Pyroglutamic Acid Research Articles

Study of the Pollen Grain Metabolome under Deposition of Nitrogen and Phosphorus in Taxus baccata L. and Juniperus communis L.

Nitrogen plays an important role in both quantitative and qualitative aspects of plant reproduction, including pollen grain compounds and seed production. Recent studies have pointed out that pollen grains produced by male plants of T. baccata and J. communis subjected to a long period of fertilizer supplementation have lower in vitro germination ability and higher nitrogen content. To gain molecular insights into these observations, we conducted GC-MS analysis of both species to characterize the metabolomes of dry, mature pollen grains, which allowed for the identification and quantification of more than 200 metabolites. The results demonstrated that fertilizer supplementation impacts the relative content of 14 metabolites in J. communis (9 downregulated and 5 upregulated) and 21 in T. baccata (6 downregulated and 15 upregulated). Although plants showed little similarity in patterns, in metabolite profiles, both up and down fold-changes were observed. This is the first report on the gymnosperm pollen grain metabolomic profile and changes induced by long-term nitrogen and phosphorus supplementation. Pollen grains produced by fertilizer-supplemented male individuals had significantly lower relative content of linolenic acid, 5,6-dihydrouracil, maltotriose, galactonic acid, D-xylulose, and glycerol-α-phosphate but higher content of sorbitol, glucosamine, and 1,5-anhydro-D-glucitol as well as n-acetyl-d-hexosamine, dimethyl phthalate, glycine, galactose-6-phosphate, D-fructose-6-phosphate, pyroglutamic acid, and 3-(3-hydroxyphenyl)-3-hydroxypropionic acid. Thus, in pollen grain samples earlier shown to have different germination abilities, the presence of different metabolites indicates a significant environmental impact on the quality of gymnosperm pollen grains.

Proline, hydroxyproline, and pyrrolidone carboxylic acid derivatives as highly efficient but reversible transdermal permeation enhancers

Overcoming the skin barrier properties efficiently, temporarily, and safely for successful transdermal drug delivery remains a challenge. We synthesized three series of potential skin permeation enhancers derived from natural amino acid derivatives proline, 4-hydroxyproline, and pyrrolidone carboxylic acid, which is a component of natural moisturizing factor. Permeation studies using in vitro human skin identified dodecyl prolinates with N-acetyl, propionyl, and butyryl chains (Pro2, Pro3, and Pro4, respectively) as potent enhancers for model drugs theophylline and diclofenac. The proline derivatives were generally more active than 4-hydroxyprolines and pyrrolidone carboxylic acid derivatives. Pro2–4 had acceptable in vitro toxicities on 3T3 fibroblast and HaCaT cell lines with IC50 values in tens of µM. Infrared spectroscopy using the human stratum corneum revealed that these enhancers preferentially interacted with the skin barrier lipids and decreased the overall chain order without causing lipid extraction, while their effects on the stratum corneum protein structures were negligible. The impacts of Pro3 and Pro4 on an in vitro transepidermal water loss and skin electrical impedance were fully reversible. Thus, proline derivatives Pro3 and Pro4 have an advantageous combination of high enhancing potency, low cellular toxicity, and reversible action, which is important for their potential in vivo use as the skin barrier would quickly recover after the drug/enhancer administration is terminated.

Real-Time Infrared Spectroscopic Measurement of Natural Moisturizing Factor

Real-Time Infrared Spectroscopic Measurement of Natural Moisturizing Factor

The moisturizing effect of Capparis spinosa fruit extract targeting filaggrin synthesis and degradation.

