Malate dehydrogenase (MDH) catalyzes a reversible NAD⁺-dependent-dehydrogenase reaction involved in central metabolism and redox homeostasis between organelle compartments. To explore the role of ...mitochondrial MDH (mMDH) in Arabidopsis (Arabidopsis thaliana), knockout single and double mutants for the highly expressed mMDH1 and lower expressed mMDH2 isoforms were constructed and analyzed. A mmdh1mmdh2 mutant has no detectable mMDH activity but is viable, albeit small and slow growing. Quantitative proteome analysis of mitochondria shows changes in other mitochondrial NAD-linked dehydrogenases, indicating a reorganization of such enzymes in the mitochondrial matrix. The slow-growing mmdh1mmdh2 mutant has elevated leaf respiration rate in the dark and light, without loss of photosynthetic capacity, suggesting that mMDH normally uses NADH to reduce oxaloacetate to malate, which is then exported to the cytosol, rather than to drive mitochondrial respiration. Increased respiratory rate in leaves can account in part for the low net CO₂ assimilation and slow growth rate of mmdh1mmdh2. Loss of mMDH also affects photorespiration, as evidenced by a lower postillumination burst, alterations in CO₂ assimilation/intercellular CO₂ curves at low CO₂, and the light-dependent elevated concentration of photorespiratory metabolites. Complementation of mmdh1mmdh2 with an mMDH cDNA recovered mMDH activity, suppressed respiratory rate, ameliorated changes to photorespiration, and increased plant growth. A previously established inverse correlation between mMDH and ascorbate content in tomato (Solanum lycopersicum) has been consolidated in Arabidopsis and may potentially be linked to decreased galactonolactone dehydrogenase content in mitochondria in the mutant. Overall, a central yet complex role for mMDH emerges in the partitioning of carbon and energy in leaves, providing new directions for bioengineering of plant growth rate and a new insight into the molecular mechanisms linking respiration and photosynthesis in plants.
Pathophysiological consequences of focal non-convulsive status epilepticus (fNCSE) have been difficult to demonstrate in humans. In rats fNCSE pathology has been identified in the eyes. Here we ...evaluated the use of high-resolution 7 T structural T1-weighted magnetic resonance imaging (MRI) and 9.4 T diffusion tensor imaging (DTI) for detecting hippocampal fNCSE-induced retinal pathology ex vivo in mice. Seven weeks post-fNCSE, increased number of Iba1+ microglia were evident in the retina ipsilateral to the hemisphere with fNCSE, and morphologically more activated microglia were found in both ipsi- and contralateral retina compared to non-stimulated control mice. T1-weighted intensity measurements of the contralateral retina showed a minor increase within the outer nuclear and plexiform layers of the lateral retina. T1-weighted measurements were not performed in the ipsilateral retina due to technical difficulties. DTI fractional anisotropy(FA) values were discretely altered in the lateral part of the ipsilateral retina and unaltered in the contralateral retina. No changes were observed in the distal part of the optic nerve. The sensitivity of both imaging techniques for identifying larger retinal alteration was confirmed ex vivo in retinitis pigmentosa mice where a substantial neurodegeneration of the outer retinal layers is evident. With MR imaging a 50 % decrease in DTI FA values and significantly thinner retina in T1-weighted images were detected. We conclude that retinal pathology after fNCSE in mice is subtle and present bilaterally. High-resolution T1-weighted MRI and DTI independently did not detect the entire pathological retinal changes after fNCSE, but the combination of the two techniques indicated minor patchy structural changes.
Purpose: To assess the additional value of subtraction ictal single‐photon emission computed tomography (SPECT) coregistered to MRI (SISCOM) for localization of the epileptogenic zone in patients ...with drug‐resistant epilepsy scheduled for invasive video‐EEG (VEEG) before epilepsy surgery by a descriptive study from clinical practice.
Methods: Forty‐nine consecutive epilepsy patients between January 2000 and March 2006 were included. Thirty‐six of the 49 patients were offered surgery, and 34 underwent resective surgery during the study period. Localizing and outcome data are presented from 31 patients with a follow‐up period of ≥12 months. Successful ictal SPECT was performed in 26 patients, and SISCOM showed significant hyperperfusions with 3.5 SD above reference. Twenty patients had SISCOM‐guided electrode placement, invasive monitoring, and 1‐year postsurgical follow‐up data. Two independent epileptologists evaluated whether SISCOM results (a) altered the hypothesis and extended the strategy for electrode placement at invasive recording, or (b) were confirmatory of other localizing data and did not alter the strategy. We defined that SISCOM had an impact on seizure outcome if the seizure‐onset zone was seen in electrodes overlying a brain region with a significant hyperperfusion. When SISCOM was concordant with ictal onset in the extended electrodes, SISCOM was considered a prerequisite for the outcome at postoperative follow‐up.
