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Introduction

The community composition and toxigenicity of the diatom Pseudo-nitzschia in the open South Atlantic Ocean were characterized during the austral spring of 2007 by Guannel et al. (). Multiple morphological types of Pseudo-nitzschia were detected in coastal and in open-ocean waters. The toxin produced by Pseudo-nitzschia, DA, was present in at least 10 species in the South Atlantic (Gayoso, ; Reguera, ). Even though Pseudo-nitzschia sp. blooms occur in different environmental conditions, Marchetti et al. () and Wells et al., ) showed a positive correlation between these blooming events and high levels of Fe(III) and NO3-, and low levels of PO43- and SiO44-. Even more, differences in temperature, salinity, pH, high irradiance, and long-term photoperiod can affect the formation of Pseudo-nitzschia blooms (Lelong et al., ). Woods () suggested a possible photo-oxidative stress regulation on DA's higher production under high irradiance. Cellular stress produced by these conditions may favor the production of toxins during HAB events. The main objective of this opinion article is to briefly report the toxicological implications of the harmful marine phycotoxin DA and its intrinsic properties. Special focus will be made on the reported oxidative stress status in marine algae in relation to the exposure to DA. Moreover, because Fe presence is known to be implicated in oxidative stress generation, and its occurrence seems to be a fundamental factor in the production of DA, the oxidative effects of the biotoxin will be discussed in relation to its capacity to bind Fe.

Discussion

On the other hand, DA production starts in late EXP and ST phase of the Pseudo-nitzschia growth cycle (Lelong et al., ). In the late ST phase, cells are physiologically impaired by injury in the structure of the wall and in the membrane (Pan et al., ). Therefore, DA tends to be released into the culture medium, along with ROS generated by the cells (as shown by Cabrera et al., ,). Because of the short life span of ROS, this increased production could not be very effective in damaging other organisms in the aquatic community. However, the release of DA could allopathically affect (protect or injure) other species of non–toxin-producing photosynthetic microalgae. Using a highly sensitive adsorptive cathodic stripping voltammetric technique, Rue and Bruland () showed that DA forms Fe chelates with a conditional stability constant of KcondFeDA,Fe(III) = 108.7±0.5 M−1. The degree to which the DA acts as a trace metal chelator reveals that it has the potential to modify the availability of these metals in the seawater. Thus, the physiological role of DA for toxigenic Pseudo-nitzschia species may be tied closely to the acquisition of Fe in coastal waters. Among several other postulations, the formation of a Fe–DA complex could be a very attractive hypothesis to explain the increased generation of ROS both in producing and non–phycotoxin-generating species. Chemical aspects of the Fe chelators, such as Fe affinity, Fe selectivity, molecular weight, and lipophilicity, in addition to stability and redox properties of the resultant Fe complex, drastically change the ability of the Fe complex to catalyze radical generation. The presence of a catalytically active Fe–DA complex could favor lipid peroxidation and radical generation in a significative amount, by affecting the composition of the labile Fe content within the cells. This is a novel point of view that should be further explored. The capacity of the antioxidant defense in each species will be the main factor to determine the hormetic (beneficial) or the damaging effect of the triggering of the burst of oxidative species.

Discussion

However, other alternatives could be considered under changing environmental conditions that can modify DA-dependent active species generation in aquatic organisms. Hypoxia is a common condition in aquatic environments, and exposure to hypoxia followed by reoxygenation is often believed to induce oxidative stress and activation of relevant signaling molecules such as the hypoxia-inducible factor 1α. This transcription factor is a potent coordinator of acclimation processes in various stress conditions. Eutrophication and extensive algal blooms caused by anthropogenic activity (input of nutrients, fertilizers, and human waste) deplete the ocean and lake bottoms of O2, i.e., make them hypoxic. In the seas, the large hypoxic areas are also known as hypoxic dead zones, and they are expanding quickly (Conley et al., ). Hypoxic bottoms release phosphorus from the aquatic sediments, which feed the algae even further. Hypoxia has an acute effect on the benthic fauna, and if the hypoxic environments are allowed to expand further, the whole ecosystem will be disturbed (Conley et al., ; Conley, ). Recently, Borowiec and Scott () reported that hypoxia acclimation of killifish leads to adjustments in ROS homeostasis and oxidative status that do not reflect oxidative stress, but may instead be part of the suite of responses used to cope with chronic hypoxia. Thus, in situ studies could show the alterations due to other transitory changes in the environment that may occur during the HABs. Overall, the specific cellular response to the increased oxidative stress triggered by DA will be one of the important factors to allow survival of each organism that contribute to determine how the composition of the community will be affected by an increasing magnitude of the HAB season produced by the climate global change.

