Intermediate Amino Research Articles

Enzymes in Synergy: Bacteria Specific Molecular Probe for Locoregional Imaging of Urinary Tract Infection in vivo

AbstractUropathogenic Escherichia coli (UPECs) is a leading cause for urinary tract infections (UTI), accounting for 70–90 % of community or hospital‐acquired bacterial infections owing to high recurrence, imprecision in diagnosis and management, and increasing prevalence of antibiotic resistance. Current methods for clinical UPECs detection still rely on labor‐intensive urine cultures that impede rapid and accurate diagnosis for timely UTI therapeutic management. Herein, we developed a first‐in‐class near‐infrared (NIR) UPECs fluorescent probe (NO−AH) capable of specifically targeting UPECs through its collaborative response to bacterial enzymes, enabling locoregional imaging of UTIs both in vitro and in vivo. Our NO−AH probe incorporates a dual protease activatable moiety, which first reacts with OmpT, an endopeptidase abundantly present on the outer membrane of UPECs, releasing an intermediate amino acid residue conjugated with a NIR hemicyanine fluorophore. Such liberated fragment would be subsequently recognized by aminopeptidase (APN) within the periplasm of UPECs, activating localized fluorescence for precise imaging of UTIs in complex living environments. The peculiar specificity and selectivity of NO−AH, facilitated by the collaborative action of bacterial enzymes, features a timely and accurate identification of UPECs‐infected UTIs, which could overcome misdiagnosis in conventional urine tests, thus opening new avenues towards reliable UTI diagnosis and personalized antimicrobial therapy management.

Early life exposure to vitamin D deficiency impairs molecular mechanisms that regulate liver cholesterol biosynthesis, energy metabolism, inflammation, and detoxification.

Emerging data suggests liver disease may be initiated during development when there is high genome plasticity and the molecular pathways supporting liver function are being developed. Here, we leveraged our Collaborative Cross mouse model of developmental vitamin D deficiency (DVD) to investigate the role of DVD in dysregulating the molecular mechanisms underlying liver disease. We defined the effects on the adult liver transcriptome and metabolome and examined the role of epigenetic dysregulation. Given that the parental origin of the genome (POG) influences response to DVD, we used our established POG model [POG1-(CC011xCC001)F1 and POG2-(CC001xCC011)F1] to identify interindividual differences. We found that DVD altered the adult liver transcriptome, primarily downregulating genes controlling liver development, response to injury/infection (detoxification & inflammation), cholesterol biosynthesis, and energy production. In concordance with these transcriptional changes, we found that DVD decreased liver cell membrane-associated lipids (including cholesterol) and pentose phosphate pathway metabolites. Each POG also exhibited distinct responses. POG1 exhibited almost 2X more differentially expressed genes (DEGs) with effects indicative of increased energy utilization. This included upregulation of lipid and amino acid metabolism genes and increased intermediate lipid and amino acid metabolites, increased energy cofactors, and decreased energy substrates. POG2 exhibited broader downregulation of cholesterol biosynthesis genes with a metabolomics profile indicative of decreased energy utilization. Although DVD primarily caused loss of liver DNA methylation for both POGs, only one epimutation was shared, and POG2 had 6.5X more differentially methylated genes. Differential methylation was detected at DEGs regulating developmental processes such as amino acid transport (POG1) and cell growth & differentiation (e.g., Wnt & cadherin signaling, POG2). These findings implicate a novel role for maternal vitamin D in programming essential offspring liver functions that are dysregulated in liver disease. Importantly, impairment of these processes was not rescued by vitamin D treatment at weaning, suggesting these effects require preventative measures. Substantial differences in POG response to DVD demonstrate that the parental genomic context of exposure determines offspring susceptibility.

Moderate Elevation of Homocysteine Induces Endothelial Dysfunction through Adaptive UPR Activation and Metabolic Rewiring.

