Physiological response in E. coli to YdgR overexpression depends on whether the protein has an intact function.

Membrane transport proteins are essential for the transport of a wide variety of molecules across the cell membrane to maintain cellular homeostasis. Generally, these transport proteins can be overexpressed in a suitable host (bacteria, yeast, or mammalian cells), and it is well documented that overexpression of membrane proteins alters the global metabolomic and proteomic profiles of the host cells. In the present study, we investigated the physiological consequences of overexpression of a membrane transport protein YdgR that belongs to the POT/PTR family from E. coli by using the lab strain BL21 (DE3)pLysS in its functional and attenuated mutant YdgR-E33Q. We found significant differences between the omics (metabolomics and proteomics) profiles of the cells expressing functional YdgR as compared to cells expressing attenuated YdgR, e.g., upregulation of several uncharacterized y-proteins and enzymes involved in the metabolism of peptides and amino acids. Furthermore, molecular network analysis suggested a relatively higher presence of proline-containing tripeptides in cells expressing functional YdgR. We envisage that an in-depth investigation of physiological alterations due to protein over-expression may be used for the deorphanization of the y-gene transportome.

The Y-ome Conundrum: Insights into Uncharacterized Genes and Approaches for Functional Annotation.

The ever-increasing availability of genome sequencing data has revealed a substantial number of uncharacterized genes without known functions across various organisms. The first comprehensive genome sequencing of E. coli K12 revealed that more than 50% of its open reading frames corresponded to transcripts with no known functions. The group of protein-coding genes without a functional description and/or a recognized pathway, beginning with the letter "Y", is classified as the "y-ome". Several efforts have been made to elucidate the functions of these genes and to recognize their role in biological processes. This review provides a brief update on various strategies employed when studying the y-ome, such as high-throughput experimental approaches, comparative omics, metabolic engineering, gene expression analysis, and data integration techniques. Additionally, we highlight recent advancements in functional annotation methods, including the use of machine learning, network analysis, and functional genomics approaches. Novel approaches are required to produce more precise functional annotations across the genome to reduce the number of genes with unknown functions.

Bioactive C17 and C18 Acetylenic Oxylipins from Terrestrial Plants as Potential Lead Compounds for Anticancer Drug Development.

Bioactive C17 and C18 acetylenic oxylipins have shown to contribute to the cytotoxic, anti-inflammatory, and potential anticancer properties of terrestrial plants. These acetylenic oxylipins are widely distributed in plants belonging to the families Apiaceae, Araliaceae, and Asteraceae, and have shown to induce cell cycle arrest and/or apoptosis of cancer cells in vitro and to exert a chemopreventive effect on cancer development in vivo. The triple bond functionality of these oxylipins transform them into highly alkylating compounds being reactive to proteins and other biomolecules. This enables them to induce the formation of anti-inflammatory and cytoprotective phase 2 enzymes via activation of the Keap1-Nrf2 signaling pathway, inhibition of proinflammatory peptides and proteins, and/or induction of endoplasmic reticulum stress, which, to some extent, may explain their chemopreventive effects. In addition, these acetylenic oxylipins have shown to act as ligands for the nuclear receptor PPARγ, which play a central role in growth, differentiation, and apoptosis of cancer cells. Bioactive C17 and C18 acetylenic oxylipins appeartherefore, to constitute a group of promising lead compounds for the development of anticancer drugs. In this review, the cytotoxic, anti-inflammatory and anticancer effects of C17 and C18 acetylenic oxylipins from terrestrial plants are presented and their possible mechanisms of action and structural requirements for optimal cytotoxicity are discussed.

Biomarkers for the Detection of Prenatal Alcohol Exposure: A Review.

Alcohol exposure during pregnancy can cause adverse effects to the fetus, because it interferes with fetal development, leading to later physical and mental impairment. The most common clinical tool to determine fetal alcohol exposure is maternal self-reporting. However, a more objective and useful method is based on the use of biomarkers in biological specimens alone or in combination with maternal self-reporting. This review reports on clinically relevant biomarkers for detection of prenatal alcohol exposure (PAE). A systematic search was performed to ensure a proper overview in existing literature. Studies were selected to give an overview on clinically relevant neonatal and maternal biomarkers. The direct biomarkers fatty acid ethyl esters (FAEEs), ethyl glucuronide (EtG), ethyl sulfate, and phosphatidylethanol (PEth) were found to be the most appropriate biomarkers in relation to detection of PAE. To review each biomarker in a clinical context, we have compared the advantages and disadvantages of each biomarker, in relation to its window of detectability, ease of collection, and the ease and cost of analysis of each biomarker. The biomarkers PEth, FAEEs, and EtG were found to be applicable for detection of even low levels of alcohol exposure. Meconium is an accessible matrix for determination of FAEEs and EtG, and blood an accessible matrix for determination of PEth.

