mRNA Expression Abundance Research Articles

Promotion effect on liver tumor progression of microcystin-LR at environmentally relevant levels in female krasV12 transgenic zebrafish

Microcystin-LR (MC-LR) is a kind of natural toxin which exists widely in aquatic environments and has been reported to be hepatotoxic and carcinogenic. At present, the promoting mechanism of MC-LR on hepatocellular carcinoma (HCC) remains largely unexplored. In this study, the hepatocellular promoting effect of MC-LR was described in KrasV12 transgenic zebrafish, a doxycycline (DOX) inducible HCC model. Our results showed that MC-LR could aggravate the progression of HCC at an environmentally relevant concentration (3 μg/L), which was accompanied by the decreased activity and down-regulated transcription level of serine/threonine phosphatase 2A (PP2A). Using TMT labeling quantitative phosphoproteomics, we found that the 1049 phosphopeptides were significantly changed (508 up-regulated and 541 down-regulated) in liver from combined exposure to DOX and 3 μg/L MC-LR group compared to the DOX group. Enriched pathways by KEGG analysis suggested that differentially phosphorylated proteins were mainly related to Wnt signaling pathway. Furthermore, the mRNA expression and protein abundance of β-Catenin in Wnt signaling pathway were significantly up-regulated following exposure to MC-LR. In short, our results suggested that MC-LR significantly inhibited the activity of PP2A, which in turn activated Wnt signaling, eventually resulting in progression of liver tumor in transgenic zebrafish.

Genome-wide Mendelian randomization identifies actionable novel drug targets for psychiatric disorders.

Psychiatric disorders impose tremendous economic burden on society and are leading causes of disability worldwide. However, only limited drugs are available for psychiatric disorders and the efficacy of most currently used drugs is poor for many patients. To identify novel therapeutic targets for psychiatric disorders, we performed genome-wide Mendelian randomization analyses by integrating brain-derived molecular quantitative trait loci (mRNA expression and protein abundance quantitative trait loci) of 1263 actionable proteins (targeted by approved drugs or drugs in clinical phase of development) and genetic findings from large-scale genome-wide association studies (GWASs). Using transcriptome data, we identified 25 potential drug targets for psychiatric disorders, including 12 genes for schizophrenia, 7 for bipolar disorder, 7 for depression, and 1 (TIE1) for attention deficit and hyperactivity. We also identified 10 actionable drug targets by using brain proteome data, including 4 (HLA-DRB1, CAMKK2, P2RX7, and MAPK3) for schizophrenia, 1 (PRKCB) for bipolar disorder, 6 (PSMB4, IMPDH2, SERPINC1, GRIA1, P2RX7 and TAOK3) for depression. Of note, MAPK3 and HLA-DRB1 were supported by both transcriptome and proteome-wide MR analyses, suggesting that these two proteins are promising therapeutic targets for schizophrenia. Our study shows the power of integrating large-scale GWAS findings and transcriptomic and proteomic data in identifying actionable drug targets. Besides, our findings prioritize actionable novel drug targets for development of new therapeutics and provide critical drug-repurposing opportunities for psychiatric disorders.

RNA-sequencing and mass-spectrometry proteomic time-series analysis of T-cell differentiation identified multiple splice variants models that predicted validated protein biomarkers in inflammatory diseases.

Profiling of mRNA expression is an important method to identify biomarkers but complicated by limited correlations between mRNA expression and protein abundance. We hypothesised that these correlations could be improved by mathematical models based on measuring splice variants and time delay in protein translation. We characterised time-series of primary human naïve CD4+ T cells during early T helper type 1 differentiation with RNA-sequencing and mass-spectrometry proteomics. We performed computational time-series analysis in this system and in two other key human and murine immune cell types. Linear mathematical mixed time delayed splice variant models were used to predict protein abundances, and the models were validated using out-of-sample predictions. Lastly, we re-analysed RNA-seq datasets to evaluate biomarker discovery in five T-cell associated diseases, further validating the findings for multiple sclerosis (MS) and asthma. The new models significantly out-performing models not including the usage of multiple splice variants and time delays, as shown in cross-validation tests. Our mathematical models provided more differentially expressed proteins between patients and controls in all five diseases. Moreover, analysis of these proteins in asthma and MS supported their relevance. One marker, sCD27, was validated in MS using two independent cohorts for evaluating response to treatment and disease prognosis. In summary, our splice variant and time delay models substantially improved the prediction of protein abundance from mRNA expression in three different immune cell types. The models provided valuable biomarker candidates, which were further validated in MS and asthma.

