Circulating tumor DNA methylation marker MYO1-G for diagnosis and monitoring of colorectal cancer.

BACKGROUND: Circulating tumor DNA (ctDNA) is a promising diagnostic and prognostic marker for many cancers and has been actively investigated in recent years. Previous studies have already demonstrated the potential use of ctDNA methylation markers in the diagnosis and prognostication of colorectal cancer (CRC). This retrospective study validated the value of methylation biomarker MYO1-G (cg10673833) in CRC diagnosis and disease monitoring using digital droplet PCR (ddPCR), a biomarker selected from our previous study due to its highest diagnostic efficiency. METHODS: Blood samples of CRC and control samples from tumor-free individuals at two institutions were collected to quantify the methylation ratio using ddPCR. Area under curve (AUC) was calculated after constructing receiver operating characteristic curve (ROC) for CRC diagnosis. Sensitivity and specificity were estimated and comparisons of methylation ratio in different groups were performed. RESULTS: We collected 673 blood samples from 272 patients diagnosed with stage I-IV CRC and 402 normal control samples. The methylation biomarker discriminated patients with CRC from normal controls with high accuracy (area under curve [AUC] = 0.94) and yielded a sensitivity of 84.3% and specificity of 94.5%. Besides, methylation ratio of MYO1-G was associated with tumor burden and treatment response. The methylation ratio was significantly lower in patients after their radical operation than when compared with those before surgeries (P < 0.001). Methylation ratio was significantly higher in patients with disease progression than those with stable disease (P = 0.002) and those with complete response or partial response (P = 0.009). CONCLUSIONS: Together, our study indicated that this methylation marker can serve as a potential biomarker for diagnosing and monitoring CRC.

Assessment of muscle fiber adaptation in footballers using a new ELISA assay of myosin isoforms.

BACKGROUND: To measure the impact of training models on injury incidence, data of health and performance were integrated to study fiber adaptation during a competitive season. We studied football players over a season, analyzing hours of exposure to sport by serum changes in fast and slow myosin, creatine kinase and lactate dehydrogenase. METHODS: A new assay was developed to measure the myosin isoforms in 49 non-sporting volunteers and in 27 professional football players. RESULTS: Myosin isoforms in volunteers with mean ages of 30±8 were 1553 µg/L fast and 1284 µg/L slow; in the group with of 56±7 were 1426 µg/L fast and 1046 µg/L slow. Slow myosin was significantly lower in older subjects (-18%). Samples from the players in preseason had lower mean scores for fast myosin (1123 µg/L) and higher for slow myosin (2072 µg/L) than reference volunteers. During the season, myosins reached the maximum with the maximum load (1537 µg/L fast, 2195 µg/L slow but decreased and adapted to the high level of demand (425 µg/L fast, 1342 µg/L slow). CK and LDH were maximal at the pre-season (227 U/L, 333 U/L) while myosin levels were maximal at the beginning of season (1537 µg/L, 2195 µg/L). CONCLUSIONS: Measuring serum myosin isoforms we identify the type and amount of damage caused by training and matches, making it a new control tool capable of advising training towards a minimum of blood slow myosin but controlling the fast fiber participating and be able to improve the performance of the players.

Statin treatment before stroke reduces pro-inflammatory cytokine levels after stroke.

OBJECTIVE: In this clinical case-control study, we investigated statin treatment in stroke patients on a range of inflammatory effectors in peripheral blood. We focus on RhoA GTPase and its downstream effectors as a future inflammatory target in stroke treatment. METHODS: Data from 10 patients already on statins at stroke onset (Pre-S group) was compared with data from both 29 patients starting statin treatment right after stroke onset (Post-S group) and with 8 healthy controls. In T-cells isolated from stroke patients, we analyzed the activity of the main cytoskeletal regulator RhoA GTPase and its downstream effectors: rho-associated protein kinase (ROCK), myosin phosphatase targeting protein subunit 1 (pMYPT1), myosin light chain kinase (pMLC) and cofilin. In the blood samples, we further determined levels of 12 key plasma cytokines as well as C-reactive protein (CRP) and kallikrein. RESULTS: Compared to healthy controls, the Post-S group achieved significantly higher RhoA and ROCK activities, while the Pre-S did not differ from controls. Levels of pMYPT1, pMLC and cofilin did not differ from controls in the Pre-S and Post-S groups. At day 90 after stroke, interferon γ and IL-18 were significantly increased in the Post-S group compared to the Pre-S group. We found a positive correlation between CRP and NIHSS, whereas kallikrein levels showed no correlation with NIHSS at any of the days. CONCLUSION: Stroke induces changes in the RhoA-ROCK pathway in T-cells. CRP and NIHSS score correlated positively in the study. Statins may have an anti-inflammatory effect as statin treatment before stroke reduces post-stroke pro-inflammatory levels. RhoA GTPase and its downstream effectors are possibly the key to improve statin treatment in stroke.

