Critical Issues on the Surface Functionalization of Plasmonic Au-Ag/TiO2 Thin Films with Thiolated Oligonucleotide-Based Biorecognition Elements.

This work reports on the surface functionalization of a nanomaterial supporting localized surface plasmon resonances (LSPRs) with (synthetic) thiolated oligonucleotide-based biorecognition elements, envisaging the development of selective LSPR-based DNA biosensors. The LSPR thin-film transducers are composed of noble metal nanoparticles (NPs) embedded in a TiO2 dielectric matrix, produced cost-effectively and sustainably by magnetron sputtering. The study focused on the immobilization kinetics of thiolated oligonucleotide probes as biorecognition elements, followed by the evaluation of hybridization events with the target probe. The interaction between the thiolated oligonucleotide probe and the transducer's surface was assessed by monitoring the LSPR signal with successive additions of probe solution through a microfluidic device. The device was specifically designed and fabricated for this work and adapted to a high-resolution LSPR spectroscopy system with portable characteristics. Benefiting from the synergetic characteristics of Ag and Au in the form of bimetallic nanoparticles, the Au-Ag/TiO2 thin film proved to be more sensitive to thiolated oligonucleotide binding events. Despite the successful surface functionalization with the biorecognition element, the detection of complementary oligonucleotides revealed electrostatic repulsion and steric hindrance, which hindered hybridization with the target oligonucleotide. This study points to an effect that is still poorly described in the literature and affects the design of LSPR biosensors based on nanoplasmonic thin films.

Cytokine production by bovine adipose tissue stromal vascular fraction cells upon Neospora caninum stimulation.

In bovines few studies addressed the contribution of adipose tissue to the host immune response to infection. Here we evaluated the in vitro response of bovine adipose tissue stromal vascular fraction (SVF) cells to the protozoan parasite Neospora caninum, using live and freeze-killed tachyzoites. Live N. caninum induced the production of IL-6, IL-1ß and IL-10 by SVF cells isolated from subcutaneous adipose tissue (SAT), while in mesenteric adipose tissue (MAT) SVF cell cultures only IL-1ß and IL-10 production was increased, showing slight distinct responses between adipose tissue depots. Whereas a clear IL-8 increase was detected in peripheral blood leucocytes (PBL) culture supernatants in response to live N. caninum, no such increase was observed in SAT or MAT SVF cell cultures. Nevertheless, in response to LPS, increased IL-8 levels were detected in all cell cultures. IL-10 levels were always increased in response to stimulation (live, freeze-killed N. caninum and LPS). Overall, our results show that bovine adipose tissue SVF cells produce cytokines in response to N. caninum and can therefore be putative contributors to the host immune response against this parasite.

Candida albicans chitinase 3 with potential as a vaccine antigen: production, purification, and characterisation.

Chitinases are widely studied enzymes that have already found widespread application. Their continued development and valorisation will be driven by the identification of new and improved variants and/or novel applications bringing benefits to industry and society. We previously identified a novel application for chitinases wherein the Candida albicans cell wall surface chitinase 3 (Cht3) was shown to have potential in vaccine applications as a subunit antigen against fungal infections. In the present study, this enzyme was investigated further, developing production and purification protocols, enriching our understanding of its properties, and advancing its application potential. Cht3 was heterologously expressed in Pichia pastoris and a 4-step purification protocol developed and optimised: this involves activated carbon treatment, hydrophobic interaction chromatography, ammonium sulphate precipitation, and gel filtration chromatography. The recombinant enzyme was shown to be mainly O-glycosylated and to retain the epitopes of the native protein. Functional studies showed it to be highly specific, displaying activity on chitin, chitosan, and chito-oligosaccharides larger than chitotriose only. Furthermore, it was shown to be a stable enzyme, exhibiting activity, and stability over broad pH and temperature ranges. This study represents an important step forward in our understanding of Cht3 and contributes to its development for application.

Effects of Zn-ZnO Core-Shell Nanoparticles on Antimicrobial Mechanisms and Immune Cell Activation.

