Semi-quantitative RT Research Articles

Physiological roles of Arabidopsis MCA1 and MCA2 based on their dynamic expression patterns

Determining the mechanisms by which plants sense and respond to mechanical stimuli is crucial for unraveling the detailed processes by which plants grow and develop. Mechanosensitive (MS) channels, including MCA1 and its paralog MCA2 in Arabidopsis thaliana, may be essential for these processes. Although significant progress has been made in elucidating the physiological roles of MS channels, comprehensive insights into their expression dynamics remain elusive. Here, we summarize recent advancements and new data on the spatiotemporal expression patterns of the MCA1 and MCA2 genes, revealing their involvement in various developmental processes. Then, we describe findings from our study, in which the expression profiles of MCA1 and MCA2 were characterized in different plant organs at various developmental stages through histochemical analyses and semiquantitative RT‒PCR. Our findings revealed that MCA1 and MCA2 are preferentially expressed in young tissues, suggesting their pivotal roles in processes such as cell division, expansion, and mechanosensing. Lastly, we discuss the differential expression patterns observed in reproductive organs and trichomes, hinting at their specialized functions in response to mechanical cues. Overall, this review provides valuable insights into the dynamic expression patterns of MCA1 and MCA2, paving the way for future research on the precise roles of these genes in planta.

Microarray-based detection and expression analysis of drug resistance in an animal model of peritoneal metastasis from colon cancer

Chemotherapy drugs efficiently eradicate rapidly dividing differentiated cells by inducing cell death, but poorly target slowly dividing cells, including cancer stem cells and dormant cancer cells, in the later course of treatment. Prolonged exposure to chemotherapy results in a decrease in the proportion of apoptotic cells in the tumour mass. To investigate and characterize the molecular basis of this phenomenon, microarray-based expression analysis was performed to compare tHcred2-DEVD-EGFP-caspase 3-sensor transfected C-26 tumour cells that were harvested after engraftment into mice treated with or without 5-FU. Peritoneal metastasis was induced by intraperitoneal injection of C-26 cells, which were subsequently reisolated from omental metastatic tumours after the mice were sacrificed by the end of the 10th day after tumour injection. The purity of reisolated tHcred2-DEVD-EGFP-caspase 3-sensor-expressing C-26 cells was confirmed using FLIM, and total RNA was extracted for gene expression profiling. The validation of relative transcript levels was carried out via real-time semiquantitative RT‒PCR assays. Our results demonstrated that chemotherapy induced the differential expression of mediators of cancer cell dormancy and cell survival-related genes and downregulation of both intrinsic and extrinsic apoptotic signalling pathways. Despite the fact that some differentially expressed genes, such as BMP7 and Prss11, have not been thoroughly studied in the context of chemoresistance thus far, they might be potential candidates for future studies on overcoming drug resistance.

Показатели нового полуколичественного экспресс-теста на прокальцитонин у пациентов с внебольничной пневмонией при наличии и отсутствии артериальной гипертензии

The presence of cardiovascular diseases can be an independent risk factor for an unfavorable prognosis of community-acquired pneumonia (CAP) and cause its erased course, which significantly complicates the timely diagnosis. It has been reported that assessment of plasma procalcitonin (PCT) levels may be useful in the differential diagnosis of lung diseases. Aim. To study the levels of a new semi-quantitative rapid PCT test (PCT RT) of plasma in therapeutic patients with CAP depending on the presence or absence of arterial hypertension (AH). Design. The simultaneous comparative study. Materials and methods. The study included 123 patients hospitalized with a diagnosis of CAP: 41 patients (mean age 69.8 ± 11.9 years) with hypertension and 82 patients (mean age 39.3 ± 16.7 years) without hypertension, who made up the comparison group. Using a new semi-quantitative RT and the method of enzyme-linked immunosorbent assay, the plasma PCT level was determined before the start of antibiotic therapy. Results. In patients with CAP and hypertension, positive PCT RT results were registered 1.8 times more often than in the comparison group — 19 (46.3%) people and 21 (25.6 %) people, respectively (p = 0.018), borderline values of PCT RT — 2.2 times more often: in 13 (31.7%) people and 12 (14.6%) people (p = 0.001) patients, respectively. The comparison group was characterized by negative PCT RT values, which were recorded in 61 (74.4%) of cases (p = 0.001). Quantitative PCT scores did not have statistically significant differences in the groups studied. In the group of patients with CAP and hypertension, positive correlations of PCT RT with body temperature (r = 0.329; p = 0.038), quantitative PCT (r = 0.505; p = 0.001), plasma glucose levels (r = 0.325; p = 0.041) and potassium (0.325; p = 0.041) were established. Conclusion. Patients with CAP and hypertension in comparison with those without hypertension are characterized by positive values of the new PCT RT, which have positive correlations with body temperature, quantitative PCT values, and plasma glucose and potassium levels. Keywords: community-acquired pneumonia, arterial hypertension, diagnostics, procalcitonin, procalcitonin rapid test.

