Thiamin pyrophosphokinase is required for thiamin cofactor activation in Arabidopsis

Thiamin pyrophosphate (TPP) is an essential enzyme cofactor required for the viability of all organisms. Whether derived from exogenous sources or through de novo synthesis, thiamin must be pyrophosphorylated for cofactor activation. The enzyme thiamin pyrophosphokinase (TPK) catalyzes the conversion of free thiamin to TPP in plants and other eukaryotic organisms and is central to thiamin cofactor activation. While TPK activity has been observed in a number of plant species, the corresponding gene/protein has until now not been identified or characterized for its role in thiamin metabolism. Here we report the functional identification of two Arabidopsis TPK genes, AtTPK1 and AtTPK2 and the enzymatic characterization of the corresponding proteins. AtTPK1 and AtTPK2 are biochemically redundant cytosolic proteins that are similarly expressed throughout different plant tissues. The essential nature of TPKs in plant metabolism is reflected in the observation that while single gene knockouts of either AtTPK1 or AtTPK2 were viable, the double mutant possessed a seedling lethal phenotype. HPLC analysis revealed the double mutant is nearly devoid of TPP and instead accumulates the precursor of the TPK reaction, free thiamin. These results suggest that TPK activity provides the sole mechanism by which exogenous and de novo derived thiamin is converted to the enzyme cofactor TPP.     

Lacrimo-auriculo-dento-digital syndrome is caused by reduced activity of the fibroblast growth factor 10 (FGF10)-FGF receptor 2 signaling pathway

Lacrimo-auriculo-dento-digital (LADD) syndrome is characterized by abnormalities in lacrimal and salivary glands, in teeth, and in the distal limbs. Genetic studies have implicated heterozygous mutations in fibroblast growth factor 10 (FGF10) and in FGF receptor 2 (FGFR2) in LADD syndrome. However, it is not clear whether LADD syndrome mutations (LADD mutations) are gain- or loss-of-function mutations. In order to reveal the molecular mechanism underlying LADD syndrome, we have compared the biological properties of FGF10 LADD and FGFR2 LADD mutants to the activities of their normal counterparts. These experiments show that the biological activities of three different FGF10 LADD mutants are severely impaired by different mechanisms. Moreover, haploinsufficiency caused by defective FGF10 mutants leads to LADD syndrome. We also demonstrate that the tyrosine kinase activities of FGFR2 LADD mutants expressed in transfected cells are strongly compromised. Since tyrosine kinase activity is stimulated by ligand-induced receptor dimerization, FGFR2 LADD mutants may also exert a dominant inhibitory effect on signaling via wild-type FGFR2 expressed in the same cell. These experiments underscore the importance of signal strength in mediating biological responses and that relatively small changes in receptor signaling may influence the outcome of developmental processes in cells or organs that do not possess redundant signaling pathway.     

Design,synthesis and SAR of a novel series of heterocyclic phenylpropanoic acids as GPR120 agonists

A novel series of 5-membered heterocycle-containing phenylpropanoic acid derivatives was discovered as potent GPR120 agonists with low clearance, high oral bioavailability and in vivo antidiabetic activity in rodents.     

Computational Tension Mapping of Adherent Cells Based on Actin Imaging

Forces transiting through the cytoskeleton are known to play a role in adherent cell activity. Up to now few approaches haves been able to determine theses intracellular forces. We thus developed a computational mechanical model based on a reconstruction of the cytoskeleton of an adherent cell from fluorescence staining of the actin network and focal adhesions (FA). Our custom made algorithm converted the 2D image of an actin network into a map of contractile interactions inside a 2D node grid, each node representing a group of pixels. We assumed that actin filaments observed under fluorescence microscopy, appear brighter when thicker, we thus presumed that nodes corresponding to pixels with higher actin density were linked by stiffer interactions. This enabled us to create a system of heterogeneous interactions which represent the spatial organization of the contractile actin network. The contractility of this interaction system was then adapted to match the level of force the cell truly exerted on focal adhesions; forces on focal adhesions were estimated from their vinculin expressed size. This enabled the model to compute consistent mechanical forces transiting throughout the cell. After computation, we applied a graphical approach on the original actin image, which enabled us to calculate tension forces throughout the cell, or in a particular region or even in single stress fibers. It also enabled us to study different scenarios which may indicate the mechanical role of other cytoskeletal components such as microtubules. For instance, our results stated that the ratio between intra and extra cellular compression is inversely proportional to intracellular tension.     