The deposition of zinc-zinc oxide nanoparticles (Zn-ZnO NPs) onto porous Ta2O5 surfaces enriched with calcium phosphate by DC magnetron sputtering was investigated to improve the surface antimicrobial activity without triggering an inflammatory response. Different sizes and amounts of Zn NPs obtained by two optimized different depositions and an additional thin carbon (C) layer deposited over the NPs were explored. The deposition of the Zn NPs and the C layer mitigates the surface porosity, increasing the surface hydrophobicity and decreasing the surface roughness. The possible antimicrobial effect and immune system activation of Zn-ZnO NPs were investigated in Candida albicans and macrophage cells, respectively. It was found that the developed surfaces displayed a fungistatic behavior, as they impair the growth of C. albicans between 5 and 24 h of culture. This behavior was more evident on the surfaces with bigger NPs and the highest amounts of Zn. The same trend was observed in both reactive oxygen species (ROS) generation and loss of C. albicans' membrane integrity. After 24 h of culture, cell toxicity was also dependent on the amount of the NPs. Cell toxicity was observed in surfaces with the highest amount of Zn NPs and with the C layer, while cells were able to grow without any signs of cytotoxicity in the porous surfaces with the lowest amount of NPs. The same Zn-dose-dependent behavior was noticed in the TNF-α production. The Zn-containing surfaces show a vastly inferior cytokine secretion than the lipopolysaccharide (LPS)-stimulated cells, indicating that the modified surfaces do not induce an inflammatory response from macrophage cells. This study provides insights for understanding the Zn amount threshold that allows a simultaneous inhibition of the fungi growth with no toxic effect and the main antimicrobial mechanisms of Zn-ZnO NPs, contributing to future clinical applications.

Design of a lipid nano-delivery system containing recombinant Candida albicans chitinase 3 as a potential vaccine against fungal infections.

Opportunistic fungi cause lethal systemic infections and impose high medical costs to health systems. The World Health Organization has recognized the importance of fungal infections, including them in its global priority list guiding research, development, and discovery of new therapeutic approaches. Fungal vaccine development has been proposed as one of the treatment and prevention strategies in the last decade. In this study, we present the design of a lipid antigen delivery system based on Dioctadecyldimethylammonium bromide: Monoolein (DODAB: MO) containing recombinant Candida albicans Chitinase 3 (Cht3) for modulation the immune response against fungal infections. Several DODAB:MO liposomes containing Cht3 were prepared and those prepared by the incubation method and containing 5 µg/mL Cht3 were selected due to their favorable size, ζ-potential and stability, suited for antigen delivery applications. The encapsulation of Cht3 in these liposomes resulted in a significant increase in cellular uptake compared to empty liposomes, demonstrating their efficacy in delivering the antigen. Moreover, the liposomes proved to be safe for use in immunization procedures. Subcutaneous administration of Cht3 liposomes elicited a Th1/Th17 immune response profile, associated with the production of high levels of antibodies against Cht3. These antibodies recognized both the native and the recombinant forms of the protein, opsonizing mother-yeast at the cell scars, which has the potential to disrupt cell separation and hinder yeast growth. The findings suggest that the designed lipid antigen delivery system shows promise as a potential candidate for enhancing immune responses against fungal infections, offering a valuable strategy for future fungal vaccine development.

Valorisation of the Invasive Macroalgae Undaria pinnatifida (Harvey) Suringar for the Green Synthesis of Gold and Silver Nanoparticles with Antimicrobial and Antioxidant Potential.

Bacterial and fungal infections are a challenging global problem due to the reported increasing resistance of pathogenic microorganisms to conventional antimicrobials. Nanomaterials are a promising strategy to fight infections caused by multidrug-resistant microbes. In this work, gold (Au@UP) and silver (Ag@UP) nanoparticles were produced for the first time by green synthesis using an aqueous extract of the invasive macroalgae Undaria pinnatifida (UP). The nanoparticles were characterized by a wide range of physicochemical techniques. Au@UP and Ag@UP demonstrated to be spherical and crystalline with an average size of 6.8 ± 1.0 nm and 14.1 ± 2.8 nm, respectively. Carbohydrates and proteins of the UP extract may participate in the synthesis and capping of the nanoparticles. The UP extract, Ag@UP, and Au@UP were assessed for their antimicrobial activity against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Candida auris. Ag@UP showed the highest antimicrobial activity with very low MIC and MBC values for all the tested bacteria, and Au@UP demonstrated to be very effective against biofilm-producing bacteria. The antifungal properties of both Ag@UP and Au@UP were remarkable, inhibiting hyphae formation. This study points towards a very promising biomedical exploitation of this invasive brown algae.