Elevation of the intermediate amino acid metabolite Homocysteine (Hcy) causes Hyperhomocysteinemia (HHcy), a metabolic disorder frequently associated with mutations in the methionine-cysteine metabolic cycle as well as with nutritional deficiency and aging. The previous literature suggests that HHcy is a strong risk factor for cardiovascular diseases. Severe HHcy is well-established to correlate with vascular pathologies primarily via endothelial cell death. Though moderate HHcy is more prevalent and associated with an increased risk of cardiovascular abnormalities in later part of life, its precise role in endothelial physiology is largely unknown. In this study, we report that moderate elevation of Hcy causes endothelial dysfunction through impairment of their migration and proliferation. We established that unlike severe elevation of Hcy, moderate HHcy is not associated with suppression of endothelial VEGF/VEGFR transcripts and ROS induction. We further showed that moderate HHcy induces a sub-lethal ER stress that causes defective endothelial migration through abnormal actin cytoskeletal remodeling. We also found that sub-lethal increase in Hcy causes endothelial proliferation defect by suppressing mitochondrial respiration and concomitantly increases glycolysis to compensate the consequential ATP loss and maintain overall energy homeostasis. Finally, analyzing a previously published microarray dataset, we confirmed that these hallmarks of moderate HHcy are conserved in adult endothelial cells as well. Thus, we identified adaptive UPR and metabolic rewiring as two key mechanistic signatures in moderate HHcy-associated endothelial dysfunction. As HHcy is clinically associated with enhanced vascular inflammation and hypercoagulability, identifying these mechanistic pathways may serve as future targets to regulate endothelial function and health.

Diet-Induced Severe Hyperhomocysteinemia Promotes Atherosclerosis Progression and Dysregulates the Plasma Metabolome in Apolipoprotein-E-Deficient Mice.

Atherosclerosis and resulting cardiovascular disease are the leading causes of death in the US. Hyperhomocysteinemia (HHcy), or the accumulation of the intermediate amino acid homocysteine, is an independent risk factor for atherosclerosis, but the intricate biological processes mediating this effect remain elusive. Several factors regulate homocysteine levels, including the activity of several enzymes and adequate levels of their coenzymes, including pyridoxal phosphate (vitamin B6), folate (vitamin B9), and methylcobalamin (vitamin B12). To better understand the biological influence of HHcy on the development and progression of atherosclerosis, apolipoprotein-E-deficient (apoE-/- mice), a model for human atherosclerosis, were fed a hyperhomocysteinemic diet (low in methyl donors and B vitamins) (HHD) or a control diet (CD). After eight weeks, the plasma, aorta, and liver were collected to quantify methylation metabolites, while plasma was also used for a broad targeted metabolomic analysis. Aortic plaque burden in the brachiocephalic artery (BCA) was quantified via 14T magnetic resonance imaging (MRI). A severe accumulation of plasma and hepatic homocysteine and an increased BCA plaque burden were observed, thus confirming the atherogenic effect of the HHD. Moreover, a decreased methylation capacity in the plasma and aorta, indirectly assessed by the ratio of S-adenosylmethionine to S-adenosylhomocysteine (SAM:SAH) was detected in HHD mice together with a 172-fold increase in aortic cystathionine levels, indicating increased flux through the transsulfuration pathway. Betaine and its metabolic precursor, choline, were significantly decreased in the livers of HHD mice versus CD mice. Widespread changes in the plasma metabolome of HHD mice versus CD animals were detected, including alterations in acylcarnitines, amino acids, bile acids, ceramides, sphingomyelins, triacylglycerol levels, and several indicators of dysfunctional lipid metabolism. This study confirms the relevance of severe HHcy in the progression of vascular plaque and suggests novel metabolic pathways implicated in the pathophysiology of atherosclerosis.

