Individual and population diversity of 20 representative olfactory receptor genes in pigs.

Understanding the influence of genetic variations in olfactory receptor (OR) genes on the olfaction-influenced phenotypes such as behaviors, reproduction, and feeding is important in animal biology. However, our understanding of the complexity of the OR subgenome is limited. In this study, we analyzed 1120 typing results of 20 representative OR genes belonging to 13 OR families on 14 pig chromosomes from 56 individuals belonging to seven different breeds using a sequence-based OR typing method. We showed that the presence of copy number variations, conservation of locus-specific diversity, abundance of breed-specific alleles, presence of a loss-of-function allele, and low-level purifying selection in pig OR genes could be common characteristics of OR genes in mammals. The observed nucleotide sequence diversity of pig ORs was higher than that of dogs. To the best of our knowledge, this is the first report on the individual- or population-level characterization of a large number of OR family genes in livestock species.

Metabolic and cell cycle shift induced by the deletion of Dnm1l attenuates the dissolution of pluripotency in mouse embryonic stem cells.

Mitochondria are versatile organelles that continuously change their morphology via fission and fusion. However, the detailed functions of mitochondrial dynamics-related genes in pluripotent stem cells remain largely unclear. Here, we aimed to determine the effects on energy metabolism and differentiation ability of mouse embryonic stem cells (ESCs) following deletion of the mitochondrial fission-related gene Dnml1. Resultant Dnm1l-/- ESCs maintained major pluripotency characteristics. However, Dnm1l-/- ESCs showed several phenotypic changes, including the inhibition of differentiation ability (dissolution of pluripotency). Notably, Dnm1l-/- ESCs maintained the expression of the pluripotency marker Oct4 and undifferentiated colony types upon differentiation induction. RNA sequencing analysis revealed that the most frequently differentially expressed genes were enriched in the glutathione metabolic pathway. Our data suggested that differentiation inhibition of Dnm1l-/- ESCs was primarily due to metabolic shift from glycolysis to OXPHOS, G2/M phase retardation, and high level of Nanog and 2-cell-specific gene expression.

Origin and population structure of native dog breeds in the Korean peninsula and East Asia.

To study the ancestry and phylogenetic relationships of native Korean dog breeds to other Asian dog populations, we analyzed nucleotide variations in whole-genome sequences of 205 canid individuals. Sapsaree, Northern Chinese indigenous dog, and Tibetan Mastiff were largely related to West Eurasian ancestry. Jindo, Donggyeongi, Shiba, Southern Chinese indigenous (SCHI), Vietnamese indigenous dogs (VIET), and Indonesian indigenous dogs were related to Southeast and East Asian ancestry. Among East Asian dog breeds, Sapsaree presented the highest haplotype sharing with German Shepherds, indicating ancient admixture of European ancestry to modern East Asian dog breeds. SCHI showed greater haplotype sharing with New Guinea singing dogs, VIET, and Jindo than with other Asian breeds. The predicted divergence time of East Asian populations from their common ancestor was approximately 2,000 to 11,000 years ago. Our results expand understanding of the genetic history of dogs in the Korean peninsula to the Asian continent and Oceanic region.

Population differentiated copy number variation between Eurasian wild boar and domesticated pig populations.

Sus scrofa is a globally distributed livestock species that still maintains two different ways of life: wild and domesticated. Herein, we detected copy number variation (CNV) of 328 animals using short read alignment on Sscrofa11.1. We compared CNV among five groups of porcine populations: Asian domesticated (AD), European domesticated (ED), Asian wild (AW), European wild (EW), and Near Eastern wild (NEW). In total, 21,673 genes were identified on 154,872 copy number variation region (CNVR). Differences in gene copy numbers between populations were measured by considering the variance-based value [Formula: see text] and the one-way ANOVA test followed by Scheffe test. As a result, 111 genes were suggested as copy number variable genes. Abnormally gained copy number on EEA1 in all populations was suggested the presence of minor CNV in the reference genome assembly, Sscrofa11.1. Copy number variable genes were related to meat quality, immune response, and reproduction traits. Hierarchical clustering of all individuals and mean pairwise [Formula: see text] in breed level were visualized genetic relationship of 328 individuals and 56 populations separately. Our findings have shown how the complex history of pig evolution appears in genome-wide CNV of various populations with different regions and lifestyles.

Genetic Diversity and Sequence Conservation of Peptide-Binding Regions of MHC Class I Genes in Pig, Cattle, Chimpanzee, and Human.

