Anti-Obesity and Anti-Adipogenic Effects of Administration of Arginyl-Fructose-Enriched Jeju Barley (Hordeum vulgare L.) Extract in C57BL/6 Mice and in 3T3-L1 Preadipocytes Models.

In our previous study, we reported that arginyl-fructose (AF), one of the Amadori rearrangement compounds (ARCs) produced by the heat processing of Korean ginseng can reduce carbohydrate absorption by inhibiting intestinal carbohydrate hydrolyzing enzymes in both in vitro and in vivo animal models. This reduced absorption of carbohydrate might be helpful to control body weight gain due to excessive carbohydrate consumption and support induced calorie restriction. However, the weight management effect, except for the effect due to anti-hyperglycemic action, along with the potential mechanism of action have not yet been determined. Therefore, the efforts of this study are to investigate and understand the possible weight management effect and mechanism action of AF-enriched barley extracts (BEE). More specifically, the effect of BEE on lipid accumulation and adipogenic gene expression, body weight gain, body weight, plasma lipids, body fat mass, and lipid deposition were evaluated using C57BL/6 mice and 3T3-L1 preadipocytes models. The formation of lipid droplets in the 3T3-L1 treated with BEE (500 and 750 µg/mL) was significantly blocked (p < 0.05 and p < 0.01, respectively). Male C57BL/6 mice were fed a high-fat diet (30% fat) for 8 weeks with BEE (0.3 g/kg-body weight). Compared to the high fat diet control (HFD) group, the cells treated with BEE significantly decreased in intracellular lipid accumulation with concomitant decreases in the expression of key transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (CEBP/α), the mRNA expression of downstream lipogenic target genes such as fatty acid binding protein 4 (FABP4), fatty acid synthase (FAS), and sterol regulatory element-binding protein 1c (SREBP-1c). Supplementation of BEE effectively lowered the body weight gain, visceral fat accumulation, and plasma lipid concentrations. Compared to the HFD group, BEE significantly suppressed body weight gain (16.06 ± 2.44 g vs. 9.40 ± 1.39 g, p < 0.01) and increased serum adiponectin levels, significantly, 1.6-folder higher than the control group. These results indicate that AF-enriched barley extracts may prevent diet-induced weight gain and the anti-obesity effect is mediated in part by inhibiting adipogenesis and increasing adiponectin level.

Calcium fructoborate regulate colon cancer (Caco-2) cytotoxicity through modulation of apoptosis.

Sugar-borate esters have recently been reported to have anti-cancer potential. Among the sugar-borate esters, calcium fructoborate (CaFB) possesses beneficial effects on human health. Despite the beneficial effects of CaFB, there is a lack of knowledge about their mode of action in cancer. The potential cytotoxic effects of CaFB were investigated on colon cancer cells (Caco-2). The mode of action was determined through the evaluation of Fyn and Hck expression levels together with Bcl-2, Bax, and PI3K/Akt pathway proteins. CaFB treatment was found to be most effective on Caco-2 cells at 10 mM concentration for 24 h. Decreased Bcl-2 levels and increased Bax levels at 10 mM were evaluated as an indicator of apoptotic effects of CaFB. Akt, p70S6K, and 4EBP1 levels, in general, tend to decrease following CaFB, while PTEN and TSC2 levels have been found to increase. Furthermore, CaFB upregulated Hck expression and downregulated Fyn expression. In conclusion, our results indicated that CaFB treatment at 10 mM concentration, the IC50 dose found in our study, might prevent colon cancer cell proliferation both by inducing apoptosis and presumably by activating autophagy.

Potential roles of 1,5-anhydro-D-fructose in modulating gut microbiome in mice.