Small molecular natural products, such as betaine, have unique moisturizing advantages. Capparis spinosa L. fruit is rich in quaternary ammonium alkaloids such as betaine and stachydrine. However, few studies investigated its efficacy and mechanism on human skin. Polysaccharides-free C. spinosa fruit extract (CS) was obtained to study its moisturizing effect and mechanisms focusing on filaggrin (FLG) synthesis and degradation. The clinical moisturizing test was carried out on human arms, calves, and faces after CS treatment for 0.5-6h. The change in the level of FLG, caspase 14, loricrin, and transglutaminase 5 (TGM 5) was measured by immunofluorescence after CS treatment for 4 and 24 h in a reconstructed epidermis model. Also, the content of pyrrolidone carboxylic acid (PCA) in the stratum corneum was tested by high-performance liquid chromatography (HPLC) both in the epidermis model and human calves. Compared with glycerin (positive control), 5% CS showed a strong skin hydration effect on arms and calves when applied for 0.5-6h. Also, the face hydration increased at 0.5 and 4 h. In addition, 3% CS applied to the recombinant epidermis model under low humidity promoted the immunodetected levels of caspase 14 and PCA content but reduced the levels of FLG at 4h, however, the levels of FLG, loricrin, and TGM 5 were promoted at 24 h. Meanwhile, CS treatment for 4h in human calves increased the PCA content in the stratum corneum by 29.9%. Topical application of CS on human skin showed an instant and long-lasting increase in skin hydration by regulating the FLG network. It promoted FLG degradation to form PCA at 4h both in vivo and in vitro, increasing FLG synthesis after 24 h, potentially reforming the FLG monomer reservoir to alleviate the skin's dry condition.

Role of pyroglutamic acid in cumulus cells of women with polycystic ovary syndrome.

Polycystic ovary syndrome is a complex heterogeneous endocrine disorder associated with established metabolic abnormalities and is a common cause of infertility in females. Glutathione metabolism in the cumulus cells (CCs) of women with PCOS may be correlated to the quality of oocytes for infertility treatment; therefore, we used a metabolomics approach to examine changes in CCs from women with PCOS and oocyte quality. Among 135 women undergoing fertility treatment in the present study, there were 43 women with PCOS and 92 without. CCs were collected from the two groups and levels of pyroglutamic acid were measured using LC-MS/MS followed by qPCR and Western blot analysis to examine genes and proteins involved in pyroglutamic acid metabolism related to glutathione synthesis. Women with PCOS showed increased levels of L-pyroglutamic acid, L-glutamate, and L-phenylalanine and decreased levels of Cys-Gly and N-acetyl-L-methionine. Gene expression of OPLAH, involved in pyroglutamic synthesis, was significantly increased in women with PCOS compared with those without. Gene expression of GSS was significantly decreased in women with PCOS and synthesis of glutathione synthetase protein was decreased. Expression of nuclear factor erythroid 2-related factor 2, involved in resistance to oxidative stress, was significantly increased in women with PCOS. CCs of women with PCOS showed high concentrations of pyroglutamic acid and reduced glutathione synthesis, which causes oxidative stress in CCs, suggesting that decreased glutathione synthesis due to high levels of pyroglutamic acid in CCs may be related to the quality of oocytes in women with PCOS.

RF22 | PMON10 Exposure to a Real Life Environmental Chemical Mixture Alters the Metabolite Profile in the Plasma of Sheep: An Untargeted Metabolomics Study.