Electrospinnability of Poly Lactic-co-glycolic Acid (PLGA): the Role of Solvent Type and Solvent Composition.

PURPOSE: In this study, the electrospinnability of poly(lactic-co-glycolic acid) (PLGA) solutions was investigated, with a focus on understanding the influence of molecular weight of PLGA, solvent type and solvent composition on the physical properties of electrospun nanofibers. METHOD: Various solvents were tested to dissolve two PLGA grades (50 KDa-RG755, 100 KDa-RG750). The viscoelasticity, surface tension, and evaporation rate of the PLGA solutions were characterized prior to the electrospinning process. The resulting electrospun nanofibers were characterized with respect to the morphology and mechanical properties. RESULTS: Two pairs of solvent mixtures, i.e. dimethylformamide (DMF)-tetrahydrofuran (THF) and DMF-chloroform (CHL), were identified to provide a stable cone-jet. Within the polymer concentration range studied (10-30%, w/v), RG750 solutions could be electrospun into uniform fibers at 30% (w/v) or at 20% (w/v) when modifying the solvent composition. In comparison to DMF-THF solution, fibers had larger diameter, higher stiffness and tensile strength when electrospun from DMF-CHL solution. CONCLUSION: The high molecular weight polymer could ensure sufficient intermolecular interaction to generate uniform fibers. The solvent could influence the morphology and mechanical properties of the electrospun fibers by altering the properties of PLGA solution, and drying rate of fibers in the electrospinning process.

Effect of resveratrol on experimental non-alcoholic fatty liver disease depends on severity of pathology and timing of treatment.

BACKGROUND AND AIM: Non-alcoholic steatohepatitis (NASH) is a leading cause of chronic liver disease with few therapeutic options. Resveratrol (RSV) prevents the development of steatosis in a number of experimental fatty liver (non-alcoholic fatty liver [NAFL]) models, but the preventive or therapeutic effects on experimental NASH are not yet clarified, and clinical results on non-alcoholic fatty liver disease are ambiguous. Thus, we aimed to compare the RSV-mediated preventive and therapeutic effects on experimental NAFL and NASH. METHODS: We used a high-fat (HF) diet to generate a rat NAFL model and a high-fat, high-cholesterol (HFC) diet to generate a rat NASH model. The preventive and therapeutic potential of RSV was tested by adding RSV to the HF and HFC diet from study start or after 1 week of the diets. Animals were sacrificed after 8 weeks with appropriate controls. Blood and liver were harvested for analysis, including measurement of RSV metabolites. RESULTS: Resveratrol reduced the development of histological steatosis (P = 0.03) and partly triglyceride accumulation (fold change reduced from 3.6 to 2.4, P = 0.08) in the male NAFL model, although effects were moderate. In NASH prevention, RSV reduced the accumulation of triglyceride in hepatic tissue (P < 0.01), while there was no effect on biochemical, histopathological, or transcriptional NASH changes. Further, RSV had no therapeutic effect on established NASH. We found RSV metabolites but no parent RSV in serum or liver tissue, confirming low bioavailability. CONCLUSIONS: These experimental findings suggest that a weak hepatic benefit of RSV treatment is seen in prevention of steatosis only.

Effect of resveratrol on experimental non-alcoholic steatohepatitis.

