Training in hypoxia fails to further enhance endurance performance and lactate clearance in well-trained men and impairs glucose metabolism during prolonged exercise.

The aim of this study was to investigate the synergistic effects of endurance training and hypoxia on endurance performance in normoxic and hypoxic conditions (approximately 3000 m above sea level) as well as on lactate and glucose metabolism during prolonged exercise. For this purpose, 14 well-trained cyclists performed 12 training sessions in conditions of normobaric hypoxia (HYP group, n = 7) or normoxia (NOR group, n = 7) over 4 weeks. Before and after training, lactate and glucose turnover rates were measured by infusion of exogenous lactate and stable isotope tracers. Endurance performance was assessed during incremental tests performed in normoxia and hypoxia and a 40 km time trial performed in normoxia. After training, performance was similarly and significantly improved in the NOR and HYP groups (training, P < 0.001) in normoxic conditions. No further effect of hypoxic training was found on markers of endurance performance in hypoxia (training x hypoxia interaction, n.s.). In addition, training and hypoxia had no significant effect on lactate turnover rate. In contrast, there was a significant interaction of training and hypoxia (P < 0.05) on glucose metabolism, as follows: plasma insulin and glucose concentrations were significantly increased; glucose metabolic clearance rate was decreased; and the insulin to glucagon ratio was increased after training in the HYP group. In conclusion, our results show that, compared with training in normoxia, training in hypoxia has no further effect on endurance performance in both normoxic and hypoxic conditions or on lactate metabolic clearance rate. Additionally, these findings suggest that training in hypoxia impairs blood glucose regulation in endurance-trained subjects during exercise.

Anthropogenic disturbance as a driver of microspatial and microhabitat segregation of cytotypes of Centaurea stoebe and cytotype interactions in secondary contact zones.

BACKGROUND AND AIMS: In a mixed-ploidy population, strong frequency-dependent mating will lead to the elimination of the less common cytotype, unless prezygotic barriers enhance assortative mating. However, such barriers favouring cytotype coexistence have only rarely been explored. Here, an assessment is made of the mechanisms involved in formation of mixed-ploidy populations and coexistence of diploid plants and their closely related allotetraploid derivates from the Centaurea stoebe complex (Asteraceae). METHODS: An investigation was made of microspatial and microhabitat distribution, life-history and fitness traits, flowering phenology, genetic relatedness of cytotypes and intercytotype gene flow (cpDNA and microsatellites) in six mixed-ploidy populations in Central Europe. KEY RESULTS: Diploids and tetraploids were genetically differentiated, thus corroborating the secondary origin of contact zones. The cytotypes were spatially segregated at all sites studied, with tetraploids colonizing preferentially drier and open microhabitats created by human-induced disturbances. Conversely, they were rare in more natural microsites and microsites with denser vegetation despite their superior persistence ability (polycarpic life cycle). The seed set of tetraploid plants was strongly influenced by their frequency in mixed-ploidy populations. Triploid hybrids originated from bidirectional hybridizations were extremely rare and almost completely sterile, indicating a strong postzygotic barrier between cytotypes. CONCLUSIONS: The findings suggest that tetraploids are later immigrants into already established diploid populations and that anthropogenic activities creating open niches favouring propagule introductions were the major factor shaping the non-random distribution and habitat segregation of cytotypes at fine spatial scale. Establishment and spread of tetraploids was further facilitated by their superior persistence through the perennial life cycle. The results highlight the importance of non-adaptive spatio-temporal processes in explaining microhabitat and microspatial segregation of cytotypes.

Temperature-responsive sensing regulates biocontrol factor expression in Pseudomonas fluorescens CHA0.

