Acid Analysis Research Articles

Hot isopropanol quenching procedure for automated microtiter plate scale 13C-labeling experiments

BackgroundCurrently, the generation of genetic diversity for microbial cell factories outpaces the screening of strain variants with omics-based phenotyping methods. Especially isotopic labeling experiments, which constitute techniques aimed at elucidating cellular phenotypes and supporting rational strain design by growing microorganisms on substrates enriched with heavy isotopes, suffer from comparably low throughput and the high cost of labeled substrates.ResultsWe present a miniaturized, parallelized, and automated approach to 13C-isotopic labeling experiments by establishing and validating a hot isopropanol quenching method on a robotic platform coupled with a microbioreactor cultivation system. This allows for the first time to conduct automated labeling experiments at a microtiter plate scale in up to 48 parallel batches. A further innovation enabled by the automated quenching method is the analysis of free amino acids instead of proteinogenic ones on said microliter scale. Capitalizing on the latter point and as a proof of concept, we present an isotopically instationary labeling experiment in Corynebacterium glutamicum ATCC 13032, generating dynamic labeling data of free amino acids in the process.ConclusionsOur results show that a robotic liquid handler is sufficiently fast to generate informative isotopically transient labeling data. Furthermore, the amount of biomass obtained from a sub-milliliter cultivation in a microbioreactor is adequate for the detection of labeling patterns of free amino acids. Combining the innovations presented in this study, isotopically stationary and instationary automated labeling experiments can be conducted, thus fulfilling the prerequisites for 13C-metabolic flux analyses in high-throughput.

Analysis of acid\u2013base disorders in an ICU cohort using a computer script

Background/aimsAcid–base status is important for understanding pathophysiology, making a diagnosis, planning effective treatment and monitoring progress of critically ill patients. Manual calculations are cumbersome, easily result in wrong conclusions. We wanted to develop an automated assessment of acid–base status.MethodsA simplified adaptive MATLAB script processing all available theory to date was created, evaluated and used on blood gas analyses drawn immediately after admission to ICU. The script was compared to golden standard, calculating manually by two experienced ICU physicians.ResultsResults from the script correlated completely with detailed manual calculations of randomly chosen 100 blood gas results and it was able to deliver complex data on cohort level with advanced graphics. The initial blood gas analyses from 8875 admissions constituted the cohort, of which 4111 (46.3%) were normal. Respiratory acidosis was the primary disturbance in 2753 (31.0%) and metabolic acidosis in 464 (5.2%). Respiratory alkalosis was the primary disturbance in 1501 (17.0%) and metabolic alkalosis in 46 (0.5%). Of the disturbances 74.7% were mixed with two and 2.1% with three simultaneous disturbances. Acidoses were less compensated compared to alkaloses.ConclusionsAcid–base theories are developed on ideal models and not on critical care patients, they require inputs that might not be available, and therefore, estimations are needed. In our cohort, it was difficult to develop a working script based on Stewart, whereas Boston/Copenhagen worked better. Acidoses were more common and more deviated compared to alkaloses.

Soil Organic Matter in Soils of Suburban Landscapes of Yamal Region: Humification Degree and Mineralizing Risks

Various research issues related to soil organic matter in permafrost soils are still poorly investigated. At the same time, numerous investigations have shown the importance of permafrost soils, as they serve as a huge reservoir of organic matter. This work is aimed at the investigation of permafrost-affected soils in the southern part of the Yamal region, namely at the assessment of composition (fractional, elemental and molecular) of soil organic matter in topsoils formed under different biogeoclimatogenic conditions in tundra and forest tundra. Special attention was given to assessment of potential vulnerability of soil organic matter in the context of Arctic warming. Results showed the predominance of fulvic acids in the humus of the studied soils, which indicates mineralization risks in the humic substances system of Arctic soils under conditions of further warming. The 13C-NMR analysis of humic acids revealed that all the studied soils are characterized by higher portions of aliphatic groups of carbon and decreased portions of aromatic groups and revealed early stages of the humification process in studied soils. These results contribute to scarcely distributed research of soil organic matter in permafrost soils of the Arctic. Moreover, our research provided new data on the vulnerability of soil organic matter and its possible mineralization risks under pronounced climate change in the Arctic using the modern instrumental technique.

