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Background/aims: Due to rapid metabolic and growth rates during the first two years of life, the nutritional needs of young children are high. Given the small portion sizes consumed by children between the ages of 6 and 24 months, it is necessary to improve diets to meet the nutritional needs of this age group. Therefore, the analysis of lysine content is an important parameter in the evaluation of enriched foods., Methods: The utilization of an enzymatic sensor employing lysine-α-oxidase (LOx) as a biorecognition element represents an alternative to the existing methods. This sensor was optimized for quantifying the lysine content in flour mixtures: Quinoa-Lablab purpureus rye - Lablab purpureus, and pole beans - Lablab purpureus, with a maximum ratio of 85g/100g., Results: The addition of lablab purpureus significantly increased the lysine concentration in the enriched samples. When 30 percent was substituted in quinoa, it reached a 143 percent increase. And when 15 percent was substituted in the rye flour, the final concentration of this amino acid increased by 64 percent. In order to quantify the lysine concentration, it was necessary to optimize various parameters during the use of the sensor, e.g. a potentiometric signal was detected upon the depletion of oxygen present during the oxidation of lysine in the samples, and the sensor response was recorded at 2 s. This was possible due to the modification of the pH and the thickness of the membrane. The oxidation of lysine is catalyzed by LOx using molecular oxygen as the electron acceptor. The corresponding acidic compounds and hydrogen peroxide were formed in the reaction medium., Conclusion: It was possible to increase and verify the concentration of lysine in all the flours tested through the use of the biosensor, which turned out to be a valid method for controlling the nutritional quality of flours., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

To investigate the impact of extrusion parameters on the formation of N ε -(carboxymethyl)lysine (CML), N ε -(carboxyethyl)lysine (CEL) and acrylamide in plant-based meat analogues (PBMAs), the content changes and the correlations of compounds related to their formation were studied. The extrusion promoted CML, CEL and acrylamide formation, with more CEL being formed than CML. Variations in the moisture level and barrel temperature exerted a greater influence on the CML, CEL, acrylamide and α-dicarbonyl compounds than the screw speed and the feed rate. An increase in the moisture content led to a decrease in the CEL content, whereas it enhanced CML formation. The impact of moisture on acrylamide formation varied depending on whether low- or high-moisture extrusion was applied. Elevated temperatures promoted the accumulation of CEL, methylglyoxal and 2,3-butanedione while diminishing the accumulation of CML, acrylamide, glyoxal and 3-deoxyglucosone. CML and CEL were positively correlated with glyoxal and methylglyoxal, respectively. CEL and methylglyoxal were negatively correlated with protein and water content, whereas CML, glyoxal and 3-deoxyglucosone displayed positive correlations. In summary, higher moisture levels and feed rates and lower screw speeds and barrel temperatures are advantageous for producing PBMAs with lower CEL and total advanced glycation end-products contents, while lower or higher moisture contents, a lower feed rate and a higher barrel temperature are beneficial to reducing the acrylamide content.

Advanced glycation end products (AGEs) are complex and heterogeneous compounds closely associated with various chronic diseases. The changes in N ε -carboxymethyllysine (CML), N ε -carboxyethyllysine (CEL), N ε -(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1), and fluorescent AGEs (F-AGEs) in fried shrimp during frying (170 °C, 0-210 s) were described by kinetic models. Besides,the correlations between AGEs contents and physicochemical indicators were analyzed to reveal their intrinsic relationship. Results showed that the changes of four AGEs contents followed the zero-order kinetic, and their rate constants were ranked as k CML  < k CEL  ≈ k MG-H1  < k F-AGEs . Oil content and lipid oxidation were critical factors that affected the AGEs levels of the surface layer. Protein content and Maillard reaction were major factors in enhancing the CML and CEL levels of the interior layer. Furthermore, the impact of temperature on the generation of CML and CEL was greater than that of MG-H1 and F-AGEs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Pasteurisation and spray drying are critical steps to ensure the safety and shelf-life of formulae, but these treatments also induce formation of some potentially harmful Maillard reaction products. In this study, the occurrence of potentially harmful Maillard reaction products and proximate compositions in different commercial formulae were analysed. Our results showed that infant formulae had significantly higher concentrations of furosine, N ε -(carboxymethyl)lysine (CML) and N ε -(carboxyethyl)lysine (CEL) than follow-on/toddler formula. Specialty formulae had higher concentrations of glyoxal and CML than other types of formulae. Correlation analysis indicated that concentrations of 5-hydroxymethylfurfural, 3-deoxyglucosone, CML and CEL were closely related to fat contents. These results provided insight into concentrations of potentially harmful Maillard reaction products in different types of formulae and provide a theoretical basis for further optimisation of processing., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: In-cell NMR is a valuable technique for investigating protein structure and function in cellular environments. However, challenges arise due to highly crowded cellular environment, where nonspecific interactions between the target protein and other cellular components can lead to signals broadening or disappearance in NMR spectra., Results: We implemented chemical reduction methylation to selectively modify lysine residues on protein surfaces aiming to weaken charge interactions and recover obscured NMR signals. This method was tested on six proteins varying in molecular size and lysine content. While methylation did not disrupt the protein's native conformation, it successful restored some previously obscured in-cell NMR signals, particularly for proteins with high isoelectric points that decreased post-methylation., Significance: This study affirms lysine methylation as a feasible approach to enhance the sensitivity of in-cell NMR spectra for protein studies. By mitigating signal loss due to nonspecific interactions, this method expands the utility of in-cell NMR for investigating proteins in their natural cellular environment, potentially leading to more accurate structural and functional insights., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Periodontitis is an inflammatory condition of supporting structures of teeth leading to attachment and bone loss. Cigarette smoking is the single most important and modifiable risk factor with 5 to 20-fold susceptibility for periodontal diseases. Reverse smoking is a peculiar habit of smoking where the lit end is kept inside the mouth, which is predominant in the northern coastal districts of Andhra Pradesh. Polyamines are biologically active amines involved in tissue regeneration and modulation of inflammation. The study aimed to evaluate polyamines and check their utility as a marker in detection of periodontitis among different groups. Total polyamine levels showed significant increase in reverse smokers with periodontitis when compared to the other groups. Qualitative analysis by thin layer chromatography showed three polyamine bands with varying intensity among the different groups. Mass spectrometric and NMR analyses of the three bands identified them as N1, N8-diacetyl spermidine, N-acetyl cadaverine and lysine. Most significantly elevated levels of lysine was observed in the smoker and reverse smoker periodontitis groups when compared to healthy and non-smoker periodontitis groups. The significantly elevated levels of N-acetyl cadaverine could be responsible for the more destruction of periodontium in the reverse smoker group. Antioxidant potential decreased significantly in different smoker periodontitis groups. The present study suggests that the quantitative analysis of salivary polyamines, lysine and N-acetyl cadaverine can aid as an easy noninvasive diagnostic method for assessing the periodontal status, especially in smokers., (© 2024. The Author(s).)

Advanced glycation end products (AGEs) are formed within the body as a part of normal metabolism and are also the by-products of cooking food. The elevated levels of AGEs in the body are considered pathogenic. The modern diets contain high levels of AGEs which are getting incorporated into the body AGEs pool and contribute to post-diabetic and age-related complications. The objective of the present study is to estimate the cross-linked AGEs (AGE-fluorescence) and the more stable carboxymethyl-lysine (CML) by spectrofluorimetry and ELISA in 58 kinds of foods in India. It was evident from the results that the foods cooked at higher temperatures showed high levels of AGEs. Among the studied foods, the highest fluorescence was observed in Biscuits 2 (362 AU), and the highest level of carboxymethyl lysine (CML) was found in Soya milk (659.3 ng/g). However, there was less correlation between the AGE-fluorescence and the CML content of the food samples. Processed food such as tomato sauce, chilli sauce, and cheese, along with western foods like chicken nuggets, pizza, and biscuits like Biscuits 2, are known to contain high levels of AGEs. In the present study a preliminary database of AGE-fluorescence and CML content of 58 foods was developed, which is the first attempt among Indian foods. Furthermore, elaborated database can be developed including maximum consumed foods in India which will help in suggesting a better diet for the diabetic population., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Metal clusters have drawn considerable research attention over the years due to their fascinating optical properties. Owing to their appealing photophysical characteristics, these materials have drawn attention as potential candidates for various application in diverse fields, including disease detection, biosensing, chemical sensing, and the fabrication of light-harvesting materials. Presently, there is an increasing research focus on the use of clusters in biomedical research, both as biodetection platform and as bioimaging agents. Of special interest are chiral clusters, which can selectively interact with chiral biomolecules owing to their optical activity. Herein, we showcase the use of a pair of chiroptically active copper clusters for the enantioselective detection of lysine, an amino acid of vast biological relevance. Two techniques are concurrently employed for the detection of lysine at varying concentrations. Circular dichroism serves as a potent tool for detecting lysine at low concentrations, whereas luminescence is effectively employed as a detection method for high analyte concentrations. The combined electronic impact of clusters and lysine resulted in the emergence of an enhanced enantioselective Cotton effect at specific wavelength., (© 2024 Wiley Periodicals LLC.)

