Skeletal muscle AMPK is not activated during 2 h of moderate intensity exercise at ~65% VO2peak in endurance trained men

Key points: AMP-activated protein kinase (AMPK) is considered a major regulator of skeletal muscle metabolism during exercise. However, we previously showed that, although AMPK activity increases by 8–10-fold during ∼120 min of exercise at ∼65% (Formula presented.) in untrained individuals, there is no increase in these individuals after only 10 days of exercise training (longitudinal study). In a cross-sectional study, we show that there is also a lack of activation of skeletal muscle AMPK during 120 min of cycling exercise at 65% (Formula presented.) in endurance-trained individuals. These findings indicate that AMPK is not an important regulator of exercise metabolism during 120 min of exercise at 65% (Formula presented.) in endurance trained men. It is important that more energy is directed towards examining other potential regulators of exercise metabolism. Abstract: AMP-activated protein kinase (AMPK) is considered a major regulator of skeletal muscle metabolism during exercise. Indeed, AMPK is activated during exercise and activation of AMPK by 5-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR) increases skeletal muscle glucose uptake and fat oxidation. However, we have previously shown that, although AMPK activity increases by 8–10-fold during ∼120 min of exercise at ∼65% (Formula presented.) in untrained individuals, there is no increase in these individuals after only 10 days of exercise training (longitudinal study). In a cross-sectional study, we examined whether there is also a lack of activation of skeletal muscle AMPK during 120 min of cycling exercise at 65% (Formula presented.) in endurance-trained individuals. Eleven untrained (UT; (Formula presented.) = 37.9 ± 5.6 ml.kg−1 min−1) and seven endurance trained (ET; (Formula presented.) = 61.8 ± 2.2 ml.kg−1 min−1) males completed 120 min of cycling exercise at 66 ± 4% (Formula presented.) (UT: 100 ± 21 W; ET: 190 ± 15 W). Muscle biopsies were obtained at rest and following 30 and 120 min of exercise. Muscle glycogen was significantly (P < 0.05) higher before exercise in ET and decreased similarly during exercise in the ET and UT individuals. Exercise significantly increased calculated skeletal muscle free AMP content and more so in the UT individuals. Exercise significantly (P < 0.05) increased skeletal muscle AMPK α2 activity (4-fold), AMPK αThr172 phosphorylation (2-fold) and ACCβ Ser222 phosphorylation (2-fold) in the UT individuals but not in the ET individuals. These findings indicate that AMPK is not an important regulator of exercise metabolism during 120 min of exercise at 65% (Formula presented.) in endurance trained men

The Generation of Successive Unmarked Mutations and Chromosomal Insertion of Heterologous Genes in Actinobacillus pleuropneumoniae Using Natural Transformation

We have developed a simple method of generating scarless, unmarked mutations in Actinobacillus pleuropneumoniae by exploiting the ability of this bacterium to undergo natural transformation, and with no need to introduce plasmids encoding recombinases or resolvases. This method involves two successive rounds of natural transformation using linear DNA: the first introduces a cassette carrying cat (which allows selection by chloramphenicol) and sacB (which allows counter-selection using sucrose) flanked by sequences to either side of the target gene; the second transformation utilises the flanking sequences ligated directly to each other in order to remove the cat-sacB cassette. In order to ensure efficient uptake of the target DNA during transformation, A. pleuropneumoniae uptake sequences are added into the constructs used in both rounds of transformation. This method can be used to generate multiple successive deletions and can also be used to introduce targeted point mutations or insertions of heterologous genes into the A. pleuropneumoniae chromosome for development of live attenuated vaccine strains. So far, we have applied this method to highly transformable isolates of serovars 8 (MIDG2331), which is the most prevalent in the UK, and 15 (HS143). By screening clinical isolates of other serovars, it should be possible to identify other amenable strains

Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel

Group recommended a panel of mutations and variants that should be tested to determine carrier status within the CFTR gene as a part of population screening programs.1,2 This was initially done in response to the recommendations of an NIH CF Consensus Conference that CF carrier screening be consid-ered by all couples for use before conception or prenatally.3 At that time, the Working Group recognized limitations in our understanding of the population frequencies of several CF al-leles and proposed to review mutation distribution data after the first two years of the program. In 2002, as part of an ongo-ing effort to ensure that the cystic fibrosis carrier screening programs are current with respect to the scientific literature and other available data and practices, we initiated a second review of data on the distribution of mutations in different ethnic groups and we began to assess whether providers wer

