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Chondroitin is a glycosaminoglycan that has gained widespread use in nutraceuticals and pharmaceuticals, mainly for treating osteoarthritis. Traditionally, it has been extracted from animal cartilage but recently, biotechnological processes have emerged as a commercial alternative to avoid the risk of viral or prion contamination and offer a vegan-friendly source. Typically, these methods involve producing the chondroitin backbone using pathogenic bacteria and then modifying it enzymatically through the action of sulfotransferases. Despite the challenges of expressing active sulfotransferases in bacteria, the use of eukaryotic microorganisms is still limited to a few works using Pichia pastoris. To create a safer and efficient biotechnological platform, we constructed a biosynthetic pathway for chondroitin production in S. cerevisiae as a proof-of-concept. Up to 125 mg/L and 200 mg/L of intracellular and extracellular chondroitin were produced, respectively. Furthermore, as genome-scale models are valuable tools for identifying novel targets for metabolic engineering, a stoichiometric model of chondroitin-producing S. cerevisiae was developed and used in optimization algorithms. Our research yielded several novel targets, such as uridine diphosphate (UDP)-N-acetylglucosamine pyrophosphorylase (QRI1), glucosamine-6-phosphate acetyltransferase (GNA1), or N-acetylglucosamine-phosphate mutase (PCM1) overexpression, that might enhance chondroitin production and guide future experimental research to develop more efficient host organisms for the biotechnological production process.

ABSTRACTChronic obstructive pulmonary disease (COPD), a condition primarily linked to oxidative stress, poses significant health burdens worldwide. Recent evidence has shed light on the association between the dysbiosis of gut microbiota and COPD, and their metabolites have emerged as potential modulators of disease progression through the intricate gut-lung axis. Here, we demonstrate the efficacy of oral administration of the probiotic Pediococcus pentosaceus SMM914 (SMM914) in delaying the progression of COPD by attenuating pulmonary oxidative stress. Specially, SMM914 induces a notable shift in the gut microbiota toward a community structure characterized by an augmented abundance of probiotics producing short-chain fatty acids and antioxidant metabolisms. Concurrently, SMM914 synthesizes L-tryptophanamide, 5-hydroxy-L-tryptophan, and 3-sulfino-L-alanine, thereby enhancing the tryptophan-melatonin pathway and elevating 6-hydroxymelatonin and hypotaurine in the lung environment. This modulation amplifies the secretion of endogenous anti-inflammatory factors, diminishes macrophage polarization toward the M1 phenotype, and ultimately mitigates the oxidative stress in mice with COPD. The demonstrated efficacy of the probiotic intervention, specifically with SMM914, not only highlights the modulation of intestine microbiota but also emphasizes the consequential impact on the intricate interplay between the gastrointestinal system and respiratory health.

Epilactose is a disaccharide composed of galactose and mannose, and it is currently considered an “under development” prebiotic. In this study, we described the prebiotic potential of epilactose by in vitro fermentation using human fecal inocula from individuals following a Mediterranean diet (DM) or a Vegan diet (DV). The prebiotic effect of epilactose was also compared with lactulose and raffinose, and interesting correlations were established between metabolites and microbiota modulation. The production of several metabolites (lactate, short-chain fatty acids, and gases) confirmed the prebiotic properties of epilactose. For both donors, the microbiota analysis showed that epilactose significantly stimulated the butyrate-producing bacteria, suggesting that its prebiotic effect could be independent of the donor diet. Butyrate is one of the current golden metabolites due to its benefits for the gut and systemic health. In the presence of epilactose, the production of butyrate was 70- and 63-fold higher for the DM donor, when compared to lactulose and raffinose, respectively. For the DV donor, an increase of 29- and 89-fold in the butyrate production was obtained when compared to lactulose and raffinose, respectively. In conclusion, this study suggests that epilactose holds potential functional properties for human health, especially towards the modulation of butyrate-producing strains.

Abstract Zymomonas mobilis is a well-recognized ethanologenic bacterium with outstanding characteristics which make it a promising platform for the biotechnological production of relevant building blocks and fine chemicals compounds. In the last years, research has been focused on the physiological, genetic, and metabolic engineering strategies aiming at expanding Z. mobilis ability to metabolize lignocellulosic substrates toward biofuel production. With the expansion of the Z. mobilis molecular and computational modeling toolbox, the potential of this bacterium as a cell factory has been thoroughly explored. The number of genomic, transcriptomic, proteomic, and fluxomic data that is becoming available for this bacterium has increased. For this reason, in the forthcoming years, systems biology is expected to continue driving the improvement of Z. mobilis for current and emergent biotechnological applications. While the existing molecular toolbox allowed the creation of stable Z. mobilis strains with improved traits for pinpointed biotechnological applications, the development of new and more flexible tools is crucial to boost the engineering capabilities of this bacterium. Novel genetic toolkits based on the CRISPR-Cas9 system and recombineering have been recently used for the metabolic engineering of Z. mobilis. However, they are mostly at the proof-of-concept stage and need to be further improved. Graphical Abstract