Chemosensing of fat digestion by the expression pattern of GPR40, GPR120, CD36 and enteroendocrine profile in sheep

Gastrointestinal tract (GIT) epithelial cells detect nutrients in the lumen via G-protein coupled receptors (GPRs) located in the gut epithelial cells especially in enteroendocrine cells. Dietary free fatty acids (FFA) are the major energy source and also acts as signalling molecules for FFA receptors. Long chain fatty acids (LCFA) activate LCFA receptors, GPR40/FFAR1 and GPR120/FFAR4 which trigger intracellular signalling and release gut hormones or modifies gene expression that facilitate fat digestion and absorption. However, there is a paucity of information on chemosensing of nutrients and digestion in ruminants. Hence, present study was aimed to evaluate chemosensing of fat digestion and absorption by the expression pattern of GPR40, GPR120, chylomicron forming genes, fatty acid translocase (CD36/FAT), microsomal triglyceride transfer protein (MTTP) and apolipoprotein B (APOB) in the various segments of GIT in sheep supplemented with calcium salts of long chain fatty acids (CSLCFAs) along with the secretory patterns of gut peptides cholecystokinin (CCK) and peptide tyrosine tyrosine (PYY). The study was carried out for a period 60 days with eighteen adult ewes of 8–12 months of age and they were divided into three groups with six animals each as group-I, group-II and group-III. All the experimental animals were stall fed with a basal diet and maintained as per animal husbandry standards. Group-II and group-III were supplemented additionally with 3% and 5% CSLCFAs, respectively on dry matter intake. The results from the study indicated that the supplementation of CSLCFAs upregulated (P < 0.05) the relative mRNA expression of GPR40 and GPR120 in the various segments of GIT of sheep in correspondence to level of dietary fat. Abundance of mRNA expression of CD36, MTTP and APOB increased (P < 0.05) in the GIT of sheep in accordance to quantity of LCFAs in the diet where these genes facilitate fatty acid uptake. Feeding of CSLCFAs enhanced (P < 0.05) pre-feeding level of CCK from day 15 onwards, whereas, post-feeding CCK and PYY increased in all the experimental sheep. However, the increase was higher (P < 0.05) in sheep supplemented with CSLCFAs by 10.80 ± 1.45% and 14.25 ± 1.17%, respectively in comparison to group-I. The comprehensive results of the study concluded that feeding of additional CSLCFAs upregulated the expression of GPR40, GPR120, CD36, and chemosensing of LCFAs by these genes triggered the signalling transduction that enhanced CCK and PYY levels to facilitate fat digestion and absorption in accordance with quantity of dietary fat. This was further evident from the significant upregulation of MTTP and APOB in the various segments of GIT supported the high content of dietary fat at cellular fat metabolism in the gut that regulates the fatty acid uptake.

Dietary supplementation of salidroside alleviates liver lipid metabolism disorder and inflammatory response to promote hepatocyte regeneration via PI3K/AKT/Gsk3-β pathway

Fatty liver hemorrhagic syndrome (FLHS) is a chronic hepatic disease which occurs when there is a disorder in lipid metabolism. FLHS is often observed in caged laying hens and characterized by a decrease in egg production and dramatic increase of mortality. Salidroside (SDS) is an herbal drug which has shown numerous pharmacological activities, such as protecting mitochondrial function, attenuating cell apoptosis and inflammation, and promoting antioxidant defense system. We aimed to determine the therapeutic effects of SDS on FLHS in laying hens and investigate the underlying mechanisms through which SDS operates these functions. We constructed oleic acid (OA)-induced fatty liver model in vitro and high-fat diet-induced FLHS of laying hens in vivo. The results indicated that SDS inhibited OA-induced lipid accumulation in chicken primary hepatocytes, increased hepatocyte activity, elevated the mRNA expression of proliferation related genes PCNA, CDK2, and cyclinD1 and increased the protein levels of PCNA and CDK2 (P < 0.05), as well as decreased the cleavage levels of Caspase-9, Caspase-8, and Caspase-3 and apoptosis in hepatocytes (P < 0.05). Moreover, SDS promoted the phosphorylation levels of PDK1, AKT, and Gsk3-β, while inhibited the PI3K inhibitor (P < 0.05). Additionally, we found that high-fat diet-induced FLHS hens had heavier body weight, liver weight, and abdominal fat weight, and severe steatosis in histology, compared with the control group (Con). However, hens fed with SDS maintained lighter body weight, liver weight, and abdominal fat weight, as well as normal liver without hepatic steatosis. In addition, high-fat diet-induced FLHS hens had high levels of serum total cholesterol (TC), triglyceride (TG), alanine transaminase (ALT), and aspartate aminotransferase (AST) compared to the Con group, however, in the Model+SDS group, the levels of TC, TG, ALT, and AST decreased significantly, whereas the level of superoxide dismutase (SOD) increased significantly (P < 0.05). We also found that SDS significantly decreased the mRNA expression abundance of PPARγ, SCD, and FAS in the liver, as well as increased levels of PPARα and MTTP, and decreased the mRNA expression of TNF-α, IL-1β, IL-6, and IL-8 in the Model+SDS group (P < 0.05). In summary, this study showed that 0.3 mg/mL SDS attenuated ROS generation, inhibited lipid accumulation and hepatocyte apoptosis, and promoted hepatocyte proliferation by targeting the PI3K/AKT/Gsk3-β pathway in OA-induced fatty liver model in vitro, and 20 mg/kg SDS alleviated high-fat-diet-induced hepatic steatosis, oxidative stress, and inflammatory response in laying hens in vivo.