Transdermal Influenza Immunization with Vaccine-Coated Microneedle Arrays

Influenza is a contagious disease caused by a pathogenic virus, with outbreaks all over the world and thousands of hospitalizations and deaths every year. Due to virus antigenic drift and short-lived immune responses, annual vaccination is required. However, vaccine coverage is incomplete, and improvement in immunization is needed. The objective of this study is to investigate a novel method for transdermal delivery using metal microneedle arrays (MN) coated with inactivated influenza virus to determine whether this route is a simpler and safer approach than the conventional immunization, capable to induce robust immune responses and confer protection against lethal virus challenge.Inactivated A/Aichi/2/68 (H3N2) influenza virus was coated on metal microneedle arrays and applied to mice as a vaccine in the caudal dorsal skin area. Substantial antibody titers with hemagglutination inhibition activity were detected in sera collected two and four weeks after a single vaccine dose. Challenge studies in mice with 5 x LD(50) of mouse adapted Aichi virus demonstrated complete protection. Microneedle vaccination induced a broad spectrum of immune responses including CD4+ and CD8+ responses in the spleen and draining lymph node, a high frequency of antigen-secreting cells in the lung and induction of virus-specific memory B-cells. In addition, the use of MN showed a dose-sparing effect and a strong Th2 bias when compared to an intramuscular (IM) reference immunization.The present results show that delivery of inactivated influenza virus through the skin using metal microneedle arrays induced strong humoral and cellular immune responses capable of conferring protection against virus challenge as efficiently as intramuscular immunization, which is the standard vaccination route. In view of the convenience of delivery and the potential for self-administration, vaccine-coated metal microneedles may provide a novel and highly effective immunization method

Intradermal influenza vaccination of healthy adults using a new microinjection system: a 3-year randomised controlled safety and immunogenicity trial

<p>Abstract</p> <p>Background</p> <p>Intradermal vaccination provides direct and potentially more efficient access to the immune system via specialised dendritic cells and draining lymphatic vessels. We investigated the immunogenicity and safety during 3 successive years of different dosages of a trivalent, inactivated, split-virion vaccine against seasonal influenza given intradermally using a microinjection system compared with an intramuscular control vaccine.</p> <p>Methods</p> <p>In a randomised, partially blinded, controlled study, healthy volunteers (1150 aged 18 to 57 years at enrolment) received three annual vaccinations of intradermal or intramuscular vaccine. In Year 1, subjects were randomised to one of three groups: 3 μg or 6 μg haemagglutinin/strain/dose of inactivated influenza vaccine intradermally, or a licensed inactivated influenza vaccine intramuscularly containing 15 μg/strain/dose. In Year 2 subjects were randomised again to one of two groups: 9 μg/strain/dose intradermally or 15 μg intramuscularly. In Year 3 subjects were randomised a third time to one of two groups: 9 μg intradermally or 15 μg intramuscularly. Randomisation lists in Year 1 were stratified for site. Randomisation lists in Years 2 and 3 were stratified for site and by vaccine received in previous years to ensure the inclusion of a comparable number of subjects in a vaccine group at each centre each year. Immunogenicity was assessed 21 days after each vaccination. Safety was assessed throughout the study.</p> <p>Results</p> <p>In Years 2 and 3, 9 μg intradermal was comparably immunogenic to 15 μg intramuscular for all strains, and both vaccines met European requirements for annual licensing of influenza vaccines. The 3 μg and 6 μg intradermal formulations were less immunogenic than intramuscular 15 μg. Safety of the intradermal and intramuscular vaccinations was comparable in each year of the study. Injection site erythema and swelling was more common with the intradermal route.</p> <p>Conclusion</p> <p>An influenza vaccine with 9 μg of haemagglutinin/strain given using an intradermal microinjection system showed comparable immunogenic and safety profiles to a licensed intramuscular vaccine, and presents a promising alternative to intramuscular vaccination for influenza for adults younger than 60 years.</p> <p>Trial registration</p> <p>Clinicaltrials.gov NCT00703651.</p

