Screening of Proliferation-Related Genes and Pathological Changes in Thiram-Induced Tibial Dyschondroplasia.

Materials and Methods: Three hundred sixty (n = 360) broiler chickens were equally divided into control (C) and thiram (T) groups. Furthermore, the C and T groups were dividedinto 8-, 9-, 11-, and 13-day-old chickens. Results: Clinically, it was observed that broiler chickens of group T had abnormal posture, gait, and lameness, and histopathological results revealed dead and abnormal chondrocytes of T group on day 6. Real-time qPCR results showed that HDAC1, MTA1, H4, and PCNA genes were significantly expressed (P < 0.05). HDAC1 was upregulated on days 1, 2, 4, and 6 (P < 0.01); MTA1 was upregulated on days 1 and 2 (P < 0.01); H4 was upregulated on days 2 and 4 (P < 0.01), and PCNA was downregulated on days 1, 2, and 4 (P < 0.01). Furthermore, IHC results of HDAC1 protein were significantly (P < 0.01) expressed in proliferative zone of day 1 and hypertrophic zone of day 6. MTA1 protein was significantly (P < 0.01) expressed on days 1, 2, and 6 in all zones, except prehypertrophic zone of day 2. Conclusion: In conclusion, the mRNA expressions of HDAC1, MTA1, H4, and PCNA were differentially expressed in the chondrocytes of thiram-induced TD chickens. HDAC1 and MTA1 protein expression found involved and responsible in the abnormal chondrocytes' proliferation of broiler chicken.

Transcriptome-based biomarker gene screening and evaluation of the extracellular fatty acid-binding protein (Ex-FABP) on immune and angiogenesis-related genes in chicken erythrocytes of tibial dyschondroplasia.

BACKGROUND: Tibial dyschondroplasia (TD) is a bone disorder in which dead chondrocytes accumulate as a result of apoptosis and non-vascularization in the tibial bone of broiler chickens. The pathogenicity of TD is under extensive research but is yet not fully understood. Several studies have linked it to apoptosis and non-vascularization in the tibial growth plate (GP). We conceived the idea to find the differentially expressed genes (DEGs) in chicken erythrocytes which vary in expression over time using a likelihood-ratio test (LRT). Thiram was used to induce TD in chickens, and then injected Ex-FABP protein at 0, 20, and 50 µg.kg-1 to evaluate its therapeutic effect on 30 screened immunity and angiogenesis-related genes using quantitative PCR (qPCR). The histopathology was also performed in TD chickens to explore the shape, circularity, arrangements of chondrocytes and blood vessels. RESULTS: Clinical lameness was observed in TD chickens, which decreased with the injection of Ex-FABP. Histopathological findings support Ex-FABP as a therapeutic agent for the morphology and vascularization of affected chondrocytes in TD chickens. qPCR results of 10 immunity (TLR2, TLR3, TLR4, TLR5, TLR7, TLR15, IL-7, MyD88, MHCII, and TRAF6) and 20 angiogenesis-related genes (ITGAV, ITGA2, ITGB2, ITGB3, ITGA5, IL1R1, TBXA2R, RPL17, F13A1, CLU, RAC2, RAP1B, GIT1, FYN, IQGAP2, PTCH1, NCOR2, VAV-like, PTPN11, MAML3) regulated when Ex-FABP is injected to TD chickens. CONCLUSION: Immunity and angiogenesis-related genes can be responsible for apoptosis of chondrocytes and vascularization in tibial GP. Injection of Ex-FABP protein to thiram induced TD chickens decrease the chondrocytes damage and improves vascularization.

Identification of genetic variants the CCKAR gene and based on body measurement and carcass quality characteristics in Qinchuan beef cattle (Bos taurus)