Abstract Humans are exposed to a mixture of endocrine disrupting chemicals (EDCs) in everyday life, believed to contribute to a range of non-communicable diseases. While the adverse health impacts of exposure to individual EDCs have been extensively investigated, studies on effects of real-world exposures characterized by chronic low doses of a mixture of chemicals, are only recently emerging. Biosolids derived from human wastes, exemplify both the array and dosage of chemicals humans are exposed to in real life and represents a novel model to investigate the risk posed by the EDCs. Previous studies in sheep have demonstrated aberrant reproductive and metabolic phenotypic effects in offsprings, as a result of maternal biosolid exposure. We hypothesize that EDC-mediated disruption in the maternal metabolic milieu is a potential mediator of the aberrant phenotypic outcomes in offsprings in the biosolid-exposure model. Recent untargeted metabolomic studies provide evidence of the technique's ability to unravel biomarkers of both exposure and effect by identifying the disrupted endogenous metabolite profile and xenobiotic mixture, respectively. In this study, we sought to identify biosolid exposure-induced maternal metabolic fingerprints that could influence fetal programming. Ewes were grazed on either organic fertilizer or biosolid-treated pastures from 1 month before mating until close to parturition. Plasma from blood samples collected on day 90 of the 147-day gestation period, from ewes carrying singleton pregnancies (Control n=15, Treatment n=15) were subjected to untargeted metabolomics using liquid chromatography-quadrupole time-of-flight mass spectrometry. Metabolites were identified by matching the retention time, mass and isotope profile to authentic standards or metabolome databases using Agilent's MassHunter Find by Molecular Feature workflow (v7.0). Untargeted metabolomics identified 1006 and 1295 annotated features from the negative and positive mode respectively. Principal component analysis and orthogonal signal correction partial least-squares discriminant analysis discriminated the metabolomes of control sheep and sheep grazed in biosolid-treated pasture. The key differential metabolites identified by variable importance in projection score primarily belonged to the super-class organic acids, fatty acids, benzenoids, glycerophospholipids and organoheterocyclic compounds. These differentially expressed metabolites are implicated in metabolic pathways including carnitine synthesis, alanine metabolism, glucose alanine cycle, phosphatidyl choline biosynthesis and glutathione metabolism. Some of the top differential metabolites- oleamide, pyroglutamic acid and 15-HETrE have been implicated in fetal development. Oleamide, an endocannabinoid, has growth inhibitory properties. 15-HETrE modulates arachidonic acid metabolism and upregulate PPARγ expression, which plays a pivotal role in fetal development and metabolic outcomes. These findings indicate that biosolids exposure results in alterations in the maternal metabolome which likely contributes to the aberrant phenotypic outcomes reported in offspring of biosolid-exposed mothers. This is the first study to apply metabolomics to validate the effect of real-life EDC exposure in sheep, a translationally relevant precocial model with a developmental trajectory similar to humans. Presentation: Sunday, June 12, 2022 1:00 p.m. - 1:05 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Untargeted Metabolomics Reveals New Markers of Food Processing for Strawberry and Apple Purees.

In general, food processing and its conditions affect nutrients, bioactive compounds, and sensory characteristics of food products. This research aims to use a non-targeted metabolomics approach based on UPLC-ESI-QTOF-MS to determine how fruit processing can affect the metabolic profile of fruits and, through a comprehensive metabolic analysis, identify possible markers to assess their degree of processing. The present study uses a real case from the food industry to evaluate markers of the processing of strawberry and apple purees industrially elaborated with different processing techniques and conditions. The results from the multivariate analysis revealed that samples were grouped according to the type of processing, evidencing changes in their metabolic profiles and an apparent temperature-dependent effect. These metabolic profiles showed changes according to the relevance of thermal conditions but also according to the exclusively cold treatment, in the case of strawberry puree, and the pressure treatment, in the case of apple puree. After data analysis, seven metabolites were identified and proposed as processing markers: pyroglutamic acid, pteroyl-D-glutamic acid, 2-hydroxy-5-methoxy benzoic acid, and 2-hydroxybenzoic acid β-d-glucoside in strawberry and di-hydroxycinnamic acid glucuronide, caffeic acid and lysoPE(18:3(9Z,12Z,15Z)/0:0) in apple purees. The use of these markers may potentially help to objectively measure the degree of food processing and help to clarify the controversial narrative on ultra-processed foods.

Discovery and Validation of Potential Serum Biomarkers with Pro-Inflammatory and DNA Damage Activities in Ulcerative Colitis: A Comprehensive Untargeted Metabolomic Study.

