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Pemphigus foliaceus (PF) is a blistering autoimmune skin disease rare in most of the world but endemic in certain regions of Brazil. PF is characterized by the detachment of epidermal cells and the presence of autoantibodies against desmoglein 1. In previous studies, we have shown that genetic polymorphisms and variable expression levels of certain leucocyte receptor complex (LRC) genes were associated with PF. However, the role of the LRC on PF susceptibility remained to be investigated. Here, we analysed 527 tag single nucleotide polymorphisms (SNPs) distributed within the 1·5 Mb LRC. After quality control, a total of 176 SNPs were analysed in 229 patients with PF and 194 controls. Three SNPs were associated with differential susceptibility to PF. The intergenic variant rs465169 [odds ratio (OR) = 1·50; P = 0·004] is located in a region that might regulate several immune-related genes, including VSTM1, LILRB1/2, LAIR1/2, LILRA3/4 and LENG8. The rs35336528 (OR = 3·44; P = 0·009) and rs1865097 (OR = 0·57; P = 0·005) SNPs in LENG8 and FCAR genes, respectively, were also associated with PF. Moreover, we found four haplotypes with SNPs within the KIR3DL2/3, LAIR2 and LILRB1 genes associated with PF (P < 0·05), which corroborate previously reported associations. Thus, our results confirm the importance of the LRC for differential susceptibility to PF and reveal new markers that might influence expression levels of several LRC genes, as well as candidates for further functional studies., (© 2018 John Wiley & Sons Ltd.)

Background: Despite treatment with effective antimalarial drugs, the mortality rate is still high in severe cases of the disease, highlighting the need to find adjunct therapies that can inhibit the adhesion of Plasmodium falciparum-infected erythrocytes (Pf-iEs)., Objectives: In this context, we evaluated a new heparan sulfate (HS) from Nodipecten nodosus for antimalarial activity and inhibition of P. falciparum cytoadhesion and rosetting., Methods: Parasite inhibition was measured by SYBR green using a cytometer. HS was assessed in rosetting and cytoadhesion assays under static and flow conditions using Chinese hamster ovary (CHO) and human lymphatic endothelial cell (HLEC) cells expressing intercellular adhesion molecule-1 (ICAM1) and chondroitin sulfate A (CSA), respectively., Findings: This HS inhibited merozoite invasion similar to heparin. Moreover, mollusk HS decreased cytoadherence of P. falciparum to CSA and ICAM-1 on the surface of endothelial cells under static and flow conditions. In addition, this glycan efficiently disrupted rosettes., Conclusions: These findings support a potential use for mollusk HS as adjunct therapy for severe malaria.

Anthrax is an infectious disease caused by Bacillus anthracis, a bioterrorism agent that develops resistance to clinically used antibiotics. Therefore, alternative mechanisms of action remain a challenge. Herein, we disclose deoxy glycosides responsible for specific carbohydrate-phospholipid interactions, causing phosphatidylethanolamine lamellar-to-inverted hexagonal phase transition and acting over B. anthracis and Bacillus cereus as potent and selective bactericides. Biological studies of the synthesized compound series differing in the anomeric atom, glycone configuration and deoxygenation pattern show that the latter is indeed a key modulator of efficacy and selectivity. Biomolecular simulations show no tendency to pore formation, whereas differential metabolomics and genomics rule out proteins as targets. Complete bacteria cell death in 10 min and cellular envelope disruption corroborate an effect over lipid polymorphism. Biophysical approaches show monolayer and bilayer reorganization with fast and high permeabilizing activity toward phosphatidylethanolamine membranes. Absence of bacterial resistance further supports this mechanism, triggering innovation on membrane-targeting antimicrobials.

Neurospora crassa is a non-pathogenic filamentous fungus widely used as a multicellular eukaryotic model. Recently, the biophysical properties of the plasma membrane of N. crassa conidia were thoroughly characterized. They evolve during conidial germination at a speed that depends on culture conditions, suggesting an important association between membrane remodeling and the intense membrane biogenesis that takes place during the germinative process. Staurosporine (STS) is a drug used to induce programmed cell death in various organisms. In N. crassa , STS up-regulates the expression of the ABC transporter ABC-3, which localizes at the plasma membrane and pumps STS out. To understand the role of plasma membrane biophysical properties in the fungal drug response, N. crassa was subjected to STS treatment during early and late conidial development stages. Following 1 h treatment with STS, there is an increase in the abundance of the more ordered, sphingolipid-enriched, domains in the plasma membrane of conidia. This leads to higher fluidity in other membrane regions. The global order of the membrane remains thus practically unchanged. Significant changes in sphingolipid-enriched domains were also observed after 15 min challenge with STS, but they were essentially opposite to those verified for the 1 h treatment, suggesting different types of drug responses. STS effects on membrane properties that are more dependent on ergosterol levels also depend on the developmental stage. There were no alterations on 2 h-grown cells, clearly contrasting to what happens at longer growth times. In this case, the differences were more marked for longer STS treatment, and rationalized considering that the drug prevents the increase in the ergosterol/glycerophospholipid ratio that normally takes place at the late conidial stage/transition to the mycelial stage. This could be perceived as a drug-induced development arrest after 5 h growth, involving ergosterol, and pointing to a role of lipid rafts possibly related with an up-regulated expression of the ABC-3 transporter. Overall, our results suggest the involvement of membrane ordered domains in the response mechanisms to STS in N. crassa .

