Direct observation by time-resolved infrared spectroscopy of the bright and the dark excited states of the [Ru(phen)2(dppz)]2+ light-switch compound in solution and when bound to DNA

The [Ru(phen)2(dppz)]2+ complex (1) is non-emissive in water but is highly luminescent in organic solvents or when bound to DNA, making it a useful probe for DNA binding. To date, a complete mechanistic explanation for this “light-switch” effect is still lacking. With this in mind we have undertaken an ultrafast time resolved infrared (TRIR) study of 1 and directly observe marker bands between 1280–1450 cm-1, which characterise both the emissive “bright” and the non-emissive “dark” excited states of the complex, in CD3CN and D2O respectively. These characteristic spectral features are present in the [Ru(dppz)3]2+ solvent light-switch complex but absent in [Ru(phen)3]2+, which is luminescent in both solvents. DFT calculations show that the vibrational modes responsible for these characteristic bands are predominantly localised on the dppz ligand. Moreover, they reveal that certain vibrational modes of the “dark” excited state couple with vibrational modes of two coordinating water molecules, and through these to the bulk solvent, thus providing a new insight into the mechanism of the light-switch effect. We also demonstrate that the marker bands for the “bright” state are observed for both L- and D enantiomers of 1 when bound to DNA and that photo-excitation of the complex induces perturbation of the guanine and cytosine carbonyl bands. This perturbation is shown to be stronger for the L enantiomer, demonstrating the different binding site properties of the two enantiomers and the ability of this technique to determine the identity and nature of the binding site of such intercalators.

Bone Remodeling Adjacent to Morse Cone-Connection Implants with Platform Switch: A Fluorescence Study in the Dog Mandible

Purpose: The aim of this study was to evaluate, through fluorescence analysis, the effect that different interimplant distances, after prosthetic restoration, will have on bone remodeling in submerged and nonsubmerged implants restored with a ""platform switch."" Materials and Methods: Fifty-six Ankylos implants were placed 1.5 mm subcrestally in seven dogs. The implants were placed so that two fixed prostheses, with three interimplant contacts separated by 1-mm, 2-mm, and 3-mm distances, could be fabricated for each side of the mandible. The sides and the positions of the groups were selected randomly. To better evaluate bone remodeling, calcein green was injected 3 days before placement of the prostheses at 12 weeks postimplantation. At 3 days before sacrifice (8 weeks postloading), alizarin red was injected. The amounts of remodeled bone within the different interimplant areas were compared statistically before and after loading in submerged and nonsubmerged implants. Results: Statistically significant differences existed in the percentage of remodeled bone seen in the different regions. Mean percentages of remodeled bone in the submerged and nonsubmerged groups, respectively, were as follows: for the 1-mm distance, 23.0% +/- 0.05% and 23.1% +/- 0.03% preloading and 27.0% +/- 0.03% and 25.2% +/- 0.04% postloading, for the 2-mm distance, 18.2% +/- 0.05% and 18.1% +/- 0.04% preloading and 21.3% +/- 0.07% and 19.9% +/- 0.03% postloading, for the 3-mm distance, 18.3% +/- 0.03% and 18.3% +/- 0.03% preloading and 18.8% +/- 0.04% and 19.8% +/- 0.04% postloading, for distal-extension regions, 16.6% +/- 0.02% and 17.4% +/- 0.04% preloading and 17.0% +/- 0.04% and 18.4% +/- 0.04% postloading. Conclusions: Based upon this animal study, loading increases bone formation for submerged or nonsubmerged implants, and the interimplant distance of 1 mm appears to result in more pronounced bone remodeling than the 2-mm or 3-mm distances in implants with a ""platform switch."" INT J ORAL MAXILLOFAC IMPLANTS 2009;24:257-266

A Single Mutation at the Sheet Switch Region Results in Conformational Changes Favoring lambda 6 Light-Chain Fibrillogenesis

Systemic amyloid light-chain (LC) amyloidosis is a disease process characterized by the pathological deposition of monoclonal LCs in tissue. All LC subtypes are capable of fibril formation although lambda chains, particularly those belonging to the lambda 6 type, are overrepresented. Here, we report the thermodynamic and in vitro fibrillogenic properties of several mutants of the lambda 6 protein 6aJL2 in which Pro7 and/or His8 was substituted by Ser or Pro. The H8P and H8S mutants were almost as stable as the wildtype protein and were poorly fibrillogenic. In contrast, the P7S mutation decreased the thermodynamic stability of 6aJL2 and greatly enhanced its capacity to form amyloid-like fibrils in vitro. The crystal structure of the P7S mutant showed that the substitution induced both local and long-distance effects, such as the rearrangement of the V(L) (variable region of the light chain)-V(L) interface. This mutant crystallized in two orthorhombic polymorphs, P2(1)2(1)2(1) and C222(1). In the latter, a monomer that was not arranged in the typical Bence-Jones dimer was observed for the first time. Crystal-packing analysis of the C222(1) lattice showed the establishment of intermolecular beta-beta interactions that involved the N-terminus and beta-strand B and that these could be relevant in the mechanism of LC fibril formation. Our results strongly suggest that Pro7 is a key residue in the conformation of the N-terminal sheet switch motif and, through long-distance interactions, is also critically involved in the contacts that stabilized the V(L) interface in lambda 6 LCs. (C) 2009 Elsevier Ltd. All rights reserved.

