Indigo Formation and Rapid NADPH Consumption Provide Robust Prediction of Raspberry Ketone Synthesis by Engineered Cytochrome P450 BM3

Natural raspberry ketone has a high value in the flavor, fragrance and pharmaceutical industries. Its extraction is costly, justifying the search for biosynthetic routes. We hypothesized that cytochrome P450 BM3 (P450 BM3) could be engineered to catalyze the hydroxylation of 4‐phenyl‐2‐butanone, a naturally sourceable precursor, to raspberry ketone. The synthesis of indigo by variants of P450 BM3 has previously served as a predictor of promiscuous oxidation reactions. To this end, we screened 53 active‐site variants of P450 BM3 using orthogonal high‐throughput workflows to identify the most streamlined route to all indigo‐forming variants. Among the three known and 13 new indigo‐forming variants, eight hydroxylated 4‐phenyl‐2‐butanone to raspberry ketone. Previously unreported variant A82Q displayed the highest initial rates and coupling efficiencies in synthesis of indigo and of raspberry ketone. It produced the highest total concentration of raspberry ketone despite producing less total indigo than previously reported variants. Its productivity, although modest, clearly demonstrates the potential for development of a biocatalytic route to raspberry ketone. In addition to validating indigo as a robust predictor of this promiscuous activity, we demonstrate that monitoring rapid NADPH consumption serves as an alternative predictor of a promiscuous reactivity in P450 BM3

Recovery, assessment, and molecular characterization of minor olive genotypes in Tunisia

Olive is one of the oldest cultivated species in the Mediterranean Basin, including Tunisia, where it has a wide diversity, with more than 200 cultivars, of both wild and feral forms. Many minor cultivars are still present in marginal areas of Tunisia, where they are maintained by farmers in small local groves, but they are poorly characterized and evaluated. In order to recover this neglected germplasm, surveys were conducted in different areas, and 31 genotypes were collected, molecularly characterized with 12 nuclear microsatellite (simple sequence repeat (SSR)) markers, and compared with 26 reference cultivars present in the Tunisian National Olive collection. The analysis revealed an overall high genetic diversity of this olive’s germplasm, but also discovered the presence of synonymies and homonymies among the commercialized varieties. The structure analysis showed the presence of different gene pools in the analyzed germplasm. In particular, the marginal germplasm from Ras Jbal and Azmour is characterized by gene pools not present in commercial (Nurseries) varieties, pointing out the very narrow genetic base of the commercialized olive material in Tunisia, and the need to broaden it to avoid the risk of genetic erosion of this species in this country

Rhaponticum acaule (L) DC essential oil: chemical composition, in vitro antioxidant and enzyme inhibition properties

Background: α-glucosidase is a therapeutic target for diabetes mellitus (DM) and α-glucosidase inhibitors play a vital role in the treatments for the disease. Furthermore, xanthine oxidase (XO) is a key enzyme that catalyzes hypoxanthine and xanthine to uric acid which at high levels can lead to hyperuricemia which is an important cause of gout. Pancreatic lipase (PL) secreted into the duodenum plays a key role in the digestion and absorption of fats. For its importance in lipid digestion, PL represents an attractive target for obesity prevention. Methods: The flowers essential oil of Rhaponticum acaule (L) DC (R. acaule) was characterized using gas chromatography-mass spectrometry (GC-MS). The antioxidant activities of R. acaule essential oil (RaEO) were also determined using 2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), reducing power, phosphomolybdenum, and DNA nicking assays. The inhibitory power of RaEO against α-glucosidase, xanthine oxidase and pancreatic lipase was evaluated. Enzyme kinetic studies using Michaelis-Menten and the derived Lineweaver-Burk (LB) plots were performed to understand the possible mechanism of inhibition exercised by the components of this essential oil. Results: The result revealed the presence of 26 compounds (97.4%). The main constituents include germacrene D (49.2%), methyl eugenol (8.3%), (E)-β-ionone (6.2%), β-caryophyllene (5.7%), (E,E)-α-farnesene (4.2%), bicyclogermacrene (4.1%) and (Z)-α-bisabolene (3.7%). The kinetic inhibition study showed that the essential oil demonstrated a strong α-glucosidase inhibiton and it was a mixed inhibitor. On the other hand, our results evidenced that this oil exhibited important xanthine oxidase inhibitory effect, behaving as a non-competitive inhibitor. The essential oil inhibited the turkey pancreatic lipase, with maximum inhibition of 80% achieved at 2 mg/mL. Furthermore, the inhibition of turkey pancreatic lipase by RaEO was an irreversible one. Conclusion: The results revealed that the RaEO is a new promising potential source of antioxidant compounds, endowed with good practical applications for human health. Keywords: α-glucosidase, Antioxidant activity, Chemical composition, Pancreatic lipase inhibition, Rhaponticum acaule essential oil, Xanthine oxidase