Comparative analyses of MHC gene diversity and evolution across different species could offer valuable insights into the evolution of MHC genes. Intra- and inter-species sequence diversity and conservation of 12 classical major histocompatibility complex (MHC) class I genes from cattle, chimpanzees, pigs, and humans was analyzed using 20 representative allelic groups for each gene. The combined analysis of paralogous loci for each species revealed that intra-locus amino-acid sequence variations in the peptide-binding region (PBR) of MHC I genes did not differ significantly between species, ranging from 8.44% for SLA to 10.75% for BoLA class I genes. In contrast, intraspecies differences in the non-PBRs of these paralogous genes were more pronounced, varying from 4.59% for SLA to 16.89% for HLA. Interestingly, the Shannon diversity index and rate of nonsynonymous substitutions for PBR were significantly higher in SLA and BoLA than those in Patr and HLA. Analysis of peptide-binding pockets across all analyzed MHC class I genes of the four species indicated that pockets A and E showed the lowest and highest diversity, respectively. The estimated divergence times suggest that primate and artiodactyl MHC class I genes diverged 60.41 Mya, and BoLA and SLA genes diverged 35.34 Mya. These results offer new insights into the conservation and diversity of MHC class I genes in various mammalian species.

Genomewide Analysis and Biological Characterization of Cathelicidins with Potent Antimicrobial Activity and Low Cytotoxicity from Three Bat Species.

Cathelicidins are potent antimicrobial peptides with broad spectrum antimicrobial activity in many vertebrates and an important component of the innate immune system. However, our understanding of the genetic variations and biological characteristics of bat cathelicidins is limited. In this study, we performed genome-level analysis of the antimicrobial peptide cathelicidins from seven bat species in the six families, listed 19 cathelicidin-like sequences, and showed that the number of functional cathelicidin genes differed among bat species. Based on the identified biochemical characteristics of bat cathelicidins, three cathelicidins, HA-CATH (from Hipposideros armiger), ML-CATH (from Myotis lucifugus), and PD-CATH (from Phyllostomus discolor), with clear antimicrobial signatures were chemically synthesized and evaluated antimicrobial activity. HA-CATH showed narrow-spectrum antibacterial activity against a panel of 12 reference bacteria, comprising 6 Gram-negative and 6 Gram-positive strains. However, ML-CATH and PD-CATH showed potent antibacterial activity against a broad spectrum of Gram-negative and Gram-positive bacteria with minimum inhibitory concentration (MIC) of 1 and 3 µg/mL, respectively, against Staphylococcus aureus. ML-CATH and PD-CATH also showed antifungal activities against Candida albicans and Cryptococcus cuniculi with MIC of 5 to 40 µg/mL, respectively, and 80% inhibition of the metabolism of Mucor hiemalis hyphae at 80 µg/mL, while displaying minimal cytotoxicity to HaCaT cells. Taken together, although the spectrum and efficacy of bat cathelicidins were species-dependent, the antimicrobial activity of ML-CATH and PD-CATH was comparable to that of other highly active cathelicidins in vertebrates while having negligible cytotoxicity to mammalian cells. ML-CATH and PD-CATH can be exploited as promising candidates for the development of antimicrobial therapeutics.

Sequence polymorphisms of PR39 cathelicidins and extensive copy variations in commercial pig breeds.

Copy number polymorphisms (CNPs) of antimicrobial peptides (AMPs) in livestock can influence the innate immune response of individuals. We conducted a high-resolution analysis of the genomic variations of porcine cathelicidin PR39 using cloned PR39 amplicons corresponding to the 5' untranslated region (UTR) to 3' UTR from four individuals of three different pig breeds. We identified 15 different sequences corresponding to 9 different coding domain sequences (CDSs), encoding 7 different protein sequences consisting of 3 functional and 4 non-functional forms. Subsequently, we developed a PR39 CNP typing method using real-time polymerase chain reaction (PCR) and analyzed the PR39 copy numbers from 44 pigs of six breeds. Significant variations in PR39 copies ranging from 2 to 10 copies, with a mean copy number of 5, were observed among all commercial breeds, except the wild boar. Among the different breeds, the PR39 copy number was highest (10) in Korean native pigs. Gene expression analysis showed that PR39 expression was correlated with the copy number. Moreover, the comparative analysis of the cathelicidin cluster-containing region among eight mammalian species showed the complete evolutionary conservation of the region, except for differences in the degree of cathelicidin expansion in each species. Therefore, characterization of CNPs in AMP genes could aid in improving the genetic potential of innate immune responses in livestock animals.