Chondroitin sulfate (CS) is a glycosaminoglycan with a broad range of applications being a popular dietary supplement for osteoarthritis. Usually, CS is extracted from animal sources. However, the known risks of animal products use have been driving the search for alternative methods and sources to obtain this compound. Several pathogenic bacteria naturally produce chondroitin-like polysaccharides through well-known pathways and, therefore, have been the basis for numerous studies that aim to produce chondroitin using non-pathogenic hosts. However, the yields obtained are not enough to meet the high demand for this glycosaminoglycan. Metabolic engineering strategies have been used to construct improved heterologous hosts. The identification of metabolic bottlenecks and regulation points, and the screening for efficient enzymes are key points for constructing microbial cell factories with improved chondroitin yields to achieve industrial CS production. The recent advances on enzymatic and microbial strategies to produce non-animal chondroitin are herein reviewed. Challenges and prospects for future research are also discussed.

Microbial exopolysaccharides (EPS) are promising alternatives to synthetic polymers in a variety of applications. Their high production costs, however, limit their use despite their outstanding properties. The use of low-cost substrates such as agro-industrial wastes in their production, can help to boost their market competitiveness. In this work, an alternative low-cost culture medium (CSLM) was developed for EPS production by Rhizobium viscosum CECT908, containing sugarcane molasses (60 g/L) and corn steep liquor (10 mL/L) as sole ingredients. This medium allowed the production of 6.1 ± 0.2 g EPS/L, twice the amount produced in the standard medium (Syn), whose main ingredients were glucose and yeast extract. This is the first report of EPS production by R. viscosum using agro-industrial residues as sole substrates. EPSCSLM and EPSSyn exhibited a similar carbohydrate composition, mainly 4-linked galactose, glucose and mannuronic acid. Although both EPS showed a good fit to the Herschel–Bulkley model, EPSCSLM displayed a higher yield stress and flow consistency index when compared with EPSSyn, due to its higher apparent viscosity. EPSCSLM demonstrated its potential use in Microbial Enhanced Oil Recovery by enabling the recovery of nearly 50% of the trapped oil in sand-pack column experiments using a heavy crude oil.

Coumarins and furanocoumarins are plant secondary metabolites with known biological activities. As they are present in low amounts in plants, their heterologous production emerged as a more sustainable and efficient approach to plant extraction. Although coumarins biosynthesis has been positively established, furanocoumarin biosynthesis has been far more challenging. This study aims to evaluate if Escherichia coli could be a suitable host for furanocoumarin biosynthesis. The biosynthetic pathway for coumarins biosynthesis in E. coli was effectively constructed, leading to the production of umbelliferone, esculetin and scopoletin (128.7, 17.6, and 15.7 µM, respectively, from tyrosine). However, it was not possible to complete the pathway with the enzymes that ultimately lead to furanocoumarins production. Prenyltransferase, psoralen synthase, and marmesin synthase did not show any activity when expressed in E. coli. Several strategies were tested to improve the enzymes solubility and activity with no success, including removing potential N-terminal transit peptides and expression of cytochrome P450 reductases, chaperones and/or enzymes to increase dimethylallylpyrophosphate availability. Considering the results herein obtained, E. coli does not seem to be an appropriate host to express these enzymes. However, new alternative microbial enzymes may be a suitable option for reconstituting the furanocoumarins pathway in E. coli. Nevertheless, until further microbial enzymes are identified, Saccharomyces cerevisiae may be considered a preferred host as it has already been proven to successfully express some of these plant enzymes.

This work reports for the first time a colorimetric based biosensor using nanocellulose (NC) based supports drop-deposited onto a cellulose paper substrate for glucose detection in point-of-care. For this purpose, microcrystalline cellulose (MCC) samples were oxidized with 2,2,6,6-tetramethylpiperidine-N-oxyl radical (TEMPO), sodium hypochlorite, and potassium bromide, to produce carboxylated NC. For the characterization, we used several methods: TEM, FTIR and conductometric titration. In all samples, the primary alcohol groups were selectively oxidized into carboxyl groups, provided the sodium hypochlorite is added dropwise and the reaction is performed at constant pH 10. Carboxyl- NC was further casted on a cellulose substrate and used as support for glucose oxidase (GOx), horseradish peroxidase (HRP) and 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) reactions, aiming to yield a coloured detection system for glucose.The sensing system was generated by integrating GOx on the carboxyl- NC /cellulose substrate. Upon reaction with glucose, the enzyme produced hydrogen peroxide, which was converted into a blue-coloured product by reaction with HRP and the chromogenic reagent ABTS. The test-strip was calibrated by incubating it in different concentrations of glucose. The colours obtained were further analysed by a suitable image analysis software. Linear response for glucose ranged from 1.5 to 13.0 mM.Overall, this new test-strip used renewable material for glucose determination, which is an advantage when compared to other systems that require more complex technological approaches. Moreover, it was found that carboxyl- NC improved the colour homogeneity of the test-strip and the intrinsic linear response of concentration range.

