Effect of different 3’ flanking regions on the activity of the Vitis vinifera alcohol dehydrogenase 2 promoter

3’untranslated regions (3' UTR) are isogene specific regions which contain sequences likely playing an important role in gene expression. To evaluate the importance of these regions on Vitis vinifera alcohol dehydrogenase 2 (VvAdh2) expression, we designed expression cassettes containing the luciferase reporter gene under the VvAdh2 or CaMV 35S promoters and flanked by different 3’ UTRs. Luciferase activity monitoring was performed through transient expression experiments, using biolistic on Cabernet Sauvignon suspension cells. Results showed that absence of the 3’ region had a strong down-regulating effect on the VvAdh2 promoter activity (but not on the CaMV 35S promoter activity). The nature of the flanking 3' UTR was shown to influence expression cassette activity. Whatever the promoter, VvAdh1 and VvAdh2 terminators had similar effect on expression of luciferase in air leading to an activity level very close to that of CaMV 35S terminator. Under anaerobiosis, luciferase expression was strongly increased with all terminators, VvAdh6 3'-end inducing the highest level of expression. Functional constructs with VvAdh2 promoter and VvAdh terminators designed in this study could be used wherever grapevine-homologous, stress-stimulated cassettes should be of interest

Analysis of the transcript levels of VvAdh1, VvAdh2 and VvGrip4, three genes highly expressed during Vitis vinifera L. berry development

Well defined promoter sequences are required for the targeted expression of genes in transgenic grapevines. This paper describes a detailed study of the expression of three genes with potentially useful promoters. We have used real-time RT-PCR to evaluate the transcript levels of VvAdh1, VvAdh2 and VvGrip4 genes in various tissues (root, bud, tendril, inflorescence, fruit and embryogenic callus) of Vitis vinifera L. at different stages of development. Transcript levels of the three genes were highest in berry tissues but each had a distinct pattern of expression. VvAdh1 showed higher transcript levels during the early stages of berry development, while levels of VvAdh2 and VvGrip4 were higher during ripening. However, none of these genes was expressed in a strictly fruit-specific manner. In particular, significant levels of VvAdh1 and VvGrip4 transcripts were observed during late tendril development and in the inflorescence, respectively. Transcript levels of all three genes were similar in both flesh and skin, indicating no tissue-specificity within the berry. Promoter sequences of the VvAdh1, VvAdh2 and VvGrip4 genes could be very useful to drive ectopic gene expression in berries of transgenic plants

Ethylene and other stimuli affect expression of the UDP glucose-flavonoid 3-O-glucosyltransferase in a non-climacteric fruit

The UDP glucose-flavonoid 3-O-glucoslyltransferase (UFGT) is a key enzyme for biosynthesis and stability of anthocyanin pigments of red grapes. Understanding factors affecting expression of this enzyme is thus important for the control of grape colour. A 1640 bp promoter region of the grapevine ufgt gene was cloned and sequenced. Sequence analysis revealed seven putative ethylene-responsive cis-elements and others related to three major signals known to induce anthocyanin accumulation in plant tissues: light, sugar, and abscisic acid. In order to evaluate the ability of ethylene and other signals to drive expression from the ufgt promoter, we ran transient expression experiments using an anthocyanin-rich grape cell culture, with very low green auto-fluorescence. After biolistic bombardment, the cells were treated with various combinations of the four signals on gfp expression (green fluorescent protein). The comparison of fluorescent light intensity in cells subjected to the various treatments showed that ethylene better stimulates expression of the ufgt promoter in the dark than under light. In addition, results showed that there may be a positive interaction between ethylene and abscisic acid. This system, a promoter of interest driving the gfp expression in cells with low auto-fluorescence, may be a good tool for studies about synergistic or antagonist roles of transcription factors. Moreover, treatment of grape berries with a specific inhibitor of ethylene receptors (1-methylcyclopropene) inhibited ufgt mRNA accumulation. This confirms that the ethylene signal is likely a regulator of grape UFGT expression in a non-climacteric fruit.

Cellular localisation of VvRops and VvRabA5e, small GTPases developmentally regulated in grape berries

VvRops, in particular VvRop9, and VvRabA5e are small GTPases which are developmentally regulated in grape berries. In an attempt to help elucidate the role of these proteins during fruit development and ripening, we investigated their localisation in the fruit by immunocytofluorescence. These proteins were observed at a perinuclear location, at cell periphery and around vesicles. In particular VvRops were found to be located in the nucleus and likely on the plasma membrane. VvRop9 and VvRabA5e cDNAs were introduced separately into S. cerevisiae mutants with RHO1 and YPT31/YPT32 defective genes respectively. Neither cDNAs could complement these temperature-sensitive mutants, suggesting that the functions of the VvRop9 and VvRabA5e genes in grapevine likely differ from the functions of RHO1 and YPT31/YPT32 genes in yeast.