This review is focused on sphingolipid backbone hydroxylation, a small but widespread structural feature with profound impact on membrane biophysical properties. We start by summarizing sphingolipid metabolism in mammalian cells, yeast and plants, focusing on how distinct hydroxylation patterns emerge in different eukaryotic kingdoms. Then, a comparison of the biophysical properties in membrane model systems and cellular membranes from diverse organisms is made. From an integrative perspective, these results can be rationalized considering that superficial hydroxyl groups in the backbone of sphingolipids (by intervening in the H-bond network) alter the balance of favorable interactions between membrane lipids. They may strengthen the bonding or compete with other hydroxyl groups, in particular the one of membrane sterols. Different sphingolipid hydroxylation patterns can stabilize/disrupt specific membrane domains or change whole plasma membrane properties, and therefore be important in the control of protein distribution, function and lateral diffusion and in the formation and overtime stability of signaling platforms. The recent examples explored throughout this review unveil a potentially key role for sphingolipid backbone hydroxylation in both physiological and pathological situations, as it can be of extreme importance for the proper organization of cell membranes in mammalian cells, yeast and, most likely, also in plants., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Genome-wide and fine mapping studies have shown that more than 90% of genetic variants associated with autoimmune diseases (AID) are located in non-coding regions of the human genome and especially in regulatory sequences, including microRNAs (miRNA) target sites. MiRNAs are small endogenous noncoding RNAs that modulate gene expression at the post-transcriptional level. Single nucleotide polymorphisms (SNPs) located within the 3' untranslated region of their target mRNAs (miRSNP) can alter miRNA binding sites. Yet, little is known about their effect on regulation by miRNA and the consequences for AID. Conversely, it is well known that two or more AID may share part of their genetic background. Here, we hypothesized that miRSNPs could be associated with more than one AID. To identify miRSNPs associated with AID, we integrated results from three different prediction tools (Polymirts, miRSNP, and miRSNPscore) using a naïve Bayes classifier approach to identify miRSNPs predicted to affect binding sites of miRNAs. Further, to detect miRSNPs associated with two or more AID, we integrated predictions with summary statistics from 12 AID studies. In addition, to prioritize miRSNPs, miRNAs and AID-associated target genes, we used public expression quantitative trait locus (eQTL) data and mRNA-seq and small RNA-seq data. We identified 34 miRNSPs associated with at least two AID. Furthermore, we found 86 miRNAs predicted to target 18 of the associated gene's mRNAs. Our integrative approach revealed new insights into miRNAs and AID associated target genes. Thus, it helped to prioritize AID noncoding risk SNPs that might be involved in the causal mechanisms, providing valuable information for further functional studies.

Skin blisters of pemphigus foliaceus (PF) present concomitant deposition of autoantibodies and components of the complement system (CS), whose gene polymorphisms are associated with susceptibility to different autoimmune diseases. To investigate these in PF, we evaluated 992 single-nucleotide polymorphisms (SNPs) of 44 CS genes, genotyped through microarray hybridization in 229 PF patients and 194 controls. After excluding SNPs with minor allele frequency <1%, out of Hardy-Weinberg equilibrium in controls or in strong linkage disequilibrium ( r 2  ≥ 0.8), 201 SNPs remained for logistic regression. Polymorphisms of 11 genes were associated with PF. MASP1 encodes a crucial serine protease of the lectin pathway (rs13094773: OR = 0.5, p  = 0.0316; rs850309: OR = 0.23, p  = 0.03; rs3864098: OR = 1.53, p  = 0.0383; rs698104: OR = 1.52, p  = 0.0424; rs72549154: OR = 0.55, p  = 0.0453). C9 (rs187875: OR = 1.46, p  = 0.0189; rs700218: OR = 0.12, p  = 0.0471) and C8A (rs11206934: OR = 4.02, p  = 0.0323) encode proteins of the membrane attack complex (MAC) and C5AR1 (rs10404456: OR = 1.43, p  = 0.0155), a potent anaphylatoxin-receptor. Two encode complement regulators: MAC-blocking CD59 (rs1047581: OR = 0.62, p  = 0.0152) and alternative pathway-blocking CFH (rs34388368: OR = 2.57, p  = 0.0195). One encodes opsonin: C3 (rs4807895: OR = 2.52, p  = 0.0239), whereas four encode receptors for C3 fragments: CR1 (haplotype with rs6656401: OR = 1.37, p  = 0.0382), CR2 (rs2182911: OR = 0.23, p  = 0.0263), ITGAM (CR3, rs12928810: OR = 0.66, p  = 0.0435), and ITGAX (CR4, rs11574637: OR = 0.63, p  = 0.0056). Associations reinforced former findings, regarding differential gene expression, serum levels, C3, and MAC deposition on lesions. Deregulation of previously barely noticed processes, e.g., the lectin and alternative pathways and opsonization-mediated phagocytosis, also modulate PF susceptibility. The results open new crucial avenues for understanding disease etiology and may improve PF treatment through additional therapeutic targets.