Results: SISCOM findings altered and extended the strategy for electrode placement at invasive recording in 15 patients (group A). SISCOM was a prerequisite for seizure outcome in all six patients with favorable outcomes. Nine patients had poor results from surgery in this group; SISCOM was concordant with invasive VEEG in six patients, and discordant with invasive VEEG in three patients. SISCOM findings were confirmatory with other localizing data and did not alter the strategy at invasive recording in five patients (group B). Two patients had favorable surgical outcomes. In this group, three patients had poor results; SISCOM and other localizing findings were concordant with invasive VEEG in one patient and discordant with invasive VEEG in two patients.
Conclusions: SISCOM is valuable for the identification of the epileptogenic zone in patients with drug‐resistant epilepsy scheduled for invasive VEEG. SISCOM analysis was either a prerequisite for favorable result or concordant with other localizing methods in all patients with favorable seizure outcome at 1 year of follow‐up 40%; confidence interval (CI), 19–64).
In plants, an individually darkened leaf initiates senescence much more rapidly than a leaf from a whole darkened plant. Combining transcriptomic and metabolomic approaches in Arabidopsis ...(Arabidopsis thaliana), we present an overview of the metabolic strategies that are employed in response to different darkening treatments. Under darkened plant conditions, the perception of carbon starvation drove a profound metabolic readjustment in which branched-chain amino acids and potentially monosaccharides released from cell wall loosening became important substrates for maintaining minimal ATP production. Concomitantly, the increased accumulation of amino acids with a high nitrogen-carbon ratio may provide a safety mechanism for the storage of metabolically derived cytotoxic ammonium and a pool of nitrogen for use upon returning to typical growth conditions. Conversely, in individually darkened leaf, the metabolic profiling that followed our 13C-enrichment assays revealed a temporal and differential exchange of metabolites, including sugars and amino acids, between the darkened leaf and the rest of the plant. This active transport could be the basis for a progressive metabolic shift in the substrates fueling mitochondrial activities, which are central to the catabolic reactions facilitating the retrieval of nutrients from the senescing leaf. We propose a model illustrating the specific metabolic strategies employed by leaves in response to these two darkening treatments, which support either rapid senescence or a strong capacity for survival.
The functions of mitochondria during leaf senescence, a type of programmed cell death aimed at the massive retrieval of nutrients from the senescing organ to the rest of the plant, remain elusive. ...Here, combining experimental and analytical approaches, we showed that mitochondrial integrity in Arabidopsis (Arabidopsis thaliana) is conserved until the latest stages of leaf senescence, while their number drops by 30%. Adenylate phosphorylation state assays and mitochondrial respiratory measurements indicated that the leaf energy status also is maintained during this time period. Furthermore, after establishing a curated list of genes coding for products targeted to mitochondria, we analyzed in isolation their transcript profiles, focusing on several key mitochondrial functions, such as the tricarboxylic acid cycle, mitochondrial electron transfer chain, iron-sulfur cluster biosynthesis, transporters, as well as catabolic pathways. In tandem with a metabolomic approach, our data indicated that mitochondrial metabolism was reorganized to support the selective catabolism of both amino acids and fatty acids. Such adjustments would ensure the replenishment of α-ketoglutarate and glutamate, which provide the carbon backbones for nitrogen remobilization. Glutamate, being the substrate of the strongly up-regulated cytosolic glutamine synthase, is likely to become a metabolically limiting factor in the latest stages of developmental leaf senescence. Finally, an evolutionary age analysis revealed that, while branched-chain amino acid and proline catabolism are very old mitochondrial functions particularly enriched at the latest stages of leaf senescence, auxin metabolism appears to be rather newly acquired. In summation, our work shows that, during developmental leaf senescence, mitochondria orchestrate catabolic processes by becoming increasingly central energy and metabolic hubs.
Chlorine is a toxic industrial chemical that has been used as a chemical weapon in recent armed conflicts. Confirming human exposure to chlorine has proven challenging, and there is currently no ...established method for analyzing human biomedical samples to unambiguously verify chlorine exposure. In this study, two chlorine-specific biomarkers: palmitoyl-oleoyl phosphatidylglycerol chlorohydrin (POPG-HOCl) and the lipid derivative oleoyl ethanolamide chlorohydrin (OEA-HOCl) are shown in bronchoalveolar lavage fluid (BALF) samples from spontaneously breathing pigs after chlorine exposure. These biomarkers are formed by the chemical reaction of chlorine with unsaturated phospholipids found in the pulmonary surfactant, which is present at the gas-liquid interface within the lung alveoli. Our results strongly suggest that lipid chlorohydrins are promising candidate biomarkers in the development of a verification method for chlorine exposure. The establishment of verified methods capable of confirming the illicit use of toxic industrial chemicals is crucial for upholding the principles of the Chemical Weapons Convention (CWC) and enforcing the ban on chemical weapons. This study represents the first published dataset in BALF revealing chlorine biomarkers detected in a large animal. Furthermore, these biomarkers are distinct in that they originate from molecular chlorine rather than hypochlorous acid.