Bovine lactoferrin (bLF) has been extensively described as a wide spectrum antimicrobial protein. bLF bactericidal activity has been mainly attributed to two different mechanisms: environmental iron depletion and cell membrane destabilization. Due to its antimicrobial properties, bLF has been included in the formulation nutraceutical food products and edible active packages. This work comprises the experimental evidence of the requirement of bLF unrestricted mobility (“free bLF”) to effectively perform its bactericidal action. To assess the unrestricted and restricted bLF activity, a nontoxic matrix of bacterial nanocellulose (BNC) was used as carrier, and as an anchoring scaffold, respectively. Therefore, BNC was functionalized with bLF through two different methodologies: (i) bLF was embedded within the three-dimensional structure of BNC and; (ii) bLF was covalently bounded to the nanofibrils of BNC. bLF efficiency was tested against two bacteria isolated from clinical specimens, Escherichia coli and Staphylococcus aureus. bLF concentration after covalent binding to BNC was two-fold higher in comparison to the embedding method. Nevertheless, only the embedded bLF exhibited a significant bactericidal activity, due to bLF ability to permeate the BNC matrix and execute its bactericidal action.

Curcuminoids are well-known for their therapeutic properties. However, their extraction from natural sources is environmentally unfriendly, expensive and limited by seasonal variability, highlighting the need for alternative production processes. We propose an optimized artificial biosynthetic pathway to produce curcuminoids, including curcumin, in Escherichia coli. This pathway involves six enzymes, tyrosine ammonia lyase (TAL), 4-coumarate 3-hydroxylase (C3H), caffeic acid O-methyltransferase (COMT), 4-coumarate-CoA ligase (4CL), diketide-CoA synthase (DCS), and curcumin synthase (CURS1). Curcuminoids pathway was divided in two modules, the first module included TAL, C3H and COMT and the second one 4CL, DCS and CURS1. Optimizing the first module of the pathway, from tyrosine to ferulic acid, enabled obtaining the highest ferulic acid titer reported so far (1325.1 μM). Afterward, ferulic acid was used as substrate to optimize the second module of the pathway. We achieved the highest concentration of curcumin ever reported (1529.5 μM), corresponding to a 59.4% increase. Subsequently, curcumin and other curcuminoids were produced from tyrosine (using the whole pathway) in mono-culture. The production increased comparing to a previously reported pathway that used a caffeoyl-CoA O-methyltransferase enzyme (to convert caffeoyl-CoA to feruloyl-CoA) instead of COMT (to convert caffeic to ferulic acid). Additionally, the potential of a co-culture approach was evaluated to further improve curcuminoids production by reducing cells metabolic burden. We used one E. coli strain able to convert tyrosine to ferulic acid and another able to convert the hydroxycinnamic acids produced by the first one to curcuminoids. The co-culture strategies tested led to 6.6 times increase of total curcuminoids (125.8 μM) when compared to the mono-culture system. The curcuminoids production achieved in this study corresponds to a 6817% improvement. In addition, by using an inoculation ratio of 2:1, although total curcuminoids production decreased, curcumin production was enhanced and reached 43.2 μM, corresponding to an improvement of 160% comparing to mono-culture system. To our knowledge, these values correspond to the highest titers of curcuminoids obtained to date. These results demonstrate the enormous potential of modular co-culture engineering to produce curcumin, and other curcuminoids, from tyrosine.

Lactoferrin (Lf) is a milk-derived protein with well-recognized potential as a therapeutic agent against a wide variety of cancers. This natural protein exhibits health-promoting effects and has several interesting features, including its selectivity towards cancer cells, good tolerability in humans, worldwide availability, and holding a generally recognized as safe (GRAS) status. To prompt the rational clinical application of this promising anticancer compound, previous works aimed to unveil the molecular mechanisms underlying its selective anticancer activity, where plasmalemmal V-ATPase was identified as an Lf target in cancer cells. V-ATPase is a proton pump critical for cellular homeostasis that migrates to the plasma membrane of highly metastatic cancer cells contributing to the acidity of the tumor microenvironment. Cancer cells were found to be susceptible to Lf only when this proton pump is present at the plasma membrane. Plasmalemmal V-ATPase can thus be an excellent biomarker for driving treatment decisions and forecasting clinical outcomes of Lf-based anticancer strategies. Future research endeavors should thus seek to validate this biomarker by thorough preclinical and clinical studies, as well as to develop effective methods for its detection under clinical settings.

