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

Proposed guidelines for the diagnosis and management of methylmalonic and propionic acidemia.

Methylmalonic and propionic acidemia (MMA/PA) are inborn errors of metabolism characterized by accumulation of propionic acid and/or methylmalonic acid due to deficiency of methylmalonyl-CoA mutase (MUT) or propionyl-CoA carboxylase (PCC). MMA has an estimated incidence of ~ 1: 50,000 and PA of ~ 1:100\u27000 -150,000. Patients present either shortly after birth with acute deterioration, metabolic acidosis and hyperammonemia or later at any age with a more heterogeneous clinical picture, leading to early death or to severe neurological handicap in many survivors. Mental outcome tends to be worse in PA and late complications include chronic kidney disease almost exclusively in MMA and cardiomyopathy mainly in PA. Except for vitamin B12 responsive forms of MMA the outcome remains poor despite the existence of apparently effective therapy with a low protein diet and carnitine. This may be related to under recognition and delayed diagnosis due to nonspecific clinical presentation and insufficient awareness of health care professionals because of disease rarity

PheMaDB: A solution for storage, retrieval, and analysis of high throughput phenotype data

<p>Abstract</p> <p>Background</p> <p>OmniLog™ phenotype microarrays (PMs) have the capability to measure and compare the growth responses of biological samples upon exposure to hundreds of growth conditions such as different metabolites and antibiotics over a time course of hours to days. In order to manage the large amount of data produced from the OmniLog™ instrument, PheMaDB (Phenotype Microarray DataBase), a web-based relational database, was designed. PheMaDB enables efficient storage, retrieval and rapid analysis of the OmniLog™ PM data.</p> <p>Description</p> <p>PheMaDB allows the user to quickly identify records of interest for data analysis by filtering with a hierarchical ordering of Project, Strain, Phenotype, Replicate, and Temperature. PheMaDB then provides various statistical analysis options to identify specific growth pattern characteristics of the experimental strains, such as: outlier analysis, negative controls analysis (signal/background calibration), bar plots, pearson's correlation matrix, growth curve profile search, <it>k</it>-means clustering, and a heat map plot. This web-based database management system allows for both easy data sharing among multiple users and robust tools to phenotype organisms of interest.</p> <p>Conclusions</p> <p>PheMaDB is an open source system standardized for OmniLog™ PM data. PheMaDB could facilitate the banking and sharing of phenotype data. The source code is available for download at <url>http://phemadb.sourceforge.net</url>.</p

The remnants of galaxy formation from a panoramic survey of the region around M31

In hierarchical cosmological models, galaxies grow in mass through the continual accretion of smaller ones. The tidal disruption of these systems is expected to result in loosely bound stars surrounding the galaxy, at distances that reach $10 - 100$ times the radius of the central disk. The number, luminosity and morphology of the relics of this process provide significant clues to galaxy formation history, but obtaining a comprehensive survey of these components is difficult because of their intrinsic faintness and vast extent. Here we report a panoramic survey of the Andromeda galaxy (M31). We detect stars and coherent structures that are almost certainly remnants of dwarf galaxies destroyed by the tidal field of M31. An improved census of their surviving counterparts implies that three-quarters of M31's satellites brighter than $M_V < -6$ await discovery. The brightest companion, Triangulum (M33), is surrounded by a stellar structure that provides persuasive evidence for a recent encounter with M31. This panorama of galaxy structure directly confirms the basic tenets of the hierarchical galaxy formation model and reveals the shared history of M31 and M33 in the unceasing build-up of galaxies.Comment: Published in Nature. Supplementary movie available at https://www.astrosci.ca/users/alan/PANDAS/Latest%20news%3A%20movie%20of%20orbit.htm

The physical scale of the far-infrared emission in the most luminous submillimetre galaxies II: evidence for merger-driven star formation

