Towards fully automated segmentation of rat cardiac MRI by leveraging deep learning frameworks.

Automated segmentation of human cardiac magnetic resonance datasets has been steadily improving during recent years. Similar applications would be highly useful to improve and speed up the studies of cardiac function in rodents in the preclinical context. However, the transfer of such segmentation methods to the preclinical research is compounded by the limited number of datasets and lower image resolution. In this paper we present a successful application of deep architectures 3D cardiac segmentation for rats in preclinical contexts which to our knowledge has not yet been reported. We developed segmentation models that expand on the standard U-Net architecture and evaluated models separately trained for systole and diastole phases (2MSA) and a single model trained for all phases (1MSA). Furthermore, we calibrated model outputs using a Gaussian process (GP)-based prior to improve phase selection. The resulting models approach human performance in terms of left ventricular segmentation quality and ejection fraction (EF) estimation in both 1MSA and 2MSA settings (Sørensen-Dice score 0.91 ± 0.072 and 0.93 ± 0.032, respectively). 2MSA achieved a mean absolute difference between estimated and reference EF of 3.5 ± 2.5%, while 1MSA resulted in 4.1 ± 3.0%. Applying GPs to 1MSA enabled automating systole and diastole phase selection. Both segmentation approaches (1MSA and 2MSA) were statistically equivalent. Combined with a proposed cardiac phase selection strategy, our work presents an important first step towards a fully automated segmentation pipeline in the context of rat cardiac analysis.

Diastolic dysfunction and impaired cardiac output reserve in dysmetabolic nonhuman primate with proteinuria.

BACKGROUND: Cardiorenal complications are common in patients with dysmetabolism and diabetes. The present study aimed to examine if a nonhuman primate (NHP) model with spontaneously developed metabolic disorder and diabetes develops similar complications to humans, such as proteinuria and cardiac dysfunction at resting condition or diminished cardiac functional reserve following dobutamine stress echocardiography (DSE). METHODS AND RESULTS: A total of 66 dysmetabolic and diabetic cynomolgus (Macaca fascicularis) NHPs were enrolled to select 19 NHPs (MetS) with marked metabolic disorders and diabetes (fasting blood glucose: 178 ±â€¯18 vs. 61 ±â€¯3 mg/dL) accompanied by proteinuria (ACR: 134 ±â€¯34 vs. 1.5 ±â€¯0.4 mg/mmol) compared to 8 normal NHPs (CTRL). Under resting condition, MetS NHPs showed mild left ventricular (LV) diastolic dysfunction (E/A: 1 ±â€¯0.06 vs. 1.5 ±â€¯0.13), but with preserved ejection fraction (EF: 65 ±â€¯2 vs. 71 ±â€¯3%) compared to CTRL. DSE with an intravenous infusion of dobutamine at ascending doses (5, 10, 20, 30 and 40 µg/kg/min, 7 min for each dose) resulted in a dose-dependent increase in cardiac function, however, with a significantly diminished magnitude at the highest dose of dobutamine infusion (40 µg/kg/min) in both diastole (E/A: -12 ±â€¯3 vs. -38 ±â€¯5%) and systole (EF: 25 ±â€¯3 vs. 33 ±â€¯5%) as well as ~42% reduced cardiac output reserve (COR: 63 ±â€¯8 vs. 105 ±â€¯18%, p < 0.02) in the MetS compared to CTRL NHPs. CONCLUSION: These data demonstrate that MetS NHPs with cardiorenal complications: proteinuria, LV diastolic dysfunction and preserved LV systolic function under resting conditions displayed compromised cardiac functional reserve under dobutamine stress. Based on these phenotypes, this NHP model of diabetes with cardiorenal complications can be used as a highly translational model mimic human disease for pharmaceutical research.

Prevalence of probable sarcopenia in community-dwelling older Swiss people - a cross-sectional study.

