It takes acid, rather than ice, to freeze glucose.

Plasma glucose levels provide the cornerstone of diabetes evaluation. Unfortunately, glucose levels drop in vitro due to glycolysis. Guidelines provide suitable conditions which minimize glycolysis, such as immediate centrifugation or the use of ice/water slurry storage containers. For obvious practical reasons, most laboratories use blood collection tubes containing glycolysis inhibitors. We describe the effect of a variety of commonly used blood collection tubes on in vitro stability of glucose. Furthermore, we looked at the validity of the assumption that glycolytic activity is minimal when blood is kept in an ice/water slurry. Sodium fluoride alone does not reduce in vitro glycolysis in the first 120 minutes after phlebotomy. Addition of citrate almost completely prevented in vitro glycolysis, but showed a positive bias (0.2 mmol/l) compared to control. This is partly due to a small drop in glucose level in control blood, drawn according to the current guidelines. This drop occurs within 15 minutes, in which glycolysis has been described to be minimal and acceptable. NaF-EDTA-citrate based test tubes provide the best pre-analytical condition available. Furthermore, glucose levels are not stable in heparinized blood placed in an ice/water slurry. We strongly advise the use of NaF-EDTA-citrate based test tubes in diabetes research.

Influence of short-term interruption of antithyroid drugs on the outcome of radioiodine therapy of Graves' disease: results of a prospective study.

AIM: The factors influencing success of treating Graves' disease with radioiodine ( (131)I) are discussed controversially. This study analyses prospectively the influence of discontinuing antithyroid drugs (ATD) immediately prior to treatment with radioiodine on the therapeutic outcome. METHODS: We studied 141 patients with Graves' disease. In 73 of them (group A) treatment was performed under medication with ATD, in 68 patients (group B) ATD were discontinued for 3 - 7 days starting at the time of therapy. We performed a statistical analysis of the influence of ATD and other factors potentially influencing treatment results. RESULTS: In group A 49/73 patients were treated successfully (67 %) vs. 58/68 (85 %) in group B (p < 0.01). Characteristic changes in the kinetics of radioiodine were observed: after discontinuing ATD specific uptake was higher (2.0 %/ml in group A vs. 2.6 %/ml in group B, p = 0.004), and the effective half life was longer (5.1 +/- 1.3 d in group A vs. 5.5 +/- 1.1 d in group B, p = 0.076) resulting in a significantly higher radiation dose in group B (200 +/- 61 Gy in group A vs. 236 +/- 72 Gy in group B, p = 0.002). CONCLUSION: We conclude that short-term interruption of ATD improves the success rate of treating Graves' disease with radioiodine significantly.

Mechanism of thyroid-hormone regulated expression of the SERCA genes in skeletal muscle: implications for thermogenesis.

Thyroid hormone increases the Ca2+-ATPase activity of the sarcoplasmic reticulum (SR) in skeletal muscle, thereby increasing the energy-turnover associated with Ca2+-cycling during contraction and rest. The fast-muscle isoform of the Ca2+-ATPase (SERCA1) and the slow-muscle isoform (SERCA2a), are encoded by two genes that are transcriptionally regulated by T3. The SERCA1 isoform can be expressed to considerably higher levels than the SERCA2a isoform. The stimulation of transcription of the SERCA1 gene by T3 is mediated by two thyroid hormone response elements, located in the promoter of this gene. The intracellular [Ca2+] can modulate the effect of T3. The increase in SR Ca2+-ATPase activity seen when T3-levels rise above normal, results from the induction of SERCA1 expression in slow muscle fibers. Concomitant high levels of Ca2+-ATPase activity are associated with down-regulation of SERCA2a expression in these fibers. The observed T3-dependent increase in SERCAI expression and associated Ca2+ATPase activity will increase the overall metabolic rate of the organism significantly under normal conditions, because of the high average level of contractile activity of slow fibers. Given the rise in serum T3-levels during prolonged cold exposure, these data suggest that fiber-specific stimulation of SERCA1 expression contributes to the thermogenic response in non-shivering thermogenesis. This mechanism may be particularly relevant in larger mammals, which have a relatively high percentage of slow fibers in skeletal muscle, and which need to rely on tissues other than brown fat for the generation of extra heat.

