The Combined Anti-Aging Effect of Hydrolyzed Collagen Oligopeptides and Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells on Human Skin Fibroblasts.

Stem cell-derived exosomes (SC-Exos) are used as a source of regenerative medicine, but certain limitations hinder their uses. The effect of hydrolyzed collagen oligopeptides (HCOPs), a functional ingredient of SC-Exos is not widely known to the general public. We herein evaluated the combined anti-aging effects of HCOPs and exosomes derived from human umbilical cord mesenchymal stem cells (HucMSC-Exos) using a senescence model established on human skin fibroblasts (HSFs). This study discovered that cells treated with HucMSC-Exos + HCOPs enhanced their proliferative and migratory capabilities; reduced both reactive oxygen species production and senescence-associated ß-galactosidase activity; augmented type I and type III collagen expression; attenuated the expression of matrix-degrading metalloproteinases (MMP-1, MMP-3, and MMP-9), interleukin 1 beta (IL-1ß), and tumor necrosis factor-alpha (TNF-α); and decreased the expression of p16, p21, and p53 as compared with the cells treated with HucMSC-Exos or HCOPs alone. These results suggest a possible strategy for enhancing the skin anti-aging ability of HucMSC-Exos with HCOPs.

Expression patterns and correlation analyses of muscle-specific genes in the process of sheep myoblast differentiation.

The purpose of this study was to establish a system for the isolation, culture, and differentiation of sheep myoblasts, and to explore the expression patterns as well as mutual relationships of muscle-specific genes. Sheep fetal myoblasts (SFMs) were isolated by two-step enzymatic digestion, purified by differential adhesion and identified using immunofluorescence techniques. Two percent horse serum was used to induce differentiation in SFMs. Real-time quantitative and Western blot analyses were respectively used to detect the mRNA and protein expressions of muscle-specific genes including MyoD, MyoG, Myf5, Myf6, PAX3, PAX7, myomaker, desmin, MYH1, MYH2, MYH4, MYH7, and MSTN during the differentiation of SFMs. Finally, the correlation between muscle-specific genes was analyzed by the Pearson correlation coefficient method. The results showed that the isolated and purified SFMs could form myotubes after the induction for differentiation. The marker factors including MyoD, MyoG, myomaker, desmin, and MyHC were positively stained in SFMs. The mRNA expressions of MyoD, MyoG, and myomaker increased and then decreased, while Myf5, PAX3, and PAX7 decreased; Myf6, desmin, MYH1, MYH2, MYH4, and MYH7 increased; and MSTN fluctuated up and down during the differentiation of SFMs. The expression patterns of protein were basically consistent with those of mRNA except MSTN. There existed significant or highly significant correlations at mRNA or protein level among some genes. Some transcription factor proteins (MyoD, Myf5, Myf6, PAX3, PAX7) showed significant or highly significant correlations with the mRNA level of some other genes and/or themselves. In conclusion, SFMs with good myogenic differentiation ability were successfully isolated, and the expression patterns and correlations of muscle-specific genes during SFM differentiation were revealed, which laid an important foundation for elucidating the gene regulation mechanism of sheep myogenesis.

Iron and zinc micronutrients and soil inoculation of Trichoderma harzianum enhance wheat grain quality and yield.

Malnutrition is mainly caused by iron and zinc micronutrient deficiencies affecting about half of the world's population across the globe. Biofortification of staple crops is the right approach to overcome malnutrition and enhance nutrient contents in the daily food of humans. This study aimed to evaluate the role of foliar application of iron and zinc in Trichoderma harzianum treated soil on various growth characteristics, quality, and yield of wheat varieties. Plants were examined in the absence/presence of T. harzianum, and iron and zinc micronutrients in both optimal and high-stress conditions. Although the symbiotic association of T. harzianum and common wheat is utilized as an effective approach for wheat improvement because of the dynamic growth promoting the ability of the fungus, this association was found tremendously effective in the presence of foliar feeding of micronutrients for the enhancement of various growth parameters and quality of wheat. The utilization of this approach positively increased various growth parameters including spike length, grain mass, biomass, harvest index, and photosynthetic pigments. The beneficial role of T. harzianum in combination with zinc and iron in stimulating plant growth and its positive impact on the intensities of high molecular weight glutenin subunits (HMW-GS) alleles make it an interesting approach for application in eco-friendly agricultural systems. Further, this study suggests a possible alternative way that does not merely enhances the wheat yield but also its quality through proper biofortification of iron and zinc to fulfill the daily needs of micronutrients in staple food.

