Proinsulin C-peptide interferes with insulin fibril formation

Insulin aggregation can prevent rapid insulin uptake and cause localized amyloidosis in the treatment of type-1 diabetes. In this study, we investigated the effect of C-peptide, the 31-residue peptide cleaved from proinsulin, on insulin fibrillation at optimal conditions for fibrillation. This is at low pH and high concentration, when the fibrils formed are regular and extended. We report that C-peptide then modulates the insulin aggregation lag time and profoundly changes the fibril appearance, to rounded clumps of short fibrils, which, however, still are Thioflavine T-positive. Electrospray ionization mass spectrometry also indicates that C-peptide interacts with aggregating insulin and is incorporated into the aggregates. Hydrogen/deuterium exchange mass spectrometry further reveals reduced backbone accessibility in insulin aggregates formed in the presence of C-peptide. Combined, these effects are similar to those of C-peptide on islet amyloid polypeptide fibrillation and suggest that C-peptide has a general ability to interact with amyloidogenic proteins from pancreatic β-cell granules. Considering the concentrations, these peptide interactions should be relevant also during physiological secretion, and even so at special sites post-secretory or under insulin treatment conditions in vivo.     

Bone Marrow Involvement in a Patient with Paracoccidioidomycosis: A Rare Presentation of Juvenile Form

Paracoccidioides brasiliensis rarely shows bone marrow involvement and its response to treatment with itraconazole in children needs further assessment. We describe here a child with a juvenile disseminated form of paracoccidioidomycosis, which showed reticuloendothelial system involvement and the presence of Paracoccidioides brasiliensis in the bone marrow. The patient showed an effective and rapid response to itraconazole therapy.     

Environmental and neighbourhood effects on tree fern distributions in a neotropical lowland rain forest

Questions: To what extent are the distributions of tropical rain forest tree ferns (Cyatheaceae) related to environmental variation, and is habitat specialization likely to play a role in their local coexistence? Location: Lowland rain forest at La Selva Biological Station, Costa Rica. Methods: Generalized linear (GLM) and generalized additive (GAM) logistic regression were used to model the incidence of four tree fern species in relation to environmental and neighbourhood variables in 1154 inventory plots regularly distributed across 6 km² of old‐growth forest. Small and large size classes of the two most abundant species were modelled separately to see whether habitat associations change with ontogeny. Results: GLM and GAM model results were similar. All species had significant distributional biases with respect to micro‐habitat. Environmental variables describing soil variation were included in the models most often, followed by topographic and forest structural variables. The distributions of small individuals were more strongly related to environmental variation than those of larger individuals. Significant neighbourhood effects (spatial autocorrelation in intraspecific distributions and non‐random overlaps in the distributions of certain species pairs) were also identified. Overlaps between congeners did not differ from random, but there was a highly significant overlap in the distributions of the two most common species. Conclusions: Our results support the view that habitat specialization is an important determinant of where on the rain forest landscape tree ferns grow, especially for juvenile plants. However, other factors, such as dispersal limitation, may also contribute to their local coexistence.     

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank''s Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1°C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.     

The study of adhesion of Candida albicans to the selected acrylic resins

The study has been performed on 3 acrylic resins used to fabricate removable dentures. Aim of the study was to detect possible differences in Candida albicans' adhesion within particular materials. Polished and non-polished samples were made, than these samples were sunk in precipitates containing Candida albicans material. Adhesion of Candida albicans to the surface of the materials occured within a concentration of 10. After 24 hours of incubation differences were found concerning the number of the plate cultures. Most of all plate cultures were observed on Lucitone 199, fewer on Zhermacryl, the poorest one was found on Palaxpress resin. Considerable number of plate cultures occured on non-polished samples relating to polished ones. After 48 hours of incubation further development of Candida albicans took place, with differences concerning various materials.     

Assessment of the required performance and the development of corresponding program decision rules for neglected tropical diseases diagnostic tests: Monitoring and evaluation of soil-transmitted helminthiasis control programs as a case study

Recently, the World Health Organization established the Diagnostic Technical Advisory Group to identify and prioritize diagnostic needs for neglected tropical diseases, and to ultimately describe the minimal and ideal characteristics for new diagnostic tests (the so-called target product profiles (TPPs)). We developed two generic frameworks: one to explore and determine the required sensitivity (probability to correctly detect diseased persons) and specificity (probability to correctly detect persons free of disease), and another one to determine the corresponding samples sizes and the decision rules based on a multi-category lot quality assurance sampling (MC-LQAS) approach that accounts for imperfect tests. We applied both frameworks for monitoring and evaluation of soil-transmitted helminthiasis control programs. Our study indicates that specificity rather than sensitivity will become more important when the program approaches the endgame of elimination and that the requirements for both parameters are inversely correlated, resulting in multiple combinations of sensitivity and specificity that allow for reliable decision making. The MC-LQAS framework highlighted that improving diagnostic performance results in a smaller sample size for the same level of program decision making. In other words, the additional costs per diagnostic tests with improved diagnostic performance may be compensated by lower operational costs in the field. Based on our results we proposed the required minimal and ideal diagnostic sensitivity and specificity for diagnostic tests applied in monitoring and evaluating of soil-transmitted helminthiasis control programs.     