The gut microbiota has tremendous potential to affect the host's health, in part by synthesizing vitamins and generating nutrients from food that is otherwise indigestible by the host. 1,5-Anhydro-D-fructose (1,5-AF) is a monosaccharide with a wide range of bioactive potentials, including anti-oxidant, anti-inflammatory, and anti-microbial effects. Based on its potential benefits and minimal toxicity, it is anticipated that 1,5-AF will be used as a dietary supplement to support general health. However, the effects of 1,5-AF on the gut microbiota are yet to be clarified. Here, using an unbiased metagenomic approach, we profiled the bacterial taxa and functional genes in the caecal microbiota of mice fed a diet containing either 2% 1,5-AF or a reference sweetener. Supplementation with 1,5-AF altered the composition of the gut microbiota, enriching the proportion of Faecalibacterium prausnitzii. 1,5-AF also altered the metabolomic profile of the gut microbiota, enriching genes associated with nicotinamide adenine dinucleotide biosynthesis. These findings support the potential benefits of 1,5-AF, but further studies are required to clarify the impact of 1,5-AF on health and disease.

1,5-Anhydro-D-fructose Protects against Rotenone-Induced Neuronal Damage In Vitro through Mitochondrial Biogenesis.

Mitochondrial functional abnormalities or quantitative decreases are considered to be one of the most plausible pathogenic mechanisms of Parkinson's disease (PD). Thus, mitochondrial complex inhibitors are often used for the development of experimental PD. In this study, we used rotenone to create in vitro cell models of PD, then used these models to investigate the effects of 1,5-anhydro-D-fructose (1,5-AF), a monosaccharide with protective effects against a range of cytotoxic substances. Subsequently, we investigated the possible mechanisms of these protective effects in PC12 cells. The protection of 1,5-AF against rotenone-induced cytotoxicity was confirmed by increased cell viability and longer dendritic lengths in PC12 and primary neuronal cells. Furthermore, in rotenone-treated PC12 cells, 1,5-AF upregulated peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) expression and enhanced its deacetylation, while increasing AMP-activated protein kinase (AMPK) phosphorylation. 1,5-AF treatment also increased mitochondrial activity in these cells. Moreover, PGC-1α silencing inhibited the cytoprotective and mitochondrial biogenic effects of 1,5-AF in PC12 cells. Therefore, 1,5-AF may activate PGC-1α through AMPK activation, thus leading to mitochondrial biogenic and cytoprotective effects. Together, our results suggest that 1,5-AF has therapeutic potential for development as a treatment for PD.

Metabolic engineering of Pseudomonas putida for production of the natural sweetener 5-ketofructose from fructose or sucrose by periplasmic oxidation with a heterologous fructose dehydrogenase.

5-Ketofructose (5-KF) is a promising low-calorie natural sweetener with the potential to reduce health problems caused by excessive sugar consumption. It is formed by periplasmic oxidation of fructose by fructose dehydrogenase (Fdh) of Gluconobacter japonicus, a membrane-bound three-subunit enzyme containing FAD and three haemes c as prosthetic groups. This study aimed at establishing Pseudomonas putida KT2440 as a new cell factory for 5-KF production, as this host offers a number of advantages compared with the established host Gluconobacter oxydans. Genomic expression of the fdhSCL genes from G. japonicus enabled synthesis of functional Fdh in P. putida and successful oxidation of fructose to 5-KF. In a batch fermentation, 129 g l-1 5-KF were formed from 150 g l-1 fructose within 23 h, corresponding to a space-time yield of 5.6 g l-1 h-1 . Besides fructose, also sucrose could be used as substrate for 5-KF production by plasmid-based expression of the invertase gene inv1417 from G. japonicus. In a bioreactor cultivation with pulsed sucrose feeding, 144 g 5-KF were produced from 358 g sucrose within 48 h. These results demonstrate that P. putida is an attractive host for 5-KF production.

Immune Modulatory Activities of Arginyl-Fructose (AF) and AF-Enriched Natural Products in In-Vitro and In-Vivo Animal Models.