Follow-up in neuroblastoma: comparison of metaiodobenzylguanidine and a chimeric anti-GD2 antibody for detection of tumor relapse and therapy response.

Early and correct diagnosis of local tumor recurrence, occurrence of metastases, and therapy response are essential in patients with neuroblastoma stage IV. The aim of the study was to evaluate the diagnostic value of metaiodobenzylguanidine (mIBG) and a chimeric GD2 antibody in the follow-up of patients with neuroblastoma. In a prospective study, mIBG (N = 31 scans) and immunoscintigraphy were compared with a chimeric antiganglioside antibody, ch14.18 (MAb) (N = 31 scans), labeled with technetium Tc 99m in the follow-up of 18 patients with stage IV neuroblastoma. The findings were compared with histologic findings, other imaging examinations, and clinical changes over the course of 4 to 6 years. For the diagnosis of local tumor recurrences, sensitivity was 80% for MAb and 70% for mIBG. Specificity was 93% for MAb and 72% for mIBG. The MAb was superior for the detection of skeletal metastases, with a sensitivity of 82% compared with 72% for mIBG. Specificity was 100% for both techniques. Also, for soft tissue/lymph node metastases, sensitivity for MAb was higher (50%) than for mIBG (31%). Specificity was 100% for each technique. In sequential studies, metastases were detected earlier with MAb (mean: 2.3 m for skeletal metastases, 3.6 m for soft tissue metastases) than with mIBG. After therapy, tumor uptake was visualized longer with mIBG (mean 6.3 m) than with MAb. The chimeric antibody ch14.18 is likely to be valuable for follow-up examinations and for assessment of therapy response because of earlier detection of new metastases.

Eating and body image concerns among obese and average-weight children.

Research compared obese and average-weight children with regard to concerns about being or becoming overweight, history of dieting, concerns about the effects of eating food, and perceived discrepancy between real and ideal body image. Participants included 526 obese and average-weight elementary-age school children to whom questionnaires were administered. Gender (male/female), obesity status (obese/average-weight), and grade level (lower elementary/upper elementary) were considered. Obese children were significantly more likely to engage in dieting behaviors, to express concern about their weight, to restrain their eating, and to exhibit more dissatisfaction with their body image than average-weight children. Girls were more likely to exhibit these behaviors than were boys. These findings suggest the importance of studying the emergence of disordered eating habits in childhood.

Fear of intimacy among dating couples.

Fear of intimacy among heterosexual dating couples was examined with the Fear-of-Intimacy Scale (FIS) and the Personal Assessment of Intimacy in Relationships (PAIR). Following a 6-month interval, couples were again contacted to determine whether they continued to date. Males reported higher FIS scores than females, and FIS scores were positively correlated within couples. Also, FIS scores of males and females were significantly correlated with indices of actual and desired intimacy; however, for females, correlations of FIS scores with desired intimacy were significantly lower than correlations with actual intimacy. Females who were no longer in the dating relationship at the 6-month follow-up had higher FIS scores than those who were continuing in their dating relationship. These findings increase our understanding of fear of intimacy, especially gender differences in dating couples.

Examination of regional lymph nodes by sentinel node biopsy and molecular analysis provides new staging facilities in primary cutaneous melanoma.

Histopathologic parameters of the primary tumor, such as Breslow's tumor thickness and Clark's level of invasion are the current basis for prognostic classifications of primary cutaneous melanoma. Once patients develop regional node metastasis, histopathologic features of the primary melanoma no longer contribute significantly to survival prediction. In this tumor stage, the extent of lymph node involvement is the main prognostic factor. This study addresses the question whether application of a highly sensitive molecular biology assay for detection of submicroscopic melanoma cells in sentinel lymph nodes may be suitable to improve melanoma staging. One hundred and sixteen patients with primary cutaneous melanoma with a total of 214 sentinel lymph nodes were enrolled. Sentinel lymph nodes were analyzed by histopathology including immunohistochemistry and by reverse transcription-polymerase chain reaction for tyrosinase. Patients were examined for tumor recurrences during a follow-up period of 19 mo (median). Disease-free survival probabilities were calculated and independent prognostic factors were determined by multivariate analysis. Using histopathology, micrometastatic nodal involvement was detected in 15 patients (13%). Of the 101 patients with histopathologically negative sentinel lymph nodes, 36 were reclassified by positive tyrosinase reverse transcription-polymerase chain reaction and 65 patients were still negative by reverse transcription-polymerase chain reaction. Recurrences were observed in 23 (20%) of 116 patients. These tumor recurrences were demonstrated in 10 patients (67%) with histopathologically positive sentinel lymph nodes, in nine patients (25%) with submicroscopic tumor cells detected by reverse transcription-polymerase chain reaction, and in four patients (6%) negative by both methods. The differences in recurrence rates were statistically significant (p = 0.01). In a multivariate analysis, histopathologic and reverse transcription-polymerase chain reaction status of the sentinel lymph node were demonstrated to be the only significant prognostic factors for predicting disease-free survival. Tyrosinase reverse transcription-polymerase chain reaction for the detection of minimal residual melanoma in sentinel lymph nodes is a powerful tool to determine patients who are at increased risk for subsequent metastasis. Moreover, a group of patients with high tumor thickness was identified by negative reverse transcription-polymerase chain reaction to be at low risk for recurrent disease. These data may have an impact on future tumor classifications of primary cutaneous melanoma.

