Identification of two novel chicken GPR133 variants and their expression in different tissues.

GPR133 (G protein-coupled receptor 133) plays significant roles in various physiological processes. Alternatively splicing (AS) variants of GPR133 in many species have been predicted in multiple databases, but there is no available information about the AS events of chicken GPR133 (cGPR133). In the present study, two chicken GPR133 variants, cGPR133-va and cGPR133-vb, were identified by a combination of reverse transcription PCR (RT-PCR) and rapid amplification of cDNA 5'-ends (5' RACE). Sequence analysis shows that cGPR133-va and cGPR133-vb are resulting from different AS modules and their sequences are predicted to encode two distinct putative proteins, respectively. Quantitative real-time PCR (qRT-PCR) analysis revealed that cGPR133-va and cGPR133-vb are widely expressed in different tissues, while exhibiting specific expression profile. Altogether, our results first demonstrate the existence of novel cGPR133 variants and illustrate its transcriptional diversity and their widespread distribution, which provides a foundation for the further research of GPR133.

Thallium(I and III) exposure leads to liver damage and disorders of fatty acid metabolism in mice.

Thallium (Tl), a highly toxic and priority pollutant heavy metal, exposure can damage mitochondria and disrupt their function. The liver is the central organ that controls lipid homeostasis and contains a large number of mitochondria. So far, there is no study investigating the effects of Tl exposure on hepatic fatty acid metabolism. Here, we showed that 10 ppm of Tl(I) and Tl(III) exposures for two weeks did not significantly affect the body weight and water/food intake in mice. However, it decreased the ratio of liver/weight and induced hepatic sinus congestion and hepatocyte necrosis. Inductively coupled plasma-mass spectrometry (ICP-MS) analysis revealed Tl accumulation in the liver. Gas chromatography-mass spectrometry (GC-MS) results showed that Tl(I) exposure significantly increased hepatic C18:0 concentration, while significantly decreased the concentrations of C16:1n-7, C20:1n-9, C18:3n-6, and C20:2n-9. Tl(III) exposure significantly reduced hepatic concentrations of C20:0, C22:0, C20:1n-9, C18:3n-6, and C20:3n-6. In addition, Tl(I) exposure upregulated the genes related to antioxidation (HO-1, GPX1, and GPX4), fatty acid synthesis (FADS2 and Elovl2), and fatty acid oxidation pathway (PPARα, ACADM, ACADVL, ACAA2, and CPT1A) in the liver. Tl(III) exposure did not significantly affect the transcript levels of liver antioxidative/metabolic enzymes and fatty acid synthesis-related genes, but upregulated fatty acid oxidation pathway-related genes (CYP4A10 and CPT1A). These results suggest that Tl(I) and Tl(III) exposures can cause liver damage and disrupt hepatic fatty acid metabolism, which provide new insights into Tl exposure-induced energy depletion from the perspective of fatty acid metabolism.

ANI analysis of poxvirus genomes reveals its potential application to viral species rank demarcation.

Average nucleotide identity (ANI) is a prominent approach for rapidly classifying archaea and bacteria by recruiting both whole genomic sequences and draft assemblies. To evaluate the feasibility of ANI in virus taxon demarcation, 685 poxviruses were assessed. Prior to the analysis, the fragment length and threshold of the ANI value were optimized as 200 bp and 98 per cent, respectively. After ANI analysis and network visualization, the resulting sixty-one species (ANI species rank) were clustered and largely consistent with the groupings found in National Center for Biotechnology Information Virus [within the International Committee on Taxonomy of Viruses (ICTV) Master Species List]. The species identities of thirty-four other poxviruses (excluded by the ICTV Master Species List) were also identified. Subsequent phylogenetic analysis and Guanine-Cytosine (GC) content comparison done were found to support the ANI analysis. Finally, the BLAST identity of concatenated sequences from previously identified core genes showed 91.8 per cent congruence with ANI analysis at the species rank, thus showing potential as a marker gene for poxviruses classification. Collectively, our results reveal that the ANI analysis may serve as a novel and efficient method for poxviruses demarcation.

The Insights of Genomic Synteny and Codon Usage Preference on Genera Demarcation of Iridoviridae Family.