Obesity represents a continuously growing global epidemic and is associated with the development of type 2 diabetes mellitus. The etiology of type 2 diabetes is related to the resistance of insulin-sensitive tissues to its action leading to impaired blood glucose regulation. Photobiomodulation (PBM) therapy might be a non-pharmacological, non-invasive strategy to improve insulin resistance. It has been reported that PBM therapy in combination with physical exercise reduces insulin resistance. Therefore, the aim of this study was to investigate the effects of PBM therapy on insulin resistance in obese mice. Male Swiss albino mice received low-fat control diet (n = 16, LFC) or high-fat diet (n = 18, HFD) for 12 weeks. From 9th to 12th week, the mice received PBM therapy (LASER) or Sham (light off) treatment and were allocated into four groups: LFC Sham (n = 8), LFC PBM (n = 8), HFD Sham (n = 9), and HFD PBM (n = 9). The PBM therapy was applied in five locations: to the left and right quadriceps muscle, upper limbs and center of the abdomen, during 40 s at each point, once a day, 5 days a week, for 4 weeks (780 nm, 250 mW/cm 2 , 10 J/cm 2 , 0.4 J per site; 2 J total dose per day). Insulin signaling pathway was evaluated in the epididymal adipose tissue. PBM therapy improved glucose tolerance and phosphorylation of Akt (Ser473) and reversed the HFD-induced reduction of GLUT4 content and phosphorylation of AS160 (Ser588). Also, PBM therapy reversed the increased area of epididymal and mesenteric adipocytes. The results showed that chronic PBM therapy improved parameters related to obesity and insulin resistance in HFD-induced obesity in mice.

We report an immunization technique that can update the production of monoclonal antibodies (mAbs): the multiple tolerization subtractive immunization (MTSI). A total of 10 BALB/C mice were used. Animals in group 1 received one inoculation of RWPE-1 cells (nontumoral), followed by cyclophosphamide, and then received serial inoculations of nonirradiated PC3 cells (tumoral). Animals in group 2 received our MTSI protocol, as follows: one inoculation of RWPE-1 cells, followed by cyclophosphamide (Cy). This whole tolerization step was repeated three other times, with 14-day intervals between the last Cy exposure and the next RWPE-1 cell inoculation. Finally, the animals received the same nonirradiated PC3 cell exposure as group 1. Blood was taken from each animal, and their polyclonal sera individually tested against the nontumoral RWPE-1 cells in flow cytometry. We found out that, after the MTSI was employed, the serum of the immunized animals, in group 2, contained considerably less antibodies that reacted against the tolerogenic cells, compared with the serum of the animals that underwent regular subtractive immunization. We showed that, by repeating the tolerization cycles, the polyclonal antibodies produced by mice have a reduced specificity toward common/immunodominant epitopes present at nontumoral cells, and thus this technique can be readily used by others in studies involving murine mAb protocols.

Phenolic acids have been associated to a wide range of important health benefits underlain by a common molecular mechanism of action. Considering that significant membrane permeation is prevented by their hydrophilic character, we hypothesize that their main effects result from the interplay with cell membrane surface. This hypothesis was tested using the paradigmatic caffeic acid (CA) and two of its ester derivatives, rosmarinic (RA) and chlorogenic (CGA) acids, for which we predict, based on molecular dynamics simulations, a shallow location in phospholipid bilayers dependent on the protonation-state. Using complementary experimental approaches, an interaction with the membrane was definitely revealed for the three compounds, with RA exhibiting the highest lipid bilayer partition, and the redox signals of membrane-bound RA and CA being clearly detected. Cholesterol decreased the compounds bilayer partition, but not their ability to lower membrane dipole potential. In more complex membrane models containing also sphingomyelin, with liquid disordered (l d )/ liquid ordered (l o ) phases coexistence, mimicking domains in the external leaflet of human plasma membrane, all compounds were able to affect nanodomains lateral organization. RA, and to a lesser extent CGA, decreased the size of l o domains. The most significant effect of CA was the possible formation of a rigid gel-like phase, enriched in sphingomyelin. In addition, all phenolic acids decreased the order of l o domains. In sum, phenolic acid effects on the membrane are enhanced in cholesterol-rich l o phases, which predominate in the outer leaflet of human cell membranes and are involved in many key cellular processes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Despite fast advances in genomics and proteomics, monoclonal antibodies (mAbs) are still a valuable tool for areas such as the evolution of basic research in stem cells and cancer, for immunophenotyping cell populations, diagnosing and prognosis of diseases, and for immunotherapy. To summarize different subtractive immunization approaches successfully used for the production of highly specific antibodies, we identified scientific articles in NCBI PubMed using the following search terms: subtractive immunization, monoclonal antibody, tolerization, neonatal, high-zone tolerance, masking immunization. Patent records were also consulted. From the list of results, we included all available reports, from 1985 to present, that used any enhanced immunization technique to produce either polyclonal or monoclonal antibodies. Our examination yielded direct evidence that these enhanced immunization techniques are efficient in obtaining specific antibodies to rare epitopes, with different applications, such as to identify food contaminants or tumor cells.