Antagonistic regulatory effects of a single cis-acting expression quantitative trait locus between transcription and translation of the MRPL43 gene

BackgroundHeterogeneity of expression quantitative trait locus (eQTL) effects have been shown across gene expression processes. Knowledge on how to produce the heterogeneity is quite limited. This study aims to examine fluctuations in differential gene expression by alleles of sequence variants across expression processes.ResultsGenome-wide eQTL analyses with transcriptome-wide gene expression data revealed 20 cis-acting eQTLs associated simultaneously with mRNA expression, ribosome occupancy, and protein abundance. A 97 kb-long eQTL signal for mitochondrial ribosomal protein L43 (MRPL43) covered the gene, showing a heterogeneous effect size on gene products across expression stages. One allele of the eQTL was associated with increased mRNA expression and ribosome occupancy but decreased protein abundance. We examined the heterogeneity and found that the eQTL can be attributed to the independent functions of three nucleotide variants, with a strong linkage. NC_000010.11:g.100987606G > T, upstream of MRPL43, may regulate the binding affinity of transcription factors. NC_000010.11:g.100986746C > G, 3 bp from an MRPL43 splice donor site, may alter the splice site. NC_000010.11:g.100978794A > G, in the isoform with a long 3′-UTR, may strengthen the binding affinity of the microRNA. Individuals with the TGG haplotype at these three variants had higher levels of mRNA expression and ribosome occupancy than individuals with the GCA haplotype but lower protein levels, producing the flipped effect throughout the expression process.ConclusionsThese findings suggest that multiple functional variants in a linkage exert their regulatory functions at different points in the gene expression process, producing a complexity of single eQTLs.

Upregulated iRhom2 in the Hypothalamic Paraventricular Nucleus is Associated with TACE‐Mediated Shedding of Transforming Growth Factor‐α and Sympathetic Excitation in Heart Failure Rats

Tumor necrosis factor (TNF)‐α converting enzyme (TACE), also known as a disintegrin and metalloprotease (ADAM)17, is a key mediator of cell signaling by proteolytically cleaving extracellular domains of various cytokines and growth factors. TACE‐mediated shedding of transforming growth factor (TGF)‐α, has been shown to transactivate epidermal growth factor receptor (EGFR) to activate the mitogen‐activated protein kinase signaling pathway in the pathophysiological conditions. We previously reported that both TACE and TGF‐α are upregulated in the hypothalamic paraventricular nucleus (PVN, a critical cardiovascular and autonomic center) and contribute to the sympathetic excitation in heart failure (HF). However, the mechanisms by which TACE and TGF‐α are upregulated in the PVN in HF remain unclear. Recent evidence reveals that inactive Rhomboid proteins (iRhoms) including iRhom1 and iRhom2 are essential upstream regulators of TACE/ADAM17. The present study sought to determine the expression of iRhoms and its relationship with TACE‐mediated shedding of TGF‐α in the PVN in HF. Male rats underwent coronary artery ligation to induce HF or sham surgery (Sham). The left ventricular function and infarction size were assessed by echocardiography. Four weeks later, these animals were euthanized to collect cerebrospinal fluid (CSF), PVN tissues and blood for molecular and biochemical measurements. All values are expressed as the mean ± SD. Real‐time PCR showed abundant mRNA expression of both iRhom1 and iRhom2 in the PVN and brain cortex in Sham or HF rats. Compared with Sham rats, HF rats had significantly (*P&lt;0.05) increased mRNA expression of iRhom2 (2.00 ± 0.57* vs 1.03 ± 0.28, HF vs Sham) but not iRhom1 (1.32 ± 0.41 vs 1.02 ± 0.21) in the PVN. Additionally, HF rats exhibited marked increases in mRNA expression of TACE (2.25 ± 1.15* vs 1.04 ± 0.36) in the PVN and the plasma level of norepinephrine (NE, a marker of sympathetic excitation, 12.39 ± 4.96* vs 5.82 ± 2.76 ng/mL), along with elevated levels of TGF‐α (23.54 ± 6.25* vs 10.08 ± 4.38 pg/mL) in CSF. The levels of epidermal growth factor (9.88 ± 3.59 vs 8.15 ± 4.24 pg/mL) in the CSF did not alter significantly in HF compared with Sham rats. Moreover, the linear regression analysis indicated that the mRNA expression of iRhom2 in the PVN was positively correlated with TACE level in the PVN (R2 = 0.62, P &lt;0.01), TGF‐α level in the CSF (R2 = 0.64, P &lt;0.01) and NE level in the plasma (R2 = 0.63, P &lt;0.01). These results suggest that the upregulated expression of iRhom2 in the PVN may promote ectodomain shedding of TGF‐α by enhancing TACE expression to drive sympathetic activation in HF. Brain iRhom2 is a potential target for therapeutic intervention in HF.

