Beneficial Effects of Linseed Supplementation on Gut Mucosa-Associated Microbiota in a Physically Active Mouse Model of Crohn's Disease.

The Western diet, rich in lipids and in n-6 polyunsaturated fatty acids (PUFAs), favors gut dysbiosis observed in Crohn's disease (CD). The aim of this study was to assess the effects of rebalancing the n-6/n-3 PUFA ratio in CEABAC10 transgenic mice that mimic CD. Mice in individual cages with running wheels were randomized in three diet groups for 12 weeks: high-fat diet (HFD), HFD + linseed oil (HFD-LS-O) and HFD + extruded linseed (HFD-LS-E). Then, they were orally challenged once with the Adherent-Invasive Escherichia coli (AIEC) LF82 pathobiont. After 12 weeks of diet, total energy intake, body composition, and intestinal permeability were not different between groups. After the AIEC-induced intestinal inflammation, fecal lipocalin-2 concentration was lower at day 6 in n-3 PUFAs supplementation groups (HFD-LS-O and HFD-LS-E) compared to HFD. Analysis of the mucosa-associated microbiota showed that the abundance of Prevotella, Paraprevotella, Ruminococcus, and Clostridiales was higher in the HFD-LS-E group. Butyrate levels were higher in the HFD-LS-E group and correlated with the Firmicutes/Proteobacteria ratio. This study demonstrates that extruded linseed supplementation had a beneficial health effect in a physically active mouse model of CD susceptibility. Additional studies are required to better decipher the matrix influence in the linseed supplementation effect.

A Moderate Supplementation of Native Whey Protein Promotes Better Muscle Training and Recovery Adaptations Than Standard Whey Protein - A 12-Week Electrical Stimulation and Plyometrics Training Study.

The purpose of this study was to assess if native whey protein (NW) supplementation could promote recovery and training adaptations after an electrostimulation (ES) training program combined to plyometrics training. Participants were allocated into three groups, supplemented 5 days/week, either with 15 g of carbohydrates + 15 g of NW (n = 17), 15 g of carbohydrates + 15 g of standard whey protein (SW; n = 15), or placebo (PLA; 30 g of carbohydrates; n = 10), while undergoing a 12-week ES training program of the knee extensors. Concentric power (Pmax) was evaluated before, immediately after, as well as 30 min, 60 min, 24 h, and 48 h after the 1st, 4th and last ES training session. The maximal voluntary contraction torque (MVC), twitch amplitude, anatomical cross-sectional area (CSA) and maximal voluntary activation level (VA) were measured before (T0), and after 6 (T1) and 12 weeks of training (T2). P max recovery kinetics differed between groups (p < 0.01). P max started to recover at 30 min in NW, 24 h in SW and 48 h in PLA. Training adaptations also differed between groups: MVC increased between T0 and T2 in NW (+11.8%, p < 0.001) and SW (+7.1%, p < 0.05), but not PLA. Nevertheless, the adaptation kinetics differed: MVC increased in NW and SW between T0 and T1, but an additional gain was only observed between T1 and T2 in NW. VA declined at T1 and T2 in PLA (-3.9%, p < 0.05), at T2 in SW (-3.5%, p < 0.05), and was unchanged in NW. CSA increased, but did not differ between groups. These results suggest that NW could promote a faster recovery and neuromuscular adaptations after training than SW. However, the mechanisms underlying this effect remain to be identified.

An Engineered Aro1 Protein Degradation Approach for Increased cis,cis-Muconic Acid Biosynthesis in Saccharomyces cerevisiae.

Muconic acid (MA) is a chemical building block and precursor to adipic and terephthalic acids used in the production of nylon and polyethylene terephthalate polymer families. Global demand for these important materials, coupled to their dependence on petrochemical resources, provides substantial motivation for the microbial synthesis of MA and its derivatives. In this context, the Saccharomyces cerevisiae yeast shikimate pathway can be sourced as a precursor for the formation of MA. Here we report a novel strategy to balance MA pathway performance with aromatic amino acid prototrophy by destabilizing Aro1 through C-terminal degron tagging. Coupling of a composite MA production pathway to degron-tagged Aro1 in an aro3Δ aro4Δ mutant background led to the accumulation of 5.6 g/liter protocatechuic acid (PCA). However, metabolites downstream of PCA were not detected, despite the inclusion of genes mediating their biosynthesis. Because CEN.PK family strains of S. cerevisiae lack the activity of Pad1, a key enzyme supporting PCA decarboxylase activity, chromosomal expression of intact PAD1 alleviated this bottleneck, resulting in nearly stoichiometric conversion (95%) of PCA to downstream products. In a fed-batch bioreactor, the resulting strain produced 1.2 g/liter MA under prototrophic conditions and 5.1 g/liter MA when supplemented with amino acids, corresponding to a yield of 58 mg/g sugar.IMPORTANCE Previous efforts to engineer a heterologous MA pathway in Saccharomyces cerevisiae have been hindered by a bottleneck at the PCA decarboxylation step and the creation of aromatic amino acid auxotrophy through deleterious manipulation of the pentafunctional Aro1 protein. In light of these studies, this work was undertaken with the central objective of preserving amino acid prototrophy, which we achieved by employing an Aro1 degradation strategy. Moreover, resolution of the key PCA decarboxylase bottleneck, as detailed herein, advances our understanding of yeast MA biosynthesis and will guide future strain engineering efforts. These strategies resulted in the highest titer reported to date for muconic acid produced in yeast. Overall, our study showcases the effectiveness of careful tuning of yeast Aro1 activity and the importance of host-pathway dynamics.