A Critical Overview of Enzyme-Based Electrochemical Biosensors for L-Dopa Detection in Biological Samples

L-Dopa is an intermediate amino acid in the biosynthesis of endogenous catecholamines, such as dopamine. It is currently considered to be the optimal dopaminergic treatment for Parkinson’s disease, a neurodegenerative disorder affecting around 1% of the population. In an advanced stage of the disease, complications such as dyskinesia and psychosis are caused by fluctuations in plasma drug levels. Real-time monitoring of L-Dopa levels would be advantageous for properly adjusting drug dosing, thus improving therapeutic efficacy. Electrochemical methods have advantages such as easy-to-use instrumentation, fast response time, and high sensitivity, and are suitable for miniaturization, enabling the fabrication of implantable or wearable devices. This review reports on research papers of the past 20 years (2003–2023) dealing with enzyme-based biosensors for the electrochemical detection of L-Dopa in biological samples. Specifically, amperometric and voltammetric biosensors, whose output signal is a measurable current, are discussed. The approach adopted includes an initial study of the steps required to assemble the devices, i.e., electrode modification and enzyme immobilization. Then, all issues related to their analytical performance in terms of sensitivity, selectivity, and capability to analyze real samples are critically discussed. The paper aims to provide an assessment of recent developments while highlighting limitations such as poor selectivity and long-term stability, and the laborious and time-consuming fabrication protocol that needs to be addressed from the perspective of the integrated clinical management of Parkinson’s disease.

Mechanism of homocysteine-mediated endothelial injury and its consequences for atherosclerosis.

Homocysteine (Hcy) is an intermediate amino acid formed during the conversion from methionine to cysteine. When the fasting plasma Hcy level is higher than 15 μmol/L, it is considered as hyperhomocysteinemia (HHcy). The vascular endothelium is an important barrier to vascular homeostasis, and its impairment is the initiation of atherosclerosis (AS). HHcy is an important risk factor for AS, which can promote the development of AS and the occurrence of cardiovascular events, and Hcy damage to the endothelium is considered to play a very important role. However, the mechanism by which Hcy damages the endothelium is still not fully understood. This review summarizes the mechanism of Hcy-induced endothelial injury and the treatment methods to alleviate the Hcy induced endothelial dysfunction, in order to provide new thoughts for the diagnosis and treatment of Hcy-induced endothelial injury and subsequent AS-related diseases.

Total plasma homocysteine measurement: Evaluation of the Abbott immunoassay, comparison with the JEOL ion exchange chromatography and investigation of its clinical utility

ObjectivesHomocysteine is an intermediary amino acid formed in methionine metabolism, with elevated total homocysteine (tHCY) being a biomarker of cardiovascular and cerebrovascular diseases. We evaluated the Abbott ARCHITECT tHCY immunoassay, compared it with the current established JEOL ion exchange chromatography (IEC) method and evaluated its clinical utility. Design and methodsFollowing immunoassay method verification, plasma samples of 91 patients were analysed for tHCY using immunoassay and IEC. ResultsFor the Abbott immunoassay, accuracy was assessed, with UK NEQAS EQA specimens, by the correlation of our Abbott immunoassay measurements to the Abbott ARCHITECT immunoassay mean (bias = 1.6%), and to the overall immunoassay mean (bias = 2.0%). The total imprecision was 2.7% (11.00 μmol/L), 2.4% (16.80 μmol/L) and 2.8% (24.30 μmol/L) respectively. Bias in linearity assessment was 0.12%–2.58%. The inter-method correlation was strong in Passing-Bablok regression: immunoassay = IEC x0.857 + 2.445 (95% CI: slope = [0.742,0.947], intercept = [1.340,3.582]), with Spearman correlation = 0.803 (p < 0.001). The Bland-Altman plot showed an average difference of −0.284 μmol/L (95% CI: [-1.043,0.474]) with limits of agreement (mean ± 1.96SD) from −7.425 μmol/L to 6.857 μmol/L.No significant difference in tHCY was found using both methods in patients with cerebrovascular diseases and cardiovascular diseases. Most tHCY measurements were within the reference ranges of both methods. All homocystinuria patients had tHCY values above the reference ranges of both methods. ConclusionsThe immunoassay demonstrated robust performance in its verification and showed good comparability with the IEC but with some biases so caution is needed if both are used interchangeably. The immunoassay offers an automated alternative to IEC in the assessment of hyperhomocysteinaemia.

