[Modified minimally invasive video-assisted lateral neck dissection for papillary thyroid carcinoma: a series of 130 cases].

Objective: To discuss the feasibility, safety and oncologic completeness of modified minimally invasive video-assisted lateral neck dissection (MIVALND) for papillary thyroid carcinoma. Methods: Data of 130 patients from Department of Head and Neck Surgery, Sir Run Run Shaw Hospital, Medical School, Zhejiang University undergoing MIVALND from January 2013 to September 2015 were reviewed retrospectively. There were 31 male and 99 female patients with the mean age of (39±11) years. The thyroidectomy and central compartment dissection were performed under a direct visual field or video-assisted (VA) approach, lateral neck dissection was performed via the VA approach. Serum thyroglobulin and thyroglobulin antibody levels were measured every 6 months after surgery. Ulrasonography was performed to assess the thyroid bed and lateral neck compartment every 6 months after surgery. The mean operation time for MIVALND, mean postoperative hospital stay, size of primary tumor, number of retrieved lymph nodes, complication rates, and postoperative serum thyroglobulin levels were analyzed retrospectively. The patients were followed up by outpatient review and until March 2016. Results: Beside 1 case was converted to open procedure, 129 (99.2%) patients successfully underwent MIVALND. The mean operative time was (74±17) min (ranging from 40 to 120 min) for MIVALND. The mean postoperative hospital stay was (4.9±2.1) days (range 2 to 14 days). The mean size of primary tumor was (1.3±0.7) cm (range 0.3 to 4.0 cm). The mean number of lymph nodes removed was 42±13 (range 15 to 79) in lateral compartment. Postoperative complications included 19 transient hypoparathyroidism, 7 transient recurrent laryngeal nerve (RLN) palsy and 3 permanent RLN palsy (tumor invasion and the RLN was resected en bloc with the tumor in 2 cases), 2 (1.5%) transient palsy of spinal accessory nerve, 1 (0.8%) transient palsy of marginal mandibular branch of the facial nerve, 1 (0.8%) seroma, and 4 (3.1%) minor chyle leak. The mean follow-up period was (19±10) months (ranged 6 to 36 months). The mean serum thyroglobulin level was 0.10 µg/L during follow-up. No evidence of local residual or recurrent disease was observed at postoperative follow-up. Conclusion: The modified MIVALND is a safe and feasible approach in selected papillary thyroid carcinoma patients.

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.

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.

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.

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.

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.

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.

Chicken domestication: an updated perspective based on mitochondrial genomes.

Domestic chickens (Gallus gallus domesticus) fulfill various roles ranging from food and entertainment to religion and ornamentation. To survey its genetic diversity and trace the history of domestication, we investigated a total of 4938 mitochondrial DNA (mtDNA) fragments including 2843 previously published and 2095 de novo units from 2044 domestic chickens and 51 red junglefowl (Gallus gallus). To obtain the highest possible level of molecular resolution, 50 representative samples were further selected for total mtDNA genome sequencing. A fine-gained mtDNA phylogeny was investigated by defining haplogroups A-I and W-Z. Common haplogroups A-G were shared by domestic chickens and red junglefowl. Rare haplogroups H-I and W-Z were specific to domestic chickens and red junglefowl, respectively. We re-evaluated the global mtDNA profiles of chickens. The geographic distribution for each of major haplogroups was examined. Our results revealed new complexities of history in chicken domestication because in the phylogeny lineages from the red junglefowl were mingled with those of the domestic chickens. Several local domestication events in South Asia, Southwest China and Southeast Asia were identified. The assessment of chicken mtDNA data also facilitated our understanding about the Austronesian settlement in the Pacific.

A study of association between genetic markers in candidate genes and reproductive traits in one generation of a commercial broiler breeder hen population.

Markers of alleles for three physiological candidate genes for reproductive traits, growth hormone (GHR), gonadotropin-releasing hormone receptor (GNRHR) and neuropeptide Y (NPY) were assessed for the association with the total egg production, number of double-yolked eggs and age at first egg in a single generation of a broiler breeder (Gallus gallus) pedigree dam line. Single-nucleotide polymorphisms and deletions were detected in the GHR, GNRHR and NPY genes. Genotypes were identified using a PCR-RFLP assay. The frequency of restriction enzyme+/-alleles in the population was for GHR 0.68 (NspI-) and 0.32 (NspI+), for NPY 0.78 (DraI+) and 0.22 (DraI-) and for GNRHR 0.54 (Bpu1102I+) and 0.46 (Bpu1102I-). Trait data from a total of 772 hens in 67 sire families from one generation of the pedigree dam line were recorded. However, the analysis used only the offspring of heterozygous sires to reduce the influence of selection and genetic background (n=33 sire families for GHR; n=14 sire families for NPY; n=36 sire families for GNRHR). A dominance effect of NPY on age at first egg and an additive effect of GNRHR on the number of double-yolked eggs were found (P<0.05).