Abstract

Mushroom irradiation has been considered a sustainable process to generate high amounts of vitamin D2 due to the role of this vitamin for human health and of the global concerns regarding its deficient or inadequate intake. Mushrooms are also receiving increasing interest due to their nutritional and medicinal properties. However, there is still a knowledge gap regarding the effect of UV irradiation on mushroom bioactive compounds. In this study, two of the most cultivated mushroom species worldwide, Agaricus bisporus and Pleurotus ostreatus, were irradiated with UV-B, and the effect of processing was investigated on the contents of vitamin D2 as well as on antioxidant and antiglycation activities. UV irradiation increased vitamin D2 up to 57 µg/g d.w, which is an adequate level for the fortification of a number of target foods. UV irradiation decreased the antioxidant activity when measured by the Folin–Ciocalteu reagent, the 2,2-diphenyl-1-(2,4,6 trinitrophenyl) hydrazyl radical assay and the ferric ion-reducing antioxidant power assay, but did not decrease the mushroom’s ability to inhibit glycation of a target protein. These results open up a new area of investigation aimed at selecting mushroom species with high nutraceutical benefits for irradiation in order to maintain their potential properties to inhibit oxidative and glycation processes responsible for human diseases.

2.2. Mushrooms

P. ostreatus and A. bisporus were purchased from the market. The irradiation treatment was performed as described previously []. In brief, about 10 kg of body fruits were used for each mushroom type, cut into 4 mm slices, spread on an oven rack in a single layer and air-dried at 37 °C for 48 h. During the first 24 h of air-drying, the mushrooms were irradiated using two fluorescent lamps installed at 30 cm above the mushroom layer. In accordance to most of the previous studies so far performed [,], a UV-B source was chosen for the irradiation. The lamps delivered a UV-B (280–315 nm) irradiance of 0.4 W/m2. At the end of the process, about 1 kg of dried mushroom was obtained from each mushroom type and the product was grinded to a fine powder using a Thermomix TM 31 (Vorwerk Contempora S.r.l., Milan, Italy). Control P. ostreatus and A. bisporus powders were obtained by processing mushrooms with the same drying and grinding procedure without UV irradiation.

3.1. The Effect of Mushroom Irradiation on Vitamin D2 Content

Initial content of vitamin D2 was low in both commercial mushrooms considered. This result is consistent with previous studies and could be associated with the conditions used for industrial cultivation of mushrooms that generally occurs with low exposure to sunlight [,]. UV irradiation led to an increase in vitamin D2 content from 3.1 to 37 µg/g d.w. and from 0.3 to 57 µg/g d.w. in P. ostreatus and A. bisporus, respectively (). The initial contents of ergosterol were 3600 and 5700 µg/g d.w. in P. ostreatus and A. bisporus, respectively (). These values fall in the range previously reported [] and remained high after the generation of significant amounts of vitamin D2. This result may suggest that upon UV irradiation. the residual amount of ergosterol is still relevant for drug development and could contribute to the health-promoting effects of mushrooms [].