4,7-Didehydro-neophysalin B Protects Rat Lung Epithelial Cells against Hydrogen Peroxide-Induced Oxidative Damage through Nrf2-Mediated Signaling Pathway.

The administration of 4,7-didehydro-neophysalin B is expected to be a promising strategy for mitigating oxidative stress in respiratory diseases. This study was aimed at investigating the efficacy of 4,7-didehydro-neophysalin B for apoptosis resistance of rat lung epithelial cells (RLE-6TN) to oxidative stress and evaluating its underlying mechanism of action. The RLE-6TN cells treated with hydrogen peroxide (H2O2) were divided into five groups, and 4,7-didehydro-neophysalin B was administered into it. To evaluate its mechanism of action, the expression of oxidative stress and apoptotic proteins was investigated. 4,7-Didehydro-neophysalin B significantly inhibited H2O2-induced RLE-6TN cell damage. It also activated the Nrf2 signaling pathway which was evident from the increased transcription of antioxidant responsive of KLF9, NQO1, Keap-1, and HO-1. Nrf2 was found to be a potential target of 4,7-didehydro-neophysalin B. The protein levels of Bcl-2 and Bcl-xL were increased while Bax and p53 were decreased significantly. Flow cytometry showed that 4,7-didehydro-neophysalin B protected RLE-6TN cells from apoptosis and has improved the oxidative damage. This study provided a promising evidence that 4,7-didehydro-neophysalin B can be a therapeutic option for oxidative stress in respiratory diseases.

A novel strategy for optimal component formula of anti-PRRSV from natural compounds using tandem mass tag labeled proteomic analyses.

BACKGROUND: Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most important porcine viral diseases which have been threatening the pig industry in China. At present, most commercial vaccines fail to provide complete protection because of highly genetic diversity of PRRSV strains. This study aimed to optimize a component formula from traditional Chinese medicine(TCM)compounds with defined chemical characteristics and clear mechanism of action against PRRSV. METHODS: A total of 13 natural compounds were screened for the anti-PRRSV activity using porcine alveolar macrophages (PAMs). Three compounds with strong anti-PRRSV activity were selected to identify their potential protein targets by proteomic analysis. The optimal compound formula was determined by orthogonal design based on the results of proteomics. MTT assay was used to determine the maximum non-cytotoxic concentration (MNTC) of each compound using PAMs. QPCR and western blot were used to investigate the PRRSV N gene and protein expression, respectively. The Tandem Mass Tag (TMT) technique of relative quantitative proteomics was used to detect the differential protein expression of PAMs treated with PRRSV, matrine (MT), glycyrrhizic acid (GA) and tea saponin (TS), respectively. The three concentrations of these compounds with anti-PRRSV activity were used for orthogonal design. Four formulas with high safety were screened by MTT assay and their anti-PRRSV effects were evaluated. RESULTS: MT, GA and TS inhibited PRRSV replication in a dose-dependent manner. CCL8, IFIT3, IFIH1 and ISG15 were the top four proteins in expression level change in cells treated with MT, GA or TS. The relative expression of IFIT3, IFIH1, ISG15 and IFN-ß mRNAs were consistent with the results of proteomics. The component formula (0.4 mg/mL MT + 0.25 mg/mL GA + 1.95 µg/mL TS) showed synergistic anti-PRRSV effect. CONCLUSIONS: The component formula possessed anti-PRRSV activity in vitro, in which the optimal dosage on PAMs was 0.4 mg/mL MT + 0.25 mg/mL GA + 1.95 µg/mL TS. Compatibility of the formula was superposition of the same target with GA and TS, while different targets of MT. IFN-ß may be one of the targets of the component formula possessed anti-PRRSV activity.