Uridine diphosphate-glucose dehydrogenase (UGD) is an enzyme that produces uridine diphosphate-glucuronic acid (UDP-GlcA), which is an intermediate in glycosaminoglycans (GAGs) production pathways. GAGs are generally extracted from animal tissues. Efforts to produce GAGs in a safer way have been conducted by constructing artificial biosynthetic pathways in heterologous microbial hosts. This work characterizes novel enzymes with potential for UDP-GlcA biotechnological production. The UGD enzymes from Zymomonas mobilis (ZmUGD) and from Lactobacillus johnsonii (LbjUGD) were expressed in Escherichia coli. These two enzymes and an additional eukaryotic one from Capra hircus (ChUGD) were also expressed in Saccharomyces cerevisiae strains. The three enzymes herein studied represent different UGD phylogenetic groups. The UGD activity was evaluated through UDP-GlcA quantification in vivo and after in vitro reactions. Engineered E. coli strains expressing ZmUGD and LbjUGD were able to produce in vivo 28.4 µM and 14.9 µM UDP-GlcA, respectively. Using S. cerevisiae as the expression host, the highest in vivo UDP-GlcA production was obtained for the strain CEN.PK2-1C expressing ZmUGD (17.9 µM) or ChUGD (14.6 µM). Regarding the in vitro assays, under the optimal conditions, E. coli cell extract containing LbjUGD was able to produce about 1800 µM, while ZmUGD produced 407 µM UDP-GlcA, after 1 h of reaction. Using engineered yeasts, the in vitro production of UDP-GlcA reached a maximum of 533 µM using S. cerevisiae CEN.PK2-1C_pSP-GM_LbjUGD cell extract. The UGD enzymes were active in both prokaryotic and eukaryotic hosts, therefore the genes and expression chassis herein used can be valuable alternatives for further industrial applications.

Most biosurfactants are obtained using costly culture media and purification processes, which limits their wider industrial use. Sustainability of their production processes can be achieved, in part, by using cheap substrates found among agricultural and food wastes or byproducts. In the present study, crude glycerol, a raw material obtained from several industrial processes, was evaluated as a potential low-cost carbon source to reduce the costs of surfactin production by Bacillus subtilis #309. The culture medium containing soap-derived waste glycerol led to the best surfactin production, reaching about 2.8 g/L. To the best of our knowledge, this is the first report describing surfactin production by B. subtilis using stearin and soap wastes as carbon sources. A complete chemical characterization of surfactin analogs produced from the different waste glycerol samples was performed by liquid chromatography–mass spectrometry (LC-MS) and Fourier transform infrared spectroscopy (FTIR). Furthermore, the surfactin produced in the study exhibited good stability in a wide range of pH, salinity and temperatures, suggesting its potential for several applications in biotechnology.

Saccharomyces cerevisiae has been for a long time a common model for fundamental biological studies and a popular biotechnological engineering platform to produce chemicals, fuels, and pharmaceuticals due to its peculiar characteristics. Both lines of research require an effective editing of the native genetic elements or the inclusion of heterologous pathways into the yeast genome. Although S. cerevisiae is a well-known host with several molecular biology tools available, a more precise tool is still needed. The clustered, regularly interspaced, short palindromic repeats–associated Cas9 (CRISPR-Cas9) system is a current, widespread genome editing tool. The implementation of a reprogrammable, precise, and specific method, such as CRISPR-Cas9, to edit the S. cerevisiae genome has revolutionized laboratory practices. Herein, we describe and discuss some applications of the CRISPR-Cas9 system in S. cerevisiae from simple gene knockouts to more complex processes such as artificial heterologous pathway integration, transcriptional regulation, or tolerance engineering.

Lactosucrose is a synthetic trisaccharide composed of galactose, glucose and fructose. This compound is obtained through enzymatic synthesis using lactose and sucrose as substrates. The enzymes involved in the process are able to catalyse both hydrolysis and transfer reactions. The yield and productivity of the process are usually affected by the occurrence of parallel hydrolysis of the newly formed product (lactosucrose). Therefore, it is important to find efficient strategies to avoid or minimize product degradation. Furthermore, in the last decades the demand for lactosucrose has significantly increased. This compound is considered a potential prebiotic and several beneficial effects associated to its consumption have been described. As a result, it has been included in the formulations of functional foods. This review covers the most relevant information about lactosucrose, including its synthesis and purification, beneficial effects at physiological level, and also its potential applications.

Polyphenols are plant secondary metabolites with diverse biological and potential therapeutic activities such as antioxidant, anti-inflammatory and anticancer, among others. However, their extraction from the native plants is not enough to satisfy the increasing demand for this type of compounds. The development of microbial cell factories to effectively produce polyphenols may represent the most attractive solution to overcome this limitation and produce high amounts of these bioactive molecules. With the advances in the synthetic biology field, the development of efficient microbial cell factories has become easier, largely due to the development of the molecular biology techniques and by the identification of novel isoenzymes in plants or simpler organisms to construct the heterologous pathways. Furthermore, efforts have been made to make the process more profitable through improvements in the host chassis. In this review, advances in the production of polyphenols by genetically engineered Saccharomyces cerevisiae as well as by synthetic biology and metabolic engineering approaches to improve the production of these compounds at industrial settings are discussed.