The immune system plays an important role in maintaining body homeostasis. Recent studies on the immune-enhancing effects of ginseng saponins have revealed more diverse mechanisms of action. Maillard reaction that occurs during the manufacturing processes of red ginseng produces a large amount of Amadori rearrangement compounds (ARCs), such as arginyl-fructose (AF). The antioxidant and anti-hyperglycemic effects of AF have been reported. However, the possible immune enhancing effects of non-saponin ginseng compounds, such as AF, have not been investigated. In this study the effects of AF and AF-enriched natural product (Ginofos, GF) on proliferation of normal mouse splenocytes were evaluated in vitro and male BALB/c mice models. The proliferation of splenocytes treated with mitogens (concanavalin A, lipopolysaccharide) were further increased by addition of AF (p < 0.01) or GF (p < 0.01), in a dose dependent manner. After the 10 days of oral administration of compounds, changes in weights of spleen and thymus, serum immunoglobulin, and expression of cytokines were measured as biomarkers of immune-enhancing potential in male BALB/c mice model. The AF or GF treated groups had higher weights of the thymus (0.94 ± 0.25 and 0.86 ± 0.18, p < 0.05, respectively) than that of cyclophosphamide treated group (0.59 ± 0.18). This result indicates that AF or AF-enriched extract (GF) increased humoral immunity against CY-induced immunosuppression. In addition, immunoglobulin contents and expression of cytokines including IgM (p < 0.01), IgG (p < 0.05), IL-2 (p < 0.01), IL-4 (p < 0.01), IL-6 (p < 0.01), and IFN-γ (p < 0.05) were also significantly increased by supplementation of AF or GF. These results indicate that AF has immune enhancing effects by activation of adaptive immunity via increase of expression of immunoglobulins and cytokines such as IgM, IgG, IL-2, IL-4, IL-6 and thereby proliferating the weight of thymus. Our findings provide a pharmacological rationale for AF-enriched natural products such as ginseng and red ginseng that can possibly have immune-enhancement potential and should be further evaluated.

Degradation of the low-calorie sugar substitute 5-ketofructose by different bacteria.

There is an increasing public awareness about the danger of dietary sugars with respect to their caloric contribution to the diet and the rise of overweight throughout the world. Therefore, low-calorie sugar substitutes are of high interest to replace sugar in foods and beverages. A promising alternative to natural sugars and artificial sweeteners is the fructose derivative 5-keto-D-fructose (5-KF), which is produced by several Gluconobacter species. A prerequisite before 5-KF can be used as a sweetener is to test whether the compound is degradable by microorganisms and whether it is metabolized by the human microbiota. We identified different environmental bacteria (Tatumella morbirosei, Gluconobacter japonicus LMG 26773, Gluconobacter japonicus LMG 1281, and Clostridium pasteurianum) that were able to grow with 5-KF as a substrate. Furthermore, Gluconobacter oxydans 621H could use 5-KF as a carbon and energy source in the stationary growth phase. The enzymes involved in the utilization of 5-KF were heterologously overproduced in Escherichia coli, purified and characterized. The enzymes were referred to as 5-KF reductases and belong to three unrelated enzymatic classes with highly different amino acid sequences, activities, and structural properties. Furthermore, we could show that 15 members of the most common and abundant intestinal bacteria cannot degrade 5-KF, indicating that this sugar derivative is not a suitable growth substrate for prokaryotes in the human intestine. KEY POINTS: • Some environmental bacteria are able to use 5-KF as an energy and carbon source. • Four 5-KF reductases were identified, belonging to three different protein families. • Many gut bacteria cannot degrade 5-KF.

Long-term effects of weight-reducing drugs in people with hypertension.

