Anhydrobiosis in yeasts: Psychrotolerant yeasts are highly resistant to dehydration.

Yeast cells are able to transition into a state of anhydrobiosis (temporary reversible suspension of metabolism) under conditions of desiccation. One of the most efficient approaches for understanding the mechanisms underlying resistance to dehydration-rehydration is to identify yeasts, which are stable under such treatments, and compare them with moderately resistant species and strains. In the current study, we investigated the resistance to dehydration-rehydration of six psychrotolerant yeast strains belonging to two species. All studied strains of Solicoccozyma terricola and Naganishia albida were found to be highly resistant to dehydration-rehydration. The viability of S. terricola strains was close to 100%. Such results have not been previously reported in studies of anhydrobiosis in yeasts. The plasma membrane changes, revealed by determining its permeability under various rehydration conditions, were also surprisingly minimal. Thus, the high level of resistance of psychrotolerant yeast strains might be related to the chemical composition and molecular organisation of their plasma membranes. Aside from plasma membrane characteristics, other important factors may also influence the maintenance of yeast cell viability under conditions of dehydration-rehydration.

Antitrypanosomal activity and effect in plasma membrane permeability of (-)-bornyl p-coumarate isolated from Piper cernuum (Piperaceae).

This work describes the isolation of six metabolites from leaves and branches of Piper cernuum (Piperaceae): (-)-cubebin (1), (-)-hinokinin (2), (-)-kusunokinin (3), trans-dehydroagarofuran (4), 11-hydroxi-4,5-secoeudesmane-4,5-dione (5), and (-)-bornyl p-coumarate (6). Antitrypanosomal activity and toxicity of purified compounds were performed in vitro against trypomastigote forms of Trypanosoma cruzi and NCTC cells, respectively. Compounds 2, 3 and 5 showed moderate activities with IC50 values of 33.1, 31.8 and 45.9 µM, respectively, while compounds 1 and 4 were inactive (IC50 > 100 µM). On the other hand, compound 6 displayed an IC50 value of 2.1 µM, a selectivity index (SI) of 18 and induced a considerable interference in the plasma membrane permeability (87%) in trypomastigotes of T. cruzi. Additionally, the lethal effect of compound 6 in T. cruzi could be associated to the plasma membrane permeability. Finally, experiments using scanning electron microscopy (SEM) confirmed the obtained results in which was possible to observe total alteration parasites topography after treatment with compound 6 in comparison to untreated parasites. These data indicated that the lethal action of compound 6 is directly related to structural disruption of the membrane.

Individual and synergistic effect of multi-frequency ultrasound and electro-infrared pretreatments on polyphenol accumulation and drying characteristics of edible roses.

The freeze-dried (FD) edible roses with high content of bioactive substances and superior flavor have been favored by consumers. Nevertheless, the development of freeze-dried rose industry has been plagued by a long drying time and low efficiency. This study investigated the effects of ultrasonic pretreatment (UP) in multi-frequency modes and electro-infrared pretreatment (EIP) prior to FD on polyphenol accumulation and drying characteristics of roses. The mechanism was explored by the changes in microstructure, equivalent circuit parameters, and phenol identifications of rose. The results showed that the FD time of roses decreased by 26 % after ultrasonic-infrared sequential synergistic pretreatment (UP + EIP) due to the damage of cell membrane permeability from UP. The quality attributes of UP + EIP products including color, phenols, and antioxidant activity (DPPH and ABTS radical scavenging rates) remarkably improved. UP + EIP significantly (p < 0.05) increased the content of polyphenols, namely quercetin-3ß-d-glucoside, phlorizin, procyanidin B2, gallicacid, and rutin in the FD roses quantified by ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-qTOF-MS/MS). Therefore, UP + EIP is an effective pretreatment method for shortening FD time and producing high-quality FD rose products with enhanced polyphenol content.

Evidence of a non-apoptotic mode of cell death in microglial BV-2 cells exposed to different concentrations of zinc oxide nanoparticles.

