Dried Porous Biomaterials from Mealworm Protein Gels: Proof of Concept and Impact of Drying Method on Structural Properties and Zinc Retention.

Dried porous materials can be found in a wide range of applications. So far, they are mostly prepared from inorganic or indigestible raw materials. The aim of the presented study was to provide a proof of concept for (a) the suitability of mealworm protein gels to be turned into dried porous biomaterials by either a combination of solvent exchange and supercritical drying to obtain aerogels or by lyophilization to obtain lyophilized hydrogels and (b) the suitability of either drying method to retain trace elements such as zinc in the gels throughout the drying process. Hydrogels were prepared from mealworm protein, subsequently dried using either method, and characterized via FT-IR, BET volume, and high-resolution scanning electron microscopy. Retention of zinc was evaluated via energy-dispersive X-ray spectroscopy. Results showed that both drying methods were suitable for obtaining dried porous biomaterials and that the drying method mainly influenced the overall surface area and pore hydrophobicity but not the secondary structure of the proteins in the gels or their zinc content after drying. Therefore, a first proof of concept for utilizing mealworm protein hydrogels as a base for dried porous biomaterials was successful and elucidated the potential of these materials as future sustainable alternatives to more conventional dried porous materials.

A fluorometric assay to determine labile copper(II) ions in serum.

Labile copper(II) ions (Cu2+) in serum are considered to be readily available for cellular uptake and to constitute the biologically active Cu2+ species in the blood. It might also be suitable to reflect copper dyshomeostasis during diseases such as Wilson's disease (WD) or neurological disorders. So far, no direct quantification method has been described to determine this small Cu2+ subset. This study introduces a fluorometric high throughput assay using the novel Cu2+ binding fluoresceine-peptide sensor FP4 (Kd of the Cu2+-FP4-complex 0.38 pM) to determine labile Cu2+ in human and rat serum. Using 96 human serum samples, labile Cu2+was measured to be 0.14 ± 0.05 pM, showing no correlation with age or other serum trace elements. No sex-specific differences in labile Cu2+ concentrations were noted, in contrast to the total copper levels in serum. Analysis of the effect of drug therapy on labile Cu2+ in the sera of 19 patients with WD showed a significant decrease in labile Cu2+ following copper chelation therapy, suggesting that labile Cu2+ may be a specific marker of disease status and that the assay could be suitable for monitoring treatment progress.

Effects of combined cannabidiol (CBD) and hops (Humulus lupulus) terpene extract treatment on RAW 264.7 macrophage viability and inflammatory markers.

This study investigates the potential of cannabidiol (CBD), one major cannabinoid of the plant Cannabis sativa, alone and in combination with a terpene-enriched extract from Humulus lupulus ("Hops 1"), on the LPS-response of RAW 264.7 macrophages as an established in vitro model of inflammation. With the present study, we could support earlier findings of the anti-inflammatory potential of CBD, which showed a dose-dependent [0-5 µM] reduction in nitric oxide and tumor necrosis factor-alpha (TNF-α) released by LPS-stimulated RAW 264.7 macrophages. Moreover, we observed an additive anti-inflammatory effect after combined CBD [5 µM] and hops extract [40 µg/mL] treatment. The combination of CBD and Hops 1 showed effects in LPS-stimulated RAW 264.7 cells superior to the single substance treatments and akin to the control hydrocortisone. Furthermore, cellular CBD uptake increased dose-dependently in the presence of terpenes from Hops 1 extract. The anti-inflammatory effect of CBD and its cellular uptake positively correlated with terpene concentration, as indicated by comparison with a hemp extract containing both CBD and terpenes. These findings may contribute to the postulations for the so-called "entourage effect" between cannabinoids and terpenes and support the potential of CBD combined with phytomolecules from a non-cannabinoid source, such as hops, for the treatment of inflammatory diseases.

Comparison of Three Low-Molecular-Weight Fluorescent Probes for Measuring Free Zinc Levels in Cultured Mammary Cells.

Free zinc is a critical regulator in signal transduction and affects many cellular processes relevant to cancer, including proliferation and cell death. Acting as a second messenger, altered free intracellular zinc has fundamental effects on regulating enzymes such as phosphatases and caspases. Therefore, the determination of free intracellular zinc levels is essential to assess its influence on the signaling processes involved in cancer development and progression. In this study, we compare three low-molecular-weight fluorescent probes, ZinPyr-1, TSQ, and FluoZin-3, for measuring free zinc in different mammary cell lines (MCF10A, MCF7, T47D, and MDA-MB-231). In summary, ZinPyr-1 is the most suitable probe for free Zn quantification. It responds well to calibration based on minimal fluorescence in the presence of the chelator TPEN (N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine) and maximal fluorescence by saturation with ZnSO4, resulting in the detection of free intracellular zinc in breast cancer subtypes ranging from 0.62 nM to 1.25 nM. It also allows for measuring the zinc fluxes resulting from incubation with extracellular zinc, showing differences in the zinc uptake between the non-malignant MCF10A cell line and the other cell lines. Finally, ZinPyr-1 enables the monitoring of sub-cellular distributions by fluorescence microscopy. Altogether, these properties provide a basis for the further exploration of free zinc in order to realize its full potential as a possible biomarker or even therapeutic target in breast cancer.