Comparative study of e-cigarette aerosol and cigarette smoke effect on ex vivo embryonic chick lung explants.

Electronic cigarette usage has significantly expanded among young people and pregnant women in the last decade. Although there are already some data regarding the short- and long-term consequences of e-cigarettes on human health, their effect on embryo and lung development still needs to be fully disclosed. In this sense, this study describes, for the first time, the impact of electronic cigarette aerosol on early lung development. For this purpose, ex vivo chick (Gallus gallus) embryonic lungs were cultured in vitro for 48 h in e-cigarette aerosol exposed-medium or unexposed medium. Chick lung explants were also cultured in a cigarette smoke-exposed medium for comparison purposes. Lung explants were morphologically analyzed to assess the impact on lung growth. Additionally, TNF-α levels were determined in the supernatant as a marker of pro-inflammatory response. The results suggest that electronic cigarette aerosol impairs lung growth and promotes lung inflammation. However, its impact on early lung growth seems less detrimental than conventional cigarette smoke. This work provides significant data regarding the impact of e-cig aerosol, adding to the efforts to fully understand its effect on embryo development. The validation of these effects may eventually lead to new tobacco control recommendations for pregnant women.

Polymeric nanoparticles targeting Sialyl-Tn in gastric cancer: A live tracking under flow conditions.

Drug delivery using nanoparticles (NPs) represents a potential approach for therapy in cancer, such gastric cancer (GC) due to their targeting ability and controlled release properties. The use of advanced nanosystems that deliver anti-cancer drugs specifically to tumor cells may strongly rely on the expression of cancer-associated targets. Glycans aberrantly expressed by cancer cells are attractive targets for such delivery strategy. Sialylated glycans, such as Sialyl-Tn (STn) are aberrantly expressed in several epithelial tumors, including GC, being a potential target for a delivery nanosystem. The aim of this study was the development of NPs surface-functionalized with a specific antibody targeting the STn glycan and further evaluate this nanosystem effectiveness regarding its specificity and recognition capacity. Our results showed that the NPs surface-functionalized with anti-STn antibody efficiently are recognized by cells displaying the cancer-associated STn antigen under static and live cell monitoring flow conditions. This uncovers the potential use of such NPs for drug delivery in cancer. However, flow exposure was disclosed as an important biomechanical parameter to be taken into consideration. Here we presented an innovative and successful methodology to live track the NPs targeting STn antigen under shear stress, simulating the physiological flow. We demonstrate that unspecific binding of NPs agglomerates did not occur under flow conditions, in contrast with static assays. This robust approach can be applied for in vitro drug studies, giving valuable insights for in vivo studies.

Tracking Bacterial Nanocellulose in Animal Tissues by Fluorescence Microscopy.

The potential of nanomaterials in food technology is nowadays well-established. However, their commercial use requires a careful risk assessment, in particular concerning the fate of nanomaterials in the human body. Bacterial nanocellulose (BNC), a nanofibrillar polysaccharide, has been used as a food product for many years in Asia. However, given its nano-character, several toxicological studies must be performed, according to the European Food Safety Agency's guidance. Those should especially answer the question of whether nanoparticulate cellulose is absorbed in the gastrointestinal tract. This raises the need to develop a screening technique capable of detecting isolated nanosized particles in biological tissues. Herein, the potential of a cellulose-binding module fused to a green fluorescent protein (GFP-CBM) to detect single bacterial cellulose nanocrystals (BCNC) obtained by acid hydrolysis was assessed. Adsorption studies were performed to characterize the interaction of GFP-CBM with BNC and BCNC. Correlative electron light microscopy was used to demonstrate that isolated BCNC may be detected by fluorescence microscopy. The uptake of BCNC by macrophages was also assessed. Finally, an exploratory 21-day repeated-dose study was performed, wherein Wistar rats were fed daily with BNC. The presence of BNC or BCNC throughout the GIT was observed only in the intestinal lumen, suggesting that cellulose particles were not absorbed. While a more comprehensive toxicological study is necessary, these results strengthen the idea that BNC can be considered a safe food additive.