Zinc oxide nanoparticles (ZnO NPs) possess huge application potential. However, the toxicity of ZnO NPs is a great cause of concern. Indeed, ZnO NPs have been found to cause neurotoxicity. As microglial dysfunctions have been linked to the neurotoxic potential of NPs, the physico-chemical properties of ZnO NPs were determined and their cytotoxic effects were characterised on murine microglial BV-2 cells. In-house prepared and meticulously characterised ZnO NPs exhibited narrow size distribution with an average size of around 20 nm and a zeta potential at physiological pH around 24 mV. ZnO NPs did not exhibit aggregation in the cell culture medium. When microglial BV-2 cells were exposed for 6 and 24 h to ZnO NPs (5, 10, 20, 40, and 80 µg/mL), several cell damages were observed. Cellular accumulation of NPs in microglial BV-2 cells was associated with cell growth inhibition and cell death induction, measured by the trypan blue exclusion and MTT assays. Mitochondrial dysfunction and lysosomal alteration were associated with increased plasma membrane permeability measured by staining with DiOC6(3), acridine orange, and propidium iodide, respectively. In addition, an accumulation of reactive oxygen species (ROS) was detected after staining with dihydroethidium and dihydrorhodamine 123. No apoptotic features were present: no cells with condensed and/or fragmented nuclei (Hoechst staining) characteristic of apoptotic cells, absence of subG1 cells, absence of caspase-3 cleavage, and PARP fragmentation. With ZnO NPs (80 µg/mL), with the annexin V/propidium iodide (PI) assay, few apoptotic cells (annexin V+/PI- cells) were detected whereas (annexin V+/PI+ cells) evocating necrotic cells were mainly identified. No modification of the cells in the different phases of the cell cycle was found. Altogether, our data show that ZnO NPs induce a non-apoptotic mode of cell death associated with an accumulation of ROS, mitochondrial, and lysosomal dysfunction and plasma membrane damages in microglial BV-2 cells.Graphical abstract.

Dibenzylbutane neolignans from Saururus cernuus L. (Saururaceae) displayed anti-Trypanosoma cruzi activity via alterations in the mitochondrial membrane potential.

The MeOH extract from leaves of Saururus cernuus L. (Saururaceae) displayed in vitro activity against trypomastigote forms of T. cruzi (100% of parasite death at 200 µg/mL), suggesting the presence of bioactive compounds. Thus, the bioactivity-guided fractionation was carried out, leading to the isolation of three related neolignan derivatives, identified as threo-austrobailignan-5 (1), threo-austrobailignan-6 (2), and threo-dihydroguaiaretic acid (3). Anti-T. cruzi activity of compounds 1-3 was performed against cell-derived trypomastigotes and intracellular amastigotes. Additionally, the mammalian cytotoxicity was investigated using NCTC cells. Compound 2 was the most effective against extracellular trypomastigotes with IC50 of 3.7 µM, while compound 3 showed activity in both clinically relevant forms of the parasite, trypomastigotes and amastigotes, with IC50 values of 7.0 and 16.2 µM, respectively. However, the structurally related compound 1 was inactive. Based on these results, compounds 2 and 3 were selected to evaluate the mechanism of cellular death. Compound 2 induced alteration in the plasma membrane permeability and consequently in the ROS levels after 120 min of incubation. By using flow cytometry and fluorescence microscopy, compound 3 showed alterations in the mitochondrial membrane potential (ΔΨm) of trypomastigotes. Considering the promising chemical and biological properties of neolignans 2 and 3, these compounds could be used as starting points to develop new lead compounds for Chagas disease.

The relief effects of organic acids on Scirpus triqueter L. under pyrene-lead stress.

During phytoremediation of polycyclic aromatic hydrocarbons (PAHs) and heavy metals, the phytoremediation plants are often stressed by pollutants, which would reduce the efficiency of phytoremediation. The addition of organic acids from root exudates could alleviate the stress. In this study, three organic acids (citric acid, succinic acid, glutaric acid) were added to investigate the effects of organic acids on the stress response of Scirpus triqueter L. at two pyrene-lead concentrations. The activities of reactive oxygen species, malondialdehyde, plasma membrane H+-ATPase, and vacuolar H+-ATPase and PPase activity, as well as the activities of antioxidant enzymes (SOD, POD, and CAT) in Scirpus triqueter L. were determined. The addition of organic acids could effectively reduce the activities of reactive oxygen species, malondialdehyde, plasma membrane H+-ATPase, and vacuolar H+-ATPase and PPase activities. Under higher pollution, the damage of plant plasma membrane is more serious, but the addition of citric acid can alleviate this situation and even more effective than the relief under low pollution. The effect of citric acid was more significant than that of succinic acid and glutaric acid. These results demonstrated that organic acids could attenuate the stress of pyrene and lead to Scirpus triqueter L.

Thiosulphate-induced phytoextraction of mercury in Brassica juncea: Spectroscopic investigations to define a mechanism for Hg uptake.