Ulcerative colitis is a type of non-specific inflammatory bowel disease with unclear etiology. It is considered a progressive disease with risks of bowel motility disorders, anorectal dysfunction, and even colorectal cancer. Commonly used diagnostic markers have poor specificity and cannot predict the development of ulcerative colitis. In this study, 77 serum samples (31 patients, 46 healthy controls) were analyzed using high performance liquid chromatography-quadrupole time-of-flight mass spectrometry and 31 metabolites with significant level changes were found, revealing the relationship of ulcerative colitis to disturbed glutathione metabolism and caffeine metabolism. In addition, pyroglutamic acid, a biomarker of cervical cancer and gastric cancer, was identified with elevated levels in the serum of ulcerative colitis patients. The role of pyroglutamic acid was further analyzed, and the results indicated its positive correlation with the upregulation of inflammatory factors and increased levels of phosphorylated histone H2AX (γH2AX) in IEC-6 cells, which are related to DNA damage. All these results suggest that pyroglutamic acid is not only a biomarker for distinguishing ulcerative colitis status, but that it is also a potential effective metabolite that promotes the transformation of ulcerative colitis to colorectal cancer.

The ion balance of Shotokuseki extract promotes filaggrin fragmentation and increases amino acid production and pyrrolidone carboxylic acid content in three-dimensional cultured human epidermis

Natural moisturizing factor (NMF) in the stratum corneum contributes to the retention of moisture there. The purpose of this study was to determine the penetration of ions in Shotokuseki extract (SE) into the three-dimensional cultured epidermis and the effect of NMF on the biosynthesis of amino acids and pyrrolidone carboxylic acid formation. Various ions, amino acids and pyrrolidone carboxylic acid were quantified by inductively coupled plasma mass spectrometry, fully automatic amino acid analyzer or high-performance liquid chromatography (HPLC) in three-dimensional cultured epidermis after application of SE. Gene expression levels of profilaggrin, calpain1, caspase14, and bleomycin hydrolase, which are involved in NMF production, were determined by reverse-transcription qPCR and bleomycin hydrolase activity was determined by aminopeptidase assay. The application of SE increased Na, K, Mg, Ca, Al, and Fe levels in three-dimensional cultured epidermis. The mRNA levels of the starting material of amino acid synthesis profilaggrin, and calpain1 and bleomycin hydrolase, which are involved in its fragmentation, increased. The activity of bleomycin hydrolase also increased. Furthermore, the levels of amino acids and pyrrolidone carboxylic acid increased in the three-dimensional cultured epidermis. This suggests that the ionic composition of SE may be involved in its moisturizing effect on the stratum corneum.Graphical

Metabolite profiling and identification of novel umami compounds in the chaya leaves of two species using multiplatform metabolomics

Chaya (Cnidoscolus chayamansa and C. aconitifolius) is a fast-growing medicinal plant, and its leaves exhibit a strong umami taste. Here metabolite variation and umami-related compounds in the leaves of two chaya species were determined using a multiplatform untargeted-metabolomics approach, electronic tongue, and in silico screening. Metabolite profiles varied between the leaves of the two species and among leaf maturation stages. Young leaves exhibited the highest umami taste intensity, followed by mature and old leaves. Partial least square regression and computational molecular docking analyses revealed five potent umami substances (quinic acid, trigonelline, alanyl–tyrosine, leucyl–glycyl–proline, and leucyl–aspartyl–glutamine) and three known umami compounds (l-glutamic acid, pyroglutamic acid, and 5′-adenosine monophosphate). The five substances were validated as novel umami compounds using electronic tongue assay; leucyl–glycyl–proline exhibited synergism with monosodium glutamate, thereby enhancing the umami taste. Thus, substances contributing to the taste of chaya leaves were successfully identified.

Effects of Donepezil Treatment on Brain Metabolites, Gut Microbiota, and Gut Metabolites in an Amyloid Beta-Induced Cognitive Impairment Mouse Pilot Model.

