Lactate Stress Research Articles

Genes and environmental factors that influence disease resistance to microbes in the female reproductive tract of dairy cattle.

Microbes commonly infect the female reproductive tract of cattle, causing infertility, abortion and post partum uterine diseases. When organisms reach the uterus, the resistance to disease depends on the balance between the classic triad of the virulence of the microbes, the host defence systems and the environment. The present review considers each aspect of this triad, using postpartum uterine disease as an exemplar for understanding disease resistance. The bacteria that cause postpartum uterine disease are adapted to the endometrium, and their microbial toxins cause tissue damage and inflammation. However, non-specific defence systems counter ascending infections of the female reproductive tract, and inflammatory responses in the endometrium are driven by innate immunity. Disease resistance to bacterial infection involves many genes involved in the maintenance or restoration of tissue homeostasis in the endometrium, including antimicrobial peptides, complement, cytokines, chemokines and Toll-like receptors. The most important environmental factors facilitating the development of postpartum uterine disease are related to trauma of the reproductive tract and to the metabolic stress of lactation in dairy cows. Long-term solutions for uterine disease will include genetic selection for disease resistance and optimising the care of the animal before, during and after parturition.

Abstract 3010: The role of NF-kappaB in mammary tumor initiation

Abstract Nuclear factor kappa B signaling is accepted as a major component of pro-survival signaling within breast cancer cells. However, less is known about how NF-kappaB could contribute to breast tumor initiation. Inflammatory signaling can be activated within the breast by such common factors as obesity, involution following lactation, aging, and psychological stress. Thus, understanding how activated NF-kappaB could lead to the initiation of breast tumors would provide opportunities to develop prevention strategies. We generated a doxycycline-inducible mouse model to study the effects of constitutively active IKK2 on ductal morphogenesis. In this model, Tet-o-cIKK2 mice are crossed with MMTV-rtTA transgenics to target inducible expression specifically to the mammary epithelium. Expression of cIKK2 in transplanted mammary tissue results in enlarged terminal end-buds, increased lateral branching, and intraductal hyperplasia. Transgene induction for only 3 days in a 6 week old female induces filling of ductal lumens, hypercellularity and loss of strict basal/luminal structure. Quantitative PCR of mammary tissue reveals up-regulated markers of macrophage infiltration such as CCL2 and F4/80 and markers of inflammation including Cox-2, TNF-alpha, and CXCL1. Interestingly, constitutive IKK2 activation also results in a decrease in hormone receptor levels (ERα and PR). Immunofluorescent staining shows that activation of classical NF-kappaB increases nuclear p65, as expected. However, nuclear p52 is also increased in transgene positive cells, suggesting that a subset of the effects resulting from activation of classical NF-kappaB are mediated by cross talk with the alternative pathway. Taken together, we find that activation of NF-kappaB in mammary epithelium is sufficient to induce hyperplastic growth and loss of ductal structure. Ongoing studies seek to define what additional factors, such as the right hormonal context, will cause the lesions to progress to malignancy. Citation Format: Whitney Barham, Oleg Tikhomirov, Lianyi Chen, Ryan Ortega, Halina Onishko, Linda Connelly, Fiona E. Yull. The role of NF-kappaB in mammary tumor initiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3010. doi:10.1158/1538-7445.AM2014-3010

A comparative proteomic analysis of Bacillus coagulans in response to lactate stress during the production of L-lactic acid.

The growth rate and maximum biomass of Bacillus coagulans 2-6 were inhibited by lactate; inhibition by sodium lactate was stronger than by calcium lactate. The differences of protein expressions by B. coagulans 2-6 under the lactate stress were determined using two-dimensional electrophoresis coupled with mass spectrometric identification. Under the non-stress condition, calcium lactate stress and sodium lactate stress, the number of detected protein spots was 1,571±117, 1,281±231 and 904±127, respectively. Four proteins with high expression under lactate stress were identified: lactate dehydrogenase, cysteine synthase A, aldo/keto reductase and ribosomal protein L7/L12. These proteins are thus potential targets for the reconstruction of B. coagulans to promote its resistance to lactate stress.

