Gene expression changes of single skeletal muscle fibers in response to modulation of the mitochondrial calcium uniporter (MCU)

The mitochondrial calcium uniporter (MCU) gene codifies for the inner mitochondrial membrane (IMM) channel responsible for mitochondrial Ca2 + uptake. Cytosolic Ca2 + transients are involved in sarcomere contraction through cycles of release and storage in the sarcoplasmic reticulum. In addition cytosolic Ca2 + regulates various signaling cascades that eventually lead to gene expression reprogramming. Mitochondria are strategically placed in close contact with the ER/SR, thus cytosolic Ca2 + transients elicit large increases in the [Ca2 +] of the mitochondrial matrix ([Ca2 +]mt). Mitochondrial Ca2 + uptake regulates energy production and cell survival. In addition, we recently showed that MCU-dependent mitochondrial Ca2 + uptake controls skeletal muscle trophism. In the same report, we dissected the effects of MCU-dependent mitochondrial Ca2 + uptake on gene expression through microarray gene expression analysis upon modulation of MCU expression by in vivo AAV infection. Analyses were performed on single skeletal muscle fibers at two time points (7 and 14 days post-AAV injection). Raw and normalized data are available on the GEO database (http://www.ncbi.nlm.nih.gov/geo/) (GSE60931)

MIDAW: a web tool for statistical analysis of microarray data

MIDAW (microarray data analysis web tool) is a web interface integrating a series of statistical algorithms that can be used for processing and interpretation of microarray data. MIDAW consists of two main sections: data normalization and data analysis. In the normalization phase the simultaneous processing of several experiments with background correction, global and local mean and variance normalization are carried out. The data analysis section allows graphical display of expression data for descriptive purposes, estimation of missing values, reduction of data dimension, discriminant analysis and identification of marker genes. The statistical results are organized in dynamic web pages and tables, where the transcript/gene probes contained in a specific microarray platform can be linked (according to user choice) to external databases (GenBank, Entrez Gene, UniGene). Tutorial files help the user throughout the statistical analysis to ensure that the forms are filled out correctly. MIDAW has been developed using Perl and PHP and it uses R/Bioconductor languages and routines. MIDAW is GPL licensed and freely accessible at . Perl and PHP source codes are available from the authors upon request

Inspiratory muscle training and its effect on indices of physiological and perceived stress during incremental walking exercise in normobaric hypoxia

This study evaluated the effects of inspiratory muscle training (IMT) on inspiratory muscle fatigue (IMF) and physiological and perceptual responses during trekking-specific exercise. An 8-week IMT program was completed by 21 males (age 32.4 ± 9.61 years, VO2peak 58.8 ± 6.75 mL/kg/min) randomised within matched pairs to either the IMT group (n = 11) or the placebo group [(P), n = 9]. Twice daily, participants completed 30 (IMT) or 60 (P) inspiratory efforts using a Powerbreathe initially set at a resistance of 50% (IMT) or used at 15% (P) of maximal inspiratory pressure (MIP) throughout. A loaded (12.5 kg) 39-minute incremental walking protocol (3–5 km/hour and 1–15% gradient) was completed in normobaric hypoxia (PIO2 = 110 mmHg, 3000 m) before and after training. MIP increased from 164 to 188 cmH2O (18%) and from 161 to 171 cmH2O (6%) in the IMT and P groups (P = 0.02). The 95% CI for IMT showed a significant improvement in MIP (5.21±43.33 cmH2O), but not for P. IMF during exercise (MIP) was*5%, showing no training effect for either IMT or P (P = 0.23). Rating of perceived exertion (RPE) was consistently reduced (*1) throughout exercise following training for IMT, but not for P (P = 0.03). The mean blood lactate concentration during exercise was significantly reduced by 0.26 and 0.15 mmol/L in IMT and P (P = 0.00), with no differences between groups (P = 0.34). Rating of dyspnoea during exercise decreased (*0.4) following IMT but increased (*0.3) following P (P = 0.01). IMT may attenuate the increased physiological and perceived exercise stress experienced during normobaric hypoxia, which may benefit moderate altitude expedition

Statistical Test of Expression Pattern (STEPath): a new strategy to integrate gene expression data with genomic information in individual and meta-analysis studies