Accumulated clinical and biomedical evidence indicates that the gut microbiota and their metabolites affect brain function and behavior in various central nervous system disorders. This study was performed to investigate the changes in brain metabolites and composition of the fecal microbial community following injection of amyloid β (Aβ) and donepezil treatment of Aβ-injected mice using metataxonomics and metabolomics. Aβ treatment caused cognitive dysfunction, while donepezil resulted in the successful recovery of memory impairment. The Aβ + donepezil group showed a significantly higher relative abundance of Verrucomicrobia than the Aβ group. The relative abundance of 12 taxa, including Blautia and Akkermansia, differed significantly between the groups. The Aβ + donepezil group had higher levels of oxalate, glycerol, xylose, and palmitoleate in feces and oxalate, pyroglutamic acid, hypoxanthine, and inosine in brain tissues than the Aβ group. The levels of pyroglutamic acid, glutamic acid, and phenylalanine showed similar changes in vivo and in vitro using HT-22 cells. The major metabolic pathways in the brain tissues and gut microbiota affected by Aβ or donepezil treatment of Aβ-injected mice were related to amino acid pathways and sugar metabolism, respectively. These findings suggest that alterations in the gut microbiota might influence the induction and amelioration of Aβ-induced cognitive dysfunction via the gut–brain axis. This study could provide basic data on the effects of Aβ and donepezil on gut microbiota and metabolites in an Aβ-induced cognitive impairment mouse model.

Integrating 16S RRNA gene sequencing and metabolomics to evaluate the association between gut microbiota and serum metabolites in patients with myositis.

Gut microbiota and metabolites have a profound impact on the maintenance of body health. In this study, we assessed the association between gut microbiota and serum metabolite changes in myositis using 16S rRNA gene sequencing and metabolomics to provide new ideas for screening and treating myositis. Blood and faecal samples were collected from 20 myositis patients and 20 healthy control subjects. Then, 16S rRNA gene sequencing, enzyme-linked immunosorbent assays and untargeted metabolomics study were performed to evaluate the relationship between gut microbiota and serum metabolites in patients with myositis. Compared to healthy control subjects, the blood samples from the patients with myositis had elevated levels of interleukin-4 (IL-4), tumour necrosis factor-α (TNF-α), and malondialdehyde (MDA) and decreased superoxide dismutase (SOD) levels. The increase in Bacteroidota (including Bacteroides and Parabacteroides, but not Prevotella) and the decrease in Firmicutes in the patients were accompanied by functional changes in amino acid and lipid metabolism. The gut microbiota (Bacteroides and Parabacteroides) were negatively correlated with the differential serum metabolites (glutamate and taurine). The differential serum metabolites (glutamate, pyrrolidonecarboxylic acid, and taurine) were also correlated with inflammatory factors (IL-4 and TNF-α) and oxidative stress indexes (MDA and SOD). Dysbiosis of gut microbiota in patients with myositis was accompanied by changes in inflammatory factors, oxidative stress indexes, and small molecule metabolites in serum. Blood and faecal biomarkers could be used for screening myositis.

Bloodstain Metabolite Markers: Discovery and Validation for Estimating Age of Bloodstain within 7 Days.

Metabolomic research using analytical chemistry methods has been carried out in a wide range of research fields. However, research combining forensic science and metabolomics is rare. Determining the age of bloodstains could provide key information regarding when a crime was committed. Currently, validated methods for estimating the age of bloodstains are unavailable. Metabolites are intermediate and final products of chemical reactions. Therefore, they are less likely to be degraded than other components of blood under field conditions. In this study, metabolites in bloodstains were analyzed using liquid chromatography-mass spectrometry to discover and validate metabolic markers for determining the age of bloodstains within a week post-bleeding. Nontargeted analysis of bloodstain metabolites revealed statistically significant differences over time. Quantitative analysis of identified candidates via multiple reaction monitoring confirmed the statistical significance according to the age of bloodstain. Pyroglutamic acid, l-glutamine, acetylcarnitine, and adenosine 5'-monophosphate were selected as the final markers. The content of each marker exhibited a statistically significant and consistent tendency to decrease with the age of bloodstain. Furthermore, the effect of hemolysis was considered according to the blood fraction spots of the four markers. This study is the first to identify and validate metabolite markers that may help determine the age of bloodstains within a week post-bleeding. If applied to crime scenes as indicators of the age of bloodstains, they can be used as innovative and important tools for reconstructing crime scenes, suggesting initial investigative direction. This study highlights the forensic utility of blood metabolites ex vivo.