Ultrastructure of Thyroid Gland in Hipposiderid Bat Hipposideros lankadiva (Kelaart)

In chiropterans, thyroid gland plays vital role during initial growth phase of life and in mobilizing the energy to overcome stress of puberty, pregnancy and lactation. Ultrastructural observation of the thyroid gland of hipposiderid bat Hipposideros lankadiva which undergoes the phenomenon of embryonic diapauses confirms the important role played by thyroid gland during diapause. The cuboidal follicular cells with well developed cell organelle and small follicular lumen during estrous, arousal and lactation period show active state of gland in nonpregnant female bat while flattened follicular cells with less developed cell organelle and large follicular lumen show hypothyroid condition in pregnant bat H. lankadiva undergoing diapause. Probably this maternal hypothyroid condition in pregnant animal regulates the embryonic diapause.

The effects of lactational maternal stress on neuronal behavior of adolescent offspring in mice

Objective To explore the effects of lactational maternal stress on neurobehavioral development of offsprings. Methods Maternal mice were divided into normal control, maternal deprivation and maternal stress group. A 5-min/day cold water swimming was used as maternal stress for continuously of 14 day since the 7th postnatal day. The increasing bodyweight and neurobehavior of adolescent offspring mice were assessed by forced swimming test, tail suspension test and locomotor activity in the 35th postnatal day. Results Maternal chronic stress in lactational stage significantly increased the vulnerability of offspring to acute stress, which was reflected by significantly increased immobility time of adolescent offspring in forced swimming test(female: (139±6)s, (138±9)s, (96±9)s respectively; male: (139±9)s, (112±9)s, (96±9)s respectively) and in tail suspension test(femal: (127±8)s, (123±8)s, (98±6)s respectively; male: (141±7)s, (105±5)s, (92±6)s respectively). Meanwhile, sexual difference occurred for adolescent offspring to acute stress, and the female offspring were more vulnerable to acute stress. Conclusion Lactational maternal stress may be important factors for development of adolescent depression.Our findings highlight the area of early prevention and intervention for adolescent depression . Key words: Maternal stress; Lactation; Neuronal behavior; Adolescent depression

Biomarkers of peripheral muscle fatigue during exercise

BackgroundBiomarkers of peripheral muscle fatigue (BPMFs) are used to offer insights into mechanisms of exhaustion during exercise in order to detect abnormal fatigue or to detect defective metabolic pathways. This review aims at describing recent advances and future perspectives concerning the most important biomarkers of muscle fatigue during exercise.ResultsBPMFs are classified according to the mechanism of fatigue related to adenosine-triphosphate-metabolism, acidosis, or oxidative-metabolism. Muscle fatigue is also related to an immunological response. impaired calcium handling, disturbances in bioenergetic pathways, and genetic responses. The immunological and genetic response may make the muscle susceptible to fatigue but may not directly cause muscle fatigue. Production of BPMFs is predominantly dependent on the type of exercise. BPMFs need to change as a function of the process being monitored, be stable without appreciable diurnal variations, correlate well with exercise intensity, and be present in detectable amounts in easily accessible biological fluids. The most well-known BPMFs are serum lactate and interleukin-6. The most widely applied clinical application is screening for defective oxidative metabolism in mitochondrial disorders by means of the lactate stress test. The clinical relevance of most other BPMFs, however, is under debate, since they often depend on age, gender, physical fitness, the energy supply during exercise, the type of exercise needed to produce the BPMF, and whether healthy or diseased subjects are investigated.ConclusionsThough the role of BPMFs during fatigue is poorly understood, measuring BPMFs under specific, standardised conditions appears to be helpful for assessing biological states or processes during exercise and fatigue.

Maternal low‐protein diet up‐regulates the neuropeptide Y system in visceral fat and leads to abdominal obesity and glucose intolerance in a sex‐ and time‐specific manner

