Analysis of tandem repeat units of the promoter of capsanthin/capsorubin synthase (Ccs) gene in pepper fruit.

Capsanthin/capsorubin synthase (Ccs) gene is a key gene that regulates the synthesis of capsanthin and the development of red coloration in pepper fruits. There are three tandem repeat units in the promoter region of Ccs, but the potential effects of the number of repetitive units on the transcriptional regulation of Ccs has been unclear. In the present study, expression vectors carrying different numbers of repeat units of the Ccs promoter were constructed, and the transient expression of the ß-glucuronidase (GUS) gene was used to detect differences in expression levels associated with the promoter fragments. These repeat fragments and the plant expression vector PBI121 containing the 35s CaMV promoter were ligated to form recombinant vectors that were transfected into Agrobacterium tumefaciens GV3101. A fluorescence spectrophotometer was used to analyze the expression associated with the various repeat units. It was concluded that the constructs containing at least one repeat were associated with GUS expression, though they did not differ from one another. This repeating unit likely plays a role in transcription and regulation of Ccs expression.

Steel Rack Connections: Identification of Most Influential Factors and a Comparison of Stiffness Design Methods.

Steel pallet rack (SPR) beam-to-column connections (BCCs) are largely responsible to avoid the sway failure of frames in the down-aisle direction. The overall geometry of beam end connectors commercially used in SPR BCCs is different and does not allow a generalized analytic approach for all types of beam end connectors; however, identifying the effects of the configuration, profile and sizes of the connection components could be the suitable approach for the practical design engineers in order to predict the generalized behavior of any SPR BCC. This paper describes the experimental behavior of SPR BCCs tested using a double cantilever test set-up. Eight sets of specimens were identified based on the variation in column thickness, beam depth and number of tabs in the beam end connector in order to investigate the most influential factors affecting the connection performance. Four tests were repeatedly performed for each set to bring uniformity to the results taking the total number of tests to thirty-two. The moment-rotation (M-θ) behavior, load-strain relationship, major failure modes and the influence of selected parameters on connection performance were investigated. A comparative study to calculate the connection stiffness was carried out using the initial stiffness method, the slope to half-ultimate moment method and the equal area method. In order to find out the more appropriate method, the mean stiffness of all the tested connections and the variance in values of mean stiffness according to all three methods were calculated. The calculation of connection stiffness by means of the initial stiffness method is considered to overestimate the values when compared to the other two methods. The equal area method provided more consistent values of stiffness and lowest variance in the data set as compared to the other two methods.

Effect of ethyl methyl sulfonate concentration and different treatment conditions on germination and seedling growth of the cucumber cultivar Chinese long (9930).

We attempted to create a new germplasm of cucumber cultivar Chinese long (9930) using different doses of ethyl methyl sulfonate (EMS) to induce variability. We tested EMS concentration (0, 0.5, 1.0, 1.5, 2, 3% v/v) with post-treatment (0.1 M Na2S2O3 and water), EMS concentration (0, 0.5, 1.0, 1.5% v/v) over different treatment times (8, 16, 24 h), and EMS concentration (0, 0.5, 1.0, 1.5% v/v) with different treatment temperatures (20 and 28°C). In all experiments with increasing EMS concentration, germination percent, index, and rate were decreased. After addition of stop solution (0.1 M Na2S2O3), post-treatment mutated seeds showed higher germination (84.44%) and rate (37.5%) than seeds treated with water (80 and 34.07%, respectively), while the germination index was high in seeds treated with water. At 20°C, the germination index (4.13) and rate (56.25%) were affected to a greater extent than at 28°C (7.68 and 91.31%, respectively). Treatment times of 16 and 24 h showed similar results for germination percent and rate, while the germination index was decreased over time. There were significant differences in seedling height, fresh true leaf weight, seedling weight, and plant survival with increasing EMS concentration and time. Higher variations in the form of dwarf seedlings were recorded after treatment with 1.5% EMS for 24 h. Based on germination and morphological data, an EMS concentration of 1.5% for 24 h at 20°C and post-treatment with stop solution (0.1 M Na2S2O3) efficiently caused mutation.

Kill curve analysis and response of first generation Capsicum annuum L. B12 cultivar to ethyl methane sulfonate.