There is little data on directly measured carboxymethyl lysine (CML) content in Indonesian foods. This study aimed to generate a database of CML values in foods commonly consumed in West Java and West Sumatra. The results were to be used to update our previous estimated CML values. CML values in food samples were measured using high-pressure liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Food protein content was analyzed by Kjeldahl's method or inferred from the nutrition facts' label. A total of 210 food samples were examined, with the food groups of meat and poultry (1.06 mg CML/100 g edible food), and starchy foods (0.21 mg/100 g edible food) having the highest and lowest mean CML levels, respectively. We found that the foods with the top three highest CML content were fried starch dough ( cimol ), fried fish crackers, and chicken gulai. The mean of the estimated values (0.80 mg CML/100 g edible food) was higher than the directly measured values (0.66 mg CML/100 g edible food), [ p < 0.035]. Conclusion: This database provides information on CML values in Indonesian foods, and can be further used to make a guide policy for the selection of foods to reduce non-communicable diseases. Further measurements are needed on Indonesian dishes to complete the database.

Studies on the interactions and links between aroma and hazardous compounds were inadequately investigated. A complete analysis was conducted on the key aroma compounds, typical hazardous compounds and their precursors in 25 samples of roasted meats. Forty-nine aroma compounds were identified as essential odorants with odor-activity values exceeds 1. N ε -carboxymethyl lysine (CML, 11.78-49.32 μg/g) and N ε -carboxyethyl lysine (CEL, 8.48-171.00 μg/g) were identified as representative advanced glycation end products (AGEs) of meats with high concentrations. Harman and Norharman were typical heterocyclic aromatic amines. Meanwhile, correlation analysis indicated that aldehyde and alcohols showed a negative correlation with AGEs (p < 0.01), while pyrazines might affect the formation of Harman and Norharman. The furaldehyde, 1-hexanol, 2, 4-Decadienal, AGEs, and creatine were regarded as potential biomarkers that distinguished different roasted meat products. Therefore, the study could provide new insights for synergistic regulation of aroma and hazardous compounds in roasted meat products., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Objective: The objective of this study was to establish a methodology for determining carboxymethyl lysine (CML) and carboxyethyl lysine (CEL) concentrations in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The test results were also used for clinical aging research., Methods: Human plasma samples were incubated with aqueous perfluorovaleric acid (NFPA), succeeded by precipitation utilizing trichloroacetic acid, hydrolysis facilitated by hydrochloric acid, nitrogen drying, and ultimate re-dissolution utilizing NFPA, followed by filtration. Cotinine-D 3 was added as an internal standard. The separation was performed on an Agela Venusil ASB C 18 column (50 mm × 4.6 mm, 5 μm) with a 5 mmol/L NFPA and acetonitrile/water of 60:40 (v/v) containing 0.15% formic acid. The multiple reaction monitoring mode was used for detecting CML, CEL, and cotinine-D 3 , with ion pairs m/z 205.2 > 84.1 (for quantitative) and m/z 205.2 > m/z 130.0 for CML, m/z 219.1 > 84.1 (for quantitative) and m/z 219.1 > m/z 130.1 for CEL, and m/z 180.1 > 80.1 for cotinine-D 3 , respectively., Results: The separation of CML and CEL was accomplished within a total analysis time of 6 minutes. The retention times of CML, CEL, and cotinine-D 3 were 3.43 minutes, 3.46 minutes, and 4.50 minutes, respectively. The assay exhibited linearity in the concentration range of 0.025-1.500 μmol/L, with a lower limit of quantification of 0.025 μmol/L for both compounds. The relative standard deviations of intra-day and inter-day were both below 9%, and the relative errors were both within the range of ±4%. The average recoveries were 94.24% for CML and 97.89% for CEL., Conclusion: The results indicate that the developed methodology is fast, highly sensitive, highly specific, reproducible, and suitable for the rapid detection of CML and CEL in clinical human plasma samples. The outcomes of the clinical research project on aging underscored the important indicative significance of these two indicators for research on human aging., (© 2024. The Author(s).)

During the last years, a strong increase in the sales volume and consumption of plant-based drinks was observed, which were partly used as an alternative to cow's milk. As milk is a relevant protein source in many countries, we have investigated the protein bioaccessibility and digestibility of soy, almond, and oat drinks in comparison to milk using the tiny-TIMsg gastrointestinal model. The relative protein digestibility of all products was between 81% (soy drink) and 90% (milk). The digestible indispensable amino acid score (DIAAS) in vitro method was used to estimate the protein nutritional quality. The highest DIAAS values were obtained for milk in tryptophan (117%) and soy drink in sulfur containing amino acids (100%). Oat drink was limited in lysine (73%), almond drink in lysine (34%) and the sulfur containing amino acids (56%). Additionally, the antioxidant activity of the bioaccessible fractions was analyzed using Trolox equivalent antioxidative capacity and oxygen radical absorbance capacity assays, revealing a higher antioxidative potential of milk and soy drink compared to oat and almond drink.

Objective: The paper reports an attempt to develop and validate a HILIC UPLC/ QTof MS method for quantifying N-ε-carboxymethyl-L-lysine (CML) in vitro , testing N-ε- carboxy[D 2 ]methyl-L-lysine (d 2 -CML), and N-ε-carboxy[4,4,5,5-D 4 ]methyl-L-lysine (d 4 -CML) as internal standards., Methods: During the method development, several challenging questions occurred that hindered the successful completion of the method. The study emphasizes the impact of issues, generally overlooked in the development of similar analytical protocols. For instance, the use of glassware and plasticware was critical for the accurate quantification of CML. Moreover, the origin of atypical variation in the response of the deuterated internal standards, though widely used in other experimental procedures, was investigated., Result: A narrative description of the systematic approach used to address the various drawbacks during the analytical method development and validation is presented., Conclusion: Reporting those findings can be considered beneficial while bringing an insightful notion about critical factors and potential interferences. Therefore, some conclusion and ideas can be drawn from these trouble-shooting questions, which might help other researchers to develop more reliable bioanalytical methods, or to raise their awareness of stumbling blocks along the way., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

During blood storage, red blood cells (RBCs) undergo physical, chemical, and metabolic changes that may contribute to post-transfusion complications. Due to the hyperglycemic environment of typical solutions used for RBC storage, the formation of advanced glycation endproducts (AGEs) on the stored RBCs has been implicated as a detrimental chemical change during storage. Unfortunately, there are limited studies involving quantitative determination and differentiation of carboxymethyl-lysine (CML) and carboxyethyl-lysine (CEL), two commonly formed AGEs, and no reported studies comparing these AGEs in experimental storage solutions. In this study, CML and CEL were identified and quantified on freshly drawn blood samples in two types of storage solutions, standard additive solution 1 (AS-1) and a normoglycemic version of AS-1 (AS-1N). To facilitate detection of the AGEs, a novel method was developed to reliably extract AGEs from RBCs, provide Food and Drug Administration (FDA) bioanalytical guidance criteria, and enable acceptable selectivity for these analytes. Ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) was utilized to identify and quantify the AGEs. Results show this method is accurate, precise, has minimal interferences or matrix effects, and overcomes the issue of detecting AGE byproducts. Importantly, AGEs can be detected and quantified in both types of blood storage solutions (AS-1 and AS-1N), thereby enabling long-term (6 weeks) blood storage related studies.