Neuropeptide Y (NPY) mediates stress-induced obesity in adult male mice by activating its Y2 receptor (Y2R) in visceral adipose tissue (VAT). Here, we studied whether the NPY-Y2R system is also activated by maternal low-protein diet (LPD) and linked to obesity in offspring. Prenatal LPD offspring had lower birth weights compared to normal-protein diet (NPD) offspring. Female prenatal and lactation stress (PLS) offspring from mothers fed an LPD developed abdominal adiposity and glucose intolerance associated with a 5-fold up-regulation of NPY mRNA and a 6-fold up-regulation of Y2R mRNA specifically in VAT, in addition to elevated platelet-rich-plasma (PRP) NPY, compared to control females fed a high-fat diet (HFD). Conversely, PLS male offspring showed lower NPY in PRP, a 10-fold decrease of Y2R mRNA in VAT, lower adiposity, and improved glucose tolerance compared to control males. Interestingly, prenatal LPD offspring cross-fostered to control lactating mothers had completely inverse metabolic and NPY phenotypes. Taken together, these findings suggested that maternal LPD activates the VAT NPY-Y2R system and increases abdominal adiposity and glucose intolerance in a sex- and time-specific fashion, suggesting that the peripheral NPY system is a potential mediator of programming for the offspring's vulnerability to obesity and metabolic syndrome.

Anti-NMDA receptor antibodies in a case of MELAS syndrome

A 42-year-old man presented with new-onset headache, global aphasia, anteroand retrograde memory deficits, stereotypic behavior, agitation and an inappropriate foolish affect in July 2007. Immediate MRI revealed a T2w lesion in the left temporal lobe with cortical and subcortical involvement, compatible with encephalitis (Fig. 1). CSF analysis showed normal cell count and negative herpes simplex virus type I PCR while protein and lactate levels were elevated (protein: 1.36 g/l; normal: 0.15–0.45 g/l; lactate: 5.6 mmol/l; normal: 0.55–2.2 mmol/l). Repeated history taking revealed progressive bilateral hearing loss for 8 years. Additional MRI investigations showed a hyperintense appearance of the observed lesion in diffusion weighted images (DWI), mixed increased and decreased diffusion on apparent diffusion coefficient (ADC) maps, and a strong lactate peak in MR spectroscopy (Fig. 1). Further work-up revealed myopathic changes in several muscles on electromyography, elevated serum lactate during lactate stress test, hypertrophic cardiomyopathy, and signs of mitochondrial disease in muscle biopsy specimens (increased fuchsinophilia and COX-negative fibers). Family history was negative for mitochondrial disorders, but genetic analysis from leucocytes revealed a heteroplasmic m.3243A[G point mutation in mitochondrial DNA (heteroplasmy rate, 30%), compatible with a diagnosis of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). No other family members were tested. During the next few years, the patient developed focal epilepsy and progressive cognitive deficits. In 2010, he sustained a stroke-like episode of the left occipital cortex causing right homonymous hemianopia (Fig. 1). At this time, the initial clinical presentation was reconsidered and archived CSF/serum samples obtained 6 days after symptom onset in 2007 were analyzed for autoimmune antibodies. Antibodies were determined by means of a recombinant immunofluorescence assay employing NMDAR transfected HEK cells as described previously [1]. IgG-antibodies against NMDAR were detected in the patient’s serum at a titer of 1:32 (score 2 according to Irani et al. [4]). No antinuclear antibodies (ANA) or antibodies against further neuronal surface antigens (i.e., AMPA receptor, GABAb-receptor) or VGKC-complex were detected in the patient’s serum. On follow-up in 2010, NMDAR antibodies were negative both in serum and CSF. A body-CT revealed no malignancies. Taken together, our patient presented with symptoms compatible with a first manifestation of MELAS syndrome in 2007 [2]. The patient’s lesion in the left temporal lobe likely accounts for his aphasia, memory disturbances, and behavioral changes. Imaging characteristics suggest a stroke-like lesion associated with MELAS syndrome [3]. At the same time, however, the patient tested positive for anti-NMDAR antibodies that are considered highly specific C. Finke (&) H. Pruss F. Ostendorf L. Harms C. J. Ploner Department of Neurology, Charite-Universitatsmedizin Berlin, Chariteplatz 1, 10117 Berlin, Germany e-mail: carsten.finke@charite.de

Effect of season, month of parturition and lactation on estrus behavior and ovarian activity in Barki x Rahmani crossbred ewes under subtropical conditions