BACKGROUND: This is the third update of this review, first published in July 2009. All major guidelines on treatment of hypertension recommend weight loss; anti-obesity drugs may be able to help in this respect. OBJECTIVES: Primary objectives: To assess the long-term effects of pharmacologically-induced reduction in body weight in adults with essential hypertension on all-cause mortality, cardiovascular morbidity, and adverse events (including total serious adverse events, withdrawal due to adverse events, and total non-serious adverse events).. Secondary objectives: To assess the long-term effects of pharmacologically-induced reduction in body weight in adults with essential hypertension on change from baseline in systolic and diastolic blood pressure, and on body weight reduction. SEARCH METHODS: For this updated review, the Cochrane Hypertension Information Specialist searched the following databases for randomised controlled trials up to March 2020: the Cochrane Hypertension Specialised Register, CENTRAL, MEDLINE (from 1946), Embase (from 1974), the World Health Organization International Clinical Trials Registry Platform, and ClinicalTrials.gov. The searches had no language restrictions. We contacted authors of relevant papers about further published and unpublished work. SELECTION CRITERIA: Randomised controlled trials of at least 24 weeks' duration in adults with hypertension that compared approved long-term weight-loss medications to placebo.  DATA COLLECTION AND ANALYSIS: Two review authors independently selected studies, assessed risks of bias, and extracted data. Where appropriate and in the absence of significant heterogeneity between studies (P > 0.1), we pooled studies using a fixed-effect meta-analysis. When heterogeneity was present, we used the random-effects method and investigated the cause of the heterogeneity. MAIN RESULTS: This third update of the review added one new trial, investigating the combination of naltrexone/bupropion versus placebo. Two medications, which were included in the previous versions of this review (rimonabant and sibutramine) are no longer considered relevant for this update, since their marketing approval was withdrawn in 2010 and 2009, respectively. The number of included studies in this review update is therefore six (12,724 participants in total): four RCTs comparing orlistat to placebo, involving a total of 3132 participants with high blood pressure and a mean age of 46 to 55 years; one trial comparing phentermine/topiramate to placebo, involving 1305 participants with high blood pressure and a mean age of 53 years; and one trial comparing naltrexone/bupropion to placebo, involving 8283 participants with hypertension and a mean age of 62 years. We judged the risks of bias to be unclear for the trials investigating orlistat or naltrexone/bupropion. and low for the trial investigating phentermine/topiramate. Only the study of naltrexone/bupropion included cardiovascular mortality and morbidity as predefined outcomes. There were no differences in the rates of all-cause or cardiovascular mortality, major cardiovascular events, or serious adverse events between naltrexone/bupropion and placebo. The incidence of overall adverse events was significantly higher in participants treated with naltrexone/bupropion. For orlistat, the incidence of gastrointestinal side effects was consistently higher compared to placebo. The most frequent side effects with phentermine/topiramate were dry mouth and paraesthesia. After six to 12 months, orlistat reduced systolic blood pressure compared to placebo by mean difference (MD) -2.6 mm Hg (95% confidence interval (CI) -3.8 to -1.4 mm Hg; 4 trials, 2058 participants) and diastolic blood pressure by MD -2.0 mm Hg (95% CI -2.7 to -1.2 mm Hg; 4 trials, 2058 participants). After 13 months of follow-up, phentermine/topiramate decreased systolic blood pressure compared to placebo by -2.0 to -4.2 mm Hg (1 trial, 1030 participants) (depending on drug dosage), and diastolic blood pressure by -1.3 to -1.9 mm Hg (1 trial, 1030 participants) (depending on drug dosage). There was no difference in the change in systolic or diastolic blood pressure between naltrexone/bupropion and placebo (1 trial, 8283 participants). We identified no relevant studies investigating liraglutide or lorcaserin in people with hypertension. AUTHORS' CONCLUSIONS: In people with elevated blood pressure, orlistat, phentermine/topiramate and naltrexone/bupropion reduced body weight; the magnitude of the effect was greatest with phentermine/topiramate. In the same trials, orlistat and phentermine/topiramate, but not naltrexone/bupropion, reduced blood pressure. One RCT of naltrexone/bupropion versus placebo showed no differences in all-cause mortality or cardiovascular mortality or morbidity after two years. The European Medicines Agency refused marketing authorisation for phentermine/topiramate due to safety concerns, while for lorcaserin the application for European marketing authorisation was withdrawn due to a negative overall benefit/risk balance. In 2020 lorcaserin was also withdrawn from the US market. Two other medications (rimonabant and sibutramine) had already been withdrawn from the market in 2009 and 2010, respectively.

Calcium Fructoborate Prevents Skin Cancer Development in Balb-c Mice: Next Part, Reverse Inflammation, and Metabolic Alteration.