In the plant-beneficial, root-colonizing strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively regulates the synthesis of biocontrol factors (mostly antifungal secondary metabolites) and contributes to oxidative stress response via the stress sigma factor RpoS. The backbone of this pathway consists of the GacS/GacA two-component system, which activates the expression of three small regulatory RNAs (RsmX, RsmY, RsmZ) and thereby counters translational repression exerted by the RsmA and RsmE proteins on target mRNAs encoding biocontrol factors. We found that the expression of typical biocontrol factors, that is, antibiotic compounds and hydrogen cyanide (involving the phlA and hcnA genes), was significantly lower at 35 degrees C than at 30 degrees C. The expression of the rpoS gene was affected in parallel. This temperature control depended on RetS, a sensor kinase acting as an antagonist of the GacS/GacA system. An additional sensor kinase, LadS, which activated the GacS/GacA system, apparently did not contribute to thermosensitivity. Mutations in gacS or gacA were epistatic to (that is, they overruled) mutations in retS or ladS for expression of the small RNAs RsmXYZ. These data are consistent with a model according to which RetS-GacS and LadS-GacS interactions shape the output of the Gac/Rsm pathway and the environmental temperature influences the RetS-GacS interaction in P. fluorescens CHA0.

Different types of cell death are involved in brain damage of methylmalonic aciduria and glutaric aciduria type I

We previously showed that exposure of 3D organotypic rat brain cell cultures to 1mM 2-methylcitrate (2-MCA) or 3-hydroxyglutarate (3- OHGA) every 12h over three days (DIV11-DIV14) results in ammonium accumulation and cell death. The aim of this study was to define the time course (every 24h) of the observed effects. Ammonium in culture medium already increased at DIV12 staying stable on the following days under 3-OHGA exposure, while it increased consecutively up to much higher levels under 2-MCA exposure. Lactate increase and glucose decrease were observed from DIV13 and DIV14, respectively. We conclude that ammonium accumulation precedes alterations of energy metabolism. As observed by immunohistochemistry glial cells were the predominant dying cells. Immunoblotting and immunohistochemistry with cell death specific markers (caspase-3, alpha-fodrin, LC3) showed that 2-MCA exposure significantly increased apoptosis on DIV14, but did not alter autophagy or necrosis. In contrast, 3-OHGA exposure substantially increased necrosis already from DIV13, while no change was observed for apoptosis and autophagy. In conclusion, ammonium accumulation, secondary disturbance of energy metabolism and glial cell death are involved in the neuropathogenesis ofmethylmalonic aciduria and glutaric aciduria type I. Interestingly, brain cells are dying by necrosis under 3-OHGA exposure and by apoptosis under 2-MCA exposure.

Behavior of nuclear matrix proteins during camptothecin-induced apoptosis in HL-60 human leukemia cells.

In this study we focused our attention on the behavior of four nuclear matrix proteins during the various stages of apoptosis in the HL-60 cell line exposed to the DNA topoisomerase I inhibitor, camptothecin. We have examined the following antigens by immunocytochemical techniques: (i) the 180-kDa nucleolar isoform of DNA topoisomerase II; (ii) a 126-kDa polypeptide of nuclear bodies; (iii) a 125-kDa protein; and (iv) a 160-kDa polypeptide which are known to be components of the matrix inner network. Indirect immunofluorescence experiments were performed to follow these nuclear matrix antigens during apoptosis. Moreover, the ultrastructural localization of both 125- and 160-kDa proteins was investigated by electron microscope immunocytochemistry with gold-conjugated secondary antibodies. While the antibody to the nucleolar isoform of DNA topoisomerase II gave a fluorescent pattern that was well-maintained until the late phases of apoptosis, the other three nuclear antigens showed marked modifications in their distribution. A common feature, particularly evident for 125- and 160-kDa proteins, was their absence from cap-shaped chromatin marginations, whereas they were present in the areas of remaining decondensed chromatin. The 126-kDa polypeptide concentrated progressively in an irregular mass at the opposite side of the crescentic caps and then broke up in fine spots. The 125- and 160-kDa proteins localized in the nucleolus and precisely within certain granules which are known to appear in the nucleolar area after camptothecin administration. These results show that, in addition to the well-known chromatin changes, nuclear organization undergoes other rearrangements during the apoptotic process.

Tn5-directed cloning of pqq genes from Pseudomonas fluorescens CHA0: mutational inactivation of the genes results in overproduction of the antibiotic pyoluteorin.