Predictors of body image dissatisfaction in elementary-age school girls.

Concerns about weight, shape, body image, dieting, and eating are evident among elementary-age school girls [Int. J. Eating Disord. 10 (1991) 199; J. Adolesc. Health 12 (1991) 307; Pediatrics 84 (1989) 482; J. Clin. Psychol. 21 (1992) 41; Addict. Behav. (2000).]. Body image dissatisfaction, found to predict the formation of eating disturbances in longitudinal studies [Eating Disord.: J. Treat. Prev. 2 (1994) 114; Int. J. Eating Disord. 18 (1995) 221.], is likely a necessary precursor of eating disturbance development [J. Abnorm. Psychol. 102 (1993) 438.]. Turning back the etiological clock, this study examines the association between body image dissatisfaction and four potential areas of influence, including peer relationships, family characteristics, personality features, and body mass index, among girls in grades three, four, and five. Each conceptual area significantly contributed to the prediction of body image dissatisfaction. The combination of statistically significant variables, including teasing, peer modeling of weight and shape concerns, daughters' perceptions of parental influence to lose or control weight, appearance comparison, sensitivity, and body mass index, accounted for 50% of the variance in general body image dissatisfaction and 45% in weight and shape dissatisfaction. Implications for prevention, treatment, and future research are discussed.

Detection of melanoma micrometastasis in sentinel nodes by reverse transcription-polymerase chain reaction correlates with tumor thickness and is predictive of micrometastatic disease in the lymph node basin.

The sentinel node has been reported to be representative for the presence or absence of metastatic melanoma in the draining lymph node basin. In this study, for the first time sentinel nodes and adjoining nonsentinel nodes were analyzed for micrometastatic disease using tyrosinase reverse transcription-polymerase chain reaction (RT-PCR) in comparison with standard immunohistochemistry. Successful identification of the sentinel nodes using a gamma probe-guided surgery was achieved in 73 (92%) of 79 patients with cutaneous stage I and II melanoma (tumor thickness > or =0.75 mm). A total of 794 regional lymph nodes, 148 sentinel nodes, and 646 adjoining nonsentinel nodes were evaluated. Tyrosinase RT-PCR was shown to increase the sensitivity for melanoma cell detection in sentinel nodes significantly (49% positivity) as compared with immunohistochemistry using antibodies against HMB-45 antigen and S-100 protein (18% positivity). Examination of sentinel nodes was highly predictive in determining the presence of regional lymph node micrometastasis by immunohistochemistry (99%) and RT-PCR (89%). Interestingly, detection of nodal micrometastasis by RT-PCR showed a strong positive correlation with tumor thickness of primary cutaneous melanoma. These results suggest the clinical significance and emphasize the importance of tyrosinase RT-PCR for detection of melanoma micrometastasis in sentinel nodes.

Correlation of imaging techniques to histopathology in patients with diabetic foot syndrome and clinical suspicion of chronic osteomyelitis. The role of high-resolution ultrasound.