A Simple but Highly Effective Approach to Evaluate the Prognostic Performance of Gene Expression Signatures

BACKGROUND: Highly parallel analysis of gene expression has recently been used to identify gene sets or 'signatures' to improve patient diagnosis and risk stratification. Once a signature is generated, traditional statistical testing is used to evaluate its prognostic performance. However, due to the dimensionality of microarrays, this can lead to false interpretation of these signatures. PRINCIPAL FINDINGS: A method was developed to test batches of a user-specified number of randomly chosen signatures in patient microarray datasets. The percentage of random generated signatures yielding prognostic value was assessed using ROC analysis by calculating the area under the curve (AUC) in six public available cancer patient microarray datasets. We found that a signature consisting of randomly selected genes has an average 10% chance of reaching significance when assessed in a single dataset, but can range from 1% to ∼40% depending on the dataset in question. Increasing the number of validation datasets markedly reduces this number. CONCLUSIONS: We have shown that the use of an arbitrary cut-off value for evaluation of signature significance is not suitable for this type of research, but should be defined for each dataset separately. Our method can be used to establish and evaluate signature performance of any derived gene signature in a dataset by comparing its performance to thousands of randomly generated signatures. It will be of most interest for cases where few data are available and testing in multiple datasets is limited

Satellite-based terrestrial production efficiency modeling

Production efficiency models (PEMs) are based on the theory of light use efficiency (LUE) which states that a relatively constant relationship exists between photosynthetic carbon uptake and radiation receipt at the canopy level. Challenges remain however in the application of the PEM methodology to global net primary productivity (NPP) monitoring. The objectives of this review are as follows: 1) to describe the general functioning of six PEMs (CASA; GLO-PEM; TURC; C-Fix; MOD17; and BEAMS) identified in the literature; 2) to review each model to determine potential improvements to the general PEM methodology; 3) to review the related literature on satellite-based gross primary productivity (GPP) and NPP modeling for additional possibilities for improvement; and 4) based on this review, propose items for coordinated research

Anchored enrichment dataset for true flies (order Diptera) reveals insights into the phylogeny of flower flies (family Syrphidae)

Background: Anchored hybrid enrichment is a form of next-generation sequencing that uses oligonucleotide probes to target conserved regions of the genome flanked by less conserved regions in order to acquire data useful for phylogenetic inference from a broad range of taxa. Once a probe kit is developed, anchored hybrid enrichment is superior to traditional PCR-based Sanger sequencing in terms of both the amount of genomic data that can be recovered and effective cost. Due to their incredibly diverse nature, importance as pollinators, and historical instability with regard to subfamilial and tribal classification, Syrphidae (flower flies or hoverflies) are an ideal candidate for anchored hybrid enrichment-based phylogenetics, especially since recent molecular phylogenies of the syrphids using only a few markers have resulted in highly unresolved topologies. Over 6200 syrphids are currently known and uncovering their phylogeny will help us to understand how these species have diversified, providing insight into an array of ecological processes, from the development of adult mimicry, the origin of adult migration, to pollination patterns and the evolution of larval resource utilization. Results: We present the first use of anchored hybrid enrichment in insect phylogenetics on a dataset containing 30 flower fly species from across all four subfamilies and 11 tribes out of 15. To produce a phylogenetic hypothesis, 559 loci were sampled to produce a final dataset containing 217,702 sites. We recovered a well resolved topology with bootstrap support values that were almost universally >95 %. The subfamily Eristalinae is recovered as paraphyletic, with the strongest support for this hypothesis to date. The ant predators in the Microdontinae are sister to all other syrphids. Syrphinae and Pipizinae are monophyletic and sister to each other. Larval predation on soft-bodied hemipterans evolved only once in this family. Conclusions: Anchored hybrid enrichment was successful in producing a robustly supported phylogenetic hypothesis for the syrphids. Subfamilial reconstruction is concordant with recent phylogenetic hypotheses, but with much higher support values. With the newly designed probe kit this analysis could be rapidly expanded with further sampling, opening the door to more comprehensive analyses targeting problem areas in syrphid phylogenetics and ecology.Peer reviewe