Ethionine-mediated reduction of S-adenosylmethionine is responsible for the neural tube defects in the developing mouse embryo-mediated m6A modification and is involved in neural tube defects via modulating Wnt/ß-catenin signaling pathway.

Neural tube defects (NTDs) remain one of the most life-threatening birth defects affecting infants. Most patients with NTDs eventually develop lifelong disability, which cause significant morbidity and mortality and seriously reduce the quality of life. Our previous study has found that ethionine inhibits cell viability by disrupting the balance between proliferation and apoptosis, and preventing neural stem cells from differentiating into neurons and astrocytes. However, how ethionine participates in the pathogenesis of neural tube development through N6-methyladenosine (m6A) modification remains unknown. This study aims to investigate METTL3- and ALKBH5-mediated m6A modification function and mechanism in NTDs. Herein, our results demonstrate that SAM play not only a compensatory role, it also leads to changes of m6A modification in neural tube development and regulation. Additionally, these data implicate that METTL3 is enriched in HT-22 cells, and METTL3 knockdown reduces cell proliferation and increases apoptosis through suppressing Wnt/ß-catenin signaling pathway. Significantly, overexpression of ALKBH5 can only inhibit cell proliferation, but cannot promote cell apoptosis. This research reveals an important role of SAM in development of NTDs, providing a good theoretical basis for further research on NTDs. This finding represents a novel epigenetic mechanism underlying that the m6A modification has profound and lasting implications for neural tube development.

miR-222-3p is involved in neural tube closure by directly targeting Ddit4 in RA induced NTDs mouse model.

Previously our results showed miR-222-3p was significantly downregulated in retinoic acid-induced neural tube defect (NTD) mouse model through transcriptome. Down-regulation of miR-222-3p may be a causative biomarker in NTDs. In this study, RNA was extracted from mouse embryos at E8.5, E9.5 and E10.5, and the expression level of miR-222-3p was measured by quantitative real-time PCR analysis. The preliminary mechanism of miR-222-3p in NTDs involved in cell proliferation, apoptosis and migration was investigated in mouse HT-22 cell line. The expression of miR-222-3p was significantly decreased at E8.5, E9.5 and E10.5 developed in mouse embryos which were consistent with our transcriptome sequencing. Suppression of miR-222-3p in HT-22 cells resulted in the inhibition of cell proliferation and migration, cell cycle and apoptosis. Moreover, DNA damage transcript 4 (Ddit4) was identified as a direct and functional target of miR-222-3p. miR-222-3p is negatively regulated by Ddit4. The mutation of binding site of Ddit4 3'UTR abrogated the responsiveness of luciferase reporters to miR-222-3p and showed that Ddit4 expression partially attenuated the function of miR-222-3p. We preliminatively confirmed that low expression of miR-222-3p has reduced the expression of ß-catenin, TCF4 and other related genes in the Wnt/ß-catenin signaling pathway.Collectively, these results demonstrated that miR-222-3p regulates the Wnt/ß-catenin signaling pathway through Ddit4 inhibition in HT-22 cells, resulted in cell proliferation and apoptosis imbalance, and thus led to neural tube defects.

Damage to intestinal barrier integrity in piglets caused by porcine reproductive and respiratory syndrome virus infection.

Porcine reproductive and respiratory syndrome (PRRS) induces respiratory disease and reproductive failure accompanied by gastroenteritis-like symptoms. The mechanism of intestinal barrier injury caused by PRRSV infection in piglets has yet to be investigated. An in vivo PRRSV-induced model was established in 30-day-old piglets by the intramuscular injection of 2 mL of 104 TCID50/mL PRRSV for 15 days. Observations of PRRSV replication and histology were conducted in the lungs and intestine, and goblet cell counts, relative MUC2 mRNA expression, and tight junction protein, proinflammatory cytokine, TLR4, MyD88, IκB and p-IκB expression were measured. PRRSV replicated in the lungs and small intestine, as demonstrated by absolute RT-qPCR quantification, and the PRRSV N protein was detected in the lung interstitium and jejunal mucosa. PRRSV infection induced both lung and gut injury, markedly decreased villus height and the villus to crypt ratio in the small intestine, and obviously increased the number of goblet cells and the relative expression of MUC2 mRNA in the jejunum. PRRSV infection aggravated the morphological depletion of tight junction proteins and increased IL-1ß, IL-6, IL-8 and TNF-α expression by activating the NF-κB signalling pathway in the jejunum. PRRSV infection impaired intestinal integrity by damaging physical and immune barriers in the intestine by inducing inflammation, which may be related to the regulation of the gut-lung axis. This study also provides a new hypothesis regarding the pathogenesis of PRRSV-induced diarrhoea.