In vivo tests of a novel wound dressing based on agar aerogel.

Aerogels, especially bio-based ones, present a promising option for wound dressing; specifically, because of their low toxicity, high stability, bio-compatibility, and good biological performance. In this study, agar aerogel was prepared and evaluated as novel wound dressing material in an in vivo rat study. Agar hydrogel was prepared by thermal gelation, after that the water inside the gel was exchanged with ethanol, and finally the alcogel was dried by supercritical CO2. The textural and rheological properties of the prepared aerogel were characterized, showing that the prepared agar aerogels possess high porosity (97-98 %), high surface area (250-330 m2g-1) as well as good mechanical properties and easiness of removal from the wound site. The results of the in vivo experiments macroscopically demonstrate the tissue compatibility of the aerogels in dorsal interscapular injured rat tissue and a shorter wound healing time comparable to that of gauze-treated animals. The histological analysis underpins the reorganisation and healing of the tissue for the injured skin of rats treated with agar aerogel wound dressing within the studied time frame.

Quantitative Analysis and 2D/3D Elemental Imaging of Cocoa Beans Using X-ray Fluorescence Techniques.

As an important raw material for the confectionery industry, the cocoa bean (Theobroma cacao L.) has to meet certain legal requirements in terms of food safety and maximum contaminant levels in order to enter the cocoa market. Understanding the enrichment and distribution of essential minerals but also toxic metals is of utmost importance for improving the nutritional quality of this economically important raw food material. We present three X-ray fluorescence (XRF) techniques for elemental bio-imaging of intact cocoa beans and one additional XRF technique for quantitative analysis of cocoa pellets. The interrelation of all the methods presented gives a detailed picture of the content and 3D-resolved distribution of elements in complete cocoa beans for the first time.

Systematic Studies on the Antioxidant Capacity and Volatile Compound Profile of Yellow Mealworm Larvae (T. molitor L.) under Different Drying Regimes.

The yellow mealworm (Tenebrio molitor L., Coleoptera: Tenebrionidae) is an edible insect and due to its ubiquitous occurrence and the frequency of consumption, a promising candidate for the cultivation and production on an industrial scale. Moreover, it is the first insect to be approved by EFSA 2021 following the Novel Food Regulation. Industrial production of mealworms necessitates optimized processing techniques, where drying as the first postharvest procedure is of utmost importance for the quality of the final product. The focus of the present study was to analyse the chemical composition, antioxidant capacity, volatile compound profile and colouring of mealworm larvae dried in various regimes (freeze-drying, microwave drying, infrared drying, rack-oven drying and high-frequency drying). Proximate composition and fatty acid profile were similar for all dried larvae. Freeze dried larvae were predominantly marked by lipid oxidation with significantly higher peroxide values, secondary/tertiary oxidation products in the headspace GC-MS profiles and lower antioxidant capacity. High-temperature treatment in the rack oven-and to some extent also infrared or microwave drying-led to mealworm larvae darkening and the appearance of volatile Maillard secondary products such as 2-methylpropanoic acid, 2-/3-methylbutanoic acid and alkylpyrazines. High-frequency drying as a new emerging technology in insect processing was the most cost-effective method with energy costs of solely 0.09 Є/kg T. molitor L. leading to final larval material characterized by both lipid oxidation and nonenzymatic Maillard-browning.

Zinc availability from zinc-enriched yeast studied with an in vitro digestion/Caco-2 cell culture model.

BACKGROUND: Organic zinc sources for the treatment of zinc deficiency or as a supplement to a specific diet are increasingly needed. Zinc-enriched yeast (ZnYeast) biomass is a promising nutritional supplement for this essential micronutrient. However, these products are not yet authorized in the European Union and a clear position from the European Food Safety Authority on the use of ZnYeast as a zinc supplement is pending, demanding more data on its bioavailability. OBJECTIVE: The study aimed to produce a ZnYeast based on a Saccharomyces genus (S. pastorianus Rh), characterize its zinc enrichment quota, cellular distribution of zinc, and evaluate its zinc bioavailability after human digestion by comparing it to commonly used inorganic and organic zinc supplements (ZnO, ZnSO4, zinc gluconate, and zinc aspartate). METHOD AND MAIN FINDINGS: The zinc-enriched S. pastorianus Rh contained 5.9 ± 1.0 mg zinc/g yeast, which was predominantly localized on the cell surface according to its characterization on the microscale with scanning electron microscopy (SEM) with energy-dispersive X-ray (EDX). Combined experiments with a human in vitro digestion model and the in vitro intestinal cell model Caco-2 showed that intestinal zinc bioavailability of digested yeast biomass was comparable to the other zinc supplements, apart from ZnO, which was somewhat less bioavailable. Moreover, zinc released from digested ZnYeast was available for biological processes within the enterocytes, leading to mRNA upregulation of metallothionein, a biomarker of intestinal zinc status, and significantly elevated the cellular labile zinc pool. CONCLUSIONS: Our findings demonstrated that ZnYeast represents a suitable nutritional source for organically bound zinc and highlighted optimization strategies for future production of dietary ZnYeast.