Optimization of a Quantitative PCR Methodology for Detection of Aspergillus spp. and Rhizopus arrhizus.

INTRODUCTION: Multiplex quantitative polymerase chain reaction (qPCR) methods for the detection of Aspergillus spp. based only on SYBR Green and melting curve analysis of PCR products are difficult to develop because most targets are located within ITS regions. The aim of this study was to adapt our previously developed methodology based on a multiplex PCR assay coupled with GeneScan analysis to provide a qPCR method. METHODS: A SYBR Green-based real-time PCR assay was optimized to detect A. fumigatus, A. flavus, A. niger, A. terreus, and R. arrhizus in a multiplex assay and applied to cultured fungi and spiked plasma. RESULTS: Different melting temperatures allowed identification of all five pathogens and discrimination between them, even in samples with low amounts of fungal gDNA (from 1.3 to 33.0 pg/µL), which has been reported previously as problematic. No false-positive results were obtained for non-target species, including bacteria and human DNA. This method allowed detection of fungal pathogens in human plasma spiked with fungal DNA and in coinfections of A. niger/R. arrhizus. DISCUSSION: This work provides evidence for the use of a qPCR multiplex method based on SYBR Green and melting curve analysis of PCR products for the detection of A. fumigatus, A. flavus, A. niger, A. terreus, and R. arrhizus. The proposed method is simpler and less expensive than available kits based on fluorescent probes and can be used for aiding diagnosis of the most relevant invasive filamentous fungi, particularly in low-income health care institutions.

Immobilization of Streptavidin on a Plasmonic Au-TiO2 Thin Film towards an LSPR Biosensing Platform.

Optical biosensors based on localized surface plasmon resonance (LSPR) are the future of label-free detection methods. This work reports the development of plasmonic thin films, containing Au nanoparticles dispersed in a TiO2 matrix, as platforms for LSPR biosensors. Post-deposition treatments were employed, namely annealing at 400 °C, to develop an LSPR band, and Ar plasma, to improve the sensitivity of the Au-TiO2 thin film. Streptavidin and biotin conjugated with horseradish peroxidase (HRP) were chosen as the model receptor-analyte, to prove the efficiency of the immobilization method and to demonstrate the potential of the LSPR-based biosensor. The Au-TiO2 thin films were activated with O2 plasma, to promote the streptavidin immobilization as a biorecognition element, by increasing the surface hydrophilicity (contact angle drop to 7°). The interaction between biotin and the immobilized streptavidin was confirmed by the detection of HRP activity (average absorbance 1.9 ± 0.6), following a protocol based on enzyme-linked immunosorbent assay (ELISA). Furthermore, an LSPR wavelength shift was detectable (0.8 ± 0.1 nm), resulting from a plasmonic thin-film platform with a refractive index sensitivity estimated to be 33 nm/RIU. The detection of the analyte using these two different methods proves that the functionalization protocol was successful and the Au-TiO2 thin films have the potential to be used as an LSPR platform for label-free biosensors.

Cellulose-based hydrogel on quantum dots with molecularly imprinted polymers for the detection of CA19-9 protein cancer biomarker.

Molecularly imprinted polymers MIPs were successfully assembled around quantum dots (QDs), for the detection of the protein biomarker CA19-9 associated to pancreatic cancer (PC). These imprinted materials MIP@QDs were incorporated within the cellulose hydrogel with retention of its conformational structure inside the binding cavities. The concept is to use MIPs which function as the biorecognition elements, conjugated to cadmium telluride QDs as the sensing system. The excitation wavelength was set to 477 nm and the fluorescence signal was measured at its maximum intensity, with an emission range between 530 and 780 nm. The fluorescence quenching of the imprinted cellulose hydrogels occurred with increasing concentrations of CA19-9, showing linearity in the range 2.76 × 10 -2 - 5.23 × 10 2 U/ml, in a 1000-fold diluted human serum. Replicates of the imprinted hydrogel show a linear response below the cut-off values for pancreatic cancer diagnosis (< 23 U/ml), a limit of detection of 1.58 × 10 -3 U/ml and an imprinting factor (IF) of 1.76. In addition to the fact that the imprinted cellulose hydrogel displays good stability and selectivity towards CA19-9 when compared with the non-imprinted controls, the conjugation of MIPs to QDs increases the sensitivity of the system for an optical detection method towards ranges within clinical significance. This fact shows potential for the imprinted hydrogel to be applied as a sensitive, low-cost format for point-of-care tests (PoCTs).

