Expression of GroES TB antigen in tobacco and potato

As the world races towards a plant-based bioeconomy, plants known to be ideal and economical bioreactors are being harnessed for the production of recombinant proteins. The major immunodominant 10 kDa GroES TB antigen (Chaperonin 10) gene from Mycobacterium tuberculosis was selected for expression in plants as a putative tuberculosis (TB) subunit vaccine candidate. Two crops, tobacco and potato, were engineered by stable plant transformation for expression of the 10 kDa GroES TB antigen using non-viral binary vectors. The integration of the GroES TB gene into the genomes of tobacco and potato was confirmed by PCR and Southern blotting. The expression of the GroES TB antigen in tobacco was 0.04–1.2 % of the total soluble protein (TSP). However, the expression of the same TB antigen in the Indian potato cv. Kufri bahar was comparatively low (0.033 % of TSP). The recombinant GroES plant derived protein was characterised and confirmed by MALDI-TOF–TOF and ELISA. This is the first report of the expression of the 10 kDa chaperonin in tobacco and potato.     

Transarterial chemoembolization combined with sorafenib for unresectable hepatocellular carcinoma: a systematic review and meta-analysis

Sorafenib in combination with Transarterial chemoembolization (TACE) is increasingly used in patients with unresectable hepatocellular carcinoma (HCC), but the current evidence is still controversial. The aim of this systematic review was to evaluate the effectiveness and safety of TACE plus sorafenib versus TACE alone for unresectable HCC. We searched PubMed, EMBASE and the Cochrane Library for clinical trials comparing TACE plus sorafenib with TACE alone for unresectable HCC. The study outcomes included overall survival (OS), time to progression (TTP), objective response and adverse events (AEs). Six studies including 1,181 patients were included. Meta-analysis of all studies suggested that the combination therapy group had significant longer OS than TACE group [hazard ratio (HR) = 0.64, 95 % confidence interval (CI) = 0.43–0.97], but the pooled HR of randomized controlled trials (RCTs) failed to achieve statistical significance. For TTP, meta-analysis in both RCTs subgroup and retrospective studies subgroup suggested that combination therapy was superior to TACE group. The combination therapy was also associated with better response to treatment (risk ratio = 1.45, 95 % CI = 1.04–2.02) when both RCTs and retrospective studies were pooled. However, the sorafenib associated AEs were more frequent in the combination therapy group. In conclusion, the combination of TACE and sorafenib is likely to improve OS, TTP and response to treatment when compared with TACE monotherapy. The combination group is also associated with more sorafenib-related AEs.     

Molecular cloning,structural analysis,and expression of zona pellucida glycoprotein ZP3 gene from Chinese zokor,Myospalax fontanierii

The zona pellucida 3 (ZP3) plays a crucial role in reproductive immunology. We obtained a full-length cDNA encoding Chinese zokor ZP3, using rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The cDNA contains an open reading frame of 1269 nucleotides encoding a polypeptide of 422 amino acid residues. The amino acid sequence has a high degree of homology with those of hamster (78%), mouse (76%), and rat (74%). XhoI and SacI sites after restriction give an1158 bp fragment of zokor ZP3 cDNA, excluding the signal sequence and transmembrane-like domain, which was cloned under the phage T7 promoter-lac operator control in the pET-28a(+) vector. Recombinant pET-zokorZP3(r-ZP3) was expressed as a poly-histidine fusion protein in E. coli strain BL21 (DE3). Optimum expression of r-ZP3 was observed at 28°C, 1 mM IPTG and 2 h of inducing. The purified protein was tested by Western blot.     

A Systems Biology Approach to Study the Biology Characteristics of Esophageal Squamous Cell Carcinoma by Integrating microRNA and Messenger RNA Expression Profiling

Esophageal squamous cell carcinoma (ESCC) is one of the most malignant tumors. The aim of this study was to investigate the biology characteristics of ESCC by analyzing microRNA and mRNA expression profile. We used BRB-array tools to analyze the deregulated microRNA and mRNA between esophageal squamous cell carcinomas and paired normal adjacent tissues. We used miRTrail and protein–protein interaction methods to explore the related pathways and networks of deregulated microRNA and mRNA. By combining the results of pathways and networks, we found that the deregulated microRNA and their deregulated target mRNA are enriched in the following pathways: DNA replication, cell cycle, ECM-receptor interaction, focal adhesion, mismatch repair, and pathways in cancer. The results showed that many deregulated microRNAs and mRNAs may play a vital role in the pathogenesis of ESCC, and the systems biology approach is very helpful to explore molecular mechanism of ESCC.     