Non-alcoholic fatty liver disease and non-alcoholic steatohepatitis (NASH) are increasing clinical problems for which effective treatments are required. The polyphenol resveratrol prevents the development of fatty liver disease in a number of experimental studies. We hypothesized that it could revert steatohepatitis, including hepatic inflammation and fibrosis, in an experimental NASH model. To induce hepatic steatohepatitis, a 65% fat, 2% cholesterol and 0.5% cholate (HFC) diet was fed to rats for 1 or 16 weeks, prior to treatment. Subsequently, the diet was supplemented with resveratrol (approx. 100mg/rat/day) to three intervention groups; week 2-4, 2-7 or 17-22. Treated animals were sacrificed at the end of each intervention period with appropriate control and HFC diet controls. Blood and liver were harvested for analysis. When commenced early, resveratrol treatment partially mitigated transaminase elevations, hepatic enlargement and TNFα induced protein-3 protein expression, but generally resveratrol treatment had no effect on elevated hepatic triglyceride levels, histological steatohepatitis or fibrosis. We observed a slight reduction in Collagen1α1 mRNA expression and no reduction in the mRNA expression of other markers of fibrosis, inflammation or steatosis (TGFß, TNFα, α2-MG, or SREBP-1c). Resveratrol metabolites were detected in serum, including trans-resveratrol-3-O-sulphate/trans-resveratrol-4'-O-sulphate (mean concentration 7.9 µg/ml). Contrary to the findings in experimental steatosis, resveratrol treatment had no consistent therapeutic effect in alleviating manifest experimental steatohepatitis.

Investigation of the homogeneity of methacrylate allergens in commercially available patch test preparations.

BACKGROUND: The homogeneity of methacrylates in commercial patch test preparations has not yet been investigated. Inhomogeneous patch test preparations may give rise to false-negative or false-positive patch test results in patients suspected of having methacrylate allergy. OBJECTIVES: To investigate the homogeneity of methacrylates in commercial patch test preparations. METHODS: Fresh commercial patch test preparations of methyl methacrylate (MMA) and 2-hydroxyethyl methacrylate (2-HEMA) from three test material suppliers in Europe were analysed quantitatively by means of normal-phase high-performance liquid chromatography. RESULTS: The initial concentration of MMA in all six patch test preparations was lower than stated on the label, whereas four of six patch test preparations of 2-HEMA were in accordance with the stated concentrations. The concentration of MMA increased markedly from the top segment close to the tip of the syringe to the bottom segment adjacent to the piston in four syringes (3-6). In contrast, syringes with 2-HEMA maintained a constant concentration throughout the test preparation, apart from two syringes (11 and 12). CONCLUSION: Variations in concentration and heterogeneous distribution of MMA and 2-HEMA in patch test preparations may be an additional cause of variation in patch test results, besides other technical details and reading.

Carrot Juice Intake Affects the Cytokine and Chemokine Response in Human Blood after Ex Vivo Lipopolysaccharide-Induced Inflammation.

In vitro and animal studies have shown that carrot juice containing bioactive natural products, such as falcarinol (FaOH) and falcarindiol (FaDOH), can affect inflammation. The present study was designed to test whether oral intake of carrot juice containing the bioactive acetylenic oxylipins FaOH and FaDOH affects mediators of acute inflammation or the innate immune response in human blood. Carrot juice (500 mL) was administered orally to healthy volunteers, and blood samples were drawn before and 1 h after juice intake. Next, the blood samples were split in two, and one sample was stimulated ex vivo with lipopolysaccharide (LPS) and incubated at 37 °C for 24 h. The concentrations of 44 inflammatory cytokines and chemokines were examined using multiplex electrochemiluminescence analysis. In blood samples not stimulated with LPS, a significant increase in IL-15 was measured 1 h after carrot juice intake. Cytokines like IFN-É£, IL-12/IL-23(p40), IL-23, IL-17A, IL-17B, IL-17D, and IL-22 were significantly increased in LPS-stimulated blood samples after carrot juice intake. The upregulation of the immunostimulating cytokines belonging to the IL-23/IL-17 Th17 axis suggests that carrot juice intake could benefit diseases where inflammation plays a role, like in the early stages of diabetes or cancers.

Effects of Chokeberries (Aronia spp.) on Cytoprotective and Cardiometabolic Markers and Semen Quality in 109 Mildly Hypercholesterolemic Danish Men: A Prospective, Double-Blinded, Randomized, Crossover Trial.