Aberrant Gcm1 expression mediates Wnt/ß-catenin pathway activation in folate deficiency involved in neural tube defects.

Wnt signaling plays a major role in early neural development. An aberrant activation in Wnt/ß-catenin pathway causes defective anteroposterior patterning, which results in neural tube closure defects (NTDs). Changes in folate metabolism may participate in early embryo fate determination. We have identified that folate deficiency activated Wnt/ß-catenin pathway by upregulating a chorion-specific transcription factor Gcm1. Specifically, folate deficiency promoted formation of the Gcm1/ß-catenin/T-cell factor (TCF4) complex formation to regulate the Wnt targeted gene transactivation through Wnt-responsive elements. Moreover, the transcription factor Nanog upregulated Gcm1 transcription in mESCs under folate deficiency. Lastly, in NTDs mouse models and low-folate NTDs human brain samples, Gcm1 and Wnt/ß-catenin targeted genes related to neural tube closure are specifically overexpressed. These results indicated that low-folate level promoted Wnt/ß-catenin signaling via activating Gcm1, and thus leaded into aberrant vertebrate neural development.

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.

Effects of Osthole on Progesterone Secretion in Chicken Preovulatory Follicles Granulosa Cells.

Osthole (Ost) is an active constituent of Cnidium monnieri (L.) Cusson which possesses anti-inflammatory and anti-oxidative properties. It also has estrogen-like activity and can stimulate corticosterone secretion. The present study was aimed to check the role of Ost on progesterone (P4) secretion in cultured granulosa cells obtained from hen preovulatory follicles. Different concentrations (5, 2.5, and 1.25 µg/mL) of Ost was added to granulosa cells for 6, 12, 18, and 24 h to investigate the level of progesterone secretions using enzyme linked immunosorbent assay (ELISA). The results showed that progesterone secretion was significantly increased in cells treated with Ost at 2.5 µg/mL. Also, qRT-PCR showed that mRNA expression of steroidogenic acute regulatory protein (StAR) was significantly up-regulated by Ost at 2.5 µg/mL concentration. Cytochrome P450 side-chain cleavage (P450scc) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) was significantly up-regulated by Ost. However, no significant differences were observed for the expression of proliferating cell nuclear antigen (PCNA). The protein expression of StAR, P450scc and 3ß-HSD were significantly up-regulated by Ost treatment. The concentration of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) in cell lysates showed no change with Ost treatment at 2.5 µg/mL by ELISA. An ROS kit showed non-significant difference in the level of reactive oxygen species (ROS). In conclusion, Ost treatment at a concentration of 2.5 µg/mL for 24 h had significantly up-regulated P4 secretion by elevating P450scc, 3ß-HSD and StAR at both gene and protein level in granulosa cells obtained from hen preovulatory follicles.

Excessive apoptosis and ROS induced by ethionine affect neural cell viability and differentiation.