Unraveling metabolic patterns and molecular mechanisms underlying storability in sugar beet

BackgroundSugar beet is an important crop for sugar production. Sugar beet roots are stored up to several weeks post-harvest waiting for processing in the sugar factories. During this time, sucrose loss and invert sugar accumulation decreases the final yield and processing quality. To improve storability, more information about post-harvest metabolism is required. We investigated primary and secondary metabolites of six sugar beet varieties during storage. Based on their variety-specific sucrose loss, three storage classes representing well, moderate, and bad storability were compared. Furthermore, metabolic data were visualized together with transcriptome data to identify potential mechanisms involved in the storage process.ResultsWe found that sugar beet varieties that performed well during storage have higher pools of 15 free amino acids which were already observable at harvest. This storage class-specific feature is visible at harvest as well as after 13 weeks of storage. The profile of most of the detected organic acids and semi-polar metabolites changed during storage. Only pyroglutamic acid and two semi-polar metabolites, including ferulic acid, show higher levels in well storable varieties before and/or after 13 weeks of storage. The combinatorial OMICs approach revealed that well storable varieties had increased downregulation of genes involved in amino acid degradation before and after 13 weeks of storage. Furthermore, we found that most of the differentially genes involved in protein degradation were downregulated in well storable varieties at both timepoints, before and after 13 weeks of storage.ConclusionsOur results indicate that increased levels of 15 free amino acids, pyroglutamic acid and two semi-polar compounds, including ferulic acid, were associated with a better storability of sugar beet taproots. Predictive metabolic patterns were already apparent at harvest. With respect to elongated storage, we highlighted the role of free amino acids in the taproot. Using complementary transcriptomic data, we could identify potential underlying mechanisms of sugar beet storability. These include the downregulation of genes for amino acid degradation and metabolism as well as a suppressed proteolysis in the well storable varieties.

Amadori Reaction Products of Theanine and Glucose: Formation, Structure, and Analysis in Tea.

Amadori rearrangement products (ARPs) derived from the Maillard reaction between theanine and glucose (ARP 1), as well as pyroglutamic acid and glucose (ARP 2), were identified by liquid chromatograph tandem mass spectroscopy methods. The effects of initial reactant ratio, temperature, pH, and heating time on ARP generation were analyzed. The formation of both ARPs was most favored under 100 °C, while an alkaline environment slightly promoted the generation of ARP 1 and acidic conditions contributed more to ARP 2 formation. The decomposition of ARP 1 was suggested to be the predominant formation mechanism of ARP 2. Preparation, purification, and structure identification of ARP 1 were conducted, with its structure confirmed as 1-deoxy-1-l-theanino-d-fructose. The contents of ARP 1 in green, black, dark, white, yellow, and Oolong teas were quantitatively determined, of which black teas contained the highest levels of ARP 1, possibly due to the high glucose content and processing techniques.

