Geobacter’s unique ability to perform extracellular electron transfer (EET) to electrodes in Microbial Fuel Cells (MFCs) has sparked the implementation of sustainable production of electrical energy. However, the electrochemical performance of Geobacter’s biofilms in MFCs remains challenging to implement industrially. Multiple approaches are being investigated to enhance MFC technologies. Protein engineering of multihaem cytochromes, key components of Geobacter’s EET pathways, can, conceivably, be pursued to improve the EET chain. The periplasmic cytochrome PpcA bridges ET from the inner to the outer membrane and its deletion impairs this crucial step. The functional characterisation of PpcA homologs from G. sulfurreducens (Gs) and G. metallireducens (Gm) revealed a significantly different redox behaviour even though they only differ by thirteen amino acids. In a previous study, we found that the single replacement of a tryptophan residue by methionine (W45M) in PpcAGm shifted the reduction potential value 33% towards that of PpcAGs. In this work, we expanded our investigation to include other non-conserved residues by conducting five mutation rounds. We identified the most relevant residues controlling the redox properties of PpcAGm. With just four mutations (K19, G25, N26, W45) the reduction potential value of PpcAGm was shifted 71% toward that of PpcAGs. Additionally, in the quadruple mutant, it was possible to replicate the haem oxidation order and the functional mechanisms of PpcAGs, which differ from those in PpcAGm. Overall, the mutants exhibit diverse redox and functional mechanisms that could be explored as a library for the future design of minimal, synthetic, ET chains in Geobacter.

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.

Three-dimensional (3D) cell culture using tumor spheroids provides a crucial platform for replicating tissue microenvironments. However, effective gene modulation via nanoparticle-based transfection remains a challenge, often facing delivery hurdles. Gold nanoparticles (AuNPs) with their tailored synthesis and biocompatibility, have shown promising results in two-dimensional (2D) cultures, nevertheless, they still require a comprehensive evaluation before they can reach its full potential on 3D models. While 2D cultures offer simplicity and affordability, they lack physiological fidelity. In contrast, 3D spheroids better capture in vivo conditions, enabling the study of cell interactions and nutrient distribution. These models are essential for investigating cancer behavior, drug responses, and developmental processes. Nevertheless, transitioning from 2D to 3D models demands an understanding of altered internalization mechanisms and microenvironmental influences. This study assessed ASO-AuNP conjugates for silencing the c-MYC oncogene in 2D cultures and 3D tumor spheroids, revealing distinctions in gene silencing efficiency and highlighting the microenvironment’s impact on AuNP-mediated gene modulation. Herein, we demonstrate that increasing the number of AuNPs per cell by 2.6 times, when transitioning from a 2D cell model to a 3D spheroid, allows to attain similar silencing efficiencies. Such insights advance the development of targeted gene therapies within intricate tissue-like contexts.

Despite extensive efforts to unravel tumor behavior and develop anticancer therapies, most treatments fail when advanced to clinical trials. The main challenge in cancer research has been the absence of predictive cancer models, accurately mimicking the tumoral processes and response to treatments. The tumor microenvironment (TME) shows several human-specific physical and chemical properties, which cannot be fully recapitulated by the conventional 2D cell cultures or the in vivo animal models. These limitations have driven the development of novel in vitro cancer models, that get one step closer to the typical features of in vivo systems while showing better species relevance. This review introduces the main considerations required for developing and exploiting tumor spheroids and organoids as cancer models. We also detailed their applications in drug screening and personalized medicine. Further, we show the transition of these models into novel microfluidic platforms, for improved control over physiological parameters and high-throughput screening. 3D culture models have provided key insights into tumor biology, more closely resembling the in vivo TME and tumor characteristics, while enabling the development of more reliable and precise anticancer therapies.

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.

Herein, we describe the synthesis and characterization of a series of thiosemicarbazone platinacycles. Their activity towards HCT116 and A2780 cancer cell lines as well as normal fibroblasts was explored and conclusions about the influence of their structures were drawn based on the results. Ligands L1-3, tetranuclear compounds [Pt(L1-3)]4, [Pt(L1-3)(PPh3)], and [Pt(L1-L3)2{Ph2P(CH2)4PPh2}], and phosphine derivatives, were deemed unpromising owing to their lack of activity. However, mono-coordinated diphosphine complexes [Pt(L1-L3)(Ph2PCH2PPh2-P)] showed high selectivity and low IC50 values, and their antiproliferative activity was further studied. The three studied derivatives 3a, 3b and 3c showed a fast internalization of HCT116 colorectal cancer cells with similar IC50 values, which induced a depolarization of mitochondrial membrane potential, with the subsequent triggering of apoptosis and autophagy in the case of 3c. In the case of compounds 3a and 3b, cell death mechanisms (extrinsic and intrinsic apoptosis, respectively) were triggered via the induction of reactive oxygen species (ROS). The three compounds were not toxic to a chicken embryo in vivo (after 48 h), and, importantly, showed an anti-angiogenic potential after exposure to the IC50 of compounds 3a, 3b and 3c.