SARS-CoV-2 Mutant Spectra at Different Depth Levels Reveal an Overwhelming Abundance of Low Frequency Mutations.

Populations of RNA viruses are composed of complex and dynamic mixtures of variant genomes that are termed mutant spectra or mutant clouds. This applies also to SARS-CoV-2, and mutations that are detected at low frequency in an infected individual can be dominant (represented in the consensus sequence) in subsequent variants of interest or variants of concern. Here we briefly review the main conclusions of our work on mutant spectrum characterization of hepatitis C virus (HCV) and SARS-CoV-2 at the nucleotide and amino acid levels and address the following two new questions derived from previous results: (i) how is the SARS-CoV-2 mutant and deletion spectrum composition in diagnostic samples, when examined at progressively lower cut-off mutant frequency values in ultra-deep sequencing; (ii) how the frequency distribution of minority amino acid substitutions in SARS-CoV-2 compares with that of HCV sampled also from infected patients. The main conclusions are the following: (i) the number of different mutations found at low frequency in SARS-CoV-2 mutant spectra increases dramatically (50- to 100-fold) as the cut-off frequency for mutation detection is lowered from 0.5% to 0.1%, and (ii) that, contrary to HCV, SARS-CoV-2 mutant spectra exhibit a deficit of intermediate frequency amino acid substitutions. The possible origin and implications of mutant spectrum differences among RNA viruses are discussed.

Review on the intermediate amino acids and their enantiomers during the anaerobic digestion: the distribution, biofunctions and mechanisms

Review on the intermediate amino acids and their enantiomers during the anaerobic digestion: the distribution, biofunctions and mechanisms

Insulin Sensitivity and Metabolic Flexibility Parallel Plasma TCA Levels in Early Chronotype With Metabolic Syndrome.

People characterized as late chronotype have elevated type 2 diabetes and cardiovascular disease risk compared to early chronotype. It is unclear how chronotype is associated with insulin sensitivity, metabolic flexibility, or plasma TCA cycle intermediates concentration, amino acids (AA), and/or beta-oxidation. This study examined these metabolic associations with chronotype. The Morningness-Eveningness Questionnaire (MEQ) was used to classify adults with metabolic syndrome (ATP III criteria) as either early (n = 15 [13F], MEQ = 64.7 ± 1.4) or late (n = 19 [16F], MEQ = 45.5 ± 1.3) chronotype. Fasting bloods determined hepatic (HOMA-IR) and adipose insulin resistance (Adipose-IR) while a 120-minute euglycemic clamp (40 mU/m2/min, 5 mmoL/L) was performed to test peripheral insulin sensitivity (glucose infusion rate). Carbohydrate (CHOOX) and fat oxidation (FOX), as well as nonoxidative glucose disposal (NOGD), were also estimated (indirect calorimetry). Plasma tricarboxylic acid cycle (TCA) intermediates, AA, and acyl-carnitines were measured along with VO2max and body composition (DXA). There were no statistical differences in age, BMI, fat-free mass, VO2max, or ATP III criteria between groups. Early chronotype, however, had higher peripheral insulin sensitivity (P = 0.009) and lower HOMA-IR (P = 0.02) and Adipose-IR (P = 0.05) compared with late chronotype. Further, early chronotype had higher NOGD (P = 0.008) and greater insulin-stimulated CHOOX (P = 0.02). While fasting lactate (P = 0.01), TCA intermediates (isocitrate, α-ketoglutarate, succinate, fumarate, malate; all P ≤ 0.04) and some AA (proline, isoleucine; P = 0.003-0.05) were lower in early chronotype, other AA (threonine, histidine, arginine; all P ≤ 0.05) and most acyl-carnitines were higher (P ≤ 0.05) compared with late chronotype. Greater insulin sensitivity and metabolic flexibility relates to plasma TCA concentration in early chronotype.