Pseudomonas fluorescens CHA0 produces several secondary metabolites, e.g., the antibiotics pyoluteorin (Plt) and 2,4-diacetylphloroglucinol (Phl), which are important for the suppression of root diseases caused by soil-borne fungal pathogens. A Tn5 insertion mutant of strain CHA0, CHA625, does not produce Phl, shows enhanced Plt production on malt agar, and has lost part of the ability to suppress black root rot in tobacco plants and take-all in wheat. We used a rapid, two-step cloning-out procedure for isolating the wild-type genes corresponding to those inactivated by the Tn5 insertion in strain CHA625. This cloning method should be widely applicable to bacterial genes tagged with Tn5. The region cloned from P. fluorescens contained three complete open reading frames. The deduced gene products, designated PqqFAB, showed extensive similarities to proteins involved in the biosynthesis of pyrroloquinoline quinone (PQQ) in Klebsiella pneumoniae, Acinetobacter calcoaceticus, and Methylobacterium extorquens. PQQ-negative mutants of strain CHA0 were constructed by gene replacement. They lacked glucose dehydrogenase activity, could not utilize ethanol as a carbon source, and showed a strongly enhanced production of Plt on malt agar. These effects were all reversed by complementation with pqq+ recombinant plasmids. The growth of a pqqF mutant on ethanol and normal Plt production were restored by the addition of 16 nM PQQ. However, the Phl- phenotype of strain CHA625 was due not to the pqq defect but presumably to a secondary mutation. In conclusion, a lack of PQQ markedly stimulates the production of Plt in P. fluorescens.

Spatial distribution and biomass of aquatic rooted macrophytes and their relevance in the metabolism of a Mediterranean coastal lagoon

This work aims to characterise the current autotrophic compartment of the Albufera des Grau coastal lagoon (Menorca, Balearic Islands) and to assess the relationship between the submerged macrophytes and the limnological parameters of the lagoon. During the study period the submerged vegetation was dominated by the macrophyte Ruppia cirrhosa, which formed dense extensive meadows covering 79% of the surface. Another macrophyte species, Potamogeton pectinatus, was also observed but only forming small stands near the rushing streams. Macroalgae were only occasionally observed. Macrophyte biomass showed a clear seasonal trend, with maximum values in July. The biomass of R. cirrhosa achieved 1760 g DW m-2, the highest biomass ever reported for this species in the literature. The seasonal production-decomposition cycle of the macrophyte meadows appears to drive the nutrient dynamics and carbon fluxes in the lagoon. Despite the significant biomass accumulation and the absence of a washout of nutrients and organic matter to the sea, the lagoon did not experience a dystrophic collapse. These results indicate that internal metabolism is more important than exchange processes in the lagoon.

Decreased behavioral impairments in an Alzheimer mice model by interfering with TNF-alpha metabolism.

The performance of mice expressing PDAPP (+/+ or +/-) was studied in the Morris place navigation task. Different lines of questions were investigated using PDAPP+/- mice in which the activity of the cytokine Tumor Necrosing Factor alpha (TNFalpha) was attenuated by chronic treatment with anti-TNF or deleting TNFalpha (TNF-/-). Two different categories of behavior were analyzed in adult (6 months) and middle aged (15 months) subjects. Classically, the cognitive performance was assessed from the escape efficacy and quantitative bias toward the training position in a Morris water maze. Second, stereotyped circling was quantified, along with more qualitative behavioral impairments such as self-mutilation or increased reactivity. Our results can be summarized as follows. (1) All of the PDAPP mice expressed reduced cognitive performance in the Morris task, but only those with a clear-cut amyloid burden in the hippocampus showed behavioral abnormalities such as stereotyped circling. (2) Chronic treatment with anti-TNF prevented the development of pathological circling in the 6-month-old mice but not in the 15-month-old mice and had no significant effect on amyloid burden. (3) The absence of TNFalpha prevented the development of stereotyped circling in 6- and 15-month-old mice but increased amyloid burden after 15 months. These data indicate that PDAPP mice express cognitive impairments disregarding absence of TNF. The pathological behavioral anomalies related to the PDAPP mutation seem reduced by treatments interfering with TNFalpha.