Resistance Mutation R292K Is Induced in Influenza A(H6N2) Virus by Exposure of Infected Mallards to Low Levels of Oseltamivir

Resistance to neuraminidase inhibitors (NAIs) is problematic as these drugs constitute the major treatment option for severe influenza. Extensive use of the NAI oseltamivir (Tamiflu®) results in up to 865 ng/L of its active metabolite oseltamivir carboxylate (OC) in river water. There one of the natural reservoirs of influenza A, dabbling ducks, can be exposed. We previously demonstrated that an influenza A(H1N1) virus in mallards (Anas platyrhynchos) exposed to 1 µg/L of OC developed oseltamivir resistance through the mutation H274Y (N2-numbering). In this study, we assessed the resistance development in an A(H6N2) virus, which belongs to the phylogenetic N2 group of neuraminidases with distinct functional and resistance characteristics. Mallards were infected with A(H6N2) while exposed to 120 ng/L, 1.2 µg/L or 12 µg/L of OC in their sole water source. After 4 days with 12 µg/L of OC exposure, the resistance mutation R292K emerged and then persisted. Drug sensitivity was decreased ≈13,000-fold for OC and ≈7.8-fold for zanamivir. Viral shedding was similar when comparing R292K and wild-type virus indicating sustained replication and transmission. Reduced neuraminidase activity and decrease in recovered virus after propagation in embryonated hen eggs was observed in R292K viruses. The initial, but not the later R292K isolates reverted to wild-type during egg-propagation, suggesting a stabilization of the mutation, possibly through additional mutations in the neuraminidase (D113N or D141N) or hemagglutinin (E216K). Our results indicate a risk for OC resistance development also in a N2 group influenza virus and that exposure to one NAI can result in a decreased sensitivity to other NAIs as well. If established in influenza viruses circulating among wild birds, the resistance could spread to humans via re-assortment or direct transmission. This could potentially cause an oseltamivir-resistant pandemic; a serious health concern as preparedness plans rely heavily on oseltamivir before vaccines can be mass-produced.     

Fertile transgenic pearl millet [<Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.] plants recovered through microprojectile bombardment and phosphinothricin selection of apical meristem-, inflorescence-, and immature embryo-derived embryogenic tissues

Pearl millet [ Pennisetum glaucum (L.) R. Br.] is a drought-tolerant cereal crop used for grain and forage. Novel traits from outside of the gene pool could be introduced provided a reliable gene-transfer method were available. We have obtained herbicide-resistant transgenic pearl millet plants by microprojectile bombardment of embryogenic tissues with the bar gene. Embryogenic tissues derived from immature embryos, inflorescences and apical meristems from diploid and tetraploid pearl millet genotypes were used as target tissues. Transformed cells were selected in the dark on Murashige and Skoog medium supplemented with 2 mg/l 2,4-D and 15 mg/l phosphinothricin (PPT). After 3-10 weeks in the dark, herbicide-resistant somatic embryos were induced to germinate on MS medium containing 0.1 mg/l thidiazuron and 0.1 mg/l 6-benzylaminopurine. Plants were transferred to the greenhouse after they were rooted in the presence of PPT and had passed a chlorophenol red assay (the medium turned from red to yellow). Transgenic plants were recovered from bombardments using intact pAHC25 plasmid DNA, a gel-purified bar fragment, or a mixture of pAHC25 plasmid or bar fragment and a plasmid containing the enhanced green fluorescent protein ( gfp) gene (p524EGFP.1). Analyses by the polymerase chain reaction, Southern blot hybridization, GFP expression, resistance to herbicide application, and segregation of the bar and gfp genes confirmed the presence and stable integration of the foreign DNA. Transformed plants were recovered from all three explants, although transformation conditions were optimized using only the tetraploid inflorescence. Time from culture initiation to rooted transgenic plant using the tetraploid inflorescence ranged from 3-4 months. Seven independent DNA/gold precipitations were used to bombard 52 plates, 29 of which produced an average of 5.5 herbicide-resistant plants per plate. The number of herbicide-resistant plants recovered per successful bombardment ranged from one to 28 and the frequency of co-transformation with gfp ranged from 5% to 85%.