Direct electron transfer (DET)-type bioelectrocatalysis, which couples electrode reactions and catalytic functions of redox enzymes without any redox mediator, is one of the most intriguing subjects studied over the past decades in the field of bioelectrochemistry. In order to realize the DET-type bioelectrocatalysis and to improve the performance, nanostructures of the electrode surface have to be carefully tuned for each enzyme. In addition, enzymes can also be tuned by protein engineering approach for the DET-type reaction. This review summarizes the resent progresses in this field of the research, while taking into consideration of the importance of nanostructure of electrodes as well as redox enzymes. Described are basic concepts and theoretical aspects of DET-type bioelectrocatalysis, significance of nanostructures as scaffolds for DET-type reactions, protein engineering approached for DET-type reactions, and concepts and facts of bidirectional DET-type reactions, from a cross-disciplinary viewpoint.

Glioblastoma, a deadly brain tumor, shows limited response to standard therapies like te-mozolomide (TMZ). Recent findings from the REGOMA trial underscore a significant survival improvement offered by regorafenib (REGO) in recurrent glioblastoma. Our study aimed to propose a 3D ex vivo drug response precision medicine approach to investigate recurrent glio-blastoma sensitivity to REGO and elucidate the underlying molecular mechanisms involved in tumor resistance or responsiveness to treatment. 3D glioblastoma organoids (GB-EXPs) obtained from 18 patients’ resected recurrent glioblastoma tumors were treated with TMZ and REGO. Drug responses were evaluated using NAD(P)H FLIM, stratifying tumors as responders (Resp) or non-responders (NR). Whole-exome sequencing was performed on 16 tissue samples, and whole-transcriptome analysis on 13 GB-EXPs treated and untreated. We found 35% (n=9) and 77% (n=20) of tumors responded to TMZ and REGO, respectively, with no instances of TMZ-Resp be-ing REGO-NR. Exome analysis revealed a unique mutational profile in REGO-Resp tumors compared to NR tumors. Transcriptome analysis identified distinct expression patterns in Resp and NR tumors, impacting Rho GTPase and NOTCH signaling, known to be involved in drug response. In conclusion, recurrent glioblastoma tumors were more responsive to REGO com-pared to TMZ treatment. Importantly, our approach enables a comprehensive longitudinal ex-ploration of the molecular changes induced by treatment, unveiling promising biomarkers in-dicative of drug response.

In the context of the development of bioprocesses for the production of biofuels and bulk chemicals, microbial cells are rationally engineered to produce such molecules at high yield and titres in order to develop new biological methods that satisfy economic constraints. The redox and energetic balances of such strains play crucial roles in performance. Processes performed in strict anaerobes have a limited amount of energy available compared to that in aerobic organisms. This energy is obtained through fermentation and/or ion gradient-driven phosphorylation. Such anaerobic organisms have developed energy conservation mechanisms to increase ATP yields. This paper presents the properties of one of these mechanisms catalysed by the Rnf complex, an ion-translocating membrane complex with a ferredoxin NAD+ oxidoreductase activity. The Rnf complex performs the transfer of electrons from reduced ferredoxin to NAD+ coupled with an ion-motive transport. Ferredoxin is a common electron carrier for anaerobic bacteria and, with NAD+, is involved in several pathways of interest for the production of biofuels. This complex was first identified in Rhodobacter capsulatus and found to be involved in nitrogen fixation. It was then found to be involved in energy conservation in multiple anaerobic organisms, from acetobacteria such as Acetobacterium woodii to sulfate-reducing bacteria such as Desulfovibrio alaskensis and autotrophic bacteria such as Clostridium ljungdahlii and Clostridium aceticum. The Rnf complex triggers two types of ion transports: it can be either a sodium or a proton transporter. Both of these transports create a gradient of ions, generating a membrane potential that is then used by ATPase to produce ATP and thus serving as an energy conservation mechanism. In this review, the available information on the Rnf complex from genetic organization up to its in vivo and in vitro activities in several microorganisms is summarized, with a special focus on the proton-motive Rnf complex.

The molecular entity that catalyzes ferredoxin (Fd)-dependent cyclic electron flow around photosystem I (PSI) (Fd-CEF) remains unknown. To elucidate the in vivo molecular mechanism of Fd-CEF, the evaluation of Fd reduction‒oxidation kinetics is a good indicator of Fd-CEF activity. Recent research showed that the expression of Fd-CEF activity requires the oxidation of plastoquinone, and furthermore, chloroplast NAD(P)H dehydrogenase does not catalyze Fd-CEF in Arabidopsis thaliana. In the present research, the effect of reduced Fd on Fd-CEF activity was analyzed by comparing wild-type and pgr5-deficient mutants (pgr5hope1). PGR5 has been proposed to be the mediator of Fd-CEF, and pgr5hope1 showed the same CO2 assimilation rate and the same reduction‒oxidation level of PQ as WT, but P700 oxidation with Fd was highly reduced, unlike WT. As expected, the activity of Fd-CEF was enhanced in pgr5hope1 compared to the wild type, and its activity was also enhanced with the oxidation of PQ by the increase in the CO2 assimilation rate. The present in vivo research clearly shows that the expression of Fd-CEF activity requires reduced Fd in addition to oxidized PQ, and PGR5 does not catalyze Fd-CEF.