Tracing the Origin of the RSPO2 Long-Hair Allele and Epistatic Interaction between FGF5 and RSPO2 in Sapsaree Dog.

Genetic analysis of the hair-length of Sapsaree dogs, a Korean native dog breed, showed a dominant mode of inheritance for long hair. Genome-Wide Association Study (GWAS) analysis and subsequent Mendelian segregation analysis revealed an association between OXR1, RSPO2, and PKHD1L1 on chromosome 13 (CFA13). We identified the previously reported 167 bp insertion in RSPO2 3' untranslated region as a causative mutation for hair length variations. The analysis of 118 dog breeds and wolves revealed the selection signature on CFA13 in long-haired breeds. Haplotype analysis showed the association of only a few specific haplotypes to the breeds carrying the 167 bp insertion. The genetic diversity in the neighboring region linked to the insertion was higher in Sapsarees than in other Asian and European dog breeds carrying the same variation, suggesting an older history of its insertion in the Sapsaree genome than in that of the other breeds analyzed in this study. Our results show that the RSPO2 3' UTR insertion is responsible for not only the furnishing phenotype but also determining the hair length of the entire body depending on the genetic background, suggesting an epistatic interaction between FGF5 and RSPO2 influencing the hair-length phenotype in dogs.

High-Quality Nucleic Acid Isolation from Hard-to-Lyse Bacterial Strains Using PMAP-36, a Broad-Spectrum Antimicrobial Peptide.

The efficiency of existing cell lysis methods to isolate nucleic acids from diverse bacteria varies depending on cell wall structures. This study tested a novel idea of using broad-spectrum antimicrobial peptides to improve the lytic efficiency of hard-to-lyse bacteria and characterized their differences. The lysis conditions of Staphylococcus aureus using recombinant porcine myeloid antimicrobial peptide 36 (PMAP-36), a broad-spectrum pig cathelicidin, was optimized, and RNA isolation was performed with cultured pellets of ten bacterial species using various membranolytic proteins. Additionally, three other antimicrobial peptides, protegrin-1 (PG-1), melittin, and nisin, were evaluated for their suitability as the membranolytic agents of bacteria. However, PMAP-36 use resulted in the most successful outcomes in RNA isolation from diverse bacterial species. The amount of total RNA obtained using PMAP-36 increased by ~2-fold compared to lysozyme in Salmonella typhimurium. Streptococci species were refractory to all lytic proteins tested, although the RNA yield from PMAP-36 treatment was slightly higher than that from other methods. PMAP-36 use produced high-quality RNA, and reverse transcription PCR showed the efficient amplification of the 16S rRNA gene from all tested strains. Additionally, the results of genomic DNA isolation were similar to those of RNA isolation. Thus, our findings present an additional option for high quality and unbiased nucleic acid isolation from microbiomes or challenging bacterial strains.

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.

SLA-1 Genetic Diversity in Pigs: Extensive Analysis of Copy Number Variation, Heterozygosity, Expression, and Breed Specificity.

Swine leukocyte antigens play indispensable roles in immune responses by recognizing a large number of foreign antigens and thus, their genetic diversity plays a critical role in their functions. In this study, we developed a new high-resolution typing method for pig SLA-1 and successfully typed 307 individuals from diverse genetic backgrounds including 11 pure breeds, 1 cross bred, and 12 cell lines. We identified a total of 52 alleles including 18 novel alleles and 9 SLA-1 duplication haplotypes, including 4 new haplotypes. We observed significant differences in the distribution of SLA-1 alleles among the different pig breeds, including the breed specific alleles. SLA-1 duplication was observed in 33% of the chromosomes and was especially high in the biomedical model breeds such as SNU (100%) and NIH (76%) miniature pigs. Our analysis showed that SLA-1 duplication is associated with the increased level of SLA-1 mRNA expression in porcine cells compared to that of the single copy haplotype. Therefore, we provide here the results of the most extensive genetic analysis on pig SLA-1.

Protegrin-1 cytotoxicity towards mammalian cells positively correlates with the magnitude of conformational changes of the unfolded form upon cell interaction.

