Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) incidence is rising, and prognosis remains poor due to late diagnosis and limited effective therapies. Currently, patients are treated based on TNM staging, without molecular tumor characterization. This study aimed to validate a technique that combines the amplification refractory mutation system (ARMS) with high-resolution melting analysis (HRMA) for detecting mutations in codon 12 of KRAS in tumor and plasma, and to assess its prognostic value. Methods: Prospective study including patients with newly diagnosed PDAC with tumor and plasma samples collected before treatment. Mutations in codon 12 of KRAS (G12D, G12V, G12C, and G12R) were detected using ARMS–HRMA and compared to Sanger sequencing (SS). Univariate and multivariate analyses were used to evaluate the prognostic significance of these mutations. Results: A total of 88 patients, 93% with ECOG-PS 0–1, 57% with resectable disease. ARMS–HRMA technique showed a higher sensitivity than SS, both in tumor and plasma (77% vs. 51%; 25 vs. 0%, respectively). The most frequent mutation was G12D (n = 32, 36%), followed by G12V (n = 22, 25%). On multivariate analysis, patients with G12D and/or G12C mutations, either in tumor or plasma, had lower PFS (HR 1.792, 95% CI 1.061–3.02

Cu(II) complexes with 2,2′:6′,2″-terpyridines (terpy) and 2,6-bis(thiazol-2-yl)pyridines (dtpy) with 1- or 2-naphtyl and methoxy-naphtyl were synthesized to elucidate the impact of the triimine core, naphtyl linking mode, and presence of methoxy groups on the antiproliferative activity of [CuCl2(Ln)]. Their antiproliferative effect was analyzed in ovarian (A2780) and colorectal (HCT116) carcinomas and colorectal carcinoma resistant to doxorubicin (HCT116-DoxR) cell lines and in normal human fibroblasts. Among all complexes, the 1- and 2-naphtyl substituted terpy Cu(II) complexes (Cu1a and Cu1b) showed the strongest cytotoxicity, namely, in HCT116-DoxR 2Dcells and were also capable of inducing the loss of cell viability in 3D HCT116-DoxR spheroids. Their intracellular localization, capability to increase reactive oxygen species (ROS), and interaction with DNA (nonintercalative mode) trigger oxidative DNA cleavage leading to cell death by apoptosis and autophagy. Cu1a and Cu1b do not show in vivo toxicity in a chicken embryo and can interact with bovine serum albumin (BSA).

Cu(II) complexes with 2,2':6',2″-terpyridines (terpy) and 2,6-bis(thiazol-2-yl)pyridines (dtpy) with 1- or 2-naphtyl and methoxy-naphtyl were synthesized to elucidate the impact of the triimine core, naphtyl linking mode, and presence of methoxy groups on the antiproliferative activity of [CuCl 2(L n )]. Their antiproliferative effect was analyzed in ovarian (A2780) and colorectal (HCT116) carcinomas and colorectal carcinoma resistant to doxorubicin (HCT116-DoxR) cell lines and in normal human fibroblasts. Among all complexes, the 1- and 2-naphtyl substituted terpy Cu(II) complexes ( Cu1a and Cu1b) showed the strongest cytotoxicity, namely, in HCT116-DoxR 2Dcells and were also capable of inducing the loss of cell viability in 3D HCT116-DoxR spheroids. Their intracellular localization, capability to increase reactive oxygen species (ROS), and interaction with DNA (nonintercalative mode) trigger oxidative DNA cleavage leading to cell death by apoptosis and autophagy. Cu1a and Cu1b do not show in vivo toxicity in a chicken embryo and can interact with bovine serum albumin (BSA).

Molybdenum- or tungsten-dependent formate dehydrogenases have emerged as significant catalysts for the chemical reduction of CO${\sb 2}$ to formate, with biotechnological applications envisaged in climate-change mitigation. The role of Met405 in the active site of ıt Desulfovibrio vulgaris} formate dehydrogenase AB (ıt Dv}FdhAB) has remained elusive. However, its proximity to the metal site and the conformational change that it undergoes between the resting and active forms suggests a functional role. In this work, the M405S variant was engineered, which allowed the active-site geometry in the absence of methionine S${\sp {$δ$}}$ interactions with the metal site to be revealed and the role of Met405 in catalysis to be probed. This variant displayed reduced activity in both formate oxidation and CO${\sb 2}$ reduction, together with an increased sensitivity to oxygen inactivation.

