Genome-wide single nucleotide polymorphism array and whole-genome sequencing reveal the inbreeding progression of Banna minipig inbred line.

Inbred pigs are promising animal models for biomedical research and xenotransplantation. Established in 1980, the Banna minipig inbred (BMI) line originated from a sow and its own male offspring. It was selected from a small backcountry minority Lahu village, where records show that no other pig breed has ever been introduced. During the inbreeding process, we perfomed extreme inbreeding over 23 consecutive generations using full-sibling or parent-offspring mating. In order to investigate the inbreeding effects in BMI pigs across generations over the past 40 years, in this study we conducted a genome-wide SNP genotyping of the last 10 generations, representing generations 14-23. In total, we genotyped 57,746 SNPs, corresponding to an average decrease in heterozygosity rate of 0.0078 per generation. Furthermore, we were only able to identify 18,216 polymorphic loci with a MAF larger than 0.05, which is substantially lower than the values in previous reports on other pig breeds. In addition, we sequenced the genome of the first pig in the twenty-third generation (inbreeding coefficient 99.28%) to an average coverage of 12.4× to evaluate at the genome level the impact of advanced inbreeding. ROH analysis indicates that BMI pigs have longer ROHs than Wuzhishan and Duroc pigs. Those long ROH regions in BMI pigs are enriched for distinct functions compared with the highly polymorphic regions. Our study reveals a genome-wide allele diversity loss during the progress of inbreeding in BMI pigs and characterizes ROH and polymorphic regions as a result of inbreeding. Overall, our results indicate the successful establishment of the BMI line, which paves the way for further in-depth studies.

Cloning, sequence characterization, and expression patterns of members of the porcine TSSK family.

Testis-specific serine kinases (TSSKs) are a family of serine/threonine kinases highly expressed in the testes that are responsible for regulating many spermatogenesis-related protein activities. Mutations in this family have a positive relationship with oligospermia and azoospermia in human and mouse. Here, five members of the TSSK family from a Banna mini-pig inbred line (BMI) were cloned, sequenced, and characterized. The full-length coding sequences of BMI TSSKs varied from 807 (TSSK3) to 1095 bp (TSSK1) and encoded 268 to 364 amino acids with molecular weights in the range 30.11 to 41.34 kDa. Following comparison with TSSK4 genes in other species, BMI TSSK4 was found to contain three alternatively spliced variants, inform1, inform 3, and inform 4. BMI TSSK1 and TSSK2 are co-localized on the Sus scrofa chromosome (SSC) 14, and consist of a single exon; TSSK3, TSSK4, and TSSK6 are on SSC6, SSC7, and SSC2, and consist of two, four, and one exon, respectively. Multiple protein sequence alignment and phylogenetic analysis showed that the regions spanning the S_TKc domains were more conserved between pig and other animals: with TSSK1 and TSSK2 and TSSK3 and TSSK6 displaying the greatest degree of homology across species, and the TSSK4 protein clearly distinct from other members. Multi-tissue RT-PCR showed BMI TSSK1, TSSK3, and TSSK4 were only expressed in the testes and seminal vesicle, TSSK2 was confined to testes only, while TSSK6 was expressed widely in adult tissues but was highest in the testes.

Identification, molecular characterization, and tissue expression of parathyroid hormone-related protein gene (PTHrP) from water buffalo (Bubalus bubalis).

Parathyroid hormone-related protein (PTHrP) is involved in the deposition of milk calcium in mammal lactation, but its role in buffalo is unclear. In this study, the full-length coding sequence of the water buffalo PTHrP gene was first isolated using reverse transcription-polymerase chain reaction. The protein was then subjected to molecular characterization using bioinformatic methods, and the tissue expression pattern was further assayed by semi-quantitative reverse-transcription polymerase chain reaction. The water buffalo PTHrP gene contains an open reading frame of 534 base pairs encoding a polypeptide of 177 amino acid residues, a theoretical molecular weight of 20.32 kDa, and an isoelectric point of 10.00. In addition, water buffalo PTHrP was predicted to contain a signal peptide, a typical hydrophobic region with no hydrophobic transmembrane regions, and to exert its function in the cell nucleus. A conserved domain of parathyroid superfamily from amino acids 34-114 was observed in the polypeptide. Sequence comparison and the phylogenetic analysis showed that the sequence of the water buffalo PTHrP protein shared high homology with that of other mammals, particularly cattle and goat. Among the 16 tissues examined, the PTHrP gene was only expressed in adipose tissue, placenta, uterine wall, hypophysis, and mammary gland tissue, but gene expression levels were higher in the uterus wall and adipose tissue. The results of this study suggest that the PTHrP gene plays an important role in the deposition of milk calcium of water buffalo.