The correlations between some meteorological parameters and fertility data were evaluated in Barki x Rahmani crossbred ewes using the records of five consecutive years (2003–2007). Additionally, estrus detection and ultrasonic evaluation were applied on eighteen mature dry ewes during breeding and non-breeding seasons. The effect of lactation was evaluated by monitoring estrus behavior in ninety four lactating ewes from 40 to 120 d after parturition. Moreover, ultrasonography was used to identify ovarian activity in six cyclic and six acyclic non-lactating ewes. Results revealed that relative estrus occurrence and fertile mating were positively correlated (P <0.05) with high temperature and long photoperiod (conditions of summer season), and were negatively correlated (P < 0.01) with rainfall (condition of winter season). During breeding season, estrus rate, serum progesterone concentration, and diameter of largest follicle were significantly (P < 0.05) higher than those observed during the non-breeding season. Furthermore, month of parturition had a significant effect (P < 0.05) on estrus rate of lactating ewes where ewes that lambed in August, September, and October recorded higher estrus rate than those lambed in November and December. However, the lactational strength did not exert any deleterious effect on the reproductive performance of lactating ewes.In conclusion, in Egypt under subtropical conditions, Barki x Rahmani crossbred ewes exerted optimum estrus behavior and fertile mating during summer season. The reduction in estrus activity during lactation was due to the seasonal effect rather than lactational stress.

Production of l-lactic acid by a thermophilic Bacillus mutant using sodium hydroxide as neutralizing agent

A sodium lactate tolerant mutant strain named Bacillus sp. Na-2 was obtained and applied to sodium hydroxide-based l-lactic acid (LA) production process. The influences of aeration and pH were investigated to further improve the resistance of strain Na-2 against sodium lactate stress and to obtain the most efficient l-LA production process. Although mild aeration was favorable for cell growth and l-LA production, vigorous aeration resulted in a metabolic shift from homolactic to mixed-acid/acetoin fermentation. Therefore, a two-stage aeration control strategy was employed. Optimum pH was found to be 6.0. A total of 106.0 g/l l-LA was produced in 30 h by Bacillus sp. Na-2 using sodium hydroxide as neutralizing agent. Productivity, conversion rate and optical purity were 3.53 g/l/h, 94% and 99.5%, respectively. The remarkable fermentation traits of Bacillus sp. Na-2 and the environment-friendly characteristics of NaOH-based process represent new insight for industrial scale production of l-LA.

Nuclear magnetic resonance and mass spectrometry-based milk metabolomics in dairy cows during early and late lactation

Milk production in dairy cows has dramatically increased over the past few decades. The selection for higher milk yield affects the partitioning of available nutrients, with more energy being allocated to milk synthesis and less to physiological processes essential to fertility and fitness. In this study, the abundance of numerous milk metabolites in early and late lactation was systematically investigated, with an emphasis on metabolites related to energy metabolism. The aim of the study was the identification and correlation of milk constituents to the metabolic status of the cows. To investigate the influence of lactation stage on physiological and metabolic variables, 2 breeds of different productivity were selected for investigation by high-resolution nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. We could reliably quantify 44 different milk metabolites. The results show that biomarkers such as acetone and β-hydroxybutyrate are clearly correlated to the metabolic status of the individual cows during early lactation. Based on these data, the selection of cows that cope well with the metabolic stress of early lactation should become an option.

Mitochondriale Myopathien

The organ most frequently affected in mitochondrial disorders is the skeletal muscle (mitochondrial myopathy). Mitochondrial myopathies may be part of syndromic as well as non-syndromic mitochondrial disorders. Involvement of the skeletal muscle may remain subclinical, may manifest as isolated elevation of the creatine-kinase, or as weakness and wasting of one or several muscle groups. The course of mitochondrial myopathies is usually slowly progressive and only rarely rapidly progressive leading to restriction of mobility and requirement of a wheel chair or even muscular respiratory insufficiency. Frequently reported symptoms of mitochondrial myopathies are permanent tiredness, easy fatigability, muscle aching at rest or already after moderate exercise, muscle cramps, muscle stiffness, fasciculations and muscle weakness. The diagnosis is based on the history, clinical neurologic examination, blood chemical investigations, lactate stress test, electromyography, magnetic resonance imaging, magnetic resonance spectroscopy, muscle biopsy, biochemical investigations of the skeletal muscles, and genetic investigations. Only symptomatic therapy is available and includes physiotherapy and orthopedic supportive devices, diet, symptomatic drug therapy (analgetics, cramp-releasing drugs, antioxidants, lactate-lowering drugs, alternative energy sources, co-factors), avoidance of mitochondrion-toxic drugs, surgery (correction of ptosis or orthopedic problems), and invasive or non-invasive mechanical ventilation. General anesthesia needs to be performed in the same way as in patients with susceptibility for malignant hyperthermia.