This study aimed to investigate the effects of injecting l-arginine and l-lysine solution before freezing and after thawing on the emulsifying and gelling properties of myofibrillar proteins (MPs) of frozen porcine longissimus dorsi. The results showed that the pre-freezing injections were more effective in alleviating the decrease in emulsifying properties of MPs compared with the post-thawing injections, as evidenced by higher emulsion creaming index, oil droplet size, interfacial absorptive protein amount, and viscoelasticity. Additionally, the pre-freezing injections could effectively mitigate the damage to the gelling properties of MPs, as evidenced by the formation of a homogeneous and compact gel network with stronger water retention, strength and chemical forces, as well as a higher proportion of non-flowing water, whereas the post-thawing injections could not. These results demonstrated that the injection of l-arginine and l-lysine solution before freezing could delay freezing-induced damage to the emulsifying and gelling properties of MPs, keeping the processing characteristics of frozen porcine., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The inhibitory effects of liquiritigenin, liquiritin and glycyrrhizic acid against the hazards during the preparation of thermal reaction beef flavoring were investigated using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Liquiritigenin(1.5 mM) inhibited N ε -carboxymethyl-L-lysine and N ε -carboxyethyl-L-lysine by up to 38.69 % and 61.27 %, respectively; 1.5 mM liquiritin inhibited 4-methylimidazole by up to 48.28 %; and 1.5 mM liquiritigenin and 1.0 mM liquiritin inhibited hydroxymethylfurfural by up to 61.20 % and 59.31 %, respectively. The results of the model system showed that the inhibitory effect of the 3 inhibitors could be extended to other thermal reaction flavoring systems. The 3 inhibitors can effectively block key intermediates in beef flavoring, and liquiritigenin can inhibit up to 22.97 % of glyoxal and 22.89 % of methylglyoxal. In addition, liquiritigenin and liquiritin can directly eliminate up to 25.87 % and 21.01 % of methylglyoxal by addition and other means. Free radicals in the simultaneous formation model system were measured using electron spin resonance (ESR), and the results showed that liquiritigenin, liquiritin and glycyrrhizic acid could scavenge free radicals in the system in a dose-dependent manner, with scavenging rates of up to 44.88-57.09 %. Therefore, the inhibitory effects of the 3 inhibitors can be attributed to the intermediate blocking and free radical scavenging pathways., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Advanced glycation end products (AGEs) and heterocyclic amines (HAs) are two kinds of important harmful products formed simultaneously during the thermal processing of proteinaceous food. In this paper, the effect of roasting conditions on the formation of AGEs and HAs, as well as active carbonyl intermediates in common peanut (C-peanut) and high-oleic acid peanut (HO-peanut) was studied simultaneously for the first time. In general, with the increase in roasting temperature (160-200 °C) and time, the contents of AGEs, HAs and active carbonyl intermediates (i.e., glyoxal (GO) and methylglyoxal (MGO)) significantly increased in peanuts. Four kinds of HAs (i.e., AαC, DMIP, Harman and Norharman) were observed in roasted peanuts, of which Harman and Norharman accounted for about 93.0% of the total HAs content after roasting for 30 min at 200 °C. Furthermore, a correlation analysis among AGEs (i.e., N ε -(1-Carboxymethyl)-L-lysine (CML) and N ε -(1-Carboxyethyl)-L-lysine (CEL)), HAs, GO and MGO was conducted. Most of these compounds showed an excellent positive linear relationship ( p ≤ 0.001) with each other. The evident increase in GO and MGO contents implied an increase in not only the content of AGEs but also HAs. However, contents of AGEs and HAs showed no significant difference between roasted HO-peanut and C-peanut. This study would provide a theoretical basis for simultaneously controlling the levels of AGEs and HAs in thermal processed peanut foods.

Brown fermented milk (BFM) is favored by consumers in the dairy market for its unique burnt flavor and brown color. However, Maillard reaction products (MRP) from high-temperature baking are also noteworthy. In this study, tea polyphenols (TP) were initially developed as potential inhibitors of MRP formation in BFM. The results showed that the flavor profile of BFM did not change after adding 0.08% (wt/wt) of TP, and its inhibition rates on 5-hydroxymethyl-2-furaldehyde (5-HMF), glyoxal (GO), methylglyoxal (MGO), N ε -carboxymethyl lysine (CML), and N ε -carboxyethyl lysine (CEL) were 60.8%, 27.12%, 23.44%, 57.7%, and 31.28%, respectively. After 21 d of storage, the levels of 5-HMF, GO, MGO, CML, and CEL in BFM with TP were 46.3%, 9.7%, 20.6%, 5.2%, and 24.7% lower than the control group, respectively. Moreover, a smaller change occurred in their color and the browning index was lower than that of the control group. The significance of this study was to develop TP as additives to inhibit the production of MRP in brown fermented yogurt without changing color and flavors, thereby making dairy products safer for consumers., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Lysine, the first limiting essential amino acid, the deficiency of which seriously affects the health of human and animals. In this study, quinoa germination significantly increased the nutrients, especially lysine content. To better understanding the underlying molecular mechanism of lysine biosynthesis, isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics, RNA-sequencing (RNA-Seq) technology and liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) platform-based phytohormones analyses were conducted. Through proteome analyses, a total of 11,406 differentially expressed proteins were identified, which were mainly related to secondary metabolites. The lysine-rich storage globulins and endogenous phytohormones probably contributed the increased lysine content in quinoa during germination. Furthermore, aspartic acid semialdehyde dehydrogenase is essential for lysine synthesis in addition to aspartate kinase and dihydropyridine dicarboxylic acid synthase. Protein-protein interaction analysis indicated lysine biosynthesis is associated with "amino metabolism" and "starch and sucrose metabolism". Above all, our study screens the candidate genes participated in lysine accumulation and explores the factors affected lysine biosynthesis by multi-omics analysis. These information not only paves a foundation for breeding lysine-rich quinoa sprouts but also provides valuable multi-omics resource to explore the characteristic of nutrients during quinoa germination., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Modifying dietary amino acids has been proposed as a strategy to improve eggshell quality by slowing down increases in egg weight (EW). This study aimed to investigate the effects of different levels of digestible lysine (dLYS) and ratios of digestible sulfur amino acids (dTSAA) to dLYS on performance and eggshell quality in ISA brown hens. A total of 288 hens were individually housed and assigned to 8 treatments, which combined 2 levels of dLYS (5.9 and 5.5 g/kg) with 4 ratios of dTSAA:dLYS (90, 85, 80, and 75) in a factorial arrangement. The study lasted 12 wk, starting at 62 wk of age. The number of eggs was not affected by the interaction between dLYS and dTSAA:dLYS or their main effect. However, the interaction between dLYS and dTSAA:dLYS showed that reducing the dTSAA:dLYS ratio from 85 to 75 when hens were fed 5.5 g/kg of dLYS resulted in a lower EW. Conversely, when hens were fed 5.9 g/kg of dLYS, no significant difference was found in EW among the different ratios of dTSAA:dLYS. Although there was no interaction between the levels of dLYS and dTSAA:dLYS on eggshell quality, reducing the dLYS level from 5.9 to 5.5 slowed down the deterioration in eggshell-breaking strength and eggshell thickness, regardless of the dTSAA:dLYS ratio. These findings suggest that adjusting dietary dLYS while maintaining the dTSAA:dLYS ratio of no less than 85 may be an effective strategy for decelerating the deterioration of eggshell quality in laying hen operations without impacting the egg production rate., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Maillard reaction browning is one of the quality deterioration in dried fruit products, but how pectin affects Maillard reaction in the fruit drying and storage process is not clear. This study aimed at investigating the mechanism of pectin variation impact on the browning of Maillard reaction by using simulated system (l-lysine, d-fructose and pectin) in thermal (60 °C and 90 °C for 8 h) and storage (37 °C for 14 days) process. Results showed that apple pectin (AP) and sugar beet pectin (SP) significantly enhanced the browning index (BI) of the Maillard reaction system by 0.01 to 134.51 in the thermal and storage processes, respectively, which were methylation degree of pectin-dependent. The pectin depolymerization product participated Maillard reaction by reacting with l-lysine, and increasing the 5-hydroxymethyl furfural (5-HMF) content (1.25-11.41-fold) and Abs 420nm (0.01-0.09). It also produced a new product (m/z 225.1245), which finally increased browning level of the system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