The members of the family Iridoviridae are large, double-stranded DNA viruses that infect various hosts, including both vertebrates and invertebrates. Although great progress has been made in genomic and phylogenetic analyses, the adequacy of the existing criteria for classification within the Iridoviridae family remains unknown. In this study, we redetermined 23 Iridoviridae core genes by re-annotation, core-pan analysis and local BLASTN search. The phylogenetic tree based on the 23 re-annotated core genes (Maximum Likelihood, ML-Tree) and amino acid sequences (composition vector, CV-Tree) were found to be consistent with previous reports. Furthermore, the information provided by synteny analysis and codon usage preference (relative synonymous codon usage, correspondence analysis, ENC-plot and Neutrality plot) also supports the phylogenetic relationship. Collectively, our results will be conducive to understanding the genera demarcation within the Iridoviridae family based on genomic synteny and component (codon usage preference) and contribute to the existing taxonomy methods for the Iridoviridae family.

Genomic analysis of Poxviridae and exploring qualified gene sequences for phylogenetics.

The members of the Poxviridae family are globally distributed all over the world and can cause infectious diseases. Although genome sequences are publicly available for representative isolates of all genera, studies on the criteria for genome-based classification within the Poxviridae family have rarely been reported. In our study, 60 Poxviridae genomes were re-annotated using Prokka. By using BLAST filtration and MCScanX, synteny and similarity of whole genomic amino acid sequences were visualized. According to the analysis pattern, the Chordopoxvirinae and Entomopoxvirinae subfamilies can be subdivided into five and two categories respectively, which is consistent with the phylogenetic tree constructed based on whole genomic amino acid sequences and Poxvirus core genes. Finally, four genes (Early transcription factor, DNA-directed RNA polymerase, RNA polymerase-associated transcription-specificity factor and DNA-dependent RNA polymerase) were selected from Poxvirus core genes by substitution saturation analysis and phylogenetic tree verification. Phylogenetic trees constructed based on single gene and concatenated sequences of the four selected genes showed that the classification of subgroups was consistent with the phylogenetic trees based on genome. Conclusion: a new method based on the similarity of whole genomic amino acid sequences was proposed for Poxviridae taxon demarcation, and the use of the four selected qualified genes will help make phylogenic identification of newly discovered Poxviridae isolates more convenient and accurate.

Genomic analysis of Ranavirus and exploring alternative genes for phylogenetics.

Ranaviruses can infect both captive and wild cold-blooded vertebrates, leading to significant economic and environmental losses. With the cases of ranavirus infection increasing, many ranavirus genomic sequences were published, but little is known about ranavirus taxonomy on a whole-genome level. In this study, 44 ranaviruses core genes were identified in 32 ranaviruses genome sequences by using PanX. The neighbour-joining phylogenetic trees (NJ-tree) based on 44 ranaviruses core genes and 24 iridoviridae core genes and composition vector phylogenetic tree (CV-Tree) based on whole genome were constructed. The three of phylogenetic trees showed that 32 ranavirus isolates can be divided into 4 different subgroups including SGIV-like, EHNV-like, FV3-like and CMTV-like, and subgroups taxonomic position of three phylogenetic trees were consistent. However, the phylogenetic position of ToRV could not be determined if it belongs to FV3-like or CMTV-like group. Subsequently, we carried out dot plot analysis and confirmed that ToRV should belong to CMTV-like group. Based on dot plot analysis and phylogenetic trees, the taxonomic classification of ranaviruses was confirmed. Finally, four genes which are suitable for the construction of phylogenetic tree were selected from ranavirus core genes by recombination analysis, substitution saturation analysis and single-gene phylogenetic analysis. Phylogenetic tree based on concatenated sequences of the four selected genes showed that the classification of subgroups was identical with three of the phylogenetic trees. Conclusion: Our results confirmed taxonomic identification of ranaviruses; the four selected genes used in phylogenetic analysis will make taxonomic identification more convenient and accurate.

LncRNA NEAT1 promotes gastric cancer progression via miR-1294/AKT1 axis.

Long non-coding RNAs (lncRNAs) were reported to promote the development of gastric cancer (GC). Nuclear-enriched abundant transcript 1 (NEAT1) played a great role in diverse cancers, but the mechanism of NEAT1 in GC remains indistinct. NEAT1 and AKT1 were distinctly up-regulated and miR-1294 was down-regulated in GC tissues and cells. Cell proliferation and metastasis were refrained but apoptosis was promoted in GC cells after knockdown of NEAT1. NEAT1 negatively regulated miR-1294 expression, and the miR-1294 inhibitor reverted the si-NEAT1-induced effect on GC cells. NEAT1 modulated AKT1 expression through miR-1294, and the si-NEAT1-induced effect was relieved by AKT1. NEAT1 affected phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway via regulating miR-1294 and AKT1. NEAT1 could modulate cell proliferation, apoptosis, and metastasis in GC cells by regulating the PI3K/AKT/mTOR signaling pathway via the miR-1294/AKT1 axis, showing the great potential for NEAT1 as a valid biomarker in the progression and treatment of GC.

