Innate Immune Cells Speak Manganese.

Some metal ions, such as Ca2+ and Zn2+, are involved in intracellular communication in immune cells by acting as second messengers. In this issue of Immunity, Wang et al. (2018) present findings implying a similar role for an additional metal ion, by showing that manganese participates in the recognition of cytoplasmic DNA.

Quantification of Ergot Alkaloids via Lysergic Acid Hydrazide-Development and Comparison of a Sum Parameter Screening Method.

Ergot alkaloids are a group of mycotoxins occurring in products derived from various grasses (e.g., rye) and have been regulated in the EU recently. The new maximum levels refer to the sum of the six most common ergot alkaloids in their two stereoisomeric forms in different food matrices. Typically, these twelve compounds are individually quantified via HPLC-MS/MS or -FLD and subsequently summed up to evaluate food safety in a time-consuming process. Since all these structures share the same ergoline backbone, we developed a novel sum parameter method (SPM) targeting all ergot alkaloids simultaneously via lysergic acid hydrazide. After extraction and clean-up, in analogy to the current European standard method EN 17425 (ESM) for ergot alkaloid quantitation, the samples were derivatized by an optimized hydrazinolysis protocol, which allowed quantitative conversion after 20 min at 100 °C. The new SPM was evaluated against another established HPLC-FLD-based method (LFGB) and the HPLC-MS/MS-based ESM using six naturally contaminated rye and wheat matrix reference materials. While the SPM provided comparable values to the ESM, LFGB showed deviating results. Determined recovery rates, limits of detection and quantification of all three employed methods confirm that the new SPM is a promising alternative to the classical approaches for ergot alkaloid screening in food.

An element of life: competition for zinc in host-pathogen interaction.

Zinc homeostasis plays a plethora of different roles in the immune response. In this issue of Immunity, Vignesh et al. (2013) demonstrate that stimulation of macrophages with GM-CSF deprives intracellular Histoplasma capsulatum of zinc, improving pathogen clearance.

Arylphosphonate-Tethered Porphyrins: Fluorescence Silencing Speaks a Metal Language in Living Enterocytes*.

We report the application of a highly versatile and engineerable novel sensor platform to monitor biologically significant and toxic metal ions in live human Caco-2 enterocytes. The extended conjugation between the fluorescent porphyrin core and metal ions through aromatic phenylphosphonic acid tethers generates a unique turn off and turn on fluorescence and, in addition, shifts in absorption and emission spectra for zinc, cobalt, cadmium and mercury. The reported fluorescent probes p-H8 TPPA and m-H8 TPPA can monitor a wide range of metal ion concentrations via fluorescence titration and also via fluorescence decay curves. Cu- and Zn-induced turn off fluorescence can be differentially reversed by the addition of common chelators. Both p-H8 TPPA and m-H8 TPPA readily pass the mammalian cellular membrane due to their amphipathic character as confirmed by confocal microscopic imaging of living enterocytes.

The CRECHE study: testing the urban myth that chocolate Santa Clauses are re-wrapped Easter Bunnies.

OBJECTIVE: To test the urban myth that surplus chocolate Easter Bunnies are re-packaged as Santa Clauses for the following Christmas holiday season. DESIGN: Prospective radiographic cohort study of seasonal chocolate figurines, supplemented by anonymous 5-item questionnaire survey of belief in the re-wrapping myth (Generic Risk Items Noted by Chocolate consumers in Health care settings; GRINCH). SETTING: Two tertiary referral trauma centres in Germany (Berlin and Duisburg). PARTICIPANTS: Eighteen chocolate Easter Bunnies and 15 chocolate Santa Clauses from different manufacturers purchased during 2020; 502 randomly selected people passing through the entrance halls of the two hospitals during 16 September - 12 October 2020. MAIN OUTCOME MEASURES: Whole body computed tomography (WBCT) images of chocolate Easter Bunnies and Santa Clauses assessed by four independent, board-certified radiologists using a visual contour resemblance scale (CRS); survey participants' views on statements related to the re-wrapping myth. RESULTS: Expert examiners clearly distinguished the WBCT images of chocolate Easter Bunnies and Santa Clauses; the mean difference in CRS was 84.2 points (95% CI, 78.5-90.0 points), with excellent inter-observer agreement (mean intra-class correlation coefficient, 0.99; 95% CI, 0.99-1.00). A total of 214 survey participants (43%) disagreed and 145 (29%) agreed with the proposition that seasonal chocolate figurines are re-packaged and re-sold the following season. CONCLUSION: Although about one-third of our survey respondents did not rule out the possibility of seasonal sweets being re-used, WBCT imaging found no similarity between chocolate foil-wrapped Easter and Christmas figurines, providing solid evidence against this urban myth. Chocolate Santa Clauses are unlikely to pose a significant threat to hospital food hygiene requirements. TRIAL REGISTRATION: Current Controlled Trials, ISRCTN16847363 (prospective).