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•Detection of two phospholipid biomarkers, intact POPG-HOCl and its derivate OEA-HOCl, in BALF from pig after chlorine exposure•The chlorine specific biomarkers were detectable despite mild acute lung injury•These findings is one step closer the goal of a method for verification of chlorine exposure in humans
Leaf senescence can be induced by stress or aging, sometimes in a synergistic manner. It is generally acknowledged that the ability to withstand senescence-inducing conditions can provide plants with ...stress resilience. Although the signaling and transcriptional networks responsible for a delayed senescence phenotype, often referred to as a functional stay-green trait, have been actively investigated, very little is known about the subsequent metabolic adjustments conferring this aptitude to survival. First, using the individually-darkened leaf (IDL) experimental setup, we compared IDLs of wild-type (WT) Arabidopsis (Arabidopsis thaliana) to several stay-green contexts, i.e., IDLs of two functional stay-green mutant lines, oresara1-2 (ore1-2) and an allele of phytochrome-interacting factor 5 (pif5), as well as to leaves from a WT plant entirely darkened (DP). We provide compelling evidence that arginine and ornithine, which accumulate in all stay-green contexts - likely due to the lack of induction of amino acids transport - can delay the progression of senescence by fueling the Krebs cycle or the production of polyamines (PAs). Secondly, we show that the conversion of putrescine to spermidine is controlled in an age-dependent manner. Thirdly, we demonstrate that spermidine represses senescence via interference with ethylene signaling by stabilizing the ETHYLENE BINDING FACTOR1 and 2 (EBF1/2) complex. Taken together, our results identify arginine and ornithine as central metabolites influencing the stress- and age-dependent progression of leaf senescence. We propose that the regulatory loop between the pace of the amino acid export and the progression of leaf senescence provides the plant with a mechanism to fine-tune the induction of cell death in leaves, which, if triggered unnecessarily, can impede nutrient remobilization and thus plant growth and survival.
Riot Control Agents (RCAs) are chemicals used in law enforcement for non-lethal riot control and use in conflicts between states that violates the Chemical Weapons Convention. OPCW's Scientific ...Advisory Board has identified sixteen potential RCAs including capsaicinoids, CS, and CR. RCAs may be misused for criminal purposes, so methods for detecting such misuse are needed. This study therefore evaluates the feasibility of a rapid, high throughput screening method of RCAs on surfaces (particularly clothing surfaces) by Direct Analysis in Real Time with a thermal desorption unit coupled to high-resolution mass spectrometry (DART-TD-HRMS).
A broadly applicable method for detecting potential RCAs was developed and tested on cotton fabric samples sprayed with self-defence sprays from an in-house reference stock. The feasibility of detecting RCAs by direct analysis of surface wipe samples placed in the DART source was also investigated.
The method detected all sixteen RCAs and contaminated clothing were successfully screened for active agents in a reference collection of self-defence sprays. A pilot study also showed that RCAs can be detected by holding a sample directly in front of the DART source.
DART-TD-HRMS enables rapid and simple screening of RCAs on fabric samples enabling a high sample throughput.
Chlorine is a toxic chemical that has been used as a chemical warfare agent in recent armed conflicts. There is an urgent need for methods to verify alleged uses of chlorine, and phospholipid ...chlorohydrins (PL-HOCl) derived from the pulmonary surfactant of exposed victims have previously been proposed as biomarkers of chlorine exposure. Here, we describe an improved protocol for the chemical analysis of these biomarkers and its applicability to biomedical samples from chlorine-exposed animals. By the use of a polymeric solid-phase-supported transesterification of PL-HOCl using ethanolamine, a common biomarker, oleoyl ethanolamide chlorohydrin (OEA-HOCl), was derived from all the diverse oleoyl PL-HOCl that may be formed by chlorine exposure. Compared to native lipid biomarkers, OEA-HOCl represents a larger biomarker pool and is better suited for nano-liquid chromatography--tandem mass spectrometry (nLC-MS-MS analysis), generating 3 amol Limit of Detection (LOD) and a reduced sample carry-over. With the improved protocol, significantly elevated levels of OEA-HOCl were identified in bronchoalveolar lavage fluid (BALF) of chlorine-exposed rats, 2-48 hours after exposure. The difficulty of BALF sampling from humans limits the methods usefulness as a verification tool of chlorine exposure. Conversely, nasal lavage fluid (NLF) is readily collected without advanced equipment. In NLF from chlorine-exposed rats, PL-HOCl were identified and significantly elevated levels of the OEA-HOCl biomarker were detected 2-24 hours after exposure. In order to test the potential of NLF as a biomedical sample for verification of human exposure to chlorine, in-vitro chlorination of human NLF samples was performed. All human in-vitro chlorinated NLF samples exhibited elevated OEA-HOCl biomarker levels, following sample derivatization. These data indicate the potential of human NLF as a biomedical sample for the verification of chlorine exposure, but further work is required to develop and validate the method for the use on real-world samples.
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