Synthesis and Acid Catalytic Activity of Al‐Doped Spiky‐Shaped Niobium Pentoxide

Niobium pentoxide (Nb2O5) is a promising heterogeneous acid catalyst because of its water‐tolerant acidity. However, the reactivity of Nb2O5 is limited to the surface of a bulk material; thus, its catalytic activity is generally lower than homogeneous catalysts. Nano‐sized Nb2O5 shows superior activity but has low stability and recoverability. We develop spiky‐shaped Nb2O5 composed of radially grown nanorods, which show high stability but low acidity. In this study, an Al‐doping method and the doping effect on the acidity and nanostructure of the spiky‐shaped Nb2O5 are demonstrated. Al of 1–6 mol% is doped in Nb2O5 while maintaining the spiky nanostructure using an Nb–Al composite oxalate as a precursor. Scanning transmission electron microscope analysis results show that the doped Al ions are homogeneously dispersed in the structure. Al doping increases the catalytic activity, where 4 mol% Al‐doped Nb2O5 shows the highest production rate of 2‐phenyl‐2‐phenylaminoacetonitrile in a Strecker reaction. From the acid analysis and Raman spectroscopy, it can be concluded that the Al doping is effective in adjusting acidity and in enhancement of catalytic activity due to creation of NbO4 and distorted NbO6. These are produced by defect formation and lattice distortion caused by differences in valence and ionic radius between Al and Nb.

Spatial distribution of live gut microbiota and bile acid metabolism in various parts of human large intestine

Gut microbiomics is based on analysis of both live and dead cells in the stool. However, to understand the ecology of gut microbiota and their symbiotic relationships with hosts, spatial distribution of live bacteria must be examined. Here, we analyzed the live composition of luminal microbiota (LM) and mucosa-associated microbiota (MAM) in the ascending and descending colons and the rectums of 10 healthy adults and compared it with the total composition. The abundance of Lachnospiraceae in live LM decreased along the gut length and was significantly lower than that in total LM. Contrastingly, the abundance of Bacteroidaceae and Bifidobacteriaceae in live LM was higher than that in total LM, suggesting differences in death rate during gut migration. Live Enterobacteriaceae levels in MAM were significantly higher in rectum than in the ascending and descending colons and in LM. High-performance liquid chromatographic analysis of luminal bile acids revealed that 7α-dehydroxylation occurred towards the rectum. In live LM where a bile acid-inducible gene could be detected, 7α-dehydroxylation rates were higher than those in the group without the gene. Overall, we showed differences in live bacteria composition among three gut sites and between LM and MAM, highlighting the importance of understanding their spatial distribution.

