Effect of gamma radiation on the expression of mRNA growth factors in glycerol cryopreserved human amniotic membrane

Human amniotic membrane (HAM) due to its high biocompatibility, low immunogenicity, anti-microbial, anti-viral properties as well as the presence of growth factors has been used in various clinical applications. The growth factors play an important role in wound healing. The current study aimed to explore the effect of 15 kGy gamma radiation dose on selected growth factors and receptors mRNA present in HAM. Eight growth factors, namely, EGF, HGF, KGF, TGF-α, TGF-β1, TGF-β2, TGF-β3 and bFGF and two growth factor receptors, HGFR and KGFR were evaluated in this study. The total RNA was extracted and converted to complimentary DNA using commercial kits. Subsequently, the mRNA expressions of these growth factors were evaluated using real-time PCR and the results were statistically analyzed using REST-MCS software. This study confirmed the presence of these mRNA growth factors and receptors in fresh, glycerol cryopreserved and irradiated glycerol cryopreserved HAM. In glycerol cryopreserved HAM, the results showed up-regulation of HGF and bFGF and down-regulation of EGF, HGFR, KGF, KGFR, TGF-α, TGF-β1, TGF-β2 and TGF-β3 relative to the fresh HAM which acted as the control, whereas in irradiated glycerol cryopreserved HAM, the results showed up-regulation of EGF, HGF, KGF, KGFR, TGF-β1, TGF-β2 and TGF-β3 and down-regulation of HGFR, TGF-α and bFGF relative to the glycerol cryopreserved HAM which acted as the control. However, these mRNA expressions did not show any statistical significant difference compared to the control groups. This study concluded that a dose of 15 kGy of gamma radiation did not affect the mRNA expression for the growth factors’ and receptors’ in the glycerol cryopreserved HAM.     

Interferon-mediated antiviral activities of Angelica tenuissima Nakai and its active components

Angelica tenuissima Nakai is a widely used commodity in traditional medicine. Nevertheless, no study has been conducted on the antiviral and immune-modulatory properties of an aqueous extract of Angelica tenuissima Nakai. In the present study, we evaluated the antiviral activities and the mechanism of action of an aqueous extract of Angelica tenuissima Nakai both in vitro and in vivo. In vitro, an effective dose of Angelica tenuissima Nakai markedly inhibited the replication of Influenza A virus (PR8), Vesicular stomatitis virus (VSV), Herpes simplex virus (HSV), Coxsackie virus, and Enterovirus (EV-71) on epithelial (HEK293T/HeLa) and immune (RAW264.7) cells. Such inhibition can be described by the induction of the antiviral state in cells by antiviral, IFNrelated gene induction and secretion of IFNs and pro-inflammatory cytokines. In vivo, Angelica tenuissima Nakai treated BALB/c mice displayed higher survivability and lower lung viral titers when challenged with lethal doses of highly pathogenic influenza A subtypes (H1N1, H5N2, H7N3, and H9N2). We also found that Angelica tenuissima Nakai can induce the secretion of IL-6, IFN-λ, and local IgA in bronchoalveolar lavage fluid (BALF) of Angelica tenuissima Nakai treated mice, which correlating with the observed prophylactic effects. In HPLC analysis, we found the presence of several compounds in the aqueous fraction and among them; we evaluated antiviral properties of ferulic acid. Therefore, an extract of Angelica tenuissima Nakai and its components, including ferulic acid, play roles as immunomodulators and may be potential candidates for novel anti-viral/anti-influenza agents.     

Bone marrow mesenchymal stem cell transplantation improves radiation-induced heart injury through DNA damage repair in rat model

Radiotherapy is an effective form of therapy for most thoracic malignant tumors. However, myocardial injury resulting from the high doses of radiation is a severe complication. Here we aimed to study the possibility of reducing radiation-induced myocardial injury with mesenchymal stem cell (MSC) transplantation. We used MSCs extracted from bone marrow (BMSCs) to transplant via the tail vein into a radiation-induced heart injury (RIHI) rat model. The rats were divided into six groups: a Sham group, an IRR (irradiation) group, and four IRR + BMSCs transplantation groups obtained at different time points. After irradiation, BMSC transplantation significantly enhanced the cardiac function in rats. By analyzing the expression of PPAR-α, PPAR-γ, TGF-β, IL-6, and IL-8, we found that BMSC transplantation alleviated radiation-induced myocardial fibrosis and decreased the inflammatory reaction. Furthermore, we found that expression of γ-H2AX, XRCC4, DNA ligase4, and TP53BP1, which are associated with DNA repair, was up-regulated, along with increased secretion of growth factors SDF-1, CXCR4, VEGF, and IGF in rat myocardium in the IRR + BMSCs transplantation groups compared with the IRR group. Thus, BMSC transplantation has the potential to improve RIHI via DNA repair and be a new therapeutic approach for patients with myocardial injury.     