Prefoldin proteins 2/6, and HMG20B are regulated by HDAC1, HDAC3 and are novel therapeutic and prognostic biomarkers in hepatocellular carcinoma

Epigenetic mechanisms, such as histone deacetylases (HDACs), play an important role in the commencement and development of hepatocellular carcinoma (HCC). Previously, we have identified proteins with binds with HDAC1 and HDAC3 in liver cancer cells and also have shown that depletion of either HDAC1 or HDAC3 suppressed the expression of HDAC1/3 interacting proteins, including the prefoldin protein 2/6 (PFDN2/6), CR4-NOT transcription complex subunit 1 (CNOT1), and high mobility group 20B (HMG20b). In this study, online databases were utilized to analyze the expression of HDAC1/3, PFDN2/6, CNOT1, and HMG20b in a large panel of liver cancer cell lines, cancer tissues, and human normal and tumor liver tissues. These databases are “RNA Expression Atlas (https://www.ebi.ac.uk/gxa/home), Cancer Genome Atlas (TCGA), gene expression profiling interactive analysis (GEPIA), integrative molecular databases of hepatocellular carcinoma (HCCDB), human protein atlas, and Kaplan-Meier Plotter for RNA sequences in liver cancer (http://kmplot.com/analysis/index.php?p=service&cancer=liver_rnaseq#). The expression of these genes was verified experimentally in human HepG2 cells via semi-quantitative RT PCR. The results showed that all these genes are expressed in twenty-three human liver cancer cell lines, higher expression in human liver cancer than normal tissues. However, HDAC1 and PFDN2 are expressed at higher levels than other genes analyzed in this study. The analysis of these six genes in 364 human liver cancer patients by Kaplan Meier plotter predicted HDAC1 and PFDN2 as poor, while HMG20b as a favorable prognostic biomarker in hepatocellular carcinoma. PFDN2 and HMG20b are novel prognostic markers of hepatocellular carcinoma, identified first in this study. Further clinical studies are needed to verify the expression and patient survival concerning the expresion of PFDN2 and HMG20b in human hepatocellular carcinoma patients.

Unravelling lncRNA mediated gene expression as potential mechanism for regulating secondary metabolism in Citrus limon

Plant long non-coding RNAs (lncRNAs) are emerging as important regulators of gene expression through various mechanisms viz. binding to double-stranded DNA (dsDNA), physically targeting mRNAs and also act as microRNA (miRNA) sponges. Characteristic secondary metabolites and their content determine the market value of Citrus plant products for culinary and industrial uses. Though lncRNAs are emerging as important molecular players that regulate secondary metabolites in plants but the roles of lncRNAs are yet unknown for the regulation of secondary metabolites in Citrus limon. In this study, a total of 11814 lncRNAs were identified from four RNA-seq datasets comprising of data from four different tissues of C. limon viz. leaf, bud, fruit and peel. Co-expression analysis of whole set of genes with identified lncRNAs revealed a total of 632 lncRNAs correlating with 5810 genes. Functional annotation of genes correlating with lncRNAs unveiled the association of 379 genes with the biosynthesis of secondary metabolite pathways and among them, 36 and 12 genes are from terpenoids and flavonoids metabolism/biosynthesis pathways respectively. Expression correlation analysis unfolded the possible role of 113 lncRNAs in terpenoids metabolism and 29 lncRNAs in the flavonoids metabolic pathway. Seventy nine lncRNAs were predicted to take part in miRNA-mediated gene regulation, out of which 10 lncRNAs were predicted to act as endogenous target mimics (eTMs) for miRNAs and 74 lncRNAs were found to act as miRNA targets. Expression of six predicted lncRNAs and seven correlated genes involved in terpenoids and flavonoids metabolism were validated using semi-quantitative RT- PCR.