BACKGROUND: The European Working Group on Sarcopenia in Older People has recently defined new criteria for identifying "(probable) sarcopenia" (EWGSOP2). However, the prevalence of probable sarcopenia, defined by these guidelines, has not been determined extensively, especially in the oldest old. This study aims to determine the prevalence of probable sarcopenia in older, community-living people and its association with strength-related determinants. METHODS: Handgrip strength and reported determinants (age, height, weight, osteoarthritis of hands, medications, fall history, physical activity, activities of daily living (ADL) and global cognitive function) were collected in a cross-sectional study of 219 community-living Swiss people (75 years and over). Probable sarcopenia was estimated based on cut-off values for handgrip strength as recommended by EWGSOP2. Spearman correlations, binary-regression analyses and contingency tables were used to explore relationships between variables. RESULTS: The prevalence of probable sarcopenia in women (n = 137, age 84.1 ± 5.7 years) and men (n = 82, age 82.6 ± 5.2 years) was 26.3 and 28.0%, respectively. In women, probable sarcopenia correlated positively with age and falls (rs range 0.332-0.195, p < .05), and negatively with weight, cognition, physical activity, using stairs regularly, participating in sports activities and ADL performance (rs range = - 0.141 - -0.409, p < .05). The only significant predictor of probable sarcopenia at the multivariate level was ADL performance (Wald(1) = 5.51, p = .019). In men, probable sarcopenia was positively correlated with age (rs = 0.33, p < .05) and negatively with physical activity, participation in sports and ADL performance (rs range - 0.221 - - 0.353, p < .05). ADL performance and age (Wald(1) = 4.46, p = .035 and Wald(1) = 6.30, p = .012) were the only significant predictors at the multivariate level. Men and women with probable sarcopenia were 2.8 times more likely to be dependent in ADL than those without. CONCLUSION: Probable sarcopenia affected one in every four community-living, oldest old people and was independently associated with impaired ADL performance in both sexes. This highlights the importance of detection of handgrip strength in this age group in clinical practice. Although prospective studies are required, independence in ADL might help to protect against probable sarcopenia.

Handgrip strength in old and oldest old Swiss adults - a cross-sectional study.

BACKGROUND: Handgrip strength is indicative of overall physical health and mobility in the elderly. A reduction in strength below a certain threshold severely increases the risk of mobility limitations and is predictive for adverse outcomes such as dependence in daily activities and mortality. An overview of age- and geography- specific handgrip strength values in older adults provide a reference for further investigations and measures in clinical practice to identify people at risk for clinically meaningful weakness. The aim of this study was to evaluate handgrip strength in the Swiss-German population aged 75 and over. METHODS: In a cross-sectional study, maximal isometric handgrip strength of the dominant hand was evaluated in 244 Swiss people aged 75 years and over (62.7% women), with mean age (SD) of 84.5 (5.6) years in men and 83.1 (5.9) years in women. Demographic data and information about comorbidities, medication, fall history, global cognitive function, self-reported physical activity and dependence in activities of daily living were collected, and correlated with grip strength measures. Age- and gender specific grip strength values are reported as means, standard deviations and standard error of mean. RESULTS: Sex-stratified handgrip strength was significantly lower with advancing age in men (p < .01), from 37.7 (6.5) kg to 25.6 (7.6) kg and in women (p < .01) from 22.2 (4.0) kg to 16.5 (4.7) kg. Handgrip strength in our sample was significantly higher than in Southern European countries. Handgrip strength was independently associated with age, height and ADL dependence in men and women. Overall, 44% of men and 53% of women had handgrip strength measures that were below the clinically relevant threshold for mobility limitations. CONCLUSION: This study reports the age- and sex-stratified reference values for handgrip strength in a representative sample of the Swiss population, aged 75-99 years. Although grip strength decreased with advancing age in both sexes; the relative decline was greater in men than women. Nonetheless men had significantly higher grip strength in all age groups. While the Swiss population sampled had greater grip strength than that reported in other European countries, about 50% were still classified as at risk of mobility limitations.

Investigating the loss of work productivity due to symptomatic leiomyoma.