Corrigendum to "Polymorphisms in the Chicken Growth Differentiation Factor 9 Gene Associated with Reproductive Traits".

[This corrects the article DOI: 10.1155/2018/9345473.].

Polymorphisms in the Chicken Growth Differentiation Factor 9 Gene Associated with Reproductive Traits.

The aim of the study was to investigate GDF9 gene polymorphisms and their association with reproductive traits in chicken using DNA sequencing. A total of 279 Dongxiang blue-shelled (DX) chickens and 232 Luhua (LH) chickens were used for validation. We detected 15 single nucleotide polymorphisms (SNPs): nine SNPs were previously unreported in chicken, two were missense mutations, and only three exhibited significant associations with reproductive traits. G.17156387C>T was significantly associated with age at first egg (AFE) and weight of first egg (WFE) in both breeds. Birds carrying the CC genotype exhibited higher AFE and WFE values than those with the TT genotype. The SNP g.17156427A>G exhibited an association with egg weight at 300 days of age (EWTA) in DX but not in LH chickens. The SNP g.17156703A>C affected the AFE and EN (total number of eggs at 300 days of age) in DX chickens. In addition, certain diplotypes significantly affected AFE, BWTA (body weight at 300 days of age), and EN in both breeds. RT-PCR results showed that the GDF9 gene was highly expressed in stroma with cortical follicles (STR) and prehierarchal follicles. These results provided further evidence that the GDF9 gene is involved in determining reproductive traits in chicken.

Whole-transcriptome analysis of atrophic ovaries in broody chickens reveals regulatory pathways associated with proliferation and apoptosis.

Broodiness in laying hens results in atrophy of the ovary and consequently decreases productivity. However, the regulatory mechanisms that drive ovary development remain elusive. Thus, we collected atrophic ovaries (AO) from 380-day-old broody chickens (BC) and normal ovaries (NO) from even-aged egg-laying hens (EH) for RNA sequencing. We identified 3,480 protein-coding transcripts that were differentially expressed (DE), including 1,719 that were down-regulated and 1,761 that were up-regulated in AO. There were 959 lncRNA transcripts that were DE, including 56 that were down-regulated and 903 that were up-regulated. Among the116 miRNAs that were DE, 79 were down-regulated and 37 were up-regulated in AO. Numerous DE protein-coding transcripts and target genes for miRNAs/lncRNAs were significantly enriched in reproductive processes, cell proliferation, and apoptosis pathways. A miRNA-intersection gene-pathway network was constructed by considering target relationships and correlation of the expression levels between ovary development-related genes and miRNAs. We also constructed a competing endogenous RNA (ceRNA) network by integrating competing relationships between protein-coding genes and lncRNA transcripts, and identified several lncRNA transcripts predicted to regulate the CASP6, CYP1B1, GADD45, MMP2, and SMAS2 genes. In conclusion, we discovered protein-coding genes, miRNAs, and lncRNA transcripts that are candidate regulators of ovary development in broody chickens.

Identification of Three Novel Splicing Variants and Expression Analysis of Chicken GPR1 Gene.

GPR1 is a G protein-coupled receptor that plays critical roles in eukaryotic cells: typically, response to glucose stimulation, lipid accumulation, and transmitting nutrition signals to cAMP pathway. However, the alternative splicing of the GPR1 gene and its expression pattern in chicken tissues and ovarian follicles were unknown. In our current study, we used RACE-PCR to identify three GPR1 variants, including the full-length variant (GPR1-va1) and two alternatively spliced variants (GPR1-va2, GPR1-vb). Quantitative real-time PCR examined the expression pattern of GPR1 mRNA in chicken tissues and ovarian follicles. The result reveals that the coding sequence of the three variants cDNA is 1053, 1053, and 627 bp in length, encoding 350, 350, and 208 amino acids, respectively. The three variants of GPR1 show similar tissue distributions; GPR1 expression was abundant in the abdominal fat, lung, and heart. With the follicular development, the expression of GPR1 gene gradually increased, and GPR1-va1 and GPR1-va2 spliced variants expression in F2 were significantly higher than in F5, F4, and prehierarchical follicles (P < 0.05). Taken together, we found three novel variants of GPR1, and the results of GPR1 expression profiling in adipose tissues and ovarian follicles suggest that GPR1 may play a significant role in the lipid accumulation and progression of follicular development.