Background: Chokeberries (Aronia spp.) are known to exhibit both direct and indirect antioxidant properties and have been associated with beneficial effects on human health, including cardiovascular risk factors (inflammation, serum lipids, sugars, blood pressure), oxidative stress, and semen quality. This prospective, double-blinded, randomized, crossover clinical trial was conducted to elucidate the effects of Aronia supplementation on these health targets in mildly hypercholesterolemic men. Methods: The standardized Aronia supplementation comprised three wild Aronia spp. (A. arbutifolia, A prunifolia and A. melanocarpa) and the Aronia hybrid × Sorbaronia mitschurinii (standardized to 150 mg anthocyanins daily). Participants (n = 109) were healthy men with respect to all outcome targets except for the total cholesterol level (5.0−7.0 mM). Participants were randomized to supplementation with either Aronia or placebo for 90 days, followed by a wash-out period and lastly the complementary supplementation. Effects on the health parameters were compared among both the whole group of men and in subgroups according to age, body mass index (BMI), lifestyle, dietary habits, and serum glutathione levels at baseline. The study is registered in ClinicalTrials.gov.: NCT03405753. Results: Glutathione levels were significantly improved after 90 days intake of Aronia supplementation compared to placebo in the subgroup of men with a low level of glutathione at baseline (p = 0.038) and a high coffee intake (p = 0.045). A significant decrease in levels of sperm DNA fragmentation and an increase in the percentage of motile sperm were observed in men aged >40 and in men with BMI > 25. Further, these parameters were significantly improved in the dietary subgroup defined by a high level of coffee intake. Total cholesterol and low-density lipoprotein-cholesterol levels decreased significantly in men <40 years after Aronia supplementation. No statistically significant effects were observed regarding blood pressure, markers of blood sugar regulation, hemoglobin A1c, superoxide dismutase, catalase, isoprostane levels, high sensitivity C reactive protein, or other semen parameters. Conclusions: This study demonstrated a significant increase in glutathione levels and improvement of cytoprotective targets following Aronia supplementation in specific subgroups of men >40 years of age and BMI > 25 but did not demonstrate a significant effect in the overall analysis. The observed concurrent increase in glutathione levels and improvement of cytoprotective targets following Aronia supplementation in subgroups of men, suggests that the endogenous phase II antioxidant glutathione is involved in the modulation of the observed cytoprotective effects. This study is a good foundation for further investigation of these cytoprotective effects in groups with oxidative stress in a dose−response study.

Silicon-induced changes in antifungal phenolic acids, flavonoids, and key phenylpropanoid pathway genes during the interaction between miniature roses and the biotrophic pathogen Podosphaera pannosa.

Application of 3.6 mm silicon (Si+) to the rose (Rosa hybrida) cultivar Smart increased the concentration of antimicrobial phenolic acids and flavonoids in response to infection by rose powdery mildew (Podosphaera pannosa). Simultaneously, the expression of genes coding for key enzymes in the phenylpropanoid pathway (phenylalanine ammonia lyase, cinnamyl alcohol dehydrogenase, and chalcone synthase) was up-regulated. The increase in phenolic compounds correlated with a 46% reduction in disease severity compared with inoculated leaves without Si application (Si-). Furthermore, Si application without pathogen inoculation induced gene expression and primed the accumulation of several phenolics compared with the uninoculated Si- control. Chlorogenic acid was the phenolic acid detected in the highest concentration, with an increase of more than 80% in Si+ inoculated compared with Si- uninoculated plants. Among the quantified flavonoids, rutin and quercitrin were detected in the highest concentrations, and the rutin concentration increased more than 20-fold in Si+ inoculated compared with Si- uninoculated plants. Both rutin and chlorogenic acid had antimicrobial effects on P. pannosa, evidenced by reduced conidial germination and appressorium formation of the pathogen, both after spray application and infiltration into leaves. The application of rutin and chlorogenic acid reduced powdery mildew severity by 40% to 50%, and observation of an effect after leaf infiltration indicated that these two phenolics can be transported to the epidermal surface. In conclusion, we provide evidence that Si plays an active role in disease reduction in rose by inducing the production of antifungal phenolic metabolites as a response to powdery mildew infection.

The effect of Artemisia annua on broiler performance, on intestinal microbiota and on the course of a Clostridium perfringens infection applying a necrotic enteritis disease model.

