Toxin-induced pore formation is hindered by intermolecular hydrogen bonding in sphingomyelin bilayers

Sticholysin I and II (StnI and StnII) are pore-forming toxins that use sphingomyelin (SM) for membrane binding. We examined how hydrogen bonding among membrane SMs affected the StnI- and StnII-induced pore formation process, resulting in bilayer permeabilization. We compared toxin-induced permeabilization in bilayers containing either SM or dihydro-SM (lacking the trans 4 double bond of the long-chain base), since their hydrogen-bonding properties are known to differ greatly. We observed that whereas both StnI and StnII formed pores in unilamellar vesicles containing palmitoyl-SM or oleoyl-SM, the toxins failed to similarly form pores in vesicles prepared from dihydro-PSM or dihydro-OSM. In supported bilayers containing OSM, StnII bound efficiently, as determined by surface plasmon resonance. However, StnII binding to supported bilayers prepared from dihydro-OSM was very low under similar experimental conditions. The association of the positively charged StnII (at pH 7.0) with unilamellar vesicles prepared from OSM led to a concentration-dependent increase in vesicle charge, as determined from zeta-potential measurements. With dihydro-OSM vesicles, a similar response was not observed. Benzyl alcohol, which is a small hydrogen-bonding compound with affinity to lipid bilayer interfaces, strongly facilitated StnII-induced pore formation in dihydro-OSM bilayers, suggesting that hydrogen bonding in the interfacial region originally prevented StnII from membrane binding and pore formation. We conclude that interfacial hydrogen bonding was able to affect the membrane association of StnI- and StnII, and hence their pore forming capacity. Our results suggest that other types of protein interactions in bilayers may also be affected by hydrogen-bonding origination from SMs

Solution-based synthesis and processing of Sn- and Bi-doped Cu3SbSe4 nanocrystals, nanomaterials and ring-shaped thermoelectric generators

Copper-based chalcogenides that comprise abundant, low-cost, and environmental friendly elements are excellent materials for a number of energy conversion applications, including photovoltaics, photocatalysis, and thermoelectrics (TE). In such applications, the use of solution-processed nanocrystal (NC) to produce thin films or bulk nanomaterials has associated several potential advantages, such as high material yield and throughput, and composition control with unmatched spatial resolution and cost. Here we report on the production of Cu3SbSe4 (CASe) NCs with tuned amounts of Sn and Bi dopants. After proper ligand removal, as monitored by nuclear magnetic resonance and infrared spectroscopies, these NCs were used to produce dense CASe bulk nanomaterials for solid state TE energy conversion. By adjusting the amount of extrinsic dopants, dimensionless TE figures of merit (ZT) up to 1.26 at 673 K were reached. Such high ZT values are related to an optimized carrier concentration by Sn doping, a minimized lattice thermal conductivity due to efficient phonon scattering at point defects and grain boundaries, and to an increase of the Seebeck coefficient obtained by a modification of the electronic band structure with the Bi doping. Nanomaterials were further employed to fabricate ring-shaped TE generators to be coupled to hot pipes and which provided 20 mV and 1 mW per TE element when exposed to a 160 °C temperature gradient. The simple design and good thermal contact associated with the ring geometry and the potential low cost of the material solution processing may allow the fabrication of TE generators with short payback times.Peer ReviewedPostprint (author's final draft

Pterostilbene Reduces Liver Steatosis and Modifies Hepatic Fatty Acid Profile in Obese Rats

Excessive fat accumulation within the liver is known as "simple hepatic steatosis", which is the most benign form of non-alcoholic fatty liver disease (NAFLD). The aim of the present study was to determine whether pterostilbene improves this hepatic alteration in Zucker (fa/fa) rats. Animals were distributed in two experimental groups (n = 10) and fed a standard laboratory diet. Rats in the pterostilbene group were given a dose of 30 mg/kg body weight/d for six weeks. After sacrifice, serum glucose, transaminase, and insulin concentrations were quantified and the liver triacylglycerol content and fatty acid profile was analyzed. Different pathways of triacylglycerol metabolism in liver were studied, including fatty acid synthesis and oxidation, triglyceride assembly, fatty acid uptake, and glucose uptake. With pterostilbene administration, a reduction in insulin concentrations (consequently in the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR)) and hepatic triacylglycerol content were observed. No effects were observed in pterostilbene-treated rats in the activity of de novo lipogenesis enzymes. An improvement in the fatty acid profile was observed in pterostilbene-treated rats. In conclusion, pterostilbene is a useful molecule to reduce liver steatosis. Its delipidating effect is due, at least in part, to reduced fatty acid availability and triacylglycerol synthesis, as well as to an increased very low-density lipoprotein assembly and fatty acid oxidation.This research was funded by Ministerio de Economía y Competitividad-Fondo Europeo de Desarrollo Regional, grant number AGL-2015-65719-R; Instituto de Salud Carlos III CIBERobn, grant number, CB12/03/30007; Government of the Basque Country, grant number IT-572-13; Biodonostia Institute, grant number Biodonostia-CIBEREHD

