Anterograde transport of late endosomes or lysosomes (LE/Lys) is crucial for proper axon growth. However, the role of energetic nutrients has been poorly explored. Malonyl-CoA is a precursor of fatty ...acids, and its intracellular levels highly fluctuate depending on glucose availability or the energy sensor AMP-activated protein kinase (AMPK). We demonstrate in HeLa cells that carnitine palmitoyltransferase 1C (CPT1C) senses malonyl-CoA and enhances LE/Lys anterograde transport by interacting with the endoplasmic reticulum protein protrudin and facilitating the transfer of Kinesin-1 from protrudin to LE/Lys. In cultured mouse cortical neurons, glucose deprivation, pharmacological activation of AMPK or inhibition of malonyl-CoA synthesis decreases LE/Lys abundance at the axon terminal, and shortens axon length in a CPT1C-dependent manner. These results identify CPT1C as a new regulator of anterograde LE/Lys transport in response to malonyl-CoA changes, and give insight into how axon growth is controlled by nutrients.
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The combination of large volume injection and mixed-mode chromatography was performed for direct ultra-trace LC-MS/MS analysis of seven artificial sweeteners with varying ...physicochemical properties in surface water samples.•The injection volume was raised from 10 µL to 500 µL, while the overall analysis time was only increased by ≈5 min compared to the initial method.•Online column head refocusing and concentration of analytes enabled detection in sub-ng L−1 concentration range without elaborate sample preparation steps.•Relative standard deviations <7% despite multiple injection into the loop.
Due to the low iron solubility in alkaline soils, plants have evolved different iron acquisition strategies, which are either based on ferric iron reduction (strategy I) or complexation by ...phytosiderophores (strategy II). Recently, a prominent role of coumarins for iron acquisition has been discovered, but details of the respective mechanism remain unclear. Since coumarins may act as iron-binding ligands but also as reductants, various reaction sequences are possible, resulting in different iron species and oxidized coumarins. In this context, it is often overlooked that oxidized coumarins are not just byproducts of iron(III) reduction, but may be actively involved in further steps of iron mobilization. In order to verify this active role of oxidized coumarins in Fe(hydr)oxide dissolution, we complemented iron dissolution data with data of single coumarins (esculetin, scopoletin, fraxetin) and their oxidation products, as a function of time, pH, and mineral (goethite, lepidocrocite). Our results demonstrate that there are four different routes for coumarin oxidation, leading to quinones, dimers, hydroxylated coumarins, demethylated coumarins, and combinations of these. The time-dependent species pattern differs with respect to mineral, pH, and coumarin molecule. Oxidized coumarins are often more reactive than the original coumarins, explaining unexpected iron mobilization by scopoletin, which is demethylated to esculetin. Also oxidative hydroxylation and dimerization increase the number of phenolic groups and yield new chelating properties. Several iron-species are identified for the three coumarins. Since oxidation reactions are initiated directly at mineral surfaces, they are often very effective—but this does not always result in more iron mobilization.
An HPLC-charged-aerosol detection method was developed for the quantification of rhamnolipid biosurfactants.Rhamnolipids were quantified in fermentation supernatants from Pseudomonas ...putida.Results were evaluated in terms of labor, selectivity and sensitivity against existing methods.Quantification even without rhamnolipid standards is possible by HPLC-charged-aerosol detection.
A method using high performance liquid chromatography coupled to charged-aerosol detection (HPLC-CAD) was developed for the quantification of rhamnolipid biosurfactants. Qualitative sample composition was determined by liquid chromatography coupled to tandem mass spectrometry (LC⿿MS/MS). The relative quantification of different derivatives of rhamnolipids including di-rhamnolipids, mono-rhamnolipids, and their precursors 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) differed for two compared LC⿿MS instruments and revealed instrument dependent responses. Our here reported HPLC-CAD method provides uniform response.
An inverse gradient was applied for the absolute quantification of rhamnolipid congeners to account for the detector⿿s dependency on the solvent composition. The CAD produces a uniform response not only for the analytes but also for structurally different (nonvolatile) compounds. It was demonstrated that n-dodecyl-β-d-maltoside or deoxycholic acid can be used as alternative standards. The method of HPLC-ultra violet (UV) detection after a derivatization of rhamnolipids and HAAs to their corresponding phenacyl esters confirmed the obtained results but required additional, laborious sample preparation steps. Sensitivity determined as limit of detection and limit of quantification for four mono-rhamnolipids was in the range of 0.3⿿1.0 and 1.2⿿2.0μg/mL, respectively, for HPLC-CAD and 0.4 and 1.5μg/mL, respectively, for HPLC-UV. Linearity for HPLC-CAD was at least 0.996 (R2) in the calibrated range of about 1⿿200μg/mL. Hence, the here presented HPLC-CAD method allows absolute quantification of rhamnolipids and derivatives.
