Mechanisms of expression of pyruvate dehydrogenase deficiency caused by an E1alpha subunit mutation

OBJECTIVE: To characterize the biochemical mechanisms of expression of the pyruvate dehydrogenase (PDH) E1alpha subunit exon 10 R302C missense mutation. BACKGROUND: Mutations in the X-linked E1alpha subunit gene are responsible for most cases of PDH deficiency, an important cause of neurodevelopmental defects and neurodegeneration with primary lactic acidemia. Although the disease shows extreme allelic heterogeneity, the R302C mutation has been defined in several unrelated cases. METHODS: Cell lines expressing selectively either the mutant or wild-type E1alpha alleles against identical genetic backgrounds were generated from the fibroblasts of a female heterozygous for the R302C mutation. Enzyme activity, mRNA, polypeptide expression, and turnover were studied in each. RESULTS: The residual PDH activity was below measurable levels in the cell line (B5) expressing only the mutant allele and normal in the wild-type polypeptide expressing (A10) cell line, confirming that the R302C mutation alone is sufficient to cause a severe PDH deficiency. The mutant polypeptide was less stable than the wild-type polypeptide, but the steady-state level of the mutant E1alpha protein was reduced only two- to threefold. CONCLUSIONS: The primary mechanism of expression of the R302C mutation must be limitation of catalytic efficiency. We speculate that catalysis may be inhibited in the mutant polypeptide because conformational changes are induced near serine 300, a residue that is particularly important as a regulatory phosphorylation site in the wild-type polypeptide.     

Sentic Computing for social media marketing

In a world in which millions of people express their opinions about commercial products in blogs, wikis, fora, chats and social networks, the distillation of knowledge from this huge amount of unstructured information can be a key factor for marketers who want to create an image or identity in the minds of their customers for their product, brand or organization. Opinion mining for product positioning, in fact, is getting a more and more popular research field but the extraction of useful information from social media is not a simple task. In this work we merge AI and Semantic Web techniques to extract, encode and represent this unstructured information. In particular, we use Sentic Computing, a multi-disciplinary approach to opinion mining and sentiment analysis, to semantically and affectively analyze text and encode results in a semantic aware format according to different web ontologies. Eventually we represent this information as an interconnected knowledge base which is browsable through a multi-faceted classification website.     

Characterization of Urine Stem Cell-Derived Extracellular Vesicles Reveals B Cell Stimulating Cargo

Elucidation of the biological functions of extracellular vesicles (EVs) and their potential roles in physiological and pathological processes is an expanding field of research. In this study, we characterized USC–derived EVs and studied their capacity to modulate the human immune response in vitro. We found that the USC–derived EVs are a heterogeneous population, ranging in size from that of micro–vesicles (150 nm–1 μm) down to that of exosomes (60–150 nm). Regarding their immunomodulatory functions, we found that upon isolation, the EVs (60–150 nm) induced B cell proliferation and IgM antibody secretion. Analysis of the EV contents unexpectedly revealed the presence of BAFF, APRIL, IL–6, and CD40L, all known to play a central role in B cell stimulation, differentiation, and humoral immunity. In regard to their effect on T cell functions, they resembled the function of mesenchymal stem cell (MSC)–derived EVs previously described, suppressing T cell response to activation. The finding that USC–derived EVs transport a potent bioactive cargo opens the door to a novel therapeutic avenue for boosting B cell responses in immunodeficiency or cancer.     

The Prominin-1-Derived Peptide Improves Cardiac Function Following Ischemia

Myocardial infarction (MI) remains the leading cause of death in the western world. Despite advancements in interventional revascularization technologies, many patients are not candidates for them due to comorbidities or lack of local resources. Non-invasive approaches to accelerate revascularization within ischemic tissues through angiogenesis by providing Vascular Endothelial Growth Factor (VEGF) in protein or gene form has been effective in animal models but not in humans likely due to its short half-life and systemic toxicity. Here, we tested the hypothesis that PR1P, a small VEGF binding peptide that we developed, which stabilizes and upregulates endogenous VEGF, could be used to improve outcome from MI in rodents. To test this hypothesis, we induced MI in mice and rats via left coronary artery ligation and then treated animals with every other day intraperitoneal PR1P or scrambled peptide for 14 days. Hemodynamic monitoring and echocardiography in mice and echocardiography in rats at 14 days showed PR1P significantly improved multiple functional markers of heart function, including stroke volume and cardiac output. Furthermore, molecular biology and histological analyses of tissue samples showed that systemic PR1P targeted, stabilized and upregulated endogenous VEGF within ischemic myocardium. We conclude that PR1P is a potential non-invasive candidate therapeutic for MI.     