Circulating MicroRNA Biomarkers for Glioma and Predicting Response to Therapy

The need for glioma biomarkers with improved sensitivity and specificity has sparked research into short non-coding RNA known as microRNA (miRNA). Altered miRNA biogenesis and expression in glioma plays a vital role in important signaling pathways associated with a range of tumor characteristics including gliomagenesis, invasion, and malignancy. This review will discuss current research into the role of miRNA in glioma and altered miRNA expression in biofluids as candidate biomarkers with a particular focus on glioblastoma, the most malignant form of glioma. The isolation and characterization of miRNA using cellular and molecular biology techniques from the circulation of glioma patients could potentially be used for improved diagnosis, prognosis, and treatment decisions. We aim to highlight the links between research into miRNA function, their use as biomarkers, and how these biomarkers can be used to predict response to therapy. Furthermore, increased understanding of miRNA in glioma biology through biomarker research has led to the development of miRNA therapeutics which could restore normal miRNA expression and function and improve the prognosis of glioma patients. A panel of important miRNA biomarkers for glioma in various biofluids discovered to date has been summarized here. There is still a need, however, to standardize techniques for biomarker characterization to bring us closer to clinically relevant miRNA-based diagnostic and therapeutic signatures. A clinically validated biomarker panel has potential to improve time to diagnosis, predicting response to treatment and ultimately the prognosis of glioma patients.     

GABA Pharmacology: The Search for Analgesics

Decades of research have been devoted to defining the role of GABAergic transmission in nociceptive processing. Much of this work was performed using rigid, orthosteric GABA analogs created by Povl Krogsgaard-Larsen and his associates. A relationship between GABA and pain is suggested by the anatomical distribution of GABA receptors and the ability of some GABA agonists to alter nociceptive responsiveness. Outlined in this report are data supporting this proposition, with particular emphasis on the anatomical localization and function of GABA-containing neurons and the molecular and pharmacological properties of GABA_A and GABA_B receptor subtypes. Reference is made to changes in overall GABAergic tone, GABA receptor expression and activity as a function of the duration and intensity of a painful stimulus or exposure to GABAergic agents. Evidence is presented that the plasticity of this receptor system may be responsible for the variability in the antinociceptive effectiveness of compounds that influence GABA transmission. These findings demonstrate that at least some types of persistent pain are associated with a regionally selective decline in GABAergic tone, highlighting the need for agents that enhance GABA activity in the affected regions without compromising GABA function over the long-term. As subtype selective positive allosteric modulators may accomplish these goals, such compounds might represent a new class of analgesic drugs.     

Inhibition of the JAK/STAT pathway with ruxolitinib overcomes cisplatin resistance in non-small-cell lung cancer NSCLC

This study was aimed to elucidate the roles of inhibition of related JAK/STAT pathways in regulating cytotoxicity induced by cisplatin in non-small-cell lung cancer (NSCLC) cell. We treated five non-small-cell lung cancer cell lines with cisplatin alone or with cisplatin and Jak2 inhibitor (ruxolitinib) and assessed cell viability, expression of Jak2 and STAT3 and cell apoptosis. We also investigated the effect of combination treatment inhibited tumor xenograft growth in two human NSCLC xenograft models bearing the cisplatin resistant (H1299) and sensitive (A549) cells. Different cell lines with different genetic background showed half-maximal inhibitory concentrations (IC₅₀) of cisplatin from 4.66 to 68.28 µmol/L. They could be divided into cisplatin intrinsic resistant and cisplatin sensitive cell lines. In cisplatin-resistant cells with higher Jak2 and STAT3 expression, cisplatin and ruxolitinib combination dramatically suppressed the cell growth, down-regulated the expression of phosphorylated STAT3 and induced cleaved caspase-3 expression. Moreover combination with cisplatin and ruxolitinib also significantly inhibited the growth of resistant cell H1299, A549/DDP and H2347 in soft agar model. Finally, combination group significant inhibited the tumor growth and induced the caspase-3 expression compared with either single agent alone (P < 0.05) on the resistant cell xenografts model. The present study indicates that further study is warranted to determine the effectiveness of combination treatment with cisplatin and Jak2/stat3 pathway inhibitor for platinum-resistant NSCLC.     