Pepper seeds (Capsicum annuum L.) var. B12 were mutagenized by four presoaking treatments in ten concentrations of ethyl methane sulfonate (EMS) to determine the sensitivity of the first generation (M1) to mutagens. The spectrum of mutations and induced variability for various quantitative traits, including germination, percent plant height, injury occurrence, survival ratio, first three fruits weight, and number of seeds per first fruit, were observed in the M1 generation. Our results indicated that all of the test parameters decreased with increasing EMS concentration, except for seedling injury. There were significant differences in germination ratio, LD50, plant height, percent injury, and survival ratio among the tested presoaking treatment. The LD50 was 1% EMS in seeds that were not presoaked (T1) and seeds presoaked for 12 h before treating with EMS (T3). In contrast, the LD50 was 0.5% EMS in seeds presoaked for 6 h (T2) and seeds presoaked in water for 6 h then incubated at 28°C for 12 h before EMS treatment (T4). Five dwarf plants were observed in mutagenized seeds without presoaking as compared to control seeds (at the maturity stage of the control plant).

Exon skipping restores dystrophin expression, but fails to prevent disease progression in later stage dystrophic dko mice.

Antisense therapy with both chemistries of phosphorodiamidate morpholino oligomers (PMOs) and 2'-O-methyl phosphorothioate has demonstrated the capability to induce dystrophin expression in Duchenne muscular dystrophy (DMD) patients in phase II-III clinical trials with benefit in muscle functions. However, potential of the therapy for DMD at different stages of the disease progression is not understood. In this study, we examined the effect of peptide-conjugated PMO (PPMO)-mediated exon skipping on disease progression of utrophin-dystrophin-deficient mice (dko) of four age groups (21-29, 30-39, 40-49 and 50+ days), representing diseases from early stage to advanced stage with severe kyphosis. Biweekly intravenous (i.v.) administration of the PPMO restored the dystrophin expression in nearly 100% skeletal muscle fibers in all age groups. This was associated with the restoration of dystrophin-associated proteins including functional glycosylated dystroglycan and neuronal nitric synthase. However, therapeutic outcomes clearly depended on severity of the disease at the time the treatment started. The PPMO treatment alleviated the disease pathology and significantly prolonged the life span of the mice receiving treatment at younger age with mild phenotype. However, restoration of high levels of dystrophin expression failed to prevent disease progression to the mice receiving treatment when disease was already at advanced stage. The results could be critical for design of clinical trials with antisense therapy to DMD.

Pluronic-PEI copolymers enhance exon-skipping of 2'-O-methyl phosphorothioate oligonucleotide in cell culture and dystrophic mdx mice.

A series of small-size polyethylenimine (PEI)-conjugated pluronic polycarbamates (PCMs) have been investigated for the ability to modulate the delivery of 2'-O-methyl phosphorothioate RNA (2'-OMePS) in vitro and in dystrophic mdx mice. The PCMs retain strong binding capacity to negatively charged oligomer as demonstrated by agarose gel retardation assay, with the formation of condensed polymer/oligomer complexes at a wide-range weight ratio from 1:1 to 20:1. The condensed polymer/oligomer complexes form 100-300 nm nanoparticles. Exon-skipping effect of 2'-OMePS was dramatically enhanced with the use of the most effective PCMs in comparison with 2'-OMePS alone in both cell culture and in vivo, respectively. More importantly, the effective PCMs, especially those composed of moderate size (2k-5kDa) and intermediate hydrophilic-lipophilic balance (7-23) of pluronics, enhanced exon-skipping of 2'-OMePS with low toxicity as compared with Lipofectamine-2000 in vitro or PEI 25k in vivo. The variability of individual PCM for delivery of antisense oligomer and plasmid DNA indicate the complexity of interaction between polymer and their cargos. Our data demonstrate the potential of PCMs to mediate delivery of modified antisense oligonucleotides to the muscle for treating muscular dystrophy or other appropriate myodegenerative diseases.

The Chennai Declaration: a roadmap to tackle the challenge of antimicrobial resistance.