Expression pattern of transcriptional enhanced associate domain family member 1 (Tead1) in developing mouse molar tooth

The Hippo pathway is essential for determining organ size by regulating cell proliferation. Previous reports have shown that impairing this pathway causes abnormal tooth development. However, the precise expression profile of the members of the transcriptional enhanced associate domain family (Tead), which are key transcription factors mediating Yap function, during tooth development is unclear. In this study, among the four isoforms of Tead (Tead1 - 4), only the expression of Tead1 mRNA was observed using semiquantitative RT- PCR in murine developing tooth germ at E16.5. The expression level of Tead1 mRNA in the excised murine mandibular molar tooth germ was significantly higher at E16.5 than at other developmental stages, as determined using quantitative PCR. We found that the mRNA expression of connective tissue growth factor (Ctgf), which is one of the Yap target genes directly controlling cell growth, changed consistently with that of Tead1 in developing molars. Fluorescent immunostaining revealed that Tead1 protein was expressed in both epithelial cells and mesenchymal cells of the dental lamina and dental epithelium, including the primary enamel knot during the cap stage. During the early bell stage (E16.5), Tead1 was expressed intensely in the inner and outer enamel epithelium, including the secondary enamel knot and the neighboring mesenchymal cells. Tead1 then specifically localized to the inner and outer enamel epithelium, which is responsible for enamel formation during the bell stage. These expression patterns were consistent with those of Yap, Taz, and Ctgf protein in developing molars. These results suggest that Tead1 acts as a mediator of the biological functions of Yap, such as the morphogenesis of cusp formation, during tooth development.

Characterization and Expression Analysis of Resistance Gene Analogues in Elite Sugarcane Genotypes.

Resistance Gene Analogues (RGAs) are an important source of disease resistance in crop plants and have been extensively studied for their identification, tagging and mapping of Quantitative Trait Loci (QTLs). Tracking these RGAs in sugarcane can be of great help for the selection and screening of disease resistant clones. In the present study expression of different Resistance Gene Analogues (RGAs) was assessed in indigenous elite sugarcane genotypes which include resistant, highly resistant, susceptible and highly susceptible to disease infestation. Total cellular DNA and RNA were isolated from fourteen indigenous elite sugarcane genotypes. PCR, semi-quantitative RT PCR and real time qPCR analyses were performed. The resultant amplicons were sequence characterized, chromosomal localization and phylogenetic analysis were performed. All of the 15 RGA primers resulted in amplification of single or multiple fragments from genomic DNA whereas only five RGA primers resulted in amplification from cDNA. Sequence characterization of amplified fragments revealed 86-99% similarity with disease resistance proteins indicating their potential role in disease resistance response. Phylogenetic analysis also validated these findings. Further, expression of RGA-012, RGA-087, RGA-118, RGA-533 and RGA-542 appeared to be upregulated and down regulated in disease resistant and susceptible genotypes, respectively, after inoculation with Colletotrichum falcatum. RGAs are present in most of our indigenous genotypes. Anyhow, differential expression of five RGAs indicated that they have some critical role in disease resistance. So, the retrieved results can not only be employed to devise molecular markers for the screening of disease resistant genotypes but can also be used to develop disease resistant plants through transgenic technology.

Identification of novel fusion transcripts in meningioma.