The aerial parts of the plant Artemisia annua contain essential oils having antimicrobial properties against Clostridium perfringens Type A, the causal agent for necrotic enteritis in broilers. In two experiments, the influence of increasing dietary concentrations of dried A. annua leaves (0, 5, 10 and 20 g/kg) and n-hexane extract from fresh A. annua leaves (0, 125, 250 and 500 mg/kg) on broiler performance was investigated. Dried plant material decreased feed intake and body weight in a dose-dependent manner, and 10 and 20 g/kg diet tended to improve the feed conversion ratio. The n-hexane extract also reduced feed intake, but broiler weight tended to decrease only at the highest dietary concentration. The feed conversion ratio tended to improve when birds received 250 and 500 mg/kg n-hexane extract. In a third experiment, a necrotic enteritis disease model was applied to investigate the effect of the dietary addition of dried A. annua leaves (10 g/kg on top) or n-hexane extract of A. annua (250 mg/kg) on the severity of the disease in broilers. The addition of n-hexane extract reduced the intestinal C. perfringens numbers and the severity of the disease-related small intestinal lesions. Over the infection period from day 17 to day 27, birds supplemented with the n-hexane extract gained more weight than both the challenged control birds and birds receiving dried plant material. The results indicate that n-hexane extracts derived from A. annua can modulate the course of necrotic enteritis and compensate to a certain extent for the disease-associated weight losses.

Stability of selected volatile contact allergens in different patch test chambers under different storage conditions.

BACKGROUND: Patch test preparations of volatile substances may evaporate during storage, thereby giving rise to reduced patch test concentrations. OBJECTIVES: To investigate the stability of selected acrylates/methacrylates and fragrance allergens in three different test chambers under different storage conditions. METHODS: Petrolatum samples of methyl methacrylate (MMA), 2-hydroxyethyl methacrylate (2-HEMA), 2-hydroxypropyl acrylate (2-HPA), cinnamal and eugenol in patch test concentrations were stored in three different test chambers (IQ chamber™, IQ Ultimate™, and Van der Bend® transport container) at room temperature and in a refrigerator. The samples were analysed in triplicate with high-performance liquid chromatography. RESULTS: The decrease in concentration was substantial for all five allergens under both storage conditions in IQ chamber™ and IQ Ultimate™, with the exception of 2-HEMA during storage in the refrigerator. For these two chamber systems, the contact allergen concentration dropped below the stability limit in the following order: MMA, cinnamal, 2-HPA, eugenol, and 2-HEMA. In the Van der Bend® transport container, the contact allergens exhibited acceptable stability under both storage conditions, whereas MMA and 2-HPA required cool storage for maintenance of the limit. CONCLUSION: The Van der Bend® transport container was the best device for storage of samples of volatile contact allergens.

Strong and Bitter Vegetables from Traditional Cultivars and Cropping Methods Improve the Health Status of Type 2 Diabetics: A Randomized Control Trial.

Vegetables rich in bitter-tasting phytochemicals may exert enhanced beneficial effects against key factors associated with type two diabetes (T2D). This study investigates whether selected cultivars of bitter and strong-tasting (BST) Brassica and root vegetables exert greater health benefits on T2D patients compared to equivalent modern mild and sweet tasting (MST) vegetables. A 12-week randomized, controlled, parallel intervention study involved 92 T2D patients, who were allocated three different diets: (1) 500 g daily of bitter and strong-tasting (BST) vegetables; (2) 500 g daily of mild and sweet-tasting (MST) vegetables; (3) 120 g daily MST normal diet (control). Both vegetable diets contained root vegetables and cabbages selected based on sensory differences and content of phytochemicals. Prior to and after the study, all participants underwent an oral glucose tolerance test (OGTT), 24 h blood pressure measurements, DEXA scans, and fasted blood samples. Both diets high in vegetables significantly reduced the participants' BMI, total body fat mass, and HbA1c levels compared to control, but in the BST group, significant differences were also found regarding incremental area under the curve glucose 240 min (OGTT) and fasting glucose levels. A high daily intake of root vegetables and cabbages showed significant health improvements in both vegetable groups. BST vegetables had the greatest impact on insulin sensitivity, body fat mass, and blood pressure compared to control; moreover, they further improved glycemic control compared to MST vegetables.

Carrot Intake and Risk of Colorectal Cancer: A Prospective Cohort Study of 57,053 Danes.