Synthesis of alkyl isothiazolidine-1,1-dioxide 3-carboxylates via the intramolecular carbo-Michael reaction strategy

Herein we present short and cost-effective synthesis of alkyl isothiazolidine-1,1-dioxide 3-carboxylates starting from commercially available α-amino acid ester hydrochlorides. These compounds were designed as sulfonamide-containing bioisosteres of known pharmacological template – pyroglutamic acid (pGlu). Specifically, α-amino acid ester hydrochlorides were sulfonylated with (2-chloroethyl)sulfonyl chloride providing in one-pot manner the corresponding alkyl 2-((vinylsulfonyl)amino)carboxylates. The obtained vinyl sulfonamides possessing SO2NH functionality were alkylated with either MeI or MeOCH2Cl (MOMCl) prior to cyclization step. At the same time, vinyl sulfonamides derived from N-monosubstituted and cyclic amino acid esters were directly involved in NaH-mediated intramolecular carbo-Michael reaction affording the target alkyl isothiazolidine-1,1-dioxide 3-carboxylates. Further acid-promoted cleavage of MOM-protecting group led to NH-unsubstituted target compounds.

Urinary Microbial and Metabolomic Profiles in Kidney Stone Disease.

BackgroundKidney stones or nephrolithiasis is a chronic metabolic disease characterized by renal colic and hematuria. Currently, a pathogenetic mechanism resulting in kidney stone formation remains elusive. We performed a multi-omic study investigating urinary microbial compositions and metabolic alterations during nephrolithiasis.MethodUrine samples from healthy and individuals with nephrolithiasis were collected for 16S rRNA gene sequencing and liquid chromatography-mass spectroscopy. Microbiome and metabolome profiles were analyzed individually and combined to construct interactome networks by bioinformatic analysis.ResultsDistinct urinary microbiome profiles were determined in nephrolithiasis patients compared with controls. Thirty-nine differentially abundant taxa between controls and nephrolithiasis patients were identified, and Streptococcus showed the most significant enrichment in nephrolithiasis patients. We also observed significantly different microbial compositions between female and male nephrolithiasis patients. The metabolomic analysis identified 112 metabolites that were differentially expressed. Two significantly enriched metabolic pathways, including biosynthesis of unsaturated fatty acids and tryptophan metabolism, were also identified in nephrolithiasis patients. Four potentially diagnostic metabolites were also identified, including trans-3-hydroxycotinine, pyroglutamic acid, O-desmethylnaproxen, and FAHFA (16:0/18:2), and could function as biomarkers for the early diagnosis of nephrolithiasis. We also identified three metabolites that contributed to kidney stone size. Finally, our integrative analysis of the urinary tract microbiome and metabolome identified distinctly different network characteristics between the two groups.ConclusionsOur study has characterized important profiles and correlations among urinary tract microbiomes and metabolomes in nephrolithiasis patients for the first time. These results shed new light on the pathogenesis of nephrolithiasis and could provide early clinical biomarkers for diagnosing the disease.

Analysis of serum fatty acid, amino acid, and organic acid profiles in gestational hypertension and gestational diabetes mellitus via targeted metabolomics.

This study aimed to characterize metabolite differences and correlations between hypertensive disorders of pregnancy (HP) and gestational diabetes mellitus (GDM) using univariate, multivariate analyses, RF, and pathway analyses in a cross-sectional study. Dietary surveys were collected and targeted metabolomics was applied to measure levels of serum fatty acids, amino acids, and organic acids in 90 pregnant women at 24–28 weeks gestation at the First Affiliated Hospital of Harbin Medical University. Principal components analysis (PCA) and partial least squares-discriminatory analysis (PLS-DA) models were established to distinguish HP, GDM, and healthy, pregnant control individuals. Univariate and multivariate statistical analyses and Random Forest (RF) were used to identify and map co-metabolites to corresponding pathways in the disease states. Finally, risk factors for the disease were assessed by receiver operating characteristics (ROC) analysis. Dietary survey results showed that HP and GDM patients consumed a high-energy diet and the latter also consumed a high-carbohydrate and high-fat diet. Univariate analysis of clinical indices revealed HP and GDM patients had glycolipid disorders, with the former possessing more severe organ dysfunction. Subsequently, co-areas with significant differences identified by basic discriminant analyses and RF revealed lower levels of pyroglutamic acid and higher levels of 2-hydroxybutyric acid and glutamic acid in the GDM group. The number of metabolites increased in the HP group as compared to the healthy pregnant control group, including pyroglutamic acid, γ-aminobutyric acid (GABA), glutamic acid, oleic acid (C18:1), and palmitic acid (C16:0). ROC curves indicated that area under curve (AUC) for pyroglutamic acid in the GDM group was 0.962 (95% CI, 0.920–1.000), and the AUC of joint indicators, including pyroglutamic acid and GABA, in the HP group was 0.972 (95% CI, 0.938–1.000). Collectively, these results show that both GDM and HP patients at mid-gestation possessed dysregulated glucose and lipid metabolism, which may trigger oxidative stress via glutathione metabolism and biosynthesis of unsaturated fatty acids.