Meningiomas are the most common primary intracranial tumor. Recent next generation sequencing analyses have elaborated the molecular drivers of this disease. We aimed to identify and characterize novel fusion genes in meningiomas. We performed a secondary analysis of our RNA sequencing data of 145 primary meningioma from 140 patients to detect fusion genes. Semi-quantitative rt-PCR was performed to confirm transcription of the fusion genes in the original tumors. Whole exome sequencing was performed to identify copy number variations within each tumor sample. Comparative RNA seq analysis was performed to assess the clonality of the fusion constructs within the tumor. We detected six fusion events (NOTCH3-SETBP1, NF2-SPATA13, SLC6A3-AGBL3, PHF19-FOXP2 in two patients, and ITPK1-FBP2) in five out of 145 tumor samples. All but one event (NF2-SPATA13) led to extremely short reading frames, making these events de facto null alleles. Three of the five patients had a history of childhood radiation. Four out of six fusion events were detected in expression type C tumors, which represent the most aggressive meningioma. We validated the presence of the RNA transcripts in the tumor tissue by semi-quantitative RT PCR. All but the two PHF19-FOXP2 fusions demonstrated high degrees of clonality. Fusion genes occur infrequently in meningiomas and are more likely to be found in tumors with greater degree of genomic instability (expression type C) or in patients with history of cranial irradiation.

Early intervention with breast milk mesenchymal stem cells attenuates the development of diabetic-induced testicular dysfunction via hypothalamic Kisspeptin/Kiss1r-GnRH/GnIH system in male rats

Diabetic male infertility and sub fertility are major complications that may implicate both central and peripheral pathways as well as mechanisms controlling reproduction. This study was an attempt to explore the potential effect of breast milk mesenchymal stem cells (Br-MSCs) as a therapeutic tool for diabetic induced reproductive dysfunction at molecular level. Forty-five adult male Sprague Dawely rats were divided into 3 groups (n = 15); control group, diabetic group, and Br-MSCs treated diabetic group. The homing ability of Br-MSCs in diabetic treated rat testicles was confirmed via semi-quantitative RT- PCR analysis of a human specific Gapdh mRNA expression level. Our result showed that type1 diabetic rats exerted an elevation in blood glucose level and a reduction in body weight, fasting serum insulin, FSH, LH, and total testosterone levels, relative and absolute testicular weights, sperm count, motility, and live ratio. In addition, downregulation in the hypothalamic kisspeptin-GnRH system, HPG axis and testicular steroidogenesis compared to control group was noticed. Moreover, upregulation of testicular proinflammatory and apoptotic markers relative mRNA expression compared to control group was observed. Furthermore, a decrease in testicular tissue antioxidant activity (CAT, SOD, GSH) and an increase in lipid peroxidation (MDA) compared to control group was shown. However, Br-MSCs administration restored or even exceeded the normal physiological tone in most of these parameters to the point where a potential therapeutic role for Br-MSCs in type1diabetic induced infertility can be suggested.

The mechanism and tumor inhibitory study of Lagopsis supine ethanol extract on colorectal cancer in nude mice

BackgroundThis study was aimed to determination the tumor inhibitory effect and explore the potential mechanisms of Lagopsis supine ethanol extract (Ls) on colorectal cancer.MethodsThe cell growth inhibition experiment of Ls in colorectal cancer cell lines was determined by MTT method in the time course of 24, 48 and 72 h in four gradient drug concentrations. The protein expression levels of pSTAT3, pJAK2, STAT3, JAK2, Bcl-2 and caspase 3 were measured by Western blot method. The mRNA levels of the downstream genes of STAT3 were detected through semi-quantitative RT PCR. Sixty Balb/c-nude mice were xenograft with HCT116 colorectal cancer cells through subcutaneously. The xenografts were divided into five groups: model group, positive group (capecitabine 300 mg/kg) and three dosages of Ls treated groups (75, 150 and 300 mg/kg). Tumor size and tumor weight were calculated for evaluation the anti-tumor effects. H & E staining and immunohistochemical analysis were used to determine the histopathological changes and the levels of pSTAT3 and pJAK2 in the tumor tissues.ResultsLs exhibited a significant anti-proliferation effect in HCT116 and SW480 cells in vitro. The protein levels of pSTAT3, pJAK2 and Bcl-2, and the mRNA levels of Bcl-2 and Bak notably reduced with a dose-dependent manner. While the protein levels of caspase 3, and mRNA levels of Bax and caspase-3 remarkably increased in the gradient dosage of Ls in HCT116 cells. HCT116 in vivo xenografts experiment showed that the growth of the tumors significantly inhibited by Ls administration, which with no any significant body weight changes in each experiment group. The histopathology analysis displayed that Ls significantly reduced the inflammatory cells in tumor tissue. Furthermore, Ls also significantly down-regulate the protein levels of pSTAT3 and pJAK2 in the tumor tissues, compared with the model group.ConclusionsThis work shows that Ls inhibited the cell proliferation of colorectal cancer in vitro and significantly reduced the tumor growth in HCT116 xenografts in vivo, which is probably related with the JAK/STAT signal pathway.

