Detection of Hypertension-Induced Changes in Erythrocytes by SERS Nanosensors

Surface-enhanced Raman spectroscopy (SERS) is a promising tool that can be used in the detection of molecular changes triggered by disease development. Cardiovascular diseases (CVDs) are caused by multiple pathologies originating at the cellular level. The identification of these deteriorations can provide a better understanding of CVD mechanisms, and the monitoring of the identified molecular changes can be employed in the development of novel biosensor tools for early diagnostics. We applied plasmonic SERS nanosensors to assess changes in the properties of erythrocytes under normotensive and hypertensive conditions in the animal model. We found that spontaneous hypertension in rats leads (i) to a decrease in the erythrocyte plasma membrane fluidity and (ii) to a decrease in the mobility of the heme of the membrane-bound hemoglobin. We identified SERS parameters that can be used to detect pathological changes in the plasma membrane and submembrane region of erythrocytes.

Sonodynamic Treatment Induces Selective Killing of Cancer Cells in an In Vitro Co-Culture Model

Sonodynamic Therapy (SDT) is a new anticancer strategy based on ultrasound (US) technique and is derived from photodynamic therapy (PDT); SDT is still, however, far from clinical application. In order to move this therapy forward from bench to bedside, investigations have been focused on treatment selectivity between cancer cells and normal cells. As a result, the effects of the porphyrin activation by SDT on cancer (HT-29) and normal (HDF 106-05) cells were studied in a co-culture evaluating cell cytotoxicity, reactive oxygen species (ROS) production, mitochondrial function and plasma membrane fluidity according to the bilayer sonophore (BLS) theory. While PDT induced similar effects on both HT-29 and HDF 106-05 cells in co-culture, SDT elicited significant cytotoxicity, ROS production and mitochondrial impairment on HT-29 cells only, whereas HDF 106-05 cells were unaffected. Notably, HT-29 and HDF 106-05 showed different cell membrane fluidity during US exposure. In conclusion, our data demonstrate a marked difference between cancer cells and normal cells in co-culture in term of responsiveness to SDT, suggesting that this different behavior can be ascribed to diversity in plasma membrane properties, such as membrane fluidity, according to the BLS theory.

Unexpected Role of Sterol Synthesis in RNA Stability and Translation in Leishmania

Leishmania parasites are trypanosomatid protozoans that cause leishmaniasis affecting millions of people worldwide. Sterols are important components of the plasma and organellar membranes. They also serve as precursors for the synthesis of signaling molecules. Unlike animals, Leishmania does not synthesize cholesterol but makes ergostane-based sterols instead. C-14-demethylase is a key enzyme involved in the biosynthesis of sterols and an important drug target. In Leishmania parasites, the inactivation of C-14-demethylase leads to multiple defects, including increased plasma membrane fluidity, mitochondrion dysfunction, hypersensitivity to stress and reduced virulence. In this study, we revealed a novel role for sterol synthesis in the maintenance of RNA stability and translation. Sterol alteration in C-14-demethylase knockout mutant leads to increased RNA degradation, reduced translation and impaired heat shock response. Thus, sterol biosynthesis in Leishmania plays an unexpected role in global gene regulation.

MPK6 Kinase Regulates Plasma Membrane H+-ATPase Activity in Cold Acclimation

Cold and freezing stresses severely affect plant growth, development, and survival rate. Some plant species have evolved a process known as cold acclimation, in which plants exposed to temperatures above 0 °C trigger biochemical and physiological changes to survive freezing. During this response, several signaling events are mediated by transducers, such as mitogen activated protein kinase (MAPK) cascades. Plasma membrane H+-ATPase is a key enzyme for the plant cell life under regular and stress conditions. Using wild type and mpk3 and mpk6 knock out mutants in Arabidopsis thaliana, we explored the transcriptional, translational, and 14-3-3 protein regulation of the plasma membrane H+-ATPase activity under the acclimation process. The kinetic analysis revealed a differential profiling of the H+-ATPase activity depending on the presence or absence of MPK3 or MPK6 under non-acclimated or acclimated conditions. Negative regulation of the plasma membrane H+-ATPase activity was found to be exerted by MPK3 in non-acclimated conditions and by MPK6 in acclimated conditions, describing a novel form of regulation of this master ATPase. The MPK6 regulation involved changes in plasma membrane fluidity. Moreover, our results indicated that MPK6 is a critical regulator in the process of cold acclimation that leads to freezing tolerance and further survival.