Malaria parasite evades mosquito immunity by glutaminyl cyclase–mediated posttranslational protein modification

Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of Plasmodium QC. We show that sporozoites of QC-null mutants of rodent and human malaria parasites are recognized by the mosquito immune system and melanized when they reach the hemocoel. Detailed analyses of rodent malaria QC-null mutants showed that sporozoite numbers in salivary glands are reduced in mosquitoes infected with QC-null or QC catalytically dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito melanization or phagocytosis by hemocytes. Mutation of a single QC-target glutamine of the major sporozoite surface protein (circumsporozoite protein; CSP) of the rodent parasite Plasmodium berghei also results in melanization of sporozoites. These findings indicate that QC-mediated posttranslational modification of surface proteins underlies evasion of killing of sporozoites by the mosquito immune system.

Structural and Functional Analysis of CEX Fractions Collected from a Novel Avastin® Biosimilar Candidate and Its Innovator: A Comparative Study.

Avastin® is a humanized recombinant monoclonal antibody used to treat cancer by targeting VEGF-A to inhibit angiogenesis. SIMAB054, an Avastin® biosimilar candidate developed in this study, showed a different charge variant profile than its innovator. Thus, it is fractionated into acidic, main, and basic isoforms and collected physically by Cation Exchange Chromatography (CEX) for a comprehensive structural and functional analysis. The innovator product, fractionated into the same species and collected by the same method, is used as a reference for comparative analysis. Ultra-Performance Liquid Chromatography (UPLC) ESI-QToF was used to analyze the modifications leading to charge heterogeneities at intact protein and peptide levels. The C-terminal lysine clipping and glycosylation profiles of the samples were monitored by intact mAb analysis. The post-translational modifications, including oxidation, deamidation, and N-terminal pyroglutamic acid formation, were determined by peptide mapping analysis in the selected signal peptides. The relative binding affinities of the fractionated charge isoforms against the antigen, VEGF-A, and the neonatal receptor, FcRn, were revealed by Surface Plasmon Resonance (SPR) studies. The results show that all CEX fractions from the innovator product and the SIMAB054 shared the same structural variants, albeit in different ratios. Common glycoforms and post-translational modifications were the same, but at different percentages for some samples. The dissimilarities were mostly originating from the presence of extra C-term Lysin residues, which are prone to enzymatic degradation in the body, and thus they were previously assessed as clinically irrelevant. Another critical finding was the presence of different glyco proteoforms in different charge species, such as increased galactosylation in the acidic and afucosylation in the basic species. SPR characterization of the isolated charge variants further confirmed that basic species found in the CEX analyses of the biosimilar candidate were also present in the innovator product, although at lower amounts. The charge variants’ in vitro antigen- and neonatal receptor-binding activities varied amongst the samples, which could be further investigated in vivo with a larger sample set to reveal the impact on the pharmacokinetics of drug candidates. Minor structural differences may explain antigen-binding differences in the isolated charge variants, which is a key parameter in a comparability exercise. Consequently, such a biosimilar candidate may not comply with high regulatory standards unless the binding differences observed are justified and demonstrated not to have any clinical impact.