The non-coding RNA rprA can increase the resistance to ampicillin in Escherichia coli

ObjectivesThe non-coding RNA rprA can increase the resistance to ampicillin in Escherichia coli. MethodsBacterial DNA was extracted by boiling method and then amplified using polymerase chain reaction (PCR) with two different primer sets. Recombinant pET28a/rprA-sense and -antisense plasmids were separately transferred into the competent E. coli BL21 (DE3) by chemical methods using heat shock. The expression was analyzed at the RNA level using Semi quantitative RT PCR. The turbidity difference between the bacteria was checked by Broth Dilution method. ResultsThe statistical analysis showed that the turbidity difference between the up regulated and control bacteria is significant (p value < 0.0001). The ANOVA test also showed the significant difference between the down regulated and control bacteria (p value < 0.0001). ConclusionConsidering this mechanism, there are some reports indicating the role of rprA in antibiotic resistance. However, the role of rprA in ampicillin resistance is remained to be unknown. The aim of this study was to analyze the up regulation and down regulation of rprA and check their effects on ampicillin resistance in Escherichia coli. It was found that the up regulation and down regulation of rprA can lead into more antibiotics resistance and susceptibility, respectively. Our results showed the potential role of rprA expression in the response to ampicillin stress in E. coli.

Differentially expressed alternatively spliced genes in skeletal muscle from cancer patients with cachexia.

BackgroundAlternative splicing (AS) is a post‐transcriptional gene regulatory mechanism that contributes to proteome diversity. Aberrant splicing mechanisms contribute to various cancers and muscle‐related conditions such as Duchenne muscular dystrophy. However, dysregulation of AS in cancer cachexia (CC) remains unexplored. Our objectives were (i) to profile alternatively spliced genes (ASGs) on a genome‐wide scale and (ii) to identify differentially expressed alternatively spliced genes (DASGs) associated with CC.MethodsRectus abdominis muscle biopsies obtained from cancer patients were stratified into cachectic cases (n = 21, classified based on International consensus diagnostic framework for CC) and non‐cachectic controls (n = 19, weight stable cancer patients). Human transcriptome array 2.0 was used for profiling ASGs using the total RNA isolated from muscle biopsies. Representative DASG signatures were validated using semi‐quantitative RT–PCR.ResultsWe identified 8960 ASGs, of which 922 DASGs (772 up‐regulated and 150 down‐regulated) were identified at ≥1.4 fold‐change and P < 0.05. Representative DASGs validated by semi‐quantitative RT–PCR confirmed the primary findings from the human transcriptome arrays. Identified DASGs were associated with myogenesis, adipogenesis, protein ubiquitination, and inflammation. Up to 10% of the DASGs exhibited cassette exon (exon included or skipped) as a predominant form of AS event. We also observed other forms of AS events such as intron retention, alternate promoters.ConclusionsOverall, we have, for the first time, conducted global profiling of muscle tissue to identify DASGs associated with CC. The mechanistic roles of the identified DASGs in CC pathophysiology using model systems is warranted, as well as replication of findings in independent cohorts.

Correlative Studies Unravelling the Possible Mechanism of Cell Death in Tideglusib-Treated Human Ovarian Teratocarcinoma-Derived PA-1 Cells.

