Antileishmanial Activity of Ziziphus spina-christi Leaves Extract and Its Possible Cellular Mechanisms

This experimental investigation was designed to assess the in vitro and in vivo antileishmanial effects of Z. spina-christi methanolic extract (ZSCME) and also aims to assess some of the antileishmanial mechanisms such as the NO production, apoptosis, and plasma membrane permeability. We assessed the in vitro leishmanicidal effects of ZSCME (10–200 µg/mL) against intracellular amastigote stage of the Leishmania major (MRHO/IR/75/ER) and, then, in vivo examined male BALB/c mice infected by L. major. In addition, the rate of infectivity, Caspase 3 activity, nitric oxide (NO) production, the plasma membrane permeability, and the cytotoxic effects of ZSCME were studied. The primary phytochemical analysis of ZSCME revealed the existence of high amounts of flavonoids, tannins, glycosides, alkaloids, and saponin in this plant. The findings exhibited that ZSCME meaningfully (p < 0.001) reduced the viability of amastigotes of L. major, whereas it prompted the creation and release of NO, apoptosis, and the plasma membrane permeability (p < 0.05) and indicated no cytotoxicity in macrophage cells. The in vivo results also demonstrated that ZSCME significantly decreased the parasite load and the diameter of the lesions in the infected mice. Our results demonstrate the promising in vitro and in vivo antileishmanial effects of ZSCME against of L. major. Although the findings of the present study showed some possible antileishmanial mechanisms of ZSCME, such as stimulating NO production, apoptosis, and increasing plasma membrane permeability, additional investigations are required to confirm these results.

Single-particle cryo-EM reveals conformational variability of the oligomeric VCC β-barrel pore in a lipid bilayer

Vibrio cholerae cytolysin (VCC) is a water-soluble, membrane-damaging, pore-forming toxin (PFT) secreted by pathogenic V. cholerae, which causes eukaryotic cell death by altering the plasma membrane permeability. VCC self-assembles on the cell surface and undergoes a dramatic conformational change from prepore to heptameric pore structure. Over the past few years, several high-resolution structures of detergent-solubilized PFTs have been characterized. However, high-resolution structural characterization of small β-PFTs in a lipid environment is still rare. Therefore, we used single-particle cryo-EM to characterize the structure of the VCC oligomer in large unilamellar vesicles, which is the first atomic-resolution cryo-EM structure of VCC. From our study, we were able to provide the first documented visualization of the rim domain amino acid residues of VCC interacting with lipid membrane. Furthermore, cryo-EM characterization of lipid bilayer–embedded VCC suggests interesting conformational variabilities, especially in the transmembrane channel, which could have a potential impact on the pore architecture and assist us in understanding the pore formation mechanism.

Altitudinal Effects on Osmotic Regulation and Plasma Membrane Permeability of Lonicera caerulea Leaves on the Northern Slope of Changbai Mountain, China

Background: Lonicera caerulea is a perennial deciduous shrub of medical and edible value that is widely distributed on the northern slope of Changbai Mountain. Soil properties and climate parameters at different elevations affect plant growth, but thus far no studies have been conducted on Lonicera caerulea in different elevation gradients on the northern slope of Changbai Mountain. Here, the leaves of Lonicera caerulea, collected from different elevations (800–1800 m) on the northern slope of Changbai Mountain in China, were used as test materials. The aim was to determine the changes in the leaves’ soluble protein content, soluble sugar content, malondialdehyde (MDA) content, superoxide anion content, and plasma membrane permeability along the altitudinal gradient. The leaf-level data were statistically analyzed with respect to various environmental factors (soil properties and climate parameters) to explore the physiological and biochemical mechanisms of their adaptation to mountainous habitats. Results: The soluble protein and soluble sugar contents initially increased and then decreased with greater altitude. The soluble protein content reached its maximum value of 1.84 mg/g at 1400 m while soluble sugar content peaked at 37.40 mg/g at 1600 m. The soluble protein content of the leaves was mainly affected by the total K content of the soil, while the total K content of the soil, total P content of the soil, and organic matter were the main factors explaining their soluble sugar content. The MDA content increased at first, then decreased, and then increased with altitude, for which the lowest values of 5.76 mol/g and 6.29 mol/g occurred at 1000 m and 1600 m, respectively; the MDA content was mainly influenced by hydrolysis N in soil. The superoxide anion content initially decreased and then increased with greater elevation, for which the minimum value of 26 μg/g occurred at 1200 m. The superoxide anion content was mainly driven by the total K and total P contents. The leaves’ plasma membrane permeability initially decreased and then increased with greater elevation; values were lowest at the intermediate elevations, and the minimum value of 0.30% occurred at 1000 m. Plasma membrane permeability was mainly affected by the total K content. Based on their relationships with the environmental factors (soil properties and climate parameters), the soluble protein, soluble sugar, malondialdehyde (MDA), and superoxide anion contents as well as the plasma membrane permeability of Lonicera caerulea leaves are very sensitive to environmental changes. For plants established at differing elevations, the leaf traits appear able to correspondingly adapt to the local habitat. Conclusions: In general, the growth of Lonicera caerulea at low and high altitude areas on the northern slope of Changbai Mountain is easily restricted by various environmental factors, resulting in its poor growth condition there. By contrast, this shrub grows well at mid-elevations; thus, the planting area of Lonicera caerulea should be expanded to those areas to increase this shrub’s fruit yield and quality.

