Quantitative Real-time Transcription PCR Research Articles

Nanobiostimulant action of trigolic formulated zinc sulfide nanoparticles (ZnS-T NPs) on rice seeds by triggering antioxidant defense network and plant growth specific transcription factors

Under a changing climate, nanotechnological interventions for climate resilience in crops are critical to maintaining food security. Prior research has documented the affirmative response of nano zinc sulfide (nZnS) on physiological traits of fungal-infested rice seeds. Here, we propose an application of trigolic formulated zinc sulfide nanoparticles (ZnS-T NPs) on rice seeds as nanobiostimulant to improve physiological parameters by triggering antioxidative defense system, whose mechanism was investigated at transcriptional level by differential expression of genes in germinated seedlings. Nanopriming of healthy rice seeds with ZnS-T NPs (50 μg/ml), considerably intensified the seed vitality factors, including germination percentage, seedling length, dry weight and overall vigor index. Differential activation of antioxidant enzymes, viz. SOD (35.47%), APX (33.80%) and CAT (45.94%), in ZnS-T NPs treated seedlings reduced the probability of redox imbalance and promoted the vitality of rice seedlings. In gene expression profiling by reverse transcription quantitative real time PCR (qRT-PCR), the notable up-regulation of target antioxidant genes (CuZn SOD, APX and CAT) and plant growth specific genes (CKX and GRF) in ZnS-T NPs treated rice seedlings substantiates their molecular role in stimulating both antioxidant defenses and plant growth mechanisms. The improved physiological quality parameters of ZnS-T NPs treated rice seeds under pot house conditions corresponded well with in vitro findings, which validated the beneficial boosted impact of ZnS-T NPs on rice seed development. Inclusively, the study on ZnS-T NPs offers fresh perspectives into biochemical and molecular reactions of rice, potentially positioning them as nanobiostimulant capable of eliciting broad-spectrum immune and growth-enhancing responses.

Resveratrol affects ccRCC cell senescence and macrophage polarization by regulating the stability of CCNB1 by RBM15.

Aim: The present study sought to investigate the therapeutic effect of resveratrol on clear cell renal cell carcinoma. Materials & methods: Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine assays were used to verify the cell proliferation. Transwell, real-time quantitative transcription PCR, western blot and β-galactosidase staining were used to verify the migration, macrophage polarization and senescence. The tumor inhibitory effect of resveratrol on clear cell renal cell carcinoma was verified in vivo. Results: This study confirmed that resveratrol could affect the stability of CCNB1 mRNAmediated by RBM15 and inhibit the cancer process by inhibiting the expression of EP300/CBP from the perspective of cell senescence. Conclusion: Resveratrol is able to treat clear cell renal cell carcinoma through RBM15-induced cell senescence.

Circulating miRNA-16 in inflammatory bowel disease and some clinical correlations - a cohort study in Bulgarian patients.

The pathogenesis of the inflammatory bowel disease (IBD) includes chronic inflammation and altered immune reactions. There are several publications, reporting that micro ribonucleic acids (miRNAs) may serve as a diagnostic biomarker with a potential to assess inflammation severity and treatment response1,2 in IBD patients. The objective of the study is to assess and correlate the serum expression of circulating miR-16 in IBD patients with some clinical parameters, such as extent, activity and severity of the disease. 70 IBD patients [35 with ulcerative colitis (UC) and 35 with Crohn's disease (CD)] were included in the study. Serum miR-16 expression in both IBD diseases was assessed using reverse transcription quantitative real time PCR (RT-qPCR). Circulating miR-16 levels were also correlated with disease extent, activity and severity indices [Crohn's Disease Activity Index (CDAI), Montreal classification, Partial Mayo score]. Serum expression of miR-16 in the 70 patients was also compared to miR-16 serum levels in 30 healthy control subjects. The patients' group showed mean serum miR-16 expression of 3.07 for CD, 1.97 for UC and 1.61 for the control group of healthy subjects with a significant difference in the expression between groups. There is a significant correlation between increased serum expression of miR-16 and disease activity, extent and severity. The increased miR-16 serum expression correlates with disease activity, intestinal localization of CD, stenotic and penetrating phenotype. MiR-16 could serve as a potential biomarker to assess inflammation.