Carrots are consumed worldwide. Several meta-analysis studies on carrot consumption have indicated that carrots play a central role as a protecting vegetable against development of different types of cancers. A cancer-preventive role of carrots is plausible because they are the main dietary source of the bioactive polyacetylenic oxylipins falcarinol (FaOH) and falcarindiol (FaDOH), which have shown anti-proliferative and anti-inflammatory activity in numerous in vitro studies. In addition, purified FaOH and FaDOH have, in recent studies in colorectal cancer (CRC)-primed rats, demonstrated an anti-neoplastic effect in a dose-dependent manner. The mechanisms of action for this effect appears to be due to inhibition of pro-inflammatory and transcription factor biomarkers for inflammation and cancer. However, studies of the CRC-preventive effect of carrots in a large cohort are still missing. We therefore examined the risk of being diagnosed with CRC as predicted by intake of carrots in a Danish population of 57,053 individuals with a long follow-up. Self-reported intake of raw carrots at a baseline of 2-4 carrots or more each week (>32 g/day) was associated with a 17% decrease in risk of CRC with a mean follow-up of >18 years, compared to individuals with no intake of raw carrots even after extensive model adjustments (HR 0.83 CI 95% 0.71; 0.98). An intake below 2-4 carrots each week (<32 g/day) was not significantly associated with reduced risk of CRC (HR 0.93 CI 95% 0.82; 1.06). The results of this prospective cohort study clearly support the results from studies in cancer-primed rats for CRC and hence a CRC-preventive effect of carrots.

Falcarindiol Purified From Carrots Leads to Elevated Levels of Lipid Droplets and Upregulation of Peroxisome Proliferator-Activated Receptor-γ Gene Expression in Cellular Models.

Falcarindiol (FaDOH) is a cytotoxic and anti-inflammatory polyacetylenic oxylipin found in food plants of the carrot family (Apiaceae). FaDOH has been shown to activate PPARγ and to increase the expression of the cholesterol transporter ABCA1 in cells, both of which play an important role in lipid metabolism. Thus, a common mechanism of action of the anticancer and antidiabetic properties of FaDOH may be due to a possible effect on lipid metabolism. In this study, the effect of sub-toxic concentration (5 µM) of FaDOH inside human mesenchymal stem cells (hMSCs) was studied using white light microscopy and Raman imaging. Our results show that FaDOH increases lipid content in the hMSCs cells as well as the number of lipid droplets (LDs) and that this can be explained by increased expression of PPARγ2 as shown in human colon adenocarcinoma cells. Activation of PPARγ can lead to increased expression of ABCA1. We demonstrate that ABCA1 is upregulated in colorectal neoplastic rat tissue, which indicates a possible role of this transporter in the redistribution of lipids and increased formation of LDs in cancer cells that may lead to endoplasmic reticulum stress and cancer cell death.

Association between positive patch tests to epoxy resin and fragrance mix I ingredients.

BACKGROUND: Both epoxy resin (diglycidyl ether of bisphenol A) and fragrance mix I are included in the European baseline series of contact allergens. A significant association between positive reactions to epoxy resin and fragrance mix has been reported by others. OBJECTIVE: To investigate and possibly reproduce this association with the use of TRUE((R)) test data and supplementary tests with fragrance mix ingredients from the Department of Dermatology, Odense University Hospital. MATERIALS AND METHODS: Six thousand one hundred and fifteen consecutive eczema patients tested from 1995 to 2007 were included, and test results from all patients tested with fragrance mix ingredients were analysed. RESULTS: One hundred and forty-five (2.4%) were positive to epoxy resin and 282 (4.6%) were positive to fragrance mix I. Nineteen were positive to both giving an odds ratio of 3.3, which is significant (95% CI 2.0-5.4). Analysis of association to individual fragrance mix ingredients showed a significant association to alpha-amyl cinnamal and isoeugenol. CONCLUSIONS: The significant association between positive reactions to epoxy resin and fragrance mix I was reproduced. However, the clinical implications are not clarified, and even though the association may be coincidental, the fact that it can be reproduced with a different patch test system and in a different population speaks against a random result. Further studies may help to interpret the association.

Stability of lysozyme incorporated into electrospun fibrous mats for wound healing.

