Grapevine root transformation with Agrobacterium rhizogenes

Grapevine shoots were obtained from tissue cultures of cvs Barbera, Moscato bianco and Nebbiolo; their stems were wounded and inoculated with the following strains of Agrobacterium rhizogenes. A4, 8196, NCPP 2659, and 15834. Root production at the inoculation site was obtained in about 25% of the stems of all cultivars with all the bacterial strains, with the exception of NCPP 2659, which gave a lower root proliferation rate. Cultures of roots obtained after inoculation were established and their growth was enhanced by some media and by addition of cytokinins and auxins to the medium. Root cultures obtained after inoculation with the strain 8196 were genetically transformed, as shown by opine production. In contrast, opines were seldom detected in the root cultures obtained with the other bacterial strains. Light microscope observation showed that the cortex of transformed roots has more cell layers and a larger average cell size than in normal roots

Improved control of water loss from micropropagated grapevines (Vitis vinifera cv. Nebbiolo)

Micropropagated plantlets are generally susceptible to rapid desiccation when exposed to reduced relative humidity and require a costly acclimatization procedure. Detached leaves of micropropagated Vitis vinifera, cv. Nebbiolo, plantlets were used to evaluate the relative contribution of leaf cuticle and stomata to water loss. Water loss occurred mainly from the abaxial surface of detached leaves; moreover, a large majority of stomata was still open 3 h after exposure to 63 % RH. An indirect estimation of epicuticular wax suggested a lower wax deposition for micropropagated plantlets compared to acclimatized and field-grown plants of the same clone. A previously developed method to produce hardened micropropagated plants was adopted: 1 mg(.)l(-1) paclobutrazol (PBZ) was added to the medium and culture vessels with reduced relative humidity (rRH) were used during the last stage of micropropagation. Under our experimental conditions, rRH was more effective to reduce transpiration than PBZ; a combination of both treatments improved plant survival during acclimatization.

In ovulo embryo culture of stenospermocarpic grapes

Ovules of 14 seedless cultivars, collected 41-49 d after anthesis, were excised and in vitro cultured on 2 different media: NN + 1 µM GA3 + 10 µM IAA and NN + 1 µM GA3 + 20 µM IAA + 2 g/l activated charcoal. Plant development occurred at variable rates; the best percentages were found in Flame Seedless, Perlon, Imperatrice, Carina, Perlette and Ruby Seedless. Genotype resulted to be the main factor affecting ovule response to culture, but also the medium and the interaction genotype x medium influenced it. The addition of 2 g/l activated charcoal to the medium, tested on the cv. Perlette, and the type of added auxin (IBA, IAA, NAA), tested on the cvs Perlette and Sultanina, enhanced plantlet development

High efficiency somatic embryogenesis and plant germination in grapevine cultivars Chardonnay and Brachetto a grappolo lungo

A highly efficient, reproducible method for somatic embryogenesis induction, plant recovery and embryogenic culture preservation has been developed for cvs Chardonnay and Brachetto a grappolo lungo (Vitis vinifera), starting from immature anthers and ovaries. Embryogenic induction efficiency was 2 % and 17 % in anthers for Chardonnay and Brachetto g.l., respectively, and 14 % in ovaries for both cultivars. Embryogenic cultures of both genotypes are still propagating 3.5 years after the initial induction and are still morphogenic. Embryo conversion into plantlets occurred at suitable efficiencies during a 100 d culture for both Chardonnay (37 % and 15 %) and Brachetto g.l. (30 % and 29 %), in the two media tested. Organogenesis was also obtained from cotyledonary leaves of Chardonnay.