Background and Purpose: Vasoactive intestinal peptide (VIP) is an important modulator of hippocampal synaptic transmission that influences both GABAergic synaptic transmission and glutamatergic cell excitability through activation of VPAC 1 and VPAC 2 receptors. Presynaptic enhancement of GABA release contributes to VIP modulation of hippocampal synaptic transmission., Experimental Approach: We investigated which VIP receptors and coupled transduction pathways were involved in VIP enhancement of K + -evoked [ 3 H]-GABA release from isolated nerve terminals of rat hippocampus., Key Results: VIP enhancement of [ 3 H]-GABA release was potentiated in the presence of the VPAC 1 receptor antagonist PG 97-269 but converted into an inhibition in the presence of the VPAC 2 receptor antagonist PG 99-465, suggesting that activation of VPAC 1 receptors inhibits and activation of VPAC 2 receptors enhances, GABA release. A VPAC 1 receptor agonist inhibited exocytotic voltage-gated calcium channel (VGCC)-dependent [ 3 H]-GABA release through activation of protein G i/o , an effect also dependent on PKC activity. A VPAC 2 receptor agonist enhanced both exocytotic VGCC-dependent release through protein G s -dependent, PKA-dependent and PKC-dependent mechanisms and GABA transporter 1-mediated [ 3 H]-GABA release through a G s protein-dependent and PKC-dependent mechanism., Conclusions and Implications: Our results show that VPAC 1 and VPAC 2 VIP receptors have opposing actions on GABA release from hippocampal nerve terminals through activation of different transduction pathways. As VPAC 1 and VPAC 2 receptors are located in different layers of Ammon's horn, our results suggest that these VIP receptors underlie different modulation of synaptic transmission to pyramidal cell dendrites and cell bodies, with important consequences for their possible therapeutic application in the treatment of epilepsy., (© 2017 The British Pharmacological Society.)

Osteoarthritis (OA) is the most prevalent chronic age-related arthritic disease that mainly affects the diarthrodial joints. Nevertheless, there is no treatment currently available that can effectively reduce symptoms or slow down or stop disease progression. The lack of disease-modifying therapies could be explained by the complex pathogenesis of OA, which is still not completely understood. Intertwined epigenetic mechanisms such as DNA methylation, histone modifications, and noncoding RNAs (ncRNAs) have been indicated as important cellular tools to maintain tissue homeostasis upon environmental challenges. The current review illustrates that dysfunctional epigenetic control mechanisms in the articular cartilage likely play an important role in driving OA pathophysiology., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Synthetic systems are widely used to unveil the molecular mechanisms of complex cellular events. Artificial membranes are key examples of models employed to address lipid-lipid and lipid-protein interactions. In this work, we developed a new synthetic system that more closely resembles the lysosome - the lysosome-mimicking vesicles (LMVs) - displaying stable acid-to-neutral pH gradient across the membrane. To evaluate the advantages of this synthetic system, we assessed the distinct effects of sphingosine (Sph) accumulation in membrane structure and biophysical properties of standard liposomes (no pH gradient) and in LMVs with lipid composition tuned to mimic physiological- or NPC1-like lysosomes. Ternary 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/Sphingomyelin (SM)/Cholesterol (Chol) mixtures with, respectively, low and high Chol/SM levels were prepared. The effect of Sph on membrane permeability and biophysical properties was evaluated by fluorescence spectroscopy, electrophoretic and dynamic light scattering. The results showed that overall Sph has the ability to cause a shift in vesicle surface charge, increase membrane order and promote a rapid increase in membrane permeability. These effects are enhanced in NPC1- LMVs. The results suggest that lysosomal accumulation of these lipids, as observed under pathological conditions, might significantly affect lysosomal membrane structure and integrity, and therefore contribute to the impairment of cell function.