Metabolic alterations and inflammation are regarded as hallmarks of cancer. Glycolytic flux and intermediate accumulation lead to the production of building blocks and NADPH which is important in protecting the cell from oxidative damage. Inflammation causes the release of mediators responsible for regulating molecular mechanism affecting metabolic pathways. CaFB due to its cis-diol-rich feature may have the potential to interact with molecules taking part in cancer development. This study was aimed to investigate the effects of CaFB on metabolic alterations and inflammation in 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin cancer. For this purpose, 92 Balb-c mice were distributed into 6 groups as control, CaFB, DMBA/TPA (D-T), treatment 1 (T1), 2 (T2), and 3(T3). Apart from control and CaFB in other groups, tumors initiated with 97.5-nmol DMBA and 6.5-nmol TPA. Treatment groups received 3 mg/kg/day CaFB with DMBA (T1), with TPA (T2), and after tumor formation (T3). In the D-T group, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, 6-phosphogluconate dehydrogenase (PGD), glutathione (GSH), interleukin 6 (IL-6), (IL-1ß), tumor necrosis factor-α (TNF-α) levels increased (p < 0.001) while malondialdehyde (MDA) levels decreased (p < 0.001) compared with that in control. CaFB application ameliorated DMBA-TPA effect according to the distribution time. It is noteworthy to consider CaFB as a potential preventive agent in skin cancer development.

Fructosan form Paenibacillus kribbensis PS04 enhance disease resistance against Rhizoctonia solani and tobacco mosaic virus

BACKGROUND: Rice sheath blight (caused by Rhizoctonia solani) and tobacco mosaic virus are very important plant diseases, causing a huge loss in global crop production. Paenibacillus kribbensis PS04 is a broad-spectrum biocontrol agent, used for controlling these diseases. Previously, extracellular polysaccharides (EPS) from P. kribbensis PS04 had been purified and their structure was inferred to be fructosan. This study aimed to evaluate the effects of exogenous EPS treatment on plant­pathogen interactions. RESULTS: Plant defense genes such as phenylalanine ammonia-lyase, catalase, chitinase, allene oxide synthase, and PR1a proteins were significantly induced by exogenous EPS treatment. Moreover, subsequent challenge of EPSpretreated plants with the pathogens (R. solani or tobacco mosaic virus) resulted in higher expression of defenseassociated genes. Increased activities of defense-associated enzymes, total phenols, and flavonoids were also observed in EPS pretreated plants. The contents of malondialdehyde in plants, which act as indicator of lipid peroxidation, were reduced by EPS treatment. CONCLUSIONS: This study comprehensively showed that EPS produced from P. kribbensis PS04 enhances disease resistance in plants by the activation of defense-associated genes as well as through the enhancement of activities of defense-related enzymes.

Biodistribution studies of boronophenylalanine in different types of skin melanoma.

A study of the 10B-enriched Boronophenylalanine-fructose complex(10BPA-F) infusion procedure in potential BNCT patients, including three skin melanomas of extremities, was performed. 10B concentration in tumor(T), blood(B), skin(S) were measured to determine tumor/blood(T/B) and skin/blood(S/B) ratios. T/B ratio for three melanoma patients was in the range 1.48-3.82(average 2.56 ± 0.69). S/B ratio was in the range 0.81-1.99(average 1.29 ± 0.35). Results showed that T/B ratio of nodular metastasis melanoma was higher than superficial spreading melanoma. 10B concentration in skin was higher than blood, which was helpful to avoid over-dose in normal skin.

5-Keto-D-fructose production from sugar alcohol by isolated wild strain Gluconobacter frateurii CHM 43.

GLUCONOBACTER FRATEURII: CHM 43 have D-mannitol dehydrogenase (quinoprotein glycerol dehydrogenase) and flavoprotein D-fructose dehydrogenase in the membranes. When the two enzymes are functional, D-mannitol is converted to 5-keto-D-fructose with 65% yield when cultivated on D-mannitol. 5-Keto-D-fructose production with almost 100% yield was realized with the resting cells. The method proposed here should give a smart strategy for 5-keto-D-fructose production.

The genetic and functional analysis of flavor in commercial tomato: the FLORAL4 gene underlies a QTL for floral aroma volatiles in tomato fruit.