Circular RNA circMYO9B facilitates breast cancer cell proliferation and invasiveness via upregulating FOXP4 expression by sponging miR-4316.

Recently, circular RNAs (circRNAs) have been demonstrated as essential regulators in human cancers. However, the function and mechanism of circRNAs in breast cancer (BC) remain largely unknown and require to be investigated. In the present study, we found that circMYO9B was highly expressed in BC tissues by bioinformatics analysis. And we showed that circMYO9B expression was positively correlated with patients' prognosis. Moreover, we found that circMYO9B knockdown significantly suppressed the proliferation, migration and invasion of BC cells in vitro. In vivo assays also indicated that circMYO9B silence delayed tumor growth. In mechanism, we found that circMYO9B promoted the expression of FOXP4 by sponging miR-4316 in BC cells. We showed that the expression of miR-4316 was inversely associated with that of circMYO9B or FOXP4 in BC tissues. Finally, we found that restoration of FOXP4 expression significantly reversed the effects of circMYO9B knockdown on BC cell proliferation, migration and invasion. In conclusion, our findings demonstrated a key role of circMYO9B/miR-4316/FOXP4 axis in regulating BC progression.

Effects of a Terrified-Sound Stress on Serum Proteomic Profiling in Mice.

The serum proteomic profiles of mice exposed to terrified-sound-induced stress and after stress release were investigated. Serum samples from 32 mice were divided into four groups (n = 8 each) and analyzed using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry techniques (MALDI-TOF MS) combined with magnetic bead-based weak cation-exchange chromatography. ClinProTools software identified several distinct markers that differed between the stressed and control groups and between the stress released and stressed released controls. Of 33 m/z peaks that differed among the four groups, 17 were significantly different (P < 0.05). Five peaks (m/z: 2793.37, 2924.86, 1979.90, 3492.49, 3880.24) showed significant differences in expression after exposure to terrified-sound stress and returned to control levels after stress release. These were sequence identified as peptide regions of dimethylaniline monooxygenase, myosin-9, uncharacterized protein in Rattus norvegicus, apolipoprotein C-I, and plasma serine protease inhibitor (Serpina 5). Our study provides the first evidence of significant changes in serum proteomic profiles in mice exposed to terrified-sound stress, which suggests that protein expression profiles are affected by the stress. Normal expression levels were restored after stress release, suggesting the activation of self-adjustment mechanisms for the recovery of protein expression levels altered by this stress.

Detection of antibodies to self-antigens (K-alpha 1 tubulin, collagen I, II, IV, and V, myosin, and vimentin) by enzyme-linked immunosorbent assay (ELISA).

The enzyme-linked immunosorbent assay (ELISA) is a widely used technique for detecting antibodies (Abs) and is employed in clinical laboratories to identify Abs against various self-antigens-autoAb development and quantitation. This method relies on specific antigen-Ab interactions where one of the components is immobilized on a solid surface. Using this method, the concentrations of antigens or Ab present in the serum can be quantified with high specificity and accuracy. Here, we describe the detection of autoAbs to various self-antigens with different tissue restriction patterns which includes collagens, k-α1 tubulin, vimentin, and myosin. We also discuss their relevance in monitoring for rejection following solid organ transplantation.

The clinical and diagnostic significance of anti-myosin autoantibodies in cardiac disease.

Autoimmunity is influenced by genetic, immune, hormonal, and environmental factors. Viral infections may trigger autoimmunity. It has been established that autoimmunity may be a contributing factor in the pathogenesis of heart disease. Anti-heart autoantibodies have been identified in the sera of patients with heart diseases, as well as in low titers in certain healthy individuals. Nevertheless, the role of humoral immunity in the development of autoimmune heart disease has not been fully established. Anti-myosin autoantibodies appear in several heart diseases such as myocarditis, dilated cardiomyopathy, Chagas' heart disease, Kawasaki disease, rheumatic fever, and ischemic myocardium. The pathogenic role of anti-myosin autoantibodies in heart disease is not fully understood. Moreover, little is known concerning the clinical implications of anti-myosin autoantibodies in heart disease and its prognostic significance. Anti-cardiac myosin autoantibodies were found to cross-react with the ß-adrenergic receptor. Studies have reported the effective use of the anti-myosin directed immune-modulating approach in animals with heart disease, although no specific anti-myosin autoantibody therapeutic approach has been attempted in humans. Herein, we review the current knowledge of anti-myosin autoantibodies and the use of targeted immune-modulating therapy in different heart diseases.

