Facioscapulohumeral dystrophy (FSHD) is a muscular hereditary disease with a prevalence of 1 in 20,000 caused by a partial deletion of a subtelomeric repeat array on chromosome 4q. However, very little is known about the pathogenesis as well as the molecular and biochemical changes linked to the progressive muscle degeneration observed in these patients. Several studies have investigated possible pathophysiological pathways in FSHD myoblasts and mature muscle cells but some of these reports were apparently in contradiction. The discrepancy between these studies may be explained by differences between the sources of myoblasts. Therefore, we decided to thoroughly analyze affected and unaffected muscles from patients with FSHD in terms of vulnerability to oxidative stress, differentiation capacity and morphological abnormalities. We have established a panel of primary myoblast cell cultures from patients affected with FSHD and matched healthy individuals. Our results show that primary myoblasts are more susceptible to an induced oxidative stress than control myoblasts. Moreover, we demonstrate that both types of FSHD primary myoblasts differentiate into multi-nucleated myotubes, which present morphological abnormalities. Whereas control myoblasts fuse to form branched myotubes with aligned nuclei, FSHD myoblasts fuse to form either thin and branched myotubes with aligned nuclei or large myotubes with random nuclei distribution. In conclusion, we postulate that these abnormalities could be responsible for muscle weakness in patients with FSHD and provide an important marker for FSHD myoblasts.

The phylogenetic relationships among marine Alteromonas-like bacteria of the genera Alteromonas, Pseudoalteromonas, Glaciecola, Thalassomonas, Colwellia, Idiomarina, Oceanimonas, Oceanisphaera, Shewanella, Moritella, Ferrimonas, Psychromonas and several other genera of the ‘Gammaproteobacteria’ were studied. Results of 16S rRNA gene sequence analyses revealed that some members of these genera formed several coherent groups at the family level. Characteristic signature oligonucleotides for studied taxa were defined. Signature positions are divided into three classes: (i) single compensatory mutations, (ii) double compensatory mutations and (iii) mutations affecting nucleotides not paired in the secondary structure. The 16S rRNA gene sequence similarity level within genera was 93 % or above. This value can be a useful additional criterion for genus discrimination. On the basis of this work and previous polyphasic taxonomic studies, the circumscription of the family Alteromonadaceae is limited to the genera Alteromonas and Glaciecola and the creation is proposed of the families Pseudoalteromonadaceae fam. nov. to accommodate bacteria of the genera Pseudoalteromonas and Algicola gen. nov. (formerly Pseudoalteromonas bacteriolytica) and Colwelliaceae fam. nov. to accommodate bacteria of the genera Colwellia and Thalassomonas. Bacteria of the genera Oceanimonas and Oceanisphaera formed a robust cluster and shared common signature oligonucleotides. Because of deep branching and lack of association with any other genus, the following families are proposed that include single genera: Idiomarinaceae fam. nov., Psychromonadaceae fam. nov., Moritellaceae fam. nov., Ferrimonadaceae fam. nov. and Shewanellaceae fam. nov. Finally, this study also revealed that [Hyphomicrobium] indicum should be reclassified as Photobacterium indicum comb. nov.

Quantitative PCR (Q-PCR) is a fast and efficient tool to quantify target genes. In eukaryotic cells, quantitative reverse transcription-PCR (Q-RT-PCR) is also used to quantify gene expression, with stably expressed housekeeping genes as standards. In bacteria, such stable expression of housekeeping genes does not occur, and the use of DNA standards leads to a broad underestimation. Therefore, an accurate quantification of RNA is feasible only by using appropriate RNA standards. We established and validated a Q-PCR method which enables the quantification of not only the number of copies of target genes (i.e., the number of bacterial cells) but also the number of RNA copies. The genes coding for InvA and the 16S rRNA of Salmonella enterica serovar Typhimurium were selected for the evaluation of the method. As DNA standards, amplified fragments of the target genes were used, whereas the same DNA standards were transcribed in vitro for the development of appropriate RNA standards. Salmonella cultures and environmental water samples inoculated with bacteria were then employed for the final testing. Both experimental approaches led to a sensitive, accurate, and reproducible quantification of the selected target genes and RNA molecules by Q-PCR and Q-RT-PCR. It is the first time that RNA standards have been successfully used for a precise quantification of the number of RNA molecules in prokaryotes. This demonstrates the potential of this approach for determining the presence and metabolic activity of pathogenic bacteria in environmental samples.