Tomato (Solanum lycopersicum L.) has become a popular model for genetic studies of fruit flavor in the last two decades. In this article we present a study of tomato fruit flavor, including an analysis of the genetic, metabolic and sensorial variation of a collection of contemporary commercial glasshouse tomato cultivars, followed by a validation of the associations found by quantitative trait locus (QTL) analysis of representative biparental segregating populations. This led to the identification of the major sensorial and chemical components determining fruit flavor variation and detection of the underlying QTLs. The high representation of QTL haplotypes in the breeders' germplasm suggests that there is great potential for applying these QTLs in current breeding programs aimed at improving tomato flavor. A QTL on chromosome 4 was found to affect the levels of the phenylalanine-derived volatiles (PHEVs) 2-phenylethanol, phenylacetaldehyde and 1-nitro-2-phenylethane. Fruits of near-isogenic lines contrasting for this locus and in the composition of PHEVs significantly differed in the perception of fruity and rose-hip-like aroma. The PHEV locus was fine mapped, which allowed for the identification of FLORAL4 as a candidate gene for PHEV regulation. Using a gene-editing-based (CRISPR-CAS9) reverse-genetics approach, FLORAL4 was demonstrated to be the key factor in this QTL affecting PHEV accumulation in tomato fruit.

Dimeric dihydrodiol dehydrogenase is an efficient primate 1,5-anhydro-D-fructose reductase.

1,5-Anhydro-D-fructose (AF), a metabolite of the anhydrofructose pathway of glycogen metabolism, has recently been shown to react with intracellular proteins and form advanced glycation end-products. The reactive AF is metabolized to non-reactive 1,5-anhydro-D-glucitol by AF reductase in animal tissues and human cells. Pig and mouse AF reductases were characterized, but primate AF reductase remains unknown. Here, we examined the AF-reducing activity of eleven primate NADPH-dependent reductases with broad substrate specificity for carbonyl compounds. AF was reduced by monkey dimeric dihydrodiol dehydrogenase (DHDH), human aldehyde reductase (AKR1A1) and human dicarbonyl/L-xylulose reductase (DCXR). DHDH showed the lowest KM (21 µM) for AF, and its kcat/KM value (1208 s-1mM-1) was much higher than those of AKR1A1 (1.3 s-1mM-1), DCXR (1.1 s-1mM-1) and the pig and mouse AF reductases. AF is a novel substrate with higher affinity and catalytic efficiency than known substrates of DHDH. Docking simulation study suggested that Lys156 in the substrate-binding site of DHDH contributes to the high affinity for AF. Gene database searches identified DHDH homologues (with >95% amino acid sequence identity) in humans and apes. Thus, DHDH acts as an efficient AF reductase in primates.

Hydrogen-Bond-Mediated Aglycone Delivery: Synthesis of ß-d-Fructofuranosides.

The construction of ß-d-fructofuranosidic linkages is one of the major challenges in carbohydrate chemistry. In this work, we developed an efficient method for the synthesis of ß-d-fructofuranosides by using a 6-picoloyl-protected fructofuranosyl thioglycoside as the glycosyl donor. Subsequently, we applied the approach to a wide variety of donors and acceptors. Furthermore, the successful synthesis of levantetrose confirmed its applicability in the multistep synthesis of oligosaccharides.

Obesity Treatment: A Focus on Pharmacotherapy of Weight Management.

Obesity, a chronic multifactorial disease, has been on the rise in the United States in recent years. It paves a way to other chronic conditions and related morbidity and mortality. The treatment of obesity should have a chronic approach involving lifestyle modifications from the very beginning. Along with reduced calorie diet, increased physical activity, and behavior modifications, various short- and long-term pharmacological agents are available to help with the weight loss. For qualifying patients, selection of an appropriate agent based on its mechanism, efficacy, and safety profile as well as patient preference can provide desired outcomes. This medical weight management should be a multidisciplinary approach involving nurses to provide continuous patient education and motivation.

Novel plasmid-free Gluconobacter oxydans strains for production of the natural sweetener 5-ketofructose.

