Identification of Biomarkers that are Related to the Actin Cytoskeleton
Curator and Writer: Larry H Bernstein, MD, FCAP
This is Part I in a series of articles on Calcium and Cell motility.
The Series consists of the following articles:
Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton
Larry H Bernstein, MD, FCAP
Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility
Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN
Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease
Larry H. Bernstein, MD, FCAP, Stephen J. Williams, PhD and Aviva Lev-Ari, PhD, RN
Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
Part V: Ca2+-Stimulated Exocytosis: The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Aviva Lev-Ari, PhD, RN
Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
Part IX: Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction (Ryanopathy) and Calcium as Neurotransmitter Sensor
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
Part X: Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
Part XI: Sensors and Signaling in Oxidative Stress
Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2013/11/01/sensors-and-signaling-in-oxidative-stress/
Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
In this article the Author will cover two types of biomarker on the function of actin in cytoskeleton mobility in situ.
- First, is an application in developing the actin or other component, for a biotarget and then, to be able to follow it as
(a) a biomarker either for diagnosis, or
(b) for the potential treatment prediction of disease free survival.
- Second, is mostly in the context of MI, for which there is an abundance of work to reference, and a substantial body of knowledge about
(a) treatment and long term effects of diet, exercise, and
(b) underlying effects of therapeutic drugs.
1. Cell Membrane (cytoskeletal) Plasticity
Refer to … Squeezing Ovarian Cancer Cells to Predict Metastatic Potential: Cell Stiffness as Possible Biomarker
Reporter/curator: Prabodh Kandala, PhD
Comparative gene expression analyses indicate that the reduced stiffness of highly metastatic HEY A8 cells is associated with actin cytoskeleton remodeling and microscopic examination of actin fiber structure in these cell lines is consistent with this prediction. The results suggest either of two approaches. Atomic Force Microscopy is not normally used by pathologists in diagnostics. Electron microscopy requires space for making and cutting the embedded specimen, and a separate room for the instrument. The instrument is large and the technique was not suitable for anything other than research initially until EM gained importance in Renal Pathology. It has not otherwise been used. This new method looks like it might be more justified over a spectrum of cases.
A. Atomic Force Microscopy
So the first point related to microscopy is whether AFM has feasibility for routine clinical use in the pathologists’ hands. This requires:
- suitable size of equipment
- suitable manipulation of the specimen
- The question of whether you are using overnight fixed specimen, or whether the material is used unfixed
- Nothing is said about staining of cells for identification.
- Then there is the question about whether this will increase the number of Pathologist Assistants used across the country, which I am not against. This would be the end of “house” trained PAs, and gives more credence to the too few PA programs across the country. The PA programs have to be reviewed and accredited by NAACLS (I served 8 years on the Board). A PA is represented on the Board, and programs are inspected by qualified peers. There is no academic recognition given to this for tenure and promotion in Pathology Departments, and a pathologist is selected for a medical advisory role by the ASCP, and must be a Medical Advisor to a MLS accredited Program. The fact is that PAs do gross anatomic dictation of selected specimens, and they do autopsies under the guidance of a pathologist. This is the reality of the profession today. The pathologist has to be in attendance at a variety of quality review conferences, for surgical morbidity and mortality to obstetrics review, and the Cancer Review. Cytopathology and cytogenetics are in the pathology domain. In the case of tumors of the throat, cervix, and accessible orifices, it seems plausible to receive a swab for preparation. However, sampling error is greater than for a biopsy. A directed needle biopsy or a MIS specimen is needed for the ovary.
B. identification of biomarkers that are related to the actin cytoskeleton
The alternative to the first approach is the identification of biomarkers that are related to the actin cytoskeleton, perhaps in the nature of the lipid or apoprotein isoform that gives the cell membrane deformability. The method measuring by Atomic Force Microscopy is shown with the current method of cytological screening, and I call attention to cells clustered together that have a scant cytoplasm surrounding nuclei occupying 1/2 to 3/4 of the cell radius. The cells are not anaplastic, but the clumps are suggestive of glnad forming epithelium.