"A Roadmap to Tackle the Challenge of Antimicrobial Resistance - A Joint meeting of Medical Societies in India" was organized as a pre-conference symposium of the 2 nd annual conference of the Clinical Infectious Disease Society (CIDSCON 2012) at Chennai on 24 th August. This was the first ever meeting of medical societies in India on issue of tackling resistance, with a plan to formulate a road map to tackle the global challenge of antimicrobial resistance from the Indian perspective. We had representatives from most medical societies in India, eminent policy makers from both central and state governments, representatives of World Health Organization, National Accreditation Board of Hospitals, Medical Council of India, Drug Controller General of India, and Indian Council of Medical Research along with well-known dignitaries in the Indian medical field. The meeting was attended by a large gathering of health care professionals. The meeting consisted of plenary and interactive discussion sessions designed to seek experience and views from a large range of health care professionals and included six international experts who shared action plans in their respective regions. The intention was to gain a broad consensus and range of opinions to guide formation of the road map. The ethos of the meeting was very much not to look back but rather to look forward and make joint efforts to tackle the menace of antibiotic resistance. The Chennai Declaration will be submitted to all stake holders.

Pancreatic endocrine neoplasms: a current update on genetics and imaging.

Pancreatic endocrine neoplasms are rare pancreatic tumours that may occur sporadically or as part of inherited syndromes such as multiple endocrine neoplasia-1 syndrome, von Recklinghausen disease, von Hippel-Lindau syndrome and tuberous sclerosis complex. Recent advances in the genetics and pathology of hereditary syndromes have provided valuable insights into the pathophysiology and biology of sporadic pancreatic endocrine neoplasms. Evolving molecular data on the biology of these neoplasms have the potential for diagnostic, therapeutic and prognostic use.

Preparation and evaluation of soft gellan gum gel containing paracetamol.

The objective of this study was to develop soft paracetamol gel using gellan gum as a gelling agent and sodium citrate as a source of cation. Different batches were prepared using three different concentrations of gellan gum (0.1, 0.3, and 0.5%), each with two different sodium citrate concentrations (0.3 and 0.5%). The consistency of the paracetamol gel was dependent on the concentration of gellan gum, sodium citrate and co-solute. The results of dissolution study of soft gel containing 0.3% gellan gum and 0.3% sodium citrate revealed that paracetamol was completely released in 30 min. Polyethylene glycol 400 worked as a solubilizer for paracetamol. All the gels possessed acceptable sensory characteristics when evaluated by human volunteers. Short term stability study carried out for four weeks at different temperatures revealed no considerable changes in performance characteristics of developed optimized formulation.

Quality of life in patients with age-related macular degeneration: results from the VISION study.

PURPOSE: To assess the impact of treatment with pegaptanib sodium vs usual care on vision-related quality of life (VRQoL) in patients with age-related macular degeneration (AMD). METHODS: VRQoL was a secondary end point in the trial, a prospective, randomized, double-masked, multicentre, dose-ranging study. Three doses of pegaptanib (0.3, 1, and 3 mg) were compared with usual care with respect to changes in VRQoL as indicated by the 25-item National Eye Institute Visual Function Questionnaire (NEI-VFQ 25), administered at baseline and weeks 30 and 54. Four of the NEI-VFQ 25 domains were prospectively designated as primary: near vision, distance vision, role limitations, and dependency. Between-group differences were assessed using an analysis of covariance model with age, gender, and baseline score as covariates. RESULTS: NEI-VFQ 25 data were available for 569 subjects. At week 54, improvements in the distance vision and role limitations domains were greater in pegaptanib than usual care arms. No substantial increase in ocular pain was noted in pegaptanib-treated patients. No clear superiority of any particular dosage strength of pegaptanib was demonstrated, and no significant differences or trends favoured usual care on any domain score or the NEI-VFQ 25 composite score. The greatest VRQoL benefit was seen in responders (lost<3 lines) to treatment. CONCLUSION: The VISION trial provided evidence of trends in quality-of-life benefit associated with effective treatment of AMD using pegaptanib. Treatment with pegaptanib is expected to contribute significantly to VRQoL improvement for responder patients.

A benefit-based copay for prescription drugs: patient contribution based on total benefits, not drug acquisition cost.

