Modern molecular biology and the prediction of human physiology, disease and performance

Question 1

What is ‘omics’?

Answer 1

“ome” and “omics” are suffixes that are derived from genome (as a combination of “gene” and “chromosome”) and genomics (the study of the genome)

Question 2

Why use ‘omic’ analyses?

Answer 2

o WB, RT-PCR, IF etc. are reductionist approaches:
 Permit examination of a VERY limited number of molecules
o However, MANY genes/ proteins might co-vary with (e.g.) muscular adaptation to exercise
o ‘omic’ analyses allow expression changes in hundreds-thousands of molecules to be assessed at once Provides a global (holistic) approach far beyond what is known of a limited number of well-defined molecules
o Might facilitate identification of novel biomarkers regulating (patho)physiological processes

Question 3

Can exercise be viewed as medicine?

Answer 3

o Susruta (600 B.C.) advocates exercise to restore equilibria between the humors
o Hippocrates (400 B.C.) “If there is any deficiency in food and exercise the body will fall sick.”
o Yuri Gagaran (1961) orbits Earth and functional incapacity observed with reduced gravity
o Yes, in that drugs have variable inter-individual efficacy and side-effect profiles
 i.e. some are ‘responders’ and some are ‘non-responders’
 Drug delivery programmes tailored to the individual
o Exercise should be ‘prescribed’ in the same manner

Question 4

Which behaviours are most important for you?

Answer 4

o Non-smoking? NO
o Maintenance of body mass appropriate for height (BMI < 21 units per week)? NO
o Regular ‘physical exercise’ (150 min aerobic exercise/wk)? YES
o A diet low in fat & high in plant foods? NO
o Light to moderate alcohol intake (< 21 units per week)? NO

Question 5

“It is much more important to know what kind of patient has a disease, than to know what kind of disease a patient has” Caleb Parry (Bath, UK, 1755-1822). What did Parry mean?

Answer 5

o 150 min/wk of exercise training produces NON-responders
o Every exercise intervention study has low or adverse responders…
o No training effect with undertraining, proves…?
o No double BL testing
o Arbitrary criteria for training response
o Expected non-responder frequency with 1st round of training that ‘disappears’ with 4-5 x per week
 Contradicts all other studies
 Selected population bias?
o Thus, the MEAN control of exercise gains are not always what they might seem…

Question 6

Why was there no difference for any baseline values or no relationship between baseline physiological variable & magnitude of change observed for any parameter?

Answer 6

Influence of hereditary factors

Question 7

DNA sequence – what’s the most we can expect to learn?

Answer 7

o Physiological capacity?
 Delta VO2max ~60% ‘genetic’ (HERITAGE)
 Insulin Sensitivity ~40% ‘genetic’ (HERITAGE)
o Performance?
 Knee extension ~50% genetic (154,970 sibling pairs, Silventoinen et al. 2008)
 Rodent endurance ~50% genetic contribution

Question 8

What pathways determine VO2max responses to training?

Answer 8

o Higher responders for muscle hypertrophy ‘activate’ an ‘anti-growth’ signature!!!
o Specifically, many rRNAs are down-regulated in human high gainers for lean mass
o New insights – potential role of microRNAs (miR)
 89 men and women performed 16 wk diet and exercise intervention
 High-responders (>10 % BM loss) and low-responders (<5% BM loss) identified
 Circulating miR-935 and miR-140 increased only in low responders
 These miR’s target genes associated with energy expenditure/ metabolism

Question 9

So, what are the implications for exercise prescription?