Simultaneous absence of surfactant proteins A and D increases lung inflammation and injury after allogeneic HSCT in mice

The relative contributions of the hydrophilic surfactant proteins (SP)-A and -D to early inflammatory responses associated with lung dysfunction after experimental allogeneic hematopoietic stem cell transplantation (HSCT) were investigated. We hypothesized that the absence of SP-A and SP-D would exaggerate allogeneic T cell-dependent inflammation and exacerbate lung injury. Wild-type, SP-D-deficient (SP-D(-/-)), and SP-A and -D double knockout (SP-A/D(-/-)) C57BL/6 mice were lethally conditioned with cyclophosphamide and total body irradiation and given allogeneic bone marrow plus donor spleen T cells, simulating clinical HSCT regimens. On day 7, after HSCT, permeability edema progressively increased in SP-D(-/-) and SP-A/D(-/-) mice. Allogeneic T cell-dependent inflammatory responses were also increased in SP-D(-/-) and SP-A/D(-/-) mice, but the altered mediators of inflammation were not identical. Compared with wild-type, bronchoalveolar lavage fluid (BALF) levels of nitrite plus nitrate, GM-CSF, and MCP-1, but not TNF-alpha and IFN-gamma, were higher in SP-D-deficient mice before and after HSCT. In SP-A/D(-/-) mice, day 7 post-HSCT BALF levels of TNF-alpha and IFN-gamma, in addition to nitrite plus nitrate and MCP-1, were higher compared with mice lacking SP-D alone. After HSCT, both SP-A and SP-D exhibited anti-inflammatory lung-protective functions that were not completely redundant in vivo.     

The DNAJA2 Substrate Release Mechanism Is Essential for Chaperone-mediated Folding

DNAJA1 (DJA1/Hdj2) and DNAJA2 (DJA2) are the major J domain partners of human Hsp70/Hsc70 chaperones. Although they have overall similarity with the well characterized type I co-chaperones from yeast and bacteria, they are biologically distinct, and their functional mechanisms are poorly characterized. We identified DJA2-specific activities in luciferase folding and repression of human ether-a-go-go-related gene (HERG) trafficking that depended on its expression levels in cells. Mutations in different internal domains of DJA2 abolished these effects. Using purified proteins, we addressed the mechanistic defects. A mutant lacking the region between the zinc finger motifs (DJA2-Δm2) was able to bind substrate similar to wild type but was incapable of releasing substrate during its transfer to Hsc70. The equivalent mutation in DJA1 also abolished its substrate release. A DJA2 mutant (DJA-221), which had its C-terminal dimerization region replaced by that of DJA1, was inactive but retained its ability to release substrate. The release mechanism required the J domain and ATP hydrolysis by Hsc70, although the nucleotide dependence diverged between DJA2 and DJA1. Limited proteolysis suggested further conformational differences between the two wild-type co-chaperones and the mutants. Our results demonstrate an essential role of specific DJA domains in the folding mechanism of Hsc70.     

Model uncertainty analysis using data analytics for life-cycle assessment (LCA) applications

Purpose

Objective uncertainty quantification (UQ) of a product life-cycle assessment (LCA) is a critical step for decision-making. Environmental impacts can be measured directly or by using models. Underlying mathematical functions describe a model that approximate the environmental impacts during various LCA stages. In this study, three possible uncertainty sources of a mathematical model, i.e., input variability, model parameter (differentiate from input in this study), and model-form uncertainties, were investigated. A simple and easy to implement method is proposed to quantify each source.

Methods

Various data analytics methods were used to conduct a thorough model uncertainty analysis; (1) Interval analysis was used for input uncertainty quantification. A direct sampling using Monte Carlo (MC) simulation was used for interval analysis, and results were compared to that of indirect nonlinear optimization as an alternative approach. A machine learning surrogate model was developed to perform direct MC sampling as well as indirect nonlinear optimization. (2) A Bayesian inference was adopted to quantify parameter uncertainty. (3) A recently introduced model correction method based on orthogonal polynomial basis functions was used to evaluate the model-form uncertainty. The methods are applied to a pavement LCA to propagate uncertainties throughout an energy and global warming potential (GWP) estimation model; a case of a pavement section in Chicago metropolitan area was used.

Results and discussion

Results indicate that each uncertainty source contributes to the overall energy and GWP output of the LCA. Input uncertainty was shown to have significant impact on overall GWP output; for the example case study, GWP interval was around 50%. Parameter uncertainty results showed that an assumption of ±?10% uniform variation in the model parameter priors resulted in 28% variation in the GWP output. Model-form uncertainty had the lowest impact (less than 10% variation in the GWP). This is because the original energy model is relatively accurate in estimating the energy. However, sensitivity of the model-form uncertainty showed that even up to 180% variation in the results can be achieved due to lower original model accuracies.