This study aims to unravel the use of GSK-3 inhibitors as viable apoptotic inducers for teratocarcinoma-derived ovarian PA-1 cells. MTT assay was carried out to assess inhibitory concentrations of LiCl and TDG. AO/EB staining and Hoechst 33258 staining were employed to assess the damage. Mitochondrial membrane potential (ΔΨm) and ROS generation were assessed with IC50 concentrations of LiCl and TDG. Tumor-related genes (p53, p21, IL-8, TNF-α, MMP-2, Fas-L, Cox-2, and caspase-3) were assessed with 1/4 IC50, 1/2 IC50, IC50 concentrations by semi-quantitative RT- PCR. Cell cycle analysis was performed with IC50 concentration of LiCl and TDG. Western blot analysis was performed for caspase-3, caspase-7, caspase-9, PARP to estimate the possible damage induced by GSK-3 inhibitors and regulation of GSK-3β, pGSK-3β, Cox-2. GSK-3 inhibitors demonstrated a concentration and time-dependent reduction in cell viability, exhibiting significant ROS generation and reduced ΔΨm at their IC50 values. Substantial concentration-dependent gene expression changes with significant upregulation of P21, Cox-2, TNF-α, caspase-3, Fas-L were observed. Protein expression of caspase-3 caspase-7, caspase-9, PARP exhibited significant cleavage in LiCl and TDG-treated cells. Protein expression of Cox-2 was significantly increased in IC50 concentration of TDG. Cell cycle analysis showed significant accumulation of cells at sub-G0-G1.

Downregulation of the DST Transcription Factor Using Artificial microRNA to Increase Yield, Salt and Drought Tolerance in Rice

Abiotic stresses like salinity and drought directly affect plant growth and water availability, resulting in lower yield in rice. So, a combination of stress tolerance along with enhanced grain yield is a major focus of rice breeding. It was reported earlier that loss in function of the drought and salt tolerance (DST) gene results in increase in grain production through downregulating Gn1a/OsCKX2 expression. Moreover, dst mutants also showed enhanced drought and salt tolerance in rice by regulating genes involved in ROS homeostasis. In the present study, we proceeded to test these reports by downregulating DST using artificial microRNA technology in the commercial but salt sensitive, high-yielding, BRRIdhan 28 (BR28). This cultivar was transformed with DST_artificial microRNA (DST_amiRNA) driven by the constitutive CaMV35S promoter using tissue culture independent Agrobacterium mediated in planta transformation. DST_amiRNA transgenic plants were confirmed by artificial microRNA specific PCR. Transformed plants at T0 generation showed vigorous growth with significantly longer panicle length and higher primary branching resulting in higher yield, compared to the wild type (WT) BR28. Semi-quantitative RT PCR confirmed the decrease in DST expression in the BR28 transgenic plants compared to WT. T1 transgenic plants also showed improvement in a number of physiological parameters and greater growth compared to WT after 14 days of 120 mM salt (NaCl) stress at seedling stage. Therefore, DST downregulated transgenic plants showed both higher stress tolerance as well as better yields. Furthermore, stable inheritance of the improved phenotype of the DST_amiRNA transgenics will be tested in advanced generations.

Structure, evolution, and expression of antimicrobial silk proteins, seroins in Lepidoptera