The central nervous system (CNS) diseases are still a major cause of morbidity and mortality throughout the world, which imposes heavy burden on the development of society. Ethionine is a non-proteinogenic amino acid having similar chemical structure and activity to that of methionine, with which it competes. Previous studies have confirmed that ethionine affects various cellular functions by inhibiting the biosynthesis of proteins, RNA, DNA, and phospholipids, or all of them. The relationship of ethionine with some CNS diseases, including neural tube defects, has been investigated recently. However, the detailed effects of ethionine on the nerve cell bioactivities and the underlying mechanisms have not been fully explored. Herein, we systematically investigated the influences of ethionine on the proliferation, differentiation, and apoptosis of neural stem cells (NSCs) and post-mitotic nerve cells. We demonstrated that ethionine inhibited cell viability by disrupting the balance between proliferation and apoptosis, prevented NSCs from differentiating into neurons and astrocytes, and blocked cell progression from G1 to S phase via reducing cyclin D1 function in nerve cells including NSCs, a mouse hippocampal neuron cell line (HT-22), and a mouse brain neuroma cell line (Neuro-2a). We speculated that the inhibitory effect of ethionine on cell viability and differentiation are associated with increased reactive oxygen species production. Our results also supported the concept that ethionine may be an underlying cause of abnormal folate metabolism-induced CNS diseases. Our findings may provide important direction for the application of abnormal folate metabolism-induced CNS diseases in future NSC-based therapies.

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.

Chlorogenic acid rescues zearalenone induced injury to mouse ovarian granulosa cells.

Zearalenone (ZEA), a toxic substance produced by Fusarium fungi, accumulated in cereals grain and animal feed, causes injury to humans and animals. ZEA can induce obvious reproductive toxicity with the ovarian granulosa cells (GCs) as the main target. However, the study on exploring the protective compounds against ZEA-induced mouse primary ovarian GCs damage remains less. In the current study, the protective effect of 20 compounds derived from traditional Chinese medicines (TCMs) on the injury of mouse GCs caused by ZEA were evaluated using MTT assay and the cell morphology. Our results showed that chlorogenic acid (250, 500, and 1000 µg/mL) significantly suppress ZEA-induced GCs death. Western blot analysis suggested chlorogenic acid could rescue the up-regulated apoptosis of GCs induced by ZEA via attenuating the protein expression of cleaved caspase-3, the ratio of Bax/Bcl-2 and cleaved-PARP. Our results provide strong evidence that chlorogenic acid warrants further optimization for more potent and safer compounds for against the ZEA lead toxicity to humans and animals.

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).

miRNA-183∼96∼182 regulates melanogenesis, cell proliferation and migration in B16 cells.

Melanoma is a highly invasive malignant skin tumor having high metastatic rate and poor prognosis. The biology of melanoma is controled by miRNAs. The miRNA-183 cluster, which is composed of miRNA-183∼96∼182 genes, plays an important roles in tumor development. In order to investigate the role and action of miRNA-183 cluster in B16 cells, we overexpressed and knocked down miRNA-183 cluster in B16 cells. Using bioinformatics analysis, we predicted that the key framscript factor of melangenic genes. Microphthalmia-associated transcription factor (MITF) is one of the targets of miRNA-183 cluster. The results of Luciferase activity assays confirmed that MITF was targeted by miRNA-183 cluster. Overexpression and knockdown of miRNA-183 cluster in B16 cells resulted in down and up regulation of MITF expression, respectively at both mRNA and protein levels. Furthmore, overexpression and knockdown of the miRNA-183 cluster in B16 cells decreased and increased the expression of mRNA and protein of melangenic genes tyrosinase (TYR), and tyrosinase-related protein 1 (TYRP1), dopachrome-tautomerase (DCT), as well as the production of melanins and eumelanin production, respectively. On the proliferation and migration pathway, overexpression and knockdown of miRNA-183 cluster increased and decreased, respectively the expression of mRNA and protein of mitogen-activated protein kinase 1 (MEK1), extracellular regulated protein kinases1/2 (ERK1/2) and cAMP-responsive-element binding protein (CREB). These results indicated that miRNA-183 cluster regulated melanogenesis in B16 cells as well as cell proliferation and migration by directly targeting MITF through migration pathway.

Does Cereal crops asymmetrically affect Agriculture gross domestic product in Pakistan? Using NARDL model approach / As colheitas de cereais afetam assimetricamente o produto interno bruto da agricultura no Paquistão? Usando a abordagem do modelo NARDL

ABSTRACT: This study contributes to the extant literature on the nexus among rice, maize and wheat production with agriculture gross domestic product (AGDP) of Pakistan. We use time series data from 1970 to 2017 and employ the Non-linear Autoregressive Distributed Lag (NARDL) model. Short run and long run shocks between the selected variables and result's is checked through the co-integration and nonlinear error correction model.Autoregressive distributed lag bound testing approach for co-integration and to find the relationship between variables Granger causality test is applied.Our results confirm co-integration, positive shocks results show that rice, maize and wheat production have significantly influence on AGDP. The asymmetrically positive shocks of three crops have neutral effect on AGDP. While in symmetric results show the unidirectional effect between rice, maize production with AGDP and wheat production do not have ganger causality with AGDP. Finally, results depict that wheat, maize and rice production significantly contributes to agricultural GDP in the case of Pakistan.