INTRODUCTION: Leiomyoma affects up to 50% of fertile women, leading to morbidity such as bleeding or pain. The effect of symptomatic leiomyoma on the productivity of employed women is understudied. The present study investigates productivity loss in a Swedish setting in women with symptomatic leiomyoma compared to healthy women. MATERIAL AND METHODS: Women seeking care for leiomyoma and heavy menstrual bleeding (HMB) were recruited at nine Swedish sites. Healthy controls with self-perceived mild to normal menstruation were recruited at routine visits. Cases and controls were employed without option to work from home. After recruitment, all women reported the work productivity and activity impairment (WPAI) questionnaire, the pictorial blood assessment chart (PBAC) and pain on the visual analog scale (VAS). RESULTS: Women with symptomatic leiomyoma (n = 88) missed more working time during menses compared to asymptomatic controls (n = 34): 7.6 vs 0.2% p = 0.003. The proportion of impairment while working was also significantly higher in women with symptomatic leiomyoma (43.8 vs 12.1% p<0.001). Moreover, cases reported greater activity impairment outside office hours (43.9 vs 12.1%, p<0.001). Among healthy controls, 69.5% reported symptoms of HMB (PBAC>100). CONCLUSIONS: Symptomatic leiomyoma leads to loss of working hours as well as loss of productivity during working hours, and affects women in other daily activities. Increased awareness of the impact of leiomyomas on women's lives is needed, and timely and appropriate management of the symptomatic leiomyomas could improve work productivity and quality of life.

Menopausal hormone therapy and pancreatic cancer risk in women: a population-based matched cohort study.

BACKGROUND: The role of menopausal hormone therapy (MHT) in the development of pancreatic cancer is inconclusive owing to small studies and lack of proper study design. METHODS: This population-based matched cohort study included all Swedish women who used systemic MHT between 1 July 2005 and 31 December 2012. For each user of MHT, three never-users of MHT were randomly selected, matched for childbirth, history of thromboembolic events, and previous hysterectomy, as well as for year of birth, diabetes, obesity, and smoking- or alcohol-related disorders. Multivariable conditional logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for the association between MHT use and pancreatic cancer. The effect of MHT duration on pancreatic cancer development was calculated using multivariable Poisson regression. RESULTS: There were 290,186 ever-users of MHT and 870,165 matched never-users. During the follow-up, 311 (0.0011%) ever-users of MHT and 1220 (0.0014) never-users developed pancreatic cancer. In a multivariable adjusted model, ever-users had a 23% reduced risk (OR 0.77; 95% CI: 0.68-0.87) of pancreatic cancer. This risk decreased by 35% (incidence rate ratio (IRR) 0.65; 95% CI: 0.33-1.27) in women who used MHT 1-2 years and by 60% (IRR 0.40; 95% CI: 0.18-0.88) in women who used MHT ≥ 3 years compared to women with <1 year of MHT use. The type of MHT did not change the results. CONCLUSION: Systemic MHT use might reduce the risk of pancreatic cancer.

Colorectal Cancer Prognosis Following Obesity Surgery in a Population-Based Cohort Study.

BACKGROUND: Obesity surgery involves mechanical and physiological changes of the gastrointestinal tract that might promote colorectal cancer progression. Thus, we hypothesised that obesity surgery is associated with poorer prognosis in patients with colorectal cancer. METHODS: This nationwide population-based cohort study included all patients with an obesity diagnosis who subsequently developed colorectal cancer in Sweden from 1980 to 2012. The exposure was obesity surgery, and the main and secondary outcomes were disease-specific mortality and all-cause mortality, respectively. Cox proportional hazard survival models were used to calculate hazard ratios (HRs) with 95% confidence intervals (CIs), adjusted for sex, age, calendar year and education level. RESULTS: The exposed and unexposed cohort included 131 obesity surgery and 1332 non-obesity surgery patients with colorectal cancer. There was a statistically significant increased rate of colorectal cancer deaths following obesity surgery (disease-specific HR 1.50, 95% CI 1.00-2.19). When analysed separately, the mortality rate was more than threefold increased in rectal cancer patients with prior obesity surgery (disease-specific HR 3.70, 95% CI 2.00-6.90), while no increased mortality rate was found in colon cancer patients (disease-specific HR 1.10, 85% CI 0.67-1.70). CONCLUSION: This population-based study among obese individuals found a poorer prognosis in colorectal cancer following obesity surgery, which was primarily driven by the higher mortality rate in rectal cancer.

Menopausal hormone therapy and the risk of esophageal and gastric cancer.