Unexpected Role of Sterol Synthesis in RNA Stability and Translation in Leishmania

Leishmania species are parasitic protozoans which cause leishmaniasis affecting millions of people worldwide. Sterols are important components of plasma and organellar membranes and serve as precursors for the synthesis of signaling molecules. In contrast to animals, Leishmania do not produce cholesterol but instead synthesize ergostane-based sterols. C14-demethylase is a key enzyme involved in the biosynthesis of sterols in Leishmania parasites and an important drug target. Its inactivation leads to multiple defects including increased plasma membrane fluidity, mitochondrion dysfunction, hypersensitivity to stress and reduced virulence. In this study, we revealed a novel role for sterol synthesis in the maintenance of RNA stability and translation. Sterol alteration in C14-demethylase knockout mutant leads to increased RNA degradation, reduced translation and impaired heat shock response. Thus, sterol biosynthesis plays an unexpected role in global gene regulation in Leishmania parasites.

The glycosphingolipid inhibitor eliglustat inhibits autophagy in osteoclasts to increase bone mass and reduce myeloma bone disease

Multiple myeloma (MM) is a fatal hematological malignancy, where the majority of patients are diagnosed with, or develop, destructive and debilitating osteolytic bone lesions. Current treatments for MM bone disease such as the bisphosphonate zoledronic acid can result in deleterious side effects at high doses. In this study, eliglustat, an FDA approved glycosphingolipid inhibitor, was shown to reduce MM bone disease in preclinical models of MM. Mechanistically, eliglustat alters the lipid composition and plasma membrane fluidity and acts as an autophagy flux inhibitor in bone-resorbing osteoclasts (OC). Autophagic degradation of the signaling molecule TRAF3 is key step in OC differentiation; this was prevented by eliglustat in OC precursors. In addition, eliglustat works depend on TRAF3 in vivo. Furthermore, the combination of eliglustat and zoledronic acid was found to have an additive effect to reduce MM bone disease, suggesting the potential for combination therapies that would allow for drug dose reductions. Taken together, this project identifies a novel mechanism in which glycosphingolipid inhibition reduces osteoclastogenesis via autophagy and highlights the translational potential of eliglustat for the treatment of bone loss disorders such as MM.One Sentence SummaryTranslational use of eliglustat as an autophagy inhibitor to limit bone lesions in multiple myeloma.

Melatonin added to freezing diluent improves canine (Bulldog) sperm cryosurvival

Cryopreservation compromises the capacity of sperm fertilizing due to a series of alterations in the structure and physiology of the sperm. The use of antioxidants, such as melatonin, added to freezing media, may help to reduce sperm cryoinjury. To test the effect of melatonin on Bulldog (Canis lupus familiaris) sperm cryosurvival, spermatozoa were diluted in a standard freezing medium and cooled to 5°C. Then, more freezing medium was added to obtain 200 × 106 cells/mL, and 5% glycerol. Diluted spermatozoa were treated with melatonin (0.0, 0.0005, 0.002, and 0.0035 mol/L), and packaged in 0.25 mL straws, which were further cooled to −5°C before freezing in liquid nitrogen. Thawing was carried out at 70°C for 5 s, and the progressive motility, viability, plasma membrane integrity, acrosome integrity, capacitation status, and plasma membrane fluidity of the spermatozoa (at 37°C) were assessed. Data were analyzed using ANOVA to detect differences between the melatonin doses. There were statistical differences (P < 0.05) in the percentage of sperm having hyper-fluid membranes, intact acrosome, capacitated acrosome-intact, and acrosome-reacted. The values for the high melatonin doses (0.002 and 0.0035 mol/L) were better than for the low melatonin doses (0.0 and 0.0005 mol/L). In conclusion, 0.002 and 0.0035 mol/L of melatonin improved the cryosurvival of sperm from male bulldogs. Lay summary Preservation of sperm by freezing enables breeding of individuals geographically separated; protocols for the dog may be used to preserve the semen from threatened wild canids. To improve fertility of female dogs that become pregnant with frozen and then defrosted sperm, these cells must survive that process which can be damaging whilst keeping their ability to fertilize. Antioxidants are substances capable of retarding or preventing the oxidation of any oxidizing substrate such as lipids, proteins, and DNA, which are structural compounds of the sperm. The use of antioxidants, added to freezing media, may provide the sperm the capacity to neutralize oxidative compounds, such as reactive oxygen species, produced during the freezing and thawing process. In this work we tested different levels of melatonin, a natural antioxidant, on dog (English Bulldog) sperm survival and quality after freezing. We found that adding melatonin to the freezing media improved sperm quality after thawing.