As a representative product of advanced glycation end products, Nɛ-carboxymethyllysine (CML) exists in free and bound forms in vivo and in food with different bioavailability. To thoroughly understand the bioavailability of free Nɛ-carboxymethyllysine (CML) and bovine serum albumin (BSA)-CML in vivo after intragastric administration, pharmacokinetics, biodistribution, and excretion of CML in rats were investigated by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Pharmacokinetics results revealed that free CML peaked at 1.83 h (1684.72 ± 78.08 ng/mL) and 1.33 h (1440.84 ± 72.48 ng/mL) in serum after intragastric administration of free CML and BSA-CML, demonstrating the higher absorption of free CML than BSA-CML. Besides, dietary free CML exhibited a relatively lower body clearance and tissue distribution than dietary BSA-CML based on the apparent volume of distribution and body clearance. Moreover, free CML was concentrated in the kidneys, indicating that kidneys were the target organ for the uptake of absorbed free CML. Additionally, the total excretion rate of CML in urine and feces were 37% and 60% after oral administration of free CML and BSA-CML. These results shed pivotal light on a better understanding of the biological effects of free and bound CML on health., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Soy protein isolate (SPI) is an attractive natural material for preparing wood adhesives that has found broad application. However, poor mechanical properties and unfavorable water resistance of wood composites with SPI adhesive bonds limit its more extensive utilization. The combination of lysine (Lys) with a small molecular structure as a curing agent for modified soy-based wood adhesive allows Lys to penetrate wood pores easily and can result in better mechanical strength of soy protein-based composites, leading to the formation of strong chemical bonds between the amino acid and wood interface. Scanning electron microscopy (SEM) results showed that the degree of penetration of the S/G/L-9% adhesive into the wood was significantly increased, the voids, such as ducts of wood at the bonding interface, were filled, and the interfacial bonding ability of the plywood was enhanced. Compared with the pure SPI adhesive, the corresponding wood breakage rate was boosted to 84%. The wet shear strength of the modified SPI adhesive was 0.64 MPa. When Lys and glycerol epoxy resin (GER) were added, the wet shear strength of plywood prepared by the S/G/L-9% adhesive reached 1.22 MPa, which increased by 29.8% compared with only GER (0.94 MPa). Furthermore, the resultant SPI adhesive displayed excellent thermostability. Water resistance of S/G/L-9% adhesive was further enhanced with respect to pure SPI and S/GER adhesives through curing with 9% Lys. In addition, this work provides a new and feasible strategy for the development and application of manufacturing low-cost, and renewable biobased adhesives with excellent mechanical properties, a promising alternative to traditional formaldehyde-free adhesives in the wood industry.

The field of N-terminomics has been advancing with the development of novel methods that provide a comprehensive and unbiased view of the N-terminome. Negative selection N-terminomics enables the identification of free and naturally modified protein N-termini. Here, we present a streamlined protocol that combines two negative selection N-terminomics methods, LATE and HYTANE, to increase N-terminome coverage by 1.5-fold compared to using a single methodology. Our protocol includes sample preparation and data analysis of both methods and can be applied to studying the N-terminome of diverse samples. The suggested approach enables researchers to achieve a more detailed and accurate understanding of the N-terminome., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The formation of Maillard reaction products, including Amadori compounds (determined as furosine), advanced glycation end products (AGEs), α-dicarbonyl and furfural compounds, as well as amino acid cross-links (lysinoalanine and lanthionine) was investigated in direct (DI) and indirect (IN) UHT-treated experimental liquid infant formula (IF) during storage at 40 °C. IN-IF had higher concentrations of all investigated compounds compared to DI-IF and low pasteurized IF. IN UHT treatment induced significantly higher concentrations of α-dicarbonyl compounds (glyoxal, methylglyoxal, 3-deoxyglucosone and 3-deoxygalactosone) compared to DI, which facilitated increased formation of AGEs (N-Ɛ-(carboxymethyl)lysine, methylglyoxal- and glyoxal-derived hydroimidazolones) in unstored IFs. During storage for 6 months, concentrations of furosine and AGEs increased while α-dicarbonyl compounds decreased. Principal component analysis indicated that differences between IN-IF and DI-IF disappeared after 2 months of storage. IN-IF had higher concentrations of lysinoalanine and lanthionine and lower concentrations of available lysine and arginine than DI-IF indicating higher loss of protein quality in IN-IF., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Thermal processing is used to produce most commercial pet foods and treats to improve safety, shelf life, nutritional characteristics, texture, and nutrient digestibility. However, heat treatments can degrade protein quality by damaging essential amino acids, as well as contribute to the Maillard reaction. The Maillard reaction forms melanoidins that favorably improve food qualities (e.g., color, flavor, aroma), but also form Maillard reaction products (MRP) and advanced glycation end-products that may negatively affect health. Because commercial pet diets are frequently fed to domestic cats and dogs throughout their lifetimes, it is critical to quantify MRP concentrations and understand the variables that influence their formation so future diets may be formulated with that in mind. Because few research studies on MRP in pet diets have been conducted, the goals of this study were to measure the MRP in commercial pet foods and treats, estimate pet MRP intake, and correlate MRP with dietary macronutrient concentrations. Fifty-three dry and wet dog foods, dog treats, and cat foods were analyzed for dry matter, organic matter, crude protein, acid-hydrolyzed fat, total dietary fiber, and gross energy using standard techniques. MRP were analyzed using high-performance liquid chromatography and gas chromatography-mass spectrometry. Data were analyzed using the Mixed Models procedure of SAS 9.4. Dry foods had lower reactive lysine concentrations and reactive lysine: total lysine ratios (indicator of damage) than wet foods. Wet foods had more fructoselysine (FRUC) than dry foods; however, dry dog treats contained more FRUC than wet dog treats. The greatest 5-hydroxymethyl-2-furfural (HMF) concentrations were measured in dry and wet dog foods, whereas the lowest HMF concentrations were measured in dry and wet cat foods. Based on dietary concentrations and estimated food intakes, dogs and cats fed wet foods are more likely to consume higher carboxymethyllysine and FRUC concentrations than those fed dry foods. However, dogs fed wet foods are more likely to consume higher HMF concentrations than those fed dry foods. In cats, those fed dry foods would consume higher HMF concentrations than those fed wet foods. We demonstrated that pet foods and treats contain highly variable MRP concentrations and depend on diet/treat type. In general, higher MRP concentrations were measured in wet pet foods and dry treats. While these findings are valuable, in vivo testing is needed to determine if and how MRP consumption affect pet health., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Maintaining the health of seafarers is a difficult task during long-term voyages. Little is known about the corresponding changes in the gut microbiome-host interaction. This study recruited 30 seafarers undertaking a 6-month voyage and analyzed their gut microbiota using 16S rRNA gene sequencing. Fecal untargeted metabolomics analysis was performed using liquid chromatography-mass spectrometry. Significant changes in the composition of the gut microbiota and an increased ratio of Firmicutes / Bacteroidetes at the end (day 180) of the 6-month voyage, relative to the start (day 0), were observed. At the genus level, the abundances of Holdemanella and Plesiomonas were significantly increased, while the abundance of Bacteroides was decreased. Predicted microbial functional analysis revealed significant decreases in folate biosynthesis and biotin metabolism. Furthermore, 20 differential metabolites within six differentially enriched human metabolic pathways (including arginine biosynthesis, lysine degradation, phenylalanine metabolism, sphingolipid metabolism, pentose and glucuronate interconversions, and glycine, serine, and threonine metabolism) were identified by comparing the fecal metabolites at day 0 and day 180. Spearman correlation analysis revealed close relationships between the 14 differential microbiota members and the six differential fecal metabolites that might affect specific human metabolic pathways. This study adopted a multi-omics approach and provides potential targets for maintaining the health of seafarers during long-term voyages. These findings are worthy of more in-depth exploration in future studies. IMPORTANCE Maintaining the health of seafarers undertaking long-term voyages is a difficult task. Apart from the alterations in the gut microbiome and fecal metabolites after a long-term voyage, our study also revealed that 20 differential metabolites within six differentially enriched human metabolic pathways are worthy of attention. Moreover, we found close relationships between the 14 differential microbiota members and the six differential fecal metabolites that might impact specific human metabolic pathways. Accordingly, preventative measures, such as adjusting the gut microbiota by decreasing potential pathobionts or increasing potential probiotics as well as offsetting the decrease in B vitamins and beneficial metabolites (e.g., d-glucuronic acid and citrulline) via dietary adjustment or nutritional supplements, might improve the health of seafarers during long-term sea voyages. These findings provide valuable clues about gut microbiome-host interactions and propose potential targets for maintaining the health of seafarers engaged in long-term sea voyages.

