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.

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.

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).

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.

Development and Characterization of Monoolein-Based Liposomes of Carvacrol, Cinnamaldehyde, Citral, or Thymol with Anti-Candida Activities.

There is an increasing need for novel drugs and new strategies for the therapy of invasive candidiasis. This study aimed to develop and characterize liposome-based nanoparticles of carvacrol, cinnamaldehyde, citral, and thymol with anti-Candida activities. Dioctadecyldimethylammonium bromide- and monoolein-based liposomes in a 1:2 molar ratio were prepared using a lipid-film hydration method. Liposomes were assembled with equal volumes of liposomal stock dispersion and stock solutions of carvacrol, cinnamaldehyde, citral, or thymol in dimethyl sulfoxide. Cytotoxicity was tested on RAW 264.7 macrophages. In vitro antifungal activity of liposomes with phytocompounds was evaluated according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology using clinical isolates of Candida albicans, Candida auris, Candida dubliniensis, and Candida tropicalis Finally, the ability of macrophage cells to kill Candida isolates after addition of phytocompounds and their nanoparticles was determined. Nanoparticles with 64 µg/ml of cinnamaldehyde, 256 µg/ml of citral, and 128 µg/ml of thymol had the best characteristics among the formulations tested. The highest encapsulation efficiencies were achieved with citral (78% to 83%) and carvacrol (66% to 71%) liposomes. Carvacrol and thymol in liposome-based nanoparticles were nontoxic regardless of the concentration. Moreover, carvacrol and thymol maintained their antifungal activity after encapsulation, and there was a significant reduction (∼41%) of yeast survival when macrophages were incubated with carvacrol or thymol liposomes. In conclusion, carvacrol and thymol liposomes possess high stability, low cytotoxicity, and antifungal activity that act synergistically with macrophages.

Modified high-throughput Nile red fluorescence assay for the rapid screening of oleaginous yeasts using acetic acid as carbon source.

BACKGROUND: Over the last years oleaginous yeasts have been studied for several energetic, oleochemical, medical and pharmaceutical purposes. However, only a small number of yeasts are known and have been deeply exploited. The search for new isolates with high oleaginous capacity becomes imperative, as well as the use of alternative and ecological carbon sources for yeast growth. RESULTS: In the present study a high-throughput screening comprising 366 distinct yeast isolates was performed by applying an optimised protocol based on two approaches: (I) yeast cultivation on solid medium using acetic acid as carbon source, (II) neutral lipid estimation by fluorimetry using the lipophilic dye Nile red. CONCLUSIONS: Results showed that, with the proposed methodology, the oleaginous potential of yeasts with broad taxonomic diversity and variety of growth characteristics was discriminated. Furthermore, this work clearly demonstrated the association of the oleaginous yeast character to the strain level, contrarily to the species-level linkage, as usually stated.

Coherent-hybrid STED: high contrast sub-diffraction imaging using a bi-vortex depletion beam.

Stimulated emission depletion (STED) fluorescence microscopy squeezes an excited spot well below the wavelength scale using a doughnut-shaped depletion beam. To generate a doughnut, a scale-free vortex phase modulation (2D-STED) is often used because it provides maximal transverse confinement and radial-aberration immunity (RAI) to the central dip. However, RAI also means blindness to a defocus term, making the axial origin of fluorescence photons uncertain within the wavelength scale provided by the confocal detection pinhole. Here, to reduce the uncertainty, we perturb the 2D-STED phase mask so as to change the sign of the axial concavity near focus, creating a dilated dip. By providing laser depletion power, the dip can be compressed back in three dimensions to retrieve lateral resolution, now at a significantly higher contrast. We test this coherent-hybrid STED (CH-STED) mode in x-y imaging of complex biological structures, such as the dividing cell. The proposed strategy creates an orthogonal direction in the STED parametric space that uniquely allows independent tuning of resolution and contrast using a single depletion beam in a conventional (circular polarization-based) STED setup.

Jmy regulates oligodendrocyte differentiation via modulation of actin cytoskeleton dynamics.

During central nervous system development, oligodendrocytes form structurally and functionally distinct actin-rich protrusions that contact and wrap around axons to assemble myelin sheaths. Establishment of axonal contact is a limiting step in myelination that relies on the oligodendrocyte's ability to locally coordinate cytoskeletal rearrangements with myelin production, under the control of a transcriptional differentiation program. The molecules that provide fine-tuning of actin dynamics during oligodendrocyte differentiation and axon ensheathment remain largely unidentified. We performed transcriptomics analysis of soma and protrusion fractions from rat brain oligodendrocyte progenitors and found a subcellular enrichment of mRNAs in newly-formed protrusions. Approximately 30% of protrusion-enriched transcripts encode proteins related to cytoskeleton dynamics, including the junction mediating and regulatory protein Jmy, a multifunctional regulator of actin polymerization. Here, we show that expression of Jmy is upregulated during myelination and is required for the assembly of actin filaments and protrusion formation during oligodendrocyte differentiation. Quantitative morphodynamics analysis of live oligodendrocytes showed that differentiation is driven by a stereotypical actin network-dependent "cellular shaping" program. Disruption of actin dynamics via knockdown of Jmy leads to a program fail resulting in oligodendrocytes that do not acquire an arborized morphology and are less efficient in contacting neurites and forming myelin wraps in co-cultures with neurons. Our findings provide new mechanistic insight into the relationship between cell shape dynamics and differentiation in development.

High variability within Candida albicans transcription factor RLM1: Isolates from vulvovaginal infections show a clear bias toward high molecular weight alleles.

