Oncolytic adenoviruses armed with thymidine kinase can be traced by PET imaging and show potent antitumoural effects by ganciclovir dosing

Replication-competent adenoviruses armed with thymidine kinase (TK) combine the concepts of virotherapy and suicide gene therapy. Moreover TK-activity can be detected by noninvasive positron emission-computed tomography (PET) imaging, what could potentially facilitate virus monitoring in vivo. Here, we report the generation of a novel oncolytic adenovirus that incorporates the Tat8-TK gene under the control of the Major Late Promoter in a highly selective backbone thus providing selectivity by targeting the retinoblastoma pathway. The selective oncolytic TK virus, termed ICOVIR5-TK-L, showed reduced potency compared to a non-selective counterpart. However the combination of ICOVIR5-TK-L with ganciclovir (GCV) induced a potent antitumoural effect similar to that of wild type adenovirus in a preclinical model of pancreatic cancer. Although the treatment with GCV provoked a reduction in the viral yield, both in vitro and in vivo, a two-cycle treatment of virus and GCV resulted in an enhanced antitumoral response that correlated with high TK-activity, based on microPET measurements. Thus, TK-expressing oncolytic adenoviruses can be traced by PET imaging providing real time information on the activity of the virus and its antitumoral potency can be optimized by GCV dosing.     

The role of cofactor binding in tryptophan accessibility and conformational stability of His-tagged D-amino acid oxidase from Trigonopsis variabilis

d-amino acid oxidase from Trigonopsis variabilis (TvDAAO) is a flavoenzyme with high biotechnological and industrial interest. The overexpression and purification of the apoprotein form of a recombinant His-tagged TvDAAO allowed us to go deep into the structural differences between apoenzyme and holoenzyme, and on the cofactor binding and its contribution to enzyme stability. A significant decrease in intrinsic fluorescence emission took place upon FAD binding, associated to cofactor induced conformational transitions or subunit dimerization that could affect the local environment of protein tryptophan residues. Furthermore, acrylamide-quenching experiments indicated that one of the five tryptophan residues of TvDAAO became less accessible upon FAD binding. A K(d)=1.5+/-0.1x10(-7) M for the dissociation of FAD from TvDAAO was calculated from binding experiments based on both quenching of FAD fluorescence and activity titration curves. Secondary structure prediction indicated that TvDAAO is a mixed alpha/beta protein with 8 alpha-helices and 14 beta-sheets connected by loops. Prediction results were in good agreement with the estimates obtained by circular dichroism which indicated that both the apoenzyme and the holoenzyme had the same structural component ratios: 34% alpha-helix content, 20% beta-structure content (14% antiparallel and 6% parallel beta-sheet), 15% beta-turns and 31% of random structure. Circular dichroism thermal-transition curves suggested single-step denaturation processes with apparent midpoint transition temperatures (T(m)) of 37.9 degrees C and 41.4 degrees C for the apoenzyme and the holoenzyme, respectively. A three-dimensional model of TvDAAO built by homology modelling and consistent with the spectroscopic studies is shown. Comparing our results with those reported for pig kidney (pkDAAO) and Rhodotorula gracilis (RgDAAO) d-amino acid oxidases, a "head-to-head" interaction between subunits in the TvDAAO dimer might be expected.     

Cellular transplants in amyotrophic lateral sclerosis patients: an observational study

Background aimsCytotherapy is a promising option for neurodegenerative disease treatment. Because of the fatal prognosis and imperative need for effective treatment, amyotrophic lateral sclerosis (ALS) patients request this therapy before its effectiveness has been verified. The increase in clinics offering cytotherapies but providing little scientific information has prompted considerable medical tourism. We present an observational study of Spanish ALS patients receiving cytotherapy, analyzing the experiences arising from the treatment (TX) and considering two progression markers, FVC and ALSFRS-R.MethodsTwelve ALS patients with a mean age of 48.6 years (SD 12.8) received cytotherapy 26.9 months (SD 15.8) after clinical onset. ALSFRS-R and FVC at TX were 32.3 (SD 6.8) and 63.4% (SD 15.3), respectively. TX involved transplants of olfactory ensheathing cells in three patients, and autologous mesenchymal stromal cells in the remainder.ResultsOne patient died 33 months post-TX after surviving for 49 months. Five required mechanical non-invasive home ventilation 7.4 months post-TX. Two required invasive ventilation 13 months post-TX. Five patients needed gastrostomy feeding 23.3 months post-TX. Survival between clinical onset and the study end date was 50 months (SD 17.2). No significant adverse events or changes in the decline of FVC and ALSFRS-R compared with the disease's natural history were observed.ConclusionsOur observations suggest that these therapies do not halt the course of the disease. Cytotherapy cannot yet be considered a curative treatment for ALS.     

