Large-Scale, Vertically Aligned 2D Subnanochannel Arrays by a Smectic Liquid Crystal Network for High-Performance Osmotic Energy Conversion.

The osmotic energy, an abundant renewable energy source, can be directly converted to electricity by nanofluidic devices with ion-selective membranes. 2D nanochannels constructed by nanosheets possess abundant lateral interfacial ion-exchange sites and exhibit great superiority in nanofluidic devices. However, the most accessible orientation of the 2D nanochannels is parallel to the membrane surface, undoubtedly resulting in the conductivity loss. Herein, first vertically aligned 2D subnanochannel arrays self-assembled by a smectic liquid crystal (LC) network that exhibit high-performance osmotic energy conversion are demonstrated. The 2D subnanochannel arrays are fabricated by in situ photopolymerization of monomers in the LC phase. The as-prepared membrane exhibits excellent water-resistance and mechanical strength. The 2D subnanochannels with excellent cation selectivity and conductivity show high-performance osmotic energy conversion. The power density reaches up to about 22.5 W m-2 with NaCl solution under a 50-fold concentration gradient, which is among with ultrahigh power density. This membrane design concept provides promising applications in osmotic energy conversion.

High Strength MXene/PBONF Heterogeneous Membrane with Excellent Ion Selectivity for Efficient Osmotic Energy Conversion.

Layered MXene nanofluidic membranes still face the problems of low mechanical property, poor ion selectivity, and low output power density. In this work, we successfully constructed heterostructured membranes with the combination of the layered channels of the MXene layer on the top and the nanoscale poly(p-phenylene-benzodioxazole) nanofiber (PBONF) layer on the bottom through a stepwise filtration method. The as-prepared MXene/PBONF-50 heterogeneous membrane exhibits high mechanical properties (strength of 221.6 MPa, strain of 3.2%), high ion selectivity of 0.87, and an excellent output power density of 15.7 W/m2 at 50-fold concentration gradient. Excitingly, the heterogeneous membrane presents a high power density of 6.8 W/m2 at a larger testing area of 0.79 mm2 and long-term stability. This heterogeneous membrane construction provides a viable strategy for the enhancement of mechanical properties and osmotic energy conversion of 2D materials.

A Euryhaline-Fish-Inspired Salinity Self-Adaptive Nanofluidic Diode Leads to High-Performance Blue Energy Harvesters.

The adaptability to wide salinities remains a big challenge for artificial nanofluidic systems, which plays a vital role in water-energy nexus science. Here, inspired by euryhaline fish, sandwich-structured nanochannel systems are constructed to realize salinity self-adaptive nanofluidic diodes, which lead to high-performance salinity-gradient power generators with low internal resistance. Adaptive to changing salinity, the pore morphology of one side of the nanochannel system switches from a 1D straight nanochannel (45 nm) to 3D network pores (1.9 nm pore size and ≈1013 pore density), along with three orders of magnitude change for charge density. Thus, the abundant surface charges and narrow pores render the membrane-based osmotic power generator with power density up to 26.22 Wm-2 . The salinity-adaptive membrane solves the surface charge-shielding problem caused by abundant mobile ions in high salinity and increases the overlapping degree of the electric double layer. The dynamic adaption process of the membrane to the hypersaline environment endows it with good salt endurance and stability. New routes for designing nanofluidic devices functionally adaptable to different salinities and building power generators with excellent salt endurance are demonstrated.

Unique ion rectification in hypersaline environment: A high-performance and sustainable power generator system.

The development of membrane science plays a fundamental role in harvesting osmotic power, which is considered a future clean and renewable energy source. However, the existing designs of the membrane cannot handle the low conversion efficiency and power density. Theory has predicted that the Janus membrane with ionic diode-type current would be the most efficient material. Therefore, rectified ionic transportation in a hypersaline environment (the salt concentration is at least 0.5 M in sea) is highly desired, but it still remains a challenge. Here, we demonstrate a versatile strategy for creating a scale-up Janus three-dimensional (3D) porous membrane-based osmotic power generator system. Janus membranes with tunable surface charge density and porosity were obtained by compounding two kinds of ionomers. Under electric fields or chemical gradients, the Janus membrane has ionic current rectification properties and anion selectivities in a hypersaline environment. Experiments and theoretical calculation demonstrate that abundant surface charge and narrow pore size distribution benefit this unique ionic transport behavior in high salt solution. Thus, the output power density of this membrane-based generator reaches 2.66 W/m2 (mixing seawater and river water) and up to 5.10 W/m2 at a 500-fold salinity gradient (i.e., flowing salt lake into river water). Furthermore, a generator, built by connecting a series of membranes, could power a calculator for 120 hours without obvious current decline, proving the excellent physical and chemical stabilities. Therefore, we believe that this work advances the fundamental understanding of fluid transport and materials design as a paradigm for a high-performance energy conversion generator.

3D Porous Hydrogel/Conducting Polymer Heterogeneous Membranes with Electro-/pH-Modulated Ionic Rectification.