Whey proteins are more efficient than casein in the recovery of muscle functional properties following a casting induced muscle atrophy.

The purpose of this study was to investigate the effect of whey supplementation, as compared to the standard casein diet, on the recovery of muscle functional properties after a casting-induced immobilization period. After an initial (I0) evaluation of the contractile properties of the plantarflexors (isometric torque-frequency relationship, concentric power-velocity relationship and a fatigability test), the ankle of 20 male adult rats was immobilized by casting for 8 days. During this period, rats were fed a standard diet with 13% of casein (CAS). After cast removal, rats received either the same diet or a diet with 13% of whey proteins (WHEY). A control group (n = 10), non-immobilized but pair-fed to the two other experimental groups, was also studied and fed with the CAS diet. During the recovery period, contractile properties were evaluated 7 (R7), 21 (R21) and 42 days (R42) after cast removal. The immobilization procedure induced a homogeneous depression of average isometric force at R7 (CAS: - 19.0 ± 8.2%; WHEY: - 21.7 ± 8.4%; P<0.001) and concentric power (CAS: - 26.8 ± 16.4%, P<0.001; WHEY: - 13.5 ± 21.8%, P<0.05) as compared to I0. Conversely, no significant alteration of fatigability was observed. At R21, isometric force had fully recovered in WHEY, especially for frequencies above 50 Hz, whereas it was still significantly depressed in CAS, where complete recovery occurred only at R42. Similarly, recovery of concentric power was faster at R21 in the 500-700°/s range in the WHEY group. These results suggest that recovery kinetics varied between diets, the diet with the whey proteins promoting a faster recovery of isometric force and concentric power output as compared to the casein diet. These effects were more specifically observed at force level and movement velocities that are relevant for functional abilities, and thus natural locomotion.

Characterization of a flavoprotein oxidase from opium poppy catalyzing the final steps in sanguinarine and papaverine biosynthesis.

Benzylisoquinoline alkaloids are a diverse class of plant specialized metabolites that includes the analgesic morphine, the antimicrobials sanguinarine and berberine, and the vasodilator papaverine. The two-electron oxidation of dihydrosanguinarine catalyzed by dihydrobenzophenanthridine oxidase (DBOX) is the final step in sanguinarine biosynthesis. The formation of the fully conjugated ring system in sanguinarine is similar to the four-electron oxidations of (S)-canadine to berberine and (S)-tetrahydropapaverine to papaverine. We report the isolation and functional characterization of an opium poppy (Papaver somniferum) cDNA encoding DBOX, a flavoprotein oxidase with homology to (S)-tetrahydroprotoberberine oxidase and the berberine bridge enzyme. A query of translated opium poppy stem transcriptome databases using berberine bridge enzyme yielded several candidate genes, including an (S)-tetrahydroprotoberberine oxidase-like sequence selected for heterologous expression in Pichia pastoris. The recombinant enzyme preferentially catalyzed the oxidation of dihydrosanguinarine to sanguinarine but also converted (RS)-tetrahydropapaverine to papaverine and several protoberberine alkaloids to oxidized forms, including (RS)-canadine to berberine. The K(m) values of 201 and 146 µm for dihydrosanguinarine and the protoberberine alkaloid (S)-scoulerine, respectively, suggested high concentrations of these substrates in the plant. Virus-induced gene silencing to reduce DBOX transcript levels resulted in a corresponding reduction in sanguinarine, dihydrosanguinarine, and papaverine accumulation in opium poppy roots in support of DBOX as a multifunctional oxidative enzyme in BIA metabolism.

Sequential O-methylation of tricetin by a single gene product in wheat.

Flavonoid compounds are ubiquitous in nature. They constitute an important part of the human diet and act as active principles of many medicinal plants. Their O-methylation increases their lipophilicity and hence, their compartmentation and functional diversity. We have isolated and characterized a full-length flavonoid O-methyltransferase cDNA (TaOMT2) from a wheat leaf cDNA library. The recombinant TaOMT2 protein was purified to near homogeneity and tested for its substrate preference against a number of phenolic compounds. Enzyme assays and kinetic analyses indicate that TaOMT2 exhibits a pronounced preference for the flavone, tricetin and gives rise to three methylated enzyme reaction products that were identified by TLC, HPLC and ESI-MS/MS as its mono-, di- and trimethyl ether derivatives. The sequential order of tricetin methylation by TaOMT2 is envisaged to proceed via its 3'-mono--->3',5'-di--->3',4',5'-trimethyl ether derivatives. To our knowledge, this is the first report of a gene product that catalyzes three sequential O-methylations of a flavonoid substrate.