Answer 9

o Aerobic fitness determines quality of life and is a powerful correlate of all cause mortality e.g. Myers NEJM
o In the USA ~20M people have sarcopenia – the age related decline in muscle size and function

Question 10

Quantitative immunofluorescence microscopy of subcellular GLUT4 distribution in human skeletal muscle: effects of endurance and sprint interval training (Bradley et al., 2014)

Answer 10

This study used an immunofluorescence microscopy method to investigate changes in subcellular GLUT4 distribution and content following ET and SIT. Percutaneous muscle biopsy samples were taken from the m. vastus lateralis of 16 sedentary males in the overnight fasted state before and after 6 weeks of ET and SIT. An antibody was fully validated and used to show large (> 1 lm) and smaller (< 1 lm) GLUT4-containing clusters. The large clusters likely represent trans-Golgi network stores and the smaller clusters endosomal stores and GLUT4 storage vesicles (GSVs). Density of GLUT4 clusters was higher at the fibre periphery especially in perinuclear regions. A less dense punctate distribution was seen in the rest of the muscle fibre. Total GLUT4 fluorescence intensity increased in type I and type II fibres following both ET and SIT. Large GLUT4 clusters increased in number and size in both type I and type II fibres, while the smaller clusters increased in size. The greatest increases in GLUT4 fluorescence intensity occurred within the 1 lm layer immediately adjacent to the PM. The increase in peripheral localisation and protein content of GLUT4 following ET and SIT is likely to contribute to the improvements in glucose homeostasis observed after both training modes.

Question 11

Observation of the molecular organization of calcium release sites in fast- and slow-twitch skeletal muscle with nanoscale imaging (Jayasinghe et al., 2014)

Answer 11

Our findings confirm that junctophilin-1 (JPH1), which tethers the sarcoplasmic reticulum ((SR) intracellular calcium store) to the tubular (t-) system at triads, was present throughout the RyR array, whereas JPH2 was contained within much smaller nanodomains. Similar imaging of the primary SR calcium buffer, calsequestrin (CSQ), detected less overlap of the triad with CSQ in slow-twitch muscle supporting greater spatial heterogeneity in the luminal Ca2+ buffering when compared with fast twitch muscle. Taken together, these nanoscale differences can explain the fundamentally different physiologies of fast- and slow-twitch muscle.

Question 12

Skeletal muscle proteomics: current approaches, technical challenges and emerging techniques (Ohlendieck, 2011)

Answer 12

Over the past few years, skeletal muscle proteomics has successfully catalogued the majority of abundant and soluble fibre-associated proteins. The refined proteomic analysis of isoform expression patterns and biochemical studies of dynamic posttranslational modifications has identified thousands of distinct muscle protein species. Myogenesis, muscle maturation, muscle transformation and aging-related muscle wasting have been intensively investigated by using proteomic methods and has resulted in the establishment of a comprehensive biomarker signature for major physiological adaptation processes in contractile tissues. The proteomic characterisation of common neuromuscular disorders has revealed novel disease-specific marker proteins of disuse atrophy, muscular dystrophy, obesity, type 2 diabetes, sepsis, hypokalemic myopathy, inclusion body myositis and reducing body myopathy. Thus, MS-based proteomics has decisively improved our general understanding of physiological and pathophysiological mechanisms in muscle tissues. New biomarker candidates can now be used for improving diagnostic methods, the identification of novel therapeutic targets, better comprehension of the molecular pathogenesis of muscular disorders, improved monitoring of disease progression and the judging of potential side effects of experimental drugs. Importantly, if the proteomic workflow could be successfully miniaturised, then single-cell proteomics of different fibre populations would lead to more comprehensive coverage of the skeletal muscle proteome. This depends on technical developments in the field of MS [20, 21, 22]. In the future, organelle and membrane proteomics will probably play a more prominent role in muscle biochemistry to study less abundant and more hydrophobic proteins. Filter-aided sample preparation and on-membrane digestion may be preferred for the proteomic analysis of high molecular mass membrane proteins. Once the majority of large and integral muscle proteins have been catalogued by proteomics, it will be crucial to correlate these findings with genomic, transcriptomic and metabolomic databanks [147] and establish the global relationship of biomolecules in striated voluntary muscle tissues.