The Effects of Supplementary Cr3 (Chromium(III) Propionate Complex) on the Mineral Status in Healthy Female Rats

Circulating concentration of the essential trace element selenium (Se) was significantly lower in inflammatory disorders. Although Se plays physiological roles mainly through the function of 25 selenoproteins, the response of the selenogenome in immune tissues during inflammatory reactions remains unclear. The objective of this study was to determine the Se retention and selenogenome expression in immune tissues during the lipopolysaccharide (LPS)-induced inflammatory response in porcine. A total of 12 male pigs were randomly divided into two groups and injected with LPS or saline. After 4 h postinjection, blood samples were collected and pigs were euthanized. Pigs challenged with LPS had 36.8 and 16.6 % lower (P < 0.05) Se concentrations in the serum and spleen, respectively, than those injected with saline. Moreover, the activities of GPX decreased (P < 0.05) by 23.4, 26.6, and 30.4 % in the serum, thymus, and lymph node, respectively, in the pigs injected with LPS. Furthermore, the LPS challenge altered (P < 0.05) the mRNA expression of 14, 16, 10, and 6 selenoprotein genes in the liver, spleen, thymus, and lymph node, respectively. Along with 10 previously reported selenoprotein genes, the response of Txnrd2, Txnrd3, Sep15, Selh, Seli, Seln, Selo, Selt, Selx, and Sephs2 to inflammatory reaction in immune tissues were newly illustrated in this study. In conclusion, the LPS-induced inflammatory response impaired Se metabolism and was associated with dysregulation of the selenogenome expression in immune tissues.     

Expression of <Emphasis Type="Italic">Tlr2</Emphasis>, <Emphasis Type="Italic">Defa</Emphasis>, and <Emphasis Type="Italic">Muc2</Emphasis> genes in rat duodenum epithelial cells during prolonged stomach hypoacidity and after hypoacidity correction by multiprobiotics

Expression of Tlr2, Defa, and Muc2 genes in epithelial cells of rat duodenum during prolonged stomach hypoacidity (hypochlorhydria) and after hypoacidity correction by multiprobiotics has been investigated. Elevation of Tlr2, Muc2, and Defa gene expression levels upon the intensification of lipid peroxidation processes in the epithelial cells of the villi and crypts of the rat duodenum under hypoacidic conditions has been demonstrated. Administration of a multiprobiotic under the same conditions downregulated the expression of the aforementioned genes in epithelial cells of the villi and crypts, bringing it to a nearly normal level, and exerted a similar effect on the levels of lipid peroxidation products. The data obtained may be indicative of the involvement of Tlr2, Muc2, and Defa genes in the development of duodenal inflammation induced by dysbiotic changes occurring in prolonged hypochlorhydria.     

Cytotoxic and toxicogenomic effects of silibinin in bladder cancer cells with different <Emphasis Type="Italic">TP53</Emphasis> status

Silibinin is a natural phenol found in the seeds of the milk thistle plant. Recent data have shown its effectiveness for preventing/treating bladder tumours. Therefore, in this study we investigated the cytotoxic and toxicogenetic activity of silibinin in bladder cancer cells with different TP53 statuses. Two bladder urothelial carcinoma cell lines were used: RT4 (wild-type TP53 gene) and T24 (mutated TP53 gene). Cell proliferation, clonogenic survival, apoptosis rates, genotoxicity and relative expression profile of FRAP/mTOR, FGFR3, AKT2 and DNMT1 genes and of miR100 and miR203 were evaluated. Silibinin promoted decreased proliferation and increased late apoptosis in TP53 mutated cells. Increased early apoptosis rates, primary DNA damage, and decrease of cell colonies in the clonogenic survival assay were detected in both RT4 and T24 cell lines. Down-regulation of FRAP/mTOR, AKT2, FGFR3, DNMT1 and miR100 expression occurred in RT4 cells. Modulation of miR203 was observed in both cell lines. In conclusion, despite the reduction of clone formation in both cell lines, the toxicogenomic effect of silibinin on FRAP/mTOR, AKT2, FGFR3, DNMT1 and miR100 was dependent on the TP53 status. Taken together, the data confirmed the role of silibinin as an antiproliferative compound, whose mechanism of action was related to the TP53 status.     