English: Animation showing 3-D nature of cluster. Image:Serous carcinoma 2a – cytology.jpg (Photo credit: Wikipedia)
The cell membrane, also called the plasma membrane or plasmalemma, is a semipermeable lipid bilayer common to all living cells. It contains a variety of biological molecules, primarily proteins and lipids, which are involved in a vast array of cellular processes. It also serves as the attachment point for both the intracellular cytoskeleton and, if present, the cell wall. (Photo credit: Wikipedia)
AFM non contact mode (Photo credit: Wikipedia)
C. The diagnosis of ovarian cancer can be problematic because it can have a long period of growth undetected.
Predictive Modeling
Ovarian Cancer a plot of the CA125 elimination half-life vs the Kullback-Liebler distance
Troponin(s) T, I, C and the contractile apparatus (contributed by Aviva Lev-Ari, PhD, RN)
For 2012 – 2013 Frontier Contribution in Cardiology on Gene Therapy Solutions for Improving Myocardial Contractility, see
Lev-Ari, A. 8/1/2013 Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
For explanation of Conduction prior to Myocardial Contractility, see
Lev-Ari, A. 4/28/2013 Genetics of Conduction Disease: Atrioventricular (AV) Conduction Disease (block): Gene Mutations – Transcription, Excitability, and Energy Homeostasis
The contraction of skeletal muscle is triggered by nerve impulses, which stimulate the release of Ca2+ from the sarcoplasmic reticulum—a specialized network of internal membranes, similar to the endoplasmic reticulum, that stores high concentrations of Ca2+ ions. The release of Ca2+ from the sarcoplasmic reticulum increases the concentration of Ca2+ in the cytosol from approximately 10-7 to 10-5 M. The increased Ca2+ concentration signals muscle contraction via the action of two accessory proteins bound to the actin filaments: tropomyosin and troponin (Figure 11.25). Tropomyosin is a fibrous protein that binds lengthwise along the groove of actin filaments. In striated muscle, each tropomyosin molecule is bound to troponin, which is a complex of three polypeptides: troponin C (Ca2+-binding), troponin I (inhibitory), and troponin T (tropomyosin-binding).
- When the concentration of Ca2+ is low, the complex of the troponins with tropomyosin blocks the interaction of actin and myosin, so the muscle does not contract.
- At high concentrations, Ca2+ binding to troponin C shifts the position of the complex, relieving this inhibition and allowing contraction to proceed.
Figure 11.25
Association of tropomyosin and troponins with actin filaments. (A) Tropomyosin binds lengthwise along actin filaments and, in striated muscle, is associated with a complex of three troponins: troponin I (TnI), troponin C (TnC), and troponin T (TnT). In (more…)
Contractile Assemblies of Actin and Myosin in Nonmuscle Cells
Contractile assemblies of actin and myosin, resembling small-scale versions of muscle fibers, are present also in nonmuscle cells. As in muscle, the actin filaments in these contractile assemblies are interdigitated with bipolar filaments of myosin II, consisting of 15 to 20 myosin II molecules, which produce contraction by sliding the actin filaments relative to one another (Figure 11.26). The actin filaments in contractile bundles in nonmuscle cells are also associated with tropomyosin, which facilitates their interaction with myosin II, probably by competing with filamin for binding sites on actin.
Figure 11.26
Contractile assemblies in nonmuscle cells. Bipolar filaments of myosin II produce contraction by sliding actin filaments in opposite directions.
Two examples of contractile assemblies in nonmuscle cells, stress fibers and adhesion belts, were discussed earlier with respect to attachment of the actin cytoskeleton to regions of cell-substrate and cell-cell contacts (see Figures 11.13 and 11.14). The contraction of stress fibers produces tension across the cell, allowing the cell to pull on a substrate (e.g., the extracellular matrix) to which it is anchored. The contraction of adhesion belts alters the shape of epithelial cell sheets: a process that is particularly important during embryonic development, when sheets of epithelial cells fold into structures such as tubes.
The most dramatic example of actin-myosin contraction in nonmuscle cells, however, is provided by cytokinesis—the division of a cell into two following mitosis (Figure 11.27). Toward the end of mitosis in animal cells, a contractile ring consisting of actin filaments and myosin II assembles just underneath the plasma membrane. Its contraction pulls the plasma membrane progressively inward, constricting the center of the cell and pinching it in two. Interestingly, the thickness of the contractile ring remains constant as it contracts, implying that actin filaments disassemble as contraction proceeds. The ring then disperses completely following cell division.
Figure 11.27
Cytokinesis. Following completion of mitosis (nuclear division), a contractile ring consisting of actin filaments and myosin II divides the cell in two.
http://www.ncbi.nlm.nih.gov/books/NBK9961/
2. Use of Troponin(s) in Diagnosis
Troponins T and I are released into the circulation at the time of an acute coronary syndrome (ACS). Troponin T was first introduced by Roche (developed in Germany) for the Roche platform as a superior biomarker for identifying acute myocardial infarction (AMI), because of a monoclonal specificity to the cardiac troponin T. It could not be measured on any other platform (limited license patent), so the Washington University Clinical Chemistry group developed a myocardiocyte specific troponin I that quickly became widely available to Beckman, and was adapted to other instruments. This was intended to replace the CK isoenzyme MB, that is highly elevated in rhabdomyolysis associated with sepsis or with anesthesia in special cases.