A protective effect of female sex hormones has been suggested to explain the male predominance in esophageal and gastric adenocarcinoma, but evidence is lacking. We aimed to test whether menopausal hormone therapy (MHT) decreases the risk of these tumors. For comparison, esophageal squamous cell carcinoma was also assessed. This population-based matched cohort study included all women who had ever used systemic MHT in Sweden in 2005-2012. A comparison cohort of non-users of MHT was matched to the MHT-users regarding age, parity, thrombotic events, hysterectomy, diabetes, obesity, smoking-related diseases and alcohol-related diseases. Individuals with any previous cancer were excluded. Data on MHT use, cancer, comorbidity and mortality were collected from well-established Swedish nationwide registers. Odds ratios (OR) with 95% confidence intervals (CI) were calculated using conditional logistic regression. Different MHT regimens and age groups were compared in sub-group analyses. We identified 290,186 ever-users and 870,165 non-users of MHT. Ever-users had decreased ORs of esophageal adenocarcinoma (OR = 0.62, 95% CI 0.45-0.85, n = 46), gastric adenocarcinoma (OR = 0.61, 95% CI 0.50-0.74, n = 123) and esophageal squamous cell carcinoma (OR = 0.57, 95% CI 0.39-0.83, n = 33). The ORs were decreased for both estrogen-only MHT and estrogen and progestin combined MHT, and in all age groups. The lowest OR was found for esophageal adenocarcinoma in MHT-users younger than 60 years (OR = 0.20, 95% CI 0.06-0.65). Our study suggests that MHT-users are at a decreased risk of esophageal and gastric adenocarcinoma and also of esophageal squamous cell carcinoma. The mechanisms behind these associations remain to be elucidated.

Antireflux Surgery and Risk of Esophageal Adenocarcinoma: A Systematic Review and Meta-analysis.

OBJECTIVE: To investigate the preventive effect of antireflux surgery against esophageal adenocarcinoma (EAC) compared with medical treatment of gastroesophageal reflux disease (GERD) and to the background population. BACKGROUND: GERD is causally associated with EAC. Effective symptomatic treatment can be achieved with medication and antireflux surgery; however the possible preventive effect on EAC development remains unclear. METHODS: This systematic review identified 10 studies comparing EAC risk after antireflux surgery with nonoperated GERD patients, including 7 studies of patients with Barrett's esophagus, and 2 studies comparing EAC risk after antireflux surgery to the background population. A fixed-effects Poisson meta-analysis was conducted to calculate pooled incidence rate ratios (IRR) and 95% confidence intervals (CIs). RESULTS: The pooled IRR in patients after antireflux surgery was 0.76 (95% CI 0.42-1.39) compared with medically treated GERD patients. In patients with Barrett's esophagus, the corresponding IRR was 0.46 (95% CI 0.20-1.08), and 0.26 (95% CI 0.09-0.79) when restricted to publications after 2000. There was no difference in EAC risk between antireflux surgery and medical treatment in GERD patients without known Barrett's esophagus (IRR 0.98, 95% CI 0.72-1.33). The EAC risk remained elevated in patients after antireflux surgery compared with the background population (IRR 10.78, 95% CI 8.48-13.71). Although the clinical heterogeneity of the included studies was high, the statistical heterogeneity was low. CONCLUSIONS: Antireflux surgery may prevent EAC better than medical therapy in patients with Barrett's esophagus. The EAC risk after antireflux surgery does not seem to revert to that of the background population.

Genome-Wide Copy Number Variation Scan Identifies Complement Component C4 as Novel Susceptibility Gene for Crohn's Disease.

BACKGROUND: The genetic component of Crohn's disease (CD) is well known, with 140 susceptibility loci identified so far. In addition to single nucleotide polymorphisms typically studied in genome-wide scans, copy number variation is responsible for a large proportion of human genetic variation. METHODS: We performed a genome-wide search for copy number variants associated with CD using array comparative genomic hybridization. One of the found regions was validated independently through real-time PCR. Serum levels of the found gene were measured in patients and control subjects. RESULTS: We found copy number differences for the C4S and C4L gene variants of complement component C4 in the central major histocompatibility complex region on chromosome 6p21. Specifically, we saw that CD patients tend to have lower C4L and higher C4S copies than control subjects (P = 5.00 × 10 and P = 9.11 × 10), which was independent of known associated classical HLA I and II alleles (P = 7.68 × 10 and P = 6.29 × 10). Although C4 serum levels were not different between patients and control subjects, the relationship between C4 copy number and serum level was different for patients and control subjects with higher copy numbers leading to higher serum concentrations in control subjects, compared with CD patients (P < 0.001). CONCLUSIONS: C4 is part of the classical activation pathway of the complement system, which is important for (auto)immunity. Low C4L or high C4S copy number, and corresponding effects on C4 serum level, could lead to an exaggerated response against infections, possibly leading to (auto)immune disease.