In this work, we report the disruptive use of membrane-localized magnetic hyperthermia to promote the internalization of cell-impermeant probes. Under an alternating magnetic field, magnetic nanoparticles (MNPs) immobilized on the cell membrane via bioorthogonal click chemistry act as nanoheaters and lead to the thermal disruption of the plasma membrane, which can be used for internalization of different types of molecules, such as small fluorescent probes and nucleic acids. Noteworthily, no cell death, oxidative stress and alterations of the cell cycle are detected after the thermal stimulus, although cells are able to sense and respond to the thermal stimulus through the expression of different types of heat shock proteins (HSPs). Finally, we demonstrate the utility of this approach for the transfection of cells with a small interference RNA (siRNA), revealing a similar efficacy to a standard transfection method based on the use of cationic lipid-based reagents (such as Lipofectamine), but with lower cell toxicity. These results open the possibility of developing new procedures for “opening and closing” cellular membranes with minimal disturbance of cellular integrity. This on-demand modification of cell membrane permeability could allow the direct intracellular delivery of biologically relevant (bio)molecules, drugs and nanomaterials, thus overcoming traditional endocytosis pathways and avoiding endosomal entrapment.

In recent years, organometallic complexes have attracted much attention as anticancer therapeutics aiming at overcoming the limitations of platinum drugs that are currently marketed. Still, the development of half-sandwich organometallic cobalt complexes remains scarcely explored. Four new cobalt(III)-cyclopentadienyl complexes containing N,N-heteroaromatic bidentate, and phosphane ligands were synthesized and fully characterized by elemental analysis, spectroscopic techniques, and DFT methods. The cytotoxicity of all complexes was determined in vitro by the MTS assay in colorectal (HCT116), ovarian (A2780), and breast (MDA-MB-231 and MCF-7) human cancer cell lines and in a healthy human cell line (fibroblasts). The complexes showed high cytotoxicity in cancer cell lines, mostly due to ROS production, apoptosis, autophagy induction, and disruption of the mitochondrial membrane. Also, these complexes were shown to be nontoxic in vivo in an ex ovo chick embryo yolk sac membrane (YSM) assay.

Metal-dependent formate dehydrogenases reduce CO2 with high efficiency and selectivity, but are usually very oxygen sensitive. An exception is Desulfovibrio vulgaris W/Sec-FdhAB, which can be handled aerobically, but the basis for this oxygen tolerance was unknown. Here we show that FdhAB activity is controlled by a redox switch based on an allosteric disulfide bond. When this bond is closed, the enzyme is in an oxygen-tolerant resting state presenting almost no catalytic activity and very low formate affinity. Opening this bond triggers large conformational changes that propagate to the active site, resulting in high activity and high formate affinity, but also higher oxygen sensitivity. We present the structure of activated FdhAB and show that activity loss is associated with partial loss of the metal sulfido ligand. The redox switch mechanism is reversible in vivo and prevents enzyme reduction by physiological formate levels, conferring a fitness advantage during O2 exposure.

The structural and chemical diversity of β-glucans is reflected on the variety of essential biological roles tackled by these polysaccharides. This natural heterogeneity requires an elaborate assortment of enzymatic mechanisms to assemble, degrade or modify, as well as to extract their full biotechnological potential. Recent metagenomic efforts have provided an unprecedented growth in potential new biocatalysts, most of which remain unconfirmed or uncharacterized. Here we report the first biochemical and structural characterization of two bacterial β-glucanases from the recently created glycoside hydrolase family 157 (LaGH157 and BcGH157) and investigate their molecular basis for substrate hydrolysis. Structural analysis by X-ray crystallography revealed that GH157 enzymes belong to clan GH-A, possessing a (β/α)8-barrel fold catalytic domain, two β-sandwich accessory domains and two conserved catalytic glutamates residues, with relative positions compatible with a retaining mechanism of hydrolysis. Specificity screening and enzyme kinetics suggest that the enzymes prefer mixed-linkage glucans over β-1,3-glucans. Activity screening showed that both enzymes exhibit pH optimum at 6.5 and temperature optimum for LaGH157 and BcGH157 at 25 °C and 48 °C, respectively. Product analysis with HPAEC-PAD and LC-MS revealed that both enzymes are endo-1,3(4)-β-glucanases, capable of cleaving β-1,3 and β-1,4-linked glucoses, when preceded by a β-1,3 linkage. Moreover, BcGH157 needs a minimum of 4 subsites occupied for hydrolysis to occur, while LaGH157 only requires 3 subsites. Additionally, LaGH157 possesses exohydrolytic activity on β-1,3 and branching β-1,6 linkages. This unusual bifunctional endo-1,3(4)/exo-1,3–1,6 activity constitutes an expansion on our understanding of β-glucan deconstruction, with the potential to inspire future applications.