OBJECTIVE: To investigate the role of ultrasound in the diagnosis of osteomyelitis in the diabetic foot compared with magnetic resonance imaging (MRI), bone scintigraphy (BS), and plain film radiography (PFR). RESEARCH DESIGN AND METHODS: We investigated 19 consecutive diabetic patients (2 women, 17 men, age 60.7 +/- 9.8 years, BMI 27.0 +/- 3.8 kg/m2) with clinical suspicion of bone infection of the foot. A high-resolution ultrasound system (Esaote/Biosound, Munich) with a linear array transducer up to 13.0 MHz was used. The prospective and blinded results of each method were compared with histopathology as the reference method after metatarsal resection. RESULTS: In 14 of 19 patients, histopathology confirmed osteomyelitis. Ultrasound showed a sensitivity of 79% (PFR, 69%; BS, 83%; MRI, 100%), a specificity of 80% (PFR, 80%; BS, 75%; MRI, 75%), a positive predictive value of 92% (PFR, 90%; BS, 91%; MRI, 93%), and a negative predictive value of 57% (PFR, 50%; BS, 60%; MRI, 100%). CONCLUSIONS: Our data indicate that ultrasound might have a better diagnostic power for detecting chronic osteomyelitis in the diabetic foot than PFR and has similar sensitivity and specificity as BS. MRI is superior to the other three methods. We conclude that the use of ultrasound in the management of the diabetic foot is worthy of further investigation.

Lymph node micrometastases of cutaneous melanoma: increased sensitivity of molecular diagnosis in comparison to immunohistochemistry.

The presence of regional lymph node metastases is one of the most significant prognostic factors for predicting survival in patients with clinical stage I or II cutaneous melanoma. For accurate staging of the primary tumor a sensitive technique is required to detect occult nodal micrometastases. This prospective diagnostic study was designed to evaluate the incidence of nodal micrometastases using nested reverse transcription-polymerase chain reaction (RT-PCR) for tyrosinase in comparison to immunohistochemical examination. Furthermore, the incidence of melanoma micrometastases detected by RT-PCR was analysed in correlation to major prognostic factors. A total of 466 regional lymph nodes from 79 patients with primary cutaneous melanoma (tumor thickness > 0.75 mm) were investigated. In 49 lymph nodes from 31 patients immunohistochemistry demonstrated melanoma metastases. Using tyrosinase RT-PCR, nodal micrometastases were detected in 136 lymph nodes from 52 patients including all lymph nodes positive by immunohistochemical examination. Out of the 417 lymph nodes negative by immunohistochemistry, 87 nodes (21%) were identified to express tyrosinase by the RT-PCR technique. Among the 48 patients negative by immunohistochemical assessment, 21 (44%) had nodal micrometastases (n = 40) using RT-PCR. All 68 lymph nodes from 46 non-melanoma patients serving as negative controls for tyrosinase RT-PCR were negative. The detection of melanocytic nodal micrometastases by tyrosinase RT-PCR is a highly specific method with a sensitivity significantly higher than that achieved by immunohistochemistry (p < 0.0001). Patients with nodal micrometastases identified exclusively by RT-PCR had significantly higher tumor thickness as compared to patients with negative results by RT-PCR (p < 0.01).

[Osteitis diagnosis in a group of angiology patients: a comparison of X-ray and nuclear medicine studies]. / Osteitisdiagnostik in einem angiologischen Patientengut: Vergleich von röntgen- und nuklearmedizinischen Untersuchungen.

PURPOSE: Of the study was to examine the ranking of radiographs and bone scans in the diagnosis of osteitis. PATIENTS AND METHODS: 115 patients with arterial occlusive disease in stage Fontaine i.v. were referred for examination of the peripheral skeletal parts of the lower extremity. 126 radiographs and bone scans were taken. The time between the two examinations was 10 days maximum to enable direct comparison. Leukocyte scintigraphy was employed as gold standard. RESULTS: The sensitivity of radiographs was 47% with a specificity of 91%. Sensitivity and specificity of bone scans was 78% for both values. The positive predictive values for radiographs and bone scans were 88% and 85%, the negative predictive values were between 80% and 97%. CONCLUSION: In the majority of cases osteitis can be diagnosed with relatively inexpensive methods such as conventional x-rays and bone scans. Only in unclear cases further examinations such as leukocyte scintigraphy or magnetic resonance imaging should supplement the diagnosis.

Bone marrow scintigraphy with technetium-99m anti-NCA-95 to monitor therapy in malignant osteopetrosis.