In this study, we investigated the feasibility of incorporating protein drugs into electrospun fibrous mats (EFMs) for wound healing using lysozyme as a model drug. Lysozyme nanoparticles (Lyso- NPs) were first obtained by electrospray. Lysozyme solutions were prepared with a binary solvent mixture of ethanol (EtOH)-water (H2O) at varied volume ratios. Subsequently, Lyso-NPs were suspended in poly(lactic-co-glycolic acid) (PLGA) solutions using trifluoroethanol (TFE) as a solvent. Lyso-NPs loaded EFMs were obtained by electrospinning of the aforementioned suspensions, and the bioactivity of lysozyme in the EFMs was investigated using fluorescence-based assay kit. The electrosprayed Lyso-NPs were spherical with barely altered bioactivity as compared to the untreated raw material when using EtOH- H2O (30:70, v/v) as the solvent. After the subsequent electrospinning process, more than 90% of the bioactivity of lysozyme was retained compared to the raw material. The cytotoxicity of the produced EFMs was evaluated by thiazolyl blue tetrazolium bromide (MTT) study and the proliferation and distribution of mouse fibroblast cells (L929) growing on EFMs were investigated using 4,6-diamidino-2-phenylindol dihydrochloride (DAPI) for nucleic acid staining. Nearly negligible cytotoxicity of all the EFMs was observed according to the MTT study. Furthermore, it was observed that the L929 cells grew well on the Lyso-EFMs, especially those with the modification of polyethylene glycol (PEG) that was added to improve the hydrophilicity of EFMs. This study demonstrated that the electrospray/electrospinning processes are suitable for loading biomacromolecules to produce functionalized wound dressings to promote wound healing.

Dietary Polyacetylenic Oxylipins Falcarinol and Falcarindiol Prevent Inflammation and Colorectal Neoplastic Transformation: A Mechanistic and Dose-Response Study in A Rat Model.

Falcarinol (FaOH) and falcarindiol (FaDOH) are cytotoxic and anti-inflammatory polyacetylenic oxylipins, which are commonly found in the carrot family (Apiaceae). FaOH and FaDOH have previously demonstrated a chemopreventive effect on precursor lesions of colorectal cancer (CRC) in azoxymethane (AOM)-induced rats. The purpose of the present study was to elucidate possible mechanisms of action for the preventive effect of FaOH and FaDOH on colorectal precancerous lesions and to determine how this effect was dependent on dose. Gene expression studies performed by RT-qPCR of selected cancer biomarkers in tissue from biopsies of neoplastic tissue revealed that FaOH and FaDOH downregulated NF-κß and its downstream inflammatory markers TNFα, IL-6, and COX-2. The dose-dependent anti-neoplastic effect of FaOH and FaDOH in AOM-induced rats was investigated in groups of 20 rats receiving a standard rat diet (SRD) supplemented with 0.16, 0.48, 1.4, 7 or 35 µg FaOH and FaDOH g-1 feed in the ratio 1:1 and 20 rats were controls receiving only SRD. Analysis of aberrant crypt foci (ACF) showed that the average number of small ACF (<7 crypts) and large ACF (>7 crypts) decreased with increasing dose of FaOH and FaDOH and that this inhibitory effect on early neoplastic formation of ACF was dose-dependent, which was also the case for the total number of macroscopic neoplasms. The CRC protective effects of apiaceous vegetables are mainly due to the inhibitory effect of FaOH and FaDOH on NF-κB and its downstream inflammatory markers, especially COX-2.

Effect of the dietary polyacetylenes falcarinol and falcarindiol on the gut microbiota composition in a rat model of colorectal cancer.

OBJECTIVES: (3R)-Falcarinol (FaOH) and (3R,8S)-falcarindiol (FaDOH) have previously been shown to reduce the number of neoplastic lesions and the growth rate of polyps in the colon of azoxymethane (AOM) treated rats. Based on previous investigations, it appears that different mechanisms of actions are involved in the antineoplastic effect of FaOH and FaDOH. One mechanism of action may be related to the antibacterial effect of FaOH and FaDOH and thus their effect on the gut microbiota. This study aimed to determine the effect of FaOH and FaDOH on gut microbiota composition of AOM treated rats. RESULTS: Azoxymethane treated rats were fed either a standard rat diet or a rat diet supplemented with FaOH and FaDOH. The gut microbiota of AOM-induced rats was determined by 16S rRNA gene-amplicon sequencing. Analysis of fecal cecum samples demonstrated a significant gut microbiota change in rats receiving standard rat diet supplemented with FaOH and FaDOH compared with the control group that only received the rat diet. Comparison of the gut microbiota of rats who developed large neoplasms in the colon with rats without large neoplasms showed that the gut microbiota was significantly different in rats who developed large colon neoplasms compared to rats with no macroscopic colon neoplasms.