Load balancing solution and evaluation of F5 content switch equipment

The Thesis focused on hardware based Load balancing solution of web traffic through a load balancer F5 content switch. In this project, the implemented scenario for distributing HTTPtraffic load is based on different CPU usages (processing speed) of multiple member servers.Two widely used load balancing algorithms Round Robin (RR) and Ratio model (weighted Round Robin) are implemented through F5 load balancer. For evaluating the performance of F5 content switch, some experimental tests has been taken on implemented scenarios using RR and Ratio model load balancing algorithms. The performance is examined in terms of throughput (bits/sec) and Response time of member servers in a load balancing pool. From these experiments we have observed that Ratio Model load balancing algorithm is most suitable in the environment of load balancing servers with different CPU usages as it allows assigning the weight according to CPU usage both in static and dynamic load balancing of servers.

The most severe parkinsonian patients are most improved with duodenal levodopa infusion

Objectives:To find variables correlated to improvement with intraduodenal levodopa/carbidopa infusion (Duodopa) in order to identify potential candidates for this treatment. Two clinical studies comparing Duodopa with oral treatments in patients with advanced Parkinson’s disease have shown significant improvement in percent on-time on a global treatment response scale (TRS) based on hourly and half-hourly clinical ratings and in median UPDRS scores.Methods:Data from study 1 comparing infusion with Sinemet CR (12 patients, Nyholm et al, Clin Neuropharmacol 2003; 26(3): 156-163) and study 2 comparing infusion with individually optimised conventional combination therapies (18 patients, Nyholm et al, Neurology, in press) were used. Measures of severity were defined as total UPDRS score and scores for sections II and III, percent functional on-time and mean squared error of ratings on the TRS and as mean of diary questions about mobility and satisfaction (only study 2). Absolute improvement was defined as difference in severity, and relative improvement was defined as percent absolute improvement/severity on oral treatment. Pearson correlation coefficients between measures of improvement and other variables were calculated.Results:Correlations (r2>0.28, p<0.05) between severity during oral treatment and absolute improvement on infusion were found for: Total UPDRS, UPDRS III and TRS ratings (studies 1 and 2) and for diary question 1 (mobility) and UPDRS II (study 2). Correlation to relative improvement was found for total UPDRS (study 2, r2=0.47). Figure 1 illustrates absolute improvement in total UPDRS vs. total UPDRS during oral treatment (study 2).Conclusion:Correlating different measures of severity and improvement revealed that patients with more severe symptoms were most improved and that the relation between severity and improvement was linear within the studied groups. The result, which was reproducible between two clinical studies, could be useful when deciding candidates for the treatment.

Pedal : identification of models for duodenal administration of levodopa

Backgound and aims: The main purpose of the PEDAL study is to identify and estimate sample individual pharmacokinetic- pharmacodynamic (PK/PD) models for duodenal infusion of levodopa/carbidopa (Duodopa®) that can be used for in numero simulation of treatment strategies. Other objectives are to study the absorption of Duodopa® and to form a basis for power calculation for a future larger study. PK/PD based on oral levodopa is problematic because of irregular gastric emptying. Preliminary work with data from [Gundert-Remy U et al. Eur J Clin Pharmacol 1983;25:69-72] suggested that levodopa infusion pharmacokinetics can be described by a two-compartment model. Background research led to a hypothesis for an effect model incorporating concentration-unrelated fluctuations, more complex than standard E-max models. Methods: PEDAL involved a few patients already on Duodopa®. A bolus dose (normal morning dose plus 50%) was given after a washout during night. Data collection continued until the clinical effect was back at baseline. The procedure was repeated on two non-consecutive days per patient. The following data were collected in 5 to 15 minutes intervals: i) Accelerometer data. ii) Three e-diary questions about ability to walk, feelings of “off” and “dyskinesia”. iii) Clinical assessment of motor function by a physician. iv) Plasma concentrations of levodopa, carbidopa and the metabolite 3-O-methyldopa. The main effect variable will be the clinical assessment. Results: At date of abstract submission, lab analyses were currently being performed. Modelling results, simulation experiments and conclusions will be presented in our poster.