Hypomethylation associated vitamin D receptor expression in ATP1A1 mutant aldosterone-producing adenoma

DNA methylation alteration is tissue-specific and play a pivotal role in regulating gene transcription during cell proliferation and survival. We aimed to detect genes regulated by DNA methylation, and then investigated whether the gene influenced cell proliferation or survival in adrenal cells. DNA methylation and qPCR analyses were performed in nonfunctioning adrenocortical adenoma (NFA, n = 12) and aldosterone-producing adenoma (APA, n = 35) samples. The VDR gene promoter was markedly hypomethylated in APA with ATP1A1 mutation, and the promoter methylation levels showed a significant inverse association with the transcripts in APA. ATP1A1 mutation led to VDR transcription in HAC15 cells, and VDR suppression abrogated ATP1A1 mutation-mediated cell proliferation in HAC15 cells. We demonstrated that APA with ATP1A1 mutation showed entire hypomethylation in the VDR promoter and abundant VDR mRNA and protein expression. VDR suppression abrogated ATP1A1 mutation-mediated cell proliferation in HAC15 cells. Abundant VDR expression would be essential for ATP1A1 mutation-mediated cell proliferation.

Mesangial cell-derived tenascin-C contributes to mesangial cell proliferation and matrix protein production in IgA nephropathy.

Tenascin-C (TNC), a non-structural extracellular matrix glycoprotein, is transiently expressed during development or after injury, playing an important role in injury and repair process. The potential role of TNC in the pathogenesis of IgA nephropathy (IgAN) remains to be clarified. Immunohistochemistry staining for TNC was conducted on paraffin-embedded slices from renal biopsies of 107 IgAN patients, and correlation analysis was made between mesangial TNC expression and clinic-pathological parameters. In situ hybridization for TNC mRNA was further performed to figure out the cells that express TNC within glomeruli. In vitro experiments were also carried out on mouse mesangial cells (SV40 MES13) to elucidate the effect of TNC on mesangial cells. TNC was expressed in the mesangial area of IgAN, as well as in fibrotic regions. Correlation analysis showed that higher mesangial TNC was associated with higher level of proteinuria, lower estimated glomerular filtration rate and more serious pathological lesions (MEST score). In situ hybridization revealed that abundant TNC mRNA expression was observed in the affected glomeruli of IgAN, but not in minimal change disease. Moreover, TNC mRNA co-localized with PDGFRβ mRNA, but not with PODXL mRNA, suggesting that TNC mRNA was expressed in the mesangial cells within glomeruli in IgAN. In vitro experiments showed that exogenous TNC promoted matrix protein production and mesangial cell proliferation, which was attenuated by an epidermal growth factor receptor inhibitor. Taken together, these results suggest that mesangial cell-derived TNC contributes to mesangial matrix expansion and mesangial cell proliferation, which is a potential therapeutic target in IgAN.

Expression characteristic of 4Ig B7-H3 and 2Ig B7-H3 in acute myeloid leukemia

ABSTRACT 4IgB7-H3 (4Ig) and 2IgB7-H3 (2Ig) expression characteristics in acute myeloid leukemia (AML) remain unknown. This study investigated mRNA and membrane protein expression of two B7-H3 isoforms in AML cell lines and de novo patients by using RT-PCR and flow cytometry, and analyzed the B7-H3 promoter methylation state by utilizing RQ-MSP. 4Ig was the dominant isoform. 2Ig mRNA expression rate and abundance were elevated in AML in comparison with controls (P = 0.000 and 0.000), while no significant difference in 4Ig (P = 0.802, P = 0.398). Membrane protein levels of B7-H3 isoforms in AML was higher than controls, detected by total B7-H3 expression rate (P = 0.002), total B7-H3 mean fluorescence intensity (MFI) ratio of blast cells and lymphocytes (MFI ratio) (P = 0.000), and 4Ig B7-H3 MFI ratio (P = 0.005). Compared with 2Ig low group, 2Ig high patients had older age, lower NPM1 mutation, higher FLT3-ITD mutation, and declining complete remission (CR) rates (P = 0.026, 0.012, 0.047, and 0.028). In B7-H3 high group, there was a trend toward older age, M4 and M5, worse karyotype, and lower CR rates, although with no marked difference (P > 0.05). The overall survival (OS) of 2Ig high and B7-H3 high groups were shorter than that of 2Ig low and B7-H3 low groups in the whole and non-M3 AML cohorts (P = 0.006 and 0.046; P = 0.003 and 0.032). Besides, an unmethylated B7-H3 promoter was identified. In conclusion, 2Ig mRNA and total B7-H3 membrane protein tended to have potential diagnostic value for AML. Specifically, high 2Ig mRNA and total B7-H3 membrane protein expression indicate worse OS. 4Ig and 2Ig expression are methylation-independent.