Early complications following oesophagectomy for cancer in relation to long-term healthcare utilisation: a prospective population-based cohort study.

BACKGROUND: Little is known about how early postoperative complications after oesophagectomy for cancer influence healthcare utilisation in the long-term. We hypothesised that these complications also increase healthcare utilisation long after the recovery period. METHODS: This was a prospective, nationwide Swedish population-based cohort study of patients who underwent curatively intended oesophagectomy for cancer in 2001-2005 and survived at least 1 year postoperatively (n = 390). Total days of in-hospitalisation, number of hospitalisations and number of visits to the outpatient clinic within 5 years of surgery were analysed using quasi-Poisson models with adjustment for patient, tumour and treatment characteristics and are expressed as incidence rate ratios (IRR) and 95% confidence intervals (CI). RESULTS: There was an increased in-hospitalisation period 1-5 years after surgery in patients with more than 1 complication (IRR 1.5, 95% CI 1.0-2.4). The IRR for the number of hospitalisations by number of complications was 1.1 (95% CI 0.7-1.6), and 1.2 (95% CI 0.9-1.6) for number of outpatient visits in patients with more than 1 complication. The IRR for in-hospitalisation period 1-5 years following oesophagectomy was 1.8 (95% CI 1.0-3.0) for patients with anastomotic insufficiency and 1.5 (95% CI 0.9-2.5) for patients with cardiovascular or cerebrovascular complications. We found no association with number of hospitalisations (IRR 1.2, 95% CI 0.7-2.0) or number of outpatient visits (IRR 1.3, 95% CI 0.9-1.7) after anastomotic insufficiency, or after cardiovascular or cerebrovascular complications (IRR 1.2, 95% CI 0.7-1.9) and (IRR 1.1, 95% CI 0.8-1.5) respectively. CONCLUSION: This study showed an increased total in-hospitalisation period 1-5 years after oesophagectomy for cancer in patients with postoperative complications, particularly following anastomotic insufficiency.

eXtasy: variant prioritization by genomic data fusion.

Massively parallel sequencing greatly facilitates the discovery of novel disease genes causing Mendelian and oligogenic disorders. However, many mutations are present in any individual genome, and identifying which ones are disease causing remains a largely open problem. We introduce eXtasy, an approach to prioritize nonsynonymous single-nucleotide variants (nSNVs) that substantially improves prediction of disease-causing variants in exome sequencing data by integrating variant impact prediction, haploinsufficiency prediction and phenotype-specific gene prioritization.

Microarray analysis of copy number variation in single cells.

We present a protocol for reliably detecting DNA copy number aberrations in a single human cell. Multiple displacement-amplified DNAs of a cell are hybridized to a 3,000-bacterial artificial chromosome (BAC) array and to an Affymetrix 250,000 (250K)-SNP array. Subsequent copy number calling is based on the integration of BAC probe-specific copy number probabilities that are estimated by comparing probe intensities with a single-cell whole-genome amplification (WGA) reference model for diploid chromosomes, as well as SNP copy number and loss-of-heterozygosity states estimated by hidden Markov models (HMM). All methods for detecting DNA copy number aberrations in single human cells have difficulty in confidently discriminating WGA artifacts from true genetic variants. Furthermore, some methods lack thorough validation for segmental DNA imbalance detection. Our protocol minimizes false-positive variant calling and enables uniparental isodisomy detection in single cells. Additionally, it provides quality assessment, allowing the exclusion of uninterpretable single-cell WGA samples. The protocol takes 5-7 d.

Chronic lymphocytic leukemia and prolymphocytic leukemia with MYC translocations: a subgroup with an aggressive disease course.