BACKGROUND: This study aimed to explore genetic polymorphisms of the CCKAR gene and their relationship with the growth and development of Qinchuan cattle which could be used as molecular markers for the improvement of the breeding of Qinchuan cattle. RESULTS: Here, we have identified seven single nucleotide polymorphisms (SNPs) at loci g. 1463 C>G; g. 1532 T>A; g. 1570 G>A; g. 1594 C>A; g. 1640 T>C; g. 1677 G>C; and g. 1735 C>T in the coding region of the bovine CCKAR gene. The frequencies identified on allelic and genotypic characteristics have shown that all seven SNPs diverged from the Hardy-Weinberg-Equilibrium. The SNP2, SNP3, SNP6 and SNP7 had the lowest polymorphism information content values, and remaining SNPs were found to be moderate (0.25 < PIC < 0.50). The genotype CG in SNP1 at loci g.1463 C>G had the greatest association with WH, HW, CD and CCF, while the genotype TA at the very same loci was associated with BFT, ULA and IMF content in Qinchuan cattle. The CCKAR gene expression level in adipose tissue, small intestine, liver and skeleton muscle was found to be higher, whereas, the expression level of mRNA in organs of other digestive system including reticulum, abomasum and omasum was moderate. Some expression of CCKAR mRNA was found in the large intestine, kidney and rumen. CONCLUSIONS: In summary, our finding suggested that the CCKAR gene could be used as a potential candidate for the improvement of carcass quality and body measurements of Qinchuan cattle.

Janus kinase/signal transducer and activator of transcription signaling pathway-related genes STAT3, SOCS3 and their role in thiram induced tibial dyschondroplasia chickens.

Pathogenicity of tibial dyschondroplasia (TD) in broiler chickens is not detected yet. Janus Kinase/Signal Transducer and Activator of Transcription (JAK-STAT) signaling pathway-related genes were investigated in thiram induced TD chickens. Real-time qPCR and immunohistochemical (IHC) technique were used to observe the expression changes of STAT3 and SOSC3 gene on days 1, 2, 4, 6 after feeding 100 mg·kg-1 thiram. Morphological, pathological, and histological results of this study suggested that chondrocyte cells were observed more damaged on day 6 than day 1, 2, and 4. Therefore, Lameness and damaged chondrocytes gradually increased from day 1 to 6. The mRNA expression level of STAT3 was observed insignificant (P > 0.05) in thiram induced TD chickens' group of day 1. However, on days 2, 4, and 6, the expression was significant (P < 0.05). SOCS3 increased in thiram group on days 1, 2 and 6, decreased on day 4 (P < 0.05). The p-STAT3 and SOCS3 protein's protein localization was evaluated in the control and thiram-induced TD broiler chickens through IHC, suggesting that SOSC3 protein was observed significantly higher on days 1, 2, and 6 and down-regulated on day 4. p-STAT3 protein on thiram induced group was observed significantly upregulated on days 4 and 6. In conclusion, the differential expression of STAT3 and SOCS3 showed that the JAK-STAT signaling pathway might play an important role in regulating an abnormal proliferation, differentiation, or apoptosis of chondrocytes in TD at an early stage.

Cellular, molecular and genetical overview of avian tibial dyschondroplasia.

Tibial dyschondroplasia (TD) is an intractable avian bone disease that causes severe poultry economic losses. The pathogenicity of TD is unknown. Therefore, TD disease has not been evacuated yet. Based on continuous research findings, we have gone through the molecular and cellular insight into the TD and proposed possible pathogenicity for future studies. Immunity and angiogenesis-related genes expressed in the erythrocytes of chicken, influenced the apoptosis of chicken chondrocytes to cause TD. TD could be defined as the irregular, unmineralized and un-vascularized mass of cartilage, which is caused by apoptosis, degeneration and insufficient blood supply at the site of the chicken growth plate. The failure of angiogenesis attributed improper nutrients supply to the chondrocytes; ultimately, bone development stopped, poor calcification of cartilage matrix, and apoptosis of chondrocytes occurred. Recent studies explore potential signaling pathways that regulated TD in broiler chickens, including parathyroid hormone-related peptide (PTHrP), transforming growth factor ß (TGF- ß)/bone morphogenic proteins (BMPs), and hypoxia-inducible factor (HIF). Several studies have reported many medicines to treat TD. However, recently, rGSTA3 protein (50 µg·kg-1) is considered the most proper TD treatment. The present review has summarized the molecular and cellular insight into the TD, which will help researchers in medicine development to evacuate TD completely.

Screening of toll-like receptor signaling pathway-related genes and the response of recombinant glutathione S-transferase A3 protein to thiram induced apoptosis in chicken erythrocytes.

