Different molecular and structural adaptations with eccentric and conventional strength training in elderly men and women

Reprogramming of gene expression contributes to structural and functional adaptation of muscle tissue in response to altered use. The aim of this study was to investigate mechanisms for observed improvements in leg extension strength, gain in relative thigh muscle mass and loss of body and thigh fat content in response to eccentric and conventional strength training in elderly men (n = 14) and women (n = 14; average age of the men and women: 80.1 ± 3.7 years) by means of structural and molecular analyses. Biopsies were collected from m. vastus lateralis in the resting state before and after 12 weeks of training with two weekly resistance exercise sessions (RET) or eccentric ergometer sessions (EET). Gene expression was analyzed using custom-designed low-density PCR arrays. Muscle ultrastructure was evaluated using EM morphometry. Gain in thigh muscle mass was paralleled by an increase in muscle fiber cross-sectional area (hypertrophy) with RET but not with EET, where muscle growth is likely occurring by the addition of sarcomeres in series or by hyperplasia. The expression of transcripts encoding factors involved in muscle growth, repair and remodeling (e.g., IGF-1, HGF, MYOG, MYH3) was increased to a larger extent after EET than RET. MicroRNA 1 expression was decreased independent of the training modality, and was paralleled by an increased expression of IGF-1 representing a potential target. IGF-1 is a potent promoter of muscle growth, and its regulation by microRNA 1 may have contributed to the gain of muscle mass observed in our subjects. EET depressed genes encoding mitochondrial and metabolic transcripts. The changes of several metabolic and mitochondrial transcripts correlated significantly with changes in mitochondrial volume density. Intramyocellular lipid content was decreased after EET concomitantly with total body fat. Changes in intramyocellular lipid content correlated with changes in body fat content with both RET and EET. In the elderly, RET and EET lead to distinct molecular and structural adaptations which might contribute to the observed small quantitative differences in functional tests and body composition parameters. EET seems to be particularly convenient for the elderly with regard to improvements in body composition and strength but at the expense of reducing muscular oxidative capacity.

Detecting and resolving position-dependent temperature effects in real-time quantitative polymerase chain reaction

Real-time quantitative polymerase chain reaction (qPCR) depends on precise temperature control of the sample during cycling. In the current study, we investigated how temperature variation in plate-based qPCR instruments influences qPCR results. Temperature variation was measured by amplicon melting analysis as a convenient means to assess well-to-well differences. Multiple technical replicates of several SYBR Green I-based qPCR assays allowed correlation of relative well temperature to quantification cycle. We found that inadequate template denaturation results in an inverse correlation and requires increasing the denaturation temperature, adding a DNA destabilizing agent, or pretreating with a restriction enzyme. In contrast, inadequate primer annealing results in a direct correlation and requires lowering the annealing temperature. Significant correlations were found in 18 of 25 assays. The critical nature of temperature-dependent effects was shown in a blinded study of 29 patients for the diagnosis of Prader-Willy and Angelman syndromes, where eight diagnoses were incorrect unless temperature-dependent effects were controlled. A method to detect temperature-dependent effects by pairwise comparisons of replicates in routine experiments is presented and applied. Systematic temperature errors in qPCR instruments can be recognized and their effects eliminated when high precision is required in quantitative genetic diagnostics and critical complementary DNA analyses.

Dosing algorithm to target a predefined AUC in patients with primary central nervous system lymphoma receiving high dose methotrexate

There is no consensus regarding optimal dosing of high dose methotrexate (HDMTX) in patients with primary CNS lymphoma. Our aim was to develop a convenient dosing algorithm to target AUC(MTX) in the range between 1000 and 1100 µmol l(-1) h.

General Protocol for the Culture of Cells on Plasma-Coated Electrospun Scaffolds

As opposed to culture on standard tissue-treated plastic, cell culture on three-dimensional scaffolds impedes additional challenges with respect to substrate preparation, cell seeding, culture maintenance, and analysis. We herewith present a general route for the culture of primary cells, differentiated cells, or stem cells on plasma-coated, electrospun scaffolds. We describe a method to prepare and fix the scaffolds in culture wells and discuss a convenient method for cell seeding and subsequent analysis by scanning electron microscopy or immunohistology.