Contributions of <Emphasis Type="Italic">TaSUTs</Emphasis> to grain weight in wheat under drought

Key message

The homologous genes to OsSUT1-5 in wheat were identified and detailed analysed. TaSUT1 was the predominant sucrose transporter group and it illustrated the genotypic variations towards drought during grain filling.

Abstract

Sucrose transporters (SUT) play crucial roles in wheat stem water soluble carbohydrate (WSC) remobilization to grain. To determine the major functional SUT gene groups in shoot parts of wheat during grain development, drought tolerant varieties, Westonia and Kauz, were investigated in field drought experiments. Fourteen homologous genes to OsSUT1-5 were identified on five homeologous groups, namely TaSUT1_4A, TaSUT1_4B, TaSUT1_4D; TaSUT2_5A, TaSUT2_5B, TaSUT2_5D; TaSUT3_1A, TaSUT3_1D; TaSUT4_6A, TaSUT4_6B, TaSUT4_6D; TaSUT5_2A, TaSUT5_2B, and TaSUT5_2D, and their gene structures were analysed. Wheat plants above the ground were harvested from pre-anthesis to grain maturity and the stem, leaf sheath, rachis, lemma and developing grain were used for analysing TaSUT gene expression. Grain weight, thousand grain weight, kernel number per spike, biomass and stem WSC were characterized. The study showed that among the five TaSUT groups, TaSUT1 was the predominant sucrose transporting group in all organs sampled, and the expression was particularly high in the developing grain. In contrast to TaSUT1, the gene expression levels of TaSUT2, TaSUT3 and TaSUT4 were lower, except for TaSUT3 which showed preferential expression in the lemma before anthesis. The TaSUT5 gene group was very weakly expressed in all tissues. The upregulated gene expression of TaSUT1 Westonia type in stem and grain reveal a crucial role in stem WSC remobilization to grain under drought. The high TaSUT1 gene expression and the significant correlations with thousand grain weight (TGW) and kernel number per spike demonstrated the contribution in Kauz’s high grain yield in an irrigated environment and high TGW in Westonia under drought stress. Further molecular level identification is required for gene marker development.

     

Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Gene Expression Analysis in Rice Plants Exposed to Metal Stresses

Environmental pollution by toxic heavy metals may lead to the possible contamination of the rice plant (Oryza sativa L.). Although gene expression analysis through real-time quantitative PCR (RT-qPCR) has increased our knowledge about biological responses to heavy metals, gene network that mediates rice plant responses to heavy metal stress remains elusive. In such scenario, validation of reference gene is a major requirement for successful analyzes involving RT-qPCR. In this study, we analyzed the expression stability of eight commonly used housekeeping genes (GAPDH, Actin, eIF-4α, UBQ 5, UBQ 10, UBC, EF-1α and β-TUB) in rice leaves exposed to four kinds of heavy metals (Zn, Cu, Cd and Pb). The expression stability of these genes was determined using geNorm, NormFinder, BestKeeper and RefFinder algorithms. The results showed that UBQ 10 and UBC were the most stable reference genes across all the tested samples. We measured the expression profiles of the heavy metal-inducible gene O. sativa METALLOTHIONEIN2b (OsMT2b) using the two most stable and one least stable reference genes in all samples. The relative expression of OsMT2b varied greatly according to the different reference genes. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in rice plants.     

Selection of reference genes for quantitative real-time PCR in <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> under salt stress

Real time quantitative PCR (qPCR) is widely used in gene expression analysis for its accuracy and sensitivity. Reference genes serving as endogenous controls are necessary for gene normalization. In order to select an appropriate reference gene to normalize gene expression in Casuarina equisetifolia under salt stress, 10 potential reference genes were evaluated using real time qPCR in the leaves and roots of plants grown under different NaCl concentrations and treatment durations. GeNorm, NormFinder, and BestKeeper analyses reveal that elongation factor 1-alpha (EF1α) and ubiquitin-conjugating enzyme E2 (UBC) were the most appropriate reference genes for real time qPCR under salt stress. However, β-tubulin (βTUB) and actin 7, which were widely used as reference genes in other plant species, were not always stably expressed. The combination of EF1α, UBC, uncharacterized protein 2, DNAJ homolog subfamily A member 2, and glyceraldehyde-3-phosphate dehydrogenase should be ideal reference genes for normalizing gene expression data in all samples under salt stress. It indicates the need for reference gene selection for normalizing gene expression in C. equisetifolia. In addition, the suitability of reference genes selected was confirmed by validating the expression of WRKY29-like and expansin-like B1. The results enable analysis of salt response mechanism and gene expression in C. equisetifolia.     

