3-keto-DON, but Not 3-epi-DON, Retains the in Planta Toxicological Potential after the Enzymatic Biotransformation of Deoxynivalenol.

Deoxynivalenol (DON) is a secondary fungal metabolite that is associated with many adverse toxicological effects in agriculture as well as human/animal nutrition. Bioremediation efforts in recent years have led to the discovery of numerous bacterial isolates that can transform DON to less toxic derivatives. Both 3-keto-DON and 3-epi-DON were recently shown to exhibit reduced toxicity, compared to DON, when tested using different cell lines and mammalian models. In the current study, the toxicological assessment of 3-keto-DON and 3-epi-DON using in planta models surprisingly revealed that 3-keto-DON, but not 3-epi-DON, retained its toxicity to a large extent in both duckweeds (Lemna minor L.) and common wheat (Triticum aestivum L.) model systems. RNA-Seq analysis revealed that the exposure of L. minor to 3-keto-DON and DON resulted in substantial transcriptomic changes and similar gene expression profiles, whereas 3-epi-DON did not. These novel findings are pivotal for understanding the environmental burden of the above metabolites as well as informing the development of future transgenic plant applications. Collectively, they emphasize the fundamental need to assess both plant and animal models when evaluating metabolites/host interactions.

Challenges Associated With the Formation of Recombinant Protein Inclusion Bodies in Escherichia coli and Strategies to Address Them for Industrial Applications.

Recombinant proteins are becoming increasingly important for industrial applications, where Escherichia coli is the most widely used bacterial host for their production. However, the formation of inclusion bodies is a frequently encountered challenge for producing soluble and functional recombinant proteins. To overcome this hurdle, different strategies have been developed through adjusting growth conditions, engineering host strains of E. coli, altering expression vectors, and modifying the proteins of interest. These approaches will be comprehensively highlighted with some of the new developments in this review. Additionally, the unique features of protein inclusion bodies, the mechanism and influencing factors of their formation, and their potential advantages will also be discussed.

Molecular Mechanisms Underlying the Absorption of Aglycone and Glycosidic Flavonoids in a Caco-2 BBe1 Cell Model.

The mechanisms of cellular absorption and transport underlying the differences between flavonoid aglycones and glycosides and the effect of the structural feature are not well established. In this study, aglycone, mono-, and diglycosides of quercetin and cyanidin were selected to examine the effects of the structural feature on the bioavailability of flavonoids using hexose transporters SGLT1 and GLUT2 in a Caco-2 BBe1 cell model. Cellular uptake and transport of all glycosides were significantly different. The glycosides also significantly inhibited cellular uptake of d-glucose, indicating the involvement of the two hexose transporters SGLT1 and GLUT2 in the absorption, and the potential of the glycosides in lowering the blood glucose level. The in silico prediction model also supported these observations. The absorption of glycosides, especially diglycosides but not the aglycones, was significantly blocked by SGLT1 and GLUT2 inhibitors (phloridzin and phloretin) and further validated in SGLT1 knockdown Caco-2 BBe1 cells.

Understanding the Bacterial Response to Mycotoxins: The Transcriptomic Analysis of Deoxynivalenol-Induced Changes in Devosia mutans 17-2-E-8.

Deoxynivalenol (DON) is a major fusarium toxin widely detected in cereal grains. The inadvertent exposure to this fungal secondary-metabolite gives rise to a myriad of adverse health effects including appetite loss, emesis, and suppression of the immune system. While most of the attention this mycotoxin has gained in the past four decades was related to its eukaryotic toxicity (monogastric animals and plants more precisely), recent studies have begun to reveal its negative influence on prokaryotes. Recently presented evidence indicates that DON can negatively affect many bacterial species, raising the possibility of DON-induced imbalances within the microbiota of the human and animal gut, in addition to other environmental niches. This in turn has led to a greater interest in understanding bacterial responses toward DON, and the involved mechanism(s) and metabolic pathways, in order to build a more comprehensive picture of DON-induced changes in both prokaryotes and eukaryotes alike. This study reveals the transcriptomic profiling of Devosia mutans strain 17-2-E-8 after the inclusion of DON within its growth medium. The results highlight three adaptive mechanisms involved in the response of D. mutans 17-2-E-8 to this mycotoxin, which include: (a) activation of adenosine 5'-triphosphate-binding cassette transporters; (b) engagement of a toxin-specific pyrroloquinoline quinone-dependent detoxification pathway; and finally (c) the upregulation of auxiliary coping proteins such as porins, glutathione S-transferases, and phosphotransferases. Some of the identified mechanisms are universal in nature and are shared with other bacterial genera and species.

