mediating inflammation, attenuating fibrosis, and treating cancer. However, selectively targeting ERβ over estrogen receptor alpha (ERα) has been a longstanding challenge. Recently, we developed OSU-ERβ-12, a novel carborane-based ERβ agonist that has a greater than 100-fold selectivity for ERβ over ERα. In this study, we compare the pharmacokinetics and functional activity of OSU-ERβ-12 against the clinical comparator ERβ agonist erteberel (LY500307) in multiple model systems. Pharmacokinetic profiling revealed OSU-ERβ-12 to have superior pharmacokinetics in pre-clinical models compared to LY500307 while maintaining a similar ERβ selectivity. Additionally, OSU-ERβ-12 displayed high human liver microsome stability and negligible CYP, hERG, and off-target interactions. Overall, OSU-ERβ-12 is a potent, selective, pharmacokinetically superior ERβ agonist that warrants additional study.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-22258-x.

ABSTRACTHerein we report the in silico discovery of 13 novel micromolar potent inhibitors of the SARS‐CoV‐2 NSP13 helicase validated in cellular antiviral and biophysical ThermoFluor assays. The compounds, discovered using a novel fragment‐based pharmacophore virtual screening workflow named FragmentScout, enable the advancement of novel antiviral agents. FragmentScout uses publicly accessible structural data of the SARS‐CoV‐2 NSP13 helicase, which was previously generated at the Diamond LightSource by XChem high‐throughput crystallographic fragment screening. The workflow generates a joint pharmacophore query for each binding site, thereby aggregating the pharmacophore feature information present in each experimental fragment pose. The joint pharmacophore query is then used to search 3D conformational databases using the Inte:ligand LigandScout XT software. The FragmentScout in silico workflow offers a novel tool for identifying micromolar hits from millimolar fragments in fragment‐based lead discovery. It is anticipated that this workflow will enhance systematic data mining of the growing collection of XChem datasets.

Alpha-synuclein (aSyn) is linked to Parkinson's disease (PD) through SNCA genetic mutations, phosphorylated aSyn in Lewy bodies and Lewy neurites, and most recently through evidence of aSyn aggregation in patient spinal fluid using the aSyn seed amplification assay. Therefore, understanding the biology of this protein and developing therapeutic interventions targeting pathological processing of aSyn are a key area of focus for novel treatments to slow or stop PD. Reliable preclinical models are imperative for these efforts. To this end, we developed a novel model using CRISPR/Cas9 to humanize the regions surrounding the naturally occurring threonine 53 amino acid in the Sprague Dawley rat to generate a humanized A53T aSyn rat model (aSyn A53T KI). We also generated an Snca knockout (aSyn KO) line to pair with the humanized A53T aSyn rat line to confirm that phenotypes were not due to loss of endogenous rat aSyn protein. A systematic phenotyping study was performed on these lines, assessing PD-related pathology and phenotypes at multiple timepoints. The aSyn KO rat line was profiled at 6 and 12 months of age, revealing successful aSyn protein knockout. The aSyn A53T KI model was profiled at 4, 8, 12, and 18 months of age for motor and non-motor phenotypes, nigrostriatal degeneration, and brain pathology. We confirmed the aSyn A53T KI rat expresses human aSyn while lacking endogenous rat aSyn. Motor function and non-motor function remain largely unaffected in this model, and no overt nigrostriatal degeneration or brain pathology are observed up to 18 months of age. Although the aSyn A53T KI rat lacks the ability to model PD pathology and phenotypes at baseline, it is an ideal model for investigating the impact of exogenous synuclein aggregates or environmental triggers on human aSyn in an in vivo model system.

Abstract The manufacturing of therapeutic antibodies requires expensive, complex, and frequently challenging production that keeps the cost of treatment in the clinic high. Alternatively, leveraging novel modalities such as mRNA to encode therapeutics circumvents many of the problems associated with the large-scale production of biologics, and instead relies on in vivo expression of antibodies within patients. Importantly, recent work demonstrates that therapeutic antibodies translated from mRNA in pre-clinical models can be readily detected within hours and persist up to several weeks. Peak levels of circulating mRNA-encoded antibodies are comparable to recombinant protein equivalents and have been shown to be within favorable therapeutic ranges in first-in-human phase I trials. Here we outline an mRNA-LNP based roadmap to encode and deliver therapeutic antibodies, using the standard-of-care anti-HER2 antibody Trastuzumab as a case study, and harness a plethora of pre-clinical models to validate the platform. Characterization of mRNA-expressed Trastuzumab in vitro showed robust translation in producer cell lines, retention of antigen specificity and maintenance of heavy chain-light chain integrity as expected. To evaluate in vivo expression of mRNA encoded Trastuzumab, antibody levels were measured in serum after infusing mRNA-LNPs in translational pharmacology models. Plasma concentrations of secreted mRNA-encoded trastuzumab were benchmarked against circulating levels of infused recombinant antibody equivalents and demonstrated superior pharmacokinetics as well as tolerability at lower doses compared to protein-infused groups. Additionally, bioluminescence imaging was utilized to dissect whole-animal biodistribution and sites of mRNA-translation of ALC-0315 containing LNP formulations used for mRNA antibody delivery. To demonstrate anti-tumor potency, mRNA-encoded trastuzumab was purified from producer cell lines and tested in cytotoxicity assays using human NK cells and HER2-positive tumor cell lines. mRNA-encoded trastuzumab robustly induced antibody-dependent cell cytotoxicity (ADCC) to the same magnitude as recombinant biosimilars. In mouse tumor xenograft models infused with LNPs, mRNA-encoded trastuzumab displayed superior in vivo efficacy at comparatively lower doses relative to recombinant formats by persistently reducing tumor growth and volume as well as enhancing overall survival. Overall, the case study data demonstrate that mRNA-encoded therapeutic antibodies could provide a cost effective, alternate strategy for solid tumor immunotherapy and may unlock an approach to democratize and accelerate patient access to biologics in the clinic. Citation Format: Dan Rocca, Roxana Redis, Phillipp Meyer, Ina Rohleff, Eva Oswald, Sarah L. Martin, Rachel Pooley, Matthew Benson, Namrata Jayanth, Maxim Mashrick, Christian Cobaugh, Michael Shaw, Julia Schueler, Louise S. Brackenbury, Justin Bryans. Developing an mRNA nanomedicine platform to democratise therapeutic antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3794.