Differential gene expression analyses of ten defence response genes during Fusarium wilt infection in resistant and susceptible pigeonpea cultivars

The present study was undertaken to partly understand the molecular mechanism of defence response during pigeonpea-Fusarium udum interaction. To study the response of defence related gene expression using reverse transcription quantitative real time PCR (RT-qPCR), roots of resistant and susceptible pigeonpea cultivars were infected with F. udum pathogen and samples were collected at 2, 24, 48, and 72 h after inoculation (HAI). PR2, PR10, PAL2, CHS8, IFS1, F3H, LOX, ADC, SPDS, and SPMS were amongst the ten defence response genes studied. The major changes observed in gene expression for all the ten genes are as follows: (i) Out of the ten genes, the expression of three genes, PR2, PR10, and CHS8, peaked at 48 HAI in resistant cultivars compared to susceptible cultivars (ii) the expression of two genes, IFS1 and F3H, was highly induced at all stages in resistant cultivars, while some genotypic differences were observed in susceptible cultivars (iii) the expression of the LOX gene increased only during the early stages at 2 HAI but decreased at later stages in resistant cultivars (iv) In resistant cultivars, the expression of three genes involved in polyamine synthesis, namely ADC, SPDS, and SPMS, peaked at 24 HAI compared to susceptible cultivars. Overall, increased gene expression was found mostly at earlier stages of infection, indicating that these genes are important in providing early resistance to F. udum infection in pigeon pea cultivars. The LOX gene, which codes for the lipooxygenase enzyme, appears to play a role in conferring susceptibility to pigeonea cultivars against F. udum infection, though more research is needed. Further validation studies of all ten genes using overexpression or RNAi technology will provide more information about their roles in the pigeonpea-F.udum interaction.

A High-Cholesterol Diet Increases Toll-like Receptors and Other Harmful Factors in the Rabbit Myocardium: The Beneficial Effect of Statins.

Background: A high-cholesterol diet (HCD) induces vascular atherosclerosis through vascular inflammatory and immunological processes via TLRs. The aim of this study is to investigate the mRNA expression of TLRs and other noxious biomarkers expressing inflammation, fibrosis, apoptosis, and cardiac dysfunction in the rabbit myocardium during (a) high-cholesterol diet (HCD), (b) normal diet resumption and (c) fluvastatin or rosuvastatin treatment. Methods: Forty-eight male rabbits were randomly divided into eight groups (n = 6/group). In the first experiment, three groups were fed with HCD for 1, 2 and 3 months. In the second experiment, three groups were fed with HCD for 3 months, followed by normal chow for 1 month and administration of fluvastatin or rosuvastatin for 1 month. Control groups were fed with normal chow for 90 and 120 days. The whole myocardium was removed; total RNA was isolated from acquired samples, and polymerase chain reaction, reverse transcription PCR and quantitative real-time PCR were performed. Results: mRNA of TLRs 2, 3, 4 and 8; interleukin-6; TNF-a; metalloproteinase-2; tissue inhibitor of metalloproteinase-1; tumor protein 53; cysteinyl aspartate specific proteinase-3; and brain natriuretic peptide (BNP) increased in HCD. Statins but not resumption of a normal diet decreased levels of these biomarkers and increased levels of antifibrotic factors. Conclusions: HCD increases the levels of TLRs; inflammatory, fibrotic and apoptotic factors; and BNP in the rabbit myocardium. Atherogenic diets adversely affect the myocardium at a molecular level and are reversed by statins.

LncRNA SNHG6 can regulate the proliferation and apoptosis of rat degenerate nucleus pulposus cells via regulating the expression of miR-101-3p.