Abstract

Chronic low-grade inflammation is a primary characteristic of obesity and can lead to other metabolic complications including insulin resistance and type 2 diabetes (T2D). Several anti-inflammatory dietary bioactives decrease inflammation that accompanies metabolic diseases. We are specifically interested in delta-tocotrienol, (DT3) an isomer of vitamin E, and tart cherry anthocyanins (TCA), both of which possess individual anti-inflammatory properties. We have previously demonstrated that DT3 and TCA, individually, reduced systemic and adipose tissue inflammation in rodent models of obesity. However, whether these compounds have combinatorial effects has not been determined yet. Hence, we hypothesize that a combined treatment of DT3 and TCA will have great effects in reducing inflammation in adipocytes, and that these effects are mediated via the nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB), a major inflammatory transcription factor. We used 3T3-L1 adipocytes and treated them with 1–5 µM doses of DT3 along with tart cherry containing 18–36 µg anthocyanin/mL, to assess effects on inflammation. Neither DT3 nor TCA, nor their combinations had toxic effects on adipocytes. Furthermore, pro-inflammatory markers interleukin-6 (IL-6) and p-65 (subunit of NFkB) were reduced at the protein level in media collected from adipocytes with both individual and combined treatments. Additionally, other downstream targets of NFkB including macrophage inflammatory protein 2 (Mip2), and Cyclooxygenase-2 (Cox2) were also significantly downregulated (p ≤ 0.05) when treated with individual and combined doses of DT3 and TCA with no additional combinatorial effects. In summary, DT3 and TCA individually, are beneficial in reducing inflammation with no additional combinatorial effects.

2.2. Fatty Acid Composition

The main fatty acids in olive oil are oleic (18:1), linoleic (18:2), palmitic (16:0), and stearic acid (18:0). The fatty acid composition is a quality parameter and an authenticity indicator of virgin olive oils []. As shown in , oleic acid displayed the highest content that ranged between 76.60% (Koroneiki from Lesvos) and 71.92% (Kolovi from Lesvos). Linoleic acid ranged between 11.63% (Kolovi from Lesvos) and 5.71% (Koroneiki from Chania), while the corresponding values for palmitic acid ranged between 13.61% (Koroneiki from Chania) and 9.65% (Kolovi from Lesvos). The stearic acid content ranged between 2.76% (Koroneiki from Chania) and 2.20% (Kolovi from Lesvos). The fatty acids, such as palmitoleic (16:1), linolenic (18:3), arachidic (20:0), gadoleic (20:1), and behenic (22:0) acid, were detected in smaller amounts (<0.9%). The content of myristic (14:0), margaric (17:0), heptadecenoic (17:1), and lignoceric (24:0) acid did not exceed 0.1%.

2.3. Classification of EVOOs According to Cultivar

It is evident (a) that samples produced from the Koroneiki cultivar form a distinct group (green area), which is clearly separated from that of Kolovi (red area). The two Koroneiki samples that lie outside the confidence eclipse originated from Lesvos. The two principal components explain 67.7% of the total variance. Examining the loading plot (b), samples from the Kolovi cultivar are positively correlated with the content of γ-tocopherol as well as linoleic (18:2), linolenic (18:3), and gadoleic (20:1) acid. The remaining fatty acids (16:0, 16:1, 18:0, 18:1, 20:0, and 22:0) and squalene correlated positively with the Koroneiki EVOOs. When student’s t-test was applied, it turned out that all variables were statistically significant (p < 0.05) between the two cultivars except from α-tocopherol, implying that its content is not dependent on variety.

2.3. Classification of EVOOs According to Cultivar

The results can be better visualized using a heatmap (). γ-Tocopherol, 20:1, 18:2, and 18:3 are present at higher levels in samples from the Kolovi cultivar and lower in the Koroneiki cultivar. The rest of the components display the opposite pattern, except for α-tocopherol, which does not show a specific pattern between the two cultivars.

4.1. Olive Oil Samples

A total of 68 monovarietal EVOOs were obtained during the harvesting period of 2018–2019 (). They were produced from two cultivars (Kolovi and Koroneiki) in three different geographical regions of Greece (Crete island, Lesvos island, and Peloponnese). The samples were packaged in dark-brown glass bottles and stored at 4 °C until analysis.

Methods

In this randomized, double‐blind, placebo‐controlled clinical trial, 84 PCOS patients were randomly assigned into the treatment (90 µg Menaquinone‐7 daily for 8 weeks) and placebo groups. Insulin resistance, lipid profile, endocrine biomarkers, and body composition of the participants were measured before and after the intervention. This study was performed in Ghadir Mother & Child Hospital affiliated to Shiraz University of Medical Sciences, Shiraz, Iran.