Legacy effects of anthropogenic disturbances modulate dynamics in the world's coral reefs

Rapidly changing conditions alter disturbance patterns, highlighting the need to better understand how the transition from pulse disturbances to more persistent stress will impact ecosystem dynamics. We conducted a global analysis of the impacts of 11 types of disturbances on reef integrity using the rate of change of coral cover as a measure of damage. Then, we evaluated how the magnitude of the damage due to thermal stress, cyclones, and diseases varied among tropical Atlantic and Indo-Pacific reefs and whether the cumulative impact of thermal stress and cyclones was able to modulate the responses of reefs to future events. We found that reef damage largely depends on the condition of a reef before a disturbance, disturbance intensity, and biogeographic region, regardless of the type of disturbance. Changes in coral cover after thermal stress events were largely influenced by the cumulative stress of past disturbances and did not depend on disturbance intensity or initial coral cover, which suggests that an ecological memory is present within coral communities. In contrast, the effect of cyclones (and likely other physical impacts) was primarily modulated by the initial reef condition and did not appear to be influenced by previous impacts. Our findings also underscore that coral reefs can recover if stressful conditions decrease, yet the lack of action to reduce anthropogenic impacts and greenhouse gas emissions continues to trigger reef degradation. We uphold that evidence-based strategies can guide managers to make better decisions to prepare for future disturbances.     

A Solid-Phase Method for the Synthesis of Small to Medium-Sized Cyclic Oligonucleotides

Abstract

Cyclic oligonucleotides (2- to 30-mer) are synthesized by a solid-phase method, for both chain elongation and cyclization, employing a new linker and standard phosphoramidite chemistry. Fairly pure crude products (>90% by HPLC) are obtained.     

Relationship between Tobacco,cagA and vacA i1 Virulence Factors and Bacterial Load in Patients Infected by Helicobacter pylori

MethodsBiopsies of the gastric corpus and antrum from 155 consecutive patients in whom there was clinical suspicion of infection by H. pylori were processed. In 106 patients H. pylori infection was detected. Molecular methods were used to quantify the number of microorganisms and presence of cagA and vacA i1 genes. A standardized questionnaire was used to obtain patients’ clinical data and lifestyle variables, including tobacco and alcohol consumption. Adjusted Odds Ratios (OR_adjusted) were estimated by unconditional logistic regression.ResultscagA was significantly associated with active-smoking at endoscope: OR_adjusted 4.52. Evidence of association was found for vacA i1 (OR_adjusted 3.15). Bacterial load was higher in active-smokers, although these differences did not yield statistical significance (median of 262.2 versus 79.4 copies of H. pylori per cell).ConclusionsThe association between smoking and a higher risk of being infected by a virulent bacterial population and with higher bacterial load, support a complex interaction between H. pylori infection and environmental factors.     

Effects of methamidophos on sediment processing and body mass of Capitella sp. Y from Estero del Yugo, Mazatlán, Mexico

The cosmopolitan species-complex Capitella, a deposit-feeding polychaete, is widely used as an indicator of organic pollution and plays an important role in sewage waste cycling in marine and estuarine ecosystems. The organophosphorous insecticide methamidophos is currently employed in agriculture fields to control insect infestations. Its occurrence could pose a hazard to infauna. A bioassay to investigate the effects of exposing Capitella sp. Y to sediment spiked with methamidophos (0.008, 0.016, 0.032, 0.064, 0.130 and 0.260 mg/g dry wt sediment) is described. Increasing methamidophos concentrations significantly reduced faecal pellet production and body mass. Some specimens exhibited morphological abnormalities and behaviour changes, which could be attributed to toxic effects of methamidophos not severe enough to stop feeding activity. Results using Capitella sp. Y suggest that methamidophos could affect the rate of sediment processing by polychaetes or other benthic invertebrates in zones subjected to the influence of such insecticide.