Corticosteroid suppresses urea-cycle-related gene expressions in ornithine transcarbamylase deficiency

BackgroundOrnithine transcarbamylase deficiency (OTCD) is most common among urea cycle disorders (UCDs), defined by defects in enzymes associated with ureagenesis. Corticosteroid administration to UCD patients, including OTCD patients, is suggested to be avoided, as it may induce life-threatening hyperammonemia. The mechanism has been considered nitrogen overload due to the catabolic effect of corticosteroids; however, the pathophysiological process is unclear.MethodsTo elucidate the mechanism of hyperammonemia induced by corticosteroid administration in OTCD patients, we analyzed a mouse model by administering corticosteroids to OTCspf−ash mice deficient in the OTC gene. Dexamethasone (DEX; 20 mg/kg) was administered to the OTCspf−ash and wild-type (WT) mice at 0 and 24 h, and the serum ammonia concentrations, the levels of the hepatic metabolites, and the gene expressions related with ammonia metabolism in the livers and muscles were analyzed.ResultsThe ammonia levels in Otcspf−ash mice that were administered DEX tended to increase at 24 h and increased significantly at 48 h. The metabolomic analysis showed that the levels of citrulline, arginine, and ornithine did not differ significantly between Otcspf−ash mice that were administered DEX and normal saline; however, the level of aspartate was increased drastically in Otcspf−ash mice owing to DEX administration (P < 0.01). Among the enzymes associated with the urea cycle, mRNA expressions of carbamoyl-phosphate synthase 1, ornithine transcarbamylase, arginosuccinate synthase 1, and arginosuccinate lyase in the livers were significantly downregulated by DEX administration in both the Otcspf−ash and WT mice (P < 0.01). Among the enzymes associated with catabolism, mRNA expression of Muscle RING-finger protein-1 in the muscles was significantly upregulated in the muscles of WT mice by DEX administration (P < 0.05).ConclusionsWe elucidated that corticosteroid administration induced hyperammonemia in Otcspf−ash mice by not only muscle catabolism but also suppressing urea-cycle-related gene expressions. Since the urea cycle intermediate amino acids, such as arginine, might not be effective because of the suppressed expression of urea-cycle-related genes by corticosteroid administration, we should consider an early intervention by renal replacement therapy in cases of UCD patients induced by corticosteroids to avoid brain injuries or fatal outcomes.

L-citrulline: A preclinical safety biomarker for the small intestine in rats and dogs in repeat dose toxicity studies

IntroductionGastrointestinal (GI) toxicity is still an issue within drug development, especially for novel oncology drugs. The identification of GI mucosal damage at an early stage with high sensitivity and specificity across preclinical species and humans remains difficult. To date, in preclinical studies, no qualified mechanistic, diagnostic or prognostic biomarkers exist for GI mucosal toxicity. L-citrulline is one of the most promising biomarker candidates used in clinical settings to quantify enterocyte integrity in various small intestinal diseases. L-citrulline is an intermediate metabolic amino acid produced mainly by functional enterocytes of the small intestine, whereby enterocyte loss will cause a drop in circulating L-citrulline. MethodsIn several repeat-dose toxicity studies, plasma L-citrulline has been evaluated as a potential safety biomarker for intestinal toxicity in beagle dogs and Wistar (Han) rats treated with different oncological drug candidates in drug development. Clinical observations and body weight determinations were performed during the pretreatment, treatment and treatment-free recovery period as well as toxicokinetic, gross and histopathology examinations. The quantitative determination of plasma L-citrulline levels during the pretreatment (only dogs), treatment and treatment-free recovery period were performed using an HPLC MS/MS assay. In cynomolgus monkeys, the first investigations on baseline L-citrulline levels were performed. ResultsIn dogs, a dose- and exposure-dependent decrease of up to 50% in plasma L-citrulline was seen without histopathological alterations. However, a decrease of more than 50% in comparison to the individual animal pretreatment value of L-citrulline correlated very well with histopathological findings (intestinal crypt necrosis, villus atrophy, enterocyte loss) and clinical signs (bloody faeces and diarrhoea). During a treatment-free recovery period, a trend of increasing levels was observed in dogs. In rats, absolute L-citrulline plasma levels of treated animals decreased compared to the values of the concurrent control group. This decrease also correlated with the histopathological findings in the small intestine (single cell necrosis and mucosa atrophy). Because of a large physiological variation in L-citrulline plasma levels in dogs and rats, a clear cut-off value for absolute L-citrulline levels predictive of intestinal mucosal toxicity was difficult to establish. However, a > 50% decrease in L-citrulline plasma levels during the treatment period strongly correlated with histopathological findings. DiscussionBased on the performed analysis, a longitudinal investigation of L-citrulline plasma levels for individual animals in the control and treatment groups is essential and pretreatment values of L-citrulline levels in rodents would be highly informative. Overall, further cross-species comparison (Cynomolgus monkey, mouse) and implementation in clinical trials as exploratory biomarker is essential to foster the hypothesis and to understand completely the clinical relevance of L-citrulline as a small intestine biomarker.