Correlation of angiotensin converting enzyme gene polymorphism with perioperative myocardial protection under extracorporeal circulation.

OBJECTIVE: To observe the expression of angiotensin converting enzyme (ACE), angiotensin II (Ang II), cardiac troponin (cTn I), creatine kinase isozymes (CK-MB) and muscle red protein(Myo) after cardiopulmonary bypass (CPB), and to investigate the association of polymorphisms in angiotensin converting enzyme genes and myocardial injury. METHODS: Sixty-three patients suffered from rheumatic mitral stenosis and scheduled for mitral valve replacement with CPB, were randomly divided into three groups according polymorphisms in angiotensin converting enzyme genes: type II, type ID, type DD (each=21). Blood samples were withdrawn from artery before operation (T1), at the beginning of CPB (T2), 30 min after CPB (T3), (T4) at the end of CPB (T5), 2 h after CPB (T6), 6 h after CPB (T7) to measure the expression of ACE, Ang II, cTn I, CK-MB, Myo. RESULTS: The level of ACE during and after CPB were significantly higher than those before CPB (P<0.05). As extension of CPB time, the expression of ACE was increased. The level of cTn I, CK-MB, Myo after CPB were significantly higher than those before CPB(P<0.05). The level of cTn I, CK-MB and Myo were highest at T7, T6 and T5 and T7, respectively. The level of ACE, Ang II cTn I in patients with DD genotype was significantly higher than the ID and II genotype (P< 0.05). Besides, the level of ACE, Ang II in patients with ID genotype was significantly higher than the II (P< 0.05). CONCLUSIONS: There is certain correlation between CPB perioperative midterm ACE and cTn I, Myo, CK-MB. ACE DD genotype is a susceptibility gene of the CPB perioperative myocardial injury.

Fast and slow myosins as markers of muscle injury.

OBJECTIVE: The diagnosis of muscular lesions suffered by athletes is usually made by clinical criteria combined with imaging of the lesion (ultrasonography and/or magnetic resonance) and blood tests to detect the presence of non-specific muscle markers. This study was undertaken to evaluate injury to fast and slow-twitch fibres using specific muscle markers for these fibres. METHODS: Blood samples were obtained from 51 non-sports people and 38 sportsmen with skeletal muscle injury. Western blood analysis was performed to determine fast and slow myosin and creatine kinase (CK) levels. Skeletal muscle damage was diagnosed by physical examination, ultrasonography and magnetic resonance and biochemical markers. RESULTS: The imaging tests were found to be excellent for detecting and confirming grade II and III lesions. However, grade I lesions were often unconfirmed by these techniques. Grade I lesions have higher levels of fast myosin than slow myosin with a very small increase in CK levels. Grade II and III lesions have high values of both fast and slow myosin. CONCLUSIONS: The evaluation of fast and slow myosin in the blood 48 h after the lesion occurs is a useful aid for the detection of type I lesions in particular, since fast myosin is an exclusive skeletal muscle marker. The correct diagnosis of grade I lesions can prevent progression of the injury in athletes undergoing continual training sessions and competitions, thus aiding sports physicians in their decision making.

The MYO9B gene is a strong risk factor for developing refractory celiac disease.

BACKGROUND & AIMS: Celiac disease (CD) is associated with HLA-DQ2 and HLA-DQ8 and has been linked to genetic variants in the MYO9B gene on chromosome 19. HLA-DQ2 homozygosity is associated with complications of CD such as refractory celiac disease type II (RCD II) and enteropathy-associated T-cell lymphoma (EATL). We investigated whether MYO9B also predisposes to RCD II and EATL. METHODS: Genotyping of MYO9B and molecular HLA-DQ2 typing were performed on 62 RCD II and EATL patients, 421 uncomplicated CD patients, and 1624 controls. RESULTS: One single nucleotide polymorphism in MYO9B showed a significantly different allele distribution in RCD II and EATL patients compared with controls (P = .00002). The rs7259292 T allele was significantly more frequent in RCD II and EATL patients compared with CD patients (P = .0003; odds ratio [OR], 3.61; 95% confidence interval [CI], 1.78-7.31). The frequency of the haplotype carrying the T allele of this single nucleotide polymorphism was significantly increased in RCD II and EATL patients (11%), compared with controls (2%) and CD patients (3%) (OR, 6.76; 95% CI, 3.40-13.46; P = 2.27E-09 and OR, 4.22; 95% CI, 1.95-9.11; P = .0001, respectively). Both MYO9B rs7259292 and HLA-DQ2 homozygosity increase the risk for RCD II and EATL to a similar extent when compared with uncomplicated CD patients (OR, 4.3; 95% CI, 1.9-9.8 and OR, 5.4; 95% CI, 3.0-9.6, respectively), but there was no evidence for any interaction between these 2 risk factors. CONCLUSIONS: We show that both MYO9B and HLA-DQ2 homozygosity might be involved in the prognosis of CD and the chance of developing RCD II and EATL.