<p>Abstract</p> <p>Background</p> <p>In the last decades, microarray technology has spread, leading to a dramatic increase of publicly available datasets. The first statistical tools developed were focused on the identification of significant differentially expressed genes. Later, researchers moved toward the systematic integration of gene expression profiles with additional biological information, such as chromosomal location, ontological annotations or sequence features. The analysis of gene expression linked to physical location of genes on chromosomes allows the identification of transcriptionally imbalanced regions, while, Gene Set Analysis focuses on the detection of coordinated changes in transcriptional levels among sets of biologically related genes.</p> <p>In this field, meta-analysis offers the possibility to compare different studies, addressing the same biological question to fully exploit public gene expression datasets.</p> <p>Results</p> <p>We describe STEPath, a method that starts from gene expression profiles and integrates the analysis of imbalanced region as an <it>a priori </it>step before performing gene set analysis. The application of STEPath in individual studies produced gene set scores weighted by chromosomal activation. As a final step, we propose a way to compare these scores across different studies (meta-analysis) on related biological issues. One complication with meta-analysis is batch effects, which occur because molecular measurements are affected by laboratory conditions, reagent lots and personnel differences. Major problems occur when batch effects are correlated with an outcome of interest and lead to incorrect conclusions. We evaluated the power of combining chromosome mapping and gene set enrichment analysis, performing the analysis on a dataset of leukaemia (example of individual study) and on a dataset of skeletal muscle diseases (meta-analysis approach).</p> <p>In leukaemia, we identified the Hox gene set, a gene set closely related to the pathology that other algorithms of gene set analysis do not identify, while the meta-analysis approach on muscular disease discriminates between related pathologies and correlates similar ones from different studies.</p> <p>Conclusions</p> <p>STEPath is a new method that integrates gene expression profiles, genomic co-expressed regions and the information about the biological function of genes. The usage of the STEPath-computed gene set scores overcomes batch effects in the meta-analysis approaches allowing the direct comparison of different pathologies and different studies on a gene set activation level.</p

Meta-analysis of expression signatures of muscle atrophy: gene interaction networks in early and late stages

<p>Abstract</p> <p>Background</p> <p>Skeletal muscle mass can be markedly reduced through a process called atrophy, as a consequence of many diseases or critical physiological and environmental situations. Atrophy is characterised by loss of contractile proteins and reduction of fiber volume. Although in the last decade the molecular aspects underlying muscle atrophy have received increased attention, the fine mechanisms controlling muscle degeneration are still incomplete. In this study we applied meta-analysis on gene expression signatures pertaining to different types of muscle atrophy for the identification of novel key regulatory signals implicated in these degenerative processes.</p> <p>Results</p> <p>We found a general down-regulation of genes involved in energy production and carbohydrate metabolism and up-regulation of genes for protein degradation and catabolism. Six functional pathways occupy central positions in the molecular network obtained by the integration of atrophy transcriptome and molecular interaction data. They are TGF-β pathway, apoptosis, membrane trafficking/cytoskeleton organization, NFKB pathways, inflammation and reorganization of the extracellular matrix. Protein degradation pathway is evident only in the network specific for muscle short-term response to atrophy. TGF-β pathway plays a central role with proteins SMAD3/4, MYC, MAX and CDKN1A in the general network, and JUN, MYC, GNB2L1/RACK1 in the short-term muscle response network.</p> <p>Conclusion</p> <p>Our study offers a general overview of the molecular pathways and cellular processes regulating the establishment and maintenance of atrophic state in skeletal muscle, showing also how the different pathways are interconnected. This analysis identifies novel key factors that could be further investigated as potential targets for the development of therapeutic treatments. We suggest that the transcription factors SMAD3/4, GNB2L1/RACK1, MYC, MAX and JUN, whose functions have been extensively studied in tumours but only marginally in muscle, appear instead to play important roles in regulating muscle response to atrophy.</p

New miRNA labeling method for bead-based quantification

<p>Abstract</p> <p>Background</p> <p>microRNAs (miRNAs) are small single-stranded non-coding RNAs that act as crucial regulators of gene expression. Different methods have been developed for miRNA expression profiling in order to better understand gene regulation in normal and pathological conditions. miRNAs expression values obtained from large scale methodologies such as microarrays still need a validation step with alternative technologies.</p> <p>Results</p> <p>Here we have applied with an innovative approach, the Luminex^®xMAP™ technology validate expression data of differentially expressed miRNAs obtained from high throughput arrays. We have developed a novel labeling system of small RNA molecules (below 200 nt), optimizing the sensitive cloning method for miRNAs, termed miRNA amplification profiling (mRAP). The Luminex expression patterns of three miRNAs (miR-23a, miR-27a and miR-199a) in seven different cell lines have been validated by TaqMan miRNA assay. In all cases, bead-based meas were confirmed by the data obtained by TaqMan and microarray technologies.</p> <p>Conclusions</p> <p>We demonstrate that the measure of individual miRNA by the bead-based method is feasible, high speed, sensitive and low cost. The Luminex^®xMAP™ technology also provides flexibility, since the central reaction can be scaled up with additional miRNA capturing beads, allowing validation of many differentially expressed miRNAs obtained from microarrays in a single experiment. We propose this technology as an alternative method to qRT-PCR for validating miRNAs expression data obtained with high-throughput technologies.</p