The Antifungal Drug Isavuconazole Inhibits the Replication of Human Cytomegalovirus (HCMV) and Acts Synergistically with Anti-HCMV Drugs

We recently reported that some clinically approved antifungal drugs are potent inhibitors of human cytomegalovirus (HCMV). Here, we report the broad-spectrum activity against HCMV of isavuconazole (ICZ), a new extended-spectrum triazolic antifungal drug. ICZ inhibited the replication of clinical isolates of HCMV as well as strains resistant to the currently available DNA polymerase inhibitors. The antiviral activity of ICZ against HCMV could be linked to the inhibition of human cytochrome P450 51 (hCYP51), an enzyme whose activity we previously demonstrated to be required for productive HCMV infection. Moreover, time-of-addition studies indicated that ICZ might have additional inhibitory effects during the first phase of HCMV replication. Importantly, ICZ showed synergistic antiviral activity in vitro when administered in combination with different approved anti-HCMV drugs at clinically relevant doses. Together, these results pave the way to possible future clinical studies aimed at evaluating the repurposing potential of ICZ in the treatment of HCMV-associated diseases

Adjuvant vaginal interventional radiotherapy in early-stage non-endometrioid carcinoma of corpus uteri: a systematic review

Purpose: This systematic review focused on rare histological types of corpus uteri malignancy, including uterine carcinosarcoma (UCS), uterine clear cell carcinoma (UCCC), and uterine papillary serous carcinoma (UPSC), and it is proposed to assist with clinical decision-making. Adjuvant treatment decisions must be made based on available evidences. We mainly investigated the role of vaginal interventional radiotherapy (VIRt) in UCS, UCCC, and UPSC managements. Material and methods: A systematic research using PubMed and Cochrane library was conducted to identify full articles evaluating the efficacy of VIRt in early-stage UPSC, UCCC, and UCS. A search in ClinicalTrials.gov was performed in order to detect ongoing or recently completed trials as well as in PROSPERO for ongoing or recently completed systematic reviews. Survival outcomes and toxicity rates were obtained. Results: All studies were retrospective. For UCS, the number of evaluated patients was 432. The 2- to 5-year aver- age local control (LC) was 91% (range, 74.2-96%), disease-free survival (DFS) 88% (range, 82-94%), overall survival (OS) 79% (range, 53.8-84.3%), the average 5-year cancer-specific survival (CSS) was 70% (range, 70-94%), and G3-G4 toxicity was 0%. For UCCC, the number of investigated patients was 335 (UCCC – 124, mixed – 211), with an average 5-year LC of 100%, DFS of 83% (range, 82-90%), OS of 93% (range, 83-100%), and G3-G4 toxicity of 0%. For UPSC, the number of examined patients was 1,092 (UPSC – 866, mixed – 226). The average 5-year LC was 97% (range, 87.1-100%), DFS 84% (range, 74.7-95.6%), OS 93% (range, 71.9-100%), CSS 89% (range, 78.9-94%), and G3-G4 toxicity was 0%. Conclusions: These data suggest that in adequately selected early-stage UPSC and UCCC patients, VIRt alone may be suitable in women who underwent surgical staging and received adjuvant chemotherapy. In early-stage UCS, a multidisciplinary therapeutic approach has to be planned, considering high-rate of pelvic and distant relapses

Expression of thymidylate synthase in human cells is an early G1 event regulated by CDK4 and p16INK4A but not E2F

Thymidylate synthase (TS) is the enzyme that catalyses the last step in de novo thymidylate synthesis. It is of interest clinically because it is an effective target for drugs such as 5-fluorouracil, often used in combination therapy. Despite a number of earlier reports indicating that TS is a cell cycle-dependent enzyme, this remains equivocal. Here, we show that in HCT116 cells synchronised by serum starvation, there is a clear dissociation between the expression of cyclin E (a well-characterised cell-cycle protein) and TS. Although both cyclin E and TS mRNA and protein increased during G1, TS upregulation was delayed. Moreover, TS levels did not decrease following S-phase completion while cyclin E decreased sharply. Similarly, clear differences were seen between cyclin E and TS as asynchronously growing HCT116 cells were growth-inhibited by low-serum treatment. In contrast to previous reports using rodent cells, adenovirus-mediated over-expression of E2F1 and cyclin E in three human cell lines had no effect on TS. Cell-cycle progression was blocked by treatment of cells with pharmacological inhibitors of CDK2 and CDK4 and by ectopic expression of p16INK4A. Whereas CDK2 inhibition had no effect on TS levels, inhibition of CDK4 was associated with decreased TS protein levels. These results provide the first evidence that drugs targeting CDK4 may be useful with anti-TS drugs as combination therapy for cancer