The reference liver\u2014CYP450 and UGT enzymes in healthy donor and metastatic livers: the impact of genotype

BackgroundHepatic enzymes involved in drug metabolism vary markedly in expression, abundance and activity, which affects individual susceptibility to drugs and toxicants. The present study aimed to compare mRNA expression and protein abundance of the most pharmacologically relevant drug-metabolizing enzymes in two main sources of the control liver samples that are used as the reference, i.e. organ donor livers and non-tumorous tissue from metastatic livers. An association analysis of the most common genetic variants with mRNA and protein levels was also performed.MethodsThe CYP450 and UGT enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, UGT1A1, UGT1A3, UGT2B7 and UGT2B15) were analyzed for mRNA (qPCR) and protein abundance (LC–MS/MS) in healthy donors (n = 11) and metastatic (n = 13) livers. Genotyping was performed by means of TaqMan assays and pyrosequencing.ResultsSignificantly higher protein abundance in the metastatic livers was observed in case of CYP2C9, CYP2D6, and UGT2B7, and for UGT1A3 the difference was only significant at mRNA level. For all the enzymes except CYP2E1 some significant correlation between mRNA and protein content was observed, and for UGT1A1 an inverse correlation with age was noted. CYP2C19, CYP3A5 and CYP2D6 were significantly affected by genotype.ConclusionThe selection of a control group for the study on drug-metabolizing enzymes (e.g. in pathological states) may possibly affect its conclusions on differences in mRNA and protein content. Genotyping for common functional variants of CYP450 enzymes should be performed in all studies on drug-metabolizing enzymes.

Hepatic drug-metabolizing enzymes and drug transporters in Wilson\u2019s disease patients with liver failure

BackgroundWilson’s disease is a genetic disorder inherited in a recessive manner, caused by mutations in the copper-transporter ATP7B. Although it is a well-known disease, currently available treatments are far from satisfactory and their efficacy varies in individual patients. Due to the lack of information about drug-metabolizing enzymes and drug transporters profile in Wilson’s disease livers, we aimed to evaluate the mRNA expression and protein abundance of selected enzymes and drug transporters in this liver disorder.MethodsWe analyzed gene expression (qPCR) and protein abundance (LC–MS/MS) of 14 drug-metabolizing enzymes and 16 drug transporters in hepatic tissue from Wilson’s disease patients with liver failure (n = 7, Child–Pugh class B and C) and metastatic control livers (n = 20).ResultsIn presented work, we demonstrated a downregulation of majority of CYP450 and UGT enzymes. Gene expression of analyzed enzymes ranged between 18 and 65% compared to control group and significantly lower protein content of CYP1A1, CYP1A2, CYP2C8, CYP2C9, CYP3A4 and CYP3A5 enzymes was observed in Wilson’s disease. Moreover, a general decrease in hepatocellular uptake carriers from SLC superfamily (significant at protein level for NTCP and OATP2B1) was observed. As for ABC transporters, the protein abundance of BSEP and MRP2 was significantly lower, while levels of P-gp and MRP4 transporters were significantly higher in Wilson’s disease.ConclusionsAltered hepatic expression of drug‐metabolizing enzymes and drug transporters in Wilson’s disease patients with liver failure may result in changes of drug pharmacokinetics in that group of patients.

Neither Peristaltic Pulse Dynamic Compressions nor Heat Therapy Accelerate Glycogen Resynthesis after Intermittent Running.