Background/objectives: Self-reported digestive intolerance to dairy foods is common. As dairy can be an important source of dietary protein, this study aimed to identify whether milk protein digestion is compromised in individuals with digestive intolerance., Subjects/methods: Adult women (n = 40) were enroled in this double-blinded, randomised cross-over trial, with digestive symptoms characterised using a lactose challenge and self-reported digestive symptom questionnaire. Participants were classified as either lactose intolerant (LI, n = 10), non-lactose dairy intolerant (NLDI, n = 20) or dairy tolerant (DT, n = 10). In a randomised sequence, participants consumed three different kinds of milk (750 ml); conventional milk (CON), a2 Milk™ (A2M), and lactose-free conventional milk (LF-CON). Circulatory plasma amino acid (AA) concentrations were measured at baseline and every 30 min until 3 h post-ingestion., Results: In all participants across all milk types, plasma AA concentrations (AUC 0-180 ) increased after milk ingestion with no significant differences in responses observed between milk types or participants (P > 0.05), with the exception of the suppressed lysine response in the DT group following A2M ingestion, relative to the other two groups and milk types (P < 0.05)., Conclusion: Milk protein digestion, as determined by circulatory AAs, is largely unaffected by dairy- and lactose- intolerances., (© 2022. The Author(s).)

Accumulation of advanced glycation end products (AGEs) of the Maillard reaction has been implicated in the pathogenesis of diabetes and its complications. Connarus ruber has been used as a folk remedy for several diseases, including diabetes; however, its underlying mechanism has not yet been investigated. This study investigated the effects of C. ruber extract against glycation on collagen-linked AGEs in vitro and streptozotocin-induced diabetic rats (STZ-DM rats) in vivo . The antiglycation activities of C. ruber extract and aminoguanidine (AG) were examined using a collagen glycation assay kit. Nonfluorescent AGE, N ε -carboxymethyl lysine (CML), N ω -carboxymethyl arginine, and N ε -carboxyethyl lysine levels were measured via electrospray ionization-liquid chromatography-tandem mass spectrometry. The effect of the extract on the cytotoxicity of methylglyoxal (MG), a precursor of AGEs, was examined in HL60 cells. STZ-DM rats were treated with the extract for 4 wk, and the effect was assessed using biochemical markers in the serum and CML-positive cells in renal tissues. C. ruber extract dose-dependently inhibited the glycation of collagen and formation of nonfluorescent AGEs, which was comparable to AG, and it significantly attenuated MG-induced cytotoxicity in HL60 cells. Furthermore, the glycated albumin levels in STZ-DM rats decreased, the increase in serum lipid levels was reversed, and immunohistochemistry demonstrated that CML deposition in the glomerulus of STZ-DM rats significantly decreased. Although further studies are needed, C. ruber could be a potential therapeutic for preventing and progressing many pathological conditions, including diabetes., Competing Interests: Authors Rumi Takasugi, Michi Iwai, and Taizo Taniguchi of this study are regular employees of the companies. However, the funders did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to Planta Medica policies on sharing data and materials., (Thieme. All rights reserved.)

N ε -(carboxymethyl)lysine (CML) and N ε -(carboxyethyl)lysine (CEL) have been the most extensively studied advanced glycation end-products (AGEs) in foods. Their formation mechanism, especially the latter, has not been clearly delineated in fermented food. In this work, the relative contents of CEL and CML were evaluated in a sourdough-bread and a silica solid chemical model. Lactic acid (LA) content in the sourdough increased with fermentation time that was accompanied by an increase in CEL, but not CML content in the bread. The role of LA as a precursor for CEL was supported by a positive significant correlation between LA and CEL contents, and further analysis using isotope-labeled LA (LA- 13 C 3 ) revealed that the three carbon atoms of LA were incorporated into CEL. These findings for the first time indicate LA may function as a precursor to promote CEL formation in sourdough bread that merits further investigation., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The Maillard reaction kinetics in the confined volume of the thin film produced by ESI microdroplet deposition was studied by mass spectrometry. The almost exclusive formation of the Amadori product from the reaction of D-xylose and D-glucose toward L-glycine and L-lysine was demonstrated. The thin film Maillard reaction occurred at a mild synthetic condition under which the same process in solution was not observed. The comparison of the thin film kinetics with that of the reaction performed in solution showed strong thin film rate acceleration factors.

Diisocyanates have long been a leading cause of occupational asthma. As control often relies on personal protective equipment and there is the potential for skin uptake, biological monitoring is often used to assess worker exposure. Current routine biological monitoring methods do not distinguish between a diisocyanate and the corresponding diamine exposure in urine samples; therefore, a specific urinary biomarker is desirable. Urine samples were obtained from a group of workers exposed to methylenediphenyl diisocyanate (MDI) where aerosol generation was unlikely. Lysine conjugates of MDI were extracted from urine by solid phase extraction; analysis was performed by liquid chromatography tandem mass spectrometry. Acetylated MDI-lysine (acMDI-Lys) conjugates were detected in 73% of samples tested from persons with exposure to MDI compared to 93% of samples that were positive for methylene dianiline (MDA) in hydrolysed urine. There was a weak but significant positive correlation between the two biomarkers (r 2 = 0.377). This is the first report detecting and quantifying acMDI-Lys in the urine of workers exposed to MDI, and acMDI-Lys may be a useful non-invasive biomarker in discriminating between MDI and MDA exposures.

Aflatoxin B 1 is an important toxic food contaminant and there is very little information available about its exposure and effects on the health of the Pakistani population. Therefore, children ( n  = 238) aged 1-11 years were recruited in this study to estimate the levels of aflatoxin B 1 -lysine adduct and to measure its adverse effects on growth. Blood samples were analyzed to detect AFB 1 -lysine adducts through high-performance liquid chromatography. Socio-demographic information and anthropometry measurements were also obtained. All participants had detectable levels of AFB 1 -lysine adduct with a median concentration of 10.66 pg/mg albumin (95% CI: 8.6-12.4). Differences in area of residence ( p  < 0.05) and the father's employment ( p  < 0.05) were significant predictors for aflatoxin concentration levels in ordinary least square and quantile regression models (residence in 75th quantile and father employment in 90th quantile). Children aged from 5 to 11 years in the 5th and 90th quantiles of the regression model had a significant association with aflatoxin levels. A very high (50.4%, 120/238) prevalence of growth impairment (stunting, wasting, and underweight) was also observed in this study. Although we couldn't establish the effect of aflatoxin on growth impairment, children with low serum albumin levels (OR = 0.18; 95% CI: 0.05-0.56; p  = 0.004) were likely to be at risk of wasting. Also, low birth weight was strongly associated with wasting (OR = 3.11; 95% CI: 1.36-7.03; p  = 0.006) and underweight (OR = 4.60; 95% CI: 2.21-10.05; p = <0.001), while the mother's school level education had a correlation with child stunting (OR = 1.84; 95% CI: 1.07-3.22; p  = 0.029). The high prevalence of growth impairment and high concentration of serum AFB 1 -lysine adduct levels in study participants demand immediate efforts to mitigate the adverse health outcomes in children in Pakistan.