Question 13

A quantitative atlas of mitotic phosphorylation (Dephoure et al., 2008)

Answer 13

We report the identification of >14,000 different phosphorylation events, more than half of which, to our knowledge, have not been described in the literature, along with relative quantitative data for the majority of these sites. We observed >1,000 proteins with increased phosphorylation in mitosis including many known cell cycle regulators. The majority of sites on regulated phosphopeptides lie in [S/T]P motifs, the minimum required sequence for CDKs, suggesting that many of the proteins may be CDK substrates. Analysis of non-proline site-containing phosphopeptides identified two unique motifs that suggest there are at least two undiscovered mitotic kinases.

Question 14

Proteomic investigation of changes in human vastus lateralis muscle in response to interval-exercise training (Holloway et al., 2009)

Answer 14

The differences in spot expression represent changes in post‐transcriptional or post‐translational processing. In particular, modulation of muscle creatine kinase and troponin T were prominent. Pro‐Q Diamond staining revealed these changes in expression were associated with phosphorylated protein species, which provides novel information regarding muscle adaptation to interval training.

Question 15

Precision and variance components in quantitative gel electrophoresis (Koller et al., 2005)

Answer 15

The error in quantitative gel electrophoresis/Western blotting was investigated considering the purity testing of erythropoietin. The overall error was over 35% relative standard deviation. However, an analysis of variance elucidated that the interoperator variability was the dominant error source, which already explained almost 80% of the total variance. Careful compilation and investigation of the possible error sources strongly indicates that the immunoreaction after blotting and the subsequent color reaction are the major error sources in this case.

Question 16

The Design of a Quantitative Western Blot Experiment (Taylor et al., 2014)

Answer 16

Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.

Question 17

A Defined Methodology for Reliable Quantification of Western Blot Data (Taylor et al., 2013)

Answer 17

To conclude, we propose a rigorous methodology of validating sample loading, standardizing antibody dilutions, determining the dynamic range with a sensitive, camera-based imaging system, and use of a stain-free technology to get high-quality and reliable quantitative data from western blots.

Question 18

An overview of technical considerations for Western blotting applications to physiological research (Bass et al., 2017)

Answer 18

WB has emerged as an essential tool within physiological research; nevertheless with poor understanding and implementation, any subsequent analysis can produce misleading and confusing interpretation (i.e., Ab specificity and validation). Before a sample is loaded into a gel, careful consideration must be given to often overlooked aspects such as the appropriate buffer for homogenization and extraction of the intended target protein for denaturation. Gel composition should effectively separate proteins by size, with changes to concentration giving resolution to the intended target by varying migration speed. Subsequent transfer onto an immobilizing membrane will allow the probing for one or more targets with 1°Ab and 2°Ab, with emphasis upon Ab specificity and the ability to assess PT modifications. Validation of Ab should always be undertaken, using both positive and negative controls to try to ensure specificity. Within each study design and group comparisons, QC samples should be used allowing the comparison of multiple gels. The method of detection will be ultimately be determined by the equipment available. However, fluorescent antibodies have a greater dynamic range and may be multiplexed for additional targets if desired. As sample quantity may be scarce, the ability to strip and re‐probe membranes for additional targets is desirable; however, potential issues with regard to quantification and potential signal reduction should be considered carefully and where possible mitigated. Finally, the quantification and analysis of band intensity should be evaluated consistently throughout with both single and multiple blots; as doing so can produce reliable and accurate data.

Question 19

The necessity of and strategies for improving confidence in the accuracy of western blots (Ghosh et al., 2014)

Answer 19

Western blotting is one of the most commonly used laboratory techniques for identifying proteins and semi-quantifying protein amounts; however, several recent findings suggest that western blots may not be as reliable as previously assumed. This is not surprising since many labs are unaware of the limitations of western blotting. In this manuscript, we review essential strategies for improving confidence in the accuracy of western blots. These strategies include selecting the best normalization standard, proper sample preparation, determining the linear range for antibodies and protein stains relevant to the sample of interest, confirming the quality of the primary antibody, preventing signal saturation and accurately quantifying the signal intensity of the target protein. Although western blotting is a powerful and indispensable scientific technique that can be used to accurately quantify relative protein levels, it is necessary that proper experimental techniques and strategies are employed.

