A fast, reliable and sample-sparing method to identify fibre types of single muscle fibres.
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ABSTRACT:
SUBMITTER: Christiansen D
PROVIDER: S-EPMC6482153 | biostudies-literature | 2019 Apr
REPOSITORIES: biostudies-literature
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Project description:Fibre type-specific analyses are required for broader understanding of muscle physiology, but such analyses are difficult to conduct due to the extreme time requirements of dissecting and fibre typing individual fibres. Investigations are often confined to a small number of fibres from few participants with low representativeness of the entire fibre population and the participant population. To increase the feasibility of conducting large-scale fibre type-specific studies, a valid and rapid method for high-throughput fibre typing of individually dissected fibres was developed and named THRIFTY (for high-THRoughput Immunofluorescence Fibre TYping). Employing THRIFTY, 400 fibre segments were fixed onto microscope slides with a pre-printed coordinated grid system, probed with antibodies against myosin heavy chain (MyHC)-I and MyHC-II and classified using a fluorescence microscope. The validity and speed of THRIFTY was compared to a previously validated protocol (dot blot) on a fibre-to-fibre basis. Fibre pool purity was evaluated using 'gold standard' SDS-PAGE and silver staining. A modified THRIFTY-protocol using fluorescence western blot equipment was also validated. THRIFTY displayed excellent agreement with the dot blot protocol, κ = 0.955 (95% CI: 0.928, 0.982), P < 0.001. Both the original and modified THRIFTY protocols generated type I and type II fibre pools of absolute purity. Using THRIFTY, 400 fibres were typed just under 11 h, which was approximately 3 times faster than dot blot. THRIFTY is a novel and valid method with high versatility for very rapid fibre typing of individual fibres. THRIFTY can therefore facilitate the generation of large fibre pools for more extensive mechanistic studies into skeletal muscle physiology. KEY POINTS: Skeletal muscle is composed of different fibre types, each with distinct physiological properties. To fully understand how skeletal muscle adapts to external cues such as exercise, nutrition and ageing, fibre type-specific investigations are required. Such investigations are very difficult to conduct due to the extreme time requirements related to classifying individually isolated muscle fibres. To bypass this issue, we have developed a rapid and reliable method named THRIFTY which is cheap as well as versatile and which can easily be implemented in most laboratories. THRIFTY increases the feasibility of conducting larger fibre type-specific studies and enables time-sensitive assays where measurements need to be carried out in close connection with tissue sampling. By using THRIFTY, new insights into fibre type-specific muscle physiology can be gained which may have broad implications in health and disease.
| S-EPMC9825974 | biostudies-literature
Project description:Bacteriophages, that is viruses that infect bacteria, either lyse bacteria directly or integrate their genome into the bacterial genome as so-called prophages, where they remain at a silent state. Both phages and bacteria are able to survive in this state. However, prophages can be reactivated with the introduction of chemicals, followed by the release of a high number of phage particles, which could infect other bacteria, thus harming ecosystems by a viral bloom. The basics for a fast, automatable analytical method for the detection of prophage-activating chemicals are developed and successfully tested here. The method exploits the differences in metabolic heat produced by Escherichia coli with (λ+) and without the lambda prophages (λ-). Since the metabolic heat primarily reflects opposing effects (i.e. the reduction of heat-producing cells by lysis and enhanced heat production to deliver the energetic costs for the synthesis of phages), a systematic analysis of the influence of the different conditions (experimentally and in silico) was performed and revealed anoxic conditions to be best suited. The main advantages of the suggested monitoring method are not only the possibility of obtaining fast results (after only few hours), but also the option for automation, the low workload (requires only few minutes) and the suitability of using commercially available instruments. The future challenge following this proof of principle is the development of thermal transducers which allow for the electronic subtraction of the λ+ from the λ- signal.