Intervertebral disc (IVD) degeneration (IDD) is a well-known consequence of low back pain, as characterized by aberrant cell proliferation and apoptosis of nucleus pulposus (NP) cells. In the present study, we aimed to investigate the effect of lncRNA small nucleolar RNA host gene 6 (SNHG6) on deregulated functions of degenerative NP cells. After the establishment of rat IDD models, the mRNA and protein levels of collagen-I (Col-I) and collagen II (Col-II), and mRNA level of SNHG6 were detected by using reverse transcription quantitative Real Time-PCR (RT-qPCR) and Western blot. We further investigated the role and molecular mechanisms of SNHG6 by overexpressing or silencing it in degenerative NP cells. Cell proliferation was measured by MTT assay and EdU staning, and apoptosis was measured by flow cytometry. The target of SNHG6 was identified by starBase and Dual-Luciferase reporter assay. Upregulation of SNHG6 was found in IDD NP cells than in normal cells, associated with higher level of Col-I and lower level of Col-II. Overexpression of SNHG6 inhibited cell proliferation and enhanced apoptosis, accompanied by increased expression of Bax, caspase-3, and p21, as well as decreased expression of Bcl-2, which was in reverse to the treatment of SNHG6 silencing. Moreover, miR-101-3p was indicated as a target of SNHG6, and inhibition of miR-101-3p reversed the effects on proliferation and apoptosis induced by SNHG6. SNHG6 suppressed cell proliferation and induced apoptosis by increasing expression of Bax, caspase-3, p21 and decreasing Bcl-2 through targeting miR-101-3p, which suggested that SNHG6 could be a potential target in the treatment of IDD.

Identification of Long-Distance Transmissible mRNA between Scion and Rootstock in Cucurbit Seedling Heterografts.

Grafting has been widely used to improve plant growth and tolerance in crop production, as well as for clarifying systemic mRNA signaling from donor to recipient tissues in organ-to-organ communication. In this study, we investigated graft partner interaction mechanisms of Cucumis sativus (Csa) and Cucurbita moschata (Cmo) using a large-scale endogenous mRNA transport. The results indicated that most mobile transcripts followed an allocation pathway from source to sink. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that mRNA mobility functions are universally common and individually specific. Identification of mRNA mobility between distant tissues in heterografts with RT-PCR (reverse transcription PCR), RT-qPCR (reverse transcriptional quantitative real time PCR), and clone sequencing were used to estimate 78.75% of selected mobile transcripts. Integration of bioinformatic analysis and RT-qPCR identification allowed us to hypothesize a scion-to-rootstock-to-scion feedback signal loop of Csa move-down and Cmo move-up mRNAs, where Csa scion move-down mRNAs were involved in carbon fixation and biosynthesis of amino acid pathways, and Cmo root received Csa move-down mRNA and then delivered the corresponding Cmo upward mRNA to scion to improve photosynthesis of cucumber scion. This formed a feedback signal loop of scion-to-rootstock-to scion to explain why pumpkin rootstock enhanced cucumber production in the industry, which was utilized for organ communication and mediates photosynthesis processes in heterograft cucurbit crops.

Gene expression of heat shock protein (hsp90) in Plodia interpunctella and Ephestia kuehniella (Lepidoptera: Pyralidae) larvae parasitized by Bracon hebetor wasp (Hymenoptera: Braconidae)

Venoms of parasitoid wasps injected into the host may play vital roles in successful parasitism. It could manipulate the host physiology and suppress its immune response. Heat shock proteins (hsps) could be induced by a variety of physiological stresses and might play roles in modulating the host-parasitoid relationship. Quantitative real-time transcription PCR was used to determine the changes of heat shock protein90 gene in Polidia interpunctella and Ephestia kuehniella larvae post parasitization by Bracon hebtor wasp. The results indicated that hsp90 gene expression level showed different behavior in both hosts, at all-time intervals post parasitism. Expression of the gene in P. interpunctella larvae was significantly down regulated at 12h. A high significant down regulated was observed at 24 and 72h after parasitization. Meanwhile, a high significant up-regulation was recorded after 48 h compared with control. On the other hand, the levels of hsp90 in E. kuehniella were high significantly down regulated in all treated larvae compared with control. We can conclude that the suppression hsp90 gene could be a component of parasitized hosts' manipulation strategy that regulate the host physiology and suppress the immune response. Hsp90 might play an important role in host paralysis and inhibit its development.