Mitochondrial disorder overlapping with aorto-iliac occlusive disease (Leriche-syndrome)

Objectives Diagnosing mitochondrial disorder (MCD) is challenging if symptoms and signs of a second trouble overlap with those of the MCD. Case report A 54-yo male developed muscle pain in his proximal lower and distal upper limbs, easy fatigability and exercise intolerance, and myoglobinurea. Based upon the clinical presentation, recurrent creatine-kinase elevation, highly abnormal lactate stress testing, abnormal muscle biopsy, and a positive MRI spectroscopy, MCD was diagnosed. Based upon progressing pain of the lower limbs, the development of claudication, recurrent falls, nightly, muscle cramping, gait disturbance, erectile dysfunction, the inability to sit, and an angiography revealing stenosis of the distal aorta, occlusion of the right iliac and left internal iliac and stenosis of the left external iliac artery, Leriche-syndrome was diagnosed. He profited from stenting of the left external iliac artery. Manifestations of MCD remained stable throughout the period of observation. Conclusion If the history is misleading or if manifestations of a MCD overlap with those of a Leriche-syndrome, diagnosing either condition is challenging, Atherosclerosis may be a feature of mitochondrial disease

Effects of Experience and Lactation on Lupine Consumption by Cattle

Lupines ( Lupinus spp.) are widespread range plants that are often toxic to livestock. Some reports suggest that naïve, younger animals might consume more lupine than more experienced, older cattle. Further, lactational stress might alter forage selection, and lactating cows might eat more lupine than nonlactating cows. Thus, the objectives of these trials were to examine the influence of experience and lactation on lupine intake. Both study areas were near Ritzville, Washington, on rangeland dominated by cheatgrass or downy brome ( Bromus tectorum L.), with abundant velvet lupine ( Lupinus leucophyllus Dougl.). During the first trial, six naïve and six experienced Hereford cows were grazed together for 25 d during summer. There was no difference ( P > 0.5) in consumption of lupine by naïve and experienced cows. Consumption peaked at 10%–14% of daily bites. During the second trial, six lactating and six nonlactating cows grazed a lupine-infested pasture for 18 d. There was no difference ( P > 0.6) in consumption between lactating and dry cows. Lupine consumption gradually increased and peaked at 10%–15% of the diets. Our results suggest that experience and lactation status are probably minor considerations in livestock management schemes to reduce losses to lupine. Las plantas Lupinos ( Lupinus spp.) se dan abundantemente en el pastizal y son con frecuencia tóxicas para el ganado. Algunos reportes sugieren que animales jóvenes y sin experiencia en pastoreo consumen más lupinos que animales adultos con experiencia. Además, el estrés lactacional en las hembras puede alterar la selección de forraje por lo que las vacas lactantes pueden consumir más lupinos que vacas no lactantes (secas). Estos estudios tuvieron como objetivo determinar la influencia tanto de la experiencia como del estado lactacional sobre el consumo de lupinos. El área de estudio se localiza cerca de Ritzville, WA en un pastizal dominado por Bromus tectorum L. (cheatgrass) con abundancia de lupinos ( Lupinus leucophyllus Dougl.). Durante la primera prueba, seis vacas Hereford sin experiencia y seis con experiencia estuvieron pastoreando por 25 días durante el verano. No se encontraron diferencias ( P > 0.5) en el consumo de lupinos en las vacas con y sin experiencia. El máximo consumo de lupinos se presentó del 10% al 14% del total de bocados al día. Durante la segunda prueba, seis vacas en lactación y seis vacas secas estuvieron pastoreando un potrero infestado de lupinos por 18 días. No se encontraron diferencias ( P > 0.6) en el consumo de lupinos entre vacas lactantes y secas. El consumo de lupinos gradualmente se incrementó hasta un 10%–15% de la dieta. Estos resultados sugieren que la experiencia y estado lactacional de las vacas son probablemente consideraciones menores para reducir pérdidas causadas por lupinos durante la implementación de programas de manejo del ganado en pastoreo.

