Universidad de Castilla-La Mancha

Several commercially important vinyl phenolic compounds can be produced by enzymatic decarboxylation of phenolic acids such as ferulic and p-coumaric acids which can be extracted from agro-industrial waste. Phenolic acid decarboxylase is an enzyme that acts in the decarboxylation of these acids to 4-vinylguaiacol and 4-vinylphenol, respectively. In this study, the gene encoding phenolic acid decarboxylase from Klebsiella pneumoniae TD 4.7 was isolated and identified as a 504 bp fragment, encoding a polypeptide of 167 amino acid residues. A 98 % predicted amino acid sequence identity between ferulic acid decarboxylase from other bacteria of the same genus was determined. The gene was successfully expressed in Escherichia coli BL21 (DE3), and the recombinant enzyme was purified as active in absence of cofactor. The protein had a mass of 22-kDa protein, with greater activity at pH 5.5 and 40 °C. The decarboxylase activity was inhibited by Hg^2 + , Zn²⁺, Cu²⁺, and Cd²⁺ ions and increased by 20 % in the presence of Co²⁺. The K_m and V_max values for the recombinant enzyme were estimated at 2.95 mM and 102.10 µmol min^-1 mg^-1, respectively. The enzyme's structure was modelled using the structural prediction programs AlphaFold Multimer and SWISS-MODEL, with an RMSD of just 0.7 Å, demonstrating the absence of cysteine and disulfide bonds in the homodimer, with the presence of a high number of lysine residues. The amino acids involved in the catalytic site were Tyr27, Glu134, and Asn23. The Enzyme activity on substrates ferulic and p-coumaric acids extracted from sugarcane bagasse, resulted in 4-vinylguaiacol and 4-vinylphenol, respectively, with conversion yields of 43 % for ferulic acid and 55 % for p-coumaric acid. These data are important in terms of obtaining an enzyme that decarboxylates ferulic and p-coumaric acids obtained from sugarcane bagasse hydrolyzed with similar efficiency, in a single step and without the need for a cofactor, making it an excellent option for bioprocesses using lignocellulosic biomass derivatives.

The objective was to examine the adjuvant effect of active pulsed electromagnetic field (PEMF) versus microwave (MW) therapy, as well as sham PEMF, in addressing pain and improving functionality for treating knee osteoarthritis (KOA).

This was a double-blind, placebo-controlled, randomized clinical trial. Individuals diagnosed with KOA were assigned to an intervention combining an exercise program (EX) with active PEMF, MW, or sham PEMF. The main outcomes were pain, reported on a visual analogue scale (VAS), and functionality, assessed using the Western Ontario and McMaster Universities Arthritis (WOMAC) questionnaire, and the Timed Up and Go test (TUG). The outcomes were measured preintervention, immediately postintervention, and at 1 and 4 months of follow-up (FU).

Sixty individuals (n = 83 knees) were evaluated. Significant between-group differences were found in WOMAC stiffness (rmMANOVA (F(2,77) = 4.33, P = .017, partial η 2  = 0.10)). A notable interaction effect between group and time was found for the WOMAC pain score (rmMANOVA (F(2, 77) = 3.14, P = .049, partial η 2  = 0.07)). After 4 months, the PEMF + EX group demonstrated superior pain relief compared to the sham PEMF + EX (WOMAC pain “mean difference ± standard error between-groups”: −3.2 ± 1.2, P = .028) and MW + EX (VAS pain: −2.1 ± 0.9, P = .042) groups. PEMF + EX perceived less stiffness than did the sham PEMF + EX in both the pre-FU (WOMAC stiffness: −1.6 ± 0.6, P = .047), 1-month FU (−1.7 ± 0.6, P = .015) and 4-month FU (−1.4 ± 0.6, P = .038), with no changes in the MW + EX group. WOMAC function score showed greater improvement in the PEMF + EX group compared to the MW + EX group at 4-month of FU (−9.0 ± 3.6, P = .039). Only PEMF + EX showed a positive effect on the TUG score at 1-month FU (−1.7 ± 0.5, P = <.001) and 4-month of FU (1.9 ± 0.5, P = .020).

The application of PEMF could be a useful adjuvant treatment to exercise programs to further decrease pain and improve knee stiffness and function in individuals with KOA in the medium term compared to MW and sham PEMF.

The increasing production and application of graphene-related materials (GRMs) and other 2D nanomaterials (2D materials) raise concerns about their potential genotoxicity. This study aims to assess the genotoxic effects of these materials using a modified in vitro comet assay and double-strand break (DSB) detection via the γH2AX assay, which is crucial for determining the safety of these compounds. Chinese hamster ovary cells (CHO-K1) were exposed to graphene oxide (GO), few-layer graphene (FLG), monolayer GO (mlGO), molybdenum disulfide (MoS₂), and hexagonal boron nitride (hBN) at concentrations of 0, 0.05, 0.5, 5, 20, and 50 μg/mL for 24 h. Adaptations to the standard comet assay were implemented to address interference caused by 2D material agglomeration, which can hinder accurate DNA damage detection. Controlled, gentle mechanical shaking during incubation minimized agglomeration, improving the visualization of DNA damage. Both the modified in vitro comet assay and γH2AX DSB assay indicated that all materials tested, except mlGO, exhibited genotoxic effects at sublethal concentrations. These findings suggest that certain GRMs should be classified as genotoxic, warranting careful consideration of their potential risks, and recommending the modified comet assay as a reliable tool for 2D material genotoxicity assessment.