A 2,7-dichlorofluorescein derivative to monitor microcalcifications.

Herein, we report the crystal structure of 2,7-dichlorofluorescein methyl ester (DCF-ME) and its fluorescence response to hydroxyapatite binding. The reported fluorophore is very selective for staining the bone matrix and provides turn-on fluorescence upon hydroxyapatite binding. The reported fluorophore can readily pass the cell membrane of the C2C12 cell line, and it is non-toxic for the cell line. The reported fluorophore DCF-ME may find applications in monitoring bone remodeling and microcalcification as an early diagnosis tool for breast cancer and age-related macular degeneration.

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.

Time- and Zinc-Related Changes in Biomechanical Properties of Human Colorectal Cancer Cells Examined by Atomic Force Microscopy.

Monitoring biomechanics of cells or tissue biopsies employing atomic force microscopy (AFM) offers great potential to identify diagnostic biomarkers for diseases, such as colorectal cancer (CRC). Data on the mechanical properties of CRC cells, however, are still scarce. There is strong evidence that the individual zinc status is related to CRC risk. Thus, this study investigates the impact of differing zinc supply on the mechanical response of the in vitro CRC cell lines HT-29 and HT-29-MTX during their early proliferation (24-96 h) by measuring elastic modulus, relaxation behavior, and adhesion factors using AFM. The differing zinc supply severely altered the proliferation of these cells and markedly affected their mechanical properties. Accordingly, zinc deficiency led to softer cells, quantitatively described by 20-30% lower Young's modulus, which was also reflected by relevant changes in adhesion and rupture event distribution compared to those measured for the respective zinc-adequate cultured cells. These results demonstrate that the nutritional zinc supply severely affects the nanomechanical response of CRC cell lines and highlights the relevance of monitoring the zinc content of cancerous cells or biopsies when studying their biomechanics with AFM in the future.

Ca-Zn-Ag Alginate Aerogels for Wound Healing Applications: Swelling Behavior in Simulated Human Body Fluids and Effect on Macrophages.

Chronic non-healing wounds represent a substantial economic burden to healthcare systems and cause a considerable reduction in quality of life for those affected. Approximately 0.5-2% of the population in developed countries are projected to experience a chronic wound in their lifetime, necessitating further developments in the area of wound care materials. The use of aerogels for wound healing applications has increased due to their high exudate absorbency and ability to incorporate therapeutic substances, amongst them trace metals, to promote wound-healing. This study evaluates the swelling behavior of Ca-Zn-Ag-loaded alginate aerogels and their metal release upon incubation in human sweat or wound fluid substitutes. All aerogels show excellent liquid uptake from any of the formulas and high liquid holding capacities. Calcium is only marginally released into the swelling solvents, thus remaining as alginate bridging component aiding the absorption and fast transfer of liquids into the aerogel network. The zinc transfer quota is similar to those observed for common wound dressings in human and animal injury models. With respect to the immune regulatory function of zinc, cell culture studies show a high availability and anti-inflammatory activity of aerogel released Zn-species in RAW 264.7 macrophages. For silver, the balance between antibacterial effectiveness versus cytotoxicity remains a significant challenge for which the alginate aerogels need to be improved in the future. An increased knowledge of the transformations that alginate aerogels undergo in the course of the fabrication as well as during wound fluid exposure is necessary when aiming to create advanced, tissue-compatible aerogel products.

Dietary zinc enrichment reduces the cadmium burden of mealworm beetle (Tenebrio molitor) larvae.

