Myoblast senescence in muscular dystrophy

The limited proliferative capacity of normal diploid cells predicts that the utilization of cell divisions in vivo should reduce the lifespan of cells in culture. Because of the continuing demands for muscle regeneration in muscular dystrophy, myoblasts isolated from affected muscles should thus show a decrease in the number of cell divisions they are capable of expressing in culture. This hypothesis was tested by examining the proliferative capacity of myoblasts from different muscles for normal line 412 and dystrophic line 413 chickens of various ages. Prior to approx. 2 months of age, dystrophic myoblasts exhibited a relatively normal proliferative lifespan. By 5 months of age, myoblasts from the severely affected pectoralis major showed a 40% reduction in their proliferative potential, while myoblasts from the less affected posterior latissimus dorsi muscle showed a 25% decrease in their cultured lifespan. The time course of the appearance of a decreased proliferative capacity only after the disease has been clinically manifested strongly supports it representing a secondary response rather than it being an intrinsic property of dystrophic myoblasts. A hypothesis for manipulating the pattern of stem cell division in order to increase the mass of muscle produced from a constant number of cell divisions is presented. If myoblast senescence and the consequent failure of muscle regeneration is a contributing factor in the progressive deterioration of muscle function in the disease, then this hypothesis might provide an important therapeutic strategy for ameliorating the course of muscular dystrophy.     

Bronchoabsorption; a novel bronchoscopic technique to improve biomarker sampling of the airway

Background

Current techniques used to obtain lung samples have significant limitations and do not provide reproducible biomarkers of inflammation. We have developed a novel technique that allows multiple sampling methods from the same area (or multiple areas) of the lung under direct bronchoscopic vision. It allows collection of mucosal lining fluid and bronchial brushing from the same site; biopsy samples may also be taken. The novel technique takes the same time as standard procedures and can be conducted safely.

Methods

Eight healthy smokers aged 40–65 years were included in this study. An absorptive filter paper was applied to the bronchial mucosa under direct vision using standard bronchoscopic techniques. Further samples were obtained from the same site using bronchial brushings. Bronchoalveolar lavage (BAL) was obtained using standard techniques. Chemokine (C-C Motif) Ligand 20 (CCL20), CCL4, CCL5, Chemokine (C-X-C Motif) Ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, Interleukin 1 beta (IL-1β), IL-6, Vascular endothelial growth factor (VEGF), Matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured in exudate and BAL. mRNA was collected from the bronchial brushings for gene expression analysis.

Results

A greater than 10 fold concentration of all the biomarkers was detected in lung exudate in comparison to BAL. High yield of good quality RNA with RNA integrity numbers (RIN) between 7.6 and 9.3 were extracted from the bronchial brushings. The subset of genes measured were reproducible across the samples and corresponded to the inflammatory markers measured in exudate and BAL.

Conclusions

The bronchoabsorption technique as described offers the ability to sample lung fluid direct from the site of interest without the dilution effects caused by BAL. Using this method we were able to successfully measure the concentrations of biomarkers present in the lungs as well as collect high yield mRNA samples for gene expression analysis from the same site. This technique demonstrates superior sensitivity to standard BAL for the measurement of biomarkers of inflammation. It could replace BAL as the method of choice for these measurements. This method provides a systems biology approach to studying the inflammatory markers of respiratory disease progression.

Trial registration

NHS Health Research Authority (13/LO/0256).     

SECRETION OF LIPASES IN THE DIGESTIVE TRACT OF THE CRICKET Gryllus bimaculatus

Little is known concerning the sites and the ratios of the lipase secretions in insects, therefore we undertook an examination of the lipase secretion of fed and unfed adult female Gryllus bimaculatus. The ratio of triacylglyceride lipase, diacylglyceride lipase, and phosphatidylcholine lipase secreted by fed females in the caecum and ventriculus is 1:1.4:0.4. These activities decrease in the caecum by 30–40% in unfed females. The total lipase activity (TLA) in the caecum is about 10 times that in the ventriculus. Minimal lipase secretion occurs before and during the final moult, and remains at this level in unfed crickets, indicating a basal secretion rate. In 2‐day‐old fed females, about 10% of the TLA in the entire gut is found in the crop, about 70% in the caecum, 20% in the ventriculus, and 3% in the ileum. Lipases in the ventriculus are recycled back to the caecum and little is lost in the feces. Oleic acid stimulated in vitro lipase secretion, but lipids did not. Feeding stimulated lipase secretion, starvation reduced lipase secretion, but this does not prove a direct prandal regulation of secretion, because feeding also induced a size and volume increase of the caecum.     

A comparison of ten digestive enzymes reveals a lack of chitinase in a phasmid and the loss of two β-glucanases in a mantid

In all developmental stages, the phasmid Peruphasma schultei (Conle & Hennemann, 2005) is an obligate herbivore, whereas the mantid Hierodula membranacea (Burmeister, 1838) is an obligatory carnivore. In P. schultei, the luminal activity of all enzymes is approxximately 50% in the crop and 50% in the midgut, which corresponds to the approximate 50 : 50 ratio of volumes of these two regions. These ratios would be expected in insects with a constant feeding rate on an unvaried diet. The enzyme activity and volume ratios in Hierodula membranacea vary considerably because of the irregular feeding habits. These differences in activity ratios between phasmids and mantids are not associated with the obligate phytophagous or carnivorous diet. The ratio of membrane bound to luminal aminopeptidases and disaccharidases in the midgut of both species are not significantly different and are within the normal range of other paurometabolous insects. Cellobiase and other plant cell wall digesting enzymes, laminarinase and cellobiase, are present in the phasmid but totally lacking in the mantid. The obligate carnivorous feeding habits of mantids could represent a selective factor leading to the loss of the ability to produce β-glucanases. Chitinase is a moulting enzyme in all insects, whereas, in H. membranacea, chitinase also occurs as a luminal digestive enzyme. This modified enzyme function requires production and secretion in another tissue, namely the midgut.     