Progression of O6-methylguanine-DNA methyltransferase and temozolomide resistance in cancer research

Temozolomide (TMZ) is an alkylating agent that is widely used in chemotherapy for cancer. A key mechanism of resistance to TMZ is the overexpression of O⁶-methylguanine-DNA methyltransferase (MGMT). MGMT specifically repairs the DNA O⁶-methylation damage induced by TMZ and irreversibly inactivates TMZ. Regulation of MGMT expression and research regarding the mechanism of TMZ resistance will help rationalize the clinical use of TMZ. In this review, we provide an overview of recent advances in the field, with particular emphasis on MGMT structure, function, expression regulation, and the association between MGMT and resistance to TMZ.     

Cathepsin B is involved in the heat shock induced cardiomyocytes apoptosis as well as the anti-apoptosis effect of HSP-70

Cathepsin B is one of the major lysosomal cysteine proteases that plays an important role in apoptosis. Herein, we investigated whether Cathepsin B is involved in cardiomyocyte apoptosis caused by hyperthermic injury (HI) and heat shock protein (HSP)-70 protects these cells from HI-induced apoptosis mediated by Cathepsin. HI was produced in H9C2 cells by putting them in a circulating 43 °C water bath for 120 min, whereas preinduction of HSP-70 was produced in H9C2 cells by mild heat preconditioning (or putting them in 42 °C water bath for 30 min) 8 h before the start of HI. It was found that HI caused both cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. E-64-c, in addition to reducing Cathepsin B activity, significantly attenuated HI-induced cardiomyocyte apoptosis (evidenced by increased apoptotic cell numbers, increased tuncated Bid (t-Bid), increased cytochrome C, increased caspase-9/-3, and decreased Bcl-2/Bax) in H9C2 cells. In addition, preinduction of HSP-70 by mild heat preconditioning or inhibition of HSP-70 by Tripolide significantly attenuated or exacerbated respectively both the cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. Furthermore, the beneficial effects of pre-induction of HSP-70 by mild heat production in reducing both cardiomyocyte apoptosis and increased Cathepsin B activity caused by HI can be significantly reduced by Triptolide preconditioning. These results indicate that Cathepsin B is involved in HI-induced cardiomyocyte apoptosis in H9C2 cells and HSP-70 protects these cells from HI-induced cardiomyocyte apoptosis through Cathepsin B pathways.     

The GABAA Antagonist DPP-4-PIOL Selectively Antagonises Tonic over Phasic GABAergic Currents in Dentate Gyrus Granule Cells

GABA_A receptors mediate two different types of inhibitory currents: phasic inhibitory currents when rapid and brief presynaptic GABA release activates postsynaptic GABA_A receptors and tonic inhibitory currents generated by low extrasynaptic GABA levels, persistently activating extrasynaptic GABA_A receptors. The two inhibitory current types are mediated by different subpopulations of GABA_A receptors with diverse pharmacological profiles. Selective antagonism of tonic currents is of special interest as excessive tonic inhibition post-stroke has severe pathological consequences. Here we demonstrate that phasic and tonic GABA_A receptor currents can be selectively inhibited by the antagonists SR 95531 and the 4-PIOL derivative, 4-(3,3-diphenylpropyl)-5-(4-piperidyl)-3-isoxazolol hydrobromide (DPP-4-PIOL), respectively. In dentate gyrus granule cells, SR 95531 was found approximately 4 times as potent inhibiting phasic currents compared to tonic currents (IC₅₀ values: 101 vs. 427 nM). Conversely, DPP-4-PIOL was estimated to be more than 20 times as potent inhibiting tonic current compared to phasic current (IC₅₀ values: 0.87 vs. 21.3 nM). Consequently, we were able to impose a pronounced reduction in tonic GABA mediated current (>70 %) by concentrations of DPP-4-PIOL, at which no significant effect on the phasic current was seen. Our findings demonstrate that selective inhibition of GABA mediated tonic current is possible, when targeting a subpopulation of GABA_A receptors located extrasynaptically using the antagonist, DPP-4-PIOL.     