Results

Menaquinone‐7 supplementation, when compared to placebo, significantly decreased serum fasting insulin (p = .002), homeostasis model of assessment insulin resistance (p = .002), and homeostasis model of assessment β‐cell function (p = .02) in addition to a significant increase in quantitative insulin sensitivity check index (p = .001). Also, MK‐7 administration led to significant declines in serum triglyceride (p = .003) and dihydrotestosterone (DHT; p = .03) levels, free androgen index (p < .001), waist circumference (p = .03), and body fat mass (p < .001) as well as significant increases in skeletal muscle (p < .001) and sex hormone binding globulin (SHBG, p < .001).

Study design

Polycystic ovary syndrome participants were randomized into the control and treatment arms by block randomization with fixed block size of four by means of random allocation software (Saghaei, ). The treatment group consumed one vitamin K2 capsule, containing 90 µg of Menaquinone‐7 (MK‐7), daily for 8 weeks; the dosage was administered in a previous study (Razavi et al., ). Patients in the control group received a placebo capsule (avesil) daily for 8 weeks. The MK‐7 and placebo capsules were identical in color (white), smell, size (size 0), and weight which were prepared for this study by Arian Salamat Sina Company to blind the participants, researchers, and the Infertility Clinic staff to group allocation (Fermented soybean called natto was the food source of MK‐7 used in this study). A booklet containing PCOS dietary recommendation was given to both groups. The participants were requested not to change their regular physical activity level and diet and in the course of study.

Abstract

Chemically synthesized retinyl palmitate has been widely used in the cosmetic and biotechnology industry. In this study, we aimed to demonstrate the microbial production of retinyl palmitate and the benefits of microbial retinyl palmitate in skin physiology. A heterologous retinyl palmitate biosynthesis pathway was reconstructed in metabolically engineered Escherichia coli using synthetic expression modules from Pantoea agglomerans, Salinibacter ruber, and Homo sapiens. High production of retinyl palmitate (69.96 ± 2.64 mg/L) was obtained using a fed-batch fermentation process. Moreover, application of purified microbial retinyl palmitate to human foreskin HS68 fibroblasts led to increased cellular retinoic acid-binding protein 2 (CRABP2) mRNA level [1.7-fold (p = 0.001) at 100 μg/mL], acceleration of cell proliferation, and enhancement of procollagen synthesis [111% (p < 0.05) at 100 μg/mL], strongly indicating an anti-ageing-related effect of this substance. These results would pave the way for large-scale production of retinyl palmitate in microbial systems and represent the first evidence for the application of microbial retinyl palmitate as a cosmeceutical.

Abstract

Chemically synthesized retinyl palmitate has been widely used in the cosmetic and biotechnology industry. In this study, we aimed to demonstrate the microbial production of retinyl palmitate and the benefits of microbial retinyl palmitate in skin physiology. A heterologous retinyl palmitate biosynthesis pathway was reconstructed in metabolically engineered Escherichia coli using synthetic expression modules from Pantoea agglomerans, Salinibacter ruber, and Homo sapiens. High production of retinyl palmitate (69.96 ± 2.64 mg/L) was obtained using a fed-batch fermentation process. Moreover, application of purified microbial retinyl palmitate to human foreskin HS68 fibroblasts led to increased cellular retinoic acid-binding protein 2 (CRABP2) mRNA level [1.7-fold (p = 0.001) at 100 μg/mL], acceleration of cell proliferation, and enhancement of procollagen synthesis [111% (p < 0.05) at 100 μg/mL], strongly indicating an anti-ageing-related effect of this substance. These results would pave the way for large-scale production of retinyl palmitate in microbial systems and represent the first evidence for the application of microbial retinyl palmitate as a cosmeceutical.

1. Introduction

In this study, we aimed to reconstruct a retinyl palmitate biosynthesis pathway in a β-carotene-overproducing E. coli strain engineered for enhancement of isoprenyl pyrophosphate and β-carotene biosynthesis. High-yield retinyl palmitate was obtained using fed-batch fermentation of retinyl palmitate-producing E. coli. Moreover, we evaluated the anti-ageing-related activity of the microbial retinyl palmitate purified from the engineered E. coli in human foreskin fibroblasts.