Flow capacitive deionization (FCDI) is an emerging desalination technology at which flow electrodes (shear-thinning flowable carbon slurries) are used to remove ions from saline water. The geometry of flow electrode channels, which provide the path and ensure the distribution and mixing of the flow electrodes, is one of the most important aspects to be optimized. This work presents experimental and computational fluid dynamics (CFD) modelling analysis of the influence of the geometry of flow electrode channels on FCDI performance. Flow electrode gaskets (with open, serpentine (short) horizontal and serpentine (long) vertical channels) were 3D printed using a polyethylene terephthalate glycol (PET-G) filament. The FCDI cell with a vertical serpentine flow electrode channel exhibited the poorest performance due to channel blockage by carbon particles, while the best results were achieved with a horizontal serpentine flow electrode channel. CFD simulations aided in understanding this behaviour by showing that the channel geometry strongly affects the local shear rate, and thus the local viscosity of flow electrodes. Thus, it is recommended to design channels that induce flow disturbance aiming for increasing the shear rate and hence reducing flow electrode viscosity, therefore promoting their flowability and reducing clogging chances.

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.

Background: The delivery of therapeutic nucleic acids, such as small interfering RNA (siRNA) and antisense oligonucleotides (ASO) into cells, is widely used in gene therapy. Gold nanoparticles (AuNPs) have proved to be effective in delivering silencing moieties with high efficacy. Moreover, AuNPs offer the possibility of spatial–temporal triggering of cell uptake through light irradiation due to their unique optical properties. Our study focuses on the use of AuNPs as improved vectorisation agents through mild photothermy triggered by visible light irradiation. This method promotes the transfection of oligonucleotides for gene silencing in 2D cells and more complex 3D spheroids. Results: Improving gene silencing strategies in 3D cell cultures is crucial since it provides more effective in vitro models to study cellular responses that closely resemble the in vivo tumour microenvironment. We demonstrate the potential of mild photothermy by effectively silencing the GFP gene in 2D cell cultures: HCT116 and MCF-7. Then we showed that mild photothermy could be effectively used for silencing the c-MYC oncogene transcript, which is greatly overexpressed in cancer cells. A decrease of 25% and 30% in c-MYC expression was observed in HCT116 2D cells and 7-day 3D spheroids, respectively. Conclusions: In summary, our findings offer a novel transfection approach for gene therapy applications in 2D and 3D tumour models. This approach is based on the use of mild photothermy mediated by AuNPs combined with visible laser irradiation that might pave the way for the spatial–temporal control of gene modulation.

The bacterium Geotalea uraniireducens, commonly found in uranium-contaminated environments, plays a key role in bioremediation strategies by converting the soluble hexavalent form of uranium (UVI) into less soluble forms (e.g. UIV.). While most of the reduction and concomitant precipitation of uranium occur outside the cells, there have been reports of important reduction processes taking place in the periplasm. In any case, the triheme periplasmic cytochromes are crucial players, either by ensuring an effective interface between the cell´s interior and exterior or by directly participating in the reduction of the metal. Therefore, understanding the functional mechanism of the highly abundant G. uraniireducens’ triheme cytochromes is crucial to assist the elucidation on the respiratory pathways in this bacterium. In this work, a detailed functional characterization of the triheme cytochromes PpcA and PpcB from G. uraniireducens was conducted using NMR and visible spectroscopy techniques. Despite sharing high amino acid sequence and structural homology with their counterparts from G. sulfurreducens, the results obtained showed that the heme reduction potential values are less negative, the order of oxidation of the hemes is distinct, and the redox and redox-Bohr network of interactions revealed unprecedented functional mechanisms of the G. uraniireducens cytochromes. In these cytochromes, the reduction potential values of the three heme groups are much more similar, hence covering a narrow range of values, features that facilitate the directional electron flow from the inner membrane, thereby favouring the optimal reduction of uranium.

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.

Tumor-on-chip (ToC) is crucial to bridge the gap between traditional cell culture experiments and in vivo models, allowing to recreate an in vivo-like microenvironment in cancer research. ToC use microfluidics to provide fine-tune control over environmental factors, high-throughput screening, and reduce requirements of samples and reagents. However, creating these microfluidic devices requires skilled researchers and dedicated manufacturing equipment, making widespread adoption cumbersome and difficult. To address some bottlenecks and improve accessibility to ToC technology, innovative materials and fabrication processes are required. Polystyrene (PS) is a promising material for microfluidics due to its biocompatibility, affordability, and optical transparency. Herein, a fabrication process based on direct laser writing on thermosensitive PS, allowing the swift and economical crafting of devices with easy pattern alterations, is presented. For the first time, a device for cell culture fabricated only by PS is presented, allowing customizing and optimization for efficient cell culture approaches. These biochips support 2D and 3D cultures with comparable viability and proliferation kinetics to traditional 96-well plates. The data show that gene and protein silencing efficiencies remain consistent across both chip and plate-based cultures, either 2D culture or 3D spheroid format. Although simple, this approach might facilitate the use of customized chip-based cancer models.