Gas chromatography-mass spectrometry profiling is the most established method for the analysis of organic residues, particularly lipids, from archaeological contexts. This technique allows the ...decryption of hidden chemical information associated with archaeological artefacts, such as ceramic pottery fragments. The molecular and isotopic compositions of such residues can be used to reconstruct past resource use, and hence address major questions relating to patterns of subsistence, diet and ritual practices in the past. A targeted data analysis approach, based on previous findings reported in the literature is common but greatly depends on the investigator's prior knowledge of specific compound classes and their mass spectrometric behaviour, and poses the risk of missing unknown, potentially diagnostic compounds. Organic residues from post-prehistoric archaeological samples often lead to highly complex chromatograms, which makes manual chromatogram inspection very tedious and time consuming, especially for large datasets. This poses a significant limitation regarding the scale and interpretative scopes of such projects. Therefore, we have developed a non-targeted data mining workflow to extract a higher number of known and unknown compounds from the raw data to reduce investigator's bias and to vastly accelerate overall analysis time. The workflow covers all steps from raw data handling, feature selection, and compound identification up to statistical interpretation.
The peroxisome is a key organelle of low abundance that fulfils various functions essential for human cell metabolism. Severe genetic diseases in humans are caused by defects in peroxisome biogenesis ...or deficiencies in the function of single peroxisomal proteins. To improve our knowledge of this important cellular structure, we studied for the first time human liver peroxisomes by quantitative proteomics. Peroxisomes were isolated by differential and Nycodenz density gradient centrifugation. A label-free quantitative study of 314 proteins across the density gradient was accomplished using high resolution mass spectrometry. By pairing statistical data evaluation, cDNA cloning and in vivo colocalization studies, we report the association of five new proteins with human liver peroxisomes. Among these, isochorismatase domain containing 1 protein points to the existence of a new metabolic pathway and hydroxysteroid dehydrogenase like 2 protein is likely involved in the transport or β-oxidation of fatty acids in human peroxisomes. The detection of alcohol dehydrogenase 1A suggests the presence of an alternative alcohol-oxidizing system in hepatic peroxisomes. In addition, lactate dehydrogenase A and malate dehydrogenase 1 partially associate with human liver peroxisomes and enzyme activity profiles support the idea that NAD(+) becomes regenerated during fatty acid β-oxidation by alternative shuttling processes in human peroxisomes involving lactate dehydrogenase and/or malate dehydrogenase. Taken together, our data represent a valuable resource for future studies of peroxisome biochemistry that will advance research of human peroxisomes in health and disease.
Septic arthritis, most often caused by
, is a rapidly progressive and destructive joint disease with substantial mortality and morbidity.
lipoproteins (Lpps) are known to induce arthritis and bone ...destruction. Here, we aimed to investigate the bone resorptive effect of
Lpps in a murine arthritis model by intra-articular injection of purified
Lpps, synthetic lipopeptides, and live
strains. Analyses of the bone mineral density (BMD) of the distal femur bone were performed. Intra-articular injection of both live
and purified
Lpps were shown to significantly decrease total- and trabecular BMD. Liquid chromatography-mass spectrometry analyses revealed that the Lpps expressed by
SA113 strain contain both diacyl and triacyl lipid moieties. Interestingly, synthetic diacylated lipopeptide, Pam
CSK
, was more potent in inducing bone resorption than synthetic triacylated lipopeptide, Pam
CSK
. Modified lipoproteins lacking the lipid moiety were deprived of their bone resorptive abilities. Monocyte depletion by clodronate liposomes fully abrogated the bone resorptive capacity of
lipoproteins. Our data suggest that
Lpps induce bone resorption in locally-induced murine arthritis, an effect mediated by their lipid-moiety through monocytes/macrophages.