Molecular characteristics and cloning of two pepper genes AN2 and UPA20.

The complete coding sequences (CDSs) of "Yunnan Purple Pepper No.1" (Capsicum annuum L.) AN2 and UPA20 genes were amplified using the reverse transcriptase polymerase chain reaction on the basis of the conserved sequence information of some Solanaceae plants and known highly homologous pepper expressed sequence tags. The nucleotide sequence analysis of these 2 genes revealed that pepper AN2 gene encoded a protein of 263 amino acids that has high homology with the AN2-like protein of 4 species: tobacco, tomato, potato, and petunia. The UPA20 gene encoded a protein of 341 amino acids that has high homology with the proteins of 3 species: tobacco, petunia, and tomato. The tissue expression analysis indicated that the pepper AN2 gene was overexpressed in the pericarp and placenta; moderately in stems, flowers, and seeds; and weakly in the roots, leaves, and pericarp. The pepper UPA20 gene was overexpressed in the flowers and seeds; moderately expressed in the roots and stems; and weakly expressed in the leaves and placenta. Our findings might form the basis for further research on these 2 pepper genes.

Sequence characterization, polymorphism, and tissue expression profile of an effector immediate-early gene: activity-regulated cytoskeletal associated protein gene (Arc/Arg3.1) in swamp and river buffalo.

The activity-regulated cytoskeletal associated protein (Arc/Arg3.1) has been implicated in experience-dependent synaptic plasticity and memory formation. However, information regarding its coding gene in buffalo remains scarce. In this study, the full-length of Arc/Arg3.1 was isolated and characterized (accession No. JX491649) and genetic variations of six river buffalo and eight swamp buffalo were investigated. A tissue expression profile was obtained using semi-quantitative reverse transcription-polymerase chain reaction. The coding region sequence of Arc/Arg3.1 contained 1191 nucleotides encoding a putative protein of 396 amino acids with a theoretical isoelectric point (pI) and molecular weight (Mw) of 5.4 and 45.2 kDa, respectively. Four polymorphisms (c.63T>C, c.228T>C, c.558G>A, and c.625G>C) were found in buffalo; however, only substitution c.625G>C was non-synonymous, leading to an amino acid change from Val to Leu at the 209th position of the Arc/Arg3.1 protein sequence. Bioinformatics analysis revealed that this substitution had no significant effect on Arc/Arg3.1 function (subPSEC = -1.4039, Pdeleterious = 0.1685), which indicated that Arc/Arg3.1 was highly conserved and functionally important in buffalo. Phylogenetic analysis revealed that the gene is closely related to that of Bos taurus and Bos grunniens. The gene was moderately expressed in the hypophysis and the placenta; it was weakly expressed in the kidney, milk, mammary gland, cerebrum, lung, heart, rumen, fat, and uterus; and it was almost silent in the muscle, liver, and skin. These findings will provide further insights into the structure and function of the immediate-early gene in buffalo.

Genetic diversity of local Yunnan chicken breeds and their relationships with Red Junglefowl.

Yunnan is situated in the Southwest China and encompasses regions having high biodiversity, including habitats for several ancestral species of domestic animals such as chicken. Domestic chickens in Yunnan were kept by peoples of varied ethnic and economic backgrounds living in highly varied geographic environments. To identify the genetic background of Yunnan domestic chickens and their relationships with Red Junglefowl, we applied 28 widely used microsatellite DNA markers to genotype 340 birds from 7 chicken breeds and Red Junglefowl indigenous to Yunnan. Among a total of 342 alleles identified, 121 (35.4%) were breed specific, with Red Junglefowl harboring most microsatellite alleles (23). High levels of heterozygosity were observed within populations indicated by a mean unbiased HE value of 0.663, which was higher than the reported for most populations elsewhere. The FIS value of domestic populations ranged from -0.098-0.005, indicating a lack of inbreeding among these populations. A high proportion of significant departures (89) from the 224 HWE tests for each locus in each population reflected an excess of heterozygosity and population substructure. Individual assignment tests, high FST values (0.1757-0.3015), and Nei's DA genetic distances (0.4232-0.6950) indicated clear differentiation among these populations. These observations, along with the close genetic distance between indigenous domestic populations and Red Junglefowl, were consistent with the primitive and ancestral state of Yunnan indigenous chickens. Protecting the unique variants of these indigenous poultry varieties from contamination with commercial breeds might provide values for improving modern agricultural livestock and breeding programs. Thus, the current study may benefit breeding management and conservation efforts.