A6 MECHANISMS UNDERLYING GUT DYSFUNCTION FOLLOWING C. DIFFICILE INFECTION AND IMPLICATIONS FOR TREATMENT

BackgroundRecent evidence suggests an increasing prevalence of gut dysfunction following C. difficile infection (CDI). The accompanying prolonged antibiotic (AB) exposure likely contributes to chronic gut dysfunction and our ability to induce gut dysfunction in germ free (GF) mice colonized with microbiota from a patient with severe slow transit post CDI (PCDI) supports this notion (10.1093/jcag/gwz047.117). Furthermore, we were able to restore gut function following fecal microbial transfer from healthy murine donors. Our studies have implicated a role for macrophages in the destruction of the Interstitial Cell of Cajal (ICC) network underlying slow colonic transit in the humanized mouse model. These findings prompted us to evaluate microbiota-directed therapy in normalising gut function in this model.Aims 1)To investigate whether dietary psyllium rescues the development of slow colonic transit (SCT) through modulating host function via microbiota mediated immune mechanisms; and 2)To evaluate the mechanisms underlying the beneficial effects of psylliumMethodsGF mice were colonized with either microbiota from the PCDI patient or healthy control (HC) for 3 weeks following which PCDI mice were treated with either a control diet or a 15% psyllium diet (PSY). Colonic motility was assessed before and after the diet intervention using the bead expulsion test. Stool samples were collected for microbial profiling, and short and branched-chain fatty acids (SCFA/BCFA) analysis. Colonic muscle layers encompassing myenteric plexus (MP) were collected for gene expression analysis and to evaluate activated macrophages and ICC degeneration using immunohistochemistry.ResultsMicrobiota from a PCDI patient induced a SCT phenotype in GF mice (n=13) as compared to mice colonized with HC microbiota (p=0.0002). Psyllium rescued this SCT phenotype in mice (PCDI(n=7) vs.PSY(n=6):p=0.0014). The psyllium-induced rescue was accompanied by normalization of the ICC network and morphological alterations in infiltrating macrophages. This was supported by changes in immune-related gene expression in the MP including CD11b, NOS, Myd88, Mapk1 and NF-κB. Additionally, bacterial composition was different between PCDI and PSY group (p=0.003). SCFAs like acetic and propionic acid were increased, while BCFA like isobutyric and isovaleric acid were decreased following PSY treatment. These alterations in SCFA/BCFA were supported by fluctuations in specific bacteria like Butyricimonas, Phascolarctobacterium and Allistipes.ConclusionsOur results provide evidence that chronic gut dysfunction following CDI and AB exposure is microbiota-driven. Furthermore, microbiota-directed therapy using psyllium could serve as a novel therapeutic strategy to normalize gut function via microbiota-mediated restoration of immune homeostasis in these patients.Funding AgenciesW. Garfield Weston Foundation

A47 MICROBIAL FUNCTIONS AS BIOMARKERS OF PRO-INFLAMMATORY RESPONSE TO SELECT DIETARY FIBERS IN IBD

BackgroundDietary fibers are not digested in the bowel; they are fermented by microbes, typically promoting gut health. However, IBD patients experience sensitivity to consumption of fibers. Our previous findings offered the first mechanistic evidence demonstrating that unfermented dietary β-fructans (inulin and FOS) can induce pro-inflammatory cytokines in a subset of pediatric IBD colonic biopsies cultured ex vivo, and in the SYNERGY-1 (β-fructan) clinical study of adult remission UC patients. Incubating FOS with whole-microbiota intestinal washes from non-IBD or remission IBD patients improved fermentation and reduced pro-inflammatory responses, but not from patients with active disease. Fibre-induced immune responses correlated with microbe functions, luminal metabolites, and fibre avoidance.AimsHere we aimed to expand on our findings and define the role of microbial functions in mediating host response to β-fructans.MethodsColonic biopsies cultured ex vivo and cell lines in vitro were incubated with FOS (5g/L), or fermentation supernatants (24hr anaerobic fermentation). Immune responses (cytokine secretion [ELISA/MSD] and expression [qPCR]) were assessed. Taxonomic classification of microbial fermentation cultures was conducted with Kraken2 and metabolic profiling by HUMAnN2. HPLC and gas chromatography volatile fatty acid (CG-VFA) analysis were used to identify concentrations of remaining fibre and SCFAs following anaerobic fermentation.Results7 microbial enzymes were identified to be predictive of cytokine (IL-1β, IL23, IL-5, IL-8, MIP-1α) secretion in ex vivo colonic biopsies from pediatric Crohn disease (CD; n=38), ulcerative colitis (UC; n=20), and non-IBD (n=21) patients, in response to β-fructans; their use as biomarkers of response was determined in patient stool from the SYNERGY-1 clinical study cohort. Fermentation of FOS by whole-microbe intestinal washes from only non-IBD or remission IBD patients reduced cytokine secretion, and our findings demonstrate that this was due to a combination of reduction of β-fructan present and production of a precise combination of anti-inflammatory SCFAs.ConclusionsOur findings suggest that intolerance and avoidance of fibers in select IBD patients is associated with the inability to ferment these fibers, mediated by altered microbial functions (enzymes), leading to worsened inflammation. Data indicate that gut microbial function, not composition, predicts patient pro-inflammatory response to β-fructans, supporting our hypothesis that overall community function impacts fibre fermentation and affects associated pro-inflammatory effects. Our work highlights select disease state scenarios in which administration of fermentable fibers should be avoided and tailored dietary interventions considered.Funding AgenciesCIHRWeston Foundation, Mitacs