Translocations involving MYC are rare in chronic lymphocytic leukemia (CLL), and up to now, their prognostic significance remains unclear. We report the characteristics of 21 patients with CLL and nine patients with prolymphocytic leukemia (PLL), diagnosed in multiple centers (n = 13), which showed an MYC translocation demonstrated by fluorescence in situ hybridization. The prevalence was estimated to be <1%. Advanced age and male predominance were observed. Morphological analysis frequently revealed the presence of prolymphocytes. A typical "CLL-immunophenotype" was found in four of nine cases with PLL. Moreover, CD5 and CD23 were frequently expressed in PLL. The latter findings are atypical for PLL and may suggest transformation or progression of an underlying CLL. MYC translocations were frequently observed with concomitant adverse cytogenetic markers, such as del(11q) (n = 8/30) and/or del(17p)/monosomy 17 (n = 7/30). In addition, the presence of unbalanced translocations (n = 24 in 13/30 cases) and complex karyotype (n = 16/30) were frequent in cases with MYC translocations. Altogether, del(17p)/monosomy 17, del(11q), and/or complex karyotype were observed in 22 of 30 patients. Survival outcome was poor: the median time to treatment was only 5 months, and overall survival (OS) from clinical diagnosis and from genetic detection was 71 and 19 months, respectively. In conclusion, CLL/PLL with MYC translocations is a rare entity, which seems to be associated with adverse prognostic features and unfavorable outcome.

Single-cell copy number variation detection.

Detection of chromosomal aberrations from a single cell by array comparative genomic hybridization (single-cell array CGH), instead of from a population of cells, is an emerging technique. However, such detection is challenging because of the genome artifacts and the DNA amplification process inherent to the single cell approach. Current normalization algorithms result in inaccurate aberration detection for single-cell data. We propose a normalization method based on channel, genome composition and recurrent genome artifact corrections. We demonstrate that the proposed channel clone normalization significantly improves the copy number variation detection in both simulated and real single-cell array CGH data.

Use of femoral shaft fracture classification for predicting the risk of associated injuries.

OBJECTIVES: To investigate the hypothesis that specific fracture patterns in patients with femoral shaft fractures can predict the likelihood of associated injuries. DESIGN: Retrospective cohort study. SETTING: Level I trauma center. PATIENTS/PARTICIPANTS: Consecutive patients treated because of a traumatic diaphyseal femoral fracture. MAIN OUTCOME MEASUREMENT: We studied the association between the Orthopaedic Trauma Association (OTA) fracture classification (derived from initial radiographs) and concomitant injuries of the head, spine, chest, abdomen, and pelvis with a severity of two or more points according to the Abbreviated Injury Scale by logistic regression analysis. RESULTS: One hundred forty-three of 203 patients (80 men, 63 women; mean age 54 ± 26 years) met the inclusion criteria. All patients had unilateral diaphyseal fractures, 64 OTA 32.A (45%), 46 OTA 32.B (32%), and 33 OTA 32.C (23%). In addition, 134 associated injuries were identified in 52 patients. Increasing fracture severity, as expressed by the OTA classification (ie, A, B, C), was significantly associated with a higher likelihood of thoracic (odds ratio [OR], 5.89; 95% confidence interval [CI], 2.59-13.40), pelvic (OR, 4.55; 95% CI, 2.01-10.28), upper (OR, 2.38; 95% CI, 1.27-4.48), and lower extremity injuries (OR, 3.12; 95% CI, 1.78-5.46). Fracture severity explained between 70% and 86% of the probability of having accompanying injuries. CONCLUSION: Radiographic grading of the severity of a femoral shaft fracture may signal the presence of accompanying injuries and should contribute to the clinical decision-making process in severe trauma.

Interphase fluorescence in situ hybridization on selected plasma cells is superior in the detection of cytogenetic aberrations in plasma cell dyscrasia.

Interphase fluorescence in situ hybridization (FISH) detects nonrandom cytogenetic abnormalities in plasma cell (PC) dyscrasia according to PC burden. However, when performed on cultured whole bone marrow (BM), it often fails to detect these aberrations. We have compared this interphase FISH technique with FISH after PC purification or identification to detect recurrent aberrations. In this study, 235 BM samples were collected from patients with multiple myeloma (MM) or related PC disorders regardless of disease status. All samples were analyzed in parallel. Clonal abnormalities were detected in 34.9% of cultured samples compared with 71.0% PC selected samples (P < 0.001). Moreover, FISH on PCs allowed to detect more abnormalities per case (P < 0.001) and identified higher percentages of abnormal nuclei (P < 0.001). This study indicates that FISH on PCs is the preferred technique for routine cytogenetic investigation of MM.