Two recently published Ru(III) complexes bearing (N 2 O 2 ) tetradentate bis(aminophenolate) ligands, formulated as [Ru(III)(salan)(PPh 3 )Cl] (salan is the tetradentate ligand 6,6'-(1S,2S)-cyclohexane-1,2-diylbis(azanediyl)bis(methylene)bis(3-methoxyphenol) in complex 1, or 2,2'-(1S,2S)-cyclohexane-1,2-diylbis(azanediyl)bis(methylene)bis(4-methoxyphenol) in complex 2; PPh 3 is triphenylphosphane) and found very active against ovarian and breast adenocarcinoma human cells were studied to outline their antitumor mode of action. The human cisplatin-sensitive ovarian adenocarcinoma line A2780 was used herein as the cell model. At a 24h challenge (similarly as found before for 72h) both complexes are active, their cytotoxicity being comparable to that of cisplatin in the same conditions. As a possible target in the cell for their action, the interaction of 1 and 2 with DNA was assessed through displacement of well-established DNA fluorescent probes (ethidium bromide, EB, and 4',6-diamidino-2-phenylindole, DAPI) through steady-state and time-resolved fluorescence spectroscopy. The whole emission spectra were analyzed globally for the binary DNA-probe and ternary DNA-probe-Ru(III) complex systems. Both Ru(III) complexes can displace EB and bind to DNA with similar and moderate strong affinity with conditional stability constants of logK'=(5.05±0.01) for 1 and logK'=(4.79±0.01) for 2. The analysis of time-domain fluorescence intensity decays confirmed both qualitatively and quantitatively the model used to describe the binding and competition processes. Cell studies indicated that apoptosis is the major mechanism of cell death for both complexes, with 2 (the more active complex) promoting that process more efficiently than 1. Transmission electron micrographs revealed clear alterations on intracellular organization consistent with the induction of programmed cell death processes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Genetic variations mapping to 3' untranslated regions (3'UTRs) may overlap with microRNA (miRNA) binding sites, therefore potentially interfering with translation inhibition or messenger RNA (mRNA) degradation. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) located within the 3'UTRs of six candidate genes and predicted to interfere with miRNA ligation could account for disease-relevant differential mRNA levels. Focusing on pemphigus foliaceus (PF) - an autoimmune blistering skin condition with unique endemic patterns - we investigated whether nine 3'UTR SNPs from the CD1D, CTLA4, KLRD1, KLRG1, NKG7, and TNFSF13B genes differentially expressed in PF were disease-associated. The heterozygous genotype of the KLRG1 rs1805672 polymorphism was associated with increased predisposition to PF (A/G vs. A/A: P=0.038; OR=1.60), and a trend for augmented susceptibility was observed for carriers of the G allele (P=0.094; OR=1.44). In silico analyses suggested that rs1805672 G allele could disrupt binding of miR-584-5p, and indicated rs1805672 as an expression Quantitative Trait Locus (eQTL), with an effect on KLRG1 gene expression. Dual-luciferase assay showed that miR-584-5p mediated approximately 50% downregulation of the reporter gene's activity through the 3'UTR of KLRG1 harboring rs1805672 A allele (vs. miRNA-negative condition, P=0.006). This silencing relationship was lost after site-directed mutation to G allele (vs. miRNA-negative condition, P=0.391; vs. rs1805672 A allele, P=0.005). Collectively, these results suggest that a disease-associated SNP located within the 3'UTR of KLRG1 directly interferes with miR-584-5p binding, allowing for KLRG1 mRNA differential accumulation, which in turn may contribute to pathogenesis of autoimmune diseases, such as pemphigus., Competing Interests: Statement The authors of this study declare the absence of any conflict of interest., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The molecular processes that are crucial for cell function, such as proliferation, migration and survival, are regulated by hydrogen peroxide (H2O2). Although environmental cues, such as growth factors, regulate redox signaling, it was still unknown whether the ECM, a component of the cell microenvironment, had a function in this process. Here, we showed that the extracellular matrix (ECM) differently regulated H2O2 consumption by endothelial cells and that this effect was not general for all types of cells. The analysis of biophysical properties of the endothelial cell membrane suggested that this modification in H2O2 consumption rates was not due to altered membrane permeability. Instead, we found that the ECM regulated GPx activity, a known H2O2 scavenger. Finally, we showed that the extent of PTEN oxidation was dependent on the ECM, indicating that the ECM was able to modulate H2O2-dependent protein oxidation. Thus, our results unraveled a new mechanism by which the ECM regulates endothelial cell function by altering redox balance. These results pinpoint the ECM as an important component of redox-signaling., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

The extraction of gold, coal, nickel, uranium, copper and other earth-moving activities almost always leads to environmental damage. In metal and coal extraction, exposure of sulfide minerals to the atmosphere leads to generation of acid rock drainage (ARD) and in underground mining to acid mine drainage (AMD) due to contamination of infiltrating groundwater. This study proposes to develop a reactive cover system that inhibits infiltration of oxygen and also releases alkalinity to increase the pH of generated ARD and attenuate metal contaminants at the same time. The reactive cover system is constructed using steel slag, a waste product generated from steel industries. This study shows that this type of cover system has the potential to reduce some of the adverse effects of sulfide mine waste disposal on land. Geochemical and geotechnical characterization tests were carried out. Different proportions of sulfide mine waste and steel slag were studied in leachate extraction tests. The best proportion was 33% of steel slag in dry weight. Other tests were conducted as follows: soil consolidation, saturated permeability and soil water characteristic curve. The cover system was numerically modeled through unsaturated flux analysis using Vadose/w. The solution proposed is an oxygen transport barrier that allows rain water percolation to treat the ARD in the waste rock pile. The results showed that the waste pile slope is an important factor and the cover system must have 5 m thickness to achieve an acceptable effectiveness., (© The Author(s) 2015.)