Metabolic profile of Candida albicans and Candida parapsilosis interactions within dual-species biofilms.

Within the oral cavity, the ability of Candida species to adhere and form biofilms is well-recognized, especially when Candida albicans is considered. Lately, a knowledge gap has been identified regarding dual-species communication of Candida isolates, as a way to increase virulence, with evidences being collected to support the existence of interactions between C. albicans and Candida parapsilosis. The present work evaluated the synergistic effect of the two Candida species, and explored chemical interactions between cells, evaluating secreted extracellular alcohols and their relation with yeasts' growth and matrix composition. A total of four clinical strains of C. albicans and C. parapsilosis species, isolated from single infections of different patients or from co-infections of a same patient, were tested. It was found that dual-species biofilms negatively impacted the growth of C. parapsilosis and their biofilm matrix, in comparison with mono-species biofilms, and had minor effects on the biofilm biomass. Alcohol secretion revealed to be species- and strain-dependent. However, some dual-species cultures produced much higher amounts of some alcohols (E-nerolidol and E, E-Farnesol) than the respective single cultures, which proves the existence of a synergy between species. These results show evidence that interactions between Candida species affect the biofilm matrix, which is a key element of oral biofilms.

Anti-Inflammatory and Immunoregulatory Action of Sesquiterpene Lactones.

Sesquiterpene lactones (SL), characterized by their high prevalence in the Asteraceae family, are one of the major groups of secondary metabolites found in plants. Researchers from distinct research fields, including pharmacology, medicine, and agriculture, are interested in their biological potential. With new SL discovered in the last years, new biological activities have been tested, different action mechanisms (synergistic and/or antagonistic effects), as well as molecular structure-activity relationships described. The review identifies the main sesquiterpene lactones with interconnections between immune responses and anti-inflammatory actions, within different cellular models as well in in vivo studies. Bioaccessibility and bioavailability, as well as molecular structure-activity relationships are addressed. Additionally, plant metabolic engineering, and the impact of sesquiterpene lactone extraction methodologies are presented, with the perspective of biological activity enhancement. Sesquiterpene lactones derivatives are also addressed. This review summarizes the current knowledge regarding the therapeutic potential of sesquiterpene lactones within immune and inflammatory activities, highlighting trends and opportunities for their pharmaceutical/clinical use.

Fungal infections diagnosis - Past, present and future.

Despite the scientific advances observed in the recent decades and the emergence of new methodologies, the diagnosis of systemic fungal infections persists as a problematic issue. Fungal cultivation, the standard method that allows a proven diagnosis, has numerous disadvantages, as low sensitivity (only 50% of the patients present positive fungal cultures), and long growth time. These are factors that delay the patient's treatment and, consequently, lead to higher hospital costs. To improve the accuracy and quickness of fungal infections diagnosis, several new methodologies attempt to be implemented in clinical microbiology laboratories. Most of these innovative methods are independent of pathogen isolation, which means that the diagnosis goes from being considered proven to probable. In spite of the advantage of being culture-independent, the majority of the methods lack standardization. PCR-based methods are becoming more and more commonly used, which has earned them an important place in hospital laboratories. This can be perceived now, as PCR-based methodologies have proved to be an essential tool fighting against the COVID-19 pandemic. This review aims to go through the main steps of the diagnosis for systemic fungal infection, from diagnostic classifications, through methodologies considered as "gold standard", to the molecular methods currently used, and finally mentioning some of the more futuristic approaches.

Conformation-sensitive antibody reveals an altered cytosolic PAS/CNBh assembly during hERG channel gating.