Isolation, molecular cloning, and characterization of a novel porcine lymphotoxin beta receptor gene.

The lymphotoxin beta receptor (LTßR) is a member of the tumor necrosis factor family of receptors (TNFR). It plays a role in regulating lymphoid organogenesis and homeostasis of the immune system. In the present study, the full coding region of a putative LTßR gene of Sus scrofa was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned for the first time (accession Nos. JX457347 and AFU74012). In addition, analysis of the tissue expression profile was carried out via RT-PCR. The full-length coding region of porcine LTßR had 1266 nucleotides (molecular weight, 45.61 kDa; pI, 5.71) and encoded 421 amino acids. Bioinformatic prediction indicates that LTßR belongs to the TNFR superfamily and contains a TNFR domain. The sequence homology analysis revealed that the amino acid sequences of S. scrofa LTßR had 82.9, 82.4, 81.3, 80.5, 78.7, 74.6, and 73.0% identity with those of Equus caballus, Canis lupus, Ailuropoda melanoleuca, Oryctolagus cuniculus, Bos taurus, Mus musculus, and Homo sapiens, respectively. The phylogenetic tree based on the amino acid sequences of LTßR from 8 species revealed that S. scrofa was more closely related to E. caballus, C. lupus, and A. melanoleuca. RT-PCR analysis showed that the porcine LTßR gene was differentially expressed (e.g., high, moderate, low, or nonexistent) in various tissues (e.g., prostate, pituitary, brainstem, and esophagus, respectively). This may be related to differences in the regulation of LTßR in the different tissues.

Molecular cloning and tissue expression analyses of two novel pepper genes: heterotrimeric G protein beta 2 subunit and ArcA1.

We isolated two transcription factor genes, heterotrimeric G protein beta 2 subunit (Gß2) and ArcA1, from pepper (Capsicum annuum). The complete coding sequences were amplified using reversed transcriptase PCR based on conserved sequence information of Solanum lycopersicum and several other plant species. Nucleotide sequence analysis of these two genes revealed that the pepper Gß2 gene encodes a protein of 376 amino acids that belongs to the WD40 superfamily. Tissue expression analysis indicated that this gene is highly expressed in the pericarp, moderately expressed in stem, flower, placenta, and leaves, and weakly expressed in seed. There was no expression in the roots. The ArcA1 gene encodes a protein of 331 amino acids that also belongs to the WD40 superfamily. Tissue expression analysis indicated that the pepper ArcA1 gene is moderately expressed in the pericarp and weakly expressed in seed. There was no expression in root, stem, flower, placenta, or leaves.

Complementary DNA cloning, sequence analysis, and tissue transcription profile of a novel U2AF2 gene from the Chinese Banna mini-pig inbred line.

U2 small nuclear RNA auxiliary factor 2 (U2AF2) is an important gene for pre-messenger RNA splicing in higher eukaryotes. In this study, the Banna mini-pig inbred line (BMI) U2AF2 coding sequence (CDS) was cloned, sequenced, and characterized. The U2AF2 complete CDS was amplified using the reverse transcription-polymerase chain reaction (RT-PCR) technique based on the conserved sequence information of cattle and known highly homologous swine expressed sequence tags. This novel gene was deposited into the National Center for Biotechnology Information database (Accession No. JQ839267). Sequence analysis revealed that the BMI U2AF2 coding sequence consisted of 1416 bp and encoded 471 amino acids with a molecular weight of 53.12 kDa. The protein sequence has high sequence homology with U2AF65 of 6 species - Homo sapiens (100%), Equus caballus (100%), Canis lupus (100%), Macaca mulatta (99.8%), Bos taurus (74.4%), and Mus musculus (74.4%). The phylogenetic tree analysis revealed that BMI U2AF65 has a closer genetic relationship with B. taurus U2AF65 than with U2AF65 of E. caballus, C. lupus, M. mulatta, H. sapiens, and M. musculus. RT-PCR analysis showed that BMI U2AF2 was most highly expressed in the brain; moderately expressed in the spleen, lung, muscle, and skin; and weakly expressed in the liver, kidney, and ovary. Its expression was nearly silent in the spinal cord, nerve fiber, heart, stomach, pancreas, and intestine. Three microRNA target sites were predicted in the CDS of BMI U2AF2 messenger RNA. Our results establish a foundation for further insight into this swine gene.