Prostate cancer and osteosarcoma are the second most common type of cancer affecting men and the fifth most common malignancy among adolescents, respectively. The use of non-toxic natural or natural-derived products has been one of the current strategies for cancer therapy, owing to the reduced risks of induced-chemoresistance development and the absence of secondary effects. In this perspective, lactoferrin (Lf), a natural protein derived from milk, emerges as a promising anticancer agent due to its well-recognized cytotoxicity and anti-metastatic activity. Here, we aimed to ascertain the potential activity of bovine Lf (bLf) against highly metastatic cancer cells. The bLf effect on prostate PC-3 and osteosarcoma MG-63 cell lines, both displaying plasmalemmal V-ATPase, was studied and compared with the breast cancer MDA-MB-231 and the non-tumorigenic BJ-5ta cell lines. Cell proliferation, cell death, intracellular pH, lysosomal acidification, and extracellular acidification rate were evaluated. Results show that bLf inhibits proliferation, induces apoptosis, intracellular acidification, and perturbs lysosomal acidification only in highly metastatic cancer cell lines. By contrast, BJ-5ta cells are insensitive to bLf. Overall, our results establish a common mechanism of action of bLf against highly metastatic cancer cells exhibiting plasmalemmal V-ATPase. This study opens promising perspectives for further research on the anticancer role of Lf, which ultimately will contribute to its safer and more rational application in the human therapy of these life-threatening cancers.

Studies on the specific and nonspecific interactions of biosurfactants with proteins are broadly relevant given the potential applications of biosurfactant/protein systems in pharmaceutics and cosmetics. The aim of this study was to evaluate the interactions of divalent counterions with the biomolecular anionic biosurfactant surfactin-C15 through molecular modeling, surface tension and dynamic light scattering (DLS), with a specific focus on its effects on biotherapeutic formulations. The conformational analysis based on a semi-empirical approach revealed that Cu2+ ions can be coordinated by three amide nitrogens belonging to the surfactin-C15 cycle and one oxygen atom of the aspartic acid from the side chain of the lipopeptide. Backbone oxygen atoms mainly involve Zn2+, Ca2+ and Mg2+. Subsequently, the interactions between metal-coordinated lipopeptide complexes and bovine serum albumin (BSA) were extensively investigated by fluorescence spectroscopy and molecular docking analysis. Fluorescence results showed that metal-lipopeptide complexes interact with BSA through a static quenching mechanism. Molecular docking results indicate that the metal-lipopeptide complexes are stabilized by hydrogen bonding and van der Waals forces. The biosurfactant-protein interaction properties herein described are of significance for metal-based drug discovery hypothesizing that the association of divalent metal ions with surfactin allows its interaction with bacteria, fungi and cancer cell membranes with effects that are similar to those of the cationic peptide antibiotics.

Marine microorganisms possess unique metabolic and physiological features and are an important source of new biomolecules, such as biosurfactants. Some of these surface-active compounds synthesized by marine microorganisms exhibit antimicrobial, anti-adhesive and anti-biofilm activity against a broad spectrum of human pathogens (including multi-drug resistant pathogens), and could be used instead of existing drugs to treat infections caused by them. In other cases, these biosurfactants show anti-cancer activity, which could be envisaged as an alternative to conventional therapies. However, marine biosurfactants have not been widely explored, mainly due to the difficulties associated with the isolation and growth of their producing microorganisms. Culture-independent techniques (metagenomics) constitute a promising approach to study the genetic resources of otherwise inaccessible marine microorganisms without the requirement of culturing them, and can contribute to the discovery of novel biosurfactants with significant biological activities. This paper reviews the most relevant biosurfactants produced by marine microorganisms with potential therapeutic applications and discusses future perspectives and opportunities to discover novel molecules from marine environments.

Biosurfactant production was screened in four lactobacilli strains. The highest biosurfactant production (excreted and cell-bound biosurfactants) was achieved with Lactobacillus paracasei ssp. paracasei A20, a strain isolated from a Portuguese dairy plant, with a decrease in the surface tension of 6.4 mN m−1 and 22.0 mN m−1, respectively. Biosurfactant production by this strain was evaluated under different culture broth compositions. The use of different nitrogen sources revealed that yeast extract is essential for bacterial growth, while peptone is crucial for biosurfactant synthesis. For biosurfactant production, the use of peptone and meat extract yielded a higher production when compared to the standard medium, with a surface tension reduction of 24.5 mN m−1 Furthermore, experiments were also conducted in a reactor with pH and temperature control. Biomass and biosurfactant production in bioreactor was higher comparing with the experiments conducted in shake flaks. The optimization procedure adopted in the current work was found to improve the biosurfactant production and opened new perspectives for the use of L. paracasei ssp. paracasei A20 as a promising biosurfactant-producer.

Abstract Triple-negative breast cancer is the most aggressive subtype of invasive breast cancer with a poor prognosis and no approved targeted therapy. Hence, the identification of new and specific ligands is essential to develop novel targeted therapies. In this study, we aimed to identify new aptamers that bind to highly metastatic breast cancer MDA-MB-231 cells using the cell-SELEX technology aided by high throughput sequencing. After 8 cycles of selection, the aptamer pool was sequenced and the 25 most frequent sequences were aligned for homology within their variable core region, plotted according to their free energy and the key nucleotides possibly involved in the target binding site were analyzed. Two aptamer candidates, Apt1 and Apt2, binding specifically to the target cells with $$K_{d}$$ K d values of 44.3 ± 13.3 nM and 17.7 ± 2.7 nM, respectively, were further validated. The binding analysis clearly showed their specificity to MDA-MB-231 cells and suggested the targeting of cell surface receptors. Additionally, Apt2 revealed no toxicity in vitro and showed potential translational application due to its affinity to breast cancer tissue sections. Overall, the results suggest that Apt2 is a promising candidate to be used in triple-negative breast cancer treatment and/or diagnosis.