Grape Pomace as a Promising Antimicrobial Alternative in Feed: A Critical Review.

Antimicrobial resistance is among the most urgent global challenges facing sustainable animal production systems. The use of antibiotics as growth promoters and for infectious disease prevention in intensive animal-farming practices has translated into the selection and spread of antimicrobial resistance genes in an unprecedented fashion. Several multi-resistant bacterial strains have been isolated from food-producing animals, thus constituting an alarming food-safety issue. Many industrial byproducts with potential antimicrobial properties are currently being investigated to identify empirical and affordable solutions/alternatives that can potentially be used in feed for animals. Grape pomace is among such byproducts that gained the attention as a result of its low cost, abundance, and, most importantly, its bioactive and antibacterial properties. This review discusses the recently reported studies with regard to exploring the use of grape pomace (and its extracts) in animal production to control pathogens, along with the promotion of beneficial bacterial species in the gut to ultimately alleviate antibacterial resistance. The review further summarizes realistic expectations connected with grape pomace usage and lists the still-to-be-addressed concerns about its application in animal agriculture.

Anti-Inflammatory Effects of Different Astaxanthin Isomers and the Roles of Lipid Transporters in the Cellular Transport of Astaxanthin Isomers in Caco-2 Cell Monolayers.

The anti-inflammatory effects and cellular transport mechanisms of all- E-astaxanthin and its 9Z- and 13Z-isomers were investigated in a Caco-2 cell monolayer model. All three astaxanthin isomers at 1.2 µM significantly reduced the TNF-α-induced secretion of IL-8 by 22-27%. Z-Astaxanthins, especially 9 Z-astaxanthin exhibited greater anti-inflammatory effect than all- E-astaxanthin by down-regulating pro-inflammatory cytokines COX-2 and TNF-α gene expression to 0.88 ± 0.01-fold and 0.83 ± 0.17-fold that of the negative control (NC), respectively. The anti-inflammatory effects of astaxanthin isomers were achieved via modulating the NF-κB signaling pathway as they down-regulated TNF-α-induced phosphorylation of IκBα from 5.3 ± 0.19-fold to 3.8 ± 0.33-4.5 ± 0.27-fold of NC. The scavenger receptor class B type I protein (SR-BI) was found to facilitate the cellular uptake of astaxanthin isomers. Its inhibitor (BLT-1) and antibody (Anti-SRBI) significantly reduced cellular uptake efficiency of all- E-astaxanthin (18.9% and 16.7%, respectively) and 13Z-astaxanthin (28.8% and 30.2%, respectively), but not of 9Z-astaxanthin. The molecular docking experiment showed that 13 Z-astaxanthin had significantly higher affinity with SR-BI (atomic contact energy: -420.31) than all- E-astaxanthin and 9 Z-astaxanthin, which at least partially supports the higher bioavailability of 13 Z-astaxanthin observed in vivo by others.

Innovative drugs, chemicals, and enzymes within the animal production chain.

The alarming number of recently reported human illnesses with bacterial infections resistant to multiple antibacterial agents has become a serious concern in recent years. This phenomenon is a core challenge for both the medical and animal health communities, since the use of antibiotics has formed the cornerstone of modern medicine for treating bacterial infections. The empirical benefits of using antibiotics to address animal health issues in animal agriculture (using therapeutic doses) and increasing the overall productivity of animals (using sub-therapeutic doses) are well established. The use of antibiotics to enhance profitability margins in the animal production industry is still practiced worldwide. Although many technical and economic reasons gave rise to these practices, the continued emergence of antimicrobial resistant bacteria is furthering the need to reduce the use of medically important antibiotics. This will require improving on-farm management and biosecurity practices, and the development of effective antibiotic alternatives that will reduce the dependence on antibiotics within the animal industry in the foreseeable future. A number of approaches are being closely scrutinized and optimized to achieve this goal, including the development of promising antibiotic alternatives to control bacterial virulence through quorum-sensing disruption, the use of synthetic polymers and nanoparticles, the exploitation of recombinant enzymes/proteins (such as glucose oxidases, alkaline phosphatases and proteases), and the use of phytochemicals. This review explores the most recent approaches within this context and provides a summary of practical mitigation strategies for the extensive use of antibiotics within the animal production chain in addition to several future challenges that need to be addressed.

The Identification of DepB: An Enzyme Responsible for the Final Detoxification Step in the Deoxynivalenol Epimerization Pathway in Devosia mutans 17-2-E-8.

