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The conversion of phenolic compounds by lactobacilli in food fermentations contributes to food quality. The metabolism of phenolics by lactobacilli has been elucidated in the past years but information on the contribution of specific enzymes in food fermentations remains scarce. This study aimed to address this gap by disruption of genes coding for the hydroxycimmanic acid reductase Par1, the hydroxycinnamic acid decarboxylase Pad, the hydrocinnamic esterase EstR, and strains with disruption of all three genes in Furfurilactobacillus milii FUA3583. The conversion of phenolics by Ff. milii and its isogenic mutants in sorghum fermentations was studied by LC-UV and LC-UV-MS/MS analyses. Ff. milii FUA3583 converted hydroxycinnamic acids predominantly with Par1. Vinylphenols were detected only in mutants lacking par1. A phenotype for the estR defective mutant was not identified. The formation of pyrano-3-deoxyanthocyanidins was observed only after fermentation with strains expressing Pad. Specifically, formation of these compounds was low with Ff. milii FUA3583, substantially increased in the Par1 mutant and abolished in all mutants with disrupted pad. Competition experiments with Ff. milii FUA3583 and its isogenic mutants demonstrated that expression of one of the two metabolic pathways for hydroxycinnamic acids increases the ecological fitness of the strain. Disruption of EstR in a Δpar1Δpar2Δpad background improved ecological fitness, indirectly demonstrating a phenotype of the esterase in Ff. milii. The documentation of the functionality of genes coding for conversion of hydroxycinnamic acids may support the selection of starter cultures for improved quality of fermented cereal products., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In contrast to other species, cattle possess exceptional antibodies with ultra-long complementarity-determining regions (ulCDRs) that can consist of 40-70 amino acids. The bovine ulCDR is folded into a stalk and a disulfide-rich knob domain. The binding to the antigen is via the 3-6 kDa knob. There exists an immense sequence and structural diversity in the knob that enables binding to different antigens. Here we summarize the current knowledge of the ulCDR structure and provide an overview of the approaches to discover ulCDRs against novel antigens. Furthermore, we outline protein engineering approaches inspired by the natural ulCDRs. Finally, we discuss the enormous potential of using isolated bovine knobs, also named picobodies, as the smallest antigen-binding domains derived from natural antibodies., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Stress and everyday problems may impact memory and cognition. Therefore, many people use cognitive enhancers (CEs), sold for prescription, as over-the-counter drugs, or dietary supplements, believing they may help with everyday functioning. Our study was designed to answer whether taking CEs is common among Medical University students and to identify which substances are mainly used., Methods and Results: An anonymous online questionnaire was answered by 479 students of Medical (88%) and Dentistry (12%) Faculties in Poland. Women constituted the majority of respondents (63%). CEs were used by 53% of respondents, with the most frequent being caffeine, ginseng, nicotine, theanine, ginkgo, and lecithin. Some persons used CEs that are available only with a prescription. The most important reasons for the use of CEs were to increase arousal and improve concentration (mentioned by 81% and 73%, respectively). Over 65% of students experienced some undesired/adverse effects after taking CEs, with tachycardia being the most common, followed by sleep disturbances (reported by 51% and 40%, respectively)., Conclusions: More than half of the respondents from the Medical and Dentistry Faculties reported using CEs, despite their unproven efficacy and not-well-established safety. This raises significant concern about the knowledge of young persons regarding CEs and should encourage universities to undertake educational actions.

Lysosomes are cell organelles that degrade macromolecules to recycle their components. If lysosomal degradative function is impaired, e.g., due to mutations in lysosomal enzymes or membrane proteins, lysosomal storage diseases (LSDs) can develop. LSDs manifest often with neurodegenerative symptoms, typically starting in early childhood, and going along with a strongly reduced life expectancy and quality of life. We show here that small molecule activation of the Ca 2+ -permeable endolysosomal two-pore channel 2 (TPC2) results in an amelioration of cellular phenotypes associated with LSDs such as cholesterol or lipofuscin accumulation, or the formation of abnormal vacuoles seen by electron microscopy. Rescue effects by TPC2 activation, which promotes lysosomal exocytosis and autophagy, were assessed in mucolipidosis type IV (MLIV), Niemann-Pick type C1, and Batten disease patient fibroblasts, and in neurons derived from newly generated isogenic human iPSC models for MLIV and Batten disease. For in vivo proof of concept, we tested TPC2 activation in the MLIV mouse model. In sum, our data suggest that TPC2 is a promising target for the treatment of different types of LSDs, both in vitro and in-vivo., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Introduction: Craterostigma plantagineum and Lindernia brevidens are resurrection plants, so these plants can tolerate desiccation of their vegetative tissues. Different components and mechanisms contribute to desiccation tolerance and secondary plant metabolites, like phenolic compounds, may play a role during these processes., Objectives: Secondary plant metabolites of the two resurrection plants, C. plantagineum and L. brevidens as well as the closely related desiccation sensitive species, L. subracemosa, were investigated regarding the polyphenol profile., Material and Methods: Secondary plant compounds were extracted with acidified methanol and analysed with ultra-high-performance liquid chromatography electrospray ionisation mass spectrometry (UHPLC-ESI-MS). Phenolic compounds were identified by comparing of ultraviolet (UV) and MS n -spectra with published data. All compounds were quantified as verbascoside equivalents by external calibration at the compound specific wavelength., Results: In total, eight compounds that belong to the subclass of phenylethanoid glycosides and one flavone, luteolin hexoside pentoside, were identified. Two of these compounds exhibited a fragmentation pattern, which is closely related to phenylethanoid glycosides. The predominantly synthesised phenylethanoid in all of the three plant species and in every stage of hydration was verbascoside. The total content of phenolic compounds during the three stages of hydration, untreated, desiccated, and rehydrated revealed differences especially between C. plantagineum and L. brevidens as the latter one lost almost all phenolic compounds during rehydration., Conclusion: The amount of verbascoside correlates with the degree of desiccation tolerance and verbascoside might play a role in the protective system in acting as an antioxidant., (© 2020 The Authors. Phytochemical Analysis published by John Wiley & Sons Ltd.)

