Lifelong physical exercise delays age-associated skeletal muscle decline.

Aging is usually accompanied by a significant reduction in muscle mass and force. To determine the relative contribution of inactivity and aging per se to this decay, we compared muscle function and structure in (a) male participants belonging to a group of well-trained seniors (average of 70 years) who exercised regularly in their previous 30 years and (b) age-matched healthy sedentary seniors with (c) active young men (average of 27 years). The results collected show that relative to their sedentary cohorts, muscle from senior sportsmen have: (a) greater maximal isometric force and function, (b) better preserved fiber morphology and ultrastructure of intracellular organelles involved in Ca(2+) handling and ATP production, (c) preserved muscle fibers size resulting from fiber rescue by reinnervation, and (d) lowered expression of genes related to autophagy and reactive oxygen species detoxification. All together, our results indicate that: (a) skeletal muscle of senior sportsmen is actually more similar to that of adults than to that of age-matched sedentaries and (b) signaling pathways controlling muscle mass and metabolism are differently modulated in senior sportsmen to guarantee maintenance of skeletal muscle structure, function, bioenergetic characteristics, and phenotype. Thus, regular physical activity is a good strategy to attenuate age-related general decay of muscle structure and function (ClinicalTrials.gov: NCT01679977).

Increased levels of HPV16 E6*I transcripts in high-grade cervical cytology and histology (CIN II+) detected by rapid real-time RT-PCR amplification.

OBJECTIVE: As cervical dysplasia may regress to normal cytology or progress to cervical carcinoma, it would be valuable to have a diagnostic tool to help decide whether therapeutic conization should be performed. METHODS: Cervical samples of 301 HPV16 positive women were collected in RNAlater reagent to prevent RNA degradation. Relative levels of HPV16 DNA and HPV16 E6*I mRNA in the samples were determined using real-time polymerase chain reaction. Findings were correlated with histological diagnoses and cytological follow-up. RESULTS: HPV16 E6*I mRNA levels were significantly higher in women with cytologically diagnosed severe cervical dysplasia (mean normalized ratio = 0.25) than in those with mild-to-moderate dysplasia (mean norm. ratio = 0.12), atypical squamous cells of uncertain origin (mean norm. ratio = 0.071) or normal cytology (mean norm. ratio = 0.034). Viral DNA levels were not significantly different between severe and mild-to-moderate dysplasia (mean norm. ratios, 55.8 and 63.5, respectively). The PPV for a histological diagnosis of severe cervical dysplasia [cervical intraepithelial neoplasia (CIN) II+] increased with the amounts of E6*I mRNA to more than 90% whereas the sensitivity decreased. The absence of HPV16 E6*I transcripts as well as HPV16 DNA considerably increased the negative predictive value and the specificity. However, low concentrations (or complete absence) of E6*I mRNA did not preclude a CIN II+ diagnosis. CONCLUSIONS: Although the sensitivity is low, high levels of HPV16 E6*I mRNA are indicative of CIN II+ in cytologically diagnosed cervical dysplasia of individual patients. Thus, quantification of HPV16 E6*I mRNA could be helpful in managing follow-up and treatment in a subset of HPV16 positive women.

Ignicoccus gen. nov., a novel genus of hyperthermophilic, chemolithoautotrophic Archaea, represented by two new species, Ignicoccus islandicus sp nov and Ignicoccus pacificus sp nov. and Ignicoccus pacificus sp. nov.

Two species of novel, chemolithoautotrophic, sulfidogenic micro-organisms were isolated from submarine hydrothermal systems in the Atlantic (at the Kolbeinsey Ridge north of Iceland) and in the Pacific (at 9 degrees N, 104 degrees W). The coccoid cells grew within a temperature range of 70-98 degrees C (optimum around 90 degrees C). They gained energy by reduction of elemental sulfur using molecular hydrogen as the electron donor. 165 rDNA-based sequence comparisons revealed that the organisms are members of the crenarchaeal branch of the Archaea. They represent a new, deeply branching lineage within the family of the Desulfurococcaceae. In DNA-DNA hybridization experiments both strains exhibited low levels of hybridization to each other and to further representatives of this family. Therefore, they represent a new genus, for which the name Ignicoccus gen. nov. is proposed. At present it consists of two new species, Ignicoccus islandicus sp. nov. (type strain is Kol8T = DSM 13165T = ATCC 700957T) and Ignicoccus pacificus sp. nov. (type strain is LPC33T = DSM 13166T = ATCC 700958T).

Thermococcus acidaminovorans sp. nov., a new hyperthermophilic alkalophilic archaeon growing on amino acids.