Ion chromatography coupled to Q-Orbitrap for the analysis of formic and oxalic acid in beehive matrices: a field study

There is an increasing concern about the use of synthetic acaricides to fight the ectoparasitic mite Varroa destructor. Natural products such as formic acid (FA) and oxalic acid (OA) have emerged as a possible alternative control strategy. However, given the difficulty of analysing these highly polar compounds and the lack of robust and reliable methods, there are very few studies of the concentration and distribution of these natural acaricides in the beehive compartments. We present a reliable and simple analytical methodology, based on sample extraction with modified quick polar pesticide (QuPPe) methods followed by ion chromatography coupled to a quadrupole Orbitrap mass analyser for the analysis of FA and OA in honeybees, honey, beeswax, and beebread. The developed methods have been used in a field study for the evaluation of the presence and distribution of FA and OA in the beehive products, as well as in adult bees and bee brood samples, before, during, and up to 3 months after the application of the treatments by the beekeeper. Beebread and honey samples presented the highest concentration levels of OA and FA, respectively, mainly due to their natural presence. As expected, the organic acids showed low persistence in wax after the treatments. The natural acaricides were found in adult and developing bees at concentration levels below the reported LD50 in all the cases; however, residue levels of OA in larvae during the treatment application were very close to the reported LD50.Graphical abstract

Methodological Aspects of the Analysis of Fatty Acids in Biological Samples

Methodological Aspects of the Analysis of Fatty Acids in Biological Samples

Differential release of extracellular vesicle tRNA from oxidative stressed renal cells and ischemic kidneys

While urine-based liquid biopsy has expanded to the analyses of extracellular nucleic acids, the potential of transfer RNA (tRNA) encapsulated within extracellular vesicles has not been explored as a new class of urine biomarkers for kidney injury. Using rat kidney and mouse tubular cell injury models, we tested if extracellular vesicle-loaded tRNA and their m1A (N1-methyladenosine) modification reflect oxidative stress of kidney injury and determined the mechanism of tRNA packaging into extracellular vesicles. We determined a set of extracellular vesicle-loaded, isoaccepting tRNAs differentially released after ischemia–reperfusion injury and oxidative stress. Next, we found that m1A modification of extracellular vesicle tRNAs, despite an increase of the methylated tRNAs in intracellular vesicles, showed little or no change under oxidative stress. Mechanistically, oxidative stress decreases tRNA loading into intracellular vesicles while the tRNA-loaded vesicles are accumulated due to decreased release of the vesicles from the cell surface. Furthermore, Maf1-mediated transcriptional repression of the tRNAs decreases the cargo availability for extracellular vesicle release in response to oxidative stress. Taken together, our data support that release of extracellular vesicle tRNAs reflects oxidative stress of kidney tubules which might be useful to detect ischemic kidney injury and could lead to rebalance protein translation under oxidative stress.

Bioequivalence of long-chain omega-3 polyunsaturated fatty acids from foods enriched with a novel vegetable-based omega-3 delivery system compared to gel capsules: a randomized controlled cross-over acute trial