Feasibility study on expanded indication for endoscopic submucosal dissection of intramucosal poorly differentiated early gastric cancer.

AIM: To identify clinicopathological factors predictive of lymph node metastasis (LNM) in intramucosal poorly differentiated early gastric cancer (EGC), and further to expand the possibility of using endoscopic submucosal dissection (ESD) for the treatment of intramucosal poorly differentiated EGC. METHODS: Data for 81 surgically treated patients with intramucosal poorly differentiated EGC were collected, and the association between the clinicopathological factors and the presence of LNM was retrospectively analyzed by univariate and multivariate logistic regression analyses. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Several clinicopathologic factors were investigated to identify predictive factors for lymph nodes metastasis, including gender, age, family history of gastric cancer, number of tumors, tumor location, ulceration, tumor size, macroscopic type, lymphatic vessel involvement, and signet-ring-cell component. RESULTS: Tumor size (OR = 7.273, 95%CI: 1.246-29.918, P = 0.042), lymphatic vessel involvement (OR = 42.219, 95%CI: 1.923-97.052, P = 0.018) and signet-ring-cell component (OR = 17.513, 95%CI: 1.647-77.469, P = 0.034) that were significantly associated with LNM by univariate analysis, were found to be significant and independent risk factors for LNM by multivariate analysis. However, gender, age, family history of gastric cancer, number, location, ulceration and macroscopic type of tumor were found not to be associated with LNM. Of these 81 patients diagnosed with intramucosal poorly differentiated EGC, 7 (8.6%) had LNM. The LNM rates were 9.1%, 22.2% and 57.1%, respectively, in cases with one, two and three of the risk factors. There was no LNM in 54 patients without the three risk clinicopathological factors. CONCLUSION: Tumor size, lymphatic vessel involvement and signet-ring-cell component are independently associated with the presence of LNM in intramucosal poorly differentiated EGC. Thus, these three risk factors may be used as a simple criterion to expand the possibility of using ESD for the treatment of intramucosal poorly differentiated EGC.

Effect of Monochromatic Light on Expression of Estrogen Receptor (ER) and Progesterone Receptor (PR) in Ovarian Follicles of Chicken.

Artificial illumination is widely used in modern poultry houses and different wavelengths of light affect poultry production and behaviour. In this study, we measure mRNA and protein abundance of estrogen receptors (ERs) and progesterone receptors (PRs) in order to investigate the effect of monochromatic light on egg production traits and gonadal hormone function in chicken ovarian follicles. Five hundred and fifty-two 19-wk-old laying hens were exposed to three monochromatic lights: red (RL; 660 nm), green (GL; 560 nm), blue (BL; 480 nm) and control cool white (400-760 nm) light with an LED (light-emitting diode). There were 4 identical light-controlled rooms (n = 138) each containing 3 replicate pens (46 birds per pen). Water was supplied ad libitum and daily rations were determined according to the nutrient suggestions for poultry. Results showed that under BL conditions there was an increase in the total number of eggs at 300 days of age and egg-laying rate during the peak laying period. The BL and GL extended the duration of the peak laying period. Plasma melatonin was lowest in birds reared under BL. Plasma estradiol was elevated in the GL-exposed laying hens, and GL and BL increased progesterone at 28 wk of age. In the granulosa layers of the fifth largest preovulatory follicle (F5), the third largest preovulatory follicle (F3) and the largest preovulatory follicle (F1), ERα mRNA was increased by BL and GL. Treatment with BL increased ERß mRNA in granulosa layers of F5, F3 and F1, while GL increased ERß mRNA in F5 and F3. There was a corresponding increase in abundance of the proteins in the granulosa layers of F5, with an increase in PR-B, generated via an alternative splice site, relative to PR-A. Treatment with BL also increased expression of PR mRNA in all of the granulosa layers of follicles, while treatment with GL increased expression of PR mRNA in granulosa layers of SYF(small yellow follicle), F5 and F1. These results indicate that blue and green monochromatic lights promote egg production traits via stimulating gonadal hormone secretion and up-regulating expression of ERs and PRs. Changes in PR-B protein suggest that this form of the progesterone receptor is predominant for progesterone action in the granulosa layers of preovulatory follicles in chickens during light stimulation.