Soluble Epoxide Hydrolase Blockade after Stroke Onset Protects Normal but Not Diabetic Mice

Soluble epoxide hydrolase (sEH) is abundant in the brain, is upregulated in type 2 diabetes mellitus (DM2), and is possible mediator of ischemic injury via the breakdown of neuroprotective epoxyeicosatrienoic acids (EETs). Prophylactic, pre-ischemic sEH blockade with 4-[[trans-4-[[(tricyclo[3.3.1.13,7]dec-1-ylamino)carbonyl]amino]cyclohexyl]oxy]-benzoic acid (tAUCB) reduces stroke-induced infarct in normal and diabetic mice, with larger neuroprotection in DM2. The present study tested whether benefit occurs in normal and DM2 mice if tAUCB is administered after stroke onset. We performed 60 min middle cerebral artery occlusion in young adult male C57BL mice divided into four groups: normal or DM2, with t-AUCB 2 mg/kg or vehicle 30 min before reperfusion. Endpoints were (1) cerebral blood flow (CBF) by laser Doppler, and (2) brain infarct at 24 h. In nondiabetic mice, t-AUCB reduced infarct size by 30% compared to vehicle-treated mice in the cortex (31.4 ± 4 vs. 43.8 ± 3 (SEM)%, respectively) and 26% in the whole hemisphere (26.3 ± 3 vs. 35.2 ± 2%, both p < 0.05). In contrast, in DM2 mice, tAUCB failed to ameliorate either cortical or hemispheric injury. No differences were seen in CBF. We conclude that tAUCB administered after ischemic stroke onset exerts brain protection in nondiabetic but not DM2 mice, that the neuroprotection appears independent of changes in gross CBF, and that DM2-induced hyperglycemia abolishes t-AUCB-mediated neuroprotection after stroke onset.     

PGRP-LB: An Inside View into the Mechanism of the Amidase Reaction

Peptidoglycan recognition proteins (PGRPs) are ubiquitous among animals and play pivotal functions in insect immunity. Non-catalytic PGRPs are involved in the activation of immune pathways by binding to the peptidoglycan (PGN), whereas amidase PGRPs are capable of cleaving the PGN into non-immunogenic compounds. Drosophila PGRP-LB belongs to the amidase PGRPs and downregulates the immune deficiency (IMD) pathway by cleaving meso-2,6-diaminopimelic (meso-DAP or DAP)-type PGN. While the recognition process is well analyzed for the non-catalytic PGRPs, little is known about the enzymatic mechanism for the amidase PGRPs, despite their essential function in immune homeostasis. Here, we analyzed the specific activity of different isoforms of Drosophila PGRP-LB towards various PGN substrates to understand their specificity and role in Drosophila immunity. We show that these isoforms have similar activity towards the different compounds. To analyze the mechanism of the amidase activity, we performed site directed mutagenesis and solved the X-ray structures of wild-type Drosophila PGRP-LB and its mutants, with one of these structures presenting a protein complexed with the tracheal cytotoxin (TCT), a muropeptide derived from the PGN. Only the Y78F mutation abolished the PGN cleavage while other mutations reduced the activity solely. Together, our findings suggest the dynamic role of the residue Y78 in the amidase mechanism by nucleophilic attack through a water molecule to the carbonyl group of the amide function destabilized by Zn²⁺.     

Physicochemical Properties of Palm Oil-Based Puff Pastry Model Margarines Related to Their Baking Performance in Long-Term Storage

Developing trans-free alternative fat solutions suitable for specific applications remains a challenge in edible fats and other domains. This is particularly true for palm oil-based puff pastry margarines, which suffer from post crystallization problems, leading to dramatic loss of functionality. This research is aimed at investigating the influence of triacylglycerol (TAG) compositions of palm oil-based puff pastry margarines on the physical properties of the fat crystal network, which determine the functionality of such products. Three model puff pastry margarines are produced at pilot scale under the same crystallization conditions. They share the same fatty acid composition and close solid fat content (SFC) profiles, whereas the proportions of major TAG (tripalmitoylglycerol (PPP), 1,3-di-palmitoyl-2-oleoylglycerol (POP), 1,2-di-palmitoyl-3-oleoylglycerol (PPO), 1,2-dioleoyl-3-palmitoylglycerol (POO)) are different. Polymorphism, melting profile, hardness, microscopic structures, and baking performance (puffing effect) of the model fats are examined during a period of 6 months. The following results are obtained: 1) The TAG composition significantly influences the post crystallization processes occurring in palm oil-based margarines. 2) High amounts of POP show negative influences. 3) The proportions of POP, PPO, and PPP should be carefully balanced to prevent detrimental crystal network rearrangements, leading to textural modifications (hardness increase) and significant reduction in baking performance. Practical Applications : The results presented in this work could be helpful for edible fat products developers, especially for roll-in fat applications. This research provides an overview of the relevant properties to study for the assessment of puff pastry margarine functionality. It also highlights the importance of ensuring long-term stability of palm oil-based fat products. Finally, it emphasizes that certain combinations of fat materials should be avoided to maintain the quality of palm oil-based puff pastry margarines.     