Metal-dependent formate dehydrogenases are very promising targets for enzyme optimization and design of bio-inspired catalysts for CO2 reduction, towards innovative strategies for climate change mitigation. For effective application of these enzymes, the catalytic mechanism must be better understood, and the molecular determinants clarified. Despite numerous studies, several doubts persist, namely regarding the role played by the possible dissociation of the SeCys ligand from the Mo/W active site. Additionally, the oxygen sensitivity of these enzymes must also be understood as it poses an important obstacle for biotechnological applications. Here we present a combined biochemical, spectroscopic, and structural characterization of Desulfovibrio vulgaris FdhAB (DvFdhAB) when exposed to oxygen in the presence of a substrate (formate or CO2). This study reveals that O2 inactivation is promoted by the presence of either substrate and involves forming a different species in the active site, captured in the crystal structures, where the SeCys ligand is displaced from tungsten coordination and replaced by a dioxygen or peroxide molecule. This form was reproducibly obtained and supports the conclusion that, although W-DvFdhAB can catalyse the oxidation of formate in the presence of oxygen for some minutes, it gets irreversibly inactivated after prolonged O2 exposure in the presence of either substrate.

Extracellular electron transfer (EET) via microbial nanowires drives globally-important environmental processes and biotechnological applications for bioenergy, bioremediation, and bioelectronics. Due to highly-redundant and complex EET pathways, it is unclear how microbes wire electrons rapidly (>106 s−1) from the inner-membrane through outer-surface nanowires directly to an external environment despite a crowded periplasm and slow (<105 s−1) electron diffusion among periplasmic cytochromes. Here, we show that Geobacter sulfurreducens periplasmic cytochromes PpcABCDE inject electrons directly into OmcS nanowires by binding transiently with differing efficiencies, with the least-abundant cytochrome (PpcC) showing the highest efficiency. Remarkably, this defined nanowire-charging pathway is evolutionarily conserved in phylogenetically-diverse bacteria capable of EET. OmcS heme reduction potentials are within 200 mV of each other, with a midpoint 82 mV-higher than reported previously. This could explain efficient EET over micrometres at ultrafast (<200 fs) rates with negligible energy loss. Engineering this minimal nanowire-charging pathway may yield microbial chassis with improved performance.

In Portugal, digital transition was structured with national public policies since 2003. In 2017, initiatives for the adoption of Industry 4.0 concepts are implemented in Portugal. We analysed the diffusion and implementation of these technologies, in Portugal. Some questions were raised: has the interplay between public policies, state agencies and industrial relations players in the process been articulated, as in Germany? What have been the effects of these technologies on workers and organisations? Are the public initiatives in place enough or more is needed? Qualitative and quantitative approaches were used to collect evidence on the main features and constraints of a public policy for Industry 4.0, based on the case study of the automotive sector in Portugal. Findings suggest the need to balance regulatory policies on data related risks, and investment policies towards education, training and organisational innovation are needed to complement technology development and adoption support.