The troponins I and T had a tenfold scale difference, and the Receiver Operator Curve Generated cutoff was accurate for AMI, but had significant elevation with end-stage renal disease. The industry worked in concert to develop a high sensitivity assay for each because there were some missed AMIs just below the ROC cutoff, which could be interpreted as Plaque Rupture. However, the concept of plaque rupture had to be reconsidered, and we are left with type1 and type 2 AMI (disregarding the case of post PCI or CABG related). This led to the current establishment of 3 standard deviations above the lowest measureable level at 10% coefficient of variation. This has been discussed sufficiently elsewhere. It did introduce a problem in the use of the test as a “silver bullet” once the finer distinctions aqnd the interest in using the test for prognosis as well as diagnosis. This is where the use of another protein associated with heart failure came into play – either the B type natriuretic peptide, or its propeptide, N-terminal pro BNP. The prognostic value of using these markers, secreted by the HEART and acting on the kidneys (sodium reabsorption) has proved useful. But there has not been a multivariate refinement of the use of a two biomarker approach. In the following part D, I illustrate what can be done with an algorithmic approach to multiple markers.
Software Agent for Diagnosis of AMI
Isaac E. Mayzlin, Ph.D., David Mayzlin, Larry H. Bernstein, M.D. The so called gold standard of proof of a method is considered the Receiver-Operating Characteristic Curve, developed for detecting “enemy planes or missiles”, and adopted first by radiologists in medicine. This matches the correct “hits” to the actual calssification and it is generally taught as a plot of sensitivity vs (1 – specifity). But what if you had no “training” variable? Work inspired by Eugene Rypka’s bacterial classification led to Rosser Rudolph’s application of the Entropy of Shannon and Weaver to identify meaningful information, referring to what was Kullback-Liebler distance as “effective information”. This allowed Rudolph and Bernstein to classify using disease biomarkers obtaing the same results as the ROC curve using an apl program. The same data set was used by Bernstein, Adan et al. previously, and was again used by Izaak Mayzlin from University of Moscow with a new wrinkle. Dr. Mayzlin created a neural network (Maynet), and then did a traditional NN with training on the data, and also clustered the data using geometric distance clustering and trained on the clusters. It was interesting to see that the optimum cluster separation was closely related to the number of classes and the accuracy of classification. An earlier simpler model using the slope of the MB isoenzyme increase and percent of total CK activity was perhaps related to Burton Sobel’s work on CK-MB disappearance rate for infarct size. The main process consists of three successive steps: (1) clustering performed on training data set, (2) neural network’s training on clusters from previous step, and (3) classifier’s accuracy evaluation on testing data. The classifier in this research will be the ANN, created on step 2, with output in the range [0,1], that provides binary result (1 – AMI, 0 – not AMI), using decision point 0.5. Table 1. Effect of selection of maximum distance on the number of classes formed and on the accuracy of recognition by ANN
| Clustering Distance Factor F(D = F * R) | Number ofClasses | Number of Nodes in The Hidden Layers | Number of Misrecognized Patterns inThe TestingSet of 43 | Percent ofMisrecognized |
| 10.90.80.7 | 2414135 | 1, 02, 03, 01, 02, 03, 0 3, 2 3, 2 | 121121 1 1 | 2.34.62.32.34.62.3 2.3 2.3 |
– Creatine kinase B-subunit activity in serum in cases of suspected myocardial infarction: a prediction model based on the slope of MB increase and percentage CK-MB activity. L H Bernstein, G Reynoso Clin Chem 1983; 29(3):590-592 ICID: 825549 – Diagnosis of acute myocardial infarction from two measurements of creatine kinase isoenzyme MB with use of nonparametric probability estimation. L H Bernstein, I J Good, G I Holtzman, M L Deaton, J Babb. Clin Chem 1989; 35(3):444-447 ICID: 825570 – Information induction for predicting acute myocardial infarction. R A Rudolph, L H Bernstein, J Babb. Clin Chem 1988; 34(10):2031-2038 ICID: 825568
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- Parvin Overexpression Uncovers Tissue-Specific Genetic Pathways and Disrupts F-Actin to Induce Apoptosis in the Developing Epithelia in Drosophila (plosone.org)