Outcome of subscapularis muscle release for shoulder contracture secondary to brachial plexus palsy at birth.

Children with unresolved brachial plexus palsy frequently develop a disabling internal rotation contracture of the shoulder. Several surgical options, including soft tissue procedures such as muscle releases and/or transfers, and bone operations such as humeral osteotomy are available to correct this deformity. This study describes the effect of subscapularis muscle release performed in isolation. Thirteen patients (5 boys, 8 girls) were reviewed at an average of 3.5 years after their surgery (range, 2-7 years). Their mean age at operation was 4.7 years (range, 1-8 years). Three children had C5-C6 palsies, 8 had C5-C7 palsies, and 2 had C5-C8 palsies. Postoperatively, patients presented significant gains in shoulder active lateral rotation (+49 degrees, from 5 to 54 degrees), active abduction (+30 degrees, from 63 to 93 degrees), active flexion (+46 degrees, from 98 to 144 degrees), and active extension (+23 degrees, from 7 to 30 degrees). Gains were also observed in passive range of motion, but of a lesser degree. Subscapularis muscle release is a procedure we found to have few significant complications and was highly effective in increasing active range of motion and restoring shoulder function.

Impact of tight glycemic control on cerebral glucose metabolism after severe brain injury: a microdialysis study.

OBJECTIVES: To analyze the effect of tight glycemic control with the use of intensive insulin therapy on cerebral glucose metabolism in patients with severe brain injury. DESIGN: Retrospective analysis of a prospective observational cohort. SETTING: University hospital neurologic intensive care unit. PATIENTS: Twenty patients (median age 59 yrs) monitored with cerebral microdialysis as part of their clinical care. INTERVENTIONS: Intensive insulin therapy (systemic glucose target: 4.4-6.7 mmol/L [80-120 mg/dL]). MEASUREMENTS AND MAIN RESULTS: Brain tissue markers of glucose metabolism (cerebral microdialysis glucose and lactate/pyruvate ratio) and systemic glucose were collected hourly. Systemic glucose levels were categorized as within the target "tight" (4.4-6.7 mmol/L [80-120 mg/dL]) vs. "intermediate" (6.8-10.0 mmol/L [121-180 mg/dL]) range. Brain energy crisis was defined as a cerebral microdialysis glucose <0.7 mmol/L with a lactate/pyruvate ratio >40. We analyzed 2131 cerebral microdialysis samples: tight systemic glucose levels were associated with a greater prevalence of low cerebral microdialysis glucose (65% vs. 36%, p < 0.01) and brain energy crisis (25% vs.17%, p < 0.01) than intermediate levels. Using multivariable analysis, and adjusting for intracranial pressure and cerebral perfusion pressure, systemic glucose concentration (adjusted odds ratio 1.23, 95% confidence interval [CI] 1.10-1.37, for each 1 mmol/L decrease, p < 0.001) and insulin dose (adjusted odds ratio 1.10, 95% CI 1.04-1.17, for each 1 U/hr increase, p = 0.02) independently predicted brain energy crisis. Cerebral microdialysis glucose was lower in nonsurvivors than in survivors (0.46 +/- 0.23 vs. 1.04 +/- 0.56 mmol/L, p < 0.05). Brain energy crisis was associated with increased mortality at hospital discharge (adjusted odds ratio 7.36, 95% CI 1.37-39.51, p = 0.02). CONCLUSIONS: In patients with severe brain injury, tight systemic glucose control is associated with reduced cerebral extracellular glucose availability and increased prevalence of brain energy crisis, which in turn correlates with increased mortality. Intensive insulin therapy may impair cerebral glucose metabolism after severe brain injury.

Association of T-zone reticular networks and conduits with ectopic lymphoid tissues in mice and humans.