Lung cancer remains the top killing cancer worldwide despite advances in treatment. Seven ethanolic plant extracts were selected and evaluated for their antiproliferative activity against the two main types of lung cancers: non-small cell (A549) and small cell lung cancer cells (SHP-77). An ethanolic extract of Helichrysum odoratissimum Sweet (HO) showed significant antiproliferative activity against lung cancer, with a fifty percent inhibitory concentration (IC50) of 83.43 ± 1.60 µg/mL (A549), 49.46 ± 0.48 µg/mL (SHP-77) and 50.71 ± 2.27 µg/mL, against normal lung epithelial cells (MRC-5), resulting in a selectivity index (SI) value of 0.61 on A549 cells and 1.03 on SHP-77 cells, which was compared to the positive drug control, actinomycin D where the SI values were found to be 2 and 0.25 against A549 and SHP-77 cells, respectively. Against murine macrophages (RAW 264.7) and hepatocytes (HepG2), the HO ethanolic extract showed IC50 values of 60.15 ± 1.98 µg/mL and 23.61 ± 1.06 µg/mL, respectively. Microscopy showed that the HO ethanolic extract induced apoptosis in the A549 and HepG2 cells at 50 µg/mL and 300 µg/mL, respectively. The HO ethanolic extract, furthermore, inhibited the pro-inflammatory enzymes, cyclooxygenase 2 (COX-2) and 5-lipoxygenase (5-LOX) with IC50 values of 7.94 ± 3.84 µg/mL and 2.08 ± 1.35 µg/mL, respectively, whereas the positive controls Ibuprofen (COX-2) and Zileuton (5-LOX) showed IC50 values of 0.85 ± 0.14 µg/mL and 0.06 ± 0.05 µg/mL, respectively. The activity of NAD(P)H quinone oxidoreductase-1 (NQO1), which is a direct target of nuclear factor erythroid-2-related factor-2 (NRF2), was significantly inhibited in the A549 cells by the HO ethanolic extract (at 125 µg/mL) when compared to the positive control, brusatol (at 500 nM). Using the ex ovo yolk sac membrane (YSM) assay, the HO ethanolic extract (at 18.5 µg/egg) showed a 31.65 ± 12.80% inhibition of blood vessel formation. This is the first report of the noteworthy antiproliferative activity of the HO ethanolic extract on lung cancer cells including its potential to target several enzymes associated with inflammation and therefore, should be considered for further analysis.

Eight 2,2′:6′,2″-terpyridines, substituted at the 4′-position with aromatic groups featuring variations in π-conjugation, ring size, heteroatoms, and methoxy groups, were employed to enhance the antiproliferative potential of [Cu2Cl2(R-terpy)2](PF6)2. Assessing the cytotoxicity in A2780 (ovarian carcinoma), HCT116 (colorectal carcinoma), and HCT116DoxR (colorectal carcinoma resistant to doxorubicin) and normal primary fibroblasts revealed that Cu(II) complexes with 4-quinolinyl, 4-methoxy-1-naphthyl, 2-furanyl, and 2-pyridynyl substituents showed superior therapeutic potential in HCT116DoxR cells with significantly reduced cytotoxicity in normal fibroblasts (42-129× lower). Besides their cytotoxicity, the Cu(II) complexes are able to increase intracellular ROS and interfere with cell cycle progression, leading to cell death by apoptosis and autophagy. Importantly, they demonstrated antimetastatic and antiangiogenic properties without in vivo toxicity. In accordance with their nuclear accumulation, the Cu(II) complexes are able to cleave pDNA and interact with bovine serum albumin, which is a good indication of their ability for internalization and transport toward tumor cells.

Lung cancer (LC) is recognized as one of the most prevalent and lethal cancers worldwide, underscoring an urgent need for innovative diagnostic and therapeutic approaches. MicroRNAs (miRNAs) have emerged as promising biomarkers for several diseases and their progression, such as LC. However, traditional methods for detecting and quantifying miRNAs, such as PCR, are time-consuming and expensive. Herein, we used a molecular beacon (MB) bead-based assay immobilized in a microfluidic device to detect miR-155-3p, which is frequently overexpressed in LC. The assay relies on the fluorescence enhancement of the MB upon binding to the target miRNA via Watson and Crick complementarity, resulting in a conformational change from a stem–loop to a linear structure, thereby bringing apart the fluorophores at each end. This assay was performed on a microfluidic platform enabling rapid and straightforward target detection. We successfully detected miR-155-3p in a saline solution, obtaining a limit of detection (LOD) of 42 nM. Furthermore, we evaluated the method’s performance in more complex biological samples, including A549 cells’ total RNA and peripheral blood mononuclear cells (PBMCs) spiked with the target miRNA. We achieved satisfactory recovery rates, especially in A549 cells’ total RNA.