| S-EPMC6196395 | biostudies-literature
Project description:Background and aimNext generation sequencing (ngs) is becoming the standard for clinical diagnosis. Different steps of NGS, such as DNA extraction, fragmentation, library preparation and amplification, require handling of samples, making the process susceptible to contamination. In diagnostic environments, sample contamination with DNA from the same species can lead to errors in diagnosis. Here we propose a simple method to detect within-sample contamination based on analysis of the heterozygous single nucleotide polymorphisms allele ratio (AR).MethodsA dataset of 38000 heterozygous snps was used to estimate the ar distribution. The parameters of the reference distribution were then used to estimate the contamination probability of a sample. Validation was performed using 12 samples contaminated to different levels.ResultsResults show that the method easily detects contamination of 20% or more. The method has a limit of detection of about 10%, threshold below which the number of false positives increases significantly.ConclusionsThe method can be applied to any type of ngs analysis and is useful for quality control. Being fast and easy to implement makes it ideal for inclusion in NGS pipelines to improve quality control of data and make results more robust.
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Project description:More reliable and faster prediction methods are needed to interpret enormous amounts of data generated by sequencing and genome projects. We have developed a new computational tool, PON-P2, for classification of amino acid substitutions in human proteins. The method is a machine learning-based classifier and groups the variants into pathogenic, neutral and unknown classes, on the basis of random forest probability score. PON-P2 is trained using pathogenic and neutral variants obtained from VariBench, a database for benchmark variation datasets. PON-P2 utilizes information about evolutionary conservation of sequences, physical and biochemical properties of amino acids, GO annotations and if available, functional annotations of variation sites. Extensive feature selection was performed to identify 8 informative features among altogether 622 features. PON-P2 consistently showed superior performance in comparison to existing state-of-the-art tools. In 10-fold cross-validation test, its accuracy and MCC are 0.90 and 0.80, respectively, and in the independent test, they are 0.86 and 0.71, respectively. The coverage of PON-P2 is 61.7% in the 10-fold cross-validation and 62.1% in the test dataset. PON-P2 is a powerful tool for screening harmful variants and for ranking and prioritizing experimental characterization. It is very fast making it capable of analyzing large variant datasets. PON-P2 is freely available at http://structure.bmc.lu.se/PON-P2/.
| S-EPMC4315405 | biostudies-literature
Project description:Growing environmental concerns and stringent waste-flow regulations make the development of sustainable composites a current industrial necessity. Natural fibre reinforcements are derived from renewable resources and are both cheap and biodegradable. When they are produced using eco-friendly, low hazard processes, then they can be considered as a sustainable source of fibrous reinforcement. Furthermore, their specific mechanical properties are comparable to commonly used, non-environmentally friendly glass-fibres. In this study, four types of abundant natural fibres (jute, kenaf, curaua, and flax) are investigated as naturally-derived constituents for high performance composites. Physical, thermal, and mechanical properties of the natural fibres are examined to evaluate their suitability as discontinuous reinforcements whilst also generating a database for material selection. Single fibre tensile and microbond tests were performed to obtain stiffness, strength, elongation, and interfacial shear strength of the fibres with an epoxy resin. Moreover, the critical fibre lengths of the natural fibres, which are important for defining the mechanical performances of discontinuous and short fibre composites, were calculated for the purpose of possible processing of highly aligned discontinuous fibres. This study is informative regarding the selection of the type and length of natural fibres for the subsequent production of discontinuous fibre composites.