The expression of genes related to the Toll-like receptors (TLRs) signaling pathway were determined. Group A, B and C fed with basal diet and group D, E and F induced TD by feeding a basal diet containing 100 mg·kg-1 thiram. rGSTA3 protein was injected at 20 µg·kg-1 in group B, E and at 50 µg·kg-1 in C, F. Results suggested that lameness and death of chondrocytes were significant on day 14. TLRs signaling pathway related genes were screened based on the transcriptome enrichment, and validated on qPCR. IL-7, TLR2, 3, 4, 5, 7, 15, MyD88, MHC-II, MDA5 and TRAF6 were significantly (p < 0.05) expressed in group E and F as compared to group D on day 14 and 23. IL-7, MHCII, TRAF6, TLR3, TLR5, TLR7, and TLR15 determined insignificant in group D compared to group A on day 23. TD occur in an early phase and alleviated in the later period. rGSTA3 protein can prevent apoptosis and repair degraded chondrocytes.

Integration of gene expression profile data to screen and verify immune-related genes of chicken erythrocytes involved in Marek's disease virus.

Chicken erythrocytes participated in immunity, but the role of erythrocytes in the immunity of Marek's disease virus (MDV) has not been reported related to the immunity genes. The purpose of this study was to screen and verify the immune-related genes of chicken erythrocytes which could be proven as a biomarker in MDV. The datasets (GPL8764-Chicken Gene Expression Microarray) were downloaded from the GEO profile database for control and MDV infected chickens to obtain differentially expressed genes (DEGs) through bioinformatics methods. Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed to find enriched pathways, including Gene Ontology (GO). Based on enriched pathways, the top 19 immune-related genes were screened-out and process further to construct the protein-protein interaction (PPI) networks. The screened genes were validated on RT-PCR and qPCR. Results suggested that the mRNA transcription of Toll-like receptors 2, 3, 4, 6 (TLR2, TLR3, TLR4, TLR6), major histocompatibility complex-II (MHCII), interleukin-7 (IL-7), interferon-ßeta (IFN-ß), chicken myelomonocytic growth factor (cMGF) and myeloid differentiation primary response 88 (MyD88) were significantly up-regulated. The expression of toll-like receptor 5, 7 (TLR5, TLR7) interleukin-12 (IL-12 p40), interleukin-13 (IL-13), and interferon-αlpha (IFN-α) were significantly down-regulated in the erythrocytes of the infected group (P < 0.05). In contrast, the expression of toll-like receptor-1, 15, 21 (TLR1, TLR15, TLR21), major histocompatibility complex I (MHCI) and Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) were not significant. In conclusion, it has been verified on qRT-PCR results that 19 immune-related genes, which included TLRs, cytokines and MHC have immune functions in MDV infected chickens.

Recombinant glutathione-S-transferase A3 protein regulates the angiogenesis-related genes of erythrocytes in thiram induced tibial lesions.

Tibial dyschondroplasia (TD) is a skeletal deformity disease in broilers that occurs when vascularization in the growth plate (GP) is below normal. Although, blood vessels have been reported to contribute significantly in bone formation. Therefore, in the current study, we have examined the mRNA expression of angiogenesis-related genes in erythrocytes of thiram induced TD chickens by qRT-PCR and performed histopathological analysis to determine regulatory effect of recombinant Glutathione-S-Transferase A3 (rGSTA3) protein in response to the destructive effect of thiram following the injection of rGSTA3 protein. Histopathology results suggested that, blood vessels of GPs were damaged in thiram induced TD chicken group (D), it also affected the area and density of blood vessels. In the 20 and 50 µg·kg-1 of rGSTA3 protein-administered groups, E and F vessels appeared to be normal and improved on day 6 and 15. Furthermore, qRT-PCR results showed that rGSTA3 protein significantly (P < .05) up-regulated the expression of the most important angiogenesis-related integrin family genes ITGA2, ITGA5, ITGB2, ITGB3, ITGAV. The expression level of other genes including TBXA2R, FYN, IQGAP2, IL1R1, GIT1, RAP1B, RPL17, RAC2, MAML3, PTPN11, VAV1, PTCH1, NCOR2, CLU and ITGB3 up-regulated on dosage of rGSTA3 protein. In conclusion, angiogenesis is destroyed in thiram induced TD broilers, and rGSTA3 protein injection improved the vascularization of GPs by upregulating the angiogenesis related genes most importantly integrin family genes ITGAV, ITGA2, ITGB2, ITGB3, ITGA5.

Transcriptome-based screening of intracellular pathways and angiogenesis related genes at different stages of thiram induced tibial lesions in broiler chickens.