To investigate the effects of a single session of either peristaltic pulse dynamic leg compressions (PPDC) or local heat therapy (HT) after prolonged intermittent shuttle running on skeletal muscle glycogen content, muscle function, and the expression of factors involved in skeletal muscle remodeling. Twenty-six trained individuals were randomly allocated to either a PPDC (n = 13) or a HT (n = 13) group. After completing a 90-min session of intermittent shuttle running, participants consumed 0.3 g·kg-1 protein plus 1.0 g·kg-1 carbohydrate and received either PPDC or HT for 60 min in one randomly selected leg, while the opposite leg served as control. Muscle biopsies from both legs were obtained before and after exposure to the treatments. Muscle function and soreness were also evaluated before, immediately after, and 24 h after the exercise bout. The changes in glycogen content were similar (P > 0.05) between the thigh exposed to PPDC and the control thigh ~90 min (Control: 14.9 ± 34.3 vs PPDC: 29.6 ± 34 mmol·kg-1 wet wt) and ~210 min (Control: 45.8 ± 40.7 vs PPDC: 52 ± 25.3 mmol·kg-1 wet wt) after the treatment. There were also no differences in the change in glycogen content between thighs ~90 min (Control: 35.9 ± 26.1 vs HT: 38.7 ± 21.3 mmol·kg-1 wet wt) and ~210 min (Control: 61.4 ± 50.6 vs HT: 63.4 ± 17.5 mmol·kg-1 wet wt) after local HT. The changes in peak torque and fatigue resistance of the knee extensors, muscle soreness, and the mRNA expression and protein abundance of select factors were also similar (P > 0.05) in both thighs, irrespective of the treatment. A single 1-h session of either PPDC or local HT does not accelerate glycogen resynthesis and the recovery of muscle function after prolonged intermittent shuttle running.

Effects of Encapsulated Methionine on Skeletal Muscle Growth and Development and Subsequent Feedlot Performance and Carcass Characteristics in Beef Steers.

Simple SummaryResearch investigating the effects of amino acids in growing and finishing beef cattle is not a new topic. Many studies suggest that in cattle fed a concentrate-based diet, lysine is the most limiting amino acid followed by methionine. When considering supplementing amino acids to growing-finishing beef cattle, the amino acids must be protected from ruminal microbial consumption for the beef animal to be able to absorb the amino acids in the small intestine. The regulation of skeletal muscle growth is a multifaceted, complex process controlled through many signaling cascades. One major process involved in muscle growth is the mammalian target of the rapamycin (mTOR) signaling pathway. The objectives of these studies were to investigate the role of encapsulated methionine on feedlot growth performance and carcass characteristics and to elucidate the role of methionine supplementation on the mTOR signaling pathway and skeletal muscle development in feedlot steers during the finishing phase.Two studies were conducted to evaluate the effect of encapsulated methionine on live performance, carcass characteristics, and skeletal muscle development in feedlot steers. In Experiment 1, 128 crossbred steers (body weight [BW] = 341 ± 36.7 kg) were used in a randomized complete block design and supplemented with 0, 4, 8, or 12 g/(head day [d]) of ruminally protected methionine (0MET, 4MET, 8MET, and 12MET, respectively) for 111 d or 139 d. In Exp. 2, 20 steers (BW = 457 ± 58 kg) were stratified by BW and randomly assigned to either the 0MET or 8MET treatment; longissimus muscle (LM) biopsies were collected on d 0, 14, 28, 42, and 56, and analyzed for mRNA and protein expression. Additionally, immunohistochemical analysis was performed to measure fiber type area and distribution as well as the density of muscle nuclei and satellite cells (Myf5, Pax7, and Myf5/Pax7). In Experiment 1, no significant differences were observed for live performance (p ≥ 0.09). There was, however, a linear relationship between LM area and methionine supplementation (p = 0.04), with a 9% increase in the area when steers were supplemented with 12MET compared to 0MET. In Exp. 2, There were no treatment × day interactions (p ≥ 0.10) for expression of mRNA or protein abundance. Although mRNA expression and protein abundance of all genes were influenced by day (p ≤ 0.04), methionine supplementation did not have a significant effect (p ≥ 0.08). There was a significant treatment × day interaction for distribution of MHC-I fibers (p = 0.03), where 8MET supplemented cattle had a greater proportion of MHC-I fibers after 56 d of supplementation than did 0MET steers. Cross-sectional area was increased over time regardless of fiber type (p < 0.01) but was unaffected by treatment (p ≥ 0.36). While nuclei density was not impacted by treatment (p = 0.55), the density of myonuclei increased nearly 55% in 8MET supplemented cattle (p = 0.05). The density of Myf5 positive satellite cells tended to decrease with methionine supplementation (p = 0.10), while the density of Pax7 expressing cells tended to increase (p = 0.09). These results indicate that encapsulated methionine supplementation may influence markers of skeletal muscle growth, and potential improvements in the LM area may exist.