[Enzymatic activity of some tissues and blood serum from animals and humans exposed to microwaves and hypothesis on the possible role of free radical processes in the nonlinear effects and modification of emotional behavior of animals]. / Fermentnaia aktivnost' nekotorykh tkanei i syvorotki krovi zhivotnykh i cheloveka pri vozdeistvii mikrovoln i gipoteza o vozmozhnoi roli svobodnoradikal'nykh protesssov v nelineinykh éffektakh i modifikatsii émotsional'nogo povedeniia zhivotnykh.

The dependence of activities of actomyosin ATPase, alkaline phosphatase, aspartataminotranspherase, monoaminoxidase and that of affective rat behavior on frequency of modulation of microwaves (0.8-10 microW/cm2) was explored at short-time actions. Series of nonlinear phenomenons, inexplicable from positions of the energy approaches are revealed, The working hypothesis explaining opportunity of high performance of weak and super-weak microwaves and other revealed phenomena by resonance interaction of such electromagnetic radiofrequency radiation with paramagnetic molecules of biological tissues was proposed. This resonance interaction activate free radicals and initiate auto-supporting and auto-intensifying of chain chemical reactions. The spontaneous autocatalytic oxidation of catecholamines enlarges a common pool of free radicals, capable to participate in such enhanced generating. The protective role of monoaminoxidase is postulated. Monoaminoxidase is basically located on an outer surface of mitochondrias and it is deaminating monoamines. The deaminating prevents penetration of catecholamines inside of mitochondrias and their quinoid oxidation there with formation of free-radical semi-quinons, capable to destroy system of ATP synthesis. These inferences are obliquely confirmed by the experimentally revealed correlation between activity of monoaminoxidase and integrative activity of the rat brain.

Diagnostic implications of elevated levels of smooth-muscle myosin heavy-chain protein in acute aortic dissection. The smooth muscle myosin heavy chain study.

BACKGROUND: A rapid 30-minute assay of circulating smooth-muscle myosin heavy-chain protein has been developed as a biochemical diagnostic tool for aortic dissection. OBJECTIVE: To determine the sensitivity and specificity of this assay. DESIGN: Cross-sectional study. SETTING: 8 major cardiovascular centers in Japan. PATIENTS: 95 patients with acute aortic dissection, 48 patients with acute myocardial infarction, and 131 healthy volunteers. MEASUREMENTS: Levels of circulating smooth-muscle myosin heavy-chain protein. RESULTS: Patients with acute aortic dissection who presented within 3 hours after onset had elevated levels of circulating smooth-muscle myosin heavy-chain protein. In these patients, the assay had a sensitivity of 90.9%, a specificity of 98% compared with healthy volunteers, and a specificity of 83% compared with patients who had acute myocardial infarction; the clinical decision limit was 2.5 microgram/L. All patients with proximal lesions had elevated levels of smooth-muscle myosin heavy-chain protein, and only patients with distal lesions had decreased levels (<2.5 microgram/L). CONCLUSIONS: Levels of smooth-muscle myosin heavy-chain protein can be used to diagnose aortic dissection soon after symptom onset. The assay had the greatest diagnostic value in patients with proximal lesions.

Effect of upper gastrointestinal endoscopy on circulation in the elderly.

BACKGROUND: Upper gastrointestinal endoscopy in the elderly is increasingly becoming more common, despite the possibility that a minimal load on the circulation can cause serious complications such as shock and cardiac arrest. OBJECTIVE: The effects of endoscopy on the heart and the possibility of predicting circulatory accidents were studied using natriuretic peptide levels. METHODS: The patients were randomly chosen according to their age and divided into an elderly group (over 60 years of age, 64 patients) and a young group (under 30 years of age, 20 patients). The patients in the elderly group were further subdivided into two groups based on the presence or absence of circulatory complications (46 patients with circulatory complications and 18 without complications). The load on the heart was evaluated by measuring human atrial natriuretic peptide (hANP) and human brain natriuretic peptide (hBNP) which are secreted by the myocardial cells in response to cardiac load. Specimens were obtained before and after endoscopy. RESULTS: The hANP level was significantly higher after endoscopy in the elderly group, regardless of the presence or absence of circulatory complications. No significant difference was observed in the hBNP level. No significant increase in hANP or hBNP levels was observed after endoscopy in the young group. CONCLUSIONS: These observations suggest an increased atrial load during endoscopy in the elderly. The increase in pulse rate during endoscopy is one possible cause of atrial load. Therefore, the risk of circulatory system damage must be recognized when endoscopy is performed in the elderly. The measurement of plasma hANP and hBNP levels may provide effective indices for evaluating cardiac load during endoscopy.