Neural injuries in cerebral malaria patients are a significant cause of morbidity and mortality. Nevertheless, a comprehensive research approach to study this issue is lacking, so herein we propose an in vitro system to study human cerebral malaria using cellular approaches. Our first goal was to establish a cellular system to identify the molecular alterations in human brain vasculature cells that resemble the blood–brain barrier (BBB) in cerebral malaria (CM). Through transcriptomic analysis, we characterized specific gene expression profiles in human brain microvascular endothelial cells (HBMEC) activated by the Plasmodium falciparum parasites. We also suggest potential new genes related to parasitic activation. Then, we studied its impact at brain level after Plasmodium falciparum endothelial activation to gain a deeper understanding of the physiological mechanisms underlying CM. For that, the impact of HBMEC-P. falciparum-activated secretomes was evaluated in human brain organoids. Our results support the reliability of in vitro cellular models developed to mimic CM in several aspects. These systems can be of extreme importance to investigate the factors (parasitological and host) influencing CM, contributing to a molecular understanding of pathogenesis, brain injury, and dysfunction.

Prevention of bacterial adhesion is a way to reduce and/or avoid biofilm formation, thus restraining its associated infections. The development of repellent anti-adhesive surfaces, such as superhydrophobic surfaces, can be a strategy to avoid bacterial adhesion. In this study, a polyethylene terephthalate (PET) film was modified by in situ growth of silica nanoparticles (NPs) to create a rough surface. The surface was further modified with fluorinated carbon chains to increase its hydrophobicity. The modified PET surfaces presented a pronounced superhydrophobic character, showing a water contact angle of 156° and a roughness of 104 nm (a considerable increase comparing with the 69° and 4.8 nm obtained for the untreated PET). Scanning Electron Microscopy was used to evaluate the modified surfaces morphology, further confirming its successful modification with nanoparticles. Additionally, a bacterial adhesion assay using an Escherichia coli expressing YadA, an adhesive protein from Yersinia so-called Yersinia adhesin A, was used to assess the anti-adhesive potential of the modified PET. Contrarily to what was expected, adhesion of E. coli YadA was found to increase on the modified PET surfaces, exhibiting a clear preference for the crevices. This study highlights the role of material micro topography as an important attribute when considering bacterial adhesion.

Innovative point-of-care (PoC) diagnostic platforms are desirable to surpass the deficiencies of conventional laboratory diagnostic methods for bacterial infections and to tackle the growing antimicrobial resistance crisis. In this study, a workflow was implemented, comprising the identification of new aptamers with high affinity for the ubiquitous surface protein A2 (UspA2) of the bacterial pathogen Moraxella catarrhalis and the development of an electrochemical biosensor functionalized with the best-performing aptamer as a bioreceptor to detect UspA2. After cell-systematic evolution of ligands by exponential enrichment (cell-SELEX) was performed, next-generation sequencing was used to sequence the final aptamer pool. The most frequent aptamer sequences were further evaluated using bioinformatic tools. The two most promising aptamer candidates, Apt1 and Apt1_RC (Apt1 reverse complement), had Kd values of 214.4 and 3.4 nM, respectively. Finally, a simple and label-free electrochemical biosensor was functionalized with Apt1_RC. The aptasensor surface modifications were confirmed by impedance spectroscopy and cyclic voltammetry. The ability to detect UspA2 was evaluated by square wave voltammetry, exhibiting a linear detection range of 4.0 × 104–7.0 × 107 CFU mL−1, a square correlation coefficient superior to 0.99 and a limit of detection of 4.0 × 104 CFU mL−1 at pH 5.0. The workflow described has the potential to be part of a sensitive PoC diagnostic platform to detect and quantify M. catarrhalis from biological samples.

Calcium-doped manganese ferrite nanoparticles (NPs) are gaining special interest in the biomedical field due to their lower cytotoxicity compared with other ferrites, and the fact that they have improved magnetic properties. Magnetic hyperthermia (MH) is an alternative cancer treatment, in which magnetic nanoparticles promote local heating that can lead to the apoptosis of cancer cells. In this work, manganese/calcium ferrite NPs coated with citrate (CaxMn1−xFe2O4 (x = 0, 0.2, 1), were synthesized by the sol-gel method, followed by calcination, and then characterized regarding their crystalline structure (by X-ray diffraction, XRD), size and shape (by Transmission Electron Microscopy, TEM), hydrodynamic size and zeta potential (by Dynamic Light Scattering, DLS), and heating efficiency (measuring the Specific Absorption Rate, SAR, and Intrinsic Loss Power, ILP) under an alternating magnetic field. The obtained NPs showed a particle size within the range of 10 nm to 20 nm (by TEM) with a spherical or cubic shape. Ca0.2Mn0.8Fe2O4 NPs exhibited the highest SAR value of 36.3 W/g at the lowest field frequency tested, and achieved a temperature variation of ~7 °C in 120 s, meaning that these NPs are suitable magnetic hyperthermia agents. In vitro cellular internalization and cytotoxicity experiments, performed using the human cell line HEK 293T, confirmed cytocompatibility over 0–250 µg/mL range and successful internalization after 24 h. Based on these studies, our data suggest that these manganese-calcium ferrite NPs have potential for MH application and further use in in vivo systems.