A pharmacokinetic-pharmacodynamic model for duodenal levodopa infusion

Objective Levodopa in presence of decarboxylase inhibitors is following two-compartment kinetics and its effect is typically modelled using sigmoid Emax models. Pharmacokinetic modelling of the absorption phase of oral distributions is problematic because of irregular gastric emptying. The purpose of this work was to identify and estimate a population pharmacokinetic- pharmacodynamic model for duodenal infusion of levodopa/carbidopa (Duodopa®) that can be used for in numero simulation of treatment strategies. Methods The modelling involved pooling data from two studies and fixing some parameters to values found in literature (Chan et al. J Pharmacokinet Pharmacodyn. 2005 Aug;32(3-4):307-31). The first study involved 12 patients on 3 occasions and is described in Nyholm et al. Clinical Neuropharmacology 2003:26:156-63. The second study, PEDAL, involved 3 patients on 2 occasions. A bolus dose (normal morning dose plus 50%) was given after a washout during night. Plasma samples and motor ratings (clinical assessment of motor function from video recordings on a treatment response scale between -3 and 3, where -3 represents severe parkinsonism and 3 represents severe dyskinesia.) were repeatedly collected until the clinical effect was back at baseline. At this point, the usual infusion rate was started and sampling continued for another two hours. Different structural absorption models and effect models were evaluated using the value of the objective function in the NONMEM package. Population mean parameter values, standard error of estimates (SE) and if possible, interindividual/interoccasion variability (IIV/IOV) were estimated. Results Our results indicate that Duodopa absorption can be modelled with an absorption compartment with an added bioavailability fraction and a lag time. The most successful effect model was of sigmoid Emax type with a steep Hill coefficient and an effect compartment delay. Estimated parameter values are presented in the table. Conclusions The absorption and effect models were reasonably successful in fitting observed data and can be used in simulation experiments.

12-month results from a novel test battery used in a duodenal levodopa infusion trial

A novel test battery consisting of self-assessments and motor tests (tapping and spiral drawing) for patients with Parkinson’s disease (PD) was developed for a hand computer with touch screen in a telemedicine setting. Tests are performed four times per day in the home environment during weeklong test periods. Results are processed into scores for different dimensions of the symptom state and an ‘overall score’ reflecting the global condition of a patient during a test period. The test battery was validated in a separate study recently submitted to Mov Disord. This test battery is currently being used in an open longitudinal trial (DAPHNE, EudraCT No. 2005- 002654-21) by sixty-five patients with advanced PD at nine clinics around Sweden. On inclusion, the patients were either receiving treatment with duodenal levodopa/carbidopa infusion (Duodopa®) (n=36), or they were candidates for receiving this treatment (n=29). We now present interim results for the first twelve months. Test periods were performed in three-month intervals. During most of the periods, UPDRS ratings were performed in afternoons at the start of the week. In twenty of the patients, scores were available during individually optimized oral polypharamacy, before receiving infusion and at least one test period after having started infusion treatment. Usability and compliance with performing tests, this far are good, both with patients and clinical staff. Correlations between test periods 2 and 3 during infusion treatment (three months apart) are stronger for overall test score than for total UPDRS, indicating good reliability. The correlation between overall test score and UPDRS for all test periods is adequate (r=-0.6). In an exact Wilcoxon signed rank test, where the endpoint is the change from the first to the twelve month test period (n=25), there was no change in test results in any of the test battery dimensions for the patients already receiving infusion when included. However, in the patients entering the study before receiving infusion, there was a significant change (improvement) from the baseline to the twelve month test period in dimensions; ‘off’, ‘dyskinesia’ and ‘satisfied’ and in the ‘overall score’ (n=15). The mean improvement in overall score after infusion was 29% (p=0.015). We conclude that the test battery is able to measure a functional improvement with infusion that is sustained over at least twelve months.

Total gastrectomy with substitution of stomach by jejunal pouch with and without duodenal passage. Study in rats

A comparison was done between the F. Paulino jejunal pouch (FP) and a jejunal pouch (JP) as esophagusduodenum interpositional graft, for replacing the stomach after total gastrectomy. It was investigated the effect of the two procedures on esophagus histology, nutritional state and serum gastrin in rats. Methods: Male Wistar rats weighing 282±17g were randomly submitted to sham operation (S), FP and JP after total gastrectomy. After eight weeks the rats were killed with overdose of anesthetic and tissue was taken from the distal esophagus for histology. Serum levels of total proteins, albumin, iron, transferring, folate, cobalamine, calcium, as well as serum gastrin were determined. Survival was considered. Results: Fourty six rats were operated and thirty survived for eight weeks. Five (33.3%) died after FP and 11 (52.3%) after JP (p<0.05). Postoperative esophagitis occurred in 6 JP rats. At 8th week, no difference was observed on body weight when compared FP and JP rats (p>0.05). The JP rats had a significant decrease in serum albumin, glucose, transferrin, iron, folate and calcium, compared to sham (p<0.05). Serum gastrin, iron and calcium were significantly higher in JP rats than in FP rats (p<0.05). In FP rats, transferrin and cobalamine showed significant decrease comparing the preoperative with 8th week levels (p<0.05). Conclusion: F. Paulino pouch in rats had lower mortality than JP, and esophagitis was not detected in it. JP rats had serum gastrin, iron and calcium unaffected, possibly because of preservation of duodenal passage