PurposeTo evaluate bioavailability of omega-3 long-chain polyunsaturated fatty acids (LCPUFA) from foods enriched with novel vegetable-based encapsulated algal oil across Australian and Singaporean populations.Methods27 men (n = 12 Australian European; n = 15 Singaporean Chinese), 21–50 yr; 18–27.5 kg/m2, with low habitual intake of omega-3 LCPUFA completed a multicentre randomised controlled acute 3-way cross-over single-blind trial. They consumed, in random order 1-week apart after an overnight fast, standard breakfast meals including 400 mg docosahexanoic acid (DHA) from either extruded rice snacks or soup both containing cauliflower-encapsulated HiDHA® algal oil or gel capsules containing HiDHA® algal oil. Blood samples for analysis of plasma DHA and eicosapentaenoic acid (EPA) were taken pre-meal and after 2, 4, 6, 8 and 24 h. Primary analyses comparing 24-h incremental area under the plasma DHA, EPA and DHA + EPA concentration (µg/ml) curves (iAUC0-24 h) between test foods were performed using linear mixed models by including ethnicity as an interaction term.ResultsPlasma iAUC0-24 h did not differ significantly between test foods (adjusted mean [95% CI] plasma DHA + EPA: extruded rice snack, 8391 [5550, 11233] µg/mL*hour; soup, 8862 [6021, 11704] µg/mL*hour; capsules, 11,068 [8226, 13910] µg/mL*hour, P = 0.31) and did not differ significantly between Australian European and Singaporean Chinese (treatment*ethnicity interaction, P = 0.43).ConclusionThe vegetable-based omega-3 LCPUFA delivery system did not affect bioavailability of omega-3 LCPUFA in healthy young Australian and Singaporean men as assessed after a single meal over 24 h, nor was bioavailability affected by ethnicity. This novel delivery system may be an effective way to fortify foods/beverages with omega-3 LCPUFA.Trial registrationThe trial was registered with clinicaltrials.gov (NCT04610983), date of registration, 22 November 2020.

Comparative chemoreactomic analysis of ornithine aspartate, S-ademethionine and ursodeoxycholic acid

Purpose of the study. The pharmacological effects of ornithine are primarily due to its participation in the neutralization of ammonia and in the metabolism of amino acids. To clarify the mechanisms of action of ornithine, a chemoreactom analysis of ornithine aspartate was performed in comparison with S-ademetionine (S-AM) and ursodeoxycholic acid (UDCA). Methods. The effects of the investigated substances on the metabolism of urates (derivatives of uric acid), vasoactivity, vascular structure and cytoprotection were assessed by the method of chemoreactome analysis. Results. It was found that a decrease in blood urate levels is associated with inhibition by ornithine of the urate transporter URAT1 (ornithine – IC50 = 65 nM, S-AM – 1060 nM, UDCA – 94 nM) and xanthine oxidase (ornithine – IC50 = 910 nM, other molecules IC50 = 9655-11040 nM). Among the studied molecules, ornithine was distinguished by the most pronounced vasodilatory properties (probably associated with inhibition of adrenergic receptors: Kd = 35 nM, other molecules – 1108-1428 nM). The highest antidyslipidemic activity was also shown for ornithine (9.4 %) and S-AM (10.2 %), the lowest for UDCA (4.6 %). The antidyslipidemic effect of ornithine is associated with inhibition of endothelial lipase (IC50 = 258 nM for ornithine). The cytoprotective effects of ornithine are associated with an increase in glutathione synthesis, a decrease in the glutamatergic activity of neurons under conditions of glutamate stress, inhibition of prostaglandin D-synthase, and a decrease in the intensity of acute lymphocytic inflammation without immunosuppression. Conclusion. Thus, the results of the ornithine chemoreactom analysis indicate a significant expansion of the spectrum of the pharmacological effects of ornithine, including not only hepatoprotection, but also cardioprotection and nephroprotection.

Proteomic and lipidomics analyses of high fatty acid AhDGAT3 transgenic soybean reveals the key lipase gene associated with the lipid internal mechanism.

Vegetable oil is one of the most important components of human nutrition. Soybean (Glycine max) is an important oil crop worldwide and contains rich unsaturated fatty acids. Diacylglycerol acyltransferase (DGAT) is a key rate-limiting enzyme in the Kennedy pathway from diacylglycerol (DAG) to triacylglycerol (TAG). In this study, we conducted further research using T3 AhDGAT3 transgenic soybean. A high-performance gas chromatography flame ionization detector showed that oleic acid (18:1) content and total fatty acid content of transgenic soybean were significantly higher than those of the wild type (WT). However, linoleic acid (18:2) was much lower than that in the WT. For further mechanistic studies, 20 differentially expressed proteins (DEPs) and 119 differentially expressed metabolites (DEMs) were identified between WT (JACK) and AhDGAT3 transgenic soybean mature seeds using proteomic and lipidomics analyses. Combined proteomic and lipidomics analyses showed that the upregulation of the key DEP (lipase GDSL domain-containing protein) in lipid transport and metabolic process induced an increase in the total fatty acid and 18:1 composition, but a decrease in the 18:2 composition of fatty acids. Our study provides new insights into the deep study of molecular mechanism underlying the enhancement of fatty acids in transgenic soybeans, especially oleic acid and total fatty acid, which are enhanced by over-expression of AhDGAT3.