Thiosulphate is extensively used to enhance mercury (Hg) phytoextraction due to its efficient in prompting plant Hg uptake. However, the mechanism by which thiosulphate promotes Hg uptake is poorly understood. We determined the concentrations of Hg and potassium (K), and their spatial distribution, in the tissues of Brassica juncea grown in Hg-contaminated soils treated by thiosulphate and compared this to a non-treated soil (control). The spatial distribution of Hg and K was characterized using micro-X ray fluorescence spectroscopy. The subcellular localization and speciation of Hg in the root of plant treated by thiosulphate were elucidated using Transmission electron microscope coupled energy-dispersive X-ray (TEM-EDX) spectroscopy. Thiosulphate increased significantly the Hg concentration in the roots (mainly in the epidermis and xylem) and shoots (mainly in the vascular bundles), while Hg was accumulated in the root (mainly in the epidermis) of the control plant. Thiosulphate promoted the movement of Hg from the epidermis to the xylem of roots, with subsequent loading into the stem via vascular bundles. Thiosulphate decreased the K concentration in plant tissues, relative to the control plant, and we propose this is due to leakage of electrolyte from roots via increased plasma membrane permeability as a consequence of physiological damage caused by the added thiosulphate. Mercury was distributed mainly at the extracellular space in the roots and was shown by TEM-EDX to be predominately amorphous nano-clusters of HgS. We conclude that thiosulphate-promoted Hg accumulation in the plant may happen through increased plasma membrane permeability, a changed pathway of Hg movement within plants, and extracellular co-transportation of Hg-S complexes in the roots. Our results may underpin the ongoing development of phytomanagement as an environmental strategy for Hg contaminated soils around the world.

The Elusive P2X7 Macropore.

ATP, which is released under pathological conditions and is considered a damage-associated molecular pattern (DAMP), activates P2X7 receptors (P2X7Rs), trimeric plasma membrane ion channels selective for small cations. P2X7Rs are partners in NOD-like receptor containing a pyrin (NLRP3) inflammasome activation and promoters of tumor cell growth. P2X7R overstimulation triggers the ATP-dependent opening of a nonselective plasma membrane pore, known as a 'macropore', which allows fluxes of large hydrophilic molecules. The pathophysiological functions of P2X7R are thought to be dependent on activation of this conductance pathway, yet its molecular identity is unknown. Recent reports show that P2X7R permeability to organic solutes is an early and intrinsic property of the channel itself. A better understanding of P2X7R-dependent changes in plasma membrane permeability will allow a rationale development of novel anti-inflammatory and anticancer drugs.

Antitrypanosomal activity and evaluation of the mechanism of action of diterpenes from aerial parts of Baccharis retusa (Asteraceae).

Baccharis retusa, a medicinal Brazilian plant from Asteraceae, has been used in Brazilian folk medicine to treatment of several illnesses, including parasitic diseases. Bioactivity-guided fractionation of the n-hexane extract from the aerial parts of B. retusa resulted in the isolation and characterization of three active related diterpenes: ent-15ß-senecioyl-oxy-kaur-16-en-19-oic acid (1), ent-kaur-16-en-19-oic (2) and ent-16-oxo-17-nor-kauran-19-oic (3) acids. The structures of isolated compounds were defined by spectroscopic analysis, including NMR and HRESIMS. Antitrypanosomal activity of 1-3 was performed against cell-derived trypomastigotes using the colorimetric resazurin assay. The obtained results demonstrated that isolated compounds displayed a reduced toxicity against NCTC cells and were effective against the trypomastigote forms of T. cruzi with IC50 values of 3.8µM (1), 75.3µM (2) and 44.2µM (3). Additionally, compound 3 displayed activity against amastigote forms of T. cruzi with IC50 of 83.2µM. Compound 1 displayed the highest selectivity index (SI) when considered the trypomastigote forms, and its effect in the plasma membrane of parasite was evaluated using the fluorescent probe SYTOX Green. A considerable permeabilization (57%) in the membrane of the parasite was observed when compared to the untreated trypomastigotes. These data demonstrate, for the first time, the antitrypanosomal activity and mechanism of action of 1 and related compounds 2 and 3, obtained from aerial parts of B. retusa.

Calculated cell-specific intracellular hydrogen peroxide concentration: Relevance in cancer cell susceptibility during ascorbate therapy.