Trace element profile and incidence of type 2 diabetes, cardiovascular disease and colorectal cancer: results from the EPIC-Potsdam cohort study.

PURPOSE: We aimed to examine the prospective association between manganese, iron, copper, zinc, iodine, selenium, selenoprotein P, free zinc, and their interplay, with incident type 2 diabetes (T2D), cardiovascular disease (CVD) and colorectal cancer (CRC). METHODS: Serum trace element (TE) concentrations were measured in a case-cohort study embedded within the EPIC-Potsdam cohort, consisting of a random sub-cohort (n = 2500) and incident cases of T2D (n = 705), CVD (n = 414), and CRC (n = 219). TE patterns were investigated using principal component analysis. Cox proportional hazard models were fitted to examine the association between TEs with T2D, CVD and CRC incidence. RESULTS: Higher manganese, zinc, iodine and selenium were associated with an increased risk of developing T2D (HR Q5 vs Q1: 1.56, 1.09-2.22; HR per SD, 95% CI 1.18, 1.05-1.33; 1.09, 1.01-1.17; 1.19, 1.06-1.34, respectively). Regarding CVD, manganese, copper and copper-to-zinc ratio were associated with an increased risk (HR per SD, 95% CI 1.13, 1.00-1.29; 1.22, 1.02-1.44; 1.18, 1.02-1.37, respectively). The opposite was observed for higher selenium-to-copper ratio (HR Q5 vs Q1, 95% CI 0.60, 0.39-0.93). Higher copper and zinc were associated with increasing risk of developing CRC (HR per SD, 95% CI 1.29, 1.05-1.59 and 1.14, 1.00-1.30, respectively). Selenium, selenoprotein P and selenium-to-copper-ratio were associated to decreased risk (HR per SD, 95% CI 0.82, 0.69-0.98; 0.81, 0.72-0.93; 0.77, 0.65-0.92, respectively). Two TE patterns were identified: manganese-iron-zinc and copper-iodine-selenium. CONCLUSION: Different TEs were associated with the risk of developing T2D, CVD and CRC. The contrasting associations found for selenium with T2D and CRC point towards differential disease-related pathways.

Fluorescent Arylphosphonic Acids: Synergic Interactions between Bone and the Fluorescent Core.

Herein, we report the third generation of fluorescent probes (arylphosphonic acids) to target calcifications, particularly hydroxyapatite (HAP). In this study, we use highly conjugated porphyrin-based arylphosphonic acids and their diesters, namely 5,10,15,20-tetrakis[m-(diethoxyphosphoryl)phenyl]porphyrin (m-H8 TPPA-OEt8 ) and 5,10,15,20-tetrakis [m-phenylphosphonic acid]porphyrin (m-H8 TPPA), in comparison with their positional isomers 5,10,15,20-tetrakis[p-(diisopropoxyphosphoryl)phenyl]porphyrin (p-H8 TPPA-iPr8 ) and 5,10,15,20-tetrakis [p-phenylphosphonic acid]porphyrin (p-H8 TPPA), which have phosphonic acid units bonded to sp2 carbon atoms of the fluorescent core. The conjugation of the fluorescent core is thus extended to the (HAP) through sp2 -bonded -PO3 H2 units, which generates increased fluorescence upon HAP binding. The resulting fluorescent probes are highly sensitive towards the HAP in rat bone sections. The designed probes are readily taken up by cells. Due to the lower reported toxicity of (p-H8 TPPA), these probes could find applications in monitoring bone resorption or adsorption, or imaging vascular or soft tissue calcifications for breast cancer diagnosis etc.