From a shallow marine hydrothermal system at Vulcano (Italy), a new hyperthermophilic member of the Archaea was isolated. The cells are coccoid - shaped and possess up to five flagella. They grow between 56 degrees and 93 degrees C (optimum 85 degrees C) and pH 5.0-9.5 (optimum 9.0). The organism is strictly anaerobic and grows heterotrophically on defined amino acids and complex organic substrates such as casamino acids, yeast extract, peptone, meat extract, tryptone, and casein. Polysulfide and elemental sulfur are reduced to H2S. In the absence of polysulfide or elemental sulfur, the isolate grows at a significantly reduced rate. Growth is not influenced by the presence of H2. DNA- DNA hybridization and 16S rRNA partial sequences indicated that the new isolate belongs to the genus Thermococcus, and represents a new species, Thermococcus acidaminovorans. The type stain is isolate AEDII10 (DSM 11906).

Sulfur-inhibited Thermosphaera aggregans sp. nov., a new genus of hyperthermophilic archaea isolated after its prediction from environmentally derived 16S rRNA sequences.

Recently, a new procedure was developed which allowed for the first time the isolation of a hyperthermophilic archaeum tracked by 165 rRNA analysis from a terrestrial hot solfataric spring ('Obsidian Pool', Yellowstone National Park, WY, USA). This novel isolate is characterized here. Cells are round cocci with a diameter of 0.2-0.8 micron, occurring singly, in pairs, short chains and in grape-like aggregates. The aggregates exhibit a weak bluish-green fluorescence under UV radiation at 420 nm. The new isolate is an anaerobic obligate heterotroph, using preferentially yeast extract for growth. The metabolic products include CO2, H2, acetate and isovalerate. Growth is observed between 65 and 90 degrees C (optimum: 85 degrees C), from pH 5.0 to 7.0 (optimum: 6.5) and up to 0.7% NaCl. The apparent activation energy for growth is about 149 kJ mol-1. Elemental sulfur or hydrogen inhibits growth. The core lipids consist mainly of acyclic and cyclic glycerol diphytanyl tetraethers. The cell envelope contains a cytoplasmic membrane covered by an amorphous layer of unknown composition; there is no evidence for a regularly arrayed surface-layer protein. The G + C content is 46 mol%. On the basis of 165 rRNA sequence comparisons in combination with morphological, physiological and biochemical properties, the isolate represents a new genus within the Desulfurococcaceae, which has been named Thermosphaera. The type species is Thermosphaera aggregans, the type strain is isolate M11TLT (= DSM 11486T).

Reclassification of the crenarchael orders and families in accordance with 16S rRNA sequence data.

A phylogenetic analysis of all validly published members of the Crenarchaeota, including several new isolates from our laboratory, suggests three orders within this archaeal kingdom. The Thermoproteales consist of both the rod-shaped, hyperthermophilic, neutrophilic representatives of the Thermoproteaceae and the members of the new family Thermofilaceae. The Sulfolobales harbor all thermoacidophilic, coccoid organisms. The neutrophilic, hyperthermophilic cocci are members of a new order tentatively named "Igneococcales." This order comprises two families, the Desulfurococcaceae, characterized by maximal growth temperature of up to 100 degrees C, and the new family Pyrodictiaceae, for which optimal growth occurs at temperatures above 100 degrees C.

Pyrolobus fumarii, gen. and sp. nov., represents a novel group of archaea, extending the upper temperature limit for life to 113 degrees C.

A novel, irregular, coccoid-shaped archaeum was isolated from a hydrothermally heated black smoker wall at the TAG site at the Mid Atlantic Ridge (depth 3650 meters). It grew at between 90 degrees C and 113 degrees C (optimum 106 degrees C) and pH 4.0-6.5 (optimum 5.5) and 1%-4% salt (optimum 1.7%). The organism was a facultatively aerobic obligate chemolithoautotroph gaining energy by H2-oxidation. Nitrate, S2O3(2-), and low concentrations of O2 (up to 0.3% v/v) served as electron acceptors, yielding NH4+, H2S, and H2O as end products, respectively. Growth was inhibited by acetate, pyruvate, glucose, starch, or sulfur. The new isolate was able to form colonies on plates (at 102 degrees C) and to grow at a pressure of 25000 kPa (250 bar). Exponentially growing cultures survived a one-hour autoclaving at 121 degrees C. The GC content was 53 mol%. The core lipids consisted of glycerol-dialkyl glycerol tetraethers and traces of 2,3-di-O-phytanyl-sn-glycerol. The cell wall was composed of a surface layer of tetrameric protein complexes arranged on a p4-lattice (center-to-center distance 18.5 nm). By its 16S rRNA sequence, the new isolate belonged to the Pyrodictiaceae. Based on its GC-content, DNA homology, S-layer composition, and metabolism, we describe here a new genus, which we name Pyrolobus (the "fire lobe"). The type species is Pyrolobus fumarii (type strain 1A; DSM 11204).