The high extracellular hydrogen peroxide (H2O2) concentrations generated during pharmacological ascorbate (P-AscH-) therapy has been shown to exhibit a high flux into susceptible cancer cells leading to a decrease in clonogenic survival. It is hypothesized that the intracellular H2O2 concentration for susceptibility is independent of cell type and that the variation observed in dosing is associated with differences in the cell-specific overall steady-state intracellular H2O2 concentration values. The steady-state variation in intracellular H2O2 concentration is coupled to a number of cellular specific transport and reaction factors including catalase activity and membrane permeability. Here a lumped-parameter mathematical modeling approach, assuming a catalase-dominant peroxide removal mechanism, is used to calculate intracellular H2O2 concentration for several cell lines. Experimental measurements of critical parameters pertaining to the model are obtained. The cell lines investigated are normal pancreatic cells, H6c7, the pancreatic cancer cell line, MIA PaCa-2 and the glioblastoma cell lines, LN-229, T98G, and U-87; all which vary in susceptibility. The intracellular H2O2 concentration estimates are correlated with the clonogenic surviving fraction for each cell line, in-vitro. The results showed that, despite the fact that the experimental parameters including catalase concentration and plasma membrane permeability demonstrated significant variability across cell lines, the calculated steady-state intracellular to extracellular H2O2 concentration ratio did not vary significantly across cell lines. Thus, the calculated intracellular H2O2 concentration is not unique in characterizing susceptibility. These results imply that, although intracellular H2O2 concentration plays a key role in cellular susceptibility to P-AscH- adjuvant therapy, its overall contribution in a unifying mechanism across cell types is complex.

Neolignans from leaves of Nectandra leucantha (Lauraceae) display in vitro antitrypanosomal activity via plasma membrane and mitochondrial damages.

Chagas disease is a neglected tropical disease, caused by the protozoan parasite Trypanosoma cruzi, which affects more than eight million people in Tropical and Subtropical countries especially in Latin America. Current treatment is limited to nifurtimox and benznidazole, both with reduced effectiveness and high toxicity. In this work, the n-hexane extract from leaves of Nectandra leucantha (Lauraceae) displayed in vitro antitrypanosomal activity against T. cruzi. Using several chromatographic steps, four related neolignans were isolated and chemically characterized as dehydrodieugenol B (1), 1-(8-propenyl)-3-[3'-methoxy-1'-(8-propenyl)-phenoxy]-4,5-dimethoxybenzene (2), 1-[(7S)-hydroxy-8-propenyl]-3-[3'-methoxy-1'-(8'-propenyl)-phenoxy]-4-hydroxy-5-methoxybenzene (3), and 1-[(7S)-hydroxy-8-propenyl]-3-[3'-methoxy-1'-(8'-propenyl)-phenoxy]-4,5-dimethoxybenzene (4). These compounds were tested against intracellular amastigotes and extracellular trypomastigotes of T. cruzi and for mammalian cytotoxicity. Neolignan 4 showed the higher selectivity index (SI) against trypomastigotes (>5) and amastigotes (>13) of T. cruzi. The investigation of the mechanism of action demonstrated that neolignan 4 caused substantial alteration of the plasma membrane permeability, together with mitochondrial dysfunctions in trypomastigote forms. In silico studies of pharmacokinetics and toxicity (ADMET) properties predicted that all compounds were non-mutagenic, non-carcinogenic, non-genotoxic, weak hERG blockers, with acceptable volume of distribution (1.66-3.32 L/kg), and low rodent oral toxicity (LD50 810-2200 mg/kg). Considering some clinical events of cerebral Chagas disease, the compounds also demonstrated favorable properties, such as blood-brain barrier penetration. Unfavorable properties were also predicted as high promiscuity for P450 isoforms, high plasma protein binding affinity (>91%), and moderate-to-low oral bioavailability. Finally, none of the isolated neolignans was predicted as interference compounds (PAINS). Considering the promising chemical and biological properties of the isolated neolignans, these compounds could be used as starting points to develop new lead compounds for Chagas disease.

Effect of Soil Drought Stress on Part Physiological Activities of Masson Pine of Different Family / 浙江中医药大学学报

We detect the effect of soil drought stress on part physiological activities of Masson pine of different family.The dark respiration rate of Masson pine will decrease with different range of the decrease of leaf water potential.The photosynthesis rate will increase with the descendant leaf potential and soil potential,which demonstrates that Masson pine has much higher productivity of drought stress.Meanwhile,the relative plasma membrane permeability plasma will be enlarged and the membrane will be hurt more deeply when Masson pine meets with higher drought stress.The relationship between relative plasma membrane permeability and leaf potential shows significant linear correlation.