Molecular cloning, sequence characterization, and gene expression profiling of a novel water buffalo (Bubalus bubalis) gene, AGPAT6.

Several 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs) can acylate lysophosphatidic acid to produce phosphatidic acid. Of the eight AGPAT isoforms, AGPAT6 is a crucial enzyme for glycerolipids and triacylglycerol biosynthesis in some mammalian tissues. We amplified and identified the complete coding sequence (CDS) of the water buffalo AGPAT6 gene by using the reverse transcription-polymerase chain reaction, based on the conversed sequence information of the cattle or expressed sequence tags of other Bovidae species. This novel gene was deposited in the NCBI database (accession No. JX518941). Sequence analysis revealed that the CDS of this AGPAT6 encodes a 456-amino acid enzyme (molecular mass = 52 kDa; pI = 9.34). Water buffalo AGPAT6 contains three hydrophobic transmembrane regions and a signal 37-amino acid peptide, localized in the cytoplasm. The deduced amino acid sequences share 99, 98, 98, 97, 98, 98, 97 and 95% identity with their homologous sequences from cattle, horse, human, mouse, orangutan, pig, rat, and chicken, respectively. The phylogenetic tree analysis based on the AGPAT6 CDS showed that water buffalo has a closer genetic relationship with cattle than with other species. Tissue expression profile analysis shows that this gene is highly expressed in the mammary gland, moderately expressed in the heart, muscle, liver, and brain; weakly expressed in the pituitary gland, spleen, and lung; and almost silently expressed in the small intestine, skin, kidney, and adipose tissues. Four predicted microRNA target sites are found in the water buffalo AGPAT6 CDS. These results will establish a foundation for further insights into this novel water buffalo gene.

Cloning and bioinformatic analysis of full-length novel pepper (Capsicum annuum) genes TAF10 and TAF13.

We isolated two TATA-binding protein-associated factor (TAF) genes, TAF10 and TAF13, from pepper (Capsicum annuum). The complete coding sequences were amplified using reverse transcriptase-PCR on the basis of conserved sequence information of eggplant and several other plant species. Nucleotide sequence analysis of these two genes revealed that the pepper TAF10 gene encodes a protein of 103 amino acids that belongs to the TAF10 superfamily. The pepper TAF10 gene was highly expressed in the pericarp and placenta, moderately expressed in the stems, flowers, seeds and leaves, and weakly expressed in roots. The TAF13 gene was found to encode a protein of 130 amino acids that belongs to the TAF13 superfamily. The TAF13 gene was highly expressed in the stems, flowers and pericarp, moderately expressed in the leaves, placenta and seeds, and weakly expressed in roots.

Melatonin attenuates inflammation and cardiac dysfunction in myocardial infarction by regulating the miRNA-200b-3p/high mobility group box chromosomal protein 1 axis.

Melatonin confers protection against myocardial injury by reducing inflammation and inhibiting apoptosis. In the present study, we investigated whether melatonin regulates cardiomyocyte proliferation and improves cardiac function in rats with myocardial infarction (MI). Two MI models were established in vitro (H9c2 cells were cultured under hypoxia) and in vivo (the left anterior descending coronary artery of rats was surgically ligated). miR-200b-3p and high mobility group box 1 (HMGB1) levels were detected. Cell proliferation and apoptosis were analyzed in vitro, and cardiac function, inflammatory cytokines, and myocardial injury markers in vivo were tested. The experimental results reported that melatonin promoted proliferation and impaired apoptosis of H9c2 cells cultured in hypoxia. In vivo, melatonin improved cardiac function and inhibited the inflammation and myocardial injury of rats with MI. miR-200b-3p was downregulated and HMGB1 was upregulated in MI, while melatonin could upregulate miR-200b-3p and downregulate HMGB1. The HMGB1 was targeted by miR-200b-3p. Upregulating miR-200b-3p or downregulating HMGB1 could further promote the therapeutic effect of melatonin, and downregulating miR-200b-3p or upregulating HMGB1 could abolish the therapeutic effect of melatonin. In conclusion, melatonin alleviates inflammation and cardiac dysfunction after MI by regulating the miR-200b-3p/HMGB1 axis, offering a new therapeutic strategy for MI.