Azaindole scaffold is a privileged structure in medicinal chemistry and some derivatives have demonstrated to be potential anticancer drugs. Herein, a set of novel azaindoles, comprising the four regioisomers, bearing a morpholine (azaindoles 3a-d) and N-methyl-N-benzylamine (azaindoles 4a-d) groups were prepared. Among these compounds, azaindoles 4 exhibited higher cytotoxicity against the ovarian cancer cell line A2780 and normal dermal fibroblasts compared to azaindoles 3. Furthermore, azaindoles 4b and 4c promoted a delay in the cell cycle of the cancer cell line, inspiring an investigation into the intracellular localization of these derivatives.

The accumulation of manganese ions is crucial for scavenging reactive oxygen species (ROS) and protecting the proteome of Deinococcus radiodurans (Dr). However, metal homeostasis still needs to be tightly regulated to avoid toxicity. DR2539, a dimeric transcription regulator, plays a key role in Dr manganese homeostasis. Despite comprising three well-conserved domains: a DNA binding domain, a dimerization domain, and an ancillary domain, both the metal ion activation mechanism and the DNA recognition mechanism remain elusive. In this study, we present biophysical analyses and the structure of the dimerization and DNA binding domains of DR2539 in its holo form and in complex with the 21 bp pseudo-palindromic repeat of the dr1709 promotor region. These findings shed light into the activation and recognition mechanisms. The dimer presents eight manganese binding sites that induce structural conformations essential for DNA binding. The analysis of the protein-DNA interfaces elucidates the significance of Tyr59 and helix H3 sequence in the interaction with the DNA. Finally, the structure in solution as determined by small angle X-ray scattering experiments and supported by AlphaFold modelling provides a model illustrating the conformational changes induced upon metal binding.Competing Interest StatementThe authors have declared no competing interest.

Cu(II) complexes with 2,2′:6′,2″-terpyridines (terpy) and 2,6-bis(thiazol-2-yl)pyridines (dtpy) with 1- or 2-naphtyl and methoxy-naphtyl were synthesized to elucidate the impact of the triimine core, naphtyl linking mode, and presence of methoxy groups on the antiproliferative activity of [CuCl2(Ln)]. Their antiproliferative effect was analyzed in ovarian (A2780) and colorectal (HCT116) carcinomas and colorectal carcinoma resistant to doxorubicin (HCT116-DoxR) cell lines and in normal human fibroblasts. Among all complexes, the 1- and 2-naphtyl substituted terpy Cu(II) complexes (Cu1a and Cu1b) showed the strongest cytotoxicity, namely, in HCT116-DoxR 2Dcells and were also capable of inducing the loss of cell viability in 3D HCT116-DoxR spheroids. Their intracellular localization, capability to increase reactive oxygen species (ROS), and interaction with DNA (nonintercalative mode) trigger oxidative DNA cleavage leading to cell death by apoptosis and autophagy. Cu1a and Cu1b do not show in vivo toxicity in a chicken embryo and can interact with bovine serum albumin (BSA).

Abstract Multiheme cytochromes located in different compartments are crucial for extracellular electron transfer in the bacterium Geobacter sulfurreducens to drive important environmental processes and biotechnological applications. Recent studies have unveiled that for particular sets of electron terminal acceptors, discrete respiratory pathways selectively recruit specific cytochromes from both the inner and outer membranes. However, such specificity was not observed for the abundant periplasmic cytochromes, namely the triheme cytochrome family PpcA-E. In this work, the distinctive NMR spectroscopic signatures of these proteins in different redox states were explored to monitor pairwise interactions and electron transfer reactions between each pair of cytochromes. The results showed that the five proteins interact transiently and can exchange electrons between each other revealing intra-promiscuity within the members of this family. This discovery is discussed in the light of the establishment of an effective electron transfer network by this pool of cytochromes. This network is advantageous to the bacteria as it enables the maintenance of the functional working potential redox range within the cells.