Defining the gene expression signature of rhabdomyosarcoma by meta-analysis

BACKGROUND: Rhabdomyosarcoma is a highly malignant soft tissue sarcoma in childhood and arises as a consequence of regulatory disruption of the growth and differentiation pathways of myogenic precursor cells. The pathogenic pathways involved in this tumor are mostly unknown and therefore a better characterization of RMS gene expression profile would represent a considerable advance. The availability of publicly available gene expression datasets have opened up new challenges especially for the integration of data generated by different research groups and different array platforms with the purpose of obtaining new insights on the biological process investigated. RESULTS: In this work we performed a meta-analysis on four microarray and two SAGE datasets of gene expression data on RMS in order to evaluate the degree of agreement of the biological results obtained by these different studies and to identify common regulatory pathways that could be responsible of tumor growth. Regulatory pathways and biological processes significantly enriched has been investigated and a list of differentially meta-profiles have been identified as possible candidate of aggressiveness of RMS. CONCLUSION: Our results point to a general down regulation of the energy production pathways, suggesting a hypoxic physiology for RMS cells. This result agrees with the high malignancy of RMS and with its resistance to most of the therapeutic treatments. In this context, different isoforms of the ANT gene have been consistently identified for the first time as differentially expressed in RMS. This gene is involved in anti-apoptotic processes when cells grow in low oxygen conditions. These new insights in the biological processes responsible of RMS growth and development demonstrate the effective advantage of the use of integrated analysis of gene expression studies

TRAIT (TRAnscript Integrated Table): a knowledgebase of human skeletal muscle transcripts.

Abstract Summary: TRAIT is a knowledgebase integrating information on transcripts with related data from genome, proteins, ortholog genes and diseases. It was initially built as a system to manage an EST-based gene discovery project on human skeletal muscle, which yielded over 4500 independent sequence clusters. Transcripts are annotated using automatic as well as manual procedures, linking known transcripts to public databases and unknown transcripts to tables of predicted features. Data are stored in a MySQL database. Complex queries are automatically built by means of a user-friendly web interface that allows the concurrent selection of many fields such as ontology, expression level, map position and protein domains. The results are parsed by the system and returned in a ranked order, in respect to the number of satisfied criteria. Availability: http://muscle.cribi.unipd.it and http://muscle.cribi.unipd.it/features/querystrait.html Contact: [email protected]; [email protected] * To whom correspondence should be addressed

Development and production of an oligonucleotide MuscleChip: use for validation of ambiguous ESTs

BACKGROUND: We describe the development, validation, and use of a highly redundant 120,000 oligonucleotide microarray (MuscleChip) containing 4,601 probe sets representing 1,150 known genes expressed in muscle and 2,075 EST clusters from a non-normalized subtracted muscle EST sequencing project (28,074 EST sequences). This set included 369 novel EST clusters showing no match to previously characterized proteins in any database. Each probe set was designed to contain 20–32 25 mer oligonucleotides (10–16 paired perfect match and mismatch probe pairs per gene), with each probe evaluated for hybridization kinetics (Tm) and similarity to other sequences. The 120,000 oligonucleotides were synthesized by photolithography and light-activated chemistry on each microarray. RESULTS: Hybridization of human muscle cRNAs to this MuscleChip (33 samples) showed a correlation of 0.6 between the number of ESTs sequenced in each cluster and hybridization intensity. Out of 369 novel EST clusters not showing any similarity to previously characterized proteins, we focused on 250 EST clusters that were represented by robust probe sets on the MuscleChip fulfilling all stringent rules. 102 (41%) were found to be consistently "present" by analysis of hybridization to human muscle RNA, of which 40 ESTs (39%) could be genome anchored to potential transcription units in the human genome sequence. 19 ESTs of the 40 ESTs were furthermore computer-predicted as exons by one or more than three gene identification algorithms. CONCLUSION: Our analysis found 40 transcriptionally validated, genome-anchored novel EST clusters to be expressed in human muscle. As most of these ESTs were low copy clusters (duplex and triplex) in the original 28,000 EST project, the identification of these as significantly expressed is a robust validation of the transcript units that permits subsequent focus on the novel proteins encoded by these genes