Correlation of Utrophin Levels with the Dystrophin Protein Complex and Muscle Fibre Regeneration in Duchenne and Becker Muscular Dystrophy Muscle Biopsies

Duchenne muscular dystrophy is a severe and currently incurable progressive neuromuscular condition, caused by mutations in the DMD gene that result in the inability to produce dystrophin. Lack of dystrophin leads to loss of muscle fibres and a reduction in muscle mass and function. There is evidence from dystrophin-deficient mouse models that increasing levels of utrophin at the muscle fibre sarcolemma by genetic or pharmacological means significantly reduces the muscular dystrophy pathology. In order to determine the efficacy of utrophin modulators in clinical trials, it is necessary to accurately measure utrophin levels and other biomarkers on a fibre by fibre basis within a biopsy section. Our aim was to develop robust and reproducible staining and imaging protocols to quantify sarcolemmal utrophin levels, sarcolemmal dystrophin complex members and numbers of regenerating fibres within a biopsy section. We quantified sarcolemmal utrophin in mature and regenerating fibres and the percentage of regenerating muscle fibres, in muscle biopsies from Duchenne, the milder Becker muscular dystrophy and controls. Fluorescent immunostaining followed by image analysis was performed to quantify utrophin intensity and β-dystrogylcan and ɣ –sarcoglycan intensity at the sarcolemma. Antibodies to fetal and developmental myosins were used to identify regenerating muscle fibres allowing the accurate calculation of percentage regeneration fibres in the biopsy. Our results indicate that muscle biopsies from Becker muscular dystrophy patients have fewer numbers of regenerating fibres and reduced utrophin intensity compared to muscle biopsies from Duchenne muscular dystrophy patients. Of particular interest, we show for the first time that the percentage of regenerating muscle fibres within the muscle biopsy correlate with the clinical severity of Becker and Duchenne muscular dystrophy patients from whom the biopsy was taken. The ongoing development of these tools to quantify sarcolemmal utrophin and muscle regeneration in muscle biopsies will be invaluable for assessing utrophin modulator activity in future clinical trials.     

Appearance of different phosphatase forms and phosphorus partitioning in nodules of chickpea (Cicer arietinum L.) during development

Acid and alkaline phosphatase and phytase activities were determined in the bacteroid free fractions of chickpea (Cicer arietinum L.) nodules at 15 days intervals, from 40 days after sowing (DAS) to 85 DAS. In general, the activities and specific activity of both the acid and alkaline phosphatases declined at 55 DAS. Out of the various substrates studied, ATP was the best substrate for both phosphatases. Activities of phosphatases with glucose-6-phosphate and fructose-6-phosphate were low in comparison to these with fructose 1,6 bisphosphate. The efficiency of acid phosphatase for utilizing fructose 1,6 bis phosphate as a substrate increased with nodule development. A fructose 1,6 bis phosphate specific acid phosphatase with elution volume to void volume (Ve/Vo) ratio of around 2.0 was observed in mature nodules (80 DAS). Acid phosphatase at 40 DAS was resolved into two peaks which were eluted at Ve/Vo of about 1.5 and 1.8. However, at 60 DAS the peak with Ve/Vo of 1.5 could not be detected. With ATP as substrate, a high (Ve/Vo of 1.2) and low MM form (Ve/Vo of 2.1) alkaline phosphatases were observed at 40 DAS however at 60 DAS stage only one peak with Ve/Vo of 1.7 was detected. Although, a low activity of acid phytase was observed in nodules at all stages of development but neither alkaline phytase nor phytic acid could be detected. It appears that the nodules acquire inorganic phosphate from the roots. The higher content of water soluble organic phosphorus in mature nodules could be due to the low activities of phosphatases at maturity.     

