Characterization of wheat DArT markers: genetic and functional features

Diversity array technology (DArT) markers are largely used for mapping, genetic diversity, and association mapping studies. For years, they have been used as anonymous genomic markers, as their sequences were not known. As the sequences of 2,000 wheat DArT clones are now available, this study was designed to analyze these sequences with bioinformatic approaches, and to study the genetic features of a subset of 291 markers positioned on the A and B genomes in three durum wheat genetic maps. A set of 1,757 non-redundant sequences was identified, and used as queries for similarity searches. Analysis of the genetic positions of markers corresponding to nearly identical sequences indicates that redundancy of sequences is one of the factors that explains the clustering of these markers in specific genomic regions. Of a total of 1,124 DArT clones (64?%) that represent putatively expressed sequences, putative functions are proposed for more than 700 of them. Of note, many clones correspond to genes that are related to disease resistance, as characterized by leucine-rich repeat domains, and 40 of these clones are positioned in the three genetic maps presented in this study. Finally, DArT markers have been used to find syntenic regions in the Brachypodium and rice genomes. In conclusion, the analyses herein presented contribute to explain the main features of DArT markers observed in genetic maps, as clustering in short chromosome regions. Moreover, the attribution of putative gene functions for more than 700 sequences makes these markers an optimal tool for collinearity studies or for the identification of candidate genes.     

Novel SHOX gene mutation in a short boy with Becker muscular dystrophy: double trouble in two adjacent genes

Short stature is a well-recognized feature of Duchenne muscular dystrophy, whilst it has been reported rarely in Becker muscular dystrophy (BMD). Here we report two brothers with BMD, who exhibited a very different growth pattern. Whereas in the short brother (-2.2 SDS) molecular investigation revealed a G367A mutation in the short stature homeobox containing (SHOX) gene located in the Xp22.3 region, no abnormality was found in the brother with normal height (-0.1 SDS). Our data suggest that abnormal growth observed in a boy with BMD may be related to an additional genetic alteration, already known as correlated with short stature.     

Effects of long-term fixation on histological quality of undecalcified murine bones embedded in methylmethacrylate

While long-term fixation and storage of specimens is common and useful for many research projects, it is particularly important for space flight investigations where samples may not be returned to Earth for several months (International Space Station) or years (manned mission to Mars). We examined two critical challenges of space flight experimentation: the effect of long-term fixation on the quality of mouse bone preservation and the preservation of antigens and enzymes for both histochemical and immunohistochemical analyses, and how the animal/sample processing affects the preservation. We show that long-term fixation minimally affects standard histological staining, but that enzyme histochemistry and immunolabeling are greatly compromised. Further, we demonstrate that whole animal preservation is not as suitable as whole leg or stripped leg preservation for long-term fixation and all histological analyses. Overall, we recommend whole leg processing for long-term storage of bone specimens in fixative prior to embedding in plastic for histological examination.     

Pyrosequencing Unveils Cystic Fibrosis Lung Microbiome Differences Associated with a Severe Lung Function Decline

Chronic airway infection is a hallmark feature of cystic fibrosis (CF) disease. In the present study, sputum samples from CF patients were collected and characterized by 16S rRNA gene-targeted approach, to assess how lung microbiota composition changes following a severe decline in lung function. In particular, we compared the airway microbiota of two groups of patients with CF, i.e. patients with a substantial decline in their lung function (SD) and patients with a stable lung function (S). The two groups showed a different bacterial composition, with SD patients reporting a more heterogeneous community than the S ones. Pseudomonas was the dominant genus in both S and SD patients followed by Staphylococcus and Prevotella. Other than the classical CF pathogens and the most commonly identified non-classical genera in CF, we found the presence of the unusual anaerobic genus Sneathia. Moreover, the oligotyping analysis revealed the presence of other minor genera described in CF, highlighting the polymicrobial nature of CF infection. Finally, the analysis of correlation and anti-correlation networks showed the presence of antagonism and ecological independence between members of Pseudomonas genus and the rest of CF airways microbiota, with S patients showing a more interconnected community in S patients than in SD ones. This population structure suggests a higher resilience of S microbiota with respect to SD, which in turn may hinder the potential adverse impact of aggressive pathogens (e.g. Pseudomonas). In conclusion, our findings shed a new light on CF airway microbiota ecology, improving current knowledge about its composition and polymicrobial interactions in patients with CF.     