Deoxynivalenol (DON) is one of the most common mycotoxins found in cereal grains and grains contaminated with DON can cause health issues for both humans and animals and result in severe economic losses. Currently there is no feasible method to remediate affected grains. The development of a biological method for detoxification is becoming increasingly more plausible with the discovery of microbes which can transform DON to a relatively non-toxic stereoisomer, 3-epi-DON. Although bacteria capable of detoxifying DON have been known for some time, it is only recently an enzyme responsible was identified. In Devosia mutans 17-2-E-8 (Devosia sp. 17-2-E-8) a two-step DON epimerization (Dep) pathway, designated as the Dep system, completes this reaction. DepA was recently identified as the enzyme responsible for the conversion of DON to 3-keto-DON, and in this report, DepB, a NADPH dependent dehydrogenase, is identified as the second and final step in the pathway. DepB readily catalyzes the reduction of 3-keto-DON to 3-epi-DON. DepB is shown to be moderately thermostable as it did not lose significant activity after a heat treatment at 55°C and it is amenable to lyophilization. DepB functions at a range of pH-values (5-9) and functions equally well in multiple common buffers. DepB is clearly a NADPH dependent enzyme as it utilizes it much more efficiently than NADH. The discovery of the final step in the Dep pathway may provide a means to finally mitigate the losses from DON contamination in cereal grains through an enzymatic detoxification system. The further development of this system will need to focus on the activity of the Dep enzymes under conditions mimicking industrially relevant conditions to test their functionality for use in areas such as corn milling, fuel ethanol fermentation or directly in animal feed.

Employing immuno-affinity for the analysis of various microbial metabolites of the mycotoxin deoxynivalenol.

Deoxynivalenol (DON) is a type B trichothecene mycotoxin that is commonly detected in grains infested with Fusarium species. The maximum tolerated levels of DON in the majority of world's countries are restricted to 0.75 mg kg-1 within the human food chain and to less than 1-5 mg kg-1 in animal feed depending on the feed material and/or animal species due to DON's short and long-term adverse effects on human health and animal productivity. The ability to accurately analyze DON and some of its fungal/bacterial metabolites is increasingly gaining a paramount importance in food/feed analysis and research. In this study, we used the immuno-affinity approach to enrich and detect DON and three of its bacterial metabolites, namely 3-epi-DON, 3-keto-DON, and deepoxy-DON (DOM-1). The optimized enrichment step coupled with high performance liquid chromatography can accurately and reproducibly quantify the aforementioned metabolites in feed matrixes (silage extract as an example in this case). It minimizes any background interface and provides a fast and easy-to-operate protocol for the analytical determination of such metabolites. More importantly, the presented data demonstrates the ability of the utilized monoclonal antibody, generated originally to capture DON in Enzyme-Linked Immunosorbent Assays (ELISA), to cross react with three less/non-toxic DON metabolites. This raises the concerns about the genuine need to account for such cross-reactivity when DON contamination is assessed through an immuno-affinity based analyses using the investigated antibody.

Promising Detoxification Strategies to Mitigate Mycotoxins in Food and Feed.

Mycotoxins are secondary fungal metabolites associated with adverse human health and animal productivity consequences.[...].

An Efficient Gas Chromatography-Mass Spectrometry Approach for the Simultaneous Analysis of Deoxynivalenol and Its Bacterial Metabolites 3-keto-DON and 3- epi-DON.

Deoxynivalenol (DON) is one of the major toxic secondary metabolites produced by Fusarium fungi in cereal grains. Among the many promising strategies of DON detoxification are the microbial and enzymatic ones, which transform DON to nontoxic DON metabolites. Thus, proper analytical methods are needed for those DON metabolites. In this study, a robust gas chromatography-mass spectrometry (GC-MS) procedure was developed and validated for the simultaneous analysis of DON and two of its bacterial metabolites, 3-keto-DON and 3- epi-DON. The procedure involves a straightforward vacuum drying and derivatization step before the subsequent GC-MS analysis. Following the optimized protocol, DON and these two metabolites were separated on a capillary column within 15 min. The linear ranges for the these compounds were 10 to 2,000 ng mL-1 with correlation coefficients >0.99. For DON, 3- epi-DON, and 3-keto-DON, the limits of detection were 0.8, 3.0, and 0.05 ng mL-1, and the limits of quantification were 2.6, 10.0, and 1.0 ng mL-1, respectively. For all three compounds, the obtained relative standard deviation was 1.2 to 5.5%, and the recovery rates were 89.5 to 103.6%. The developed method was further validated by analyzing DON metabolites resulting from the biotransformation of DON initiated by cell-free lysates of the bacterium Devosia mutans 17-2-E-8. The developed protocol was sensitive, precise, accurate, and robust for the determination of DON, 3- epi-DON, and 3-keto-DON in liquid media and potentially other complex matrices without interference from other compounds.