RESUMO: Este estudo contribui para a literatura existente sobre o nexo entre a produção de arroz, milho e trigo com produto interno bruto agrícola (AGDP) do Paquistão. Utilizamos dados de séries temporais de 1970 a 2017 e empregamos o modelo NARDL (Non-linear Autoregressive Distributed Lag). Choques de curto e longo prazo entre as variáveis selecionadas e os resultados são verificados por meio do modelo de co-integração e correção não linear de erros. É aplicada uma abordagem de teste de atraso retardado distribuído autorregressivo para co-integração e para encontrar a relação entre variáveis. Nossos resultados confirmam a co-integração; os resultados de choques positivos mostram que a produção de arroz, milho e trigo influencia significativamente na AGDP. Os choques assimétricos positivos de três culturas têm efeito neutro no AGDP. Enquanto nos resultados simétricos mostram o efeito unidirecional entre o arroz, a produção de milho com AGDP e a produção de trigo não têm causalidade de ganger com AGDP. Finalmente, os resultados mostram que a produção de trigo, milho e arroz contribui significativamente para o PIB agrícola no caso do Paquistão.

The impact of agriculture trade and exchange rate on economic growth of Pakistan: an NARDL and asymmetric analysis approach / O impacto do comércio agrícola e da taxa decâmbio no crescimento econômico do Paquistão: uma abordagem de análise NARDL e assimétrica

ABSTRACT: This study contributes to the extant literature on the nexus among agriculture export, import exchange rate and economic growth in Pakistan. We used annual time series data for 1980-2017 and employ the Non-linear Autoregressive Distributed Lag (NARDL) model. The NARDL testing results affirms asymmetric co-integration among the variables. The study main results show: (i) Co-integration test for long run the positive shocks in export and import have positive significant while exchange rate has positive effect the economic growth. (ii) Co-integration test for short run the positive shocks in import has positive significant and while Export and exchange rate have negative significant effect on economic growth. The symmetrical results show: (i) Export has unidirectional granger causality (ii) Exchange rate has bidirectional granger causality (iii) Import has not ganger causality with economic growth. In addition, the results demonstrated that causality relationship can help out policy maker to design such policies which are useful to economic growth of Pakistan, which could further promote foreign trade to gain the maximum level of economic growth.

RESUMO: Este estudo contribui para a literatura existente sobre o nexo entre exportação agrícola, taxa de câmbio de importação e crescimento econômico no Paquistão. Utilizamos dados de séries temporais anuais para 1980-2017 e empregamos o modelo Não-linear Autoregressive Distributed Lag (NARDL). Os resultados dos testes NARDL afirmam a co-integração assimétrica entre as variáveis. Os principais resultados do estudo mostram: (i) no teste de co-integração de longo prazo, os choques positivos nas exportações e importações têm uma significância positiva, enquanto a taxa de câmbio afeta positivamente o crescimento econômico; (ii) Teste de co-integração para curto prazo, os choques positivos nas importações são positivos significativos e, enquanto a exportação e a taxa de câmbio têm um efeito negativo significativo no crescimento econômico. Os resultados simétricos mostram: (i) a exportação tem causalidade unidirecional sobre o crescimento ; (ii) a taxa de câmbio tem causalidade bidirecional sobre o crescimento ; (iii) a importação não tem causalidade sobre o crescimento econômico. Além disso, os resultados demonstraram que a relação de causalidade pode ajudar o formulador de políticas a elaborar políticas úteis ao crescimento econômico do Paquistão, o que poderia promover ainda mais o comércio exterior para obter o nível máximo de crescimento econômico.

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.