Polymorphic variants of SCN1A and EPHX1 influence plasma carbamazepine concentration, metabolism and pharmacoresistance in a population of Kosovar Albanian epileptic patients

Aim The present study aimed to evaluate the effects of gene variants in key genes influencing pharmacokinetic and pharmacodynamic of carbamazepine (CBZ) on the response in patients with epilepsy. Materials & Methods Five SNPs in two candidate genes influencing CBZ transport and metabolism, namely ABCB1 or EPHX1, and CBZ response SCN1A (sodium channel) were genotyped in 145 epileptic patients treated with CBZ as monotherapy and 100 age and sex matched healthy controls. Plasma concentrations of CBZ, carbamazepine-10,11-epoxide (CBZE) and carbamazepine-10,11-trans dihydrodiol (CBZD) were determined by HPLC-UV-DAD and adjusted for CBZ dosage/kg of body weight. Results The presence of the SCN1A IVS5-91G>A variant allele is associated with increased epilepsy susceptibility. Furthermore, carriers of the SCN1A IVS5-91G>A variant or of EPHX1 c.337T>C variant presented significantly lower levels of plasma CBZ compared to carriers of the common alleles (0.71±0.28 vs 1.11±0.69 μg/mL per mg/Kg for SCN1A IVS5-91 AA vs GG and 0.76±0.16 vs 0.94±0.49 μg/mL per mg/Kg for EPHX1 c.337 CC vs TT; PG showed a reduced microsomal epoxide hydrolase activity as reflected by a significantly decreased ratio of CBZD to CBZ (0.13±0.08 to 0.26±0.17, pT SNP and SCN1A 3148A>G variants were not associated with significant changes in CBZ pharmacokinetic. Patients resistant to CBZ treatment showed increased dosage of CBZ (657±285 vs 489±231 mg/day; P<0.001) but also increased plasma levels of CBZ (9.84±4.37 vs 7.41±3.43 μg/mL; P<0.001) compared to patients responsive to CBZ treatment. CBZ resistance was not related to any of the SNPs investigated. Conclusions The SCN1A IVS5-91G>A SNP is associated with susceptibility to epilepsy. SNPs in EPHX1 gene are influencing CBZ metabolism and disposition. CBZ plasma levels are not an indicator of resistance to the therapy

Brine recovery from hypersaline wastewaters from table olive processing by combination of biological treatment and membrane technologies