Antibody responses in New World camelids with tuberculosis caused by Mycobacterium microti

Antibody responses in New World camelids (NWC) infected with Mycobacterium microti were studied by two serological methods, multiantigen print immunoassay (MAPIA) and lateral-flow-based rapid test (RT). Serum samples were collected during 2004-2006 from 87 animals including 1 alpaca and 7 llamas with confirmed or suspected M. microti infection, 33 potentially exposed but clinically healthy animals from known infected herds, and 46 control NWC from herds where infection had not been previously diagnosed. The serological assays correctly identified infection status in 97% (MAPIA) or 87% (RT) cases. In three llamas with confirmed M. microti infection and one llama with gross pathology suggestive of disease, for which multiple serum samples collected over time were available, the antibody-based tests showed positive results 1-2 years prior to the onset of clinical signs or being found dead. In MAPIA, MPB83 protein was identified to be an immunodominant serological target antigen recognized in NWC infected with M. microti. With the limited number of animals tested in this study, the serological assays demonstrated the potential for convenient, rapid, and accurate diagnosis of M. microti infection in live llamas and alpacas.

Brain responses to auditory and visual stimulus offset: Shared representations of temporal edges

Edges are crucial for the formation of coherent objects from sequential sensory inputs within a single modality. Moreover, temporally coincident boundaries of perceptual objects across different sensory modalities facilitate crossmodal integration. Here, we used functional magnetic resonance imaging in order to examine the neural basis of temporal edge detection across modalities. Onsets of sensory inputs are not only related to the detection of an edge but also to the processing of novel sensory inputs. Thus, we used transitions from input to rest (offsets) as convenient stimuli for studying the neural underpinnings of visual and acoustic edge detection per se. We found, besides modality-specific patterns, shared visual and auditory offset-related activity in the superior temporal sulcus and insula of the right hemisphere. Our data suggest that right hemispheric regions known to be involved in multisensory processing are crucial for detection of edges in the temporal domain across both visual and auditory modalities. This operation is likely to facilitate cross-modal object feature binding based on temporal coincidence. Hum Brain Mapp, 2008. (c) 2008 Wiley-Liss, Inc.

Analysis of plasma tocopherols alpha, gamma, and 5-nitro-gamma in rats with inflammation by HPLC coulometric detection

Reactive nitrogen oxide species (RNOS) have been implicated as effector molecules in inflammatory diseases. There is emerging evidence that gamma-tocopherol (gammaT), the major form of vitamin E in the North American diet, may play an important role in these diseases. GammaT scavenges RNOS such as peroxynitrite by forming a stable adduct, 5-nitro-gammaT (NGT). Here we describe a convenient HPLC method for the simultaneous determination of NGT, alphaT, and gammaT in blood plasma and other tissues. Coulometric detection of NGT separated on a deactivated reversed-phase column was linear over a wide range of concentrations and highly sensitive (approximately 10 fmol detection limit). NGT extracted from blood plasma of 15-week-old Fischer 344 rats was in the low nM range, representing approximately 4% of gammaT. Twenty-four h after intraperitoneal injection of zymosan, plasma NGT levels were 2-fold higher compared to fasted control animals when adjusted to gammaT or corrected for total neutral lipids, while alpha- and gammaT levels remained unchanged. These results demonstrate that nitration of gammaT is increased under inflammatory conditions and highlight the importance of RNOS reactions in the lipid phase. The present HPLC method should be helpful in clarifying the precise physiological role of gammaT.