New point-of-care (POC) diagnosis of bacterial infections are imperative to overcome the deficiencies of conventional methods, such as culture and molecular methods. In this study, we identified new aptamers that bind to the virulence factor Yersinia adhesin A (YadA) of Yersinia enterocolitica using cell-systematic evolution of ligands by exponential enrichment (cell-SELEX). Escherichia coli expressing YadA on the cell surface was used as a target cell. After eight cycles of selection, the final aptamer pool was sequenced by high throughput sequencing using the Illumina Novaseq platform. The sequencing data, analyzed using the Geneious software, was aligned, filtered and demultiplexed to obtain the key nucleotides possibly involved in the target binding. The most promising aptamer candidate, Apt1, bound specifically to YadA with a dissociation constant (Kd) of 11 nM. Apt1 was used to develop a simple electrochemical biosensor with a two-step, label-free design towards the detection of YadA. The sensor surface modifications and its ability to bind successfully and stably to YadA were confirmed by cyclic voltammetry, impedance spectroscopy and square wave voltammetry. The biosensor enabled the detection of YadA in a linear range between 7.0 × 104 and 7.0 × 107 CFU mL−1 and showed a square correlation coefficient >0.99. The standard deviation and the limit of detection was ~2.5% and 7.0 × 104 CFU mL−1, respectively. Overall, the results suggest that this novel biosensor incorporating Apt1 can potentially be used as a sensitive POC detection system to aid the diagnosis of Y. enterocolitica infections. Furthermore, this simple yet innovative approach could be replicated to select aptamers for other (bacterial) targets and to develop the corresponding biosensors for their detection.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9) gene-editing offers exciting new therapeutic possibilities for disease treatment with a genetic etiology such as cancer, cardiovascular, neuronal, and immune disorders. However, its clinical translation is being hampered by the lack of safe, versatile, and effective nonviral delivery systems. Herein we report on the preparation and application of two cationic liposome–DNA systems (i.e., lipoplexes) for CRISPR/Cas9 gene delivery. For that purpose, two types of cationic lipids are used (DOTAP, monovalent, and MVL5, multivalent with +5e nominal charge), along with three types of helper lipids (DOPC, DOPE, and monoolein (GMO)). We demonstrated that plasmids encoding Cas9 and single-guide RNA (sgRNA), which are typically hard to transfect due to their large size (>9 kb), can be successfully transfected into HEK 293T cells via MVL5-based lipoplexes. In contrast, DOTAP-based lipoplexes resulted in very low transfection rates. MVL5-based lipoplexes presented the ability to escape from lysosomes, which may explain the superior transfection efficiency. Regarding gene editing, MVL5-based lipoplexes achieved promising GFP knockout levels, reaching rates of knockout superior to 35% for charge ratios (+/−) of 10. Despite the knockout efficiency being comparable to that of Lipofectamine 3000® commercial reagent, the non-specific gene knockout is more pronounced in MVL5-based formulations, probably resulting from the considerable cytotoxicity of these formulations. Altogether, these results show that multivalent lipid-based lipoplexes are promising CRISPR/Cas9 plasmid delivery vehicles, which by further optimization and functionalization may become suitable in vivo delivery systems.

Cancer is a major cause of mortality and morbidity worldwide. Detection and quantification of cancer biomarkers plays a critical role in cancer early diagnosis, screening, and treatment. Clinicians, particularly in developing countries, deal with high costs and limited resources for diagnostic systems. Using low-cost substrates to develop sensor devices could be very helpful. The interest in paper-based sensors with colorimetric detection increased exponentially in the last decade as they meet the criteria for point-of-care (PoC) devices. Cellulose and different nanomaterials have been used as substrate and colorimetric probes, respectively, for these types of devices in their different designs as spot tests, lateral-flow assays, dipsticks, and microfluidic paper-based devices (μPADs), offering low-cost and disposable devices. However, the main challenge with these devices is their low sensitivity and lack of efficiency in performing quantitative measurements. This review includes an overview of the use of paper for the development of sensing devices focusing on colorimetric detection and their application to cancer biomarkers. We highlight recent works reporting the use of paper in the development of colorimetric sensors for cancer biomarkers, such as proteins, nucleic acids, and others. Finally, we discuss the main advantages of these types of devices and highlight their major pitfalls.