The enzymatic detoxification of the mycotoxin deoxynivalenol: identification of DepA from the DON epimerization pathway.

The biological detoxification of mycotoxins, including deoxynivalenol (DON), represents a very promising approach to address the challenging problem of cereal grain contamination. The recent discovery of Devosia mutans 17-2-E-8 (Devosia spp. 17-2-E-8), a bacterial isolate capable of transforming DON to the non-toxic stereoisomer 3-epi-deoxynivalenol, along with earlier reports of bacterial species capable of oxidizing DON to 3-keto-DON, has generated interest in the possible mechanism and enzyme(s) involved. An understanding of these details could pave the way for novel strategies to manage this widely present toxin. It was previously shown that DON epimerization proceeds through a two-step biocatalysis. Significantly, this report describes the identification of the first enzymatic step in this pathway. The enzyme, a dehydrogenase responsible for the selective oxidation of DON at the C3 position, was shown to readily convert DON to 3-keto-DON, a less toxic intermediate in the DON epimerization pathway. Furthermore, this study provides insights into the PQQ dependence of the enzyme. This enzyme may be part of a feasible strategy for DON mitigation within the near future.

Optimizing the Maximum Recovery of Dihydromyricetin from Chinese Vine Tea, Ampelopsis grossedentata, Using Response Surface Methodology.

This work provides an optimized extraction approach intended to maximize the recovery of dihydromyricetin (DHM) from Chinese vine tea (Ampelopsis grossedentata) leaves. The presented work adopts a Box-Behnken design as a response surface methodology to understand the role and influence of specific extraction parameters including: time, temperature, and solvent composition/ethanol (%) on DHM final yields. Initially, single factor experiments were used to delineate the role of above factors (temperature, time, and solvent composition) before proceeding with three factors-three levels Box-Behnken design with 17 separate runs to assess the effect of multifactorial treatments on DHM recovery rates. The collected data shows that independent variables (solvent composition, time, and temperature) can significantly affect DHM recovery rates with maximum yields resulting from a combined 60 °C, 60% aqueous ethanol, and 180 min treatment. From the empirical point of view, the above optimized extraction protocol can substantially enhance processing and profitability margins with a minimum need of interventions or associated costs.

The enzymatic epimerization of deoxynivalenol by Devosia mutans proceeds through the formation of 3-keto-DON intermediate.

The enzymatic detoxification of deoxynivalenol (DON) is a promising mitigation strategy for addressing this mycotoxin contamination of cereal grains. A recently described bacterium, Devosia mutans 17-2-E-8, capable of transforming DON into its non-toxic stereoisomer 3-epi-DON, holds promise for the development of such applications. Earlier observations suggested that DON epimerization proceeds via a two-step catalysis with 3-keto-DON as an intermediate. The results of this study indicate that NADPH is required for DON epimerization by cell-free protein extracts of D. mutans, while high concentrations of glucose and sucrose have a suppressive effect. Chemically synthesized 3-keto-DON incubated with D. mutans protein fractions enriched by ammonium sulfate precipitation at 35-55% saturation selectively reduced 3-keto-DON to 3-epi-DON, but fell short of supporting the complete epimerization of DON. In addition, seven Devosia species investigated for DON epimerization were all able to reduce 3-keto-DON to 3-epi-DON, but only a few were capable of epimerizing DON. The above observations collectively confirm that the enzymes responsible for the oxidation of DON to 3-keto-DON are physically separate from those involved in 3-keto-DON reduction to 3-epi-DON. The enzymatic nature of DON epimerization suggests that the process could be used to develop genetically engineered crops or microorganisms, ultimately reducing foodborne exposure of consumers and farm animals to DON.

Bioaccessibility, bioavailability, and anti-inflammatory effects of anthocyanins from purple root vegetables using mono- and co-culture cell models.

SCOPE: Immune-inflammatory signaling and metabolic effects are the main pillars for bioactivity of anthocyanins derived from highly pigmented root vegetables. This study aims to assess the bioaccessibility and bioavailability of purple carrot and potato derived anthocyanins and the molecular mechanisms of their ability to ameliorate cellular inflammation in a mono- and co-culture cell models. METHODS AND RESULTS: An in vitro gastrointestinal model was used and demonstrated bioaccessibility of 44.62 and 71.8% for anthocyanins of purple carrot and potato, respectively. These accessible anthocyanins significantly inhibited cellular inflammation in Caco-2 cells. Intact cyanidinglycoside or petunidinglycoside (respectively from carrots and potatoes) were transported across a transmembrane cell model and detected by LC-MS/MS. Computational docking and glucose uptake analyses suggested uptake of anthocyanins was mediated by hexose transporters. Subsequent experiment using an inflamed Caco-2 BBe1/THP-1 co-culture cell model showed these transported anthocyanins inhibited IL-8 and TNF-α secretion,and expression of pro-inflammatory cytokines by blocking NF-κB, and MAPK mediated inflammatory cellular signaling cascades, but with varying degrees due to structural features. CONCLUSION: Anthocyanins from purple carrots and potatoes possess a promising anti-inflammatory effect in model gut system. They can be absorbed and act differently but are in general beneficial for inflammation-mediated diseases.

