Tricarboxylic acid cycle enzyme activities in a mouse model of methylmalonic aciduria

Methylmalonic acidemia (MMA) is a propionate pathway disorder caused by dysfunction of the mitochondrial enzyme methylmalonyl-CoA mutase (MMUT). MMUT catalyzes the conversion of methylmalonyl-CoA to succinyl-CoA, an anaplerotic reaction which feeds into the tricarboxylic acid (TCA) cycle. As part of the pathological mechanisms of MMA, previous studies have suggested there is decreased TCA activity due to a toxic inhibition of TCA cycle enzymes by MMA related metabolites, in addition to reduced anaplerosis. Here, we have utilized mitochondria isolated from livers of a mouse model of MMA (Mut-ko/ki) and their littermate controls (Ki/wt) to examine the amounts and enzyme functions of most of the TCA cycle enzymes. We have performed mRNA quantification, protein semi-quantitation, and enzyme activity quantification for TCA cycle enzymes in these samples. Expression profiling showed increased mRNA levels of fumarate hydratase in the Mut-ko/ki samples, which by contrast had reduced protein levels as detected by immunoblot, while all other mRNA levels were unaltered. Immunoblotting also revealed decreased protein levels of 2-oxoglutarate dehydrogenase and malate dehydrogenase 2. Interesting, the decreased protein amount of 2-oxoglutarate dehydrogenase was reflected in decreased activity for this enzyme while there is a trend towards decreased activity of fumarate hydratase and malate dehydrogenase 2. Citrate synthase, isocitrate dehydrogenase 2/3, succinyl-CoA synthase, and succinate dehydrogenase are not statistically different in terms of quantity of enzyme or activity. Finally, we found decreased activity when examining the function of methylmalonyl-CoA mutase in series with succinate synthase and succinate dehydrogenase in the Mut-ko/ki mice compared to their littermate controls, as expected. This study demonstrates decreased activity of certain TCA cycle enzymes and by corollary decreased TCA cycle function, but it supports decreased protein quantity rather than toxic inhibition as the underlying mechanism of action. SUMMARY: Methylmalonic acidemia (MMA) is an inborn metabolic disorder of propionate catabolism. In this disorder, toxic metabolites are considered to be the major pathogenic mechanism for acute and long-term complications. However, despite optimized therapies aimed at reducing metabolite levels, patients continue to suffer from late complications, including metabolic stroke and renal insufficiency. Since the propionate pathway feeds into the tricarboxylic acid (TCA) cycle, we investigated TCA cycle function in a constitutive MMA mouse model. We demonstrated decreased amounts of the TCA enzymes, Mdh2 and Ogdh as semi-quantified by immunoblot. Enzymatic activity of Ogdh is also decreased in the MMA mouse model compared to controls. Thus, when the enzyme amounts are decreased, we see the enzymatic activity also decreased to a similar extent for Ogdh. Further studies to elucidate the structural and/or functional links between the TCA cycle and propionate pathways might lead to new treatment approaches for MMA patients

Particles at oil–air surfaces : powdered oil, liquid oil marbles, and oil foam

The type of material stabilized by four kinds of fluorinated particles (sericite and bentonite platelet clays and spherical zinc oxide) in air–oil mixtures has been investigated. It depends on the particle wettability and the degree of shear. Upon vigorous agitation, oil dispersions are formed in all the oils containing relatively large bentonite particles and in oils of relatively low surface tension (γla < 26 mN m⁻¹) like dodecane, 20 cS silicone, and cyclomethicone containing the other fluorinated particles. Particle-stabilized oil foams were obtained in oils having γla > 26 mN m⁻¹ where the advancing air–oil–solid contact angle θ lies between ca. 90° and 120°. Gentle shaking, however, gives oil-in-air liquid marbles with all the oil–particle systems except for cases where θ is <60°. For oils of tension >24 mN m⁻¹ with omniphobic zinc oxide and sericite particles for which advancing θ ≥ 90°, dry oil powders consisting of oil drops in air which do not leak oil could be made upon gentle agitation up to a critical oil:particle ratio (COPR). Above the COPR, catastrophic phase inversion of the dry oil powders to air-in-oil foams was observed. When sheared on a substrate, the dry oil powders containing at least 60 wt % of oil release the encapsulated oil, making these materials attractive formulations in the cosmetic and food industries

Flow cytometry immunophenotyping for diagnostic orientation and classification of pediatric cancer based on the EuroFlow Solid Tumor Orientation Tube (STOT)