Molecular cloning, sequence characterization of a novel pepper gene NADP-ICDH and its effect on cytoplasmic male sterility.

NADP-dependent isocitrate dehydrogenase (NADP-ICDH) is an important enzyme involved in energy metabolism. The complete coding sequence of the pepper (Capsicum annuum) NADP-ICDH gene was amplified using a reverse transcriptase PCR based on the conserved sequence information of the tomato and other Solanaceae plants and known highly homologous pepper ESTs. Nucleotide sequence analysis revealed that the pepper NADP-ICDH gene encodes a protein of 415 amino acids that has high homology with the proteins of seven species, Solanum tuberosum (100%), Citrus limon (98%), Daucus carota (98%), Nicotiana tabacum (98%), Vitis vinifera (99%), Arabidopsis thaliana (97%), and Oryza sativa (98%). Tissue expression analysis demonstrated that the pepper NADP-ICDH gene is over expressed in flower, pericarp and seed, moderately in placenta, weakly in stem and leaf, hardly expressed in root. At the abortion stages, activities and expression levels of NADP-ICDH in anthers of a sterile line were strongly reduced, while those in an F(1) hybrid remained normal. Activities and expression levels of NADP-ICDH were too low to maintain balanced energy metabolism in the sterile line, which indicated that stable transcripts of NADP-ICDH are necessary to maintain energy metabolism at a normal level. When the restorer gene was transferred to the cytoplasmic male sterile line, activities and expression level of NADP-ICDH were regulated by the restorer gene and became stable. The restorer gene likely plays an important role in keeping the balance of the energy metabolism within normal levels during microspore development.

[Endoscopic selective lateral neck dissection via a chest-breast approach for papillary thyroid carcinoma: preliminary experience in 20 cases].

Objective:To explore the feasibility of endoscopic selective lateral neck dissection(SLND) via a chest-breast approach.Method:We retrospectively reviewed 20 patients who underwent endoscopic total thyroidectomy along with SLND, between January 2017 and May 2018. Result: All the 20 patients underwent total thyroidectomy, central lymph nodes dissection and selective lateral lymph nodes dissection with endoscopic surgery via chest-breast approach. In this study, lymphatic leakage, transient voice hoarseness, internal jugular vein injury and external jugular vein injury were repectively found in one patient, and 4 patients suffered from transient parathyroid hypofunction, without other serious complications.Conclusion: Endoscopic lymph node dissection including levels Ⅱ,Ⅲ and Ⅳ is feasible. It has good cosmetic effect, and haven't serious adverse events.

[A comparison of fine needle nonaspiration cytology versus fine needle aspiration for thyroid nodules: a Meta-analysis].

Objective:To evaluate the differences of smear quality and diagnostic accuracy between thyroid nodules and fine needle nonaspiration cytology (FNNAC) and fine needle aspiration cytology (FNAC).Method:Databases were used to search the literature on FNNAC and FNAC. All statistical analyses were performed using Review Manager 5.3 Software.Result:A total of 10 studies were included in the study. Meta-analysis showed no significant difference in FNNAC and FNAC between low, middle and high quality smears. There was no significant difference in diagnostic accuracy.Conclusion:There were no difference in obtaining the smear quality and diagnostic accuracy, the person doing the piercing can freely choose which way according to the habit.

[Diagnostic accuracy and safety of US-guided core needle biopsy versus fine needle aspiration biopsy of thyroid nodules: a Meta analysis].

Objective:To evaluate the efficacy and safety of core needle biopsy (CNB) and fine neon needle aspiration biopsy (FNAB) in the diagnosis of thyroid nodules.Method:The CNKI, Wanfang database, China Biomedical Literature Database (CBM), PubMed, Cochrane Library, EMBASE, Web of Science database (the deadline of February 2017) were used to search the literature on CNB and FNABCNB. Two reviewers independently screened the literature according to the inclusion and exclusion criteria, extracted the data and evaluated the quality of the literature, and used RevMan 5.3 software for Meta analysis.Result:The accuracy of the CNB group was higher than that of the FNAB group(RR= 1.14, 95%CI: 1.06-1.22, P< 0.01). Meta analysis showed that the accuracy of CNB group compared with FNAB group was statistically significant the difference was statistically significant. There was no significant difference between the two groups (RR= 0.92, 95%CI:0.67-1.25, P> 0.05).Conclusion:CNB is safe and feasible in the diagnosis of thyroid nodules under the condition of mastery of puncture technique.