A critical role of plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase in the control of plant metabolism and development

Glycolysis is a central metabolic pathway that provides energy and generates precursors for the synthesis of primary metabolites such as amino acids and fatty acids.^1–3 In plants, glycolysis occurs in the cytosol and plastids, which complicates the understanding of this essential process.¹ As a result, the contribution of each glycolytic pathway to the specific primary metabolite production and the degree of integration of both pathways is still unresolved. The glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. Both cytosolic (GAPCs) and plastidial (GAPCps) GAPDH activities have been described biochemically. But, up to now, little attention had been paid to GAPCps, probably because they have been considered as “minor isoforms” that catalyze a reversible reaction in plastids where it has been assumed that key glycolytic intermediates are in equilibrium with the cytosol. In the associated study,⁴ we have elucidated the crucial role of Arabidopsis GAPCps in the control of primary metabolism in plants. GAPCps deficiency affects amino acid and sugar metabolism and impairs plant development. Specifically, GAPCp deficiency affects the serine supply to roots, provoking a drastic phenotype of arrested root development. Also, we show that the phosphorylated serine biosynthesis pathway is critical to supply serine to non-photosynthetic organs such as roots. These studies provide new insights of the contribution of plastidial glycolysis to plant metabolism and evidence the complex interactions existing between metabolism and development.Key words: GAPDH, glycolysis, serine biosynthesis, Arabidopsis, plastid     

Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance

Introduction  

Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology.     

The pGRT1 plasmid of Pseudomonas putida DOT-T1E encodes functions relevant for survival under harsh conditions in the environment

Pseudomonas putida DOT-T1E has the capacity to grow in the presence of high concentrations of toluene. This ability is mainly conferred by an efflux pump encoded in a self-transmissible 133 kb plasmid named pGRT1. Sequence analysis of the pGRT1 plasmid revealed several key features. Most of the genes related to the plasmid maintenance functions show similarity with those encoded on pBVIE04 from Burkholderia vietnamensis G4, and knock-out mutants in several of these genes confirmed their roles. Two additional plasmid DNA fragments were incorporated into the plasmid backbone by recombination and/or transposition; in these DNA regions, apart from multiple recombinases and transposases, several stress-related and environmentally relevant functions are encoded. We report that plasmid pGRT1 not only confers the cells with tolerance to toluene but also resistance to ultraviolet light. We show here the implication of a new protein in solvent tolerance which controls the level of expression of the TtgGHI efflux pump, as well as the implication of a protein with homology to the universal stress protein in solvent tolerance and ultraviolet light resistance. Furthermore, this plasmid encodes functions that allow the cells to chemotactically respond to toluene and participate in iron scavenging.     

Microfluidic reactor for continuous cultivation of Saccharomyces cerevisiae

A diffusion-based microreactor system operated with a reaction volume of 8 μL is presented and characterized to intensify the process understanding in microscale cultivations. Its potential as screening tool for biological processes is evaluated. The advantage of the designed microbioreactor is the use for the continuous cultivation mode by integrating online measurement technique for dissolved oxygen (DO) and optical density (OD). A further advantage is the broaden application for biological systems. The bioreactor geometry was chosen to achieve homogeneous flow during continuous process operation. The device consisted of a microstructured top layer made of poly(dimethylsiloxane) (PDMS), which was designed and fabricated using UV-depth and soft lithography assembled with a glass bottom. CFD simulation data used for geometry design were verified via microparticle-image-velocimetry (μPIV). In the used microreactor geometry no concentration gradients occurred along the entire reaction volume because of rapid diffusive mixing, the homogeneous medium flow inside the growth chamber of the microreactor could be realized. Undesirable bubble formation before and during operation was reduced by using degassed medium as well as moistened and moderate incident air flow above the gas permeable PDMS membrane. Because of this a passive oxygen supply of the culture medium in the device is ensured by diffusion through the PDMS membrane. The oxygen supply itself was monitored online via integrated DO sensors based on a fluorescent dye complex. An adequate overall volumetric oxygen transfer coefficient K(L)a as well as mechanical stability of the device were accomplished for a membrane thickness of 300 μm. Experimental investigations considering measurements of OD (online) and several metabolite concentrations (offline) in a modified Verduyn medium. The used model organism Saccharomyces cerevisiae DSM 2155 tended to strong reactor wall growth resembling a biofilm.     