Question 20

Comparison of RNA-Seq and Microarray in Transcriptome Profiling of Activated T Cells (Zhao et al., 2014)

Answer 20

To demonstrate the benefits of RNA-Seq over microarray in transcriptome profiling, both RNA-Seq and microarray analyses were performed on RNA samples from a human T cell activation experiment. In contrast to other reports, our analyses focused on the difference, rather than similarity, between RNA-Seq and microarray technologies in transcriptome profiling. A comparison of data sets derived from RNA-Seq and Affymetrix platforms using the same set of samples showed a high correlation between gene expression profiles generated by the two platforms. However, it also demonstrated that RNA-Seq was superior in detecting low abundance transcripts, differentiating biologically critical isoforms, and allowing the identification of genetic variants. RNA-Seq also demonstrated a broader dynamic range than microarray, which allowed for the detection of more differentially expressed genes with higher fold-change. Analysis of the two datasets also showed the benefit derived from avoidance of technical issues inherent to microarray probe performance such as cross-hybridization, non-specific hybridization and limited detection range of individual probes. Because RNA-Seq does not rely on a pre-designed complement sequence detection probe, it is devoid of issues associated with probe redundancy and annotation, which simplified interpretation of the data. Despite the superior benefits of RNA-Seq, microarrays are still the more common choice of researchers when conducting transcriptional profiling experiments. This is likely because RNA-Seq sequencing technology is new to most researchers, more expensive than microarray, data storage is more challenging and analysis is more complex. We expect that once these barriers are overcome, the RNA-Seq platform will become the predominant tool for transcriptome analysis.

Question 21

RNA-Seq: a revolutionary tool for transcriptomics (Wang et al., 2009)

Answer 21

RNA-Seq is a recently developed approach to transcriptome profiling that uses deep-sequencing technologies. Studies using this method have already altered our view of the extent and complexity of eukaryotic transcriptomes. RNA-Seq also provides a far more precise measurement of levels of transcripts and their isoforms than other methods. This article describes the RNA-Seq approach, the challenges associated with its application, and the advances made so far in characterizing several eukaryote transcriptomes.

Question 22

Microarray technology: beyond transcript profiling and genotype analysis (Hoheisel, 2006)

Answer 22

Understanding complex functional mechanisms requires the global and parallel analysis of different cellular processes. DNA microarrays have become synonymous with this kind of study and, in many cases, are the obvious platform to achieve this aim. They have already made important contributions, most notably to gene-expression studies, although the true potential of this technology is far greater. Whereas some assays, such as transcript profiling and genotyping, are becoming routine, others are still in the early phases of development, and new areas of application, such as genome-wide epigenetic analysis and on-chip synthesis, continue to emerge.

Question 23

DNA Microarrays: a Powerful Genomic Tool for Biomedical and Clinical Research (Trevino et al., 2007)

Answer 23

Among the many benefits of the Human Genome Project are new and powerful tools such as the genome-wide hybridization devices referred to as microarrays. Initially designed to measure gene transcriptional levels, microarray technologies are now used for comparing other genome features among individuals and their tissues and cells. Results provide valuable information on disease subcategories, disease prognosis, and treatment outcome. Likewise, they reveal differences in genetic makeup, regulatory mechanisms, and subtle variations and move us closer to the era of personalized medicine. To understand this powerful tool, its versatility, and how dramatically it is changing the molecular approach to biomedical and clinical research, this review describes the technology, its applications, a didactic step-by-step review of a typical microarray protocol, and a real experiment. Finally, it calls the attention of the medical community to the importance of integrating multidisciplinary teams to take advantage of this technology and its expanding applications that, in a slide, reveals our genetic inheritance and destiny.