Sticholysin, Sphingomyelin, and Cholesterol: A Closer Look at a Tripartite Interaction

Actinoporins are a group of soluble toxic proteins that bind to membranes containing sphingomyelin (SM) and oligomerize to form pores. Sticholysin II (StnII) is a member of the actinoporin family, produced by Stichodactyla helianthus. Cholesterol (Chol) is known to enhance the activity of StnII. However, the molecular mechanisms behind this activation have remained obscure, although the activation is not Chol specific but rather sterol specific. To further explore how bilayer lipids affect or are affected by StnII, we have used a multiprobe approach (fluorescent analogs of both Chol and SM) in combination with a series of StnII tryptophan (Trp)-mutants, to study StnII/bilayer interactions. First we compared StnII bilayer permeabilization in the presence of Chol or oleoyl-ceramide (OCer). The comparison was done since both Chol and OCer have a 1-hydroxyl which help to orient the molecule in the bilayer (although OCer have additional polar functional groups). Both Chol and OCer also have increased affinity for SM, which StnII may recognize. However, our results show that only Chol was able to activate StnII-induced bilayer permeabilization – OCer failed to active. To further examine possible Chol/StnII interactions, we measured Förster resonance energy transfer (FRET) between Trp in StnII and cholestatrienol (CTL), a fluorescent analog of Chol. We could show higher FRET efficiency between CTL and Trp:s in position 100 and 114 of StnII, when compared to three other Trp positions further away from the bilayer binding region of StnII. Taken together, our results suggest that StnII was able to attract Chol to its vicinity, maybe by showing affinity for Chol. SM interactions are known to be important for StnII binding to bilayers, and Chol is known to facilitate subsequent permeabilization of the bilayers by StnII. Our results help to better understand the role of these important membrane lipids for the bilayer properties of StnII

Pore-Forming Proteins from Cnidarians and Arachnids as Potential Biotechnological Tools

Animal venoms are complex mixtures of highly specialized toxic molecules. Cnidarians and arachnids produce pore-forming proteins (PFPs) directed against the plasma membrane of their target cells. Among PFPs from cnidarians, actinoporins stand out for their small size and molecular simplicity. While native actinoporins require only sphingomyelin for membrane binding, engineered chimeras containing a recognition antibody-derived domain fused to an actinoporin isoform can nonetheless serve as highly specific immunotoxins. Examples of such constructs targeted against malignant cells have been already reported. However, PFPs from arachnid venoms are less well-studied from a structural and functional point of view. Spiders from the Latrodectus genus are professional insect hunters that, as part of their toxic arsenal, produce large PFPs known as latrotoxins. Interestingly, some latrotoxins have been identified as potent and highly-specific insecticides. Given the proteinaceous nature of these toxins, their promising future use as efficient bioinsecticides is discussed throughout this Perspective. Protein engineering and large-scale recombinant production are critical steps for the use of these PFPs as tools to control agriculturally important insect pests. In summary, both families of PFPs, from Cnidaria and Arachnida, appear to be molecules with promising biotechnological applications