Porcine protegrin-1 (PG-1) is a broad-spectrum antimicrobial peptide (AMP) with potent antimicrobial activities. We produced recombinant PG-1 and evaluated its cytotoxicity toward various types of mammalian cell lines, including embryonic fibroblasts, retinal cells, embryonic kidney cells, neuroblastoma cells, alveolar macrophage cells, and neutrophils. The sensitivity of the different mammalian cells to cytotoxic damage induced by PG-1 differed significantly among the cell types, with retinal neuron cells and neutrophils being the most significantly affected. A circular dichroism analysis showed there was a precise correlation between conformational changes in PG-1 and the magnitude of cytotoxicity among the various cell type. Subsequently, a green fluorescent protein (GFP) penetration assay using positively charged GFPs indicated there was a close correlation between the degree of penetration of charged GFP into cells and the magnitude of PG-1 cytotoxicity. Furthermore, we also showed that inhibition of the synthesis of anionic sulphated proteoglycans on the cell surface decreases the cytotoxic damage induced by PG-1 treatment. Taken together, the observed cytotoxicity of PG-1 towards different membrane surfaces is highly driven by the membrane's anionic properties. Our results reveal a possible mechanism underlying cell-type dependent differences in cytotoxicity of AMPs, such as PG-1, toward mammalian cells.

Analysis of allele-specific expression using RNA-seq of the Korean native pig and Landrace reciprocal cross.

OBJECTIVE: We tried to analyze allele-specific expression in the pig neocortex using bioinformatic analysis of high-throughput sequencing results from the parental genomes and offspring transcriptomes from reciprocal crosses between Korean Native and Landrace pigs. METHODS: We carried out sequencing of parental genomes and offspring transcriptomes using next generation sequencing. We subsequently carried out genome scale identification of SNPs in two different ways using either individual genome mapping or joint genome mapping of the same breed parents that were used for the reciprocal crosses. Using parent-specific SNPs, allele-specifically expressed genes were analyzed. RESULTS: Because of the low genome coverage (~4x) of the sequencing results, most SNPs were non-informative for parental lineage determination of the expressed alleles in the offspring and were thus excluded from our analysis. Consequently, 436 SNPs covering 336 genes were applicable to measure the imbalanced expression of paternal alleles in the offspring. By calculating the read ratios of parental alleles in the offspring, we identified seven genes showing allele-biased expression (P < 0.05) including three previously reported and four newly identified genes in this study. CONCLUSION: The newly identified allele-specifically expressing genes in the neocortex of pigs should contribute to improving our knowledge on genomic imprinting in pigs. To our knowledge, this is the first study of allelic imbalance using high throughput analysis of both parental genomes and offspring transcriptomes of the reciprocal cross in outbred animals. Our study also showed the effect of the number of informative animals on the genome level investigation of allele-specific expression using RNA-seq analysis in livestock species.

Copy number variation of PR-39 cathelicidin, and identification of PR-35, a natural variant of PR-39 with reduced mammalian cytotoxicity.

Proline-arginine-rich (PR)-39 is neutrophil antimicrobial peptide that has potent antimicrobial activity against a broad spectrum of microorganisms, including bacteria, fungi, and some enveloped viruses as a part of the innate immune system. We analyzed the nucleotide sequence variations of PR-39 exon 4, which is the mature peptide region responsible for antimicrobial activity, from 48 pigs of six breeds using sequence-based typing. The analysis identified four alleles including allele PR-35 with a 12-bp deletion near the N-terminus. Interestingly, 16.7% of individuals showed the presence of three alleles per individual, but only in the Berkshire and Duroc breeds. We further analyzed the genetic diversity of PR-39 for the entire genomic region of the gene from PR-39 exon 1 to the 3' untranslated region for different alleles by PCR amplification and cloning. The antimicrobial activity of chemically synthesized PR-35 was similar to that of PR-39, but the level of mammalian cell cytotoxicity was lower than the wild type. Better knowledge of the genetic diversity of PR-39 among different individuals and breeds may contribute to improved immune defense of pigs. PR-35, as a natural antimicrobial peptide variant, could be an interesting candidate for the development of peptide antibiotics.

The novel cathelicidin of naked mole rats, Hg-CATH, showed potent antimicrobial activity and low cytotoxicity.

We performed the in silico genome-wide identification of antimicrobial peptides against the available genome sequence of the naked mole rat Heterocephalus glaber (H. glaber). Our results showed the presence of Hg-CATH, the single cathelicidin containing the antimicrobial domain in H. glaber. We chemically synthesized a 25 amino-acid peptide (ΔHg-CATH) corresponding to the predicted antimicrobial-active core region of Hg-CATH, and evaluated its antibacterial activity against seven bacterial strains. The ΔHg-CATH peptide exhibited strong bactericidal activity against gram-negative bacteria, including a multi-drug resistant strain, while showing low toxicity towards mammalian cells, including erythrocytes. Scanning electron microscopy images of bacterial cells treated with ΔHg-CATH showed disruption of their membranes due to the formation of toroidal pores. Identifying novel antimicrobial peptides, such as Hg-CATH, may be important for identifying candidate peptides for the control of multi-drug resistant bacteria.