Blood Lactate and Heat Stress during Training in Rowers

The purpose of the present study was to test the hypothesis that large increases in blood lactate concentration ([La] (b)) and/or body temperature may occur during an endurance training on a rowing ergometer and disrupt training. The influence of an increase in air convection on the capacity to perform a prolonged exercise was also explored. Ten trained oarsmen were asked to undergo twice, in control (C) and increased air ventilation (AV) conditions, two 30-min trainings on a rowing ergometer at a work rate corresponding to 2.5 mmol . L (-1) of [La] (b) determined during a previous incremental exercise (P (2.5)). Four subjects did not complete the training session in C despite a steady state in [La] (b) in two of them. In these four subjects, the end of the exercise was associated with the highest measured rectal temperatures (T (re), 39.4 +/- 0.1 degrees C) and rate of perceived exertion (RPE, 17.8 +/- 0.3). Regarding the six other subjects, their heart rate, oxygen uptake, RPE, T (re) and water loss values were lower (p < 0.05) in AV than in C. [La] (b) displayed the same profile in C and AV. This study suggests that i) high body temperature may constitute a significant factor of perceived exertion and disrupt indoor training session, and ii) capacity to perform an endurance training on a rowing ergometer was improved by increasing air convection.

Mitochondrial Disorder Aggravated by Propranolol

Although there are indications that beta-blockers affect the skeletal muscle in therapeutic dosages, their influence on mitochondrial disorders is unknown. A 52-year-old woman developed double vision, myalgias, muscle cramps, and hip and thigh muscle stiffness. Clinical neurologic examination revealed ptosis, dysarthria, sore neck muscles, weakness and wasting of the thighs, and generally brisk tendon reflexes. Lactate stress testing was significantly abnormal. Needle electromyography was nonspecifically abnormal and myopathic. Muscle biopsy showed mild myopathic changes, target fibers, and a single COX-negative fiber. Probable mitochondrial disorder was diagnosed. The patient had been on 30 mg of propranolol during 7 years for arterial hypertension. Shortly after discontinuation of the drug, her double vision gradually disappeared, myalgias and muscle cramps gradually resolved, and the patient reported an increase in muscle mass on repeated follow-ups. Long-term administration of propranolol may aggravate a mitochondrial disorder. Discontinuation of propranolol may result in a gradual resolution of these adverse reactions.

Madarosis from mitochondriopathy

Madarosis from mitochondriopathy

The usefulness of lactate stress testing in the diagnosis of mitochondrial myopathy

The usefulness of lactate stress testing in the diagnosis of mitochondrial myopathy

Response to the letter to the Editor entitled “The usefulness of lactate stress testing in the diagnosis of itochondrial myopathy” by Dr. Finsterer

Response to the letter to the Editor entitled “The usefulness of lactate stress testing in the diagnosis of itochondrial myopathy” by Dr. Finsterer

Stress lactate in mitochondrial myopathy under constant, unadjusted workload

As it is under debate if determination of lactate during cycle ergometry (lactate stress testing, LST) under a continuous, unadjusted, low workload is a valuable diagnostic tool for mitochondrial myopathy (MMP), the present study aimed to investigate how sensitive the LST is in a large cohort of patients with indications for MMP (MMP patients). Serum lactate was determined once before, three times during, and once after a 15-min, constant 30 W-workload on a bicycle ergometer in 115 healthy controls, 166 patients with neurological disorders other than MMP, and 291 MMP patients. Serum lactate's upper reference limit at rest, 5, 10, 15 min after starting, and 15 min after finishing the exercise was 2.0, 2.0, 2.1, 2.0 and 1.7 mmol/l, respectively. Resting lactate was increased in 75 MMP patients (26%). The specificity of resting lactate determination was 84%. The sensitivity of the LST was 66% and the specificity 84%. Among the 192 MMP patients with abnormal LST, 120 (63%) had a normal resting lactate. The LST is abnormal in two thirds of the MMP patients. The sensitivity of the LST is higher than that of resting lactate determination. The LST is a simple and cheap but effective and reliable screening method for detecting the impaired oxidative metabolism in MMP.