Transgenic Mice Overexpressing PG1 Display Corneal Opacity and Severe Inflammation in the Eye.

Antimicrobial peptides (AMPs) are of interest as alternatives to antibiotics or immunomodulators. We generated and characterized the phenotypes of transgenic mice overexpressing protegrin 1 (PG1), a potent porcine cathelicidin. No obvious differences were observed between PG1 transgenic and wild-type mice in terms of growth, development, general behaviour, and the major immune cell population. However, PG1 transgenic mice intranasally infected with Staphylococcus aureus resulted in a reduction in microscopic pulmonary injury, improved clearance of bacteria, and lower proinflammatory cytokine secretion, compared to those of wild-type mice. On the other hand, approximately 25% of PG1 transgenic mice (n = 54/215) showed corneal opacity and developed inflammation in the eye, resulting ultimately in phthisis bulbi. Immunohistochemical analyses revealed that PG1 and its activator, neutrophil elastase, localized to the basal cells of the cornea and glands in eyelids, respectively. In addition, apoptosis indicated by a Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive signal was detected from flat cells of the cornea. Our study suggests that the expression regulation or localization of AMPs such as PG1 is important to prevent their adverse effects. However, our results also showed that the cytotoxic effects of PG1 on cells could be tolerated in animals, except for the eyes.

Analysis of Pig Vomeronasal Receptor Type 1 (V1R) Promoter Region Reveals a Common Promoter Motif but Poor CpG Islands.

Promoters are, generally, located immediately upstream of a transcription start site (TSS) and have a variety of regulatory motifs, such as transcription factors (TFs) and CpG islands (CGIs), that participate in the regulation of gene expression. Here analysis of the promoter region for pig vomeronasal receptor type 1 (V1R) was described. In the analysis, TSSs for pig V1R genes was first identified and five motifs (MV1, MV2, MV3, MV4, and MV5) were found that are shared by at least 50% of the pig V1R promoter input sequences from both strands. Among the five motifs, MV2 was identified as a common promoter motif shared by all (100%) pig V1R promoters. For further analysis, to better characterize and get deeper biological insight associated with MV2, TOMTOM web application was used. MV2 was compared to the known motif databases (such as JASPAR) to see if they are similar to a known regulatory motif (transcription factor). Hence, it was revealed that MV2 serves as the binding site mainly for the BetaBetaAlpha-zinc finger (BTB-ZF) transcription factor gene family to regulate expression of pig V1R genes. Moreover, it was shown that pig V1R promoters are CpG poor, suggesting that their gene expression regulation pattern is in tissue specific manner.

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.

Analysis of the vomeronasal receptor repertoire, expression and allelic diversity in swine.

Here we report a comprehensive analysis of the vomeronasal receptor repertoire in pigs. We identified a total of 25 V1R sequences consisting of 10 functional genes, 3 pseudogenes, and 12 partial genes, while functional V2R and FPR genes were not present in the pig genome. Pig V1Rs were classified into three subfamilies, D, F, and J. Using direct high resolution sequencing-based typing of all functional V1Rs from 10 individuals of 5 different breeds, a total of 24 SNPs were identified, indicating that the allelic diversity of V1Rs is much lower than that of the olfactory receptors. A high expression level of V1Rs was detected in the vomeronasal organ (VNO) and testes, while a low expression level of V1Rs was observed in all other tissues examined. Our results showed that pigs could serve as an interesting large animal model system to study pheromone-related neurobiology because of their genetic simplicity.

Deficiency in mammalian STN1 promotes colon cancer development via inhibiting DNA repair.

Despite the high lethality of colorectal cancers (CRCs), only a limited number of genetic risk factors are identified. The mammalian ssDNA-binding protein complex CTC1-STN1-TEN1 protects genome stability, yet its role in tumorigenesis is unknown. Here, we show that attenuated CTC1/STN1 expression is common in CRCs. We generated an inducible STN1 knockout mouse model and found that STN1 deficiency in young adult mice increased CRC incidence, tumor size, and tumor load. CRC tumors exhibited enhanced proliferation, reduced apoptosis, and elevated DNA damage and replication stress. We found that STN1 deficiency down-regulated multiple DNA glycosylases, resulting in defective base excision repair (BER) and accumulation of oxidative damage. Collectively, this study identifies STN1 deficiency as a risk factor for CRC and implicates the previously unknown STN1-BER axis in protecting colon tissues from oxidative damage, therefore providing insights into the CRC tumor-suppressing mechanism.