Identification and functional analysis of key members of bacterial communities in marine and estuarine environments are major challenges for obtaining a mechanistic understanding of biogeochemical processes. In the Baltic Sea basins, as in many other marine environments with anoxic bodies of water, the oxic-anoxic interface is considered a layer of high bacterial turnover of sulfur, nitrogen, and carbon compounds that has a great impact on matter balances in the whole ecosystem. We focused on autotrophic denitrification by oxidation of reduced sulfur compounds as a biogeochemically important process mediating concomitant turnover of sulfur, nitrogen, and carbon. We used a newly developed approach consisting of molecular analyses in stimulation experiments and in situ abundance. The molecular approach was based on single-strand conformational polymorphism (SSCP) analysis of the bacterial community RNA, which allowed identification of potential denitrifiers based on the sequences of enhanced SSCP bands and monitoring of the overall bacterial community during the experiments. Sequences of the SSCP bands of interest were used to design highly specific primers that enabled (i) generation of almost complete 16S rRNA gene sequences using experimental and environmental DNA as templates and (ii) quantification of the bacteria of interest by real-time PCR. By using this approach we identified the bacteria responsible for autotrophic denitrification as a single taxon, an epsilonproteobacterium related to the autotrophic denitrifier Thiomicrospira denitrificans. This finding was confirmed by material balances in the experiments that were consistent with those obtained with continuous cultures of T. denitrificans. The presence and activity of a bacterium that is phylogenetically and physiologically closely related to T. denitrificans could be relevant for the carbon budget of the central Baltic Sea because T. denitrificans exhibits only one-half the efficiency for carbon dioxide fixation per mol of sulfide oxidized and mol of nitrate reduced of Thiobacillus denitrificans hypothesized previously for this function.

Four light-yellow-pigmented, Gram-negative, short-rod-shaped, non-motile isolates were obtained from enrichment culture during degradation of the thallus of the brown alga Fucus evanescens. The isolates studied were chemo-organotrophic, alkalitolerant and mesophilic. Polar lipids were analysed and phosphatidylethanolamine was the only phospholipid identified. The predominant cellular fatty acids were 15 : 0, i15 : 0, ai15 : 0, i15 : 1 and 15 : 1(n-6). The DNA G+C contents of the four strains were 34?0-34?4 mol%. The level of DNA relatedness of the four isolates was conspecific (88-98 %), indicating that they belong to the same species. The 16S rDNA sequence of strain KMM 3553 T was determined. Phylogenetic analysis revealed that KMM 3553 T formed a distinct phyletic line in the phylum Bacteroidetes, class Flavobacteria in the family Flavobacteriaceae and that, phylogenetically, this strain could be placed almost equidistant from the genera Gelidibacter and Psychroserpens (16S rRNA gene sequence similarities of 94 %). On the basis of significant differences in phenotypic and chemotaxonomic characteristics, it is suggested that the isolates represent a novel species in a new genus; the name Formosa algae gen. nov., sp. nov. is proposed. The type strain is KMM 3553 T

We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the "Epsilonproteobacteria" related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon-and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 ؋ 10 3 to 4.4 ؋ 10 9 copies ml ؊1 or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 ؋ 10 1 to 2.2 ؋10 6 copies ml

Training-induced improvement in lipid oxidation in type 2 diabetes mellitus is related to alterations in muscle mitochondrial activity. Effect of endurance training in type 2 diabetes.. Diabetes and Metabolism, Elsevier Masson, 2008, 34 (2) AbstractAim: We investigated in type 2 diabetic patients (T2D) if an individualized training effect on whole body lipid oxidation is associated with changes in muscle oxidative capacities.Methods: Eleven T2D participated in this study. Whole body lipid oxidation during exercise was assessed by graded exercise indirect calorimetry. Blood samples for measuring blood glucose and free fatty acids during exercise and muscle oxidative capacities measured from a skeletal muscle biopsy (i.e., mitochondrial respiration and citrate synthase activity) were investigated in T2D before and after a 10-week individualized training targeted at LIPOXmax, which corresponds to the power at which the highest rate of lipids is oxidized (lipid oxidation at LIPOXmax).Results: Training induced both a shift to a higher power intensity of LIPOXmax (+9.1 ± 4.2 Watts, P<0.05) and an improvement of lipid oxidation at LIPOXmax (+51.27 ± 17.93 mg.min -1 , P<0.05). The improvement in lipid oxidation was correlated with training-induced improvement of mitochondrial respiration (r=0.78, P<0.01) and citrate synthase activity (r=0.63, P<0.05).Conclusion: This study shows that a quite moderate training protocol targeted at the LIPOXmax in T2D mellitus improves the ability to oxidize lipids during exercise, and that this improvement is associated with an enhancement of muscle oxidative capacities.

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