Countercurrent approach to the enrichment of Δ9c,11t-and Δ10t,12c-18:2 isomers by urea complexation

CLA refers to a group of geometrical and positional isomers of linoleic acid. CLA has been shown to have potentially beneficial effects on cancer, atherosclerosis, and body metabolism in animals. Mixtures containing equal amounts of these isomers are commonly used in many research studies because of their greater availability and lower cost relative to pure isomers. This has hindered progress in elucidating the biological properties of specific isomers and their relevance in animal and human biology. A method was developed that offers a compromise between cost and utility to make available enriched mixtures of either the Δ9c,11t- or Δ10t,12c-18:2 isomers for use in a wide range of experimental applications. A countercurrent approach was developed to separate the Δ9c,11t- and Δ10t,12c-18:2 isomers from an equal mixture of these two isomers by urea complexation. After three successive rounds of complexation using an equal amount of CLA and urea, a fraction enriched in Δ9c,11t-18:2 containing 42.5 and 17.4% of Δ9c,11t-and Δ10t,12c-18:2, respectively, was recovered. After a single round of complexation using 2.5 g urea/g CLA, a fraction enriched in Δ10t,12c-18:2 was recovered containing 29.7 and 69.1% of Δ9c,11t- and Δ10t,12c-18:2, respectively.     

Aromatic products of 340 °C supercritical-toluene extraction of two Turkish lignites: an n.m.r. study

Fractions of Elbistan and Seyitomer (Turkish) lignites, extracted with supercritical toluene at 340 °C and 8 MPa, have been separated by solvent extraction and silica-gel chromatography. Analyses by n.m.r. and i.r. spectroscopies and other methods have been combined in structural-analysis schemes to yield information about the average molecule in aromatic extracts. Carbon aromaticities, f_a, derived from 22.63 MHz ¹H-decoupled pulse Fourier-transform (PFT) ¹³C-n.m.r. are more widely spread for Elbistan (0.34–0.56) than for Seyitomer (0.40–0.43), and are lower than for supercritical-gas (SCG) products from bituminous coals. ¹³C-n.m.r. also reveals the presence of aromatic ether-O in polar fractions. Narrow aromatic signals in 100 MHz ¹H-n.m.r. spectra suggest the presence of single-aromatic-ring average structures. In the hexane-soluble aromatics, 27% (Elbistan) and 29% (Seyitomer) of the available sites are substituted by alkyI groups, some of which are at least eight carbon atoms long; the hexane-soluble polar and asphaltene/asphaltol fractions contain fewer such groups.     

The influence of soil properties on the effectiveness of phenylphosphorodiamidate (PPD) in reducing ammonia volatilization from surface applied urea

Urea can be an inefficient N source due to rapid hydrolysis by soil urease leading to NH₃ volatilization. The current study investigated the effect of the urease inhibitor phenylphosphorodiamidate (PPD) incorporated at two concentrations (0.5% and 1% w/w) within the fertilizer granule on NH₃ volatilization from surface applied urea. The daily rates of NH₃ loss from 20 soils of widely differing properties from Northern Ireland were measured over 14 days using ventilated enclosures under simulated spring conditions. Cumulative loss rates were calculated and fitted to a logistic model from which total NH₃ loss (Amax) and the time to maximum rate of loss (Tmax) were determined. Stepwise multiple linear regression analysis related the effectiveness of PPD in reducing NH₃ volatilization from urea to soil properties.The total cumulative loss of ammonia from unamended urea varied from 0.37 to 29.2% depending on soil type. Ammonia volatilization appeared to be greatest on a soil with a high pH (R² = 0.65), a low titratable acidity (TA) (R² = 0.63) and a soil that was drying out (R² = 0.50). Soil pH was negatively correlated with TA (r = –0.826, P < 0.001) suggesting that soils with a low TA may have received recent lime. Including cation exchange capacity (CEC) and % N as well as pH-KCl in the multiple linear regression equation explained 86% of the variance.The effectiveness of PPD in reducing Amax varied between 0% to 91% depending on soil type, the average over all 20 soils being 30 and 36% for 0.5% and 1% PPD respectively. The most important soil properties influencing the effectiveness of the urease inhibitor were soil pH-H₂O and TA accounting for 33% and 29% of the variance respectively. PPD was less effective on a soil with a high pH and low TA. These were the soil conditions that led to high NH₃ volatilization from unamended urea and may explain why PPD had limited success in reducing ammonia loss on these soils. Multiple linear regression analysis indicated that 75% of the variation in the % inhibition of NH₃ loss by PPD could be significantly accounted for by pH-H₂O, initial soil NO ₃ ^- -N concentration, % moisture content and % moisture loss.The delay in Tmax by PPD ranged from 0.19 to 7.93 days, the average over all 20 soils being 2.5 and 2.8 days for 0.5% and 1% PPD respectively. TA, % moisture content, urease activity and CEC were soil properties that significantly explained 83% of the variation in the % delay in Tmax by PPD in multiple linear regression analysis. However, none of these soil properties were significant on their own. As urea hydrolysis occurs rapidly in soil, delaying Tmax under field conditions would increase the chance of rain falling to move the urea below the soil surface and reduce NH₃ volatilization. A urease inhibitor should be more effective than PPD on soils with a high pH and low TA to be successful in reducing high NH₃ losses.