We present high-resolution 345 GHz interferometric observations of two extreme luminous (L_{IR}>10^{13} L_sun), submillimetre-selected galaxies (SMGs) in the COSMOS field with the Submillimeter Array (SMA). Both targets were previously detected as unresolved point-sources by the SMA in its compact configuration, also at 345 GHz. These new data, which provide a factor of ~3 improvement in resolution, allow us to measure the physical scale of the far-infrared in the submillimetre directly. The visibility functions of both targets show significant evidence for structure on 0.5-1 arcsec scales, which at z=1.5 translates into a physical scale of 5-8 kpc. Our results are consistent with the angular and physical scales of two comparably luminous objects with high-resolution SMA followup, as well as radio continuum and CO sizes. These relatively compact sizes (<5-10 kpc) argue strongly for merger-driven starbursts, rather than extended gas-rich disks, as the preferred channel for forming SMGs. For the most luminous objects, the derived sizes may also have important physical consequences; under a series of simplifying assumptions, we find that these two objects in particular are forming stars close to or at the Eddington limit for a starburst.Comment: 9 pages, 3 Figures, submitted to MNRA

Recommendations for diagnosing and managing individuals with glutaric aciduria type 1: Third revision

Glutaric aciduria type 1 is a rare inherited neurometabolic disorder of lysine metabolism caused by pathogenic gene variations in GCDH (cytogenic location: 19p13.13), resulting in deficiency of mitochondrial glutaryl-CoA dehydrogenase (GCDH) and, consequently, accumulation of glutaric acid, 3-hydroxyglutaric acid, glutaconic acid and glutarylcarnitine detectable by gas chromatography/mass spectrometry (organic acids) and tandem mass spectrometry (acylcarnitines). Depending on residual GCDH activity, biochemical high and low excreting phenotypes have been defined. Most untreated individuals present with acute onset of striatal damage before age 3 (to 6) years, precipitated by infectious diseases, fever or surgery, resulting in irreversible, mostly dystonic movement disorder with limited life expectancy. In some patients, striatal damage develops insidiously. In recent years, the clinical phenotype has been extended by the finding of extrastriatal abnormalities and cognitive dysfunction, preferably in the high excreter group, as well as chronic kidney failure. Newborn screening is the prerequisite for pre-symptomatic start of metabolic treatment with low lysine diet, carnitine supplementation and intensified emergency treatment during catabolic episodes, which, in combination, have substantially improved neurologic outcome. In contrast, start of treatment after onset of symptoms cannot reverse existing motor dysfunction caused by striatal damage. Dietary treatment can be relaxed after the vulnerable period for striatal damage, that is, age 6 years. However, impact of dietary relaxation on long-term outcomes is still unclear. This third revision of evidence-based recommendations aims to re-evaluate previous recommendations (Boy et al., J Inherit Metab Dis, 2017;40(1):75-101; Kolker et al., J Inherit Metab Dis 2011;34(3):677-694; Kolker et al., J Inherit Metab Dis, 2007;30(1):5-22) and to implement new research findings on the evolving phenotypic diversity as well as the impact of non-interventional variables and treatment quality on clinical outcomes

Loss of the Synaptic Vesicle Protein SV2B Results in Reduced Neurotransmission and Altered Synaptic Vesicle Protein Expression in the Retina

The Synaptic Vesicle Protein 2 (SV2) family of transporter-like proteins is expressed exclusively in vesicles that undergo calcium-regulated exocytosis. Of the three isoforms expressed in mammals, SV2B is the most divergent. Here we report studies of SV2B location and function in the retina. Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform. In mice lacking SV2B, synaptic transmission at the synapse between photoreceptors and bipolar neurons was decreased, as evidenced by a significant reduction in the amplitude of the b-wave in electroretinogram recordings. Quantitative immunoblot analyses of whole eyes revealed that loss of SV2B was associated with reduced levels of synaptic vesicle proteins including synaptotagmin, VAMP, synaptophysin and the vesicular glutamate transporter V-GLUT1. Immunolabeling studies revealed that SV2B is detected in rod photoreceptor synaptic terminals where it is the primary isoform. Thus, SV2B contributes to the modulation of synaptic vesicle exocytosis and plays a significant role in regulating synaptic protein content

Isotropic 3D Nuclear Morphometry of Normal, Fibrocystic and Malignant Breast Epithelial Cells Reveals New Structural Alterations

Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria.We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure.We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio) between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At p<0.0025 by ANOVA and Kruskal-Wallis tests, 90% of our computed descriptors statistically differentiated control from abnormal cell populations, but only 69% of these features statistically differentiated the fibrocystic from the metastatic cell populations.Our results provide a new perspective on nuclear structure variations associated with malignancy and point to the value of automated quantitative 3D nuclear morphometry as an objective tool to enable development of sensitive and specific nuclear grade classification in breast cancer diagnosis