1. Cobb and Ross broilers (200 of each sex and breed) were fed four phases of diets ad libitum formulated with balanced protein to match their amino acid requirements throughout growth. Ten birds per genotype were sampled and euthanised at two-weekly intervals from 14 to 112 d of age. All feathers were dry-plucked from each of the seven tracts (specific skin areas) and pulp (the centre of the feather filament) was removed from primary and secondary remiges.2. Daily losses of feathers were collected from an additional 20 individually-caged broilers of each breed. These feathers were separated into natal down, contour feathers, remiges and rectrices and then pooled by type, sex and genotype to quantify water and protein contents. Only those feathers collected from male Cobb 500 MX broilers were analysed for amino acid content.3. Amino acid contents of feathers from the seven tracts were measured only in Cobb males on days 1, 28 and 70; for pulp on days 28 and 70; and for the four types of moulted feathers.4. Protein content on a dry matter basis remained relatively constant over all ages and tracts during growth. Water content decreased with age in both sexes and genotype. Lysine and methionine content in feathers decreased with age while cystine, valine, leucine and serine increased. Lysine, methionine and histidine levels were higher in pulp than in mature feathers whereas cystine and valine were higher in mature feathers than in pulp.5. These results, together with information about moulting patterns in broilers, enabled the effects of age of the bird and of the type of feather, to be taken into account when determining the rate of deposition of amino acids in feathers.

This paper describes a method for the determination of lysine based on a flow injection (FI) differential potentiometry system. The flow injection manifold is composed of a support electrolyte solution channel and a water channel acting as a carrier into which the sample solution is injected. The lysine biosensor was consisted of lysine oxidase chemically immobilized on a nylon membrane and attached to an all-solid-state ammonium electrode. A circular ammonium electrode was used as a reference. Hence, the possible interference of endogenous ammonium can be partly corrected by differential potentiometry. In order to increase the sensitivity of the response, the reaction was kinetically developed following a stopped-flow method. As a result, the sensitivity increased from 20 to 40 mV per decade when comparing the FI and the stopped-flow values. Furthermore, the peak-to-peak stopped-flow signals generated can be used as a more selective analytical response for lysine. The quantification of lysine in mixture samples containing small amounts of ammonium can be achieved with an acceptable accuracy, with prediction errors lower than 4%. However, when the ammonium concentration exceeded the lysine concentration, multivariate calibration with non-linear partial least squares (PLS) regression was needed to improve the lysine quantification, with an overall prediction error around 10%. [Copyright &y& Elsevier]

Glyoxal (GO) and methylglyoxal (MGO) are highly reactive species formed in carbohydrate metabolism. N ε -Carboxymethyllysine (CML) and N ε -carboxyethyllysine (CEL) are considered to be the advanced glycation end-products (AGEs) of L-lysine (Lys) with GO and MGO, respectively. Here, we investigated the reaction of free L-lysine (Lys) with GO and MGO in phosphate buffer (pH 7.4) at 37 °C and 80 °C in detail in the absence of any other chemicals which are widely used to reduce Schiff bases. The concentrations of Lys, GO and MGO used in the experiments were 0.5, 2.5, 5.0, 7.5 and 10 mM. The reaction time ranged between 0 and 240 min. Experiments were performed in triplicate. The concentrations of remaining Lys and of CML and CEL formed in the reaction mixtures were measured by stable-isotope dilution gas chromatography-mass spectrometry (GC-MS). Our experiments showed that CML and CEL were formed at higher concentrations at 80 °C compared to 37 °C. CML was found to be the major reaction product. In mixtures of GO and MGO, MGO inhibited the formation of CML from Lys (5 mM) in a concentration-dependent manner. The highest CML concentration was about 300 µM corresponding to a reaction yield of 6% with respect to Lys. An addition of Lys to GO, MGO and their mixtures resulted in strong reversible decreases in the Lys concentration up to 50%. It is assumed that free Lys reacts rapidly with GO and MGO to form many not yet identified reaction products. Reaction mixtures of Lys and MGO were stronger colored than those of Lys and GO, notably at 80 °C, indicating higher reactivity of MGO towards Lys that leads to polymeric colored MGO species. We have a strong indication of the formation of N ε -(hydroxymethyl)-lysine (HML) as a novel reaction product of Lys methyl ester with MGO. A mechanism is proposed for the formation of HML from Lys and MGO. This mechanism may explain why Lys and GO do not react to form a related product. Preliminary analyses show that HML is formed at higher concentrations than CEL from Lys methyl ester and MGO. No Schiff bases or their hydroxylic precursors were identified as reaction products. In their reactions with Lys, GO and MGO are likely to act both as chemical oxidants on the terminal aldehyde group to a carboxylic group (i.e., R-CHO to R-COOH) and as chemical reductors on labile Schiff bases (R-CH=N-R to R-CH 2 -NH-R) presumably via disproportionation and hydride transfer. Our study shows that free non-proteinic Lys reacts with GO and MGO to form CML, CEL and HML in very low yield. Whether proteinic Lys also reacts with MGO to form HML residues in proteins remains to be investigated. The physiological occurrence and concentration of HML in biological fluids and tissues and its relation to CML and CEL are elusive and warrant further investigations in health and disease. Chemical synthesis and structural characterization of HML are expected to advance and accelerate the scientific research in this topic.

Background: The metabolism of normal prostate relies on glycolysis, with prostate cancer having reduced glycolysis and increased aerobic metabolism. Advanced glycation end products (AGEs) accumulate in tissues as a result of age and glycolytic rate. Differential AGE levels were recently observed in prostate cancer tissues. Herein we sought to quantify AGEs in benign and cancer prostate tissue in a diverse cohort of patients., Methods: Levels of the AGE Nε-(carboxylethyl)lysine (CML) were quantified by immunohistochemistry (IHC) in a tissue microarray which consisted of 3 cores from tumor and 2 cores from benign areas from 118 patients (87 African American and 31 European American). Ancestry informative markers for African Ancestry were available for 79 patients. Epithelial and stromal areas were quantified separately using an E-cadherin mask. CML levels were compared with clinical grade group and ancestry by mixed linear effect models. Age, prostate-specific antigen (PSA) levels, body mass index (BMI), and hemoglobin A1C were included as covariates., Results: CML levels were lower in areas of the tumor, for both epithelium and surrounding stroma, compared with benign, but did not significantly change with tumor grade group. Age, PSA levels, BMI, and hemoglobin A1C did not associate with CML levels. CML levels were inversely associated with the percentage of African Ancestry in all tissues., Conclusions: The low CML levels in cancer may reflect the reduced glycolytic state of the tissue. The inverse relationship between African Ancestry and CML levels in both benign and cancer areas suggests a state of reduced glycolysis. It is yet to be determined whether altered glycolysis and CML levels are bystanders or drivers of carcinogenesis., (© 2021 Wiley Periodicals LLC.)

Intravenous (IV) administration of naturally occurring adeno-associated virus (AAV) vectors are liver tropic, with a significant proportion of the total vector dose mediating gene expression in liver hepatocytes. AAV capsids that are directed toward other organs such as lung may be useful for therapy of nonliver-based diseases. Based on the knowledge that the lung capillary endothelium is the first capillary bed encountered by an intravenously administered AAV vector, and that the lung endothelium glycocalyx is enriched in negatively charged sialic acid, we hypothesized that adding positively changed lysine residues to the AAV capsid would enhance AAV biodistribution to the lung after IV administration. Using site-directed mutagenesis, two lysine residues were inserted into variable loop VIII of the AAV serotype 5 capsid (AAV5-PK2). Organ distribution of AAV5-PK2 was compared with that of AAV5, AAV2, and AAV2-7m8 4 weeks after IV administration (10 11 gc) to C57Bl/6 male mice. As predicted, after IV administration, AAV5-PK2 had the highest biodistribution in the lung ( p  < 0.02 compared with AAV5, AAV2, and AAV2-7m8). Furthermore, biodistribution to liver of AAV5-PK2 was 2 logs decreased compared with AAV5 ( p  < 10 -4 ) with a ratio of AAV5-PK2 lung to liver of 62-fold compared with AAV5 of 0.2-fold ( p  < 0.0003). The AAV5-PK2 capsid represents a lung-tropic AAV vector that is also significantly detargeted from the liver, a property that may be useful in lung-directed gene therapies.

