Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There were no indications that the tomato DNA inserts conferred autonomous replication on the plasmids. Instead, Southern blot hybridization analysis of seven kanamycin resistant calli revealed the presence of at least one kanamycin resistance locus per transformant integrated in the tomato nuclear DNA. Generally one to three truncated plasmid copies were found integrated into the tomato nuclear DNA, often physically linked to each other. For one transformant we have been able to use the bacterial ampicillin resistance marker of the vector plasmid pUC9 to 'rescue' a recombinant plasmid from the tomato genome. Analysis of the foreign sequences included in the rescued plasmid showed that integration had occurred in a non-repetitive DNA region. Calf-thymus DNA, used as a carrier in transformation procedure, was found to be covalently linked to plasmid DNA sequences in the genomic DNA of one transformant. A model is presented describing the fate of exogenously added DNA during the transformation of a plant cell. The results are discussed in reference to the possibility of isolating DNA sequences responsible for autonomous replication in tomato.

Topical immunotherapy of severe alopecia areata with diphenylcyclopropenone (DPCP): experience in an Iranian population

BACKGROUND: Highly variable results of topical diphenylcyclopropenone (DPCP) in the treatment of alopecia areata have been reported so far. The purposes of the present study were to evaluate the efficacy and tolerability of DPCP treatment in severe alopecia areata. METHODS: Twenty-eight patients (16 female and 12 male, 10–35 years old, mean age 25 years) with extensive alopecia areata were enrolled in an open-label clinical trial. After sensitization with 2% DPCP, progressively higher concentrations beginning at 0.001% were applied weekly for 6 months to one side of the scalp, after which, if terminal hair growth was noted, the entire scalp was then treated under the same weekly protocol. The maximum concentration of DPCP in acetone was 2%. RESULTS: Twenty-seven of 28 patients completed therapy. The overall response rate was 81.5% (22/27), complete remission (90%-100% terminal hair re-growth) was obtained 22.2% (6/27) and partial remission (10%-90% terminal hair re-growth) in 59.3% (16/27). In all patients an eczematous reaction consisting of erythema, itching, and scaling at the site of application were observed. During therapy, other side effects including, occipital lymphadenopathy 40.7% (11/27), severe eczema/blister formation 40.7% (11/27), hyperpigmentation 18.5% (5/27) were observed, but no hypopigmentation, vitiligo, contact urticaria, and erythema multiforme-like reaction were seen in the patients. Nineteen of 27 (70.4%) patients had at least one side effect, other than eczematous reaction. Notably, partial recurrence was observed in 50.9% (13/22) of these patients after 6 to 12 months of follow-up. During the follow-up time the maintenance DPCP immunotherapy was continued. CONCLUSION: Topical DPCP treatment for alopecia areata is an effective therapy with a slightly high relapse rate during bilateral maintenance treatment. According to the author's knowledge this is the first experience with DPCP in Iran

An unusual presentation of anetoderma: a case report

BACKGROUND: Anetoderma is a benign condition with focal loss of dermal elastic tissue resulting in localized areas of flaccid or herniated saclike skin. Currently, anetoderma is classified as either primary (idiopathic), or secondary anetoderma (which is associated with a variety of skin conditions, penicillamine use, or neonatal prematurity). Lesions appear on the upper arms, trunk, and thighs. CASE PRESENTATION: We report a 14-year-old boy, which was noticed to have had multiple, white, non-pruritic areas on the acral sites of upper and lower extremities for two years. In physical examination, the patient had normal mental development. Skin lesions consisted of scattered, white to skin-colored papules, less than 1 cm in diameter, and with central protrusion, with distribution on dorsal part of the index finger, forearms, distal portion of thighs and calves. Lesions were detected neither on the trunk nor the proximal areas of extremities. There are no sensory changes associated with the lesions. Otherwise, his general health was good. He did not have any medication consumption history. Family history was negative. Laboratory examinations were within normal limits. Skin biopsy from one of his lesions was done, that confirmed the diagnosis of anetoderma. CONCLUSIONS: In summary, we report a case of anetoderma on unusual sites of the skin. We could not find similar reports of anetoderma developing on distal extremities without involvement of the upper trunk and proximal arms, in the medical literature

Utilisation of an operative difficulty grading scale for laparoscopic cholecystectomy

Background A reliable system for grading operative difficulty of laparoscopic cholecystectomy would standardise description of findings and reporting of outcomes. The aim of this study was to validate a difficulty grading system (Nassar scale), testing its applicability and consistency in two large prospective datasets. Methods Patient and disease-related variables and 30-day outcomes were identified in two prospective cholecystectomy databases: the multi-centre prospective cohort of 8820 patients from the recent CholeS Study and the single-surgeon series containing 4089 patients. Operative data and patient outcomes were correlated with Nassar operative difficultly scale, using Kendall’s tau for dichotomous variables, or Jonckheere–Terpstra tests for continuous variables. A ROC curve analysis was performed, to quantify the predictive accuracy of the scale for each outcome, with continuous outcomes dichotomised, prior to analysis. Results A higher operative difficulty grade was consistently associated with worse outcomes for the patients in both the reference and CholeS cohorts. The median length of stay increased from 0 to 4 days, and the 30-day complication rate from 7.6 to 24.4% as the difficulty grade increased from 1 to 4/5 (both p < 0.001). In the CholeS cohort, a higher difficulty grade was found to be most strongly associated with conversion to open and 30-day mortality (AUROC = 0.903, 0.822, respectively). On multivariable analysis, the Nassar operative difficultly scale was found to be a significant independent predictor of operative duration, conversion to open surgery, 30-day complications and 30-day reintervention (all p < 0.001). Conclusion We have shown that an operative difficulty scale can standardise the description of operative findings by multiple grades of surgeons to facilitate audit, training assessment and research. It provides a tool for reporting operative findings, disease severity and technical difficulty and can be utilised in future research to reliably compare outcomes according to case mix and intra-operative difficulty

An insertional mutagenesis programme with an enhancer trap for the identification and tagging of genes involved in abiotic stress tolerance in the tomato wild-related species Solanum pennellii

Salinity and drought have a huge impact on agriculture since there are few areas free of these abiotic stresses and the problem continues to increase. In tomato, the most important horticultural crop worldwide, there are accessions of wild-related species with a high degree of tolerance to salinity and drought. Thus, the finding of insertional mutants with other tolerance levels could lead to the identification and tagging of key genes responsible for abiotic stress tolerance. To this end, we are performing an insertional mutagenesis programme with an enhancer trap in the tomato wild-related species Solanum pennellii. First, we developed an efficient transformation method which has allowed us to generate more than 2,000 T-DNA lines. Next, the collection of S. pennelli T0 lines has been screened in saline or drought conditions and several presumptive mutants have been selected for their salt and drought sensitivity. Moreover, T-DNA lines with expression of the reporter uidA gene in specific organs, such as vascular bundles, trichomes and stomata, which may play key roles in processes related to abiotic stress tolerance, have been identified. Finally, the growth of T-DNA lines in control conditions allowed us the identification of different development mutants. Taking into account that progenies from the lines are being obtained and that the collection of T-DNA lines is going to enlarge progressively due to the high transformation efficiency achieved, there are great possibilities for identifying key genes involved in different tolerance mechanisms to salinity and drought