L-citrulline: A preclinical safety biomarker for the small intestine in rats and dogs in repeat dose toxicity studies

IntroductionGastrointestinal (GI) toxicity is still an issue within drug development, especially for novel oncology drugs. The identification of GI mucosal damage at an early stage with high sensitivity and specificity across preclinical species and humans remains difficult. To date, in preclinical studies, no qualified mechanistic, diagnostic or prognostic biomarkers exist for GI mucosal toxicity. l-citrulline is one of the most promising biomarker candidates used in clinical settings to quantify enterocyte integrity in various small intestinal diseases. l-citrulline is an intermediate metabolic amino acid produced mainly by functional enterocytes of the small intestine, whereby enterocyte loss will cause a drop in circulating l-citrulline. MethodsIn several repeat-dose toxicity studies, plasma l-citrulline has been evaluated as a potential safety biomarker for intestinal toxicity in beagle dogs and Wistar (Han) rats treated with different oncological drug candidates in drug development. Clinical observations and body weight determinations were performed during the pretreatment, treatment and treatment-free recovery period as well as toxicokinetic, gross and histopathology examinations. The quantitative determination of plasma l-citrulline levels during the pretreatment (only dogs), treatment and treatment-free recovery period were performed using an HPLC MS/MS assay. In cynomolgus monkeys, the first investigations on baseline l-citrulline levels were performed. ResultsIn dogs, a dose- and exposure-dependent decrease of up to 50% in plasma l-citrulline was seen without histopathological alterations. However, a decrease of more than 50% in comparison to the individual animal pretreatment value of l-citrulline correlated very well with histopathological findings (intestinal crypt necrosis, villus atrophy, enterocyte loss) and clinical signs (bloody faeces and diarrhoea). During a treatment-free recovery period, a trend of increasing levels was observed in dogs. In rats, absolute l-citrulline plasma levels of treated animals decreased compared to the values of the concurrent control group. This decrease also correlated with the histopathological findings in the small intestine (single cell necrosis and mucosa atrophy). Because of a large physiological variation in l-citrulline plasma levels in dogs and rats, a clear cut-off value for absolute l-citrulline levels predictive of intestinal mucosal toxicity was difficult to establish. However, a > 50% decrease in l-citrulline plasma levels during the treatment period strongly correlated with histopathological findings. DiscussionBased on the performed analysis, a longitudinal investigation of l-citrulline plasma levels for individual animals in the control and treatment groups is essential and pretreatment values of l-citrulline levels in rodents would be highly informative. Overall, further cross-species comparison (Cynomolgus monkey, mouse) and implementation in clinical trials as exploratory biomarker is essential to foster the hypothesis and to understand completely the clinical relevance of l-citrulline as a small intestine biomarker.

