Genetic Technologies (Australia)

The oncolytic adenovirus Ad5-delta-24-RGD (Ad5Δ24RGD) exhibits suboptimal therapeutic efficacy in high-grade glioma. Here, we show that combining two specific potency-enhancing mutations in E3-19K and i-leader (designated 19KSS-iLQ125Ter) markedly increases the cytolytic efficiency and the size of vital dye-stained plaques. Unexpectedly, these mutations do not enhance actual spread efficiency, measured as the size of fluorescent plaques, in most tested cell lines. In contrast, the type of fiber modification (F5RGD4C, F5/3, or F5/35) strongly influences actual spread efficiency, with F5RGD4C generally conferring the greatest enhancement. In some cell lines, the fiber modification F5RGD10(2C) or verapamil treatment further improves actual spread efficiency. Nelfinavir inhibits spread and plaque formation of oncolytic adenoviruses with the 19KSS-iLQ125Ter modifications, irrespective of adenovirus death protein (ADP) expression. Expression of the reporter transgenes EGFP and Fluc or the human hyaluronidase PH20/SPAM1 from the E1B-55K region or downstream of the L3-23K or L5-Fiber region differentially affects the oncolytic potency of Ad5-delta-24-RGD-19KSS-iLQ125Ter. We identified an insertion site downstream of the L3-23K region that supports relatively high hPH20 activity while preserving the enhanced oncolytic potency of the virus. Combining 19KSS-iLQ125Ter with hPH20 expression may potentially improve therapeutic benefit in glioma.

The development of effective therapies for Alzheimer's disease remains a critical challenge in modern medicine. While monoclonal antibodies targeting beta-amyloid (Aβ) have shown the most significant clinical efficacy, their use is limited by poor blood-brain barrier permeability and side effects, which are occasionally severe. We previously devised ion-complementary to Aβ tetrapeptide HAEE, that was able to suppress amyloidogenesis in vivo. In this study, we modified HAEE by adding PGP amino acid residues to its C-terminus to improve its pharmacological properties. Pharmacokinetic analysis revealed that the 125I-HAEEPGP half-life in mouse plasma was twice that of 125I-HAEE and C-terminus modification of HAEE significantly increased its brain tissue penetration. Isothermal titration calorimetry experiments revealed that HAEEPGP formed a stable complex with Aβ and, besides, exhibited higher affinity for pathogenic isomerized Aβ than for the native peptide. 125I-HAEEPGP showed enhanced accumulation in the brain of 5xFAD transgenic mice compared to wild-type controls. Furthermore, using western blotting, ELISA, and flow cytometry, we demonstrated that HAEEPGP considerably reduced Aβ-induced astrocyte and microglia activation, prevented synaptic degeneration in 5xFAD mice, and restored lifespan of Caenorhabditis elegans overexpressing human Aβ. These features of HAEEPGP have positioned it as a promising therapeutic candidate for Aβ-related neuropathology.

Relevance: Molecular genetic testing to determine the patient's genotype and tumor molecular profile is a key component of a personalized approach to treatment and follow-up. Current research in genetic screening focuses on transitioning from phenotypic diagnostic panels and PCR testing of predisposition genes to large panels that include many identified genes or whole-genome sequencing. Multigene testing is widely used across colorectal cancer (CRC) diagnostics and therapy, where genetic components make a significant contribution. Currently, practical oncology requires a review of high-throughput sequencing systems for the genetic screening of hereditary and sporadic CRC variants and for the optimization of early diagnosis in relatives of patients. The study aimed to review the methodology and current results of next-generation sequencing (NGS) applications for genetic screening of hereditary and sporadic colorectal cancer. Methods: This analytical review included 70 original research and review articles available in open-access databases, including Google Scholar, Web of Science, SpringerLink, Scopus, ScienceDirect, PubMed, and BMJ. Results: NGS-based multigene testing enables the simultaneous analysis of multiple genes involved in carcinogenesis, the identification of germline pathogenic mutations associated with hereditary tumor syndromes, and the detection of genetic variants in less-studied regions of genes, such as introns and untranslated regions, which help identify previously unknown factors predisposing to colorectal cancer. Conclusion: Molecular genetic diagnostics facilitate personalized treatment of patients and individualized clinical examination of relatives from risk groups. However, although approximately 25% of CRC cases are familial, fewer than 5% of families are studied genetically. The analyzed data confirm the need to transition from phenotypic panels to comprehensive panels, encompassing all identified genes involved in hereditary tumor syndromes or whole-genome sequencing. In addition, identifying new variants with moderate and low penetrance, as well as those with uncertain functional significance, expands the phenotypic spectrum of CRC and necessitates further studies to determine their inclusion in diagnostic sequencing panels

Abstract Regulation of gene expression by Polycomb group (PcG) proteins orchestrates neural development and cell-type specification. However, the mechanisms by which neural progenitor cells diversify into the myriad cell types of the human brain remain poorly understood. Here, we investigate the role of Polycomb pro-teins in shaping the single-cell 3D genome, transcriptome, and chromatin binding landscape of the developing and adult human cortex. We show that PcG proteins establish a network of long-range repressive interactions encompassing loci with neuronal marker genes. These interactions occur in a neuron-type–dependent manner, selectively targeting genes that must remain silenced in a given cell type. These interactions are present in fetal neurons and increase markedly in strength after birth. Our results highlight PcG proteins as key regulators of neuronal cell fate specification in the human brain.