The metabolism of anthocyanins in humans is still not fully understood, which is partly due to the lack of reference compounds. It is known that sulfation is one way of the complex phase II biotransformation mechanism. Therefore, cyanidin-3- O -glucoside and the cyanidin aglycone were chemically converted to their sulfates by reaction with sulfur trioxide-N-triethylamine complex in dimethylformamide. The reaction products were characterized by UHPLC coupled to linear ion trap and IMS-QTOF mass spectrometry. Based on MS data, retention times, and UV-Vis spectra, the compounds could tentatively be assigned to A-, C-, or B-ring sulfates. Analysis of urine samples from two volunteers after ingestion of commercial blackberry nectar demonstrated the presence of two sulfated derivatives of the cyanidin aglycone and one sulfated derivative of the cyanidin-3- O -glucoside. It was found that both the A ring and the B ring are sulfated by human enzymes. This study marks an important step toward a better understanding of anthocyanin metabolism.

The approach presented in this study is the first for the hemisynthesis of methylated anthocyanins. It was possible to obtain cyanidin-3- O -glucoside derivatives with different degrees of methylation. Cautious identification of 4'-, 5-, and 7-OH monomethylated derivatives was also accomplished. The methylation agent used was the "green chemical" dimethyl carbonate (DMC), which is characterized by low human and ecological toxicity. The influence of the temperature, reaction time, and amount of the required diazabicyclo[5.4.0]undec-7-en (DBU) catalyst on the formation of the products was examined. Compared to conventional synthesis methods for methylated flavonoids using DMC and DBU, the conditions identified in this study result in a reduction of reaction time, and an important side reaction, so-called carboxymethylation, was minimized by using higher amounts of catalyst.

The endo-lysosomal two-pore channel (TPC2) has been established as an intracellular cation channel of significant physiological and pathophysiological relevance in recent years. For example, TPC2-/- mice show defects in cholesterol degradation, leading to hypercholesterinemia; TPC2 absence also results in mature-onset obesity, and a role in glucagon secretion and diabetes has been proposed. Infections with bacterial toxins or viruses e.g., cholera toxin or Ebola virus result in reduced infectivity rates in the absence of TPC2 or after pharmacological blockage, and TPC2-/- cancer cells lose their ability to migrate and metastasize efficiently. Finally, melanin production is affected by changes in hTPC2 activity, resulting in pigmentation defects and hair color variation. Here, we analyzed several publicly available genome variation data sets and identified multiple variations in the TPC2 protein in distinct human populations. Surprisingly, one variation, L564P, was found to be the predominant TPC2 isoform on a global scale. By applying endo-lysosomal patch-clamp electrophysiology, we found that L564P is a prerequisite for the previously described M484L gain-of-function effect that is associated with blond hair. Additionally, other gain-of-function variants with distinct geographical and ethnic distribution were discovered and functionally characterized. A meta-analysis of genome-wide association studies was performed, finding the polymorphisms to be associated with both distinct and overlapping traits. In sum, we present the first systematic analysis of variations in TPC2. We functionally characterized the most common variations and assessed their association with various disease traits. With TPC2 emerging as a novel drug target for the treatment of various diseases, this study provides valuable insights into ethnic and geographical distribution of TPC2 polymorphisms and their effects on channel activity., Competing Interests: The authors declare that they have no conflict of interest.