The complete swine olfactory subgenome: expansion of the olfactory gene repertoire in the pig genome.

BACKGROUND: Insects and animals can recognize surrounding environments by detecting thousands of chemical odorants. Olfaction is a complicated process that begins in the olfactory epithelium with the specific binding of volatile odorant molecules to dedicated olfactory receptors (ORs). OR proteins are encoded by the largest gene superfamily in the mammalian genome. RESULTS: We report here the whole genome analysis of the olfactory receptor genes of S. scrofa using conserved OR gene specific motifs and known OR protein sequences from diverse species. We identified 1,301 OR related sequences from the S. scrofa genome assembly, Sscrofa10.2, including 1,113 functional OR genes and 188 pseudogenes. OR genes were located in 46 different regions on 16 pig chromosomes. We classified the ORs into 17 families, three Class I and 14 Class II families, and further grouped them into 349 subfamilies. We also identified inter- and intra-chromosomal duplications of OR genes residing on 11 chromosomes. A significant number of pig OR genes (n = 212) showed less than 60% amino acid sequence similarity to known OR genes of other species. CONCLUSION: As the genome assembly Sscrofa10.2 covers 99.9% of the pig genome, our analysis represents an almost complete OR gene repertoire from an individual pig genome. We show that S. scrofa has one of the largest OR repertoires, suggesting an expansion of OR genes in the swine genome. A significant number of unique OR genes in the pig genome may suggest the presence of swine specific olfactory stimulation.

Genome-level identification, gene expression, and comparative analysis of porcine ß-defensin genes.

BACKGROUND: Beta-defensins (ß-defensins) are innate immune peptides with evolutionary conservation across a wide range of species and has been suggested to play important roles in innate immune reactions against pathogens. However, the complete ß-defensin repertoire in the pig has not been fully addressed. RESULT: A BLAST analysis was performed against the available pig genomic sequence in the NCBI database to identify ß-defensin-related sequences using previously reported ß-defensin sequences of pigs, humans, and cattle. The porcine ß-defensin gene clusters were mapped to chromosomes 7, 14, 15 and 17. The gene expression analysis of 17 newly annotated porcine ß-defensin genes across 15 tissues using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) showed differences in their tissue distribution, with the kidney and testis having the largest pBD expression repertoire. We also analyzed single nucleotide polymorphisms (SNPs) in the mature peptide region of pBD genes from 35 pigs of 7 breeds. We found 8 cSNPs in 7 pBDs. CONCLUSION: We identified 29 porcine ß-defensin (pBD) gene-like sequences, including 17 unreported pBDs in the porcine genome. Comparative analysis of ß-defensin genes in the pig genome with those in human and cattle genomes showed structural conservation of ß-defensin syntenic regions among these species.

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.

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.

An assessment of the potential for the development of the shale gas industry in countries outside of North America.

The revolution of shale gas in the United States (the US) has become a phenomenon at the beginning of the 21st century. It has been significantly influencing the United States' economy and the global gas market. Like America, other countries have also been searching for shale gas. However, the conditions for developing this resource are very different among regions and nations. On the other hand, there are also many doubts, debates and even strong oppositions to the development of shale gas because of the complicated issues that arise regarding its extraction, and also due to the fact that its impacts are not fully known. Therefore, at present, the development of shale gas is still a big question for regions, countries that have potential and desires to exploit such resources. Although it is difficult to identify all necessary or sufficient conditions to develop shale gas, the experiences of the United States could be instructive for other countries. In this article, the potential development of shale gas in China and Europe is analyzed, which relies on the fundamental conditions considered as important factors for the success of the shale gas industry in the US. Through these analyses and we demonstrate the difficulty of developing this resource outside North America.

Determination of complete sequence information of the human ABO blood group orthologous gene in pigs and breed difference in blood type frequencies.

The sequence information of the genomic form of the human ABO blood group orthologous gene (erythrocyte antigen A, EAA) is not complete in pigs. Therefore, we cloned and characterized the nucleotide sequence of EAA intron 7, which is critical to understand genetic difference between A and 0 blood groups in pigs, covering complete genomic sequence information of EAA excluding a ~560bp unsequencible gap. We also analyzed genetic polymorphisms within EAA intron 7 and exon 8. We found difference in A0 blood group frequencies among pig breeds. In addition, we designed a new genomic DNA-based A0 blood group typing method and improved the accuracy and simplicity of the typing.