Effect of osmo- and hydropriming of chickpea seeds on seedling growth and carbohydrate metabolism under water deficit stress

Seven-day-old seedlings obtained from seeds primed with mannitol (4%)and water showed three to four fold more growth with respect to root and shootlength in comparison with seedlings obtained from non-primed seeds. Seedlingswere grown under water deficit stress conditions created by 15% polyethyleneglycol (PEG) 6000 in the medium. Priming of chickpea seeds with NaCl and PEGwasnot effective in increasing seedling growth under these water deficit stressconditions. The activities of amylase, invertases (acid and alkaline), sucrosesynthase (SS) and sucrose phosphate synthase (SPS) were higher in shoots ofprimed seedlings. An increase in the activities of SS, and both the acid andalkaline invertases was also observed in roots of primed seedlings. The twofoldincrease in specific activity of sucrose phosphate synthase was observed incotyledons of primed seedlings. The higher amylase activity in shoots of primedseedlings enhanced the rapid hydrolysis of transitory starch of the shootleading to more availability of glucose for shoot growth and this was confirmedby the low level of starch in shoots of primed seedlings.     

Effect of water deficit on carbohydrate status and enzymes of carbohydrate metabolism in seedlings of wheat cultivars

The effect of water deficit on carbohydrate status and enzymes of carbohydrate metabolism (alpha and beta amylases, sucrose phosphate synthase, sucrose synthase, acid and alkaline invertases) in wheat (Triticum aestivum L.) was investigated in the seedlings of drought-sensitive (PBW 343) and drought-tolerant (C 306) cultivars. The water deficit was induced by adding 6% mannitol (water potential -0.815 Mpa) in the growth medium. The water deficit reduced starch content in the shoots of tolerant seedlings as compared to the sensitive ones, but increased sucrose content in the shoots and roots of tolerant seedlings, indicating their protective role during stress conditions. It also decreased the alpha-amylase activity in the endosperm of seedlings of both the cultivars, but increased alpha and beta amylase activities in the shoots of tolerant ones. Sucrose phosphate synthase (SPS) activity showed a significant increase at 6 days of seedling growth (DSG) in the shoots of stressed seedlings of tolerant cultivar. However, SPS activity in the roots of stressed seedlings of sensitive cultivar was very low at 4 DSG and appeared significantly only at day 6. Sucrose synthase (SS) activity was lower in the shoots and roots of stressed seedlings of tolerant cultivar than sensitive ones at early stage of seedling growth. Higher acid invertase activity in the shoots of seedlings of tolerant cultivar appeared to be a unique characteristic of this cultivar for stress tolerance. Alkaline invertase activity, although affected under water deficit conditions, but was too low as compared to acid invertase activity to cause any significant affect on sucrose hydrolysis. In conclusion, higher sucrose content with high SPS and low acid invertase and SS activities in the roots under water deficit conditions could be responsible for drought tolerance of C 306.     

Genetic characterization and curation of diploid A-genome wheat species

A-genome diploid wheats represent the earliest domesticated and cultivated wheat species in the Fertile Crescent and include the donor of the wheat A sub-genome. The A-genome species encompass the cultivated einkorn (Triticum monococcum L. subsp. monococcum), wild einkorn (T. monococcum L. subsp. aegilopoides (Link) Thell.), and Triticum urartu. We evaluated the collection of 930 accessions in the Wheat Genetics Resource Center (WGRC) using genotyping by sequencing and identified 13,860 curated single-nucleotide polymorphisms. Genomic analysis detected misclassified and genetically identical (>99%) accessions, with most of the identical accessions originating from the same or nearby locations. About 56% (n = 520) of the WGRC A-genome species collections were genetically identical, supporting the need for genomic characterization for effective curation and maintenance of these collections. Population structure analysis confirmed the morphology-based classifications of the accessions and reflected the species geographic distributions. We also showed that T. urartu is the closest A-genome diploid to the A-subgenome in common wheat (Triticum aestivum L.) through phylogenetic analysis. Population analysis within the wild einkorn group showed three genetically distinct clusters, which corresponded with wild einkorn races α, β, and γ described previously. The T. monococcum genome-wide F_ST scan identified candidate genomic regions harboring a domestication selection signature at the Non-brittle rachis 1 (Btr1) locus on the short arm of chromosome 3A^m at ∼70 Mb. We established an A-genome core set (79 accessions) based on allelic diversity, geographical distribution, and available phenotypic data. The individual species core set maintained at least 79% of allelic variants in the A-genome collection and constituted a valuable genetic resource to improve wheat and domesticated einkorn in breeding programs.