Conclusions

Investigating each uncertainty source of the model indicated the importance of the accurate characterization, propagation, and quantification of uncertainty. The outcome of this study proposed independent and relatively easy to implement methods that provide robust grounds for objective model uncertainty analysis for LCA applications. Assumptions on inputs, parameter distributions, and model form need to be justified. Input uncertainty plays a key role in overall pavement LCA output. The proposed model correction method as well as interval analysis were relatively easy to implement. Research is still needed to develop a more generic and simplified MCMC simulation procedure that is fast to implement.

     

Genetic polymorphism of <Emphasis Type="Italic">CYP2C19</Emphasis> in a Jordanian population: influence of allele frequencies of <Emphasis Type="Italic">CYP2C19*1</Emphasis> and <Emphasis Type="Italic">CYP2C19*2</Emphasis> on the pharmacokinetic profile of lansoprazole

To relate the pharmacokinetics of orally administered lansoprazole in healthy adult Jordanian men with CYP2C19 polymorphisms and to determine the percentage of CYP2C19 polymorphism in Jordanian population and the allelic frequency of CYP2C19*2 and CYP2C19*3. A total of 78 healthy Jordanian volunteers were included in this study from three different bioequivalence studies, one of these studies which included 26 volunteers was done on lansoprazole. Genotyping for CYP2C19*1, CYP2C19*2, CYP2C19*3 was done for all 78 volunteers, the data of genotyping of all subjects used for screening the frequency of different genotypes and the allelic frequency of different polymorphisms in healthy Jordanian men, the pharmacokinetics and genotyping data for the study of lansoprazole was matched and compared to investigate presence of statistical differences in pharmacokinetic parameters. In Jordanian subjects, the allele frequencies of the CYP2C19*2 and CYP2C19*3 mutation were 0.16 and 0, respectively. The concentration–time curves in the two groups [homozygote extensive metabolizer (homEM, n = 19) and heterozygote extensive metabolizer (homEM, n = 7)] groups were fitted to a non-compartment model. In the homEM and in the hetEM groups, the main kinetic parameters were as follows: T_max (2.1875 ± 0.777) and (2.54 ± 1.87) h, C_max (697.875 ± 335) and (833.58 ± 436.26) mg/l, t_1/2 (1.3 ± 0.43) and (2.38 ± 1.64) h, AUC_(0→∞) were (1,684.9 ± 888) and (3,609.8 ± 318) mg h l⁻¹, respectively. The Jordanian population showed similarities in CYP2C19 allele and genotype distribution pattern with Caucasians and Africans. CYP2C19 allele and poor metabolizer (PM) genotype frequencies in the Jordanian population are distinct from populations’ from East Asia such as Japanese and Koreans. Although lower pharmacokinetic parameters were found in homEM compared to hetEM but there was no significant difference between the two groups (P < 0.05).     

A roadmap to Durable BCTV Resistance Using Long-Read Genome Assembly of Genetic Stock KDH13

Plant Molecular Biology Reporter - Beet Curly Top (BCT) is a viral disease which negatively impacts crop productivity for sugar beet growers and the sugar beet industry in the western USA and dry...     

Oxidases and peroxidases in cardiovascular and lung disease: new concepts in reactive oxygen species signaling

Reactive oxygen species (ROS) are involved in numerous physiological and pathophysiological responses. Increasing evidence implicates ROS as signaling molecules involved in the propagation of cellular pathways. The NADPH oxidase (Nox) family of enzymes is a major source of ROS in the cell and has been related to the progression of many diseases and even environmental toxicity. The complexity of this family's effects on cellular processes stems from the fact that there are seven members, each with unique tissue distribution, cellular localization, and expression. Nox proteins also differ in activation mechanisms and the major ROS detected as their product. To add to this complexity, mounting evidence suggests that other cellular oxidases or their products may be involved in Nox regulation. The overall redox and metabolic status of the cell, specifically the mitochondria, also has implications on ROS signaling. Signaling of such molecules as electrophilic fatty acids has an impact on many redox-sensitive pathologies and thus, as anti-inflammatory molecules, contributes to the complexity of ROS regulation. This review is based on the proceedings of a recent international Oxidase Signaling Symposium at the University of Pittsburgh's Vascular Medicine Institute and Department of Pharmacology and Chemical Biology and encompasses further interaction and discussion among the presenters.