Early diagnosis of pediatric cancer is key for adequate patient management and improved outcome. Although multiparameter flow cytometry (MFC) has proven of great utility in the diagnosis and classification of hematologic malignancies, its application to non-hematopoietic pediatric tumors remains limited. Here we designed and prospectively validated a new single eight-color antibody combination-solid tumor orientation tube, STOT-for diagnostic screening of pediatric cancer by MFC. A total of 476 samples (139 tumor mass, 138 bone marrow, 86 lymph node, 58 peripheral blood, and 55 other body fluid samples) from 296 patients with diagnostic suspicion of pediatric cancer were analyzed by MFC vs. conventional diagnostic procedures. STOT was designed after several design-test-evaluate-redesign cycles based on a large panel of monoclonal antibody combinations tested on 301 samples. In its final version, STOT consists of a single 8-color/12-marker antibody combination (CD99-CD8/(nu)myogenin/CD4-EpCAM/CD56/GD2/(sm)CD3-CD19/(cy)CD3-CD271/CD45). Prospective validation of STOT in 149 samples showed concordant results with the patient WHO/ICCC-3 diagnosis in 138/149 cases (92.6%). These included: 63/63 (100%) reactive/disease-free samples, 43/44 (98%) malignant and 4/4 (100%) benign non-hematopoietic tumors together with 28/38 (74%) leukemia/lymphoma cases; the only exception was Hodgkin lymphoma that required additional markers to be stained.& nbsp;In addition, STOT allowed accurate discrimination among the four most common subtypes of malignant CD45(-) CD56(++) non-hematopoietic solid tumors: 13/13 (GD2(++) (nu)myogenin(-) CD271(-/+) (nu)MyoD1(-) CD99(-) EpCAM(-)) neuroblastoma samples, 5/5 (GD2(-) (nu)myogenin(++) CD271(++) (nu)MyoD1(++) CD99(-/+) EpCAM(-)) rhabdomyosarcomas, 2/2 (GD2(-/+) (nu)myogenin(-) CD271(+) (nu)MyoD1(-) CD99(+) EpCAM(-)) Ewing sarcoma family of tumors, and 7/7 (GD2(-) (nu)myogenin(-) CD271(+) (nu)MyoD1(-) CD99(-) EpCAM(+)) Wilms tumors. In summary, here we designed and validated a new standardized antibody combination and MFC assay for diagnostic screening of pediatric solid tumors that might contribute to fast and accurate diagnostic orientation and classification of pediatric cancer in routine clinical practice.Stemcel biology/Regenerative medicine (incl. bloodtransfusion

Brain energy rescue:an emerging therapeutic concept for neurodegenerative disorders of ageing

The brain requires a continuous supply of energy in the form of ATP, most of which is produced from glucose by oxidative phosphorylation in mitochondria, complemented by aerobic glycolysis in the cytoplasm. When glucose levels are limited, ketone bodies generated in the liver and lactate derived from exercising skeletal muscle can also become important energy substrates for the brain. In neurodegenerative disorders of ageing, brain glucose metabolism deteriorates in a progressive, region-specific and disease-specific manner — a problem that is best characterized in Alzheimer disease, where it begins presymptomatically. This Review discusses the status and prospects of therapeutic strategies for countering neurodegenerative disorders of ageing by improving, preserving or rescuing brain energetics. The approaches described include restoring oxidative phosphorylation and glycolysis, increasing insulin sensitivity, correcting mitochondrial dysfunction, ketone-based interventions, acting via hormones that modulate cerebral energetics, RNA therapeutics and complementary multimodal lifestyle changes

Dynamic wetting on a thin film of soluble polymer: effects of nonlinearities in the sorption isotherm.

The wetting dynamics of a solvent on a soluble substrate interestingly results from the rates of the solvent transfers into the substrate. When a supported film of a hydrosoluble polymer with thickness e is wet by a spreading droplet of water with instantaneous velocity U, the contact angle is measured to be inversely proportionate to the product of thickness and velocity, eU, over two decades. As for many hydrosoluble polymers, the polymer we used (a polysaccharide) has a strongly nonlinear sorption isotherm φ(a(w)), where φ is the volume fraction of water in the polymer and aw is the activity of water. For the first time, this nonlinearity is accounted for in the dynamics of water uptake by the substrate. Indeed, by measuring the water content in the polymer around the droplet φ at distances as small as 5 μm, we find that the hydration profile exhibits (i) a strongly distorted shape that results directly from the nonlinearities of the sorption isotherm and (ii) a cutoff length ξ below which the water content in the substrate varies very slowly. The nonlinearities in the sorption isotherm and the hydration at small distances from the line were not accounted for by Tay et al., Soft Matter 2011, 7, 6953. Here, we develop a comprehensive description of the hydration of the substrate ahead of the contact line that encompasses the two water transfers at stake: (i) the evaporation-condensation process by which water transfers into the substrate through the atmosphere by the condensation of the vapor phase, which is fed by the evaporation from the droplet itself, and (ii) the diffusion of liquid water along the polymer film. We find that the eU rescaling of the contact angle arises from the evaporation-condensation process at small distances. We demonstrate why it is not modified by the second process