Bovine cysticercosis is detected during the routine post mortem examination of carcasses by visual inspection (knife and eye method). However, the sensitivity of this procedure is several times lower than immunoassays, even when it is performed by qualified professionals. In the present study, a new generation capture antigens were screened from a phage display peptide library using antibodies from Taenia saginata-infected animals. Eight phage clones were selected, and one, Tsag 3 (VHTSIRPRCQPRAITPR), produced similar results to the T. saginata metacestode crude antigen (TsCa) when used as a capture antigen in an ELISA. The phage-displayed peptides competed with TsCa for binding sites, reducing the reactivity by approximately 30 %. Alanine scanning indicated that proline, arginine, and serine are important residues for antibody binding. Tsag 1 (HFYQITWLPNTFPAR), the most frequent affinity-selected clone, and Tsag 6 (YRWPSTPSASRQATL) shared similarity with highly conserved proteins from the Taeniidae family with known immunogenicity. Due to their epitopic or mimotopic properties, these affinity-selected phages could contribute to the rational design of an ante mortem immunodiagnosis method for bovine cysticercosis, as well as an epitope-based vaccine to interrupt the taeniosis/cysticercosis complex.

In this work, we developed a biomimetic platform where the study of membrane associated redox processes and high-resolution imaging of lipid nanodomains can be both performed, based on a new functional gold modification, l-cysteine self-assembled monolayer. This monolayer proved to be ideal for the preparation of defect-free planar supported lipid bilayers (SLBs) where nanodomains with height difference of ∼1.5 nm are clearly resolved by atomic force microscopy. Single and multicomponent lipid compositions were used, leading to the formation of different phases and domains mimicking the lateral organization of cellular membranes, and in all cases stable and continuous bilayers were obtained. These platforms were tested toward the interaction with bioelectroactive molecules, the antioxidant quercetin, and the hormone epinephrine. Despite the weak interaction detected between epinephrine and lipid bilayers, our biomimetic interface was able to sense the redox process of membrane-bound epinephrine, obtain its surface concentration (9.36 × 10(-11) mol/cm(2) for a fluid bilayer), and estimate a mole fraction membrane/water partition coefficient (Kp) from cyclic voltammetric measurements (1.13 × 10(4) for a fluid phase membrane). This Kp could be used to quantitatively describe the minute changes observed in the photophysical properties of epinephrine intrinsic fluorescence upon its interaction with liposome suspensions. Moreover, we showed that the lipid membrane stabilizes epinephrine structure, preventing its oxidation, which occurs in neutral aqueous solution, and that epinephrine partition and mobility in membranes depends on lipid phase, expanding our knowledge on hormone membrane interactions.

The very high antiproliferative activity of [Co(Cl)(H2O)(phendione)2][BF4] (phendione is 1,10-phenanthroline-5,6-dione) against three human tumor cell lines (half-maximal inhibitory concentration below 1 μM) and its slight selectivity for the colorectal tumor cell line compared with healthy human fibroblasts led us to explore the mechanisms of action underlying this promising antitumor potential. As previously shown by our group, this complex induces cell cycle arrest in S phase and subsequent cell death by apoptosis and it also reduces the expression of proteins typically upregulated in tumors. In the present work, we demonstrate that [Co(Cl)(phendione)2(H2O)][BF4] (1) does not reduce the viability of nontumorigenic breast epithelial cells by more than 85 % at 1 μM, (2) promotes the upregulation of proapoptotic Bax and cell-cycle-related p21, and (3) induces release of lactate dehydrogenase, which is partially reversed by ursodeoxycholic acid. DNA interaction studies were performed to uncover the genotoxicity of the complex and demonstrate that even though it displays K b (± standard error of the mean) of (3.48 ± 0.03) × 10(5) M(-1) and is able to produce double-strand breaks in a concentration-dependent manner, it does not exert any clastogenic effect ex vivo, ruling out DNA as a major cellular target for the complex. Steady-state and time-resolved fluorescence spectroscopy studies are indicative of a strong and specific interaction of the complex with human serum albumin, involving one binding site, at a distance of approximately 1.5 nm for the Trp214 indole side chain with log K b ~4.7, thus suggesting that this complex can be efficiently transported by albumin in the blood plasma.