Abstract

The synthetic properties of the Thiamine diphosphate (ThDP)-dependent pyruvate dehydrogenase E1 subunit from Escherichia coli (EcPDH E1) was assessed for carboligation reactions with aliphatic ketoacids. Due to its role in metabolism, EcPDH E1 was previously characterised with respect to its biochemical properties, but it was never applied for synthetic purposes. Here, we show that EcPDH E1 is a promising biocatalyst for the production of chiral α-hydroxyketones. WT EcPDH E1 shows a 180–250-fold higher catalytic efficiency towards 2-oxobutyrate or pyruvate, respectively, in comparison to engineered transketolase variants from Geobacillus stearothermophilus (TKGST). Its broad active site cleft allows for the efficient conversion of both (R)- and (S)-configured α-hydroxyaldehydes, next to linear and branched aliphatic aldehydes as acceptor substrates under kinetically controlled conditions. The alternate, thermodynamically controlled self-reaction of aliphatic aldehydes was shown to be limited to low levels of conversion, which we propose to be due to their large hydration constants. Additionally, the thermodynamically controlled approach was demonstrated to suffer from a loss of stereoselectivity, which makes it unfeasible for aliphatic substrates.

2.1.2. Optimisation of Reaction Conditions

A spectrophotometric assay utilising 2,6-dichloroindophenol (DCPIP) and pyruvate as substrates () was initially used to identify optimal conditions for EcPDH E1 catalysed reactions []. The reduction in DCPIP was followed at its isosbestic point (517 nm, ) to allow for the determination of the optimal pH. E1 retained its activity over a broad pH range from pH 5.5 to 9.5, with its highest activity at pH 7.5 (a). The enzyme showed good thermal stability with no loss of activity after two hours of incubation at 40 °C. However, higher temperatures swiftly lead to its complete inactivation (b). Notably, EcPDH E1 can be stored with no loss of activity for at least six months at −20 °C (5 mg/mL EcPDH E1 in 20 mM potassium phosphate buffer, pH 7.0).

2.2. AUCs for (6S)-5-Methyl-THF-Ca Versus Folic Acid and Methods of Indirect Comparisons with the AUCs for (6S)-5-Methyl-THF-Na

Prinz-Langenohl et al. (2009) compared the AUC0–8 h of plasma total folate between (6S)-5-Methyl-THF-Ca and folic acid in 24 young women []. shows a brief summary of the study design compared to the present study. Equimolar oral doses of (6S)-5-Methyl-THF-Na (436 µg, present study) and (6S)-5-Methyl-THF-Ca (451 µg, in Prinz-Langenohl et al.) have been tested against the same reference substance, folic acid (400 µg). Plasma concentrations of (6S)-5-Methyl-THF were selectively measured in both studies. In the study by Prinz-Langenohl et al., 2009 [], the measurements of plasma (6S)-5-Methyl-THF were conducted at VU University Medical Centre, Department of Clinical Chemistry, Amsterdam (The Netherlands), by using liquid chromatography tandem mass-spectrometer (LC-MS-MS system) using 13C5-(6S)-5-Methyl-THF as internal standard []. We here show novel data on the AUC0–8 h of plasma (6S)-5-Methyl-THF from the original study [].

2.2. Synthesis

To first synthesize tungsten-doped VEG (W-VEG), NH4VO3 (1.17 g, 10 mmol), 1-dodecanol (33.17 g, 17.8 mmol), and EG (11.05 g, 17.8 mmol) were added to a three-necked round-bottom flask and mixed with stirring. To this mixture solution, a pre-determined amount of WOCl4 (0.094 g [0.277 mmol, 2.77 mol% to NH4VO3], 0.188 g [0.553 mmol, 5.53 mol%), and 0.377 g [1.109 mmol, 11.08 mol%]) was added, and the resulting reaction mixture was heated to 160 °C at a ramping rate of 5 °C/min and stirred for 2 h. Then, the mixture with a purple color was naturally cooled down to room temperature. The precipitate was collected by centrifuging the solution at 8000 rpm for 15 min to remove the excess 1-dodecanol and EG. The collected purple powder was washed with ethanol, filtered to obtain W-VEG powder, and dried in a vacuum for 24 h at room temperature.