Comparing mRNA expression and protein abundance in MDR Mycobacterium tuberculosis: Novel protein candidates, Rv0443, Rv0379 and Rv0147 as TB potential diagnostic or therapeutic targets

Tuberculosis (TB) is a sizable public health threat in the world. This study was conducted to determine the differential protein composition between susceptible and MDRTB strains. Tuberculosis proteins were extracted by Triton™ X-114 and ammonium sulfate. Two-dimensional gel electrophoresis protein spots were selected for identification by mass spectrometry and mRNA expression levels were measured by real- time PCR.2DE-Western blot and T cell epitope prediction for identified proteins were made by the IEDB server. The result shows at least six protein spots (Rv0147, Rv3597c, Rv0379, Rv3699, Rv1392 and Rv0443) were differentially expressed in MDRTB isolates. However, difference in mRNA gene expression was not found in the six mRNA genes.2DE-Western blot procedures indicated strong reaction against MDRTB proteins corresponds to 13, 16 and 55 kDa areas that might be used as new diagnostic tools. In conclusion, these MDRTB proteins identified in this study could be reliable TB diagnostic candidates or therapeutic targets.

Artemisinin Protects Porcine Mammary Epithelial Cells against Lipopolysaccharide-Induced Inflammatory Injury by Regulating the NF-κB and MAPK Signaling Pathways.

Simple SummarySow mastitis is a serious breast disease that can cause severe inflammation, agalaxia and even lead to death of piglets. Porcine mammary epithelial cells (pMECs) are the main cell types that affect sow milk secretion, therefore, when swine mastitis occurs, the inflammatory response of pMECs directly affects the mammary gland health and sow’s lactation ability. Promoting the health of mammary gland epithelial cells is an important method for treating mastitis. Thus, in the current study, we investigated the effects of artemisinin on the inflammatory response of pMECs induced by lipopolysaccharide (LPS), and proposed a potential anti-inflammatory mechanism. We confirmed that artemisinin can reduce the inflammatory damage of pMECs induced by LPS by inhibiting MAPK and NF-κB signaling pathways. Pretreatment of pMECs with artemisinin showed enhanced anti-inflammatory activity against LPS-induced inflammation. Artemisinin could be a useful, safe and natural anti-inflammatory feed additive to prevent sow mastitis.Artemisinin performs a variety of biological functions, such as anti-cancer, anti-inflammatory, anti-viral, and anti-oxidant effects. However, the effects of artemisinin on sow mastitis have not been studied. The results of the current study showed that mRNA expression abundance and content of the inflammatory factors interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), and interleukin-6 (IL-6) were significantly increased when using 50 μg/mL LPS to stimulate pMECs for 24 h (p < 0.05). Pretreatment with 20 μM artemisinin weakened LPS-induced inflammatory damage in pMECs and decreased mRNA expression abundance and the content of inflammatory factors (IL-1β, IL-6, and TNF-α) in pMECs (p < 0.05). Mechanistically, artemisinin inhibited LPS-induced activation of the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. In summary, the pretreatment of pMECs with artemisinin showed enhanced anti-inflammatory activity against LPS-induced inflammation.

Correlation of Cry1Ac mRNA and protein abundance in transgenic Gossypium hirsutum plant.

The online version contains supplementary material available at 10.1007/s13205-021-02828-2.

799 INTESTINAL RBM47 DELETION PROMOTES SPONTANOUS POLYPOSIS THROUGH UP-REGULATED EPITHELIAL PROLIFERATION, ALTERNATIVE SPLICING OF TJP1 BUT MITIGATES COLITIS-ASSOCAITED TUMORIGENESIS VIA ANTI-OXIDATIVE ADAPTATION AND REDUCED INFLAMMATION