Methylmalonic acidemia (MMA) is a severe metabolic disorder affecting multiple organs because of a distal block in branched-chain amino acid (BCAA) catabolism. Standard of care is limited to protein restriction and supportive care during metabolic decompensation. Severe cases require liver/kidney transplantation, and there is a clear need for better therapy. Here, we investigated the effects of a small molecule branched-chain amino acid transaminase (BCAT) inhibitor in human MMA hepatocytes and an MMA mouse model. Mitochondrial BCAT is the first step in BCAA catabolism, and reduction of flux through an early enzymatic step is successfully used in other amino acid metabolic disorders. Metabolic flux analyses confirmed robust BCAT inhibition, with reduction of labeling of proximal and distal BCAA-derived metabolites in MMA hepatocytes. In vivo experiments verified the BCAT inhibition, but total levels of distal BCAA catabolite disease markers and clinical symptoms were not normalized, indicating contributions of substrates other than BCAA to these distal metabolite pools. Our study demonstrates the importance of understanding the underlying pathology of metabolic disorders for identification of therapeutic targets and the use of multiple, complementary models to evaluate them.

Passive sampling of environmental nucleic acids (eDNA and eRNA) has potential as a simple, effective, and cost-efficient method for pathogen surveillance in aquaculture. This study evaluated passive eDNA sampling for targeted detection and quantification of infectious hypodermal and haematopoietic necrosis virus (IHHNV) in giant black tiger shrimp, Penaeus monodon, aquaculture ponds. The ability of eDNA to estimate IHHNV host infection was evaluated through two investigations, comparing TaqMan qPCR detections of IHHNV from passively sampled eDNA and shrimp tissue samples from the same ponds. Passive sampling of eDNA involved timed submersion of membrane filters, within protective housings, in shrimp ponds. The effect of submersion duration on IHHNV detection was initially evaluated by submerging passive samplers for 1, 2, 4, 6 and 24 h across five ponds at 159-169 days of shrimp culture (DOC). Longer passive sampler submersion durations increased IHHNV detection from eDNA, and IHHNV loads were strongly correlated between eDNA and shrimp tissue. Passive eDNA samplers were then deployed at a farm-scale in 22 earthen shrimp ponds (125-129 DOC) for 24 h, given the higher IHHNV detection at 24 h in the initial study. The farm-scale deployment showed 95 % agreement in identifying ponds as IHHNV positive using eDNA and shrimp samples. Mean IHHNV loads in ponds were strongly correlated between eDNA and shrimp tissue ( R 2 = 0.95), indicating the potential of eDNA detection to approximate host infection levels. These findings provide evidence to advance the use of passive eDNA sampling for accurate and efficient monitoring of IHHNV in shrimp aquaculture systems. • Passive sampling captures nucleic acids on filters during environmental incubation • Passive sampling was evaluated for quantifying IHHNV eDNA in shrimp ponds • IHHNV was detected by qPCR from eDNA after 1, 2, 4, 6 and 24 h of incubation • IHHNV loads were highly correlated between pond eDNA and shrimp tissues • eDNA has high potential for estimating shrimp host infection levels

Polycystic ovary syndrome (PCOS) is among the most common disorders affecting up to 15% of the menstruating population globally. It is the leading cause of anovulatory infertility and a major risk factor for type 2 diabetes. Elevated testosterone levels are a core endophenotype. Despite that prevalence, the underlying causes remain unknown. PCOS genome-wide association studies (GWAS) have reproducibly mapped a number of susceptibility loci, including one encompassing a gene regulating androgen biosynthesis, DENND1A. Identifying the causal variants within these loci will provide fundamental insight into the precise biological pathways that are disrupted in PCOS. Here, we report the discovery of gene regulatory mechanisms that help explain genetic association with PCOS in the GATA4, FSHB and DENND1A loci using a combination of high throughput reporter assays, CRISPR-based epigenome editing, and genetic association analysis from PCOS case and control populations. In addition, we find that increasing endogenous DENND1A expression causes elevated testosterone levels in an adrenal cell model, specifically by perturbing candidate regulatory elements. These results further highlight the potential for combining genetic variant analyses with experimental approaches to fine map genetic associations with disease risk.

Ex vivo machine perfusion has been growing in utility for preserving donor organs prior to transplantation. This modality has tremendous potential for bioengineering and conditioning organs prior to transplantation using small molecule or advanced therapeutics. To safely translate potential interventions, well characterized models of disease are crucial for testing the therapeutic and possible side effects that could manifest from the interventions. Acute cellular rejection remains a significant complication in organ transplantation that affects transplant recipients with significant morbidity and mortality. This disease could potentially be mitigated with therapeutic intervention during ex vivo machine perfusion. A porcine animal model of acute rejection could be characterized in order to translate human biological processes with high fidelity. The Yucatan pig breed has been increasingly used in both biomedical research and xenotransplantation applications given its similarity to the human heart. A challenge with utilizing this pig breed for designing a model of acute rejection is its highly conserved ancestral lineage, which could make it difficult to induce acute rejection in a timely and consistent manner. We present a detailed characterization of a porcine model of acute rejection based on swine leukocyte antigen mismatching paired with a limited period of clinically relevant immunosuppression. The result is a robust and consistent protocol that results in fulminant acute rejection of an intra-abdominally transplanted heart.