| S-EPMC7254363 | biostudies-literature
Project description:BackgroundThe Pacu (Piaractus mesopotamicus) is a member of the Characiform family native to the Prata Basin (South America) and a target for the aquaculture industry. A limitation for the development of a selective breeding program for this species is a lack of available genetic information. The primary objectives of the present study were 1) to increase the genetic resources available for the species, 2) to exploit the anatomical separation of myotomal fibres types to compare the transcriptomes of slow and fast muscle phenotypes and 3) to systematically investigate the expression of Ubiquitin Specific Protease (USP) family members in fast and slow muscle in response to fasting and refeeding.ResultsWe generated 0.6 Tb of pair-end reads from slow and fast skeletal muscle libraries. Over 665 million reads were assembled into 504,065 contigs with an average length of 1,334 bp and N50 = 2,772 bp. We successfully annotated nearly 47% of the transcriptome and identified around 15,000 unique genes and over 8000 complete coding sequences. 319 KEGG metabolic pathways were also annotated and 380 putative microsatellites were identified. 956 and 604 genes were differentially expressed between slow and fast skeletal muscle, respectively. 442 paralogues pairs arising from the teleost-specific whole genome duplication were identified, with the majority showing different expression patterns between fibres types (301 in slow and 245 in fast skeletal muscle). 45 members of the USP family were identified in the transcriptome. Transcript levels were quantified by qPCR in a separate fasting and refeeding experiment. USP genes in fast muscle showed a similar transient increase in expression with fasting as the better characterized E3 ubiquitin ligases.ConclusionWe have generated a 53-fold coverage transcriptome for fast and slow myotomal muscle in the pacu (Piaractus mesopotamicus) significantly increasing the genetic resources available for this important aquaculture species. We describe significant differences in gene expression between muscle fibre types for fundamental components of general metabolism, the Pi3k/Akt/mTor network and myogenesis, including detailed analysis of paralogue expression. We also provide a comprehensive description of USP family member expression between muscle fibre types and with changing nutritional status.
| S-EPMC4372171 | biostudies-literature
Project description:It is generally accepted that carnosine (β-alanyl-L-histidine) content is higher in glycolytic than in oxidative muscle fibres, but the underlying mechanisms responsible for this difference remain to be elucidated. A first study to better understand potential mechanisms involved was undertaken (1) to determine whether differences in the expression of carnosine-related enzymes (CARNS1, CNDP2) and transporters (SLC6A6, SLC15A3, SLC15A4, SLC36A1) exist between oxidative and glycolytic myofibres and (2) to study the effect of carnosine on myoblast proliferative growth and on carnosine-related gene expression in cultured myoblasts isolated from glycolytic and oxidative muscles. Immunohistochemistry analyses were conducted to determine the cellular localization of carnosine-related proteins. Laser-capture microdissection and qPCR analyses were performed to measure the expression of carnosine-related genes in different myofibres isolated from the longissimus dorsi muscle of ten crossbred pigs. Myogenic cells originating from glycolytic and oxidative muscles were cultured to assess the effect of carnosine (0, 10, 25 and 50 mM) on their proliferative growth and on carnosine-related gene expression. The mRNA abundance of CNDP2 and of the studied carnosine transporters was higher in oxidative than in glycolytic myofibres. Since carnosine synthase (CARNS1) mRNA abundance was not affected by either the fibre type or the addition of carnosine to myoblasts, its transcriptional regulation would not be the main process by which carnosine content differences are determined in oxidative and glycolytic muscles. The addition of carnosine to myoblasts leading to a dose-dependent increase in SLC15A3 transcripts, however, suggests a role for this transporter in carnosine uptake and/or efflux to maintain cellular homeostasis.
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Project description:BackgroundBast fibres from the phloem tissues of flax are scientifically interesting and economically useful due in part to a dynamic system of secondary cell wall deposition. To better understand the molecular mechanisms underlying the process of cell wall development in flax, we extracted proteins from individually dissected phloem fibres (i.e. individual cells) at an early stage of secondary cell wall development, and compared these extracts to protein extracts from surrounding, non-fibre cells of the cortex, using fluorescent (DiGE) labels and 2D-gel electrophoresis, with identities assigned to some proteins by mass spectrometry.ResultsThe abundance of many proteins in fibres was notably different from the surrounding non-fibre cells of the cortex, with approximately 13% of the 1,850 detectable spots being significantly (> 1.5 fold, p < or = 0.05) enriched in fibres. Following mass spectrometry, we assigned identity to 114 spots, of which 51 were significantly enriched in fibres. We observed that a K+ channel subunit, annexins, porins, secretory pathway components, beta-amylase, beta-galactosidase and pectin and galactan biosynthetic enzymes were among the most highly enriched proteins detected in developing flax fibres, with many of these proteins showing electrophoretic patterns consistent with post-translational modifications.ConclusionThe fibre-enriched proteins we identified are consistent with the dynamic process of secondary wall deposition previously suggested by histological and biochemical analyses, and particularly the importance of galactans and the secretory pathway in this process. The apparent abundance of beta-amylase suggests that starch may be an unappreciated source of materials for cell wall biogenesis in flax bast fibres. Furthermore, our observations confirm previous reports that correlate accumulation proteins such as annexins, and specific heat shock proteins with secondary cell wall deposition.