Influence of localized thermal effects on the reconstitution kinetics of lactose-coated whole milk powder

Reconstitution of dairy powders is strongly influenced by the presence and physical state of fat on the particle surface. The present study investigates the effect of a micronized lactose coating on the physical state of the fat and the reconstitution kinetics of whole milk powder at four different temperatures (4/21/40/60 °C) and two stirring rates (400/800 rpm). For this purpose, two types of micronized lactose were used as coating materials: crystalline and amorphous. At 4 °C and 21 °C, the coated powders sink and are reconstituted faster than pure whole milk powder, regardless of the stirring rate applied. At 40/60 °C and 400 rpm, although the amorphous micronized lactose coating leads to a significant decrease in the reconstitution time, the crystalline coating has the opposite effect (or no effect). This discrepancy is related to the large differences in terms of dissolution enthalpy between the two micronized lactose physical states. It is posited that the dissolution of the coating material causes a temperature shift at the powder-water interface which could hamper the complete melting of surface fat and influence its viscosity, thereby affecting wetting and sinking. These differences are overcome at a high stirring rate (800 rpm) or if agglomerated whole milk powder is used as the host material

Particle surface design for enhanced reconstitution of fat-based food powders

Spray-dried milk powders are characterized by an underrepresentation of hydrophilic compounds on the particle surface compared to the bulk, which renders reconstitution unfavourable. To overcome this issue, in the present study we instigated the coating of whole milk powder with two types of micronized lactose: crystalline and amorphous. We demonstrate that deposition of micronized lactose on the surface of whole milk powder leads to an increase in the reconstitution rate at 21 °C, correlated with a decrease in the apparent contact angle of the material. We also show that the physical solid state of micronized lactose is an important factor influencing the reconstitution performance of coated whole milk powder; If micronized lactose is amorphous, increasing the coating concentration beyond a certain point has little or no further effect on the reconstitution kinetics, which could be explained by viscosity build-up effects that hamper capillary penetration and sinking

Investigation of rehydration of food powder mixtures

This study focuses on the rehydration of an amorphous powder (maltodextrin DE19) and a crystalline powder (lactose 100 M) and mixtures of these powders. The rehydration process was tracked using a Focused Beam Reflectance Measurement (FBRM) probe and a video camera. The setup allows for real-time monitoring of all rehydration steps. The dispersion behaviour of the mixtures, especially mixtures with between 40 and 60% lactose content differs from behaviour of both pure powders at an agitation rate of 600 rpm. Surprisingly, the sinking times of the powder mixtures were observed to be higher than the sinking times of component powders at higher temperatures. Increasing temperature and agitation rate improved powder wetting and rehydration. A mechanism for increased sinking time of powder mixes was proposed. Finally, regime maps were constructed to summarize the effect of mix composition, temperature and agitation rate on rehydration behaviour. This mapping approach can be used to qualitatively assess rehydration

Effect of heterogeneous hydrophobic coating on floating of insoluble particles

Food powders often exhibit hydrophobic surface areas. To understand the effect of corresponding wettability variations on particle floating, which critically influences powder reconstitution, we studied floating of glass beads after creating surface heterogeneity by spraying cocoa butter. Force-position curves were recorded to characterise contact line pinning. Floating of particles with surface heterogeneities comprised a dynamic phase, which to our knowledge has not been reported previously. Measured forces varied due to contact line pinning/depinning. The results indicate that the dynamic floating phase is caused by gradual depinning. Steady-state floating is reached once pinning is sufficiently pronounced. We also demonstrate that surface heterogeneities complicate theoretical predictions of particle floating. The use of sessile drop contact angles for modelling was identified as an important source of error, as weak pinning points can affect contact angles but are oftentimes overcome during floating. Moreover, local pinning effects are not necessarily represented by a sessile drop