Zinc Deficiency Disturbs Mucin Expression, O-Glycosylation and Secretion by Intestinal Goblet Cells.

Approximately 1 billion people worldwide suffer from zinc deficiency, with severe consequences for their well-being, such as critically impaired intestinal health. In addition to an extreme degeneration of the intestinal epithelium, the intestinal mucus is seriously disturbed in zinc-deficient (ZD) animals. The underlying cellular processes as well as the relevance of zinc for the mucin-producing goblet cells, however, remain unknown. To this end, this study examines the impact of zinc deficiency on the synthesis, production, and secretion of intestinal mucins as well as on the zinc homeostasis of goblet cells using the in vitro goblet cell model HT-29-MTX. Zinc deprivation reduced their cellular zinc content, changed expression of the intestinal zinc transporters ZIP-4, ZIP-5, and ZnT1 and increased their zinc absorption ability, outlining the regulatory mechanisms of zinc homeostasis in goblet cells. Synthesis and secretion of mucins were severely disturbed during zinc deficiency, affecting both MUC2 and MUC5AC mRNA expression with ongoing cell differentiation. A lack of zinc perturbed mucin synthesis predominantly on the post-translational level, as ZD cells produced shorter O-glycans and the main O-glycan pattern was shifted in favor of core-3-based mucins. The expression of glycosyltransferases that determine the formation of core 1-4 O-glycans was altered in zinc deficiency. In particular, B3GNT6 mRNA catalyzing core 3 formation was elevated and C2GNT1 and C2GNT3 elongating core 1 were downregulated in ZD cells. These novel insights into the molecular mechanisms impairing intestinal mucus stability during zinc deficiency demonstrate the essentiality of zinc for the formation and maintenance of this physical barrier.

Alkali Phosphonate Metal-Organic Frameworks.

A new family of porous metal-organic frameworks (MOFs), namely alkali phosphonate MOFs, is reported. [Na2 Cu(H4 TPPA)]⋅(NH2 (CH3 )2 )2 (GTUB-1) was synthesized using the tetratopic 5,10,15,20-tetrakis[p-phenylphosphonic acid] porphyrin (H8 -TPPA) linker with planar X-shaped geometrical core. GTUB-1 is composed of rectangular void channels with BET surface area of 697 m2 g-1 . GTUB-1 exhibits exceptional thermal stability. The toxicity analysis of the (H8 -TPPA) linker indicates that it is well tolerated by an intestinal cell line, suggesting its suitability for creating phosphonate MOFs for biological applications.

A Zinpyr-1-based Fluorimetric Microassay for Free Zinc in Human Serum.

Zinc is an essential trace element, making it crucial to have a reliable biomarker for evaluating an individual's zinc status. The total serum zinc concentration, which is presently the most commonly used biomarker, is not ideal for this purpose, but a superior alternative is still missing. The free zinc concentration, which describes the fraction of zinc that is only loosely bound and easily exchangeable, has been proposed for this purpose, as it reflects the highly bioavailable part of serum zinc. This report presents a fluorescence-based method for determining the free zinc concentration in human serum samples, using the fluorescent probe Zinpyr-1. The assay has been applied on 154 commercially obtained human serum samples. Measured free zinc concentrations ranged from 0.09 to 0.42 nM with a mean of 0.22 ± 0.05 nM. It did not correlate with age or the total serum concentrations of zinc, manganese, iron or selenium. A negative correlation between the concentration of free zinc and total copper has been seen for sera from females. In addition, the free zinc concentration in sera from females (0.21 ± 0.05 nM) was significantly lower than in males (0.23 ± 0.06 nM). The assay uses a sample volume of less than 10 µL, is rapid and cost-effective and allows us to address questions regarding factors influencing the free serum zinc concentration, its connection with the body's zinc status, and its suitability as a future biomarker for an individual's zinc status.

In Vitro Studies on Zinc Binding and Buffering by Intestinal Mucins.