Simultaneous determination of 3-hydroxyanthranilic and cinnabarinic acid by high-performance liquid chromatography with photometric or electrochemical detection

A convenient and rapid method for the simultaneous determination by HPLC of 3-hydroxyanthranilic acid and the dimer derived by its oxidation, cinnabarinic acid, is described. Buffers or biological samples containing these two Trp metabolites were acidified to pH 2.0 and extracted with ethyl acetate with recoveries of 96.5 +/- 0.5 and 93.4 +/- 3.7% for 3-hydroxyanthranilic and cinnabarinic acid, respectively. The two compounds were separated on a reversed-phase (C18) column combined with ion-pair chromatography and detected photometrically or electrochemically. The method was applied successfully to biological systems in which formation of either 3-hydroxyanthranilic or cinnabarinic acid had been described previously. Thus, interferon-gamma-treated human peripheral blood mononuclear cells formed and released significant amounts of 3-hydroxyanthranilic acid into the culture medium and mouse liver nuclear fraction possessed high "cinnabarinic acid synthase" activity. In contrast, addition of 3-hydroxyanthranilic acid to human erythrocytes resulted in only marginal formation of cinnabarinic acid. We conclude that the method described is specific, sensitive, and suitable for the detection of the two Trp metabolites in biological systems.

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.

Life cycle assessment of trayless dining

Universities in the United States are applying more sustainable approaches to their dining service operations. "The increase in social consciousness and environmental stewardship on college campuses has spurred an array of new and innovative sustainability programs"(ARAMARK Higher Education 2008). University residence dining is typically cafeteria style, with students using trays to carry food. Studies report that food served without trays substantially reduces food waste and water and electrical consumption associated with washing trays. Commonly, these reported results are estimates and not measurements taken under actual operating conditions. This study utilizes measurements recorded under actual dining service conditions in student residence halls at Michigan Technological University to develop the following: 1) operational-specific data on the issues and potential savings associated with a conversion to trayless dining and 2) life cycle assessment (LCA) cost and environmental impact analyses comparing dining with and without trays. For the LCA, the entire life cycle of the system is considered, from the manufacturing to the usage and disposal phases. The study shows that trayless dining reduces food waste because diners carry less food. The total savings for the diner shifts when not using trays for the standard academic year (205 days), with an average number of 700 diners, is 7,032 pounds of food waste from the pre-rinse area (33% reduction) and 3,157 pounds of food waste from the pan washing area (39% reduction). In addition, for each day of the study, the diners consumed more food during the trayless portion of the experiment. One possible explanation for the increased food consumption during this short duration study could be that the diners found it more convenient to eat the extra food on their plate rather than carrying it back for disposal. The trayless dining experiment shows a reduction in dishwasher water, steam, and electrical consumption for each day of the study. The average reduction of dishwasher water, steam, and electrical consumption over the duration of the study were 10.7%, 9.5%, and 6.4% respectively. Trayless dining implementation would result in a decrease of 4,305 gallons of consumption and wastewater discharge, 2.87 mm BTU of steam consumption, and 158 kWh of electrical consumption for the dinner shift over the academic year. Results of the LCA indicate a total savings of $190.4 when trays are not used during the dinner shift. Trayless dining requires zero CO2 eq and cumulative energy demand in the manufacturing stage, reductions of 1005 kg CO2 eq and 861 MJ eq in the usage phase, and reductions of 6458 kg CO2 eq and 1821 MJ eq in the end of the life cycle.

Statistical analysis of personal radiofrequency electromagnetic field measurements with nondetects

Exposimeters are increasingly applied in bioelectromagnetic research to determine personal radiofrequency electromagnetic field (RF-EMF) exposure. The main advantages of exposimeter measurements are their convenient handling for study participants and the large amount of personal exposure data, which can be obtained for several RF-EMF sources. However, the large proportion of measurements below the detection limit is a challenge for data analysis. With the robust ROS (regression on order statistics) method, summary statistics can be calculated by fitting an assumed distribution to the observed data. We used a preliminary sample of 109 weekly exposimeter measurements from the QUALIFEX study to compare summary statistics computed by robust ROS with a naïve approach, where values below the detection limit were replaced by the value of the detection limit. For the total RF-EMF exposure, differences between the naïve approach and the robust ROS were moderate for the 90th percentile and the arithmetic mean. However, exposure contributions from minor RF-EMF sources were considerably overestimated with the naïve approach. This results in an underestimation of the exposure range in the population, which may bias the evaluation of potential exposure-response associations. We conclude from our analyses that summary statistics of exposimeter data calculated by robust ROS are more reliable and more informative than estimates based on a naïve approach. Nevertheless, estimates of source-specific medians or even lower percentiles depend on the assumed data distribution and should be considered with caution.