Recent in vitro and in vivo studies on anthocyanins confirmed numerous health-promoting effects in humans. Daily anthocyanin intake can be estimated via food databases, but the amount absorbed by the organism still remains uncertain because anthocyanin bioavailability is yet to be elucidated in its entirety. For this purpose, suitable and validated methods of sample preparation and analysis are required. Therefore, a sample preparation method for anthocyanin metabolite analysis in plasma was successfully established and validated. The validation yielded acceptable results for the anthocyanins in terms of recovery (54-108%) and precision (coefficient of variation (CV) < 15%). The UHPLC-MS method used in the consecutive reaction monitoring (CRM) mode was sufficiently sensitive, resulting in limits of detection <2.3 ng/mL and limits of quantification < 8.1 ng/mL with associated repeatability of the MS system with CVs of <5.1%. In addition, a method for the sum parameter determination of anthocyanidins in urine comprising solely the evaporation of acidified samples was developed, validated, and successfully applied to real samples. The results showed that this method is applicable for the methylated anthocyanidins, but not for the hydroxylated anthocyanidins, due to the chosen CRM modes required for optimum selectivity., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

(1) Background: Polyphenols (PP) play an important role in the prevention of non-communicable diseases and may contribute to healthy aging. To investigate the molecular and cellular aspects of PP metabolites on longevity with a focus on mitochondrial function, we applied a pre-fermented mixture of polyphenols (Rechtsregulat ® , RR) to rodents and nematodes. (2) Methods: The lifespans of Navar Medical Research Institute (NMRI) mice and C. elegans were recorded. The heat-stress resistance (37 °C) of C. elegans N2 was measured using nucleic staining. Respiration and membrane potential (ΔΨm) were measured in isolated mitochondria. The energetic metabolites adenosine triphosphate (ATP), lactate, and pyruvate were determined in lysates. Expression levels of longevity related genes were determined using quantitative real time polymerase chain reaction (qRT-PCR). Phenolic compounds were identified using ultra high performance liquid chromatography-diode array detection-Iontrap-multiple stage mass spectrometry (UHPLC-DAD-Iontrap-MS n ). (3) Results: Several phenolic metabolites including protocatechuic acid (PCA) were identified in RR. Feeding of mice with RR resulted in a significantly increased lifespan. Heat-stress resistance (RR *** p = 0.0006; PCA **** p < 0.0001), median lifespan (NMRI: RR ** p = 0.0035; C. elegans RR * p = 0.0279; PCA **** p < 0.0001), and activity of mitochondrial respiratory chain complexes (RR * - ** p = 0.0237 - 0.0052; PCA * p = 0.019 - 0.0208) of C. elegans were significantly increased after incubation with RR (10%) or PCA (780 µM). PCA significantly improved nematodes ΔΨm (* p = 0.02058) and ATP levels (* p = 0.029). RR significantly up-regulated lactate levels, indicating enhanced glycolysis. The expression levels of longevity related genes daf-16 , sir-2.1 , and skn-1 were significantly upregulated after PCA, and partially after RR administration. (4) Conclusion: Phenolic metabolites such as PCA have the potential to enhance health and lifespan and mitochondrial function, and thus may contribute to healthy aging.

A new sample processing method for analyzing flavonol metabolites in plasma using enzymatic proteolysis was developed and validated. Four endopeptidases were examined regarding their influence on the analyte recovery of quercetin-3- O-glucuronide (Q3GlcA). Methanol was added to inactivate and precipitate the enzymes, and samples were concentrated via evaporation prior to UHPLC-MS analysis. Quercetin-3- O-rutinoside (Q3Rut) was used as an internal standard. The selectivity and accuracy of the established UHPLC-ESI-MS n method showed a coefficient of variation (CV) of the repeatability of the measuring instrument of 1.7% for Q3GlcA. The average recovery of Q3GlcA was approximately 67% with an interday method precision of 24% and r = 46.9 as its repeatability. Therefore, enzymatic proteolysis has proven to be a suitable alternative to the methods previously described in the literature, such as solid-phase extraction (SPE). Still, the method has only been validated for Q3GlcA, but its applicability to other substance classes seems possible.

The aim of this work was to obtain phase II metabolites of cyanidin-3- O-glucoside and its aglycone using porcine liver enzymes. For this purpose, anthocyanins extracted from blackberry concentrate and containing mostly cyanidin-3- O-glucoside were incubated with the S9, microsomal, and cytosolic fractions of porcine liver. The reactions were targeted to the direction of the respective phase II transformation by the addition of activated cofactors. LC-MS n and LC-IMS-QTOF-MS analyses showed that one methylated, three glucuronidated and three sulfated metabolites of cyanidin-3- O-glucoside were generated. The aglycone, cyanidin, was sulfated and glucuronidated by the liver enzymes. In addition, both were glucuronidated and methylated simultaneously. The detected compounds and the generated data like exact masses, mass spectra, and CCS values may serve as a basis in the search for metabolites formed in vivo. As their effects are largely unexplored, the described synthesis may contribute to a better understanding of the metabolism of anthocyanins.