CORRECTION: Compound-specific isotope analysis of amino acids reveals dependency on grazing rather than detritivory in mangrove food webs

CORRECTION: Compound-specific isotope analysis of amino acids reveals dependency on grazing rather than detritivory in mangrove food webs

The use of HPLC-MS/MS for the quantitative analysis of neuroactive amino acids in rat brain homogenates after derivatization with 9-fluorenylmethyl chloroformate

The use of HPLC-MS/MS for the quantitative analysis of neuroactive amino acids in rat brain homogenates after derivatization with 9-fluorenylmethyl chloroformate

In vitro Analysis of Citric Acid Crosslinked and Juglans regia Extract Loaded Hydrogel for Biomedical Applications

Herbal medicines are a familiar source of therapeutic agents for the medicaments of infections in various countries these days. In this work, biological study of citric acid crosslinked hydrogel loaded with Juglans regia leaves extract against 5 standard bacterial and fungal strains. The FTIR and FESEM results exhibited that leaves extract stick on the surface of hydrogel and it does not reacted with the functional groups of used polymers. The obtained results of MTT assay against L929 mouse fibroblasts cell line showed that hydrogel was biocompatible. Highest minimum inhibitory concentration was found in case of S. typhi, E. coli and C. albicans. P. aeruginosa and C. albicans were the most susceptible strains, while E. coli and S. typhi were the most resistant strains.

Investigation and validation of quantitative analysis of phenolic compounds and chlorogenic acid in the Vaccinium uliginosum leaves

Investigation and validation of quantitative analysis of phenolic compounds and chlorogenic acid in the Vaccinium uliginosum leaves

Raman Microscopy Techniques to Study Lipid Droplet Composition in Cancer Cells.

Raman spectroscopy using feature selection schemes has considerable advantages over gas chromatography for the analysis of fatty acids' composition changes. Here, we introduce an educational methodology to demonstrate the potential of micro-Raman spectroscopy to determine with high accuracy the unsaturation or saturation degrees and composition changes of the fatty acids found in the lipid droplets of the LNCaP prostate cancer cells that were treated with various fatty acids. The methodology uses highly discriminatory wavenumbers among fatty acids present in the sample selected by using the Support Vector Machine algorithm.

A Simple 3d Printed Microfluidic Device for Point-of-Care Analysis of Urinary Uric Acid

A Simple 3d Printed Microfluidic Device for Point-of-Care Analysis of Urinary Uric Acid

Postharvest quality and shelf life of Passiflora cincinnata BRS Sertão Forte fruits according to type of fertilization and storage

Abstract The present study aimed to evaluate the physicochemical characteristics and shelf life of P. cincinnata BRS Sertão Forte fruits produced with conventional fertilization recommended for passion fruit, conventional organic fertilization, organic fertilization enriched with biotite schist, conventional fertilization with replacement of the potassium source and chemical fertilization only and the mineral nutrition of plants stored under ambient and refrigerated conditions. Analyses of pH, soluble solids, titratable acidity, ratio, color, texture and mass loss were performed. Fertilizers differed in terms of nutrient absorption associated with protein synthesis (N, S, B, Cu, Mn, Zn), especially where soil remineralizer was applied. Treatments without fertilization and that with organic fertilization enriched with biotite schist soil remineralizer also positively influenced fruit physicochemical properties such as color and texture. Fruits stored under refrigeration conditions showed the best results for the conservation of their qualitative characteristics. Under the conditions of the present study, it could be concluded that the postharvest life of P. cincinnata fruits stored under refrigeration at 100C is at least 60 days. This period is 30 days under ambient conditions.