The industrial production of Tenebrio molitor L. requires optimized rearing and processing conditions to generate insect biomass with high nutritional value in large quantities. One of the problems arising from processing is a tremendous loss in mineral accessibility, affecting, amongst others, the essential trace element Zn. As a feasible strategy this study investigates Zn-enrichment of mealworms during rearing to meet the nutritional requirements for humans and animals. Following feeding ZnSO4-spiked wheat bran substrates late instar mealworm larvae were evaluated for essential micronutrients and human/animal toxic elements. In addition, growth rate and viability were assessed to select optimal conditions for future mass-rearing. Zn-feeding dose-dependently raised the total Zn content, yet the Znlarvae/Znwheat bran ratio decreased inversely related to its concentration, indicating an active Zn homeostasis within the mealworms. The Cu status remained stable, suggesting that, in contrast to mammals, the intestinal Cu absorption in mealworm larvae is not affected by Zn. Zn biofortification led to a moderate Fe and Mn reduction in mealworms, a problem that certainly can be overcome by Fe/Mn co-supplementation during rearing. Most importantly, Zn feeding massively reduced the levels of the human/animal toxicant Cd within the mealworm larvae, a technological novelty of outstanding importance to be implemented in the future production process to ensure the consumer safety of this edible insect species.

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.

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.

Effect of Different Drying Methods on Nutrient Quality of the Yellow Mealworm (Tenebrio molitor L.).

Yellow mealworm (Tenebrio molitor L.) represents a sustainable source of proteins and fatty acids for feed and food. Industrial production of mealworms necessitates optimized processing techniques, where drying as the first postharvest procedure is of utmost importance for the quality of the final product. This study examines the nutritional quality of mealworm larvae processed by rack oven drying, vacuum drying or freeze drying, respectively. Proximate composition and fatty acid profile were comparable between the dried larvae. In contrast, larvae color impressions and volatile compound profiles were very much dependent on processing procedure. High-temperature rack oven drying caused pronounced darkening with rather low content of volatiles, pointing toward the progress of Maillard reaction. On the other hand, vacuum drying or freeze drying led to enrichment of volatile Maillard reaction and lipid oxidation intermediates, whose actual sensory relevance needs to be clarified in the future. Beyond sensory and visual importance drying intermediates have to be considered with regard to their metal ion chelating ability; in particular for essential trace elements such as Zn2+. This study found comparable total zinc contents for the differently dried mealworm samples. However, dried larvae, in particular after rack oven drying, had only low zinc accessibility, which was between 20% and 40%. Therefore, bioaccessibility rather than total zinc has to be considered when their contribution to meeting the nutritional requirements for zinc in humans and animals is evaluated.

Sarcosine is a prostate epigenetic modifier that elicits aberrant methylation patterns through the SAMe-Dnmts axis.

DNA hypermethylation is one of the most common epigenetic modifications in prostate cancer (PCa). Several studies have delineated sarcosine as a PCa oncometabolite that increases the migration of malignant prostate cells while decreasing their doubling time. Here, we show that incubation of prostate cells with sarcosine elicited the upregulation of sarcosine N-demethylation enzymes, sarcosine dehydrogenase and pipecolic acid oxidase. This process was accompanied by a considerable increase in the production of the major methyl-donor S-adenosylmethionine (SAMe), together with an elevation of cellular methylation potential. Global DNA methylation analyses revealed increases in methylated CpG islands in distinct prostate cell lines incubated with sarcosine, but not in cells of nonprostate origin. This phenomenon was further associated with marked upregulation of DNA methyltransferases (Dnmts). Epigenetic changes were recapitulated through blunting of Dnmts using the hypomethylating agent 5-azacytidine, which was able to inhibit sarcosine-induced migration of prostate cells. Moreover, spatial mapping revealed concomitant increases in sarcosine, SAMe and Dnmt1 in histologically confirmed malignant prostate tissue, but not in adjacent or nonmalignant tissue, which is in line with the obtained in vitro data. In summary, we show here for the first time that sarcosine acts as an epigenetic modifier of prostate cells and that this may contribute to its oncometabolic role.

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.

Influencing the adhesion properties and wettability of mucin protein films by variation of the environmental pH.

Mucins, the main component of the mucus secretions of goblet and epithelial cells, are known for exhibiting a different behaviour in accordance with their surrounding environment (i.e. among others the environmental pH), which induces a drastic change in their measured mechanical properties. In this work, we have first employed Atomic Force Microscopy (AFM) in Force Spectroscopy mode to evaluate the adhesion of porcine mucin films at the nanoscale, and the changes caused in this particular factor by a pH variation between 7.0 and 4.0, both quite common values in biological conditions. Measurements also involved additional varying factors such as the indenting tip chemistry (hydrophobic vs hydrophilic), its residence time on the measured film (0, 1 and/or 2 seconds), and increasing pulling rates (ranging from 0.1 up to 10 µm/s). A second approach regarded the macroscale behaviour of the films, due to their potential applicability in the development of a new set of stimuli-responsive biomaterials. This was possible by means of complementary Wilhelmy plate method (to test the wetting properties) and cell proliferation studies on films previously exposed to the corresponding pH solution. According to our results, treatment with lowest pH (4.0) provides porcine mucin with a more hydrophilic character, showing a much stronger adhesion for analogous chemistries, as well as enhanced capability for cell attachment and proliferation, which opens new pathways for their future use and consideration as scaffold-forming material.