BACKGROUND: The Tibial dyschondroplasia (TD) in fast-growing chickens is mainly caused by improper blood circulation. The exact mechanism underlying angiogenesis and vascularization in tibial growth plate of broiler chickens remains unclear. Therefore, this research attempts to study genes involved in the regulation of angiogenesis in chicken red blood cells. Twenty-four broiler chickens were allotted into a control and thiram (Tetramethyl thiuram disulfide) group. Blood samples were collected on day 2, 6 (8- and 14-days old chickens) and 15 (23 days old chickens). RESULTS: Histopathology and hematoxylin and eosin (H&E) results showed that angiogenesis decreased on the 6th day of the experiment but started to recover on the 15th day of the experiment. Immunohistochemistry (IHC) results confirmed the expressions of integrin alpha-v precursor (ITGAV) and clusterin precursor (CLU). Transcriptome sequencing analysis evaluated 293 differentially expressed genes (DEGs), of which 103 up-regulated genes and 190 down-regulated genes were enriched in the pathways of neuroactive ligand receptor interaction, mitogen-activated protein kinase (MAPK), ribosome, regulation of actin cytoskeleton, focal adhesion, natural killer cell mediated cytotoxicity and the notch signalling pathways. DEGs (n = 20) related to angiogenesis of chicken erythrocytes in the enriched pathways were thromboxane A2 receptor (TBXA2R), interleukin-1 receptor type 1 precursor (IL1R1), ribosomal protein L17 (RPL17), integrin beta-3 precursor (ITGB3), ITGAV, integrin beta-2 precursor (ITGB2), ras-related C3 botulinum toxin substrate 2 (RAC2), integrin alpha-2 (ITGA2), IQ motif containing GTPase activating protein 2 (IQGAP2), ARF GTPase-activating protein (GIT1), proto-oncogene vav (VAV1), integrin alpha-IIb-like (ITGA5), ras-related protein Rap-1b precursor (RAP1B), tyrosine protein kinase Fyn-like (FYN), tyrosine-protein phosphatase non-receptor type 11 (PTPN11), protein patched homolog 1 (PTCH1), nuclear receptor corepressor 2 (NCOR2) and mastermind like protein 3 (MAML3) selected for further confirmation with qPCR. However, commonly DEGs were sarcoplasmic/endoplasmic reticulum calcium ATPase 3 (ATP2A3), ubiquitin-conjugating enzyme E2 R2 (UBE2R2), centriole cilia and spindle-associated protein (CCSAP), coagulation factor XIII A chain protein (F13A1), shroom 2 isoform X6 (SHROOM2), ras GTPase-activating protein 3 (RASA3) and CLU. CONCLUSION: We have found potential therapeutic genes concerned to erythrocytes and blood regulation, which regulated the angiogenesis in thiram induced TD chickens. This study also revealed the potential functions of erythrocytes. 1. Tibial dyschondroplasia (TD) in chickens were more on day 6, which started recovering on day 15. 2. The enriched pathway observed in TD chickens on day 6 was ribosome pathway, on day 15 were regulation of actin cytoskeleton and focal adhesion pathway. 3. The genes involved in the ribosome pathways was ribosomal protein L17 (RPL17). regulation of actin cytoskeleton pathway were Ras-related C3 botulinum toxin substrate 2 (RAC2), Ras-related protein Rap-1b precursor (RAP1B), ARF GTPase-activating protein (GIT1), IQ motif containing GTPase activating protein 2 (IQGAP2), Integrin alpha-v precursor (ITGAV), Integrin alpha-2 (ITGA2), Integrin beta-2 precursor (ITGB2), Integrin beta-3 precursor (ITGB3), Integrin alpha-IIb-like (ITGA5). Focal adhesion Proto-oncogene vav (Vav-like), Tyrosine-protein kinase Fyn-like (FYN).

Transcriptome analysis of MAPK signaling pathway and associated genes to angiogenesis in chicken erythrocytes on response to thiram-induced tibial lesions.