The human ERG (hERG) K+ channel has a crucial function in cardiac repolarization, and mutations or channel block can give rise to long QT syndrome and catastrophic ventricular arrhythmias. The cytosolic assembly formed by the Per-Arnt-Sim (PAS) and cyclic nucleotide binding homology (CNBh) domains is the defining structural feature of hERG and related KCNH channels. However, the molecular role of these two domains in channel gating remains unclear. We have previously shown that single-chain variable fragment (scFv) antibodies can modulate hERG function by binding to the PAS domain. Here, we mapped the scFv2.12 epitope to a site overlapping with the PAS/CNBh domain interface using NMR spectroscopy and mutagenesis and show that scFv binding in vitro and in the cell is incompatible with the PAS interaction with CNBh. By generating a fluorescently labeled scFv2.12, we demonstrate that association with the full-length hERG channel is state dependent. We detect Förster resonance energy transfer (FRET) with scFv2.12 when the channel gate is open but not when it is closed. In addition, state dependence of scFv2.12 FRET signal disappears when the R56Q mutation, known to destabilize the PAS-CNBh interaction, is introduced in the channel. Altogether, these data are consistent with an extensive structural alteration of the PAS/CNBh assembly when the cytosolic gate opens, likely favoring PAS domain dissociation from the CNBh domain.

Forecasting COVID-19 Severity by Intelligent Optical Fingerprinting of Blood Samples.

Forecasting COVID-19 disease severity is key to supporting clinical decision making and assisting resource allocation, particularly in intensive care units (ICUs). Here, we investigated the utility of time- and frequency-related features of the backscattered signal of serum patient samples to predict COVID-19 disease severity immediately after diagnosis. ICU admission was the primary outcome used to define disease severity. We developed a stacking ensemble machine learning model including the backscattered signal features (optical fingerprint), patient comorbidities, and age (AUROC = 0.80), which significantly outperformed the predictive value of clinical and laboratory variables available at hospital admission (AUROC = 0.71). The information derived from patient optical fingerprints was not strongly correlated with any clinical/laboratory variable, suggesting that optical fingerprinting brings unique information for COVID-19 severity risk assessment. Optical fingerprinting is a label-free, real-time, and low-cost technology that can be easily integrated as a front-line tool to facilitate the triage and clinical management of COVID-19 patients.

Advantages of an Automated Method Compared With Manual Methods for the Quantification of Intraepidermal Nerve Fiber in Skin Biopsy.

Intraepidermal nerve fiber density (IENFD) measurements in skin biopsy are performed manually by 1-3 operators. To improve diagnostic accuracy and applicability in clinical practice, we developed an automated method for fast IENFD determination with low operator-dependency. Sixty skin biopsy specimens were stained with the axonal marker PGP9.5 and imaged using a widefield fluorescence microscope. IENFD was first determined manually by 3 independent observers. Subsequently, images were processed in their Z-max projection and the intradermal line was delineated automatically. IENFD was calculated automatically (fluorescent images automated counting [FIAC]) and compared with manual counting on the same fluorescence images (fluorescent images manual counting [FIMC]), and with classical manual counting (CMC) data. A FIMC showed lower variability among observers compared with CMC (interclass correlation [ICC] = 0.996 vs 0.950). FIMC and FIAC showed high reliability (ICC = 0.999). A moderate-to-high (ICC = 0.705) was observed between CMC and FIAC counting. The algorithm process took on average 15 seconds to perform FIAC counting, compared with 10 minutes for FIMC counting. This automated method rapidly and reliably detects small nerve fibers in skin biopsies with clear advantages over the classical manual technique.

Waste-derived volatile fatty acids as carbon source for added-value fermentation approaches.

The establishment of a sustainable circular bioeconomy requires the effective material recycling from biomass and biowaste beyond composting/fertilizer or anaerobic digestion/bioenergy. Recently, volatile fatty acids attracted much attention due to their potential application as carbon source for the microbial production of high added-value products. Their low-cost production from different types of wastes through dark fermentation is a key aspect, which will potentially lead to the sustainable production of fuels, materials or chemicals, while diminishing the waste volume. This article reviews the utilization of a volatile fatty acid platform for the microbial production of polyhydroxyalkanoates, single cell oil and omega-3 fatty acids, giving emphasis on the fermentation challenges for the efficient implementation of the bioprocess and how they were addressed. These challenges were addressed through a research project funded by the European Commission under the Horizon 2020 programme entitled 'VOLATILE-Biowaste derived volatile fatty acid platform for biopolymers, bioactive compounds and chemical building blocks'.