Ectopic or tertiary lymphoid tissues (TLTs) are often induced at sites of chronic inflammation. They typically contain various hematopoietic cell types, high endothelial venules, and follicular dendritic cells; and are organized in lymph node-like structures. Although fibroblastic stromal cells may play a role in TLT induction and persistence, they have remained poorly defined. Herein, we report that TLTs arising during inflammation in mice and humans in a variety of tissues (eg, pancreas, kidney, liver, and salivary gland) contain stromal cell networks consisting of podoplanin(+) T-zone fibroblastic reticular cells (TRCs), distinct from follicular dendritic cells. Similar to lymph nodes, TRCs were present throughout T-cell-rich areas and had dendritic cells associated with them. They expressed lymphotoxin (LT) β receptor (LTβR), produced CCL21, and formed a functional conduit system. In rat insulin promoter-CXCL13-transgenic pancreas, the maintenance of TRC networks and conduits was partially dependent on LTβR and on lymphoid tissue inducer cells expressing LTβR ligands. In conclusion, TRCs and conduits are hallmarks of secondary lymphoid organs and of well-developed TLTs, in both mice and humans, and are likely to act as important scaffold and organizer cells of the T-cell-rich zone.

C19orf12 mutation leads to a pallido-pyramidal syndrome.

Pallido-pyramidal syndromes combine dystonia with or without parkinsonism and spasticity as part of a mixed neurodegenerative disorder. Several causative genes have been shown to lead to pallido-pyramidal syndromes, including FBXO7, ATP13A2, PLA2G6, PRKN and SPG11. Among these, ATP13A2 and PLA2G6 are inconsistently associated with brain iron deposition. Using homozygosity mapping and direct sequencing in a multiplex consanguineous Saudi Arabian family with a pallido-pyramidal syndrome, iron deposition and cerebellar atrophy, we identified a homozygous p.G53R mutation in C19orf12. Our findings add to the phenotypic spectrum associated with C19orf12 mutations.

A Sufficiency Rating System for Secondary Roads in Iowa, HR-264, Volume I, 1985

County engineers in Iowa face the dual problems of rapidly escalating costs and a decreasing rate of growth of revenues. Various priority systems are in use, ranking projects for inclusion in road improvement programs, but they generally have weaknesses when used to compare one project with another in a different location. The sufficiency rating system has proven to be a useful tool in developing a priority list of projects for primary road systems, but there are none currently in use for secondary road systems. The research reported here was undertaken to develop a sufficiency rating system which could be used for secondary roads in Iowa and to produce the necessary forms and instructions to aid county engineering personnel in their efforts to complete the ratings for roads within their county.

Protective effects of maternal nutritional supplementation with lactoferrin on growth and brain metabolism.

Background:Intrauterine growth restriction (IUGR) is a major risk factor for both perinatal and long-term morbidity. Bovine lactoferrin (bLf) is a major milk glycoprotein considered as a pleiotropic functional nutrient. The impact of maternal supplementation with bLf on IUGR-induced sequelae, including inadequate growth and altered cerebral development, remains unknown.Methods:IUGR was induced through maternal dexamethasone infusion (100 μg/kg during last gestational week) in rats. Maternal supplementation with bLf (0.85% in food pellet) was provided during both gestation and lactation. Pup growth was monitored, and Pup brain metabolism and gene expression were studied using in vivo (1)H NMR spectroscopy, quantitative PCR, and microarray in the hippocampus at postnatal day (PND)7.Results:Maternal bLf supplementation did not change gestational weight but increased the birth body weight of control pups (4%) with no effect on the IUGR pups. Maternal bLf supplementation allowed IUGR pups to recover a normalized weight at PND21 (weaning) improving catch-up growth. Significantly altered levels of brain metabolites (γ-aminobutyric acid, glutamate, N-acetylaspartate, and N-acetylaspartylglutamate) and transcripts (brain-derived neurotrophic factor (BDNF), divalent metal transporter 1 (DMT-1), and glutamate receptors) in IUGR pups were normalized with maternal bLf supplementation.Conclusion:Our data suggest that maternal bLf supplementation is a beneficial nutritional intervention able to revert some of the IUGR-induced sequelae, including brain hippocampal changes.