Cinobufagin inhibits tumor growth by inducing apoptosis through Notch signaling pathways in human cholangiocarcinoma

Background: Many studies have shown that cinobufagin has antitumor effects against many cancers. The aim of this study was to assess the effects of cinobufagin on cholangiocarcinoma (CCA) cells. Methods: Colony formation assay, cell-counting kit-8 (CCK-8) assay, and tumor xenograft experiments were applied to investigate the function of cinobufagin on human CCA cells, in vitro and in vivo . Flow cytometric analysis was performed to validate the effects of cinobufagin on cell apoptosis. Quantitative real-time reverse transcription PCR (qRT-PCR) and Western blot assays were performed to evaluate expression levels of related genes. Results: This study found that cinobufagin inhibited proliferation in both QBC939 and RBE cells. Flow cytometric analysis indicated that rate of apoptosis was significantly increased when treated with cinobufagin. Regarding mechanism analysis, cinobufagin was found to dramatically inactivate Notch signaling pathways. Animal experiment results in vivo were consistent with outcomes in vitro . Conclusions: These data suggest that cinobufagin-mediated inactivation of Notch pathways may play an important role in the induction of apoptosis in CCA.

Identification of high expression profiles of miR-31-5p and its vital role in lung squamous cell carcinoma: a survey based on qRT-PCR and bioinformatics analysis

Background: The function of miR-31-5p in lung squamous cell carcinoma (LUSC) remains unclear, therefore, a systematic study was performed for the clinical significance and molecular mechanism of miR- 31-5p in LUSC. Methods: Quantitative real-time reverse transcription PCR (qRT-PCR) was utilized to test the expression level of miR-31-5p in 88 LUSC tissue samples and their matching normal tissues. Data from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) were also utilized to confirm the expression level and clinical value of miR-31-5p in LUSC. The potential target genes of miR-31-5p were predicted by several online predicted software. Gene ontology (GO), protein-protein interaction (PPI) and pathway analysis were utilized to investigate the underlying molecular mechanism of miR-31-5p in LUSC. Results: The result from qRT-PCR found that there was significant difference of miR-31-5p between LUSC and normal tissues (P Conclusions: According to what has been discussed above, we speculated that miR-31-5p may play a vital role in the occurrence and development of LUSC.

Solamargine inhibits proliferation and promotes apoptosis of CM-319 human chordoma cells through suppression of notch pathway

Background: Solamargine (SM), which represents a natural steroid alkaloid glycoside compound and a cytotoxic agent, has been proved to enhance the sensitivity of lung cancer cells to tumor necrosis factors (TNFs). In this study, we aimed to investigate the roles and mechanisms of SM in chordoma. Methods: Cell viability, proliferation, apoptosis and cell cycle were measured by cell counting Kit-8 (CCK-8) assay, 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling and flow cytometry (FCM), respectively. Western blot and quantitative real-time reverse transcription PCR (qRT-PCR) assays were performed to detect the expressions of related mRNAs and proteins. Results: The results revealed that SM distinctly suppressed the proliferation of CM-319 cells. SM significantly induced the CM-319 cells apoptosis through up-regulating the expression levels of Caspase-3/8/9. The cell cycle of CM-319 cells was blocked by SM in G1 phase. Moreover, SM could significantly suppress the Notch pathway in CM-319 cells. Conclusions: In conclusion, SM suppressed the proliferation and enhanced the apoptosis ability of CM-319 cells via suppressing the Notch pathway. The results suggested that SM might be a novel therapeutic agent and supported the utilization of SM in chordoma.

Expression of circulating miR-200c and vascular endothelial growth factor-A (VEGF-A) mRNA as potential biomarker in human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common liver disease that causes significant public health problems throughout the world, including in Indonesia. The HCC is the six most common cancers and second cancer-related deaths among men in the world. Recently it was reported that the microRNA is an important player in hepatocarcinogenesis. The expression of MiRNA-200c is often regulated in primary HCC and HCC cell lines. Vascular endothelial growth factor-A (VEGF-A) is a regulator of angiogenesis that has been reported as miR-200c target gene. This study was conducted to measure expression levels in miR- 200c and mRNAVEGF-A and their potential role as biomarkers at HCC. A total of 36 HCC patients and 36 healthy subjects were included in this study. The relative expression of miRNA-200c and mRNA VEGF-A was quantified using reverse transcription real time quantitative PCR (qRT PCR). Relative expression was calculated using . Unpaired t-test was used to compare the expression levels of circulating miRNA-200c and mRNA VEGF-A in HCC patients and healthy subjects. Pearson test was used to determine correlation between circulating miR-200c expression and mRNA VEGF-A expression levels. The expression levels of circulating miR-200c in HCC patients were lower compared to healthy subjects although it was not significant (p = 0.258). Conversely, the expression levels of circulating mRNA VEGF-A in HCC patients were significantly higher compared to healthy subjects (p = 0.001). The relative expression levels of circulating miR-200c were negatively correlated with mRNA VEGF-A in HCC patients. In conclusion, the expression levels of mRNA VEGF-A in HCC patients are significantly deregulated in compared to that in healthy subjects. Negative correlation between circulating miRN-200c and mRNA VEGF-A expression levels are reported in HCC patients.