&amp;lt;i&amp;gt;MTHFRC677T&amp;lt;/i&amp;gt; Polymorphism and Hyperhomocysteinemia in Ischemic Stroke Patients

Background: Homocysteine is an intermediate sulfur amino acid of methionine metabolism. Hyperhomocysteinemia, characterized by increased level of homocysteine, is an independent and modifiable vascular risk factor which metabolic pathway involves vitamins B6, folate and vitamin B12. Objective: We compared the prevalence of MTHFRC677T polymorphism, homocysteine folate and vitamin B12 in ischemic stroke patient’s subgroups. Methods: We conducted a cross-sectional analytical study. The study included 128 consecutive ischemic stroke patients associated with hyperhomocysteinemia. The MTHFRC677T polymorphism was investigated by TaqMan probes (thermos Fisher Scientific) combined with polymerase chain reaction (PCR). We compared the prevalence of MTHFRC677T polymorphism and homocysteine level in ischemic stroke patient’s subgroups. We adjusted the variable homocysteine level to the covariates, MTHFR polymorphism, folate and vitamin B12 with ANCOVA. Results: The sex ratio (men/women) was 1.5 with an average age of 60 years. The prevalence of MTHFRC677T polymorphism was 19.5% with 18% CT and 1.5% TT. Homocysteine level was 29.89 µmol/l in wildtype patients, 26.54 µmol/l in patients with CT genotype, and 56.17 µmol/l in patients with TT genotype (t=2.04, p=0.033, CI 95% [0.017; 0.407]). The MTHFR polymorphism prevalence, homocysteine, folate and vitamin B12 level did not differ between large brain infarction and multiple small brain infarction patients respectively (chi square: Qobs=0.05, p=0.94, 95% CI; ttest: t=0.716, df=126, p=0.475, 95% CI). Conclusion: The MTHFRT677T genotype increases homocysteine level. MTHFR polymorphism and homocysteine did not influence the subtypes of brain ischemic stroke.

Breslow Intermediates (Amino Enols) and Their Keto Tautomers: First Gas-Phase Characterization by IR Ion Spectroscopy.

Breslow intermediates (BIs) are the crucial nucleophilic amino enol intermediates formed from electrophilic aldehydes in the course of N‐heterocyclic carbene (NHC)‐catalyzed umpolung reactions. Both in organocatalytic and enzymatic umpolung, the question whether the Breslow intermediate exists as the nucleophilic enol or in the form of its electrophilic keto tautomer is of utmost importance for its reactivity and function. Herein, the preparation of charge‐tagged Breslow intermediates/keto tautomers derived from three different types of NHCs (imidazolidin‐2‐ylidenes, 1,2,4‐triazolin‐5‐ylidenes, thiazolin‐2‐ylidenes) and aldehydes is reported. An ammonium charge tag is introduced through the aldehyde unit or the NHC. ESI‐MS IR ion spectroscopy allowed the unambiguous conclusion that in the gas phase, the imidazolidin‐2‐ylidene‐derived BI indeed exists as a diamino enol, while both 1,2,4‐triazolin‐5‐ylidenes and thiazolin‐2‐ylidenes give the keto tautomer. This result coincides with the tautomeric states observed for the BIs in solution (NMR) and in the crystalline state (XRD), and is in line with our earlier calculations on the energetics of BI keto–enol equilibria.

C=C-Ene-Reductases Reduce the C=N Bond of Oximes.

Although enzymes have been found for many reactions, there are still transformations for which no enzyme is known. For instance, not a single defined enzyme has been described for the reduction of the C=N bond of an oxime, only whole organisms. Such an enzymatic reduction of an oxime may give access to (chiral) amines. By serendipity, we found that the oxime moiety adjacent to a ketone as well as an ester group can be reduced by ene-reductases (ERs) to an intermediate amino group. ERs are well-known enzymes for the reduction of activated alkenes, as of α,β-unsaturated ketones. For the specific substrate used here, the amine intermediate spontaneously reacts further to tetrasubstituted pyrazines. This reduction reaction represents an unexpected promiscuous activity of ERs expanding the toolkit of transformations using enzymes.

Acute Coronary Syndrome in a Young Male Secondary to Hyperhomocysteinemia

Homocysteine is sulfur containing intermediary amino acid formed by the demethylation of dietary methionine to cysteine. The raised serum homocysteine level is one of the important risk factors associated with coronary artery disease. We present a 23-year-old male with no other risk factors of coronary artery disease presented to us with acute anterior wall myocardial infarction and was treated with primary percutaneous coronary intervention. During the routine work up post percutaneous coronary intervention significant elevation of serum homocysteine level was seen with markedly low serum vitamin B12 level and was treated for it along with standard therapy for acute myocardial infarction. The case illustrates the need to incorporate a plasma homocysteine level during work up of coronary artery disease especially in young patients, more importantly in those without any conventional risk factors.