Scope: After intrinsic labeling of spinach (Spinacia oleracea L., Chenopodiaceae) with 13 CO 2 , we investigated if labeled polyphenol metabolites were detectable in human plasma., Methods and Result: In a pilot intervention trial, five healthy men consumed 5 g freeze-dried 13 C labeled spinach, including a total amount of 160 μmol methoxyflavonols, including 70 μmol 5,3',4'-trihydroxy-3-methoxy-6,7-methylendioxyflavone-4'-glucuronide. Plasma samples of all subjects were analyzed with regard to their 13 C/ 12 C ratio. Additionally, 13 C labeled metabolites of patuletin, spinacetin, and 5,3',4'-trihydroxy-3-methoxy-6,7-methylendioxyflavone (TMM) were analyzed in plasma samples in a subgroup of three subjects. TMM-glucuronide, TMM-sulfate, and spinacetin-glucuronide-sulfate, the latter as 12 C 1 13 C 16 and 13 C 17 isotopologs, were tentatively identified. Plasma concentration of TMM-glucuronide and TMM-sulfate reached c max from 19.1-54.3  and 22.5-125.5 nmol L -1 , respectively, 7-9 h post-ingestion., Conclusion: It seems likely that 13 C labeled TMM-glucuronide and TMM-sulfate are phase-II metabolites which were converted after colonic transformation. Variations in plasma kinetics were observed for these two metabolites and may be attributed to the individual composition of the microbiota. We conclude that 13 C labeled polyphenol metabolites are detectable and quantifiable in human plasma., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The purpose of this postmarket surveillance registry was to document the efficacy of a vascular prosthesis coated with metallic silver in high-risk patients undergoing vascular reconstructions. Patency (primary endpoint) and freedom from graft infection (secondary endpoint) data were assessed at a minimum of 12 months in patients with significant co-morbidity and/or confirmed graft infections or infected native vessels. Between November 2006 and December 2009, 230 patients with high-risk factors underwent aortic,peripheral and/or extra-anatomic reconstructions with Silver Graft® (SG) in six German, one French and one Polish vascular center.All participating centers used the metallic silver-coated polyester graft (SG) in various diameters and lengths including tubular and bifurcate vascular grafts. Doppler ultrasound follow-ups to determine graft patency were planned at 12 months or done at an earlier time in case the patient became symptomatic. A total of 230 patients were studied. Ten of these 230 patients had graft infections at baseline whereas the remaining 220 subjects had significant risk factors such as coronary artery disease (62.7%, 138/220), vascular access in scar tissue (27.3%, 60/220), Fontaine III/IV (38.2%, 84/220), chronic renal insufficiency (26.8%, 59/220) and diabetes (21.0%,46/220). The long-term follow-up at 15.5 ± 8.3 months revealed a secondary patency rate of 93.2% (205/220) and an ‘all cause’ mortality rate of 18.6% (41/220). There was a freedom from de novo graft infection rate of 95.9% (211/220) in the high-risk group without graft infections at baseline. One regraft infection occurred distal of the revisional reconstruction in the 10 patients with graft infection at baseline. The presence of perigraft fluid at follow-up and Fontaine III/IV at baseline were found to be predictors for graft patency whereas perigraft fluid presence was the only predictor for de novo graft infections. This registry revealed favorable patency and freedom from de novo infections rates in a ‘high-risk’ population with significant co-morbidities.

As leukotriene D4 receptor CysLT1R upregulation is an early event in inflammatory processes, specific detection of CysLT1R via molecular imaging might be a promising diagnostic tool for inflammatory diseases. We coupled a specific anti-CysLT1R IgG antibody to near-infrared (NIR) hemicyanine fluorophore DY-734. The fluorophore was also coupled to unspecific rabbit-IgG antibody or corresponding Fab fragments. Expression of CysLT1R in HL-60 human promyelocytic leukemia cells in vitro could be proven by reverse transcriptase-polymerase chain reaction (PCR), real-time PCR, and flow cytometry. Detection of the probes by flow cytometry showed that CysLT1R*DY-734 probe binds distinctly stronger to HL-60 cells than IgG*DY-734. Induction of ear edema in mice was conducted to test signaling of the synthesized probes in vivo. A markedly higher fluorescence intensity was observed in the edematous region than in the healthy region by a whole-body imaging system. Semiquantitative analysis showed that CysLT1R*DY-734 and Fab-CysLT1R*DY-734 probes bind 1.9- and 1.2-fold stronger, respectively, than the unspecific probes. Biodistribution studies revealed an enrichment of full-length IgG probes in liver and spleen, whereas Fab-containing probes are mostly found in liver and kidneys. Taken together, we present an approach that might improve early diagnosis of inflammatory diseases in the long term.