Molecular Mechanisms Involved in the Impairment of Boar Sperm Motility by Peroxynitrite-Induced Nitrosative Stress

Excessive levels of reactive nitrogen species (RNS) produce nitrosative stress. Among RNS is peroxynitrite, a highly reactive free radical generated when nitric oxide reacts with superoxide anion. Peroxynitrite effects have been mainly studied in somatic cells, and in spermatozoa the majority of studies are focused in humans. The aim of this study is to investigate the in vitro peroxynitrite effect on boar spermatozoa functions and the molecular mechanisms involved. Spermatozoa were exposed to the donor 3-morpholinosydnonimine (SIN-1) in non-capacitating or capacitating medium, motility was evaluated by CASA, functional parameters by flow cytometry and sperm protein phosphorylation by Western blotting. SIN-1 treatment, that significantly increases peroxynitrite levels in boar spermatozoa, potentiates the capacitating-stimulated phosphorylation of cAMP-dependent protein kinase 1 (PKA) substrates and GSK-3α. SIN-1 induced peroxynitrite does not decrease sperm viability, but significantly reduces sperm motility, progressive motility, velocities and motility coefficients. Concomitantly, peroxynitrite does not affect mitochondrial membrane potential, plasma membrane fluidity, or A23187-induced acrosome reaction. However, peroxynitrite significantly increases sperm lipid peroxidation in both media. In conclusion, peroxynitrite compromises boar sperm motility without affecting mitochondrial activity. Although peroxynitrite potentiates the phosphorylation of pathways leading to sperm motility, it also causes oxidative stress that might explain, at least partially, the motility impairment.

Sphingolipid Effects on the Plasma Membrane Produced by Addition of Fumonisin B1 to Maize Embryos

Fumonisin B1 is a mycotoxin produced by Fusarium verticillioides that modifies the membrane properties from animal cells and inhibits complex sphingolipids synthesis through the inhibition of ceramide synthase. The aim of this work was to determine the effect of Fumonisin B1 on the plant plasma membrane when the mycotoxin was added to germinating maize embryos. Fumonisin B1 addition to the embryos diminished plasma membrane fluidity, increased electrolyte leakage, caused a 7-fold increase of sphinganine and a small decrease in glucosylceramide in the plasma membrane, without affecting phytosphingosine levels or fatty acid composition. A 20%–30% inhibition of the plasma membrane H+-ATPase activity was observed when embryos were germinated in the presence of the mycotoxin. Such inhibition was only associated to the decrease in glucosylceramide and the addition of exogenous ceramide to the embryos relieved the inhibition of Fumonisin B1. These results indicate that exposure of the maize embryos for 24 h to Fumonisin B1 allowed the mycotoxin to target ceramide synthase at the endoplasmic reticulum, eliciting an imbalance of endogenous sphingolipids. The latter disrupted membrane properties and inhibited the plasma membrane H+-ATPase activity. Altogether, these results illustrate the mode of action of the pathogen and a plant defense strategy.