BACKGROUND: 5-Ketofructose (5-KF) has recently been identified as a promising non-nutritive natural sweetener. Gluconobacter oxydans strains have been developed that allow efficient production of 5-KF from fructose by plasmid-based expression of the fructose dehydrogenase genes fdhSCL of Gluconobacter japonicus. As plasmid-free strains are preferred for industrial production of food additives, we aimed at the construction of efficient 5-KF production strains with the fdhSCL genes chromosomally integrated. RESULTS: For plasmid-free 5-KF production, we selected four sites in the genome of G. oxydans IK003.1 and inserted the fdhSCL genes under control of the strong P264 promoter into each of these sites. All four recombinant strains expressed fdhSCL and oxidized fructose to 5-KF, but site-specific differences were observed suggesting that the genomic vicinity influenced gene expression. For further improvement, a second copy of the fdhSCL genes under control of P264 was inserted into the second-best insertion site to obtain strain IK003.1::fdhSCL2. The 5-KF production rate and the 5-KF yield obtained with this double-integration strain were considerably higher than for the single integration strains and approached the values of IK003.1 with plasmid-based fdhSCL expression. CONCLUSION: We identified four sites in the genome of G. oxydans suitable for expression of heterologous genes and constructed a strain with two genomic copies of the fdhSCL genes enabling efficient plasmid-free 5-KF production. This strain will serve as basis for further metabolic engineering strategies aiming at the use of alternative carbon sources for 5-KF production and for bioprocess optimization.

Calcium Fructoborate Prevents Skin Cancer Development in Balb-c Mice.

Tumor microenvironment, genetic, and non-genetic factors are responsible for the atypical metabolic feature of cancer cells. Aberrant activity of PI3K/Akt pathway, increased glycolytic flux, and decreased intracellular pH gradient are the leading causes of this feature. Calcium Fructoborate (CaFB), a sugar-borate ester, has major benefits for human health. The aim of this study was to explore the implication of CaFB on experimentally induced skin cancer in vivo. According to the treatment, 92 female Balb-c mice are divided into six groups: control, CaFB (3 mg/kg/day), 7,12-Dimethylbenz(a)anthracene (DMBA)+12-O-tetradecanoylphorbol-13-acetate (TPA) (97.5 nmol DMBA, 6.5 nmol TPA), T1: CaFB+DMBA+TPA (3 mg/kg/day CaFB together with DMBA), T2: DMBA+CaFB+TPA (3 mg/kg/day CaFB together with TPA), T3: DMBA+TPA+CaFB (3 mg/kg/day CaFB after tumor formation). Topical DMBA and TPA application resulted in a significant increase in the protein levels, immunoreactivity, and mRNA expression of HRAS, HIF1α, Akt, and PTEN (p < 0.05). Moreover, an increase in the number of TUNEL-positive cells was observed in DMBA-TPA group compared with the control group (p < 0.05). CaFB application reduced the protein levels, immunoreactivity, and mRNA expressions of HRAS, HIF1α, Akt, and PTEN and also decreased the number of TUNEL-positive cells. Recent evidence obtained from our study validated that CaFB treatment may have skin cancer-preventing effect.

Synthesis of the alternative sweetener 5-ketofructose from sucrose by fructose dehydrogenase and invertase producing Gluconobacter strains.

A promising alternative to high-calorie sugars and artificial sweeteners is the microbially produced fructose derivative 5-ketofructose (5-KF). The key enzyme for biotransformation, fructose dehydrogenase (Fdh), was overproduced in Gluconobacter (G.) oxydans and G. japonicus LMG 26773. Furthermore, the fdh genes were integrated into the chromosome of G. oxydans (G. oxydans Δmgdh::fdh). All mutants showed high fructose oxidation rates forming 5-KF. G. japonicus LMG 26773 fdh was selected for 5-KF production from the cost-efficient and renewable feedstock sucrose because the organism possessed both, a highly active Fdh and an enzyme able to cleave sucrose. However, 5-KF yield was low because the strain formed levan and consumed 5-KF in the second growth phase. Several Gluconobacter strains were screened for sucrose-hydrolyzing enzymes. One of these proteins (Inv1417) was characterized and it was found that the enzyme showed the highest specific activity compared to all mesophilic invertases described so far (Vmax = 2295 ± 243 U mg protein-1). The corresponding gene was expressed in G. oxydans Δmgdh::fdh. The results clearly indicated that both heterologously produced enzymes Fdh and Inv1417 were active in this single-strain system for 5-KF synthesis. Overall 84 ± 2% of the available fructose units of sucrose were converted to 5-KF.