To date, it is widely accepted that microdomains do form in the biological membranes of all eukaryotic cells, and quite possibly also in prokaryotes. Those sub-micrometric domains play crucial roles in signaling, in intracellular transport, and even in inter-cellular communications. Despite their ubiquitous distribution, and the broad and lasting interest invested in those microdomains, their actual nature and composition, and even the physical rules that regiment their assembly still remain elusive and hotly debated. One of the most often considered models is the raft hypothesis, i.e., the partition of lipids between liquid disordered and ordered phases (Ld and Lo, respectively), the latter being enriched in sphingolipids and cholesterol. Although it is experimentally possible to obtain the formation of microdomains in synthetic membranes through Ld/Lo phase separation, there is an ever increasing amount of evidence, obtained with a wide array of experimental approaches, that a partition between domains in Ld and Lo phases cannot account for many of the observations collected in real cells. In particular, it is now commonly perceived that the plasma membrane of cells is mostly in Lo phase and recent data support the existence of gel or solid ordered domains in a whole variety of live cells under physiological conditions. Here, we present a model whereby seeds comprised of oligomerised proteins and/or lipids would serve as crystal nucleation centers for the formation of diverse gel/crystalline nanodomains. This could confer the selectivity necessary for the formation of multiple types of membrane domains, as well as the stability required to match the time frames of cellular events, such as intra- or inter-cellular transport or assembly of signaling platforms. Testing of this model will, however, require the development of new methods allowing the clear-cut discrimination between Lo and solid nanoscopic phases in live cells.

The structural polysaccharides contained in plant cell walls have been pointed to as a promising renewable alternative to petroleum and natural gas. Ferulic acid is a ubiquitous component of plant polysaccharides, which is found in either monomeric or dimeric forms and is covalently linked to arabinosyl residues. Ferulic acid has several commercial applications in food and pharmaceutical industries. The study herein introduces a novel feruloyl esterase from Aspergillus clavatus (AcFAE). Along with a comprehensive functional and biophysical characterization, the low-resolution structure of this enzyme was also determined by small-angle X-ray scattering. In addition, we described the production of phenolic compounds with antioxidant capacity from wheat arabinoxylan and sugarcane bagasse using AcFAE. The ability to specifically cleave ester linkages in hemicellulose is useful in several biotechnological applications, including improved accessibility to lignocellulosic enzymes for biofuel production.

Magnesium sulfate (MgSO4) is used to treat and prevent eclamptic seizures, and several anticonvulsant drugs (e.g., sodium valproate) are clinically effective antimanic drugs. Psychostimulant-induced hyperlocomotion has been proposed as an animal model for the study of antimanic drugs. The present study evaluated the effects of MgSO4 and sodium valproate (as a positive control) on hyperlocomotion induced by methylphenidate in mice. Acute MgSO4 (300-400 mg/kg), but not sodium valproate (100-300 mg/kg), prevented the increase in locomotor activity induced by methylphenidate (5.0 mg/kg). In contrast, repeated treatment (14 days) with valproate (300 mg/kg), but not MgSO4 (400 mg/kg), blocked methylphenidate-induced hyperlocomotion. Thus, acute MgSO4 exerted antimanic-like effects in this animal model.

In Saccharomyces cerevisiae, the rate of hydrogen peroxide (H(2)O(2)) diffusion through the plasma membrane decreases during adaptation to H(2)O(2) by a still unknown mechanism. Here, adaptation to H(2)O(2) was observed to modulate rapidly the expression of genes coding for enzymes involved in ergosterol and lipid metabolism. Adaptation to H(2)O(2) also alters plasma membrane lipid composition. The main changes were the following: (a) there was a decrease in oleic acid (30%) and in the ratio between unsaturated and saturated long-chain fatty acids; (b) the phosphatidylcholine:phosphatidylethanolamine ratio increased threefold; (c) sterol levels were unaltered but there was an increased heterogeneity of sterol-rich microdomains and increased ordered domains; (d) the levels of the sterol precursor squalene increased twofold, in agreement with ERG1 gene down-regulation; and (e) C26:0 became the major very long chain fatty acid owing to an 80% decrease in 2-hydroxy-C26:0 levels and a 50% decrease in C20:0 levels, probably related to the down-regulation of fatty acid elongation (FAS1, FEN1, SUR4) and ceramide synthase (LIP1, LAC1) genes. Therefore, H(2)O(2) leads to a reorganization of the plasma membrane microdomains, which may explain the lower permeability to H(2)O(2), and emerges as an important regulator of lipid metabolism and plasma membrane lipid composition.