Short-course antibiotic therapy for critically ill patients treated for postoperative intra-abdominal infection: the DURAPOP randomised clinical trial

PURPOSE: Shortening the duration of antibiotic therapy (ABT) is a key measure in antimicrobial stewardship. The optimal duration of ABT for treatment of postoperative intra-abdominal infections (PIAI) in critically ill patients is unknown. METHODS: A multicentre prospective randomised trial conducted in 21 French intensive care units (ICU) between May 2011 and February 2015 compared the efficacy and safety of 8-day versus 15-day antibiotic therapy in critically ill patients with PIAI. Among 410 eligible patients (adequate source control and ABT on day 0), 249 patients were randomly assigned on day 8 to either stop ABT immediately (n = 126) or to continue ABT until day 15 (n = 123). The primary endpoint was the number of antibiotic-free days between randomisation (day 8) and day 28. Secondary outcomes were death, ICU and hospital length of stay, emergence of multidrug-resistant (MDR) bacteria and reoperation rate, with 45-day follow-up. RESULTS: Patients treated for 8 days had a higher median number of antibiotic-free days than those treated for 15 days (15 [6-20] vs 12 [6-13] days, respectively; P < 0.0001) (Wilcoxon rank difference 4.99 days [95% CI 2.99-6.00; P < 0.0001). Equivalence was established in terms of 45-day mortality (rate difference 0.038, 95% CI - 0.013 to 0.061). Treatments did not differ in terms of ICU and hospital length of stay, emergence of MDR bacteria or reoperation rate, while subsequent drainages between day 8 and day 45 were observed following short-course ABT (P = 0.041). CONCLUSION: Short-course antibiotic therapy in critically ill ICU patients with PIAI reduces antibiotic exposure. Continuation of treatment until day 15 is not associated with any clinical benefit. CLINICALTRIALS. GOV IDENTIFIER: NCT01311765

Primary Effusion Lymphoma Cell Death Induced by Bortezomib and AG 490 Activates Dendritic Cells through CD91

To understand how cytotoxic agent-induced cancer cell death affects the immune system is of fundamental importance to stimulate immune response to counteract the high mortality due to cancer. Here we compared the immunogenicity of Primary Effusion Lymphoma (PEL) cell death induced by anticancer drug Bortezomib (Velcade) and Tyrphostin AG 490, a Janus Activated Kinase 2/signal trasducer and activator of transcription-3 (JAK2/STAT3) inhibitor. We show that both treatments were able to induce PEL apoptosis with similar kinetics and promote dendritic cells (DC) maturation. The surface expression of molecules involved in immune activation, namely calreticulin (CRT), heat shock proteins (HSP) 90 and 70 increased in dying cells. This was correlated with DC activation. We found that PEL cell death induced by Bortezomib was more effective in inducing uptake by DC compared to AG 490 or combination of both drugs. However the DC activation induced by all treatments was completely inhibited when these cells were pretreated with a neutralizing antiboby directed against the HSP90/70 and CRT common receptor, CD91. The activation of DC by Bortezomib and AG 490 treated PEL cells, as seen in the present study, might have important implications for a combined chemo and immunotherapy in such patients

A pilot study on the immunogenicity of dendritic cell vaccination during adjuvant oxaliplatin/capecitabine chemotherapy in colon cancer patients

Contains fulltext : 87604.pdf (publisher's version ) (Closed access)BACKGROUND: Dendritic cell (DC) vaccination has been shown to induce anti-tumour immune responses in cancer patients, but so far its clinical efficacy is limited. Recent evidence supports an immunogenic effect of cytotoxic chemotherapy. Pre-clinical data indicate that the combination of chemotherapy and immunotherapy may result in an enhanced anti-cancer activity. Most studies have focused on the immunogenic aspect of chemotherapy-induced cell death, but only few studies have investigated the effect of chemotherapeutic agents on the effector lymphocytes of the immune system. METHODS: Here we investigated the effect of treatment with oxaliplatin and capecitabine on non-specific and specific DC vaccine-induced adaptive immune responses. Stage III colon cancer patients receiving standard adjuvant oxaliplatin/capecitabine chemotherapy were vaccinated at the same time with keyhole limpet haemocyanin (KLH) and carcinoembryonic antigen (CEA)-peptide pulsed DCs. RESULTS: In 4 out of 7 patients, functional CEA-specific T-cell responses were found at delayed type hypersensitivity (DTH) skin testing. In addition, we observed an enhanced non-specific T-cell reactivity upon oxaliplatin administration. KLH-specific T-cell responses remained unaffected by the chemotherapy, whereas B-cell responses were diminished. CONCLUSION: The results strongly support further testing of the combined use of specific anti-tumour vaccination with oxaliplatin-based chemotherapy

Association of Inherited Variation in Toll-Like Receptor Genes with Malignant Melanoma Susceptibility and Survival

The family of Toll-like receptors (TLRs) is critical in linking innate and acquired immunity. Polymorphisms in the genes encoding TLRs have been associated with autoimmune diseases and cancer. We investigated the genetic variation of TLR genes and its potential impact on melanoma susceptibility and patient survival. The study included 763 cutaneous melanoma cases recruited in Germany and 736 matched controls that were genotyped for 47 single nucleotide polymorphisms (SNPs) in 8 TLR genes. The relationship between genotype, disease status and survival was investigated taking into account patient and tumor characteristics, and melanoma treatment. Analysis of 7 SNPs in TLR2, 7 SNPs in TLR3 and 8 SNPs in TLR4 showed statistically significant differences in distribution of inferred haplotypes between cases and controls. No individual polymorphism was associated with disease susceptibility except for the observed tendency for TLR2-rs3804099 (odds ratio OR  = 1.15, 95% CI 0.99–1.34, p = 0.07) and TLR4-rs2149356 (OR = 0.85, 95% CI 0.73–1.00, p = 0.06). Both polymorphisms were part of the haplotypes associated with risk modulation. An improved overall survival (Hazard ratio HR 0.53, 95% CI 0.32–0.88) and survival following metastasis (HR 0.55, 95% CI 0.34–0.91) were observed in carriers of the variant allele (D299G) of TLR4-rs4986790. In addition various TLR2, TLR4 and TLR5 haplotypes were associated with increased overall survival. Our results point to a novel association between TLR gene variants and haplotypes with melanoma survival. Our data suggest a role for the D299G polymorphism in the TLR4 gene in overall survival and a potential link with systemic treatment at stage IV of the disease. The polymorphic amino acid residue, located in the ectodomain of TLR4, can have functional consequences