Agonist-induced regulation of myosin phosphatase activity in human platelets through activation of Rho-kinase.

Human platelets contained about 15 times lower amounts of Rho-kinase than Ca2+/calmodulin-dependent myosin light chain (MLC) kinase. Anti-myosin-binding subunit (MBS) antibody coimmunoprecipitated Rho-kinase of human platelets, and addition of GTPgammaS-RhoA stimulated phosphorylation of the 130-kD MBS of myosin phosphatase and consequently inactivated myosin phosphatase. Two kinds of selective Rho-kinase inhibitors, HA1077 and Y-27632, reduced both GTPgammaS-RhoA-dependent MBS phosphorylation and inactivation of the phosphatase activity. Activation of human platelets with thrombin, a stable thromboxane A2 analog STA2, epinephrine, and serotonin resulted in an increase in MBS phosphorylation, and the agonist-induced MBS phosphorylation was prevented by pretreatment with the respective receptor antagonist. HA1077 and Y-27632 inhibited MBS phosphorylation in platelets stimulated with these agonists. These compounds also blocked agonist-induced inactivation of myosin phosphatase in intact platelets. In addition, HA1077 and Y-27632 inhibited 20-kD MLC phosphorylation at Ser19 and ATP secretion of platelets stimulated with STA2, thrombin (0.05 U/mL), and simultaneous addition of serotonin and epinephrine, whereas these compounds did not affect MLC phosphorylation or ATP secretion when platelets were stimulated with more than 0.1 U/mL thrombin. Thus, activation of Rho-kinase and the resultant phosphorylation of MBS is likely to be the common pathway for platelet activation induced by various agonists. These results also suggest that Rho-kinase-mediated MLC phosphorylation contributes to a greater extent to the platelet secretion induced by relatively weak agonists.

Calculation of a Gap restoration in the membrane skeleton of the red blood cell: possible role for myosin II in local repair.

Human red blood cells contain all of the elements involved in the formation of nonmuscle actomyosin II complexes (V. M. Fowler. 1986. J. Cell. Biochem. 31:1-9; 1996. Curr. Opin. Cell Biol. 8:86-96). No clear function has yet been attributed to these complexes. Using a mathematical model for the structure of the red blood cell spectrin skeleton (M. J. Saxton. 1992. J. Theor. Biol. 155:517-536), we have explored a possible role for myosin II bipolar minifilaments in the restoration of the membrane skeleton, which may be locally damaged by major mechanical or chemical stress. We propose that the establishment of stable links between distant antiparallel actin protofilaments after a local myosin II activation may initiate the repair of the disrupted area. We show that it is possible to define conditions in which the calculated number of myosin II minifilaments bound to actin protofilaments is consistent with the estimated number of myosin II minifilaments present in the red blood cells. A clear restoration effect can be observed when more than 50% of the spectrin polymers of a defined area are disrupted. It corresponds to a significant increase in the spectrin density in the protein free region of the membrane. This may be involved in a more complex repair process of the red blood cell membrane, which includes the vesiculation of the bilayer and the compaction of the disassembled spectrin network.

Dual image of myosin and actin in human neutrophils during movement by fluorescence microscopy.

It is well known that myosin and actin play important roles as a motile apparatus in cells, and as a cytoskeleton of cell structure in non-muscle cells. The purpose of this study was simultaneously to demonstrate the intracellular dynamics of myosin and actin in human neutrophils during movement. We designed a double-fluorescence labelling procedure utilising fluorescein isothiocyanate and rhodamine. Myosin was labelled with the green-fluorescence, F-actin was labelled with the red-fluorescence and the coexisting myosin and F-actin were labelled with yellow-fluorescence. We obtained the dual image of myosin and F-actin in human neutrophils during movement by this procedure through a conventional fluorescence microscope. Dual image can interpret the relationship between myosin and actin in human neutrophils during movement, however, further studies are required to elucidate the contractile mechanism in such cells.