The SARS coronavirus (SARS-CoV) envelope spike (S) glycoprotein, a Class I viral fusion protein, is responsible for the fusion between the membranes of the virus and the target cell. In the present work, we report a study of the binding and interaction with model membranes of a peptide pertaining to the putative fusion domain of SARS-CoV, SARS FP, as well as the structural changes that take place in both the phospholipid and the peptide molecules upon this interaction. From fluorescence and infrared spectroscopies, the peptide ability to induce membrane leakage, aggregation and fusion, as well as its affinity toward specific phospholipids, was assessed. We demonstrate that SARS FP strongly partitions into phospholipid membranes, more specifically with those containing negatively charged phospholipids, increasing the water penetration depth and displaying membrane-activity modulated by the lipid composition of the membrane. Interestingly, peptide organization is different depending if SARS FP is in water or bound to the membrane. These data suggest that SARS FP could be involved in the merging of the viral and target cell membranes by perturbing the membrane outer leaflet phospholipids and specifically interacting with negatively charged phospholipids located in the inner leaflet.

Recently, several indications have been found that suggest a preferential interaction between cholesterol and tryptophan residues located near the membrane-water interface. The aim of this study was to investigate by direct methods how tryptophan and cholesterol interact with each other and what the possible consequences are for membrane organization. For this purpose, we used cholesterol-containing model membranes of dimyristoylphosphatidylcholine (DMPC) in which a transmembrane model peptide with flanking tryptophans [acetyl-GWW(LA)8LWWA-amide], called WALP23, was incorporated to mimic interfacial tryptophans of membrane proteins. These model systems were studied with two complementary methods. (1) Steady-state and time-resolved Förster resonance energy transfer (FRET) experiments employing the fluorescent cholesterol analogue dehydroergosterol (DHE) in combination with a competition experiment with cholesterol were used to obtain information about the distribution of cholesterol in the bilayer in the presence of WALP23. The results were consistent with a random distribution of cholesterol which indicates that cholesterol and interfacial tryptophans are not preferentially located next to each other in these bilayer systems. (2) Solid-state 2H NMR experiments employing either deuterated cholesterol or indole ring-deuterated WALP23 peptides were performed to study the orientation and dynamics of both molecules. The results showed that the quadrupolar splittings of labeled cholesterol were not affected by an interaction with tryptophan-flanked peptides and, vice versa, that the quadrupolar splittings of labeled indole rings in WALP23 are not significantly influenced by addition of cholesterol to the bilayer. Therefore, both NMR and fluorescence spectroscopy results independently show that, at least in the model systems studied here, there is no evidence for a preferential interaction between cholesterol and tryptophans located at the bilayer interface.

A 28-mer peptide (gammaM4) corresponding to the fourth transmembrane segment of the nicotinic acetylcholine receptor (AChR) gamma-subunit, with a single tryptophan residue (Trp6), was reconstituted into lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), loaded with either high or low amounts of cholesterol, i.e., in the conjugated liquid-ordered and liquid-disordered phases, respectively, at room temperature. By making use of the Trp intrinsic fluorescence, both steady-state and time-resolved fluorescence techniques were employed, namely, red-edge excitation shift effect, decay-associated spectra (DAS), and time-resolved anisotropy. The results obtained here, together with previous studies on the same reconstituted peptide, indicate that: (i) Trp6 is strongly anchored in the bilayer with a defined transverse location; (ii) the modifications in the measured DAS are related to the complex result of a self-quenching process on the decay parameters; (iii) the wobbling movement of the indole moiety of Trp6 is fast but severely restricted in amplitude; and, (iv) in the liquid-ordered phase, the bilayer properties and the tilt angle of the peptide enhance peptide-peptide interactions, with the formation of peptide rich patches and possibly some anti-parallel helix-helix aggregates, showing different dynamics from that of the peptide in the liquid-disordered phase where the peptide is randomly distributed.

A 28-mer gammaM4 peptide, obtained by solid-state synthesis and corresponding to the fourth transmembrane segment of the nicotinic acetylcholine receptor gamma-subunit, possesses a single tryptophan residue (Trp453), making it an excellent model for studying peptide-lipid interactions in membranes by fluorescence spectroscopy. The gammaM4 peptide was reconstituted with synthetic lipids (vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, i.e., POPC) rich and poor in cholesterol and analyzed using steady-state and time-resolved fluorescence techniques. The decrease in gammaM4 intrinsic fluorescence lifetime observed upon incorporation into a cholesterol-rich lo phase could be rationalized on the basis of a dynamic self-quenching owing to the formation of peptide-rich patches in the membrane. This agrees with the low Förster type resonance energy transfer efficiency from the Trp453 residue to the fluorescent cholesterol analog, dehydroergosterol, in the lo phase. In the absence of cholesterol the gammaM4 nicotinic acetylcholine receptor peptide is randomly distributed in the POPC bilayer with its hydrophobic moiety matching the membrane thickness, whereas in the presence of cholesterol the increase in the membrane thickness and variation of the material properties favor the formation of peptide-enriched patches, i.e., interhelix interaction energy is essential for obtaining a stabilized structure. Thus, the presence of a cholesterol-rich, ordered POPC phase drives the organization of peptide-enriched patches, in which the gammaM4 peptide occupies approximately 30% of the patch area.