Produção Fecal e Fluxo Duodenal de Matéria Seca e Matéria Orgânica Estimados por Meio de Indicadores

O objetivo deste trabalho foi comparar os indicadores internos, fibra em detergente neutro e fibra em detergente ácido indigestíveis, obtidos após 144h de incubação in vitro e in situ (FDNiv; FDNis; FDAiv; FDAis) com o indicador externo, o óxido crômico (Cr2O3), para estimativas da produção fecal, do fluxo duodenal de matéria seca e matéria orgânica em novilhos mestiços (HxZ) confinados. Foram utilizadas dietas à base de silagens de milho, de raspa e de casca de mandioca, e também de cana-de-açúcar ensilada com polpa cítrica peletizada. Os novilhos foram castrados e canulados no rúmen e no duodeno. O período experimental teve 11 dias de adaptação às dietas e 8 dias de coleta. O delineamento experimental foi o quadrado latino (4x4), com quatro tratamentos, num arranjo em parcela subdividida, sendo as dietas estudadas nas parcelas, e os indicadores nas subparcelas. Os resultados obtidos em percentagem do peso vivo para a estimativa da produção fecal, utilizando-se os diferentes indicadores, mostraram que a FDAiv (0,88 %), a FDAis (0,85 %) e o Cr2O3 (0,99 %), embora com diferenças significativas, podem ser utilizados pelos resultados biologicamente consistentes. Para estimar o fluxo duodenal de matéria seca e matéria orgânica, foram utilizados os valores de produção fecal obtidos com a FDAiv. Os indicadores internos não apresentaram diferenças entre si para o fluxo duodenal de matéria seca, com média de 3,29 kg/dia, porém o óxido crômico superestimou o fluxo (4,95 kg/dia). Para o fluxo duodenal de matéria orgânica não houve diferença entre os indicadores com média de 2,73 kg/dia.

Carcinogenesis of the upper gastrointestinal tract induced by N-methyl-N '-nitro-nitrosoguanidine and reflux of duodenal contents in the rat

Purpose: To investigate the combined effects of reflux of duodenal contents through the pylorus and treatment with N-methyl-N'-nitro-nitrosoguanidine ( MNNG) on the development of lesions in the glandular stomach, at the gastrojejunal anastomosis and in the forestomach of rats. Methods: Eighty Male Wistar rats were divided into 4 groups: G1: MNNG + Reflux, G2: Reflux, G3: MNNG and G4: Gastrostomy. MNNG was given in the drinking water ( 100 mg/ml) for 12 weeks and then two groups ( G1 and G2) were submitted to a gastrojejunal anastomosis followed by section of the afferent loop and suture of both stumps to allow reflux of duodenal contents through the pylorus. The animals were sacrificed 18 and 36 weeks after surgery. The lesions obtained in the antral mucosa, at the gastrojejunal anastomosis and in the forestomach were analysed histologically. Results: Duodenal reflux induced proliferative lesions at both glandular and squamous mucosa of the stomach. In the antrum, adenomatous hyperplasia (AH) was observed in 20% and 50% of the animals at the 18(th) and 36(th) weeks respectively. Aditionally 85% of the animals presented AH at the gastrojejunal anastomosis and 60% developed squamous hyperplasia at the squamous portion of the stomach. MNNG treatment plus duodenal reflux enhanced the development of malignant tumors at both glandular and squamous mucosa, since there were 30% of antral adenocarcinomas and 45% of squamous carcinomas at the 18th week and the frequency of these malignant tumors rose to 50% in the antrum and 65% in the squamous mucosa at the 36th week. Conclusion: The reflux of duodenal contents through the pylorus enhanced the development of proliferative lesions, benign and malignant, in the glandular stomach and in the forestomach of rats.

Hymenaea stigonocarpa Mart. ex Hayne: A Brazilian medicinal plant with gastric and duodenal anti-ulcer and antidiarrheal effects in experimental rodent models

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Byrsonima intermedia A. Juss.: Gastric and duodenal anti-ulcer, antimicrobial and antidiarrheal effects in experimental rodent models

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)