The silks of silkworm and waxworm contain abundant antimicrobial proteins, including protease inhibitors and seroins. Protease inhibitors have antifungal activities, whereas seroins have antiviral and antibacterial activities. In order to obtain insights into the structure, evolution, and expression of seroins, we performed an extensive survey based on the available genome, transcriptome, and expressed sequence tags datasets. Sixty-four seroins were identified in 32 lepidopteran species. The phylogenetic and structural analyses revealed that seroins can be classified into five subfamilies: seroin 1, seroin 2, seroin 3, seroin 2 + 1, and seroin 3 + 3. It is interesting that seroin 2 + 1 contains two tandem seroin domains, seroin 2 and seroin 1, whereas seroin 3 + 3 has two tandem seroin 3 domains. Each seroin domain contains a proline-rich N-terminal motif and a conserved C-terminal motif. The transcriptome and EST data indicated that seroin 1 and seroin 2 genes were expressed in the silk gland but seroin 3 genes were not. Semi-quantitative RT–PCR and western blot analyses suggested that seroin 1 and seroin 2 were constantly accumulated in the silk gland of silkworm during the fifth instar, and then secreted into cocoon silk during spinning. Immunofluorescence analyses indicated that seroin 1 was secreted into the fibroin and sericin layers, whereas seroin 2 protein was only secreted into the sericin layer. However, the antimicrobial activity of seroin 2 was more effective than that of seroin 1. The presence of seroin 1 in the fibroin layer suggested that this protein not only acts as an antimicrobial protein, but might also play a role in the assembly and secretion of fibroins. Seroin 3, which was first identified here, might be related to pheromone synthesis or recognition, as it was highly expressed in male antennae and in the pheromone gland.

Constitutive Overexpression of the Plasma Membrane Na+/H+ Antiporter for Conferring Salinity Tolerance in Rice

At the cellular level, the Salt Overly Sensitive (SOS) signaling pathway comprising SOS3, SOS2, and SOS1 has been proposed to mediate cellular signaling under salt stress to maintain ion (Na+) homeostasis. In this regulatory pathway, both OsSOS1 encoding plasma membrane and OsNHX1 encoding vacuolar Na+/H+ antiporters are regulated by SOS3?SOS2 protein kinase complex. In the present study, the rice variety BRRI dhan28 - which is popular with farmers and high yielding, but salt sensitive, was transformed with the OsSOS1 gene isolated from salt tolerant Pokkali rice and driven by the constitutive promoter, CaMV35S. The construct was transformed through a tissue culture-independent Agrobacteriummediated in planta transformation method that circumvents the problems associated with tissue culture-based indica rice transformation methods. Integration of the foreign genes (OsSOS1) into the genome of transgenic plants was confirmed by gene-specific PCR and Southern blot analysis. The level of transgene expression (SOS1) was also quantified by semi-quantitative RT PCR and real time PCR. Genetic segregation ratio for T1 progenies was calculated and found to follow the Mendelian law of inheritance in case of positive transformants. The transformants were shown to be salt tolerant compared to wild type in molecular analysis as well as physiological screening. Future work will involve transformation of both the OsSOS1 and OsNHX1 genes together; with the expectation for enhancing the tolerance level compared to currently available transgenic rice.Plant Tissue Cult. &amp; Biotech. 25(2): 257-272, 2015 (December)

Interferon-γ Upregulates SOCS3 Expression to Reduce Cisplatin Chemoresistance in Non-Small Cell Lung Cancer A549 Cells

Abstract Background: Cisplatin (DDP)-based chemotherapy is the mainstay of first-line therapeutic strategy for the treatment of advanced non-small cell carcinoma (NSCLC). However, the anticancer efficacy of DDP is often limited by the existence or development of chemoresistance. Thus, we investigated the effect of interferon-γ on DDPresistant A549 cell. Methods: Semi-quantitative RT–PCR was used to compare the differences of SOCS3 mRNA expression in both cisplatin-resistant A549 (A549/DDP) cell and the parental A549 cell. The cellular sensitivity to cisplatin, cell viability and apoptosis were detected by MTT, flow cytometry and Western blotting. Results: Semi-quantitative RT–PCR and western blotting showed that SOCS 3 expression was significantly down-regulated in A549/DDP cell compared to the parental A549 and normal human bronchial epithelial cells BEAS-2B. IFN-γ treatment could restore SOCS3 expression, resulting in an increased sensitivity of these resistant A549 cells to DDP. In addition, p53 and Bcl-2 signaling pathways were also involved in regulating IFN-γ-induced cell death in DDP-resistant A549 cells. Conclusion: Our study indicates that SOCS 3 may play a crucial role in the progress of cisplatin resistance of A549. Moreover, IFN-γ could induce SOCS3 expression and potentiate the re-sensitization of these resistant A549 cells to DDP