| S-EPMC2408578 | biostudies-literature
Project description:Small-molecule compounds strongly affecting osteogenesis can form the basis of effective therapeutic strategies in bone regenerative medicine. A cell-based high-throughput screening system might be a powerful tool for identifying osteoblast-targeting candidates; however, this approach is generally limited with using only one molecule as a cell-based sensor that does not always reflect the activation of the osteogenic phenotype. In the present study, we used the MC3T3-E1 cell line stably transfected with the green fluorescent protein (GFP) reporter gene driven by a fragment of type I collagen promoter (Col-1a1GFP-MC3T3-E1) to evaluate a double-screening system to identify osteogenic inducible compounds using a combination of a cell-based reporter assay and detection of alkaline phosphatase (ALP) activity. Col-1a1GFP-MC3T3-E1 cells were cultured in an osteogenic induction medium after library screening of 1280 pharmacologically active compounds (Lopack1280). After 7 days, GFP fluorescence was measured using a microplate reader. After 14 days of osteogenic induction, the cells were stained with ALP. Library screening using the Col-1a1/GFP reporter and ALP staining assay detected three candidates with significant osteogenic induction ability. Furthermore, leflunomide, one of the three detected candidates, significantly promoted new bone formation in vivo. Therefore, this double-screening method could identify candidates for osteogenesis-targeting compounds more reliably than conventional methods.
| S-EPMC8962348 | biostudies-literature
Project description:1. Microsomes were isolated from rabbit fast-twitch and slow-twitch muscle and were separated into heavy and light fractions by centrifugation in a linear (0.3-2m) sucrose density gradient. The membrane origin of microsomal vesicles was investigated by studying biochemical markers of the sarcoplasmic-reticulum membranes and of surface and T-tubular membranes, as well as their freeze-fracture properties. 2. Polyacrylamide-gel electrophoresis showed differences in the Ca(2+)-dependent ATPase/calsequestrin ratio between heavy and light fractions, which were apparently consistent with their respective origin from cisternal and longitudinal sarcoplasmic reticulum, as well as unrelated differences, such as peptides specific to slow-muscle microsomes (mol.wts. 76000, 60000, 56000 and 45000). 3. Freeze-fracture electron microscopy of muscle microsomes demonstrated that vesicles truly derived from the sarcoplasmic reticulum, with an average density of 9nm particles on the concave face of about 3000/mum(2) for both fast and slow muscle, were admixed with vesicles with particle densities below 1000/mum(2). 4. As determined in the light fractions, the sarcoplasmic-reticulum vesicles accounted for 84% and 57% of the total number of microsomal vesicles, for fast and slow muscle respectively. These values agreed closely with the percentage values of Ca(2+)-dependent ATPase protein obtained by gel densitometry. 5. The T-tubular origin of vesicles with a smooth concave fracture face in slow-muscle microsomes is supported by their relative high content in total phospholipid and cholesterol, compared with the microsomes of fast muscle, and by other correlative data, such as the presence of (Na(+)+K(+))-dependent ATPase activity and of low amounts of Na(+)-dependent membrane phosphorylation. 6. Among intrinsic sarcoplasmic-reticulum membrane proteins, a proteolipid of mol.wt. 12000 is shown to be identical in the microsomes of both fast and slow muscle and the Ca(2+)-dependent ATPase to be antigenically and catalytically different, though electrophoretically homogeneous. 7. Basal Mg(2+)-activated ATPase activity was found to be high in light microsomes from slow muscle, but its identification with an enzyme different from the Ca(2+)-dependent ATPase is still not conclusive. 8. Enzyme proteins that are suggested to be specific to slow-muscle longitudinal sarcoplasmic reticulum are the flavoprotein NADH:cytochrome b(5) reductase (mol.wt. 32000), cytochrome b(5) (mol.wt. 17000) and the stearoyl-CoA desaturase, though essentially by criteria of plausibility.
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