This study was planned to investigate TD (Tibial dyschondroplasia) on the potential MAPK signaling pathway and angiogenesis related genes. Forty-eight broilers were allotted into control (C) and treatment (T) groups of 2, 6 and 15 days as C1, C2, C3, T1, T2 and T3. The histopathology results revealed that tibiotarsus bone of chickens had more lesions on day 6 (T2 group). The chondrocytes were disordered, and the size, shape and proliferation were affected. Transcriptome results revealed that differentially expressed genes (DEGs) identified were 63, 1026, 623, 130, 141 and 146 in C1 (2 days control vs 6 days control); C2 (2 days control vs 15 days control); C3 (6 days control vs 15 days control); T1 (2 days treatment vs 6 days treatment); T2 (2 days treatment vs 15 days treatment) and T3 (6 days treatment vs 15 days treatment) groups respectively. Whereas, 10 angiogenesis related-genes RHOC, MEIS2, BAIAP2, TGFBI, KLF2, CYR61, PTPN11, PLXNC1, HSPH1 and NRP2 were downregulated on day 6 in the treatment group. The pathway which was found enriched in the control and treatment groups was MAPK signaling pathway. Therefore selected 10 MAPK signaling pathway-related genes RAC2, MAP3K1, PRKCB, FLNB, IL1R1, PTPN7, RPS6KA, MAP3K6, GNA12 and HSPA8 which were found significantly downregulated in the treatment group on day 6. It is concluded that angiogenesis and MAPK signaling pathway related genes has an essential role in TD, as those top screened genes found downregulated in the thiram fed chickens when TD observed severed on day 6.

Recombinant porcine NK-lysin inhibits the invasion of hepatocellular carcinoma cells in vitro.

The therapeutics having ability to target cancer cells specifically and exhibit nominal cytopathic effect on normal healthy cells are highly significant for cancer therapeutic applications. Recombinant porcine natural killer lysin (rpNK-lysin) has proven cationic anti-bacterial and anti-tumor peptide. Herein, we report its anti-invasion and anti-metastasis effects on hepatocellular carcinoma (HCC) cells in vitro. We first investigate the maximum non-toxic concentration (MNTC) of rpNK-lysin for the normal hepato cells (L-02). Using MNTC rpNK-lysin, we explore anti-proliferative, anti-adhesive, anti-invasive and anti-metastatic effect of rpNK-lysin on three different HCC cells lines (SMMC-7721, 97-H and HepG2) through MTT, wound-healing, adhesion and invasion assay along with mRNA and protein expression. The results reveal that rpNK-lysin has potential to specifically inhibit HCC cells growth in a dose and time-dependent manner with a little cytopathic effect on the L-02 cells, effectively reduce migration, adhesion and invasion ability of HCC cells. rpNK-lysin significantly reduce Fascin1 expression, which subsequently decrease ß-catenin expression and metaloproteinases (MMP-2 and MMP9). This study suggest that MNTC rpNK-lysin has an anti-invasion and anti-metastasis effect on HCC cells in vitro through inhibition of Fascin 1 expression which regulates Wnt/ß-catenin signaling pathway by inducing ß-catenin degradation and subsequently results in suppression of MMP-2 and MMP9 expression.

Protective effects of zinc and N-acetyl-L-cysteine supplementation against cadmium induced erythrocyte cytotoxicity in Arbor Acres broiler chickens (Gallus gallus domesticus).

Cadmium (Cd) is one of the most toxic metals released into the environment. Here, we investigated the protective role of Zn2+ and/or N-acetyl-L-cysteine (NAC) against Cd cytotoxicity in the erythrocytes of Arbor Acres (AA) broiler chickens. Four hundred one-day-old AA chickens were divided into 12 groups for in vitro and in vivo studies. Zn2+ and/or NAC was given to the Cd exposed AA chickens to assess their protective roles. This was accomplished by investigating nuclear morphological abnormalities, oxidative stress (SOD, CAT, GPx, GSH and T-AOC), cell apoptosis, ROS accumulation and mitochondrial membrane potential (MMP). Results showed that Cd led to dose- and time-dependent cytotoxicity in the erythrocytes of AA chickens characterized by morphological abnormalities, nucleus damage, increased apoptosis rate and antioxidants depletion. Zn2+ or NAC significantly decreased the erythrocyte apoptosis, ROS production and mitochondrial membrane depolarization caused by Cd. SOD, CAT, GPx, GSH and T-AOC activities significantly decreased both in serum and erythrocytes of Cd exposed AA chickens. The supplementation with Zn2+ or NAC alleviated Cd induced oxidative stress through promoting SOD or GPx/GSH activities respectively. NAC presented a better role in reducing apoptosis, improving antioxidant activities more than Zn2+ in vitro. The combined use of Zn2+ and NAC enhanced cytoprotection in Cd exposed erythrocytes of AA chickens compared to Zn2+ or NAC alone. In conclusion, Zn2+ and NAC exerted remarkable protective roles in Cd exposed erythrocytes of AA chickens by inhibiting cell apoptosis and oxidative stress, and this provides a promising approach to antagonize Cd poisoning in poultry.