An Improved Total RNA Extraction Method for White Jelly Mushroom Tremella fuciformis Rich in Polysaccharides

An improved method for extracting high quality and quantity RNA from a jelly mushroom and a dimorphic fungus—Tremella fuciformis which is especially rich in polysaccharides, is described. RNA was extracted from T. fuciformis mycelium M1332 and its parental monokaryotic yeast-like cells Y13 and Y32. The A260/280 and A260/230 ratios were both approximately 2, and the RNA integrity number was larger than 8.9. The yields of RNA were between 108 and 213 μg/g fresh wt. Downstream molecular applications including reverse transcriptional PCR and quantitative real-time PCR were also performed. This protocol is reliable and may be widely applicable for total RNA extraction from other jelly mushrooms or filamentous fungi rich in polysaccharides.

Genome-wide identification and analysis of MAPK and MAPKK gene family in Chinese jujube (Ziziphus jujuba Mill.)

BackgroundChinese jujube (Ziziphus jujuba Mill.) is one of the most important members in the Rhamnaceae family. The whole genome sequence and more than 30,000 proteins of Chinese jujube have been obtained in 2014. Mitogen-activated protein kinase cascades are universal signal transduction modules in plants, which is rapidly activated under various biotic and abiotic stresses. To date, there has been no comprehensive analysis of the MAPK and MAPKK gene family in Chinese jujube at the whole genome level.ResultsBy performing a series of bioinformatics analysis, ten MAPK and five MAPKK genes were identified from the genome database of Chinese jujube, and then compared with the homologous genes from Arabidopsis. Phylogenetic analysis showed that ZjMAPKs was classified into four known groups, including A, B, C and D. ZjMAPKs contains five members of the TEY phosphorylation site and five members with the TDY motif. The ZjMAPKK family was subsequently divided into three groups, A, B and D. The gene structure, conserved motifs, functional annotation and chromosome distribution of ZjMAPKs and ZjMAPKKs were also predicted. ZjMAPKs and ZjMAPKKs were distributed on nine pseudo-chromosomes of Chinese jujube. Subsequently, expression analysis of ZjMAPK and ZjMAPKK genes using reverse transcription PCR and quantitative real-time PCR was carried out. The majority of ZjMAPK and ZjMAPKK genes were expressed in all tested organs/tissues with considerable differences in transcript levels indicating that they might be constitutively expressed. Moreover, ZjMKK5 was specific expressed in early development stage of jujube flower bud, indicating it plays some roles in reproductive organs development. The transcript expression of most ZjMAPK and ZjMAPKK genes was down-regulated in response to plant growth regulators, darkness treatment and phytoplasma infection.ConclusionsWe identified ten ZjMAPK and five ZjMAPKK genes from the genome database of Chinese jujube, the research results shown that ZjMPKs and ZjMKKs have the different expression patterns, indicating that they might play different roles in response to various treatments. The results provide valuable information for the further elucidation of physiological functions and biological roles of jujube MAPKs and MAPKKs.