The LEW.1AR1- iddm rat is an animal model of human type 1 diabetes (T1D). Previously, we have shown that combination with anti-TCR/anti-TNF-α antibody-based therapy re-established normoglycemia and increased proteinic arginine-dimethylation in the spleen, yet not in the pancreas. High blood glucose is often associated with elevated formation of advanced glycation end-products (AGEs) which act via their receptor (RAGE). Both anti-TCR and anti-TNF-α are inhibitors of RAGE. The aim of the present work was to investigate potential biochemical changes of anti-TCR/anti-TNF-α therapy in the LEW.1AR1- iddm rat. We determined by stable-isotope dilution gas chromatography-mass spectrometry (GC-MS) the content of free and proteinic AGEs and the N ε -monomethylation of lysine (Lys) residues in proteins of pancreas, kidney, liver, spleen and lymph nodes of normoglycemic control (ngCo, n = 6), acute diabetic (acT1D, n = 6), chronic diabetic (chT1D, n = 4), and cured (cuT1D, n = 4) rats after anti-TCR/anti-TNF-α therapy. Analyzed biomarkers included Lys and its metabolites N ε -carboxymethyl lysine (CML), furosine and N ε -monomethyl lysine (MML). Other amino acids were also determined. Statistical methods including ANOVA, principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to evaluate the effects. Most statistical differences between the study groups were observed for spleen, pancreas and kidney, with liver and lymph nodes showing no such differences. In the pancreas, the groups differed with respect to proteinic furosine ( p = 0.0289) and free CML ( p = 0.0023). In the kidneys, the groups differed with respect to proteinic furosine ( p = 0.0076) and CML ( p = 0.0270). In the spleen, group differences were found for proteinic furosine ( p = 0.0114) and free furosine ( p = 0.0368), as well as for proteinic CML ( p = 0.0502) and proteinic MML ( p = 0.0191). The acT1D rats had lower furosine, CML and MML levels in the spleen than the rats in all other groups. This observation corresponds to the lower citrullination levels previously measured in these rats. PCA revealed diametric associations between PC1 and PC2 for spleen ( r = -0.8271, p < 0.0001) compared to pancreas ( r = 0.5805, p = 0.0073) and kidney ( r = 0.8692, p < 0.0001). These findings underscore the importance of the spleen in this animal model of human T1D. OPLS-DA showed that in total sixteen amino acids differed in the experimental groups.

Background: The solubility limitation and poor gelation properties of myofibrillar proteins at low ionic strength are the most challenging obstacle to limit salt reduction in meat products. In the present study, five amino acids with different concentrations of 5, 10 15, and 20 mmol L -1 , l-lysine (Lys), l-arginine (Arg), l-histidine (His), l-proline (Pro) and l-glycine (Gly), were introduced into myofibrillar protein (MP) suspensions at low ionic strength to improve solubility and gelation properties., Results: The dynamic rheological analysis showed that the MPs at 100 mmol L -1 NaCl containing 15/20 mmol L -1 Lys/Arg exhibited similar gelling behaviors to MPs at 600 mmol L -1 NaCl. Similarly, 15/20 mmol L -1 Lys/Arg significantly increased the solubility of MPs and the water holding capacity (WHC) and gel strength of MP gels, which was comparable to those of MPs at 600 mmol L -1 NaCl. Furthermore, Lys and Arg promoted the formation of aggregation-type gel with a dense and compact structure observed by scanning electron microscopy. The gels containing 15/20 mmol L -1 Lys/Arg exhibited a significant increase in the proportion of immobilized water (P 21 )., Conclusion: The enhancement of WHC, gel strength, and P 21 was closely associated with the increased solubility and the dense microstructure induced by Lys and Arg with high concentrations of 15 and 20 mmol L -1 . The knowledge obtained from this study may be useful for the improvement of gelation properties of MPs at low ionic strength using l-lysine and l-arginine. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Cortical neurons exhibit extreme diversity in gene expression as well as in morphological and electrophysiological properties 1,2 . Most existing neural taxonomies are based on either transcriptomic 3,4 or morpho-electric 5,6 criteria, as it has been technically challenging to study both aspects of neuronal diversity in the same set of cells 7 . Here we used Patch-seq 8 to combine patch-clamp recording, biocytin staining, and single-cell RNA sequencing of more than 1,300 neurons in adult mouse primary motor cortex, providing a morpho-electric annotation of almost all transcriptomically defined neural cell types. We found that, although broad families of transcriptomic types (those expressing Vip, Pvalb, Sst and so on) had distinct and essentially non-overlapping morpho-electric phenotypes, individual transcriptomic types within the same family were not well separated in the morpho-electric space. Instead, there was a continuum of variability in morphology and electrophysiology, with neighbouring transcriptomic cell types showing similar morpho-electric features, often without clear boundaries between them. Our results suggest that neuronal types in the neocortex do not always form discrete entities. Instead, neurons form a hierarchy that consists of distinct non-overlapping branches at the level of families, but can form continuous and correlated transcriptomic and morpho-electrical landscapes within families., (© 2020. The Author(s).)

Type 2 diabetes mellitus (T2DM) adversely affects the normal functioning, intrinsic material properties, and structural integrity of many tissues, including bone. It is well known that the clinical utility of areal bone mineral density (aBMD) is limited to assess bone strength in individuals with T2DM. Therefore, there is a need to explore new diagnostic techniques that can better assist and improve the accuracy of assessment of bone tissue quality. The present study investigated the link between bone and fingernail material/compositional properties in type 2 diabetes mellitus (T2DM). For that, femoral head and fingernail samples were obtained from twenty-five adult female patients (with/without T2DM) with fragility femoral neck fractures undergoing hemi/total hip arthroplasty. Cylindrical cores of trabecular bone were subjected to micro-CT, and lower bone volume fraction was observed in the diabetic group than the non-diabetic group due to fewer and thinner trabeculae in individuals with T2DM. The material and compositional properties of bone/fingernail were estimated using nanoindentation and Fourier Transform Infrared Spectroscopy, respectively. Both bone/fingernails in T2DM had lower reduced modulus (Er), hardness (H), lower Amide I and Amide II area ratio (protein content), higher sugar-to-matrix ratio, and relatively high carboxymethyl-lysine (CML) content compared with non-diabetic patients. Sugar-to-matrix ratio and relative CML content were strongly and positively correlated with HbA1c for both bone/fingernail. There was a positive correlation between bone and fingernail glycation content. Our findings provide evidence that the degradation pattern of bone and fingernail properties go hand-in-hand in individuals with T2DM. Hence, the fingernail compositional/material properties might serve as a non-invasive surrogate marker of bone quality in T2DM; however, further large-scale studies need to be undertaken., Competing Interests: The authors have declared that no competing interests exist.

Background: Stunting is a condition in which a child does not reach their full growth potential due to chronic undernutrition. It arises during the first 2 years of a child's life and is associated with developmental deficiencies and life-long health problems. Current interventions provide some benefit, but new approaches to prevention and treatment grounded in a molecular understanding of stunting are needed. Epigenetic analyses are critical as they can provide insight into how signals from a poor environment lead to changes in cell function., Results: Here we profiled histone H3 acetylation on lysine 27 (H3K27ac) in peripheral blood mononuclear cells (PBMCs) of 18-week-old (n = 14) and 1-year-old children (n = 22) living in an urban slum in Dhaka, Bangladesh. We show that 18-week-old children destined to become stunted have elevated levels of H3K27ac overall, functional analysis of which indicates activation of the immune system and stress response pathways as a primary response to a poor environment with high pathogen load. Conversely, overt stunting at 1-year-of age is associated with globally reduced H3K27ac that is indicative of metabolic rewiring and downregulation of the immune system and DNA repair pathways that are likely secondary responses to chronic exposure to a poor environment with limited nutrients. Among processes altered in 1-year-old children, we identified one-carbon metabolism, the significance of which is supported by integrative analysis with results from histone H3 trimethylation on lysine 4 (H3K4me3). Together, these results suggest altered one-carbon metabolism in this population of stunted children., Conclusions: The epigenomes of stunted children undergo two global changes in H3K27ac within their first year of life, which are associated with probable initial hyperactive immune responses followed by reduced metabolic capacity. Limitation of one-carbon metabolites may play a key role in the development of stunting. Trial registration ClinicalTrials.gov NCT01375647. Registered 17 June 2011, retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT01375647 ., (© 2021. The Author(s).)