The investigation of luminal factors influencing zinc availability and accessibility in the intestine is of great interest when analyzing parameters regulating intestinal zinc resorption. Of note, intestinal mucins were suggested to play a beneficial role in the luminal availability of zinc. Their exact zinc binding properties, however, remain unknown and the impact of these glycoproteins on human intestinal zinc resorption has not been investigated in detail. Thus, the aim of this study is to elucidate the impact of intestinal mucins on luminal uptake of zinc into enterocytes and its transfer into the blood. In the present study, in vitro zinc binding properties of mucins were analyzed using commercially available porcine mucins and secreted mucins of the goblet cell line HT-29-MTX. The molecular zinc binding capacity and average zinc binding affinity of these glycoproteins demonstrates that mucins contain multiple zinc-binding sites with biologically relevant affinity within one mucin molecule. Zinc uptake into the enterocyte cell line Caco-2 was impaired by zinc-depleted mucins. Yet this does not represent their form in the intestinal lumen in vivo under zinc adequate conditions. In fact, zinc-uptake studies into enterocytes in the presence of mucins with differing degree of zinc saturation revealed zinc buffering by these glycoproteins, indicating that mucin-bound zinc is still available for the cells. Finally, the impact of mucins on zinc resorption using three-dimensional cultures was studied comparing the zinc transfer of a Caco-2/HT-29-MTX co-culture and conventional Caco-2 monoculture. Here, the mucin secreting co-cultures yielded higher fractional zinc resorption and elevated zinc transport rates, suggesting that intestinal mucins facilitate the zinc uptake into enterocytes and act as a zinc delivery system for the intestinal epithelium.

Synthesis and Structural Identification of a Biaryl Ether-Linked Zearalenone Dimer.

A new dimer of the food-relevant mycotoxin zearalenone was isolated after electrochemical and chemical oxidation. The structure was determined as a 16-O-15'-biaryl ether-linked dimer based on spectroscopic analyses (¹H- and 13C-NMR, COSY, HMBC, and HSQCAD) and high-resolution mass spectrometry analysis (Q-TOF).

Electrochemical simulation of biotransformation reactions of citrinin and dihydroergocristine compared to UV irradiation and Fenton-like reaction.

Mycotoxins occur widely in foodstuffs and cause a variety of mold-related health risks to humans and animals. Elucidation of the metabolic fate of mycotoxins and the growing number of newly discovered mycotoxins have enhanced the demand for fast and reliable simulation methods. The viability of electrochemistry coupled with mass spectrometry (EC/ESI-MS), Fenton-like oxidation, and UV irradiation for the simulation of oxidative phase I metabolism of the mycotoxins citrinin (CIT) and dihydroergocristine (DHEC) was investigated. The specific reaction products are compared with metabolites produced by human and rat liver microsomes in vitro. Depending on the applied potential between 0 and 2000 mV vs. Pd/H2 by using a flow-through cell, CIT and DHEC are oxidized to various products. Besides dehydrogenation and dealkylation reactions, several hydroxylated DHEC and CIT species are produced by EC and Fenton-like reaction, separated and analyzed by LC-MS/MS and ESI-HRMS. Compared to reaction products from performed microsomal incubations, several mono- and dihydroxylated DHEC species were found to be similar to the reaction products of EC, Fenton-like reaction, and UV-induced oxidation. Consequentially, nonmicrosomal efficient and economic simulation techniques can be useful in early-stage metabolic studies, even if one-to-one simulation is not always feasible.

Zinc and immunity: An essential interrelation.

The significance of the essential trace element zinc for immune function has been known for several decades. Zinc deficiency affects immune cells, resulting in altered host defense, increased risk of inflammation, and even death. The micronutrient zinc is important for maintenance and development of immune cells of both the innate and adaptive immune system. A disrupted zinc homeostasis affects these cells, leading to impaired formation, activation, and maturation of lymphocytes, disturbed intercellular communication via cytokines, and weakened innate host defense via phagocytosis and oxidative burst. This review outlines the connection between zinc and immunity by giving a survey on the major roles of zinc in immune cell function, and their potential consequences in vivo.