Changes in enzyme activities and amino acids and their relations with phenolic compounds contents in okra treated by LED lights of different colors

Light-emitting diodes (LEDs) of different wavelengths or colors (i.e., white, red, blue, and green) were used to treat postharvest okra, which is a rich source of phenolic compounds. Relationships between changes in the activities of key enzymes involving in the formation of phenolics (i.e., 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, chorismate mutase, anthranilate synthase, and phenylalanine ammonia lyase) and their contents upon different LED light treatments were for the first time investigated and are fully discussed. The contents of three intermediate amino acids (i.e., phenylalanine, tyrosine, and tryptophan) that formed during light treatments were also measured to confirm the enzyme activities data. White and blue light treatments increased the content of phenolics in the treated okra, while red and green lights increased the formation of other compounds. These results could be well explained by the changing levels of the measured enzyme activities and amino acids contents.

Rapid sample clean-up procedure for aminophosphonate determination by LC/MS analysis

Aminophosphonates are commonly utilised for complexing bivalent ions such as calcium and magnesium. With regard to environmental samples, the analysis of these highly polar compounds is still challenging due to matrix effects and lacking analytical standard methods. We have recently developed a LC/MS method for common aminophosphonates without derivatisation. This LC/MS method delivers precise and accurate measurement for standard samples with very low concentration of cations disturbing the analysis. However, due to matrix effects this LC/MS method requires a sample clean-up being also applicable to natural water or wastewater samples. We developed and optimised a sample clean-up procedure applying strong cation exchange resin Dowex 50WX8. This clean-up allows a single LC/MS analysis of hydroxyethelidene(diphosphonic acid) (HEDP), aminotris(methylenephosphonic acid) (ATMP), ethylenediaminetetra(methyloenephosphonic acid) (EDTMP), diethylenetriaminepenta(methylenephosphonic acid) (DTPMP) and its major intermediates amino(methylphosphonic acid) (AMPA) and iminodi(methylenephosphonic acid) (IDMP). We compared different test conditions with six aminophosphonates in either ultra pure water or tap water. The latter was used to simulate cation concentrations typical for natural waters. To elute all aminophosphonate including AMPA, ammonium acetate addition was necessary. The addition of ammonium acetate was combinable with all tested aminophosphonates and provides high sample quality for LC/MS analysis. For acceptable recovery, the smallest aminophosphonate AMPA required the highest addition of ammonium acetate (1000 mg L-1) during the sample clean-up. Finally, the optimised clean-up procedure was successfully applied to identify and quantify phosphonates from an industrial wastewater sample. The sample clean-up procedure is simple, cheap, rapid and precise and can be further combined with solid phase extraction and more sensitive LC/MS methods.

Sustainable Production of Acetonitrile from Microalgae via Catalytic Fast Pyrolysis with Ammonia over Ga/HZSM-5 Catalysts

We report on catalytic fast pyrolysis microalgae under ammonia atmosphere into acetonitrile over Ga/HZSM-5, which provides a long-term sustainable option for acetonitrile production and microalgae valorization. The effect of various catalysts, reaction conditions and species of microalgae on acetonitrile production was explored systematically. Under the optimized conditions, the maximum carbon yield of acetonitrile from Chlorella vulgaris was 23.4%, and the selectivity of acetonitrile in bio-oil was up to 77.2%. The possible reaction pathways were proposed according to the comprehensive experimental investigation on the conversion of model compounds and intermediates (proteins, carbohydrates, lipids, amino acids, carboxylic acids, furans, and amines). High-resolution transmission electron microscopy (TEM) analysis showed that Ga was present mainly at the outer surface of zeolite crystals, and a small amount of Ga was doped into the zeolite crystals.