Effect of carboxyl-terminal truncated HBx mediated down-regulation of RhoGDIα expression on the metastasis and invasion of hepatocellular carcinoma

Objective To investigate the mechanism of natural carboxyl-terminal truncated HBx (with 31 amino acids deleted at the C-terminal end) (HBxΔ31)-dependent down-regulation of Rho GDP dissociation inhibitor alpha (RhoGDIα) expression and its role in enhancing hepatocellular carcinoma (HCC) metastasis. Methods HepG2 cells with stable expression of wild type HBx and its deletion mutant HBxΔ31 protein were selected as the study subjects. The effects of HBx and HBxΔ31 on RhoGDIα expression in HepG2 cells was detected using real-time quantitative reverse transcription PCR (RT-qPCR) and Western blot analysis. A series of deleted and mutated variants of the RhoGDIα promoter were made and individually co-transfected with HBx-and HBxΔ31-expressing vectors or control empty vector into HepG2 cells. The luciferase reporter assay was used to identify the cis-regulatory elements of the RhoGDIα promoter in response to HBxΔ31 regulation. The interaction between Myc-associated zinc finger protein (MAZ) and HBxΔ31 was examined by co-immunoprecipitation experiment. Eelectrophoretic mobility shift assay (EMSA) was performed to determine interaction of MAZ with the RhoGDIα promoter in HBxΔ31-expressing HepG2 cells. The impact of reduced RhoGDIα expression by HBxΔ31 on cell-invasive activity was analyzed by the Matrigel cell invasion assay. In addition, the effects of silencing of shRNA-mediated RhoGDIα in HepG2 or introduction of RhoGDIα by transfection in HBxΔ31-expressing HepG2 cells on cell-invasion were investigated. Results HBxΔ31, but not HBx, suppressed RhoGDIα expression at transcriptional levels. Analysis of the deletion and mutation of RhoGDIα promoter showed that the HBxΔ31 repressive element localized between nt-460 and-242 bp of RhoGDIα promoter and that the transcription factor MAZ binding sites was required for RhoGDIα promoter inactivation regulated by HBxΔ31. In addition, HBxΔ31 represses RhoGDIα expression by enhancing MAZ binding to its promoter through directly associating with MAZ. The cell-migratory and cell-invasive activity were significantly increased in sh-RhoGDIα-expressing HepG2 cells, as compared to control cells (migrated cells number: 58 ± 5 vs. 98 ± 7, invaded cells number: 55 ± 6 vs. 113 ± 6, t= 18.91 and t= 20.12, both P < 0.01). However, ectopic expression of RhoGDIα in HBxΔ31-expressing HepG2 cells significantly decreased cell migration and invasion (migrated cells number: 40 ± 4 vs. 115 ± 5, invaded cells number: 42 ± 4 vs. 102 ± 4, t= 18.14 and t= 16.31, both P < 0.001). Conclusion Carboxyl-terminal truncated HBx deregulates RhoGDIα expression through MAZ, in turn which promotes the invasion and metastasis of HCC. Key words: Carcinoma, hepatocellular; Sequence deletion; Hepatitis B virus; Gnanine nucleotide dissociation inhibitors; Neoplasm metastasis

A study of changes in ET-1 gene expression in peripheral blood mononuclear cells in patients with diabetic retinopathy

Objectives The objective of this study was to assess the usefulness of relative quantification of endothelin-1 (ET-1) in peripheral blood mononuclear cells in diabetic retinopathy (DR). Background DR is one of the most serious complications of diabetes mellitus (DM) that can lead to blindness. Alterations in activity of the ET system are believed to underlie the development of chronic complications of type 2 diabetes mellitus (T2DM). Patients and methods This is a clinical study carried out at Medical Biochemistry and Ophthalmology Departments. It included 50 patients divided into three groups: group I (included 14 diabetics without DR), group II (included 26 diabetics with DR), and group III (included 10 normal healthy controls). All studied patients were subjected to full history taking, clinical and ophthalmological examination, and laboratory investigations including fasting blood glucose, glycated hemoglobin (HbA1c), lipid profile, serum creatinine, urinary albumin, and creatinine and ET-1 mRNA level in peripheral blood mononuclear cells using real-time quantitative reverse transcription PCR. Results The mRNA level of ET-1 was significantly higher in diabetics with DR than those without retinopathy and was higher in diabetics than in controls. In diabetics with DR, the blood mRNA level of ET-1 was significantly higher in patients with proliferative diabetic retinopathy than in those with nonproliferative diabetic retinopathy. Increased expression of ET-1 mRNA was associated with severity of DR, duration of DM, insulin therapy, and serum creatinine level. Decreased expression of ET-1 mRNA was associated with sulfonylureas treatment. Expression of ET-1 mRNA was not affected by age, sex, BMI, smoking, and control of blood sugar. Conclusion Expression of ET-1 is associated with severity of DR in T2DM. Expression of ET-1 is positively correlated with duration of DM, serum creatinine, and urinary albumin. A larger study is needed to confirm the role of ET-1 mRNA determination in the progression of retinal changes in patients with T2DM.