Several managerial mechanisms have been used by managed care organizations to affect prescription drug utilization and related expenditures. Some efforts have focused on monitoring clinical conditions, drug use, and compliance, whereas other efforts have focused on consumer cost sharing and changing product-mix. Efforts focusing on improving quality of care by identifying untreated patients or by enhancing compliance can lead to appropriately increased drug costs, although perhaps with reduced overall medical expenditures. In contrast, the mechanisms implemented to constrain drug costs raise concerns regarding missed opportunities to enhance clinical outcomes, and the possibility of higher medical expenditures. Cost sharing plays a critical role in defining the pharmaceutical benefit. To balance the demands for access to pharmaceuticals with pressures to constrain costs, levels of cost sharing must be set in a manner that achieves appropriate clinical and financial outcomes. Modern multitier systems often base patient contributions on drug acquisition cost, and often do not consider medical necessity as a coverage criterion. Using an alternative approach, the benefit-based copay, patient contributions are based on the potential for clinical benefit, taking into consideration the patient's clinical condition. For any given drug, patients with a high potential benefit would have lower copays than patients with a low potential benefit. Implementation of such a system would provide a financial incentive for individuals to prioritize their out-of-pocket drug expenditures based on the value of their medications, not their price.

Specific repression of beta-globin promoter activity by nuclear ferritin.

Developmental hemoglobin switching involves sequential globin gene activations and repressions that are incompletely understood. Earlier observations, described herein, led us to hypothesize that nuclear ferritin is a repressor of the adult beta-globin gene in embryonic erythroid cells. Our data show that a ferritin-family protein in K562 cell nuclear extracts binds specifically to a highly conserved CAGTGC motif in the beta-globin promoter at -153 to -148 bp from the cap site, and mutation of the CAGTGC motif reduces binding 20-fold in competition gel-shift assays. Purified human ferritin that is enriched in ferritin-H chains also binds the CAGTGC promoter segment. Expression clones of ferritin-H markedly repress beta-globin promoter-driven reporter gene expression in cotransfected CV-1 cells in which the beta-promoter has been stimulated with the transcription activator erythroid Krüppel-like factor (EKLF). We have constructed chloramphenicol acetyltransferase reporter plasmids containing either a wild-type or mutant beta-globin promoter for the -150 CAGTGC motif and have compared the constructs for susceptibility to repression by ferritin-H in cotransfection assays. We find that stimulation by cotransfected EKLF is retained with the mutant promoter, whereas repression by ferritin-H is lost. Thus, mutation of the -150 CAGTGC motif not only markedly reduces in vitro binding of nuclear ferritin but also abrogates the ability of expressed ferritin-H to repress this promoter in our cell transfection assay, providing a strong link between DNA binding and function, and strong support for our proposal that nuclear ferritin-H is a repressor of the human beta-globin gene. Such a repressor could be helpful in treating sickle cell and other genetic diseases.

Intramedullary screw fixation of proximal fifth metatarsal fractures: a biomechanical study.

Intramedullary screw fixation is a popular technique for treatment of proximal fifth metatarsal fractures. The purpose of this study was to compare the fixation rigidity of a 5.5 mm partially threaded cannulated titanium screw, with presumed superior endosteal purchase, to a similar 4.5 mm screw. Acute fifth metatarsal fractures were simulated in cadavers, stabilized with intramedullary screws, and loaded to failure in three-point bending. The initial failure loads for the metatarsals fixed with 4.5 mm and 5.5 mm screws were not significantly different (332.4 N vs. 335.2 N, respectively), nor were the ultimate failure loads (849.8 N vs. 702.2 N, respectively). Based upon our results, maximizing screw diameter does not appear to be critical for fixation rigidity and may increase the risk of intraoperative or postoperative fracture.

The post-prandial state and macrovascular disease: relevance to diabetes mellitus.

There is increasing evidence that the post-prandial state is an important contributing factor to the development of atherosclerosis. In non-diabetic subjects the atherosclerotic risk factors comprised in the categories of lipids, coagulation system and endothelial function may be adversely modified in the post-prandial phase. The generation of an oxidative stress may be the common pathway through which eating may induce these alterations. In diabetic patients these phenomena may be amplified by post-prandial hyperglycemia. There is a growing thought that diabetes is a cardiovascular disease.