Genomewide Analysis of the Antimicrobial Peptides in Python bivittatus and Characterization of Cathelicidins with Potent Antimicrobial Activity and Low Cytotoxicity.

In this study, we sought to identify novel antimicrobial peptides (AMPs) in Python bivittatus through bioinformatic analyses of publicly available genome information and experimental validation. In our analysis of the python genome, we identified 29 AMP-related candidate sequences. Of these, we selected five cathelicidin-like sequences and subjected them to further in silico analyses. The results showed that these sequences likely have antimicrobial activity. The sequences were named Pb-CATH1 to Pb-CATH5 according to their sequence similarity to previously reported snake cathelicidins. We predicted their molecular structure and then chemically synthesized the mature peptide for three putative cathelicidins and subjected them to biological activity tests. Interestingly, all three peptides showed potent antimicrobial effects against Gram-negative bacteria but very weak activity against Gram-positive bacteria. Remarkably, ΔPb-CATH4 showed potent activity against antibiotic-resistant clinical isolates and also was observed to possess very low hemolytic activity and cytotoxicity. ΔPb-CATH4 also showed considerable serum stability. Electron microscopic analysis indicated that ΔPb-CATH4 exerts its effects via toroidal pore preformation. Structural comparison of the cathelicidins identified in this study to previously reported ones revealed that these Pb-CATHs are representatives of a new group of reptilian cathelicidins lacking the acidic connecting domain. Furthermore, Pb-CATH4 possesses a completely different mature peptide sequence from those of previously described reptilian cathelicidins. These new AMPs may be candidates for the development of alternatives to or complements of antibiotics to control multidrug-resistant pathogens.

Green fluorescent protein as a scaffold for high efficiency production of functional bacteriotoxic proteins in Escherichia coli.

The availability of simple, robust, and cost-effective methods for the large-scale production of bacteriotoxic peptides such as antimicrobial peptides (AMPs) is essential for basic and pharmaceutical research. However, the production of bacteriotoxic proteins has been difficult due to a high degree of toxicity in bacteria and proteolytic degradation. In this study, we inserted AMPs into the Green fluorescent protein (GFP) in a loop region and expressed them as insoluble proteins in high yield, circumventing the inherent toxicity of AMP production in Escherichia coli. The AMPs inserted were released by cyanogen bromide and purified by chromatography. We showed that highly potent AMPs such as Protegrin-1, PMAP-36, Buforin-2, and Bactridin-1 are produced in high yields and produced AMPs showed similar activities compared to chemically synthesized AMPs. We increased the yield more than two-fold by inserting three copies of Protegrin-1 in the GFP scaffold. The immunogold electron micrographs showed that the expressed Protegrin-1 in the GFP scaffold forms large and small size aggregates in the core region of the inclusion body and become entirely nonfunctional, therefore not influencing the proliferation of E. coli. Our novel method will be applicable for diverse bacteriotoxic peptides which can be exploited in biomedical and pharmaceutical researches.

Identification of toxicity variations in a stream affected by industrial effluents using Daphnia magna and Ulva pertusa.

A comprehensive toxicity monitoring study from August to October 2011 using Daphnia magna and Ulva pertusa was conducted to identify the cause of toxicity in a stream receiving industrial effluents (IEs) from a textile and leather products manufacturing complex. Acute toxicity toward both species was observed consistently in IE, which influenced toxicity of downstream (DS) water. A toxicity identification evaluation (TIE) confirmed that both Cu and Zn were key toxicants in the IE, and that the calculated toxicity based on Cu and Zn concentrations well simulated the variation in the observed toxicity (r(2)=0.9216 and 0.7256 for D. magna and U. pertusa, respectively). In particular, U. pertusa was sensitive enough to detect acute toxicity in DS and was useful to identify Zn as a key toxicant. Activities of catalase, superoxide dismutase, glutathione peroxidase, glutathione S-transferase, and malondialdehyde were induced significantly in D. magna, although acute toxicity was not observed. In addition, higher levels of antioxidant enzymes were expressed in DS than upstream waters, likely due to the Cu and Zn from IE. Overall, TIE procedures with a battery of bioassays were effective for identifying the cause of lethal and sub-lethal toxicity in effluent and stream water.