Heterogeneous membranes composed of asymmetric structures or compositions have enormous potential in sensors, molecular sieves, and energy devices due to their unique ion transport properties such as ionic current rectification and ion selectivity. So far, heterogeneous membranes with 1D nanopores have been extensively studied. However, asymmetric structures with 3D micro-/nanoscale pore networks have never been investigated. Here, a simple and versatile approach to low-costly fabricate hydrogel/conducting polymer asymmetric heterogeneous membranes with electro-/pH-responsive 3D micro-/nanoscale ion channels is introduced. Due to the asymmetric heterojunctions between positively charged nanoporous polypyrrole (PPy) and negatively charged microscale porous hydrogel poly (acrylamide-co-acrylic acid) (P(AAm-co-AA)), the membrane can rectify ion transmembrane transport in response to both electro- and pH-stimuli. Numerical simulations based on coupled Poisson and Nernst-Plank equations are carried out to explain the ionic rectification mechanisms for the membranes. The membranes are not dependent on elaborately fabricated 1D ion channel substrates and hence can be facilely prepared in a low-cost and large-area way. The hybridization of hydrogel and conducting polymer offers a novel strategy for constructing low-cost, large-area and multifunctional membranes, expanding the tunable ionic rectification properties into macroscopic membranes with micro-/nanoscale pores, which would stimulate practical applications of the membranes.

A Charge-Density-Tunable Three/Two-Dimensional Polymer/Graphene Oxide Heterogeneous Nanoporous Membrane for Ion Transport.

The design and fabrication of a robust nanoporous membrane in large scale is still a challenge and is of fundamental importance for practical applications. Here, a robust three/two-dimensional polymer/graphene oxide heterogeneous nanoporous membrane is constructed in large scale via the self-assembly approach by chemically designing a robust charge-density-tunable nanoporous ionomer with uniform pore size. To obtain a nanoporous polymer that maintains high mechanical strength and promotes multifunctionality, we designed a series of amphiphilic copolymers by introducing a positively charged pyridine moiety into the engineered polymer polyphenylsulfone. The multiphysical-chemical properties of the membrane enable it to work as a nanogate switch with synergy between wettability and surface charge change in response to pH. Then we systematically studied the transmembrane ionic transport properties of this two-/three-dimensional porous system. By adjusting the charge density of the copolymer via chemical copolymerization through a controlled design route, the rectifying ratio of this asymmetric membrane could be amplified 4 times. Furthermore, we equipped a concentration-gradient-driven energy harvesting device with this charge-density-tunable nanoporous membrane, and a maximum power of ≈0.76 W m-2 was obtained. We expect this methodology for construction of a charge-density-tunable heterogeneous membrane by chemical design will shed light on the material design, and this membrane may further be used in energy devices, biosensors, and smart gating nanofluidic devices.

iTRAQ Mitoproteome Analysis Reveals Mechanisms of Programmed Cell Death in Arabidopsis thaliana Induced by Ochratoxin A.

Ochratoxin A (OTA) is one of the most common and dangerous mycotoxins in the world. Previous work indicated that OTA could elicit spontaneous HR-like lesions formation Arabidopsis thaliana, reactive oxygen species (ROS) play an important role in OTA toxicity, and their major endogenous source is mitochondria. However, there has been no evidence as to whether OTA induces directly PCD in plants until now. In this study, the presence of OTA in Arabidopsisthaliana leaves triggered accelerated respiration, increased production of mitochondrial ROS, the opening of ROS-dependent mitochondrial permeability transition pores and a decrease in mitochondrial membrane potential as well as the release of cytochrome c into the cytosol. There were 42 and 43 significantly differentially expressed proteins identified in response to exposure to OTA for 8 and 24 h, respectively, according to iTRAQ analysis. These proteins were mainly involved in perturbation of the mitochondrial electron transport chain, interfering with ATP synthesis and inducing PCD. Digital gene expression data at transcriptional level was consistent with the cell death induced by OTA being PCD. These results indicated that mitochondrial dysfunction was a prerequisite for OTA-induced PCD and the initiation and execution of PCD via a mitochondrial-mediated pathway.

Lipid Rafts Disruption Increases Ochratoxin A Cytotoxicity to Hepatocytes.

Lipid rafts are microdomains in plasma membrane and can mediate cytotoxicity. In this study, the role of lipid rafts in ochratoxin A-induced toxicity was investigated using Hepatoblastoma Cell Line HepG-2 cells. Disruption of cholesterol-containing lipid rafts enhanced Ochratoxin A (OTA) toxicity, as shown by increased lactate dehydrogenase leakage, increased reactive oxygen species level and reduction of superoxide dismutase activity in a time-dependent manner. Isobaric tags for relative and absolute quantitation-based proteomics of the cell membranes showed that nearly 85.5% proteins were downregulated by OTA, indicating that OTA inhibited the membrane protein synthesis. Most of altered proteins were involved in Gene Ontology "transport", "cell adhesion" and "vesicle-mediated transport". In conclusion, lipid rafts play a key role in OTA-induced cytotoxicity. This study provides insight into how OTA toxicity is regulated by the plasma membrane, especially the lipid rafts.

Arabidopsis thaliana defense response to the ochratoxin A-producing strain (Aspergillus ochraceus 3.4412).

KEY MESSAGE: OTA-producing strain Aspergillus ochraceus induced necrotic lesions, ROS accumulation and defense responses in Arabidopsis . Primary metabolic and defense-related proteins changed in proteomics. Ascorbate-glutathione cycle and voltage-dependent anion-selective channel proteins fluctuated. Mycotoxigenic fungi, as widespread contaminants by synthesizing mycotoxins in pre-/post-harvest infected plants and even stored commercial cereals, could usually induce plant-