Type IV pili are widespread among non-pathogenic Gram-positive gut bacteria with diverse carbohydrate utilization patterns

Type IV pili (T4P) are bacterial surface-exposed appendages that have been extensively studied in Gram-negative pathogenic bacteria. Despite recent sequencing efforts, little is known regarding these structures in non-pathogenic anaerobic Gram-positive species, particularly commensals of the mammalian gut. Early studies revealed that T4P in two ruminal Gram-positive species are associated with growth on cellulose, suggesting possible associations of T4P with substrate utilization patterns. In the present study, genome sequences of 118 taxonomically diverse, mainly Gram-positive, bacterial strains isolated from anaerobic (gastrointestinal) environments, have been analysed. The genes likely to be associated with T4P biogenesis were analysed and grouped according to T4P genetic organization. In parallel, consortia of Carbohydrate Active enZYmes (CAZymes) were also analysed and used to predict carbohydrate utilization abilities of selected strains. The predictive power of this approach was additionally confirmed by experimental assessment of substrate-related growth patterns of selected strains. Our analysis revealed that T4P systems with diverse genetic organization are widespread among Gram-positive anaerobic non-pathogenic bacteria isolated from different environments, belonging to two phylogenetically distantly related phyla: Firmicutes and Actinobacteria.     

Acute toxicity effects of ibuprofen on behaviour and haematological parameters of African catfish Clarias gariepinus (Burchell, 1822)

Indiscriminate discharge of pharmaceutical waste into the aquatic ecosystem may pose serious health challenges to aquatic biota. The effect of acute exposure to ibuprofen was evaluated using changes in behaviour and haematological parameters under static bio-assay method in Clarias gariepinus. Test specimens were exposed to acute concentrations of ibuprofen (0.28, 0.33, 0.38, 0.43 and 0.48 mg l^?1) for 24, 48, 72 and 96 h durations respectively. Behavioural and phenotypic changes were observed in surviving fish. There were significant (p < 0.05) concentration and duration-dependent increases in erythrocyte (RBC), haemoglobin (Hb), pack cell volume (PCV) and leukocytes (WBC) in treated fish compared to the control. Insignificant decreases (p > 0.05) in mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were observed in treated fish compared to the control. Ibuprofen elicited dose and duration- dependent decrease in neutrophil counts with the decreases being significant (p < 0.05) in the higher doses of 0.43 and 0.48 mg l^?1. Ibuprofen did not elicit any significant changes in monocytes, basophils and eosinophils. Changes observed in this study showed that ibuprofen negatively affected the health of the fish and we recommend that discharge of ibuprofen into the aquatic environment should be monitored and controlled.     

Immunochemical quantitation of enzymes in human diploid cell line WI-38

A quantitative immunochemical study was carried out of four enzymes, cathodal esterase, acid phosphatase, glucosaminidase and β-glucuronidase. In homogenates of the human diploid cell line WI-38, the relative amounts of the enzymes increased with the passage number of the culture, although great variation was found in later passages just before death of the culture.     

Glucose‐stimulated insulin release: Parallel perifusion studies of free and hydrogel encapsulated human pancreatic islets

To explore the effects immune‐isolating encapsulation has on the insulin secretion of pancreatic islets and to improve our ability to quantitatively describe the glucose‐stimulated insulin release (GSIR) of pancreatic islets, we conducted dynamic perifusion experiments with isolated human islets. Free (unencapsulated) and hydrogel encapsulated islets were perifused, in parallel, using an automated multi‐channel system that allows sample collection with high temporal resolution. Results indicated that free human islets secrete less insulin per unit mass or islet equivalent (IEQ) than murine islets and with a less pronounced first‐phase peak. While small microcapsules (d = 700 µm) caused only a slightly delayed and blunted first‐phase insulin response compared to unencapsulated islets, larger capsules (d = 1,800 µm) completely blunted the first‐phase peak and decreased the total amount of insulin released. Experimentally obtained insulin time‐profiles were fitted with our complex insulin secretion computational model. This allowed further fine‐tuning of the hormone‐release parameters of this model, which was implemented in COMSOL Multiphysics to couple hormone secretion and nutrient consumption kinetics with diffusive and convective transport. The results of these GSIR experiments, which were also supported by computational modeling, indicate that larger capsules unavoidably lead to dampening of the first‐phase insulin response and to a sustained‐release type insulin secretion that can only slowly respond to changes in glucose concentration. Bioartificial pancreas type devices can provide long‐term and physiologically desirable solutions only if immunoisolation and biocompatibility considerations are integrated with optimized nutrient diffusion and insulin release characteristics by design.