l-lysine being one of the essential amino acids is not produced by the body, but is obtained through diet. l-lysine determination is important in the food and pharmaceutical industries as well as have medical and diagnostic applications. The normal l-lysine levels in a healthy human serum sample is 150 to 250 μmol/l. There is imbalance in l-lysine levels in certain diseased conditions. So, it could be a biomarker for diagnosis. Various basic methods are available for the determination of l-lysine such as colorimetric, radioisotope dilution, chromatographic, fluorometric and voltammetric methods. These methods have certain disadvantages like sample pretreatment, costly, time consuming and requirement of skilled personnel. These drawbacks are overcome by the use of biosensors due to their high sensitivity, stability and specificity. The present review article discusses about the principles, merits and demerits of the various analytic methods for determination of l-lysine with special emphasis on biosensors. l-lysine biosensors work ideally under the optimum pH 5 to 10, potential range -0.05 to 1.5 V, temperature 25 to 40 °C, with linear range 0.01 to 5500 μM, detection limit 0.000004 to 650 μM and response time 2 to 300 s. The sensor had storage stability between 14 and 200 days., (Copyright © 2021 Elsevier B.V. All rights reserved.)

During meat processing, two typical advanced glycation end products (AGEs), N ε -carboxymethyllysine (CML) and N ε -carboxyethyllysine (CEL), are generated by free radical induction. However, the impact of peroxyl radicals on myofibrillar proteins (MPs) glycosylation and CML and CEL formation is scarcely reported. In this study, two peroxyl radicals called ROO· and LOO· derived from AAPH (2,2'-azobis (2-methylpropionamidine) dihydrochloride) and linoleic acid were exposed prior to the Maillard reaction (glucosamine incubation at 37 °C for 24 h). Levels of AGEs (CML/CEL), protein oxidation (sulfhydryl/carbonyl), free amino group, surface hydrophobicity, zeta potential, particle size, intrinsic fluorescence intensity and secondary structure were determined. Together with Pearson's correlation, the assumption that free radicals promote MPs oxidation and glycation, alter the aggregation behavior and structure modification, leading to AGEs promotion has been built. In addition, the effect of dose-dependency of peroxyl radical on AGEs has also been established with different effects of peroxyl radical induction., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Prostate cancer (PCa) is a metabolic disease. Most men are diagnosed with low grade indolent disease and differentiating these men from those who have life threatening cancer is a challenging but important clinical dilemma. There are currently limited biomarkers that can distinguish between the indolent Gleason grade 6 and higher-grade disease. Moreover, some individuals initially diagnosed with low grade disease progress to higher grade disease. Currently prostate biopsies are the only reliable methods of stratifying risk, but biopsies can cause significant morbidity, sample only a small portion of the gland and are costly. Therefore, biomarkers distinguishing between indolent and aggressive patterns of PCa are urgently required to minimize biopsy-associated morbidity, prevent over-treatment of indolent PCa and to better stratify patients for appropriate treatment., Methods: Seminal fluid samples were collected from normal individuals (n = 13) Before infertility treatment and histologically confirmed PCa patients (n = 51). 1 H Nuclear magnetic resonance spectroscopy and orthogonal partial least square discriminant analysis were used to compare the populations., Results: Alterations in amino acids levels, specifically lysine and serine and changes in glycolytic intermediates were the most significant metabolic features associated with differences between healthy controls and PCa and between Gleason grade 6 (GS6) and Gleason grade 7 (GS7) samples. Orthogonal partial least square plots discriminated healthy controls from PCa samples (R 2  = 0.54, Q 2  = 0.31; area under the receiver operating characteristics curve [AUC] = 0.96), and GS6 from GS7 samples (R 2  = 0.62, Q 2  = 0.49; AUC = 0.98) based on lysine and serine content., Conclusion: This study suggests that seminal plasma metabolomics profiling of seminal fluid is a promising means of differentiating indolent from aggressive disease. Particularly, lysine and serine levels may be able to differentiate GS6 from GS7 disease., (© 2021 Wiley Periodicals LLC.)

Robust and selective quantification methods are required to better analyze feed supplementation effectiveness with specific amino acids. In this work, a reversed-phase high-performance liquid chromatography method with fluorescence detection is proposed and validated for lysine quantification, one of the most limiting amino acids in ruminant nutrition and essential towards milk production. To assess and widen method applicability, different matrices were considered: namely Li 2 CO 3 buffer (the chosen standard reaction buffer), phosphate buffer solution (to mimic media in cellular studies), and rumen inoculum. The method was validated for all three matrices and found to be selective, accurate (92% ± 2%), and precise at both the inter- and intra-day levels in concentrations up to 225 µM, with detection and quantification limits lower than 1.24 and 4.14 µM, respectively. Sample stability was evaluated when stored at room temperature, 4 °C, and -20 °C, showing consistency for up to 48 h regardless of the matrix. Finally, the developed method was applied in the quantification of lysine on real samples. The results presented indicate that the proposed method can be applied towards free lysine quantification in ruminant feeding studies and potentially be of great benefit to dairy cow nutrition supplementation and optimization.

The impact of NaCl and tripolyphosphate (TPP)/pyrophosphate (PP) on protein oxidation and Nε-(carboxymethyl)lysine (CML) and Nε-(carboxyethyl)lysine (CEL) formation in roasted beef patties was investigated. The content of CML and CEL in patties treated with salts was approximately 1.1-1.7 and 1.2-3.2 times higher than that of the control samples, respectively. An increase in salt content caused higher oxidation of tryptophan and protein carbonylation with a decrease in Schiff bases (P < 0.05) and a slight decrease in lipid oxidation (P < 0.05). Significant correlations (P < 0.05) between CML, CEL, and protein oxidation measurements was found. The higher salts content, causing less cooking loss and higher moisture content, significantly correlated (P < 0.05) with CML, CEL content, and protein oxidation of the patties. The increase in CML and CEL content and protein oxidation in roasted patties with salts might be related to the pro-oxidation of salts, and also partly due to the temperature changes caused by the water-holding capacity of salts., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Age at death estimation in cases of human skeletal finds is an important task in forensic medicine as well as in anthropology. In forensic medicine, methods based on "molecular clocks" in dental tissues and bone play an increasing role. The question, whether these methods are applicable also in cases with post-depositional intervals far beyond the forensically relevant period, was investigated for two "protein clocks", the accumulation of D-aspartic acid (D-Asp) and the accumulation of pentosidine (Pen) in dentine. Eight teeth of skeletons from different burial sites in Austria and with post-depositional intervals between c. 1216 and c. 8775 years were analysed. The results of age at death estimation based on D-Asp and Pen in dentine were compared to that derived from a classical morphological examination. Age at death estimation based on D-Asp resulted consistently in false high values. This finding can be explained by a post-mortem accumulation of D-Asp that may be enhanced by protein degradation. In contrast, the Pen-based age estimates fitted well with the morphological age diagnoses. The described effect of post-mortem protein degradation is negligible in forensically relevant time horizons, but not for post-depositional intervals of thousands of years. That means that the "D-Asp clock" loses its functionality with increasing post-depositional intervals, whereas Pen seems to be very stable. The "Pen-clock" may have the potential to become an interesting supplement to the existing repertoire of methods even in cases with extremely long post-depositional intervals. Further investigations have to test this hypothesis.

N ε -(carboxymethyl) lysine (CML) is universally used as a marker of the occurrence of advanced glycation end products (AGEs) in foods. This study investigated the level changes of CML, chlorogenic acids (CQAs), lysine (Lys), fat and pH in coffee during roasting. The CML level went up slowly in the first 10 mins, then declined sharply during the next 2 mins, and kept increasing constantly in the following baking time, while the lowest CML level was obtained by roasting at 235 °C for 12 mins. The three CQAs isomers had different efficacy in affecting the levels of CML and other factors, indicating the CQAs isomers may play an important role in influencing the CML level. So it might be possible to regulate the formation of CML and gain better coffee quality via adjusting the levels of CQAs in baking process. This study provided important enlightenment on CML control during coffee baking., (Copyright © 2020 Elsevier Ltd. All rights reserved.)