2. Vitamin K Structure, Source, Cycle, Metabolism

Vitamin K is the term used to name a family of fat-soluble compounds, which although differ in origin and/or function, share a common 2-methyl-1,4-naphthoquinone ring, but differ in the (lipophilic) side chains linked at the 3-position. The three main forms are vitamin PK or phylloquinone (PK), vitamin K2 or menaquinones (MKn) and vitamin K3 or menadione. These three forms can be differentiated by the 3-position: PK has an isoprenoid side chain, whereas the MK form possesses a phytyl side chain and is characterized by a variable number of connected isoprenoid units (MKn), and menadione has no side chain and is a synthetic analogue [].

2. Vitamin K Structure, Source, Cycle, Metabolism

The sources of vitamin K are different depending on the vitamers. PK can be found mainly in green leafy vegetables (e.g., kale), vegetables in the Brassica genus (e.g., Brussels sprouts, broccoli), fruits (e.g., avocado, kiwi, and green grapes), herbs (e.g., cilantro, parsley), and green and herbal teas. Other dietary sources are plant oils such as soybean, canola, and olive oils [].

4. Vitamin K Recommended Intake, Deficiency, and Assessment

The recommended daily intake (RDI) or adequate intake (AI) of vitamin K is aimed at ensuring normal blood coagulation []. There is some variability of these recommended target values across various organizations. The National Academy of Medicine in the US stated the AI of vitamin PK at 120 μg/day for adult men and 90 μg/day for adult women []. The World Health Organization and the Food and Agriculture Organization recommended dosages for vitamin PK at 65 μg/day for men and 55 μg/day for women, based on a calculated requirement of 1 μg/day/kg body weight []. Finally, the European Commission has established a recommended daily allowance (RDA) for vitamin K at 75 μg/day []. In 2012, the Italian LARN (Reference Assumption Levels for Nutrients and Energy), proposed by the Human Nutrition Italian Society (SINU) suggested an intake of vitamin K stratified for age (140 or 170 μg/day for 18–59 and >60 years old, respectively) [].

2. Vitamin K Structure, Source, Cycle, Metabolism

Vitamin K acts as a cofactor for a single microsomal enzyme, namely carboxyglutamyl carboxylase (GGCX) which catalyzes the carboxylation (and thereby activation) of vitamin-K-dependent proteins (VKDPs) []. Vitamin-K-dependent carboxylase catalyzes the posttranslational γ-carboxylation of glutamic acid (Glu) residues, placed in vitamin K-dependent proteins (also known as Gla proteins), to γ-carboxylated glutamic acid (Gla) residues. Among the Gla protein family, 17 different members have been recongnized: S prothrombin, factor VII, factor IX, factor X, protein C, protein S and protein Z belonging to the coagulative cascade; (matrix Gla protein,(MGP), Osteocalcin (OC), growth arrest-specific protein 6 (Gas6), Gla-rich protein (GRP) playing a role in modulating bone and vascular mineralization; periostin and periostin-like factor, two proline-rich Gla proteins, and two transmembrane Gla proteins. Vitamin K was previously considered only an essential factor for blood coagulation [].

2. Vitamin K Structure, Source, Cycle, Metabolism

The involvement of vitamin K in bone metabolism does not occur exclusively by the γ-carboxylation reaction. MK4, binds to the nuclear receptor, steroid, and xenobiotic receptor (SXR) and its murine ortholog, pregnane X receptor (PXR), a nuclear receptor whose primary function is to regulate the expression of genes encoding enzymes involved in steroid metabolism and detoxification of xenobiotics and various drugs [,,]. Vitamin PK is not able to activate SXR directly, suggesting vitamin PK potentially contribute to this mechanism of action after being converted into MK-4 [] ().