Leptin exerts proliferative and anti-apoptotic effects on goose granulosa cells through the PI3K/Akt/mTOR signaling pathway.

Leptin was known as a pivotal regulator for the control of food intake and energy expenditure. However, leptin has also been found to be involved in the regulation of female reproductive system through interactions with pathways in the hypothalamic-hypophyseal axis and direct action at the ovarian level. In the present study, granulosa cells from goose ovarian preovulatory (F1-F3) follicles were cultured with leptin (0, 1, 10 or 100ng/ml). The proliferative and anti-apoptotic actions of leptin in granulosa cells were revealed by CCK-8, BrdU and TUNEL assays. Quantitative real-time PCR and Western blot analyses further indicated that leptin treatment led to increased expression of cyclin D1, cyclin D2, cyclin D3 and bcl-2, and decreased expression of p21 and caspase-3. The effects were involved in the activation of the PI3K/Akt/mTOR signaling pathway, as leptin treatment enhanced the expression of PI3K, Akt1, Akt2, Raptor, mTOR, S6K and p-S6K. Moreover, blockade of the PI3K/Akt/mTOR pathway attenuated the influences of leptin on proliferation and apoptosis of granulosa cells, considering that activated factors by leptin were inhibited in the presence of either 20µM LY294002 (a PI3K inhibitor) or 10µM rapamycin (an mTOR inhibitor). In addition, leptin had a modulatory effect on the expression of its receptor at the transcriptional and translational levels, and blockade of PI3K/Akt/mTOR inhibited both basal and leptin-induced Lepr gene and protein expression. These findings suggest that leptin exerts its proliferative and anti-apoptotic effects on goose granulosa cells through the PI3K/Akt/mTOR signaling pathway via interaction with its receptor.

Role of leptin in the regulation of sterol/steroid biosynthesis in goose granulosa cells.

Leptin is critical for reproductive endocrinology. The aim of this study is to assess the expression patterns of leptin receptor (Lepr) during ovarian follicle development and to reveal the mechanism by which leptin affects steroid hormone secretion in goose granulosa cells. Transcripts of Lepr were ubiquitous in all tested tissues, with pituitary and adrenal glands being the predominant sites. Goose ovarian follicles were divided into several groups by diameter including prehierarchical (4 to 6, 6 to 8, and 8 to 10 mm) and hierarchical (F5-F1) follicles. Lepr gene expression was significantly higher in granulosa cells than in theca cells from follicles of 4 to 8 mm in diameter. Expression of Lepr in granulosa cells decreased gradually as follicles developed, with fluctuating expression in F5 and F3 follicles. Lepr mRNA in theca cells underwent a slight decrease from the 6- to 8-mm cohorts to F5 follicle and then exhibited a transient increase and declined later. In vitro experiments in cultured goose granulosa cells showed that estradiol release was significantly stimulated, whereas progesterone increased slightly and testosterone decreased dramatically after leptin treatment. In accordance with the data for steroids, expression of Lepr, Srebp1, Cyp51, StAR, and Cyp19a1 were induced by the addition of leptin, and the concomitant changes in Hmgcs1, Dhcr24, Cyp11a1, 17ß-hsd, Cyp17, and 3ß-hsd gene expression were seen. These results suggested that leptin is involved in the development of goose ovarian follicles, and leptin's effect on steroid hormone secretion could be due to altered sterol/steroidogenic gene expression via interaction with its receptor.

Five novel variants of GPR103 and their expression in different tissues of goose (Anser cygnoides).

GPR103 plays an important role in various tissues, while little information is available about the alternative splicing (AS) of its mRNA. In the present study, we used genomic PCR to identify the partial genomic locus of goose (Anser cygnoides) GPR103 and rapid amplification of cDNA ends (RACE)-PCR to identify five GPR103 variants, including the full-length variant (aGPR103-n) and four alternatively spliced variants (aGPR103-va, -vb, -vc and -vd). Sequence analysis showed that aGPR103-va and -vd are less likely to undergo nonsense-mediated mRNA decay, suggesting that they may be translated into truncated proteins. Quantitative real-time PCR (qRT-PCR) analysis revealed that the five variants are widely distributed in the brain and peripheral tissues of geese and show specific expression patterns. Thus, we here provide the first account of the GPR103 genomic locus and illustrate its transcriptional diversity and widespread distribution in geese.