Muscle mass,structural and functional investigations of senescence-accelerated mouse P8 (SAMP8)

Sarcopenia is an age-related systemic syndrome with progressive deterioration in skeletal muscle functions and loss in mass. Although the senescence-accelerated mouse P8 (SAMP8) was reported valid for muscular ageing research, there was no report on the details such as sarcopenia onset time. Therefore, this study was to investigate the change of muscle mass, structure and functions during the development of sarcopenia. Besides the average life span, muscle mass, structural and functional measurements were also studied. Male SAMP8 animals were examined at month 6, 7, 8, 9, and 10, in which the right gastrocnemius was isolated and tested for ex vivo contractile properties and fatigability while the contralateral one was harvested for muscle fiber cross-sectional area (FCSA) and typing assessments. Results showed that the peak of muscle mass appeared at month 7 and the onset of contractility decline was observed from month 8. Compared with month 8, most of the functional parameters at month 10 decreased significantly. Structurally, muscle fiber type IIA made up the largest proportion of the gastrocnemius, and the fiber size was found to peak at month 8. Based on the altered muscle mass, structural and functional outcomes, it was concluded that the onset of sarcopenia in SAMP8 animals was at month 8. SAMP8 animals at month 8 should be at pre-sarcopenia stage while month 10 at sarcopenia stage. It is confirmed that SAMP8 mouse can be used in sarcopenia research with established time line in this study.     

Biochemical analysis of pistol self-cleaving ribozymes

Pistol RNAs are members of a distinct class of self-cleaving ribozymes that was recently discovered by using a bioinformatics search strategy. Several hundred pistol ribozymes share a consensus sequence including 10 highly conserved nucleotides and many other modestly conserved nucleotides associated with specific secondary structure features, including three base-paired stems and a pseudoknot. A representative pistol ribozyme from the bacterium Lysinibacillus sphaericus was found to promote RNA strand scission with a rate constant of ∼10 min⁻¹ under physiological Mg²⁺ and pH conditions. The reaction proceeds via the nucleophilic attack of a 2′-oxygen atom on the adjacent phosphorus center, and thus adheres to the same general catalytic mechanism of internal phosphoester transfer as found with all other classes of natural self-cleaving ribozymes discovered to date. Analyses of the kinetic characteristics and the metal ion requirements of the cleavage reaction reveal that members of this ribozyme class likely use several catalytic strategies to promote the rapid cleavage of RNA.     

DisCons: a novel tool to quantify and classify evolutionary conservation of intrinsic protein disorder

Background

Analyzing the amino acid sequence of an intrinsically disordered protein (IDP) in an evolutionary context can yield novel insights on the functional role of disordered regions and sequence element(s). However, in the case of many IDPs, the lack of evolutionary conservation of the primary sequence can hamper the study of functionality, because the conservation of their disorder profile and ensuing function(s) may not appear in a traditional analysis of the evolutionary history of the protein.

Results

Here we present DisCons (Disorder Conservation), a novel pipelined tool that combines the quantification of sequence- and disorder conservation to classify disordered residue positions. According to this scheme, the most interesting categories (for functional purposes) are constrained disordered residues and flexible disordered residues. The former residues show conservation of both the sequence and the property of disorder and are associated mainly with specific binding functionalities (e.g., short, linear motifs, SLiMs), whereas the latter class correspond to segments where disorder as a feature is important for function as opposed to the identity of the underlying sequence (e.g., entropic chains and linkers). DisCons therefore helps with elucidating the function(s) arising from the disordered state by analyzing individual proteins as well as large-scale proteomics datasets.

Conclusions

DisCons is an openly accessible sequence analysis tool that identifies and highlights structurally disordered segments of proteins where the conformational flexibility is conserved across homologs, and therefore potentially functional. The tool is freely available both as a web application and as stand-alone source code hosted at http://pedb.vib.be/discons.     

Construction of a virtual Mycobacterium tuberculosis consensus genome and its application to data from a next generation sequencer

Background

Although Mycobacterium tuberculosis isolates are consisted of several different lineages and the epidemiology analyses are usually assessed relative to a particular reference genome, M. tuberculosis H37Rv, which might introduce some biased results. Those analyses are essentially based genome sequence information of M. tuberculosis and could be performed in sillico in theory, with whole genome sequence (WGS) data available in the databases and obtained by next generation sequencers (NGSs). As an approach to establish higher resolution methods for such analyses, whole genome sequences of the M. tuberculosis complexes (MTBCs) strains available on databases were aligned to construct virtual reference genome sequences called the consensus sequence (CS), and evaluated its feasibility in in sillico epidemiological analyses.

Results

The consensus sequence (CS) was successfully constructed and utilized to perform phylogenetic analysis, evaluation of read mapping efficacy, which is crucial for detecting single nucleotide polymorphisms (SNPs), and various MTBC typing methods virtually including spoligotyping, VNTR, Long sequence polymorphism and Beijing typing. SNPs detected based on CS, in comparison with H37Rv, were utilized in concatemer-based phylogenetic analysis to determine their reliability relative to a phylogenetic tree based on whole genome alignment as the gold standard. Statistical comparison of phylogenic trees based on CS with that of H37Rv indicated the former showed always better results that that of later. SNP detection and concatenation with CS was advantageous because the frequency of crucial SNPs distinguishing among strain lineages was higher than those of H37Rv. The number of SNPs detected was lower with the consensus than with the H37Rv sequence, resulting in a significant reduction in computational time. Performance of each virtual typing was satisfactory and accorded with those published when those are available.

Conclusions

These results indicated that virtual CS constructed from genome sequence data is an ideal approach as a reference for MTBC studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1368-9) contains supplementary material, which is available to authorized users.     

Genome-wide survey and analysis of microsatellites in giant panda (Ailuropoda melanoleuca), with a focus on the applications of a novel microsatellite marker system

Background

The giant panda (Ailuropoda melanoleuca) is a critically endangered species endemic to China. Microsatellites have been preferred as the most popular molecular markers and proven effective in estimating population size, paternity test, genetic diversity for the critically endangered species. The availability of the giant panda complete genome sequences provided the opportunity to carry out genome-wide scans for all types of microsatellites markers, which now opens the way for the analysis and development of microsatellites in giant panda.

Results

By screening the whole genome sequence of giant panda in silico mining, we identified microsatellites in the genome of giant panda and analyzed their frequency and distribution in different genomic regions. Based on our search criteria, a repertoire of 855,058 SSRs was detected, with mono-nucleotides being the most abundant. SSRs were found in all genomic regions and were more abundant in non-coding regions than coding regions. A total of 160 primer pairs were designed to screen for polymorphic microsatellites using the selected tetranucleotide microsatellite sequences. The 51 novel polymorphic tetranucleotide microsatellite loci were discovered based on genotyping blood DNA from 22 captive giant pandas in this study. Finally, a total of 15 markers, which showed good polymorphism, stability, and repetition in faecal samples, were used to establish the novel microsatellite marker system for giant panda. Meanwhile, a genotyping database for Chengdu captive giant pandas (n = 57) were set up using this standardized system. What’s more, a universal individual identification method was established and the genetic diversity were analysed in this study as the applications of this marker system.

Conclusion

The microsatellite abundance and diversity were characterized in giant panda genomes. A total of 154,677 tetranucleotide microsatellites were identified and 15 of them were discovered as the polymorphic and stable loci. The individual identification method and the genetic diversity analysis method in this study provided adequate material for the future study of giant panda.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1268-z) contains supplementary material, which is available to authorized users.