[EN] The fermentation brines from table olive processing (FTOP) are hypersaline effluents (conductivities higher than 75 mS·cm-1) with high organic matter concentrations (COD around 10 g·L-1), which also include phenolic compounds (between 700 and 1500 mg TY·L-1). In this work, an integrated process for the FTOP reuse as brine in the table olive processing has been evaluated. This integrated process consisted of a biological treatment followed by a membrane system, which included ultrafiltration (UF) plus nanofiltration (NF). The biological treatment was carried out by 6 L laboratory sequencing batch reactor (SBR). UF and NF were performed in laboratory plants for flat membranes of 0.0125 and 0.0072 m2, respectively. Each stream generated during the FTOP treatment (SBR effluent, and UF and NF permeates) were evaluated. The SBR eliminated around 80% of COD and 71% of total phenols concentration. In the final NF permeate the COD concentration was lower than 125 mg·L-1; while the turbidity, colour and phenolic compounds, were completely removed.The authors of this work thank the financial support of CDTI (Centre for Development Technological Industrial) depending on the Spanish Ministry of Science and Innovation.Ferrer-Polonio, E.; Carbonell Alcaina, C.; Mendoza Roca, JA.; Iborra Clar, A.; Alvarez Blanco, S.; Bes-Piá, M.; Pastor Alcañiz, L. (2017). Brine recovery from hypersaline wastewaters from table olive processing by combination of biological treatment and membrane technologies. Journal of Cleaner Production. 142:1377-1386. doi:10.1016/j.jclepro.2016.11.169S1377138614

Reduced antioxidant defense in early onset first-episode psychosis: a case-control study

Background:Our objective is to determine the activity of the antioxidant defense system at admission in patients with early onset first psychotic episodes compared with a control group. Methods: Total antioxidant status (TAS) and lipid peroxidation (LOOH) were determined in plasma. Enzyme activities and total glutathione levels were determined in erythrocytes in 102 children and adolescents with a first psychotic episode and 98 healthy controls. Results: A decrease in antioxidant defense was found in patients, measured as decreased TAS and glutathione levels. Lipid damage (LOOH) and glutathione peroxidase activity was higher in patients than controls. Our study shows a decrease in the antioxidant defense system in early onset first episode psychotic patients. Conclusions: Glutathione deficit seems to be implicated in psychosis, and may be an important indirect biomarker of oxidative stress in early-onset schizophrenia. Oxidative damage is present in these patients, and may contribute to its pathophysiology

Role of DNA methylation in head and neck cancer

Head and neck cancer (HNC) is a heterogenous and complex entity including diverse anatomical sites and a variety of tumor types displaying unique characteristics and different etilogies. Both environmental and genetic factors play a role in the development of the disease, but the underlying mechanism is still far from clear. Previous studies suggest that alterations in the genes acting in cellular signal pathways may contribute to head and neck carcinogenesis. In cancer, DNA methylation patterns display specific aberrations even in the early and precancerous stages and may confer susceptibility to further genetic or epigenetic changes. Silencing of the genes by hypermethylation or induction of oncogenes by promoter hypomethylation are frequent mechanisms in different types of cancer and achieve increasing diagnostic and therapeutic importance since the changes are reversible. Therefore, methylation analysis may provide promising clinical applications, including the development of new biomarkers and prediction of the therapeutic response or prognosis. In this review, we aimed to analyze the available information indicating a role for the epigenetic changes in HNC

Regulation of cel genes of C. cellulolyticum: identification of GlyR2, a transcriptional regulator regulating cel5D gene expression

Transcription and expression regulation of some individual cel genes (cel5A, cel5I, cel5D and cel44O) of Clostridium cellulolyticum were investigated. Unlike the cip-cel operon, these genes are transcribed as monocistronic units of transcription, except cel5D. The location of the transcription initiation sites was determined using RT-PCR and the mRNA 5′-end extremities were detected using primer extension experiments. Similarly to the cip-cel operon, cel5A and cel5I expressions are regulated by a carbon catabolite repression mechanism, whereas cel44O and cel5D expressions do not seem to be submitted to this regulation. The role of the putative transcriptional regulator GlyR2 in the regulation of cel5D expression was investigated. The recombinant protein GlyR2 was produced and was shown to bind in vitro to the cel5D and glyR2 promoter regions, suggesting that besides regulating its own expression, GlyR2 may regulate cel5D expression. To test this hypothesis in vivo, an insertional glyR2 mutant was generated and the effect of this disruption on cel5D expression was evaluated. Levels of cel5D mRNAs in the mutant were 16 fold lower than that of the wild-type strain suggesting that GlyR2 acts as an activator of cel5D expression