Ethylmercury and Hg2+ induce the formation of neutrophil extracellular traps (NETs) by human neutrophil granulocytes.

Humans are exposed to different mercurial compounds from various sources, most frequently from dental fillings, preservatives in vaccines, or consumption of fish. Among other toxic effects, these substances interact with the immune system. In high doses, mercurials are immunosuppressive. However, lower doses of some mercurials stimulate the immune system, inducing different forms of autoimmunity, autoantibodies, and glomerulonephritis in rodents. Furthermore, some studies suggest a connection between mercury exposure and the occurrence of autoantibodies against nuclear components and granulocyte cytoplasmic proteins in humans. Still, the underlying mechanisms need to be clarified. The present study investigates the formation of neutrophil extracellular traps (NETs) in response to thimerosal and its metabolites ethyl mercury (EtHg), thiosalicylic acid, and mercuric ions (Hg(2+)). Only EtHg and Hg(2+) triggered NETosis. It was independent of PKC, ERK1/2, p38, and zinc signals and not affected by the NADPH oxidase inhibitor DPI. Instead, EtHg and Hg(2+) triggered NADPH oxidase-independent production of ROS, which are likely to be involved in mercurial-induced NET formation. This finding might help understanding the autoimmune potential of mercurial compounds. Some diseases, to which a connection with mercurials has been shown, such as Wegener's granulomatosis and systemic lupus erythematosus, are characterized by high prevalence of autoantibodies against neutrophil-specific auto-antigens. Externalization in the form of NETs may be a source for exposure to these self-antigens. In genetically susceptible individuals, this could be one step in the series of events leading to autoimmunity.

Differential regulation of TLR-dependent MyD88 and TRIF signaling pathways by free zinc ions.

Zinc signals are utilized by several immune cell receptors. One is TLR4, which causes an increase of free zinc ions (Zn(2+)) that is required for the MyD88-dependent expression of inflammatory cytokines. This study investigates the role of Zn(2+) on Toll/IL-1R domain-containing adapter inducing IFN-ß (TRIF)-dependent signals, the other major intracellular pathway activated by TLR4. Chelation of Zn(2+) with the membrane-permeable chelator N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine augmented TLR4-mediated production of IFN-ß and subsequent synthesis of inducible NO synthase and production of NO. The effect is based on Zn(2+) acting as a negative regulator of the TRIF pathway via reducing IFN regulatory factor 3 activation. This was also observed with TLR3, the only TLR that signals exclusively via TRIF, but not MyD88, and does not trigger a zinc signal. In contrast, IFN-γ-induced NO production was unaffected by N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine. Taken together, Zn(2+) is specifically involved in TLR signaling, where it differentially regulates MyD88 and TRIF signaling via a zinc signal or via basal Zn(2+) levels, respectively.

Alterations in zinc binding capacity, free zinc levels and total serum zinc in a porcine model of sepsis.

Zinc is crucial for immune function. In addition, the redistribution of zinc and other nutrients due to infection is an integral part of the host immune response to limit availability to pathogens. However, the major zinc binding protein albumin is down regulated during the acute phase response, implicating a decrease in zinc binding capacity. A prospective animal study with eight female German landrace pigs was conducted to investigate alterations in zinc binding capacity, total serum zinc and free zinc levels in the initial phase of sepsis. Sepsis was induced by instillation of autologous feces via midline laparotomy. Total serum zinc declined significantly after 1 h (10.89 ± 0.42 µM vs. 7.67 ± 0.41 µM, p < 0.001), total serum copper and iron reached a significant reduction at 4 h. Urinary excretion of zinc declined in line with total serum zinc. In comparison to total serum zinc, free zinc levels declined to a lesser, though significant, extent. Zinc binding capacity of serum decreased over time, whereby free zinc levels after addition of zinc correlated negatively with total serum protein and albumin levels. In addition IL-6 and TNF-α concentrations were measured and increased significantly 2 h after induction of sepsis. Hence, total serum zinc was the first marker of inflammation in our experiment, and might therefore be a promising biomarker for the early diagnosis of sepsis. Furthermore the observation of a substantially different serum free zinc homeostasis during sepsis provides valuable information for a potential therapeutic zinc supplementation, which has to take buffering capacity by serum proteins into account.