Ferroglobus placidus gen. nov., sp. nov., A novel hyperthermophilic archaeum that oxidizes Fe2+ at neutral pH under anoxic conditions.

A novel coccoid, anaerobic, Fe2+-oxidizing archaeum was isolated from a shallow submarine hydrothermal system at Vulcano, Italy. In addition to ferrous iron, H2 and sulfide served as electron donors. NO3- was used as electron acceptor. In the presence of H2, also S2O32- could serve as electron acceptor. The isolate was a neutrophilic hyperthermophile that grew between 65 degrees C and 95 degrees C. It represents a novel genus among the Archaeoglobales that we name Ferroglobus. The type species is Ferroglobus placidus (DSM 10642).

Thermococcus alcaliphilus sp. nov., a new hyperthermophilic archaeum growing on polysulfide at alkaline pH.

A novel coccoid-shaped, hyperthermophilic, heterotrophic member of the archaea was isolated from a shallow marine hydrothermal system at Vulcano Island, Italy. The isolate grew between 56 and 90 degrees C with an optimum around 85 degrees C. The pH range for growth was 6.5 to 10.5, with an optimum around 9.0. Polysulfide and elemental sulfur were reduced to H2S. Sulfur stimulated the growth rate. The isolate fermented yeast extract, peptone, meat extract, tryptone, and casein. Isovalerate, isobutyrate, propionate, acetate, CO2, NH3, and H2S (in the presence of S degrees ) were detected as end products. Growth was not inhibited by H2. Based on DNA-DNA hybridization and 16S rRNA partial sequences, the new isolate represents a new species of Thermococcus, which we named Thermococcus alcaliphilus. The type strain is isolate AEDII12 (DSM 10322).

Isolation of a hyperthermophilic archaeum predicted by in situ RNA analysis.

A variety of hyperthermophilic bacteria and archaea have been isolated from high-temperature environments by plating and serial dilutions. However, these techniques allow only the small percentage of organisms able to form colonies, or those that are predominant within environmental samples, to be obtained in pure culture. Recently, in situ 16S ribosomal RNA analyses of samples from the Obsidian hot pool at Yellowstone National Park, Wyoming, revealed a variety of archaeal sequences, which were all different from those of previously isolated species. This suggests substantial diversity of archaea with so far unknown morphological, physiological and biochemical features, which may play an important part within high-temperature ecosystems. Here we describe a procedure to obtain pure cultures of unknown organisms harbouring specific 16S rRNA sequences identified previously within the environment. It combines visual recognition of single cells by phylogenetic staining and cloning by 'optical tweezers'. Our result validates polymerase chain reaction data on the existence of large archael communities.

Isolation, taxonomy and phylogeny of hyperthermophilic microorganisms.

Hyperthermophilic Archaea and Bacteria with optimal growth temperatures between 80 and 110°C have been isolated from geo- and hydro-thermally heated terrestrial and submarine environments. 16S rRNA sequence comparisons indicate great phylogenetic diversity among the 23 different genera represented. Hyperthermophiles consist of anaerobic and aerobic chemolithoautotrophs and heterotrophs growing at neutral or acidic pH. Their outstanding heat resistance makes them as interesting objects for basic research as for biotechnology in the future.

Occurrence and Role of Di-myo-Inositol-1,1'-Phosphate in Methanococcus igneus.

Methanococcus igneus, a hyperthermophilic marine methanogen (optimum growth temperature of 88 degrees C) with a 25-min doubling time, synthesizes an unusual inositol phosphodiester which is present at high intracellular concentrations along with l-alpha-glutamate and beta-glutamate. Identification of this compound as a dimeric inositol phosphodiester (di-myo-inositol-1,1'-phosphate) was provided by two-dimensional nuclear magnetic resonance methods. The intracellular levels of all three negatively charged solutes (l-alpha-glutamate, beta-glutamate, and the inositol phosphodiester) increase with increasing levels of external NaCl, although the inositol compound shows much smaller increases with increasing NaCl levels than the glutamate isomers. The turnover of these solutes was examined by CO(2)-pulse-CO(2)-chase experiments. The results indicated that both the beta-glutamate and the inositol phosphodiester behaved as compatible solutes and were not efficiently metabolized by cells as was l-alpha-glutamate. At a fixed external NaCl concentration, lower ammonium levels increased the fraction of the inositol dimer present in extracts. The most pronounced changes in di-myo-inositol-1,1'-phosphate occurred as a function of cell growth temperature. While the organism grows over a relatively wide temperature range, the phosphodiester accumulated only when M. igneus was grown at temperatures of >/=80 degrees C. Thus, this unusual compound is a non-nitrogen-containing osmolyte preferentially synthesized at high growth temperatures.

Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes.

Two 16S rRNA-targeted oligonucleotide probes were designed for the archaeal kingdoms Euryachaeota and Crenarchaeota. Probe specificities were evaluated by nonradioactive dot blot hybridization against selected reference organisms. The successful application of fluorescent-probe derivatives for whole-cell hybridization required organism-specific optimizations of fixation and hybridization conditions to assure probe penetration and morphological integrity of the cells. The probes allowed preliminary grouping of three new hyperthermophilic isolates. Together with other group-specific rRNA-targeted oligonucleotide probes, these probes will facilitate rapid in situ monitoring of the populations present in hydrothermal systems and support cultivation attempts.

16S rRNA-targeted polymerase chain reaction and oligonucleotide hybridization to screen for Azoarcus spp., grass-associated diazotrophs.

Phylogenetic analyses after reverse transcriptase sequencing of 16S rRNA of nitrogen-fixing, grass-associated Azoarcus strains confirmed their affiliation to the beta subdivision of the Proteobacteria. Strains representing three different species formed a phylogenetically coherent unit related to Rhodocyclus purpureus, with actual percent similarities among the three sequences ranging from 93.1 to 97.3%. Within variable regions V2 and V5, we found stretches of sequences considerably conserved within the genus Azoarcus but differing from most other gram-negative bacteria, with the specificity being enhanced when different regions were combined. Genus-specific primers selected from both regions amplified fragments from all but one Azoarcus species in polymerase chain reactions (PCR) but not from any reference strain tested. Primers of lesser specificity generated fragments from members of all five Azoarcus species as well as from some reference strains. Those unspecific amplifications could be differentiated by oligonucleotide hybridization, detecting only fragments generated from Azoarcus strains except strain 6a3, which represents the same group which could not be detected by genus-specific PCR. Thus we propose the application of PCR amplification with 16S rRNA-targeted, genus-specific primers in combination with hybridization of a 16S rRNA-targeted oligonucleotide to PCR-generated fragments as diagnostic tests; this allows an initial screening for presence of members of the genus Azoarcus.

Pyrobaculum aerophilum sp. nov., a novel nitrate-reducing hyperthermophilic archaeum.

A novel rod-shaped hyperthermophilic archaeum has been isolated from a boiling marine water hole at Maronti Beach, Ischia, Italy. It grew optimally at 100 degrees C and pH 7.0 by aerobic respiration as well as by dissimilatory nitrate reduction, forming dinitrogen as a final product. Organic and inorganic compounds served as substrates during aerobic and anaerobic respiration. Growth was inhibited by elemental sulfur. The cell wall was composed of a surface layer of hexameric protein complexes arranged on a p6 lattice. The core lipids consisted mainly of glycerol diphytanyl glycerol tetraethers with various degrees of cyclization. The G+C content was 52 mol%. The new isolate resembled members of the genera Thermoproteus and Pyrobaculum by its ability to form characteristic terminal spherical bodies ("golf clubs"). On the basis of its 16S rRNA sequence, the new isolate exhibited a close relationship to the genus Pyrobaculum. It is described as a new species, which we name Pyrobaculum aerophilum (type strain: IM2; DSM 7523).

An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum.

The 16S rRNA genes of Pyrobaculum aerophilum and Pyrobaculum islandicum were amplified by the polymerase chain reaction, and the resulting products were sequenced directly. The two organisms are closely related by this measure (over 98% similar). However, they differ in that the (lone) 16S rRNA gene of Pyrobaculum aerophilum contains a 713-bp intron not seen in the corresponding gene of Pyrobaculum islandicum. To our knowledge, this is the only intron so far reported in the small subunit rRNA gene of a prokaryote. Upon excision the intron is circularized. A secondary structure model of the intron-containing rRNA suggests a splicing mechanism of the same type as that invoked for the tRNA introns of the Archaea and Eucarya and 23S rRNAs of the Archaea. The intron contains an open reading frame whose protein translation shows no certain homology with any known protein sequence.

Life in hot springs and hydrothermal vents.

Hot springs and hydrothermal systems occurring within volcanic areas are inhabited by hyperthermophilic microorganisms, some of which grow at temperatures up to 110 degrees C. Hyperthermophiles grow anaerobically or aerobically by diverse metabolic types. Within the high temperature ecosystems, primary production is independent from solar energy.