Effect of altitude on some male fertility related traits in Saudi ovine and caprine species

Abstract This study aimed to evaluate the effect of altitude on some male fertility related traits in Saudi sheep and goats. Testicular tissues were collected from a slaughterhouse in Taif governorate (1800 m above sea level) and Makkah governorate (sea level). Sperm characteristics (1 - individual motility, 2 - percent live sperm 3 - percent abnormal sperm) were examined. Semi-quantitative RT -PCR assay was used to evaluate the expression of IGF-II, StAR, LDLr and CYP11A genes. No significant effect of altitude on tested sperm parameters was revealed. Expression of IGF-II gene in both sheep and goats was significantly (P&lt;0.05) higher at sea level compared to high altitude. A similar effect of altitude on StAR gene expression was only observed in goats, while in sheep the level of effect did not reach the significance threshold. Moreover, LDLr gene expression was significantly (P&lt;0.05) higher for both sheep and goats at high altitude than at sea level. The CYP11A gene expression was significantly (P&lt;0.05) higher in high altitude sheep than in those raised at sea level, while the opposite trend was observed for goats. In conclusion, high altitude had an effect on the expression of some studied male fertility related genes, but sperm parameters were not significantly affected.

Effects of NaCl Stress on Nitrogen Metabolism of Cucumber Seedlings

To investigate the effects of NaCl stress on plant growth, nitrogen assimilation, proline and sol uble protein contents, and gene expression of enzymes of nitrogen metabolism, a hydroponic experiment using cucumber (Cucumis sativus L.) seedlings was performed. The seedlings were grown in nutrient solu tion supplemented with 0 or 84 mM NaCl for up to 9 days. Plant biomass, especially root biomass, was sig nificantly decreased under NaCl stress. Salinity significantly increased ammonium content, but decreased nitrate and soluble protein contents in leaves and roots. Salt stress caused a significant increase in proline content, which peaked on day 3 of NaCl treatment. Moreover, salt stress significantly decreased activities of nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) in the roots and leaves on the 3rd, 6th, and 9th day of NaCl treatment. A semi quantitative RT PCR approach showed that changes in NR, GS and GOGAT gene expression were consis tent with the salt induced changes in enzyme activities. These results suggest that salt stress induced growth inhibition in cucumber seedlings may involve disruption of nitrogen absorption and decreased activities of enzymes associated with nitrogen assimilation.

Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding, salicylic acid treatment, and salinity stress in safflower, Carthamus tinctorius.

Safflower (Carthamus tinctorius L.) serves as a reference dicot for investigation of defence mechanisms in Asteraceae due to abundant secondary metabolites and high resistance/tolerance to environmental stresses. In plants, phenylpropanoid and flavonoid pathways are considered as two central defence signalling cascades in stress conditions. Here, we describe the isolation of two major genes in these pathways, CtPAL (phenylalanine ammonia-lyase) and CtCHS (chalcone synthase) in safflower along with monitoring their expression profiles in different stress circumstances. The aa (amino acid) sequence of isolated region of CtPAL possesses the maximum identity up to 96% to its orthologue in Cynara scolymus, while that of CtCHS retains the highest identity to its orthologue in Callistephus chinensis up to 96%. Experiments for gene expression profiling of CtPAL and CtCHS were performed after the treatment of seedlings with 0.1 and 1 mM SA (salicylic acid), wounding and salinity stress. The results of semi-quantitative RT–PCR revealed that both CtPAL and CtCHS genes are further responsive to higher concentration of SA with dissimilar patterns. Regarding wounding stress, CtPAL gets slightly induced upon injury at 3 hat (hours after treatment) (hat), whereas CtCHS gets greatly induced at 3 hat and levels off gradually afterward. Upon salinity stress, CtPAL displays a similar expression pattern by getting slightly induced at 3 hat, but CtCHS exhibits a biphasic expression profile with two prominent peaks at 3 and 24 hat. These results substantiate the involvement of phenylpropanoid and particularly flavonoid pathways in safflower during wounding and especially salinity stress.