REGULATION OF AMYLASE,CELLULASE AND CHITINASE SECRETION IN THE DIGESTIVE TRACT OF THE TWO‐SPOTTED FIELD CRICKET,Gryllus bimaculatus

The secretion of amylase and cellulase in Gryllus bimaculatus is determined by increased food intake, whereby shortly after molting food consumption increases. About half of the standing amylase concentration (activity) in the endothelial cells can be secreted within 30 min. The peak of amylase and cellulase secretion that occurs in the photophase is related to the feeding peak in the previous scotophase. The secretion of chitinase on the other hand is primarily controlled by the molting cycle. Only amylase secretion was affected by calcium in the incubation medium, suggesting an apocrine release mechanism. Refeeding experiments (after 5 days without food) suggest that the release of amylase in response to a nutrient in the lumen (glucose) is not due to simple stimulation of exocytosis, but rather a stimulation of synthesis.     

<Emphasis Type="Italic">Burkholderia</Emphasis> Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

Background  

The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei.     

Telomere Biology and Cellular Aging in Nonhuman Primate Cells

To determine how cellular aging is conserved among primates, we analyzed the replicative potential and telomere shortening in skin fibroblasts of anthropoids and prosimians. The average telomere length of the New World primates Ateles geoffroyi (spider monkey) and Saimiri sciureus (squirrel monkey) and the Old World primates Macaca mulatta (rhesus monkey), Pongo pygmaeus (orangutan), and Pan paniscus (pigmy chimpanzee) ranged from 4 to 16 kb. We found that telomere shortening limits the replicative capacity of anthropoid fibroblasts and that the expression of human telomerase produced telomere elongation and the extension of their in vitro life span. In contrast the prosimian Lemur catta (ring-tailed lemur) had both long and short telomeres and telomere shortening did not provide an absolute barrier to immortalization. Following a transient growth arrest a subset of cells showing a reduced number of chromosomes overgrew the cultures without activation of telomerase. Here we show that the presence of continuous TTAGGG repeats at telomeres and rigorous control of replicative aging by telomere shortening appear to be conserved among anthropoid primates but is less effective in prosimian lemurs.     

Assessment of therapeutic responses to gametocytocidal drugs in Plasmodium falciparum malaria

Background

Effective mating between laboratory-reared males and wild females is paramount to the success of vector control strategies aiming to decrease disease transmission via the release of sterile or genetically modified male mosquitoes. However mosquito colonization and laboratory maintenance have the potential to negatively affect male genotypic and phenotypic quality through inbreeding and selection, which in turn can decrease male mating competitiveness in the field. To date, very little is known about the impact of those evolutionary forces on the reproductive biology of mosquito colonies and how they ultimately affect male reproductive fitness.

Methods

Here several male reproductive physiological traits likely to be affected by inbreeding and selection following colonization and laboratory rearing were examined. Sperm length, and accessory gland and testes size were compared in male progeny from field-collected females and laboratory strains of Anopheles gambiae sensu stricto colonized from one to over 25 years ago. These traits were also compared in the parental and sequentially derived, genetically modified strains produced using a two-phase genetic transformation system. Finally, genetic crosses were performed between strains in order to distinguish the effects of inbreeding and selection on reproductive traits.

Results

Sperm length was found to steadily decrease with the age of mosquito colonies but was recovered in refreshed strains and crosses between inbred strains therefore incriminating inbreeding costs. In contrast, testes size progressively increased with colony age, whilst accessory gland size quickly decreased in males from colonies of all ages. The lack of heterosis in response to crossing and strain refreshing in the latter two reproductive traits suggests selection for insectary conditions.

Conclusions

These results show that inbreeding and selection differentially affect reproductive traits in laboratory strains overtime and that heterotic ‘supermales’ could be used to rescue some male reproductive characteristics. Further experiments are needed to establish the exact relationship between sperm length, accessory gland and testes size, and male reproductive success in the laboratory and field settings.     

Effective and efficient data sampling using bitmap indices

With growing computational capabilities of parallel machines, scientific simulations are being performed at finer spatial and temporal scales, leading to a data explosion. The growing sizes are making it extremely hard to store, manage, disseminate, analyze, and visualize these datasets, especially as neither the memory capacity of parallel machines, memory access speeds, nor disk bandwidths are increasing at the same rate as the computing power. Sampling can be an effective technique to address the above challenges, but it is extremely important to ensure that dataset characteristics are preserved, and the loss of accuracy is within acceptable levels. In this paper, we address the data explosion problems by developing a novel sampling approach, and implementing it in a flexible system that supports server-side sampling and data subsetting. We observe that to allow subsetting over scientific datasets, data repositories are likely to use an indexing technique. Among these techniques, we see that bitmap indexing can not only effectively support subsetting over scientific datasets, but can also help create samples that preserve both value and spatial distributions over scientific datasets. We have developed algorithms for using bitmap indices to sample datasets. We have also shown how only a small amount of additional metadata stored with bitvectors can help assess loss of accuracy with a particular subsampling level. Some of the other properties of this novel approach include: (1) sampling can be flexibly applied to a subset of the original dataset, which may be specified using a value-based and/or a dimension-based subsetting predicate, and (2) no data reorganization is needed, once bitmap indices have been generated. We have extensively evaluated our method with different types of datasets and applications, and demonstrated the effectiveness of our approach.