Strategies and Methodologies for Developing Microbial Detoxification Systems to Mitigate Mycotoxins.

Mycotoxins, the secondary metabolites of mycotoxigenic fungi, have been found in almost all agricultural commodities worldwide, causing enormous economic losses in livestock production and severe human health problems. Compared to traditional physical adsorption and chemical reactions, interest in biological detoxification methods that are environmentally sound, safe and highly efficient has seen a significant increase in recent years. However, researchers in this field have been facing tremendous unexpected challenges and are eager to find solutions. This review summarizes and assesses the research strategies and methodologies in each phase of the development of microbiological solutions for mycotoxin mitigation. These include screening of functional microbial consortia from natural samples, isolation and identification of single colonies with biotransformation activity, investigation of the physiological characteristics of isolated strains, identification and assessment of the toxicities of biotransformation products, purification of functional enzymes and the application of mycotoxin decontamination to feed/food production. A full understanding and appropriate application of this tool box should be helpful towards the development of novel microbiological solutions on mycotoxin detoxification.

Beyond Ribosomal Binding: The Increased Polarity and Aberrant Molecular Interactions of 3-epi-deoxynivalenol.

Deoxynivalenol (DON) is a secondary fungal metabolite and contaminant mycotoxin that is widely detected in wheat and corn products cultivated around the world. Bio-remediation methods have been extensively studied in the past two decades and promising ways to reduce DON-associated toxicities have been reported. Bacterial epimerization of DON at the C3 carbon was recently reported to induce a significant loss in the bio-toxicity of the resulting stereoisomer (3-epi-DON) in comparison to the parental compound, DON. In an earlier study, we confirmed the diminished bio-potency of 3-epi-DON using different mammalian cell lines and mouse models and mechanistically attributed it to the reduced binding of 3-epi-DON within the ribosomal peptidyl transferase center (PTC). In the current study and by inspecting the chromatographic behavior of 3-epi-DON and its molecular interactions with a well-characterized enzyme, Fusarium graminearum Tri101 acetyltransferase, we provide the evidence that the C3 carbon epimerization of DON influences its molecular interactions beyond the abrogated PTC binding.

Bacterial Epimerization as a Route for Deoxynivalenol Detoxification: the Influence of Growth and Environmental Conditions.

Deoxynivalenol (DON) is a toxic secondary metabolite produced by several Fusarium species that infest wheat and corn. Food and feed contaminated with DON pose a health risk to both humans and livestock and form a major barrier for international trade. Microbial detoxification represents an alternative approach to the physical and chemical detoxification methods of DON-contaminated grains. The present study details the characterization of a novel bacterium, Devosia mutans 17-2-E-8, that is capable of transforming DON to a non-toxic stereoisomer, 3-epi-deoxynivalenol under aerobic conditions, mild temperature (25-30°C), and neutral pH. The biotransformation takes place in the presence of rich sources of organic nitrogen and carbon without the need of DON to be the sole carbon source. The process is enzymatic in nature and endures a high detoxification capacity (3 µg DON/h/10(8) cells). The above conditions collectively suggest the possibility of utilizing the isolated bacterium as a feed treatment to address DON contamination under empirical field conditions.

Insights into the Hydrocarbon Tolerance of Two Devosia Isolates, D. chinhatensis Strain IPL18T and D. geojensis Strain BD-c194T, via Whole-Genome Sequence Analysis.

Hexachlorocyclohexane (HCH) was among the most commonly used pesticides after the Second World War. The extensive use of this hydrocarbon for almost six decades has created a contamination problem on a global scale, and bioremediation methods are being extensively explored. The reported ability of some Devosia species to grow in the presence of appreciable amounts of hydrocarbons (2,000 mg/kg of contaminated soil) is attracting closer attention. Here, we report the de novo genome assembly of two hydrocarbon-tolerating Devosia isolates, D. chinhatensis strain IPL18(T) and D. geojensis strain BD-c194(T), as a first step toward understanding the metabolic pathways involved in their environmental adaptation and tolerance toward hydrocarbons.