Genotyping diploid A-genome relatives of wheat uncovered high genetic diversity and unique evolutionary relationships giving insight to the effective use of this germplasm for wheat improvement.     

Pyrrole‐coupled salicylimine‐based fluorescence “turn on” probe for highly selective recognition of Zn2+ ions in mixed aqueous media: Application in living cell imaging

Cation sensing behaviour of a pyrrole‐based derivative (2‐hydroxyl 3 methyl 6 isopropyl benzaldehyde}‐3,4‐dimethyl‐1H‐pyrrole‐2‐carbohydrazide (receptor 3) has been explored and is found to be selective towards Zn²⁺ over a variety of tested cations. The receptor 3 has shown high selectivity and sensitivity towards Zn²⁺ over the other alkali, alkaline earth and transition metal ions. In the presence of Zn²⁺, absorption band of receptor 3 has shown the red shift. The sensing behaviour has been suggested to continue via enhancement process which has further been supported by UV‐vis absorption and theoretical density functional theory (DFT) calculations indicating the formation of a 1:1 complex between the pyrrole based receptor 3 and Zn²⁺. The present work is presenting a highly selective dual channel colorimetric sensor for zinc with great sensitivity. The developed sensor was successfully applied to image intracellular Zn²⁺ in living cells. Copyright © 2015 John Wiley & Sons, Ltd.     

Effect of hydro- and osmopriming of chickpea (Cicer arietinum L.) seeds on enzymes of sucrose and nitrogen metabolism in nodules

Three year data on the effect of water- and mannitol (4%) priming of chickpea seeds (12 h at 25°C) showed higher number and biomass of nodules in the plants from primed seeds than from non-primed seeds. The biomass of nodules increased to 75 DAS but decreased by 90 DAS. Activities of sucrose metabolism enzymes (sucrose synthase (SS) and alkaline invertase) and of nitrogen metabolism (glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH)) in nodules of primed and non-primed crops during development are reported. SS and alkaline invertase activities increased to 70 DAS and then decreased. In primed plants, the higher SS activity in nodules at 60 and 70 DAS might be responsible for providing more energy and carbon skeleton for nitrogen fixation and for ammonium assimilation in primed plants. At 85 DAS, though the SS activity decreased in comparison with the earlier growth stages, it was still higher in nodules of the primed crops than the non-primed crop. Activity of alkaline invertase was maximum at 70 DAS in the nodules of primed and non-primed crops. Priming increased nodule GS activity at 70 and 85 DAS. GOGAT activity was unaffected by priming but GDH activity was greater in nodules from primed crops at 50 DAS. Elevated SS and GS nodule activities in primed chickpeas might be responsible in increasing nodule biomass and metabolic activity thereby increasing seed fill.     

Leaf litter colonization by invertebrates in the littoral zone of a small oligotrophic lake

The colonization of deciduous leaf litter by aquatic invertebrates was studied at Scott Lake in Algonquin Park, Ontario, Canada. Deciduous leaf packs were colonized after only 2 days submergence. The invertebrate community was dominated by chironomids (25–94% depending on sampling period), and to a lesser extent by oligochaetes, turbellarians, and mayflies. Collectors, such as the chironomids Dicrotendipes, Pseudochironomus, Paratanytarsus and Parakiefferiella were the dominant functional-feeding group suggesting that leaf litter is being used as habitat rather than a direct food source. Deciduous leaf litter lost a substantial amount of weight, due to leaching, after only 48 h submergence. Fall-shed beech (Fagus grandifolia) leaves decomposed more rapidly than fall-shed sugar maple (Acer saccharum) leaves with daily processing coefficients (k), determined using an exponential decay model, of 0.0058 and 0.0039, respectively. Conversely, conditioned maple leaves, defined as leaves remaining on the ground over winter, were processed faster than conditioned beech leaves, with coefficients of 0.0042 and 0.0014, respectively. It is speculated that inhibitory compounds have been leached from the maple leaves, allowing for faster leaf processing. This revised version was published online in July 2006 with corrections to the Cover Date.