Effect of cryoprotectant concentration on bovine oocyte permeability and comparison of two membrane permeability modelling approaches

The plasma membrane permeability to water and cryoprotectant (CPA) significantly impacts vitrification efficiency of bovine oocytes. Our study was designed to determine the concentration-dependent permeability characteristics for immature (GV) and mature (MII) bovine oocytes in the presence of ethylene glycol (EG) and dimethyl sulphoxide (Me2SO), and to compare two different modeling approaches: the two parameter (2P) model and a nondilute transport model. Membrane permeability parameters were determined by consecutively exposing oocytes to increasing concentrations of Me2SO or EG. Higher water permeability was observed for MII oocytes than GV oocytes in the presence of both Me2SO and EG, and in all cases the water permeability was observed to decrease as CPA concentration increased. At high CPA concentrations, the CPA permeability was similar for Me2SO and EG, for both MII and GV oocytes, but at low concentrations the EG permeability of GV oocytes was substantially higher. Predictions of cell volume changes during CPA addition and removal indicate that accounting for the concentration dependence of permeability only has a modest effect, but there were substantial differences between the 2P model and the nondilute model during CPA removal, which may have implications for design of improved methods for bovine oocyte vitrification.

Regulation Of Endocytosis By Ultrasound And Microbubble Treatment: Potential For Controlled Cellular Delivery Of Drugs To Cancer Cells

The delivery of therapeutics across biological barriers is a limiting factor in achieving ideal pharmacologic responses in patients. Modulating endocytic mechanisms with targeted, clinically-relevant interventions can increase intracellular delivery across biological barriers, and improve the efficacy of drugs. Ultrasound-microbubble (USMB) is a novel targeted delivery strategy that has shown promising potential in both diagnostic and therapeutic applications. The collective behaviour of microbubbles in the acoustic field can increase the plasma membrane permeability of surrounding cells, and enhance the delivery of therapeutics across biological barriers. USMB achieves the intracellular delivery of drugs through sonoporation and modulation of endocytic pathways, but the type of endocytic pathways and the mechanisms of activation were not known. I identified that, under distinct regulations, USMB enhances the rate of both clathrin-mediated endocytosis, as well as a non-receptor-mediated pathway responsible for internalizing bulk fluid into cells. I discovered that lysosome exocytosis and acidsphingomyelinase are required for the regulation of the clathrin-mediated pathway but not fluidphase endocytosis following USMB treatment. Given the potential of the clathrin-independent pathway to form high capacity carries for the uptake of fluids and therapeutics into cells, I aimed to identify the molecular identity of the proteins that drive the formation of non-clathrin coated vesicles following USMB treatment. I established that flotillins contribute to the USMB-induced vesicular uptake of fluid into cells, a phenomenon that depends on palmitoyltransferase DHHC5 and the Src-family kinase Fyn. Furthermore, I confirmed that USMB treatment can enhance the intracellular delivery of chemotherapeutic drugs such as cisplatin, and improve its therapeutic efficacy in a flotillin-dependent manner. This project established that both clathrin-mediated endocytosis and flotillin-dependent endocytosis can be modulated by clinically-relevant USMB treatments to enhance drug uptake and efficacy, revealing an important new strategy for targeted drug delivery in cancer treatment.

Regulation Of Endocytosis By Ultrasound And Microbubble Treatment: Potential For Controlled Cellular Delivery Of Drugs To Cancer Cells

The delivery of therapeutics across biological barriers is a limiting factor in achieving ideal pharmacologic responses in patients. Modulating endocytic mechanisms with targeted, clinically-relevant interventions can increase intracellular delivery across biological barriers, and improve the efficacy of drugs. Ultrasound-microbubble (USMB) is a novel targeted delivery strategy that has shown promising potential in both diagnostic and therapeutic applications. The collective behaviour of microbubbles in the acoustic field can increase the plasma membrane permeability of surrounding cells, and enhance the delivery of therapeutics across biological barriers. USMB achieves the intracellular delivery of drugs through sonoporation and modulation of endocytic pathways, but the type of endocytic pathways and the mechanisms of activation were not known. I identified that, under distinct regulations, USMB enhances the rate of both clathrin-mediated endocytosis, as well as a non-receptor-mediated pathway responsible for internalizing bulk fluid into cells. I discovered that lysosome exocytosis and acidsphingomyelinase are required for the regulation of the clathrin-mediated pathway but not fluidphase endocytosis following USMB treatment. Given the potential of the clathrin-independent pathway to form high capacity carries for the uptake of fluids and therapeutics into cells, I aimed to identify the molecular identity of the proteins that drive the formation of non-clathrin coated vesicles following USMB treatment. I established that flotillins contribute to the USMB-induced vesicular uptake of fluid into cells, a phenomenon that depends on palmitoyltransferase DHHC5 and the Src-family kinase Fyn. Furthermore, I confirmed that USMB treatment can enhance the intracellular delivery of chemotherapeutic drugs such as cisplatin, and improve its therapeutic efficacy in a flotillin-dependent manner. This project established that both clathrin-mediated endocytosis and flotillin-dependent endocytosis can be modulated by clinically-relevant USMB treatments to enhance drug uptake and efficacy, revealing an important new strategy for targeted drug delivery in cancer treatment.