Effect of Taurine on the expression of glucose regulated protein 78 and growth arrest and DNA damage-inducible protein 34 in zebrafish larvae brain after hypoxia/reoxygenation brain injury

Objective To investigate the expression of endoplasmic reticulum stress-related factors, glucose-regulated protein 78 (GRP78)and growth arrest and DNA damage-inducible protein 34 (GADD34)and neuronal apoptosis in the brain of zebrafish larvae after hypoxia/reoxygenation brain injury, and the neuroprotective role of Taurine. Methods The 5 day post-fertilization zebrafish larvae were randomly assigned to 3 groups: control group, hypoxia/reoxygenation model group (model group) and Taurine treatment group(Taurine group). According to the different time points for observation, each group was subdivided into 5 subgroups (1 h, 3 h, 6 h, 24 h, 48 h)with 100 zebrafish larvae each.The pathological changes in the brain tissues and cell apoptosis were detected by fluorescence terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling(TUNEL). The expression of GRP78 and GADD34 mRNA in the brain of zebrafish larvae were detected by real-time quantitative reverse transcription PCR(qRT-PCR). The changes in GRP78 and GADD34 protein were detected by Western blot. Results (1)TUNEL: apoptosis index(AI)was increased after hypoxia/reoxygenation, and reached the peak at 3 h in model group, the AI in Taurine group was decreased compared with that in the model group at the same time point (1 h: 22.83±1.80 vs 30.18±1.81, 3 h: 23.22±2.46 vs 42.97±4.01, 6 h: 16.80±1.69 vs 22.97±1.91, all P<0.05). (2) qRT-PCR: the expression of GRP78 and GADD34 mRNA was increased at 1 h after hypoxia/reoxygenation, and reached the peak at 3 h in the model group, the expression in Taurine group was decreased compared with that in the model group at the same time point (GRP78 mRNA: 1 h: 2.35±0.13 vs 5.36±0.35, 3 h: 3.08±0.33 vs 4.27±0.52, 6 h: 1.57±0.12 vs 3.00±0.13, all P<0.05; GADD34 mRNA: 1 h: 5.14±0.55 vs 7.45±0.67, 3 h: 2.79±0.58 vs 5.83±0.51, 6 h: 1.79±0.22 vs 3.67±0.30, all P<0.05). (3) Western blot: the expression of GRP78 and GADD34 protein was increased at 1 h, reached the peak at 6 h, but it was decreased in Taurine group (GRP78 protein: 1 h: 1.12±0.11 vs 1.37±0.13, 3 h: 0.79±0.11 vs 1.25±0.10, 6 h: 0.55±0.10 vs 1.52±0.14, all P<0.05; GADD34 protein: 1 h: 0.92±0.11 vs 1.11±0.13, 3 h: 0.96±0.11 vs 1.52±0.09, 6 h: 0.76±0.05 vs 1.89±0.06, all P<0.05). (4) The expression of GRP78 and GADD34 protein was positively correlated with AI in the model group(r=0.53, 0.56 respectively, all P<0.05). Conclusion One of neuroprotective mechanisms of Taurine against hypoxia/reoxygenation brain injury may down-regulate GRP78 and GADD34 expression. Key words: Endoplasmic reticulum stress; Hypoxic-ischemic brain damage; Glucose-regulated protein 78; Growth arrest and DNA damage-inducible protein 34; Apoptosis

The response of different genotypes of citrus and relatives to Citrus psorosis virus inoculation