Similar hypotensive responses to resistance exercise with and without blood flow restriction

Low intensity resistance exercise (RE) with blood flow restriction (BFR) has gained attention in the literature due to the beneficial effects on functional and morphological variables, similar to those observed during traditional RE without BFR, while the effects of BFR on post-exercise hypotension remain unclear. The aim of the present study was to compare the blood pressure (BP) response of trained normotensive individuals to RE with and without BFR. In this cross-over randomized trial, eight male subjects (23.8 ± 4 years, 74 ± 3 kg, 174 ± 4 cm) completed two exercise protocols: traditional RE (3 x 10 repetitions at 70% one-repetition maximum [1-RM]) and low intensity RE (3 x 15 repetitions at 20% 1-RM) with BFR. Blood pressure measurements were performed after 15 min of seated rest (0), immediately after and 10 min, 20 min, 30 min, 40 min, 50 min and 60 min after the experimental sessions. Similar hypotensive effects for systolic BP (SBP) were observed for both protocols (P < 0.05) after exercise, with no differences between groups (P > 0.05) and no statistically significant difference for diastolic BP (P > 0.05). These results suggest that in normotensive trained individuals, both traditional RE and RE with BFR induce hypotension for SBP, which is important to prevent cardiovascular disturbances.     

Biotransformation in double-phase systems: physiological responses of Pseudomonas putida DOT-T1E to a double phase made of aliphatic alcohols and biosynthesis of substituted catechols

Pseudomonas putida strain DOT-T1E is highly tolerant to organic solvents, with a logP(ow) (the logarithm of the partition coefficient of a solvent in a two-phase water-octanol system of > or =2.5. Solvent tolerant microorganisms can be exploited to develop double-phase (organic solvent and water) biotransformation systems in which toxic substrates or products are kept in the organic phase. We tested P. putida DOT-T1E tolerance to different aliphatic alcohols with a logP(ow) value between 2 and 4, such as decanol, nonanol, and octanol, which are potentially useful in biotransformations in double-phase systems in which compounds with a logP(ow) around 1.5 are produced. P. putida DOT-T1E responds to aliphatic alcohols as the second phase through cis-to-trans isomerization of unsaturated cis fatty acids and through efflux of these aliphatic alcohols via a series of pumps that also extrude aromatic hydrocarbons. These defense mechanisms allow P. putida DOT-T1E to survive well in the presence of high concentrations of the aliphatic alcohols, and growth with nonanol or decanol occurred at a high rate, whereas in the presence of an octanol double-phase growth was compromised. Our results support that the logP(ow) of aliphatic alcohols correlates with their toxic effects, as octanol (logP(ow) = 2.9) has more negative effects in P. putida cells than 1-nonanol (logP(ow) = 3.4) or 1-decanol (logP(ow) = 4). A P. putida DOT-T1E derivative bearing plasmid pWW0-xylE::Km transforms m-xylene (logP(ow) = 3.2) into 3-methylcatechol (logP(ow) = 1.8). The amount of 3-methylcatechol produced in an aliphatic alcohol/water bioreactor was 10- to 20-fold higher than in an aqueous medium, demonstrating the usefulness of double-phase systems for this particular biotransformation.     

Accuracy of Monoclonal Stool Tests for Determining Cure of Helicobacter pylori Infection After Treatment

Background: Studies comparing new monoclonal fecal tests for evaluating cure of Helicobacter pylori infection after treatment are scarce. The objective was to compare the diagnostic accuracy of three monoclonal stool tests: two rapid in‐office tools –RAPID Hp StAR and ImmunoCard STAT! HpSA – and an EIA test – Amplified IDEIA Hp StAR. Materials and methods: Diagnostic reliability of the three tests was evaluated in 88 patients at least 8 weeks after H. pylori treatment. Readings of immunochromatographic tests were performed by two different observers. Sensitivity, specificity, positive and negative predictive values and 95% confidence intervals were calculated. Results: All tests presented similar performance for post‐eradication testing. Sensitivity for detecting persistent infection was 100% for both Amplified IDEIA and RAPID Hp StAR and 90% for ImmunoCard STAT! HpSA. Respective specificities were 94.9%, 92.3–93.6% and 94.9%. Negative predictive values were very high (100%, 100% and 98.7% respectively). But positive predictive values were lower, ranging from 62.5 to 71.4%. Conclusion: All monoclonal fecal tests in this series presented similar performance in the post‐treatment setting. A negative test after treatment adequately predicted cure of the infection. However, nearly a third of tests were false positive, showing a poor predictive yield for persistent infection.     

The GATC-binding protein SeqA is required for bile resistance and virulence in Salmonella enterica serovar typhimurium

Disruption of the seqA gene of Salmonella enterica serovar Typhimurium causes defects similar to those described in E. coli: filament formation, aberrant nucleoid segregation, induction of the SOS response, envelope instability, and increased sensitivity to membrane-damaging agents. Differences between SeqA⁻ mutants of E. coli and S. enterica, however, are found. SeqA⁻ mutants of S. enterica form normal colonies and do not exhibit alterations in phage plaquing morphology. Lack of SeqA causes attenuation of S. enterica virulence by the oral route but not by the intraperitoneal route, suggesting a virulence defect in the intestinal stage of infection. However, SeqA⁻ mutants are fully proficient in the invasion of epithelial cells. We hypothesize that attenuation of SeqA⁻ mutants by the oral route may be caused by bile sensitivity, which in turn may be a consequence of envelope instability.