NADPH is an indispensable cofactor in a wide range of physiological processes which is generated by a family of NADPH-dehydrogenases, where the NADP-dependent malic enzyme (NADP-ME) is one member of these enzymes. Pepper (Capsicum annuum L.) fruit is a horticultural product worldwide consumed which has great nutritional and economic relevance. Besides the phenotypical changes that undergo pepper fruit during ripening, there are many associated modifications at transcriptomic, proteome ic, biochemical, and metabolic levels. Nitric oxide (NO) is a recognized signal molecule that can exert regulatory functions in diverse plant processes. To our knowledge, there is very scarce information about the number of genes encoding for NADP-ME in pepper plants and their expression during the ripening of sweet pepper fruit. Based on a data-mining approach on the pepper plant genome and fruit transcriptome (RNA-seq), five NADP-ME genes were identified, and four of them, namely CaNADP-ME2 to CaNADP-ME5, were expressed in fruit. The time-course expression analysis of these genes during different fruit ripening stages including green immature (G), breaking point (BP), and red ripe (R) showed that they were differentially modulated. Thus, while CaNADP-ME3 and CaNADP-ME5 were upregulated, CaNADP-ME2 and CaNADP-ME4 were downregulated. Exogenous NO treatment of fruit triggered the downregulation of CaNADP-ME4. Furthermore, the analysis of the cis-regulatory elements showed that CaNADP-ME2 and CaNADP-ME5 were positively regulated by the light-responsive element Box4 whereas CaNADP-ME4 was by ABRE (ACGT-containing abscisic acid response element), which is involved in the abscisic acid (ABA) responsiveness. To get deeper knowledge on this enzyme system from fruits, we obtained the 50-75% ammonium-sulfate-enriched protein fraction containing CaNADP-ME enzyme activity, and this was assayed by non-denaturing polyacrylamide gel electrophoresis (PAGE). The results allowed identifying four isozymes designated CaNADP-ME I to CaNADP-ME IV. Taken together, the data provide new pieces of information on the CaNADP-ME system with the identification of five CaNADP-ME genes and how the four genes expressed in pepper fruits are modulated during ripening and by exogenous NO gas treatment.

Capsicum is the genus where a number of species and varieties have pungent features due to the exclusive content of capsaicinoids such as capsaicin and dihydrocapsaicin. In this work, the main enzymatic and non-enzymatic systems in pepper fruits from four varieties with different pungent capacity has been investigated at two ripening stages. Thus, a sweet pepper variety (Melchor) from California type fruits, and three autochthonous Spanish varieties were used, including Piquillo, Padrón and Alegría riojana. The capsaicinoids contents were determined in pericarp and placenta from fruits showing that these phenyl-propanoids were mainly localized in placenta. The activity profile of catalase, superoxide dismutase (SOD, total and isoenzymatic), the enzymes of the ascorbate-glutathione cycle (AGC) and four NADP-dehydrogenases indicate that some interaction with the capsaicinoid metabolism seems to occur. Among the results obtained on enzymatic antioxidant, the role of an Fe-SOD and the glutathione reductase from the AGC is highlighted. Additionally, it was found that ascorbate and glutathione content were higher in those pepper fruits which displayed the greater contents of capsacinoids. Taken together, all these data indicate that antioxidants may contribute to preserve capsaicinoids metabolism to maintain their functionality in a framework where NADPH is perhaps playing an essential role.

Simple Summary: The distribution of typical bacterial redox enzymes such as pyruvate-ferredoxin oxidoreductase (PFOR) in protozoa remains interestingly puzzling. Previous studies have demonstrated diverse cellular localizations of PFOR in some amitochondriate anaerobic protozoa. PFOR is of particular pharmacological importance because it catalyzes the reductive bio-activation of nitro-based prodrugs to cytotoxic radical metabolites. Metronidazole was developed primarily as an antiprotozoal agent against infections caused by Trichomonas vaginalis. However, its antimicrobial spectrum was subsequently expanded to cover anaerobic bacterial infections. It has been shown that mutations in the genes encoding PFOR result in inherent resistance of PFOR-possessing anaerobic protozoa and bacteria to nitro-based prodrugs. Deciphering the evolutionary history of PFOR is crucial in deepening our understanding of the evolution of anaerobic pathogens and unfold new approaches for drug discovery and targeting in pathogen chemotherapy. Abstract: The present frontrunners in the chemotherapy of infections caused by protozoa are nitro-based prodrugs that are selectively activated by PFOR-mediated redox reactions. This study seeks to analyze the distribution of PFOR in selected protozoa and bacteria by applying comparative bioinformatics approaches to test the hypothesis that PFOR was most likely acquired through horizontal gene transfer (HGT) from bacteria. Furthermore, to identify other genes that were putatively acquired using proteome-wide and gene expression analyses. A plausible explanation for the patchy occurrence of PFOR in protozoa is based on the hypothesis that bacteria are potential sources of genes that enhance optimal adaptation of protozoa in hostile environments. The exclusively expressed proteins obtained from Entamoeba histolytica and the putative gene donor, Desulfovibrio vulgaris, showed an over-expression of eleven genes involved in intermediary metabolism. If these results can be reproduced in other PFOR-possessing protozoa, it would provide a more validated evidence to support the horizontal transfer of pfor from bacteria.