Development of an Immortalized Porcine Fibroblast Cell Panel With Different Swine Leukocyte Antigen Genotypes.

Immortalized cell lines are valuable resources to expand the molecular characterization of major histocompatibility complex genes and their presented antigens. We generated a panel of immortalized cell lines by transfecting human telomerase reverse transcriptase (hTERT) into primary fibroblast cells prepared from ear, fetal, and lung tissues of 10 pigs from five breeds and successfully cultured them for 30-45 passages. The cell growth characteristic of the immortalized fibroblasts was similar to that of primary fibroblast, which was unable to form colonies on soft agar. The genotypes of major swine leukocyte antigen (SLA) genes, including three classical class I (SLA-1, -2, and -3) and three class II genes (DQB1, DRB1, and DQA), were determined using high-resolution typing. A total of 58 alleles, including a novel allele for SLA-2, were identified. Each cell line was unique. A cell line derived from a National Institutes of Health miniature pig was homozygous across the six major SLA genes. The expression levels of SLA classical class I genes varied among the cell lines and were slightly upregulated in the immortalized compared to the primary cells based on semiquantitative reverse transcription polymerase chain reaction. The immortalized porcine fibroblast cell lines with diverse SLA haplotypes that were developed in this study have potential to be applied in studies regarding the molecular characteristics and genetic structure of SLA genes and epitope-major histocompatibility complex interactions in pigs.

Opossum Cathelicidins Exhibit Antimicrobial Activity Against a Broad Spectrum of Pathogens Including West Nile Virus.

This study aimed to characterize cathelicidins from the gray short-tailed opossum in silico and experimentally validate their antimicrobial effects against various pathogenic bacteria and West Nile virus (WNV). Genome-wide in silico analysis against the current genome assembly of the gray short-tailed opossum yielded 56 classical antimicrobial peptides (AMPs) from eight different families, among which 19 cathelicidins, namely ModoCath1 - 19, were analyzed in silico to predict their antimicrobial domains and three of which, ModoCath1, -5, and -6, were further experimentally evaluated for their antimicrobial activity, and were found to exhibit a wide spectrum of antimicroial effects against a panel of gram-positive and gram-negative bacterial strains. In addition, these peptides displayed low-to-moderate cytotoxicity in mammalian cells as well as stability in serum and various salt and pH conditions. Circular dichroism analysis of the spectra resulting from interactions between ModoCaths and lipopolysaccharides (LPS) showed formation of a helical structure, while a dual-dye membrane disruption assay and scanning electron microscopy analysis revealed that ModoCaths exerted bactericidal effects by causing membrane damage. Furthermore, ModoCath5 displayed potent antiviral activity against WNV by inhibiting viral replication, suggesting that opossum cathelicidins may serve as potentially novel antimicrobial endogenous substances of mammalian origin, considering their large number. Moreover, analysis of publicly available RNA-seq data revealed the expression of eight ModoCaths from five different tissues, suggesting that gray short-tailed opossums may be an interesting source of cathelicidins with diverse characteristics.

Chemical Mechanisms of Nanoparticle Radiosensitization and Radioprotection: A Review of Structure-Function Relationships Influencing Reactive Oxygen Species.

Metal nanoparticles are of increasing interest with respect to radiosensitization. The physical mechanisms of dose enhancement from X-rays interacting with nanoparticles has been well described theoretically, however have been insufficient in adequately explaining radiobiological response. Further confounding experimental observations is examples of radioprotection. Consequently, other mechanisms have gained increasing attention, especially via enhanced production of reactive oxygen species (ROS) leading to chemical-based mechanisms. Despite the large number of variables differing between published studies, a consensus identifies ROS-related mechanisms as being of significant importance. Understanding the structure-function relationship in enhancing ROS generation will guide optimization of metal nanoparticle radiosensitisers with respect to maximizing oxidative damage to cancer cells. This review highlights the physico-chemical mechanisms involved in enhancing ROS, commonly used assays and experimental considerations, variables involved in enhancing ROS generation and damage to cells and identifies current gaps in the literature that deserve attention. ROS generation and the radiobiological effects are shown to be highly complex with respect to nanoparticle physico-chemical properties and their fate within cells. There are a number of potential biological targets impacted by enhancing, or scavenging, ROS which add significant complexity to directly linking specific nanoparticle properties to a macroscale radiobiological result.

Kinase activity of ERBB3 contributes to intestinal organoids growth and intestinal tumorigenesis.

As a member of the epidermal growth factor receptor (EGFR) family, ERBB3 plays an essential role in development and disease independent of inherently inactive kinase domain. Recently, ERBB3 has been found to bind to ATP and has catalytic activity in vitro. However, the biological function of ERBB3 kinase activity remains elusive in vivo. Here we have identified the physiological function of inactivated ERBB3 kinase activity by creating Erbb3-K740M knockin mice in which ATP cannot bind to ERBB3. Unlike Erbb3 knockout mice, kinase-inactive Erbb3K740M homozygous mice were born in Mendelian ratios and showed normal development. After dextran sulfate sodium-induced colitis, the kinase-inactive Erbb3 mutant mice showed normal recovery. However, the outgrowth of ileal organoids by neuregulin-1 treatment was more attenuated in Erbb3 mutant mice than in WT mice. Moreover, in combination with the ApcMin mouse, the proportion of polyps less than 1 mm in diameter in mutant mice was higher than in control mice and an increase in the number of apoptotic cells was observed in polyps from mutant mice compared with polyps from control mice. Taken together, the ERBB3 kinase activity contributes to the outgrowth of ileal organoids and intestinal tumorigenesis, and the development of ERBB3 kinase inhibitors, including epidermal growth factor receptor family members, can be a potential way to target colorectal cancer.

ß2-microglobulin gene duplication in cetartiodactyla remains intact only in pigs and possibly confers selective advantage to the species.

Several ß2-microglobulin (B2M) -bound protein complexes undertake key roles in various immune system pathways, including the neonatal Fc receptor (FcRn), cluster of differentiation 1 (CD1) protein, non-classical major histocompatibility complex (MHC), and well-known MHC class I molecules. Therefore, the duplication of B2M may lead to an increase in the biological competence of organisms to the environment. Based on the pig genome assembly SSC10.2, a segmental duplication of ~45.5 kb, encoding the entire B2M protein, was identified in pig chromosome 1. Through experimental validation, we confirmed the functional duplication of the B2M gene with a completely identical coding sequence between two copies in pigs. Considering the importance of B2M in the immune system, we performed the phylogenetic analysis of B2M duplication in ten mammalian species, confirming the presence of B2M duplication in cetartioldactyls, like cattle, sheep, goats, pigs and whales, but non-cetartiodactyl species, like mice, cats, dogs, horses, and humans. The density of long interspersed nuclear element (LINE) at the edges of duplicated blocks (39 to 66%) was found to be 2 to 3-fold higher than the average (20.12%) of the pig genome, suggesting its role in the duplication event. The B2M mRNA expression level in pigs was 12.71 and 7.57 times (2-ΔΔCt values) higher than humans and mice, respectively. However, we were unable to experimentally demonstrate the difference in the level of B2M protein because species specific anti-B2M antibodies are not available. We reported, for the first time, the functional duplication of the B2M gene in animals. The identification of partially remaining duplicated B2M sequences in the genomes of only cetartiodactyls indicates that the event was lineage specific. B2M duplication could be beneficial to the immune system of pigs by increasing the availability of MHC class I light chain protein, B2M, to complex with the proteins encoded by the relatively large number of MHC class I heavy chain genes in pigs. Further studies are necessary to address the biological meaning of increased expression of B2M.