Background RNA-binding motif protein 47 (RBM47) is a requisite cofactor in APOBEC1-dependent C-to-U RNA editing whose RNA targets are unknown. Aims Because Apobec-1 knockout mice are protected against polyposis in the ApcMin background, we asked if intestine-specific Rbm47 knockout mice (Rbm47IKO) exhibit altered tumor susceptibility, either spontaneously or in different murine cancer models. Results 86% (6/7) of 12-month old chow-fed Rbm47IKOmice developed spontaneous intestinal and colonic polyps (Fig 1A), compared to 10% (1/10) aged Rbm47floxcontrols. RNA-seq showed upregulated proliferation, glutathione metabolism, and anti-oxidative pathways in Rbm47 IKOintestine. QPCR revealed reduced abundance of the long Tjp1 mRNA isoform (Tjp1+) in Rbm47 IKOmice (Fig 1B), which was further reduced in polyp vs uninvolved tissue (11.8±0.2 vs 15.1±1.2, P=.03) in aged Rbm47 IKOmice. In support, we observed decreased Rbm47 mRNA (–DCT 0.97±2.79 vs 3.47±2.58, P Conclusion Rbm47IKOmice demonstrate spontaneous polyposis through upregulating proliferation and modifying Tjp1 alternative RNA splicing, with increased proximal intestinal polyposis in the ApcMin/+ background. By contrast, Rbm47IKOmice are protected against AOM-DSS-driven colitis-associated colon tumorogenesis by augmenting antioxidant capacity, over-expression of Il-33, and decreasing inflammatory activity. These contrasting phenotypes suggest that RBM47 exerts pathway-specific effects on intestinal tumorigenesis as a genetic modifier of growth, proliferation and inflammatory pathways. Download : Download high-res image (81KB) Download : Download full-size image Figure 1 . A: dissecting microscope images (left) and hematoxylin and eosin staining sections (right) of spontaneous polypogenesis in small intestine and colon of 12-month old chowfed Rbm47 IKOmice. B: Relative abundance of long Tjp1 mRNA isoform (Tjp1+) by quantitative RT-PCR in young adult mice jejunum. C: Rbm47 mRNA expression and relative abundance of Tjp1+ isoform by quantitative RT-PCR in human colorectal cancer tissue vs paired normal tissue. D: Comparison of overall survival among colorectal cancer patients with different levels of Rbm47 mRNA expression in tumor tissue using the cancer genome atlas (TCGA) database. Download : Download high-res image (71KB) Download : Download full-size image Figure 2 . A: Macroscopic illustration of colitis-associated colon tumors in mice treated with AOM)-DSS. B: left; the HE stained microscopic sections illustrate abundance of crypt abscess formation (yellow arrowheads) within dysplastic lesions of colon in AOM-DSS treated mice. Middle; F4/80 immunohistochemical staining of dysplastic lesions of colon in AOM-DSS treated mice. Right: Relative mRNA expression of inflammatory genes by quantitative RTPCR in dysplastic lesions of AOM-DSS treated mice. C: Relative mRNA expression of genes related to interleukin 33 by quantitative RT-PCR in dysplastic lesions of AOM-DSS treated mice. D: Macroscopic illustration of proximal small intestine tumors in mice with Apc Min/+background.

High concentrations of calcium suppress osteogenic differentiation of human periodontal ligament stem cells in vitro

Background/purposePeriodontal ligament stem cells (PDLSCs)-based regeneration therapy has received attention for its potential alternative applications in hard tissue and tooth. However, the environmental diversity of oral cavity that regulates PDLSCs differentiation has made it difficult to develop. Therefore, we investigated how high calcium concentrations in the oral environment influence osteogenic differentiation of human PDLSCs (hPDLSCs). Materials and methodshPDLSCs collected from human molars were isolated and cultured with CaCl2. First, multi lineage differentiation potentials to osteogenic, chondrogenic, and adipogenic cells were investigated. Then, the effects of CaCl2 on both alkaline phosphatase (ALP) activity and bone mineralization were analyzed and the expression of mRNA and protein for osteogenic marker was explored. Further, luciferase assay was performed to evaluate CaCl2 could regulate the transcriptional activity on osteogenic differentiation in hPDLSCs ResultsCaCl2 treatment at normal to high concentrations showed similar suppression of ALP activity, while mineralized nodule formation was decreased by CaCl2 treatment dose-dependently without affecting proliferation or cytotoxicity in hPDLSCs. We also observed that CaCl2 treatment repressed the mRNA expression and protein abundance of osteogenic genes and transcriptional factors. Notably, repression of the Runx2 level was significant, and CaCl2 treatment inhibited Runx2-mediated transcriptional activity on the osteoblast-specific element (OSE) and ALP promoters. ConclusionHigh concentrations of calcium negatively regulate osteogenic differentiation of hPDLSCs, by repressing osteogenic gene expressions and transcriptional activity. Therefore, these conditions may be applicable to determine the physiologically appropriate concentration of calcium.

Effects of dexamethasone on the morphology, gene expression and hepatic histology in adult female mosquitofish (Gambusia affinis)

Glucocorticoids (GCs), including natural hormones as well as synthetic chemicals, can pose influences on physiological performance, development and reproduction of fish. Dexamethasone (DEX) is a synthetic glucocorticoid widely used as pharmaceutical and usually exists in effluents with varying degrees of concentrations. In this study, adult female mosquitofish (Gambusia affinis) were treated by DEX at concentrations of 0, 0.5, 5 and 50 μg/L for 60 days. Morphological parameters of anal fin and skeleton, mRNA expression abundance, and histological alterations of liver were investigated to assess effects of DEX on mosquitofish. The results showed that DEX increased number of sections of ray 3 in anal fin and decreased 16L, 15D and 16D in skeletal parameters, which indicates DEX could potentially lead to weak masculinization. Furthermore, transcriptional expression levels of ARα, ARβ, ERβ, VTGC and CYP19A genes were notably down-regulated by DEX, which will contribute to weak masculinization in females. In addition, the damage to liver tissue was also induced by DEX. Taken together, this research demonstrated that aquatic environments contaminated by DEX have negative effects on mosquitofish at a population level.