Interaction and competition for intestinal absorption by zinc, iron, copper, and manganese at the intestinal mucus layer.

Trace elements such as zinc, manganese, copper, or iron are essential for a wide range of physiological functions. It is therefore crucial to ensure an adequate supply of these elements to the body. Many previous investigations have dealt with the role of transport proteins, in particular their selectivity for, and competition between, different ions. Another so far less well investigated major factor influencing the absorption of trace elements seems to be the intestinal mucus layer. This gel-like substance covers the entire gastrointestinal tract and its physiochemical properties can be mainly assigned to the glycoproteins it contains, so-called mucins. Interaction with mucins has already been demonstrated for some metals. However, knowledge about the impact on the respective bioavailability and competition between those metals is still sketchy. This review therefore aims to summarize the findings and knowledge gaps about potential effects regarding the interaction between gastrointestinal mucins and the trace elements iron, zinc, manganese, and copper. Mucins play an indispensable role in the absorption of these trace elements in the neutral to slightly alkaline environment of the intestine, by keeping them in a soluble form that can be absorbed by enterocytes. Furthermore, the studies so far indicate that the competition between these trace elements for uptake already starts at the intestinal mucus layer, yet further research is required to completely understand this interaction.

First Synthesis of Ergotamine-13CD3 and Ergotaminine-13CD3 from Unlabeled Ergotamine.

Ergot alkaloids (EAs) formed by Claviceps fungi are one of the most common food contaminants worldwide, affecting cereals such as rye, wheat, and barley. To accurately determine the level of contamination and to monitor EAs maximum levels set by the European Union, the six most common EAs (so-called priority EAs) and their corresponding epimers are quantified using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The quantification of EAs in complex food matrices without appropriate internal standards is challenging but currently carried out in the standard method EN 17425:2021 due to their commercial unavailability. To address the need for isotopically labeled EAs, we focus on two semi-synthetic approaches for the synthesis of these reference standards. Therefore, we investigate the feasibility of the N6-demethylation of native ergotamine to yield norergotamine, which can subsequently be remethylated with an isotopically labeled methylating reagent, such as iodomethane (13CD3-I), to yield isotopically labeled ergotamine and its C8-epimer ergotaminine. Testing the isotopically labeled ergotamine/-inine against native ergotamine/-inine with HPLC coupled to high-resolution HR-MS/MS proved the structure of ergotamine-13CD3 and ergotaminine-13CD3. Thus, for the first time, we can describe their synthesis from unlabeled, native ergotamine. Furthermore, this approach is promising as a universal way to synthesize other isotopically labeled EAs.

Determination of copper status by five biomarkers in serum of healthy women.

BACKGROUND: The essential trace element copper is relevant for many important physiological processes. Changes in copper homeostasis can result from disease and affect human health. A reliable assessment of copper status by suitable biomarkers may enable fast detection of subtle changes in copper metabolism. To this end, additional biomarkers besides serum copper and ceruloplasmin (CP) concentrations are required. OBJECTIVES: The aim of this study was to investigate the emerging copper biomarkers CP oxidase (CPO) activity, exchangeable copper (CuEXC) and labile copper in serum of healthy women and compare them with the conventional biomarkers total serum copper and CP. METHOD AND MAIN FINDINGS: This observational study determined CPO activity, the non CP-bound copper species CuEXC and labile copper, total serum copper and CP in sera of 110 healthy women. Samples were collected at four time points over a period of 24 weeks. The concentrations of total serum copper and CP were within the reference ranges. The comparison of all five biomarkers provided insight into their relationship, the intra- and inter-individual variability as well as the age dependence. The correlation and Principal Component Analyses (PCA) indicated that CP, CPO activity and total copper correlated well, followed by CuEXC, while the labile copper pool was unrelated to the other parameters. CONCLUSIONS: This study suggests that the non-CP-bound copper species represent copper pools that are differently regulated from total copper or CP-bound copper, making them interesting complementary biomarkers to enable a more complete assessment of body copper status with potential relevance for clinical application.