The poplar ARGOS-LIKE gene promotes leaf initiation and cell expansion,and controls organ size

We identified a Populus nigra auxin-regulated gene involved in organ size (PnARGOS)-LIKE, encoding one organ size related protein in black poplar. It is homologous to AtARGOS and AtARGOS-LIKE genes of Arabidopsis thaliana. ABRE-like, G-box, GATA and I-box motifs were discovered in the promoter region of the poplar ARGOS-LIKE gene. In wild type aspen (Populus tremula) plants, an ortholog of the PnARGOS-LIKE gene (PtrARGOS-LIKE) was noticeably expressed in actively dividing and expanding young leaves and calli, whereas its mRNA content increased in response to exogenous 6-benzylaminopurine, 1-naphthaleneacetic acid, and 24-epibrassinolide. Expression of the PtrARGOS-LIKE gene was reduced under a salinity treatment. In addition, we generated transgenic tobacco and aspen plants with an up-regulated expression of the PnARGOS-LIKE gene. A constitutive expression of the gene contributed to an increase in size of stems and leaves of the transgenic tobacco plants. In the transgenic aspen, a constitutive expression of the PnARGOS-LIKE gene promoted an increase in the frequency of leaf initiations and in leaf length and area. The size of transgenic tobacco and aspen leaves increased due to the enlargement of individual cells. The results show the significance of the PnARGOS-LIKE gene for control of leaf initiation and organ growth by cell expansion in poplar.     

Construction of heterologous gene expression cassettes for the development of recombinant <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

Gene-expression cassettes for the construction of recombinant Clostridium beijerinckii were developed as potential tools for metabolic engineering of C. beijerinckii. Gene expression cassettes containing ColE1 origin and pAMB origin along with the erythromycin resistance gene were constructed, in which promoters from Escherichia coli, Lactococcus lactis, Ralstonia eutropha, C. acetobutylicum, and C. beijerinckii are examined as potential promoters in C. beijerinckii. Zymogram analysis of the cell extracts and comparison of lipase activities of the recombinant C. beijerinckii strains expressing Pseudomonas fluorescens tliA gene suggested that the tliA gene was functionally expressed by all the examined promoters with different expression level. Also, recombinant C. beijerinckii expressing C. beijerinckii secondary alcohol dehydrogenase by the constructed expression cassettes successfully produced 2-propanol from glucose. The best promoter for TliA expression was the R. eutropha phaP promoter while that for 2-propanol production was the putative C. beijerinckii pta promoter. Gene expression cassettes developed in this study may be useful tools for the construction of recombinant C. beijerinckii strains as host strains for the valuable chemicals and fuels from renewable resources.     

Connection between proliferation rate and temozolomide sensitivity of primary glioblastoma cell culture and expression of <Emphasis Type="Italic">YB-1</Emphasis> and <Emphasis Type="Italic">LRP/MVP</Emphasis>

Glioblastomas (GBL) are the most common and aggressive brain tumors. They are distinguished by high resistance to radiation and chemotherapy. To find novel approaches for GBL classification, we obtained 16 primary GBL cell cultures and tested them with real-time PCR for mRNA expression of several genes (YB-1, MGMT, MELK, MVP, MDR1, BCRP) involved in controlling cell proliferation and drug resistance. The primary GBL cultures differed in terms of proliferation rate, wherein a group of GBL cell cultures with low proliferation rate demonstrated higher resistance to temozolomide. We found that GBL primary cell cultures characterized by high proliferation rate and lower resistance to temozolomide expressed higher mRNA level of the YB-1 and MDR1 genes, whereas upregulated expression of MVP/LRP mRNA was a marker in the group of GBL with low proliferation rate and high resistance. A moderate correlation between expression of YB-1 and MELK as well as YB-1 and MDR1 was found. In the case of YB-1 and MGMT expression, no correlation was found. A significant negative correlation was revealed between mRNA expression of MVP/LRP and MELK, MDR1, and BCRP. No correlation in expression of YB-1 and MVP/LRP genes was observed. It seems that mRNA expression of YB-1 and MVP/LRP may serve as a marker for GBL cell cultures belonging to distinct groups, each of which is characterized by a unique pattern of gene activity.