We report a case of a 2-mo-old girl with malignant osteopetrosis. Conventional radiological investigations of the skull and left hand showed the characteristic pattern of generalized sclerosis. Bone marrow immunoscintigraphy with 99mTc-labeled antibodies against nonspecific cross-reactive antigen (NCA) 95 was performed before and after bone marrow transplantation. Before transplantation, whole-body images showed bone marrow stores exclusively in the base of the skull. The rest of the skeleton did not reveal any hematopoietic activity. The liver and spleen showed increased antibody uptake as expected in extramedullary hematopoiesis. Repeat scintigraphy after bone marrow transplantation from her haploidentical father demonstrated an almost completely normalized tracer distribution corresponding to her clinical and hematological improvement. Bone marrow immunoscintigraphy appears to be an ideal complement to radiograph diagnostics in malignant osteopetrosis. In primary diagnosis, scintigraphy demonstrates the quantitative extent of bone marrow displacement. It also proves an ideal tool in monitoring the effectiveness of therapy after bone marrow transplantation.

Fear of intimacy and attachment among rape survivors.

The purpose of the present study was to investigate attachment and fear of intimacy among rape survivors. As previous research has documented that several domains of functioning are affected by the experience of rape, it was hypothesized that the survivors may have difficulties with attachment and intimacy. Subjects were selected from an undergraduate general psychology class on the basis of responses to a questionnaire on sexual experiences. All of the subjects were female and included 44 rape survivors and 57 controls. As predicted, rape survivors reported greater fear surrounding intimacy. Survivors also differed from the controls on all of the attachment dimensions. They reported less confidence in others' dependability, less comfort with closeness, and more fear of abandonment. Trait anxiety was also controlled in the analyses, and except for fear of abandonment, it attenuated the differences between the groups such that they were no longer significantly different.

Cross-talk between transcriptional regulation by thyroid hormone and myogenin: new aspects of the Ca2+-dependent expression of the fast-type sarcoplasmic reticulum Ca2+-ATPase.

We have previously demonstrated an interaction between the major determinants of skeletal muscle phenotype by showing that continuous contractile activity represses the thyroid hormone (3,3', 5-tri-iodothyronine; T3)-dependent transcriptional activity of fast-type sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase (SERCA1), a characteristic of the fast phenotype. Both the free cytosolic Ca2+ concentration ([Ca2+]i) and the myogenic determination factors MyoD and myogenin have been implicated as mediators of the effect of contractile activity on skeletal muscle phenotype. Using L6 cells we have shown that an increase in the steady-state [Ca2+]i above the resting level of 120 nM indeed can mimic the effect of contractile activity on T3-dependent SERCA1 expression. We now show that the repressing effect of increased [Ca2+]i on T3-dependent SERCA1 expression in L6 cells is exerted at a pre-translational level and is accompanied by increased myogenin mRNA expression. Myogenin overexpression in these cells revealed that increased expression of myogenin alone strongly decreases the T3-dependent stimulation of SERCA1 promoter activity. These results suggest a pathway for the regulation of skeletal muscle phenotype in which [Ca2+]i mediates the effect of contractile activity by regulating the expression of myogenin, which in turn interferes with transcriptional regulation by T3.

Attitudes toward bulimic behaviors in two generations: the role of knowledge, body mass, gender, and bulimic symptomatology.

The perceived commonality and acceptability of symptoms of bulimia, as well as associated variables including gender, generation, knowledge of bulimia, body mass index, and bulimic symptomatology, were assessed. These views were explored in a sample of college students (N = 155 male; N = 173 female) and their parents (N = 194 mothers; N = 163 fathers). Females perceived bulimic symptoms to be more common than males, and students perceived them to be more common than their parents. Students perceived bulimic symptoms to be more acceptable than their parents. Females had higher levels of bulimic symptomatology, particularly daughters. Stepwise simultaneous regression analyses revealed that one's own level of bulimic symptomatology was the primary predictor of one's attitudes toward the commonality and acceptability of bulimic symptoms. Bulimic symptomatology was primarily predicted from perceptions of the acceptability of bulimic symptoms and body mass index. Results suggest that college-age students may be at greater risk for the development of bulimia nervosa because of their greater tolerance of these symptoms; alternatively, people endorsing higher levels of bulimic symptomatology may adjust their attitudes, perceiving bulimic symptoms to be more tolerable, in order to alleviate the dissonance between their attitudes and behaviors.