Ten-year course of treated bipolar I disorder: The role of polarity at onset

Introduction: Early-stage predictors of illness course are needed in bipolar disorder (BD). Differences among patients with a first depressive versus maniac/hypomanic episode have been stated, although in most studies, memory bias and time from onset to start of specialized treatment might interfere. The aim was to compare the first 10 years of illness course according to polarity at onset. Methods: 49 type I BD patients admitted for treatment for a first-time affective episode and a following 10-year attendance to the institution were included. A retrospective year by year comparison according to polarity at onset (depressive (DPO) or maniac (MPO)) was performed. Cramer's V and Cohen d were computed to determine effect size. Results: 59.2% (n = 29) started with MPO. Both groups were similar in demographic and social outcome characteristics, clinical features, and treatment variables. Patients with DPO reported more depressive episodes than MPO patients (U = 149.0 p < .001, Cohen's d = 0.87); both groups had a similar number of manic episodes. Only during the first year of follow-up, suicide attempts (SA) were more frequent in patients with DPO while the presence of a psychotic episode and psychiatric hospitalizations were more frequent in the MPO group. Conclusion: According to these findings, it can be concluded that illness onset is only indicative of depressive predominant polarity but is not related to other poor prognostic variables after the first year of illness onset, in treated BD. SA in the first year of an affective disorder could represent a marker of BD

Oligomerization of Sticholysins from Förster Resonance Energy Transfer

Sticholysins are pore-forming toxins produced by sea anemones that are members of the actinoporin family. They exert their activity by forming pores on membranes, provided they have sphingomyelin. To assemble into pores, specific recognition, binding, and oligomerization are required. While recognition and binding have been extensively studied, delving into the oligomerization process and the stoichiometry of the pores has been more difficult. Here, we present evidence that these toxins are capable of oligomerizing in solution and suggesting that the interaction of sticholysin II (StnII) with its isoform sticholysin I (StnI) is stronger than that of StnI with itself. We also show that the stoichiometry of the final, thermodynamically stable StnI pores is, at least, heptameric. Furthermore, our results indicate that this association maintains its oligomerization number when StnII is included, indicating that the stoichiometry of StnII is also of that order, and not tetrameric, as previously thought. These results are compatible with the stoichiometry observed for the crystallized pore of FraC, another very similar actinoporin produced by a different sea anemone species. Our results also indicate that the stoichiometry of actinoporin pores in equilibrium is conserved regardless of the particular composition of a given pore ensemble, which we have shown for mixed sticholysin pores

Structural foundations of sticholysin functionality

Actinoporins constitute a family of α pore-forming toxins produced by sea anemones. The soluble fold of these proteins consists of a β-sandwich flanked by two α-helices. Actinoporins exert their activity by specifically recognizing sphingomyelin at their target membranes. Once there, they penetrate the membrane with their N-terminal α-helices, a process that leads to the formation of cation-selective pores. These pores kill the target cells by provoking an osmotic shock on them. In this review, we examine the role and relevance of the structural features of actinoporins, down to the residue level. We look at the specific amino acids that play significant roles in the function of actinoporins and their fold. Particular emphasis is given to those residues that display a high degree of conservation across the actinoporin sequences known to date. In light of the latest findings in the field, the membrane requirements for pore formation, the effect of lipid composition, and the process of pore formation are also discussed

Solution-based synthesis and processing of Sn- and Bi-doped Cu₃SbSe₄ nanocrystals, nanomaterials and ring-shaped thermoelectric generators

Copper-based chalcogenides that comprise abundant, low-cost, and environmental friendly elements are excellent materials for a number of energy conversion applications, including photovoltaics, photocatalysis, and thermoelectrics (TE). In such applications, the use of solution-processed nanocrystals (NCs) to produce thin films or bulk nanomaterials has associated several potential advantages, such as high material yield and throughput, and composition control with unmatched spatial resolution and cost. Here we report on the production of Cu₃SbSe₄ (CASe) NCs with tuned amounts of Sn and Bi dopants. After proper ligand removal, as monitored by nuclear magnetic resonance and infrared spectroscopy, these NCs were used to produce dense CASe bulk nanomaterials for solid state TE energy conversion. By adjusting the amount of extrinsic dopants, dimensionless TE figures of merit (ZT) up to 1.26 at 673 K were reached. Such high ZT values are related to an optimized carrier concentration by Sn doping, a minimized lattice thermal conductivity due to efficient phonon scattering at point defects and grain boundaries, and to an increase of the Seebeck coefficient obtained by a modification of the electronic band structure with Bi doping. Nanomaterials were further employed to fabricate ring-shaped TE generators to be coupled to hot pipes, which provided 20 mV and 1 mW per TE element when exposed to a 160 °C temperature gradient. The simple design and good thermal contact associated with the ring geometry and the potential low cost of the material solution processing may allow the fabrication of TE generators with short payback times

Mejora de competencias profesionales en educación social a través del prácticum

Memoria ID-202. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2019-2020