Previous studies have correlated the severity of recurrent vulvovaginal Candida infections (VVC) and balanitis in patients from China with the presence of some dominant genotypes at the ORF RLM1. Here we tested VVC vs non-VVC isolates from Portugal, Brazil and Greece and, although the same genotypes were identified in VVC isolates, they were present in only five out of 150 strains. However, this analysis showed that VVC isolates presented a higher percentage of genotypes with similar high molecular weight alleles, in comparison with strains isolated from other biological sources.

Genomic and transcriptomic analysis of Saccharomyces cerevisiae isolates with focus in succinic acid production.

Succinic acid is a platform chemical that plays an important role as precursor for the synthesis of many valuable bio-based chemicals. Its microbial production from renewable resources has seen great developments, specially exploring the use of yeasts to overcome the limitations of using bacteria. The objective of the present work was to screen for succinate-producing isolates, using a yeast collection with different origins and characteristics. Four strains were chosen, two as promising succinic acid producers, in comparison with two low producers. Genome of these isolates was analysed, and differences were found mainly in genes SDH1, SDH3, MDH1 and the transcription factor HAP4, regarding the number of single nucleotide polymorphisms and the gene copy-number profile. Real-time PCR was used to study gene expression of 10 selected genes involved in the metabolic pathway of succinic acid production. Results show that for the non-producing strain, higher expression of genes SDH1, SDH2, ADH1, ADH3, IDH1 and HAP4 was detected, together with lower expression of ADR1 transcription factor, in comparison with the best producer strain. This is the first study showing the capacity of natural yeast isolates to produce high amounts of succinic acid, together with the understanding of the key factors associated, giving clues for strain improvement.

International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database--the quality controlled standard tool for routine identification of human and animal pathogenic fungi.

Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on "DNA barcoding of human and animal pathogenic fungi." A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org/ and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens.

Candida bracarensis: Evaluation of Virulence Factors and its Tolerance to Amphotericin B and Fluconazole.

Candida bracarensis is an uncommon Candida species found during an epidemiological study of candidiasis performed in Braga, Portugal. Initially, it was identified as C. glabrata, but recently detailed analyses pointed out their differences. So, little information is still available about C. bracarensis virulence factors and antifungal susceptibilities. Therefore, the main goal of this work is to evaluate the ability of C. bracarensis to form biofilms, to produce hydrolytic enzymes (proteases, phospholipases and hemolysins), as well as its susceptibility to amphotericin B and fluconazole. It was shown, for the first time, that all C. bracarensis strains were able to form biofilms and display proteinase and hemolytic activities. Moreover, although planktonic cells presented antifungal susceptibility, amphotericin B and fluconazole were unable to inhibit biofilm formation and eradicate pre-formed biofilms. Due to the propensity of C. bracarensis to display antifungal resistance and virulence attributes, the control of these emerging pathogens is recommended.

Transient complex interactions of mammalian peroxisomes without exchange of matrix or membrane marker proteins.

Peroxisomes and mitochondria show a much closer interrelationship than previously anticipated. They co-operate in the metabolism of fatty acids and reactive oxygen species, but also share components of their fission machinery. If peroxisomes - like mitochondria - also fuse in mammalian cells is a matter of debate and was not yet systematically investigated. To examine potential peroxisomal fusion and interactions in mammalian cells, we established an in vivo fusion assay based on hybridoma formation by cell fusion. Fluorescence microscopy in time course experiments revealed a merge of different peroxisomal markers in fused cells. However, live cell imaging revealed that peroxisomes were engaged in transient and long-term contacts, without exchanging matrix or membrane markers. Computational analysis showed that transient peroxisomal interactions are complex and can potentially contribute to the homogenization of the peroxisomal compartment. However, peroxisomal interactions do not increase after fatty acid or H(2) O(2) treatment. Additionally, we provide the first evidence that mitochondrial fusion proteins do not localize to peroxisomes. We conclude that mammalian peroxisomes do not fuse with each other in a mechanism similar to mitochondrial fusion. However, they show an extensive degree of interaction, the implication of which is discussed.

Intracellular trafficking of AIP56, an NF-κB-cleaving toxin from Photobacterium damselae subsp. piscicida.

AIP56 (apoptosis-inducing protein of 56 kDa) is a metalloprotease AB toxin secreted by Photobacterium damselae subsp. piscicida that acts by cleaving NF-κB. During infection, AIP56 spreads systemically and depletes phagocytes by postapoptotic secondary necrosis, impairing the host phagocytic defense and contributing to the genesis of infection-associated necrotic lesions. Here we show that mouse bone marrow-derived macrophages (mBMDM) intoxicated by AIP56 undergo NF-κB p65 depletion and apoptosis. Similarly to what was reported for sea bass phagocytes, intoxication of mBMDM involves interaction of AIP56 C-terminal region with cell surface components, suggesting the existence of a conserved receptor. Biochemical approaches and confocal microscopy revealed that AIP56 undergoes clathrin-dependent endocytosis, reaches early endosomes, and follows the recycling pathway. Translocation of AIP56 into the cytosol requires endosome acidification, and an acidic pulse triggers translocation of cell surface-bound AIP56 into the cytosol. Accordingly, at acidic pH, AIP56 becomes more hydrophobic, interacting with artificial lipid bilayer membranes. Altogether, these data indicate that AIP56 is a short-trip toxin that reaches the cytosol using an acidic-pH-dependent mechanism, probably from early endosomes. Usually, for short-trip AB toxins, a minor pool reaches the cytosol by translocating from endosomes, whereas the rest is routed to lysosomes for degradation. Here we demonstrate that part of endocytosed AIP56 is recycled back and released extracellularly through a mechanism requiring phosphoinositide 3-kinase (PI3K) activity but independent of endosome acidification. So far, we have been unable to detect biological activity of recycled AIP56, thereby bringing into question its biological relevance as well as the importance of the recycling pathway.

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.

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.

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.

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.

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.