Electrical stimulation of C2C12 myotubes induces contractions and represses thyroid-hormone-dependent transcription of the fast-type sarcoplasmic-reticulum Ca2+-ATPase gene.

Chronic low-frequency contraction of skeletal muscle, either induced by a slow motor nerve or through direct electrical stimulation, generally induces expression of proteins associated with the slow phenotype, while repressing the corresponding fast isoforms. Contractions thereby counteract the primarily transcriptional effect of thyroid hormone (T3) which results in the selective induction and stimulation of expression of fast isoforms. We studied the regulation of expression of the fast-type sarcoplasmic-reticulum Ca2+-ATPase (SERCA1), a characteristic component of the fast phenotype. Previous work suggested that reduction of SERCA1 expression by contractile activity might result from interference with the T3-dependent transcriptional stimulation of the SERCA1 gene. The present study was set up to test this unexpected mode of action of contractile activity. We show that electrical stimulation of C2C12 mouse myotubes, which results in synchronous contractions at the imposed frequency, reduces basal but virtually abolishes T3-dependent SERCA1 expression. T3-dependent expression of a reporter gene driven by the SERCA1 promoter was similarly affected by electrical stimulation. This is the first demonstration that the counteracting effects on muscle gene expression of electrically induced contractions and T3 may interact at the transcriptional level.

Characterization of the promoter of the rat sarcoplasmic endoplasmic reticulum Ca2+-ATPase 1 gene and analysis of thyroid hormone responsiveness.

Relaxation of skeletal muscle requires the re-uptake of Ca2+, which is mediated by the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Thyroid hormone (T3) stimulates the expression of the SERCA1 isoform, which is essential for fast skeletal muscle fiber phenotype. We have cloned and studied the first 962 base pairs of the 5'-flanking region of the rat SERCA1 gene. This sequence was tested for T3-regulated expression in transient transfection experiments using COS7 cells and for binding of thyroid hormone receptor (TR) alpha in mobility shift assays. A construct of the 5'-flanking region and a reporter gene was unresponsive to T3 in the absence of co-transfected thyroid hormone receptor. In the presence of TRalpha, a T3 induction ratio of almost 4.0 was found, and this induction ratio was doubled with co-transfection of an RXR expression plasmid. Analysis of progressive 5'-deletion fragments of the sequence indicated multiple regions involved in T3 responsiveness. Three regions, R1, R2, and R3, were identified that bound TR complexes in mobility shift assays and conferred T3 responsiveness to a heterologous promoter. The most potent of these thyroid hormone response elements, R3, increased the 2-fold background T3 stimulation of the thymidine kinase promoter to nearly 6-fold. Detailed analysis of this element showed that four TR-binding half-sites, comprising two independent thyroid hormone response elements, interact cooperatively to give the maximal T3 response. T3 regulation of SERCA1 expression is mediated by a complex thyroid hormone response element that may serve to provide a greater range of response in interaction with nuclear receptor partners or cell-specific transcription factors.

Expression of MyoD in cultured primary myotubes is dependent on contractile activity: correlation with phenotype-specific expression of a sarcoplasmic reticulum Ca(2+)-ATPase isoform.

Myogenic determination factors (MDF) have been implicated in the establishment and maintenance of the fast or slow phenotype in skeletal muscle, with MyoD favoring the fast and myogenin favoring the slow phenotype. Accordingly, contractility-induced changes in muscle phenotype should be accompanied by a change in the MyoD/myogenin ratio. Some reports show such changes, but limitations inherent to in vivo studies complicate interpretation of these data. Here we tested whether a relationship can be found between contractility, MDF expression, and the expression of phenotype-specific muscle proteins in a simple in vitro system of cultured primary myotubes. We show that contractions reduce the MyoD/myogenin ratio by specifically repressing MyoD mRNA expression. This is accompanied by a selective repression at a pretranslational level of the expression of fast-type sarcoplasmic reticulum Ca(2+)-ATPase. These in vitro results support a phenotype-determining role of MDFs as a function of contractile activity and show that cultured myotubes can be a useful model for the analysis of the molecular mechanism of such regulation of muscle phenotype.