Natural resources are considered a promising source of microorganisms responsible for producing biocatalysts with great relevance in several industrial areas. However, a significant fraction of the environmental microorganisms remains unknown or unexploited due to the limitations associated with their cultivation in the laboratory through classical techniques. Metagenomics has emerged as an innovative and strategic approach to explore these unculturable microorganisms through the analysis of DNA extracted from environmental samples. In this review, a detailed discussion is presented on the application of metagenomics to unravel the biotechnological potential of natural resources for the discovery of promising biocatalysts. An extensive bibliographic survey was carried out between 2010 and 2021, covering diverse metagenomic studies using soil and/or water samples from different types and locations. The review comprises, for the first time, an overview of the worldwide metagenomic studies performed in soil and water and provides a complete and global vision of the enzyme diversity associated with each specific environment.

Infectious agents, especially bacteria and viruses, account for a vast number of hospitalisations and mortality worldwide. Providing effective and timely diagnostics for the multiplicity of infectious diseases is challenging. Conventional diagnostic solutions, although technologically advanced, are highly complex and often inaccessible in resource-limited settings. An alternative strategy involves convenient rapid diagnostics which can be easily administered at the point-of-care (POC) and at low cost without sacrificing reliability. Biosensors and other rapid POC diagnostic tools which require biorecognition elements to precisely identify the causative pathogen are being developed. The effectiveness of these devices is highly dependent on their biorecognition capabilities. Naturally occurring biorecognition elements include antibodies, bacteriophages and enzymes. Recently, modified molecules such as DNAzymes, peptide nucleic acids and molecules which suffer a selective screening like aptamers and peptides are gaining interest for their biorecognition capabilities and other advantages over purely natural ones, such as robustness and lower production costs. Antimicrobials with a broad-spectrum activity against pathogens, such as antibiotics, are also used in dual diagnostic and therapeutic strategies. Other successful pathogen identification strategies use chemical ligands, molecularly imprinted polymers and Clustered Regularly Interspaced Short Palindromic Repeats-associated nuclease. Herein, the latest developments regarding biorecognition elements and strategies to use them in the design of new biosensors for pathogens detection are reviewed.

Infectious diseases are an existential health threat, potentiated by emerging and re-emerging viruses and increasing bacterial antibiotic resistance. Targeted treatment of infectious diseases requires precision diagnostics, especially in cases where broad-range therapeutics such as antibiotics fail. There is thus an increasing need for new approaches to develop sensitive and specific in vitro diagnostic (IVD) tests. Basic science and translational research are needed to identify key microbial molecules as diagnostic targets, to identify relevant host counterparts, and to use this knowledge in developing or improving IVD. In this regard, an overlooked feature is the capacity of pathogens to adhere specifically to host cells and tissues. The molecular entities relevant for pathogen–surface interaction are the so-called adhesins. Adhesins vary from protein compounds to (poly-)saccharides or lipid structures that interact with eukaryotic host cell matrix molecules and receptors. Such interactions co-define the specificity and sensitivity of a diagnostic test. Currently, adhesin-receptor binding is typically used in the pre-analytical phase of IVD tests, focusing on pathogen enrichment. Further exploration of adhesin–ligand interaction, supported by present high-throughput “omics” technologies, might stimulate a new generation of broadly applicable pathogen detection and characterization tools. This review describes recent results of novel structure-defining technologies allowing for detailed molecular analysis of adhesins, their receptors and complexes. Since the host ligands evolve slowly, the corresponding adhesin interaction is under selective pressure to maintain a constant receptor binding domain. IVD should exploit such conserved binding sites and, in particular, use the human ligand to enrich the pathogen. We provide an inventory of methods based on adhesion factors and pathogen attachment mechanisms, which can also be of relevance to currently emerging pathogens, including SARS-CoV-2, the causative agent of COVID-19.

Propionibacterium freudenreichii is a commercially relevant bacterium with probiotic potential. This bacterium can exert protective effects particularly against colorectal cancer (CRC), via the production of short chain fatty acids (SCFA), namely acetate and propionate. In this work, we aimed to evaluate the performance and adaptation capacity of P. freudenreichii to a simulated digestive stress using different culture media, namely YEL, Basal medium, Mimicking the Content of the Human Colon medium (MCHC) and DMEM. The effect of the fermented culture broth on CRC cells survival and of CRC cells conditioned media on the bacteria performance was also evaluated. Basal medium was found to be the best for P. freudenreichii to produce SCFA. MCHC medium, despite being the medium in which lower amounts of acetate and propionate were produced, showed higher acetate and propionate yields as compared to other media. We also observed that the presence of lactate in CRC cells conditioned growth medium resulting from cell metabolism, leads to an increased production of SCFA by the bacteria. The bacterial fermented broth successfully inhibited CRC cells proliferation and increased cell death. Our results showed for the first time that P. freudenreichii performance might be stimulated by extracellular lactate produced by CRC metabolic switch also known as “Warburg effect,” where cancer cells “ferment” glucose into lactate. Additionally, our results suggest that P. freudenreichii could be potentially used as a probiotic in CRC prevention at early stages of the carcinogenesis process and might help in CRC therapeutic approaches.