Bitterness and pungency are important parameters for olive oil quality. Therefore, two instrumental methods for evaluation of these taste attributes were developed. The first one is based on the ...photometric measurement of total phenolic compounds content, whereas the second one is based on the semiquantitative evaluation of hydrophilic compounds by high-performance liquid chromatography–mass spectrometry (HPLC-MS). Evaluation of total phenolic compounds content was performed by a modified method for the determination of the K 225 value using a more specific detection based on the pH value dependency of absorbance coefficients of phenols at λ = 274 nm. The latter method was not suitable for correct prediction, because no significant correlation between bitterness/pungency and total phenolic compounds content could be found. For the second method, areas of 25 peaks detected in 54 olive oil samples by a HPLC-MS profiling method were correlated with the bitterness and pungency by partial least-squares regression. Six compounds (oleuropein aglycon, ligstroside aglycon, decarboxymethyl oleuropein aglycon, decarboxymethyl ligstroside aglycon, elenolic acid, and elenolic acid methyl ester) show high correlations to bitterness and pungency. The computed model using these six compounds was able to predict bitterness and pungency of olive oil in the error margin of the sensory evaluation (±0.5) for most of the samples.
Acetamiprid (ACE) and imidacloprid (IMI) are insecticides of global importance and are used as spray and watering agents for ornamental plants to control biting and sucking insects or as topical ...medications on pets to remove and control fleas. Human biomonitoring data on ACE and IMI exposures when applying these products are limited. We investigated exposures to ACE and IMI in male volunteers after the domestic application of either an ACE-containing agent or an IMI-containing spot-on medication. Complete and consecutive urine samples were collected for up to 56 h after application. Urine samples were analyzed for ACE, IMI, and their respective metabolites (
-desmethyl-ACE, IMI-olefin, and sum of 4-/5-hydroxy-IMI) by liquid chromatography-tandem mass spectrometry. Fairly uniform concentrations of
-desmethyl-ACE could be observed before and after orchid treatment, so that an ACE exposure associated with orchid treatment can most likely be excluded. In contrast, after the application of the IMI-containing medication, elevated concentrations of IMI, 4-/5-hydroxy-IMI, and IMI-olefin were quantified in urine samples post-20 h with maximum concentrations of 3.1, 14.9, and 8.0 μg/g creatinine, respectively, well above general background levels. Nevertheless, the IMI intake (10.6 μg/kg bw), calculated from the excreted amounts, was around five times below the current European acceptable daily intake. Based on the case results here, household exposures to ACE and IMI after spray treatment of ornamental plants and anti-flea treatment of dogs can be regarded as low and safe. However, people regularly applying neonicotinoid-containing formulations, such as professional gardeners and employees in animal shelters, should be studied in more detail.
Metabolic engineering of microbial cell factories for the production of heterologous secondary metabolites implicitly relies on the intensification of intracellular flux directed toward the product ...of choice. Apart from reactions following peripheral pathways, enzymes of the central carbon metabolism are usually targeted for the enhancement of precursor supply. In Pseudomonas putida, a Gram-negative soil bacterium, central carbon metabolism, i.e., the reactions required for the synthesis of all 12 biomass precursors, was shown to be regulated at the metabolic level and not at the transcriptional level. The bacterium's central carbon metabolism appears to be driven by demand to react rapidly to ever-changing environmental conditions. In contrast, peripheral pathways that are only required for growth under certain conditions are regulated transcriptionally. In this work, we show that this regulation regime can be exploited for metabolic engineering.
We tested this driven-by-demand metabolic engineering strategy using rhamnolipid production as an example. Rhamnolipid synthesis relies on two pathways, i.e., fatty acid de novo synthesis and the rhamnose pathway, providing the required precursors hydroxyalkanoyloxy-alkanoic acid (HAA) and activated (dTDP-)rhamnose, respectively. In contrast to single-pathway molecules, rhamnolipid synthesis causes demand for two central carbon metabolism intermediates, i.e., acetyl-CoA for HAA and glucose-6-phosphate for rhamnose synthesis.
Following the above-outlined strategy of driven by demand, a synthetic promoter library was developed to identify the optimal expression of the two essential genes (rhlAB) for rhamnolipid synthesis. The best rhamnolipid-synthesizing strain had a yield of 40% rhamnolipids on sugar CmolRL/CmolGlc, which is approximately 55% of the theoretical yield. The rate of rhamnolipid synthesis of this strain was also high. Compared to an exponentially growing wild type, the rhamnose pathway increased its flux by 300%, whereas the flux through de novo fatty acid synthesis increased by 50%.
We show that the central carbon metabolism of P. putida is capable of meeting the metabolic demand generated by engineering transcription in peripheral pathways, thereby enabling a significant rerouting of carbon flux toward the product of interest, in this case, rhamnolipids of industrial interest.
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•Synthetic demand was created by the introduction of the rhamnolipid synthesis genes.•A high demand was achieved using a strong synthetic promoter.•Pseudomonas putida responded to demand by increasing flux towards required central carbon metabolites.•High rhamnolipid carbon yield of 40% was achieved.
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