Dissecting drug-induced in vitro cytotoxicity and metabolic dysfunction in conditionally immortalized human proximal tubule cells

Acute kidney injury accounts for 20% of all hospitalized adults, and 14 to 26% is drug-induced, emphasizing the importance of proper nephrotoxicity assessment. The ‘gold standard’ MTT assay is widely used to measure cell viability, but depends on cellular metabolic activity. Consequently, MTT may not be most optimal to assess cytotoxicity, as nephrotoxicity often involves mitochondrial dysfunction. We compared MTT with a direct cell death assay based on a compromised plasma membrane permeability. Mature conditionally immortalized proximal tubule epithelial cells were dose- (0.1-1,000 µM) and time- (0.5, 1, 2, 4, 8 and 24 hours) dependently exposed to a selection of prototypic nephrotoxicants. Dose-dependent reductions in cellular metabolic activity were stronger compared to declines in fluorescence-based cell death, most prominently for cisplatin (1.6 ± 2.0% and 68 ± 4% (mean ± SEM), respectively) and chloroacetaldehyde (2.13 ± 0.05% and 61.0 ± 0.8%). Similar, but more pronounced time-dependent effects were observed, particularly for sanguinarine. We show that assessing cellular metabolic activity by MTT provides a composite readout of cellular metabolic activity and cell death. A nuclear staining approach is preferable when assessing nephrotoxicity of metabolically active compounds. We recommend both assays during drug development to discriminate between metabolically active versus non-active compounds.

Simvastatin impairs myoblast proliferation and myotube formation of C2C12 myoblasts and in mouse skeletal muscle

Statins reduce cardiovascular complications in patients with high LDL-cholesterol but are associated with myopathy. We investigated the possibility that statins impair skeletal muscle regeneration by assessing simvastatin toxicity on C2C12 myoblasts and myotubes and mouse skeletal muscle. Simvastatin increased plasma membrane permeability and decreased the cellular ATP content in both myoblasts and myotubes, but with a stronger effect on myoblasts. While insulin prevented cytotoxicity up to 8 hours after addition of simvastatin to myotubes, prevention in myoblasts required simultaneous addition. Mevalonate and geranylgeraniol also prevented simvastatin-associated cytotoxicity on myoblasts and myotubes. Simvastatin impaired the phosphorylation of the insulin receptor (IR β), Akt ser473 and S6rp, and increased phosphorylation of AMPK thr172 in both myotubes and myoblasts, which was prevented by insulin and mevalonate. Simvastatin impaired oxygen consumption and increased superoxide production by myoblasts and myotubes and induced apoptosis via cytochromc c release. In addition, simvastatin impaired proliferation and fusion of myoblasts to myotubes by inhibiting the expression of the nuclear transcription factor MyoD and of the metalloprotease ADAM-12. Decreased expression of the proliferation factor Ki-67 and of ADAM-12 were also observed in gastrocnemius of mice treated with simvastatin. In conclusion, myoblasts were more susceptible to the toxic effects of simvastatin and simvastatin impaired myoblast proliferation and myotube formation. Impaired muscle regeneration represents a new mechanism of statin myotoxicity and may be important in statin-associated myopathy.

Anti-Biofilm Activity of Cannabidiol against Candida albicans

Candida albicans is a common fungal pathogen in humans. Biofilm formation is an important virulence factor of C. albicans infections. We investigated the ability of the plant-derived cannabidiol (CBD) to inhibit the formation and removal of fungal biofilms. Further, we evaluated its mode of action. Our findings demonstrate that CBD exerts pronounced time-dependent inhibitory effects on biofilm formation as well as disruption of mature biofilm at a concentration range below minimal inhibitory and fungicidal concentrations. CBD acts at several levels. It modifies the architecture of fungal biofilm by reducing its thickness and exopolysaccharide (EPS) production accompanied by downregulation of genes involved in EPS synthesis. It alters the fungal morphology that correlated with upregulation of yeast-associated genes and downregulation of hyphae-specific genes. Importantly, it represses the expression of C. albicans virulence-associated genes. In addition, CBD increases ROS production, reduces the intracellular ATP levels, induces mitochondrial membrane hyperpolarization, modifies the cell wall, and increases the plasma membrane permeability. In conclusion, we propose that CBD exerts its activity towards C. albicans biofilm through a multi-target mode of action, which differs from common antimycotic agents, and thus can be explored for further development as an alternative treatment against fungal infections.