Damage caused by Citrus psorosis virus (CPsV) could be avoided obtaining resistant cultivars by breeding, but the sensitivity of most citrus hosts to CPsV is unknown. To find potential sources of resistance we inoculated 63 cultivars and hybrids of Citrus and related genera [Citrus (36), Microcitrus (5), Fortunella (6), Eremocitrus (1), Pleiospermium (1), Atalantia (2), Severinia (1), Clausena (1), Swinglea (1), Afraegle (1), Poncirus (1) and hybrids (7)] with the CPsV isolate PB-143 and monitored symptoms and CPsV infection by ELISA. Microcitrus inodora and Fortunella hindsii were symptomless but gave high ELISA values, suggesting tolerance to CPsV, whereas Citrus depresa, Clausena excavata, Cleopatra mandarin, sour orange, Carrizo citrange and CPB4475 citrumelo were ELISA negative, suggesting resistance. Further examination of CPsV infection by reverse transcription quantitative real time PCR (RT-qPCR) in Cleopatra mandarin, Poncirus trifoliata and Carrizo citrange seedlings inoculated with CPsV isolates P-121, PB-102 and PB-143, revealed different infection rates and CPsV accumulation, depending on the isolate and citrus genotype. Bud propagation of these genotypes on CPsV-inoculated sweet orange plants incited a bud union disorder that hindered flushing and scion growth, suggesting that the high viral load in the rootstock induced a hypersensitive-like reaction in the partially resistant scion.

Transcriptional changes in three immunoglobulin isotypes of rohu, Labeo rohita in response to Argulus siamensis infection.

Immunoglobulin heavy chains of three isotypes viz., IgM, IgD and IgT/IgZ are described in teleosts. In this study, a challenge experiment with an ectoparasite Argulus siamensis was conducted to evaluate the changes in adaptive immune response by quantitation of expression of Ig heavy chains in skin, head kidney and mucus of infected rohu, Labeo rohita. Rohu were challenged with 100 metanauplii of A. siamensis/fish. Head kidney, skin and mucus samples were collected at 0 h, 12 h, 24 h, 3 d, 7 d, 15 d and 30 d by sacrificing four fish each from infected and control groups at each time point. The expression of IgM, IgD and IgZ in these tissues were measured by reverse transcription real time quantitative PCR. IgM level was found to reach its peak significantly 30 d post-infection in head kidney tissue, while IgM transcripts were below detectable range in skin and mucus at all time points. IgZ and IgD levels were significantly up-regulated post-infection in all the three tissue samples. Early up-regulation of IgD was observed in skin and mucus, compared to head kidney. This study showed that parasitic invasion can trigger varied expressions of immunoglobulin types to provide systemic as well as local protection in the host. In particular, the appearance of high level of expression of IgZ and IgD in skin and mucus will pave the way for vaccine development against A. siamensis which feeds on those tissues.

Systemic and mucosal immune response of rainbow trout to immunization with an attenuated Flavobacterium psychrophilum vaccine strain by different routes

Teleosts possess three immunoglobulin (Ig) heavy chain isotypes viz., IgM, IgT and IgD and all three isotypes are reported in rainbow trout. The expression of these Ig isotypes in response to different immunization routes was investigated and results provide a better understanding of the role these Igs in different tissues. Rainbow trout (Oncorhynchus mykiss) were immunized with an attenuated Flavobacterium psychrophilum strain, 259-93-B.17 grown under iron limiting conditions, by intraperitoneal, anal intubation and immersion routes. Serum, gill mucus, skin mucus and intestinal mucus samples were collected at 0, 3, 7, 14, 28, 42 and 56 days post immunization by sacrificing four fish from each treatment group and the unimmunized control group, and the IgM levels were estimated by an enzyme linked immunosorbent assay (ELISA). In addition, blood, gill, skin and intestinal tissue samples were collected for Ig gene expression studies. The secretory IgM, IgD and IgT gene expression levels in these tissues were estimated by reverse transcription quantitative real time PCR (RT-qPCR). Levels of IgM in serum, gill and skin mucus increased significantly by 28 days after immunization in the intraperitoneally immunized group, while no significant increase in IgM level was observed in fish groups immunized by other routes. Secretory IgD and IgT expression levels were significantly upregulated in gills of fish immunized by the immersion route. Similarly, secretory IgT and IgD were upregulated in intestines of fish immunized by anal intubation route. The results confirm mucosal association of IgT and suggest that IgD may also be specialized in mucosal immunity and contribute to immediate protection to the fish at mucosal surfaces.