NDRG1 expression is related to the progression and prognosis of gastric cancer patients through modulating proliferation,invasion and cell cycle of gastric cancer cells

N-myc downstream-regulated gene 1 (NDRG1) has been proposed as a tumor suppressor gene in many different types of tumors, but its potential function and corresponding mechanism are not yet fully elucidated. This study aims to detect the possible function of NDRG1 in gastric cancer progression. In this study, 112 paired gastric cancer tissues and corresponding nonmalignant gastric tissues were utilized to identify the differential protein expression of NDRG1 by immunohistochemistry and its clinical significance was analyzed. Furthermore, 49 of 112 paired gastric specimens were used to detect the differential mRNA expression by real-time PCR. The over expression of NDRG1 in human gastric cancer cell line AGS by PcDNA3.1–NDRG1 transfection was utilized to detect the role of NDRG1 in regulating the biological behavior of gastric cancer. NDRG1 expression was significantly decreased in primary gastric cancer tissues, compared with its corresponding nonmalignant gastric tissues (p < 0.05), and its decreased expression was significantly associated with lymph node metastasis (p < 0.01), invasion depth (p < 0.01) and differentiation (p < 0.05). Additionally, the overall survival rate of gastric cancer patients with high expression of NDRG1 was higher than those with low expression during the follow-up period. NDRG1 overexpression suppressed cells proliferation, invasion and induced a G1 cell cycle arrest in gastric cancer. Furthermore, the down-regulation of NDRG1 in gastric cancer metastatic progression was correlated to E-cadherin and MMP-9. Our results verify that NDRG1 acts as a tumor suppressor gene and may play an important role in the metastasis progression and prognosis of gastric cancer.     

A Pilot Study on the Contribution of Folate Gene Variants in the Cognitive Function of ADHD Probands

Genetic abnormalities in components important for the folate cycle confer risk for various disorders since adequate folate turnover is necessary for normal methylation, gene expression and chromosome structure. However, the system has rarely been studied in children diagnosed with attention deficit hyperactivity disorder (ADHD). We hypothesized that ADHD related cognitive deficit could be attributed to abnormalities in the folate cycle and explored functional single nucleotide polymorphisms in methylenetetrahydrofolate dehydrogenase (rs2236225), reduced folate carrier (rs1051266), and methylenetetrahydrofolate reductase (rs1801131 and rs1801133) in families with ADHD probands (N = 185) and ethnically matched controls (N = 216) recruited following the DSM-IV. After obtaining informed written consent for participation, peripheral blood was collected for genomic DNA isolation and PCR-based analysis of target sites. Data obtained was analyzed by UNPHASED. Interaction between sites was analyzed by the multi dimensionality reduction (MDR) program. Genotypic frequencies of the Indian population were strikingly different from other ethnic groups. rs1801133 “T” allele showed biased transmission in female probands (p < 0.05). Significant difference in genotypic frequencies for female probands was also noticed. rs1801131 and rs1801133 showed an association with low intelligence quotient (IQ). MDR analysis exhibited independent effects and contribution of these sites to IQ, thus indicating a role of these genes in ADHD related cognitive deficit.     

Functions of EDS1-like and PAD4 genes in grapevine defenses against powdery mildew

The molecular interactions between grapevine and the obligate biotrophic fungus Erysiphe necator are not understood in depth. One reason for this is the recalcitrance of grapevine to genetic modifications. Using defense-related Arabidopsis mutants that are susceptible to pathogens, we were able to analyze key components in grapevine defense responses. We have examined the functions of defense genes associated with the salicylic acid (SA) pathway, including ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), EDS1-LIKE 2 (EDL2), EDL5 and PHYTOALEXIN DEFICIENT 4 (PAD4) of two grapevine species, Vitis vinifera cv. Cabernet Sauvignon, which is susceptible to E. necator, and V. aestivalis cv. Norton, which is resistant. Both VaEDS1 and VvEDS1 were previously found to functionally complement the Arabidopsis eds1-1 mutant. Here we show that the promoters of both VaEDS1 and VvEDS1 were induced by SA, indicating that the heightened defense of Norton is related to its high SA level. Other than Va/VvEDS1, only VaEDL2 complemented Arabidopsis eds1-1, whereas Va/VvPAD4 did not complement Arabidopsis pad4-1. Bimolecular fluorescence complementation results indicated that Vitis EDS1 and EDL2 proteins interact with Vitis PAD4 and AtPAD4, suggesting that Vitis EDS1/EDL2 forms a complex with PAD4 to confer resistance, as is known from Arabidopsis. However, Vitis EDL5 and PAD4 did not interact with Arabidopsis EDS1 or PAD4, correlating with their inability to function in Arabidopsis. Together, our study suggests a more complicated EDS1/PAD4 module in grapevine and provides insight into molecular mechanisms that determine disease resistance levels in Vitis species native to the North American continent.