- Measurement of Phospholipids May Improve Diagnostic Accuracy in Ovarian Cancer (plosone.org)
- Cell Stiffness Of Ovarian Cancer Cells May Predict Metastatic Potential (medicalnewstoday.com)
- Ovarian Cancer Cells Trick Healthy Neighbors to Do Their Dirty Work (sciencelife.uchospitals.edu)
- LL-37 Induces Polymerization and Bundling of Actin and Affects Actin Structure (plosone.org)
- Tumor-causing cells are squishier (sciencedaily.com)
- Soluble Axoplasm Enriched from Injured CNS Axons Reveals the Early Modulation of the Actin Cytoskeleton (plosone.org)
Related articles published on this Open Access Online Scientific Journal, include the following:
- Dasatinib in Combination With Other Drugs for Advanced, Recurrent Ovarian Cancer (pharmaceuticalintelligence.com)
- BRCA1 a tumour suppressor in breast and ovarian cancer – functions in transcription, ubiquitination and DNA repair (pharmaceuticalintelligence.com)
- Squeezing Ovarian Cancer Cells to Predict Metastatic Potential: Cell Stiffness as Possible Biomarker (pharmaceuticalintelligence.com)
Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Aviva Lev-Ari, PhD, RN 8/1/2013
High-Sensitivity Cardiac Troponin Assays- Preparing the United States for High-Sensitivity Cardiac Troponin Assays
Larry Bernstein, MD, FCAP 6/13/2013
https://pharmaceuticalintelligence.com/2013/06/13/high-sensitivity-cardiac-troponin-assays/
Dealing with the Use of the High Sensitivity Troponin (hs cTn) Assays
Larry Bernstein and Aviva Lev-Ari 5/18/2013
https://pharmaceuticalintelligence.com/2013/05/18/dealing-with-the-use-of-the-hs-ctn-assays/
Acute Chest Pain/ER Admission: Three Emerging Alternatives to Angiography and PCI – Corus CAD, hs cTn, CCTA
Aviva Lev-Ari 3/10/2013
- Redberg’s conclusions are correct for the initial screening. The issue has been whether to do further testing for low or intermediate risk patients.
- The most intriguing finding that is not at all surprising is that the CCTA added very little in the suspect group with small or moderate risk.
- The ultra sensitive troponin threw the ROC out the window
- The improved assay does pick up minor elevations of troponin in the absence of MI.
Critical Care | Abstract | Cardiac ischemia in patients with septic …
Aviva Lev-Ari 6/26/2013
https://pharmaceuticalintelligence.com/2013/06/26/critical-care-abstract-cardiac-ischemia-in-patients-with-septic/
- refer to: Cardiac ischemia in patients with septic shock randomized to vasopressin or norepinephrine
Mehta S, Granton J, Gordon AC, Cook DJ, et al.
Critical Care 2013, 17:R117 http://dx.doi.org/10.1186/cc12789
Troponin and CK levels, and rates of ischemic ECG changes were similar in the VP and NE groups. In multivariable analysis
- only APACHE II was associated with 28-day mortality (OR 1.07, 95% CI 1.01-1.14, p=0.033).
Assessing Cardiovascular Disease with Biomarkers
Larry H Bernstein, MD, FCAP 12/25/2012
https://pharmaceuticalintelligence.com/2012/12/25/assessing-cardiovascular-disease-with-biomarkers/
Vascular Medicine and Biology: CLASSIFICATION OF FAST ACTING THERAPY FOR PATIENTS AT HIGH RISK FOR MACROVASCULAR EVENTS Macrovascular Disease – Therapeutic Potential of cEPCs
Aviva Lev-Ari, PhD, RN 8/24/2012
PENDING Integration
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‘Ryanopathy’: causes and manifestations of RyR2 dysfunction in heart failureCardiovasc Res. 2013;98:240-247,
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Up-regulation of sarcoplasmic reticulum Ca2+ uptake leads to cardiac hypertrophy, contractile dysfunction and early mortality in mice deficient in CASQ2Cardiovasc Res. 2013;98:297-306,
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Myocardial Delivery of Stromal Cell-Derived Factor 1 in Patients With Ischemic Heart Disease: Safe and PromisingCirc. Res.. 2013;112:746-747,
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Circulation Research Thematic Synopsis: Cardiovascular GeneticsCirc. Res.. 2013;112:e34-e50,
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Gene and cytokine therapy for heart failure: molecular mechanisms in the improvement of cardiac functionAm. J. Physiol. Heart Circ. Physiol.. 2012;303:H501-H512,
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Ryanodine Receptor Phosphorylation and Heart Failure: Phasing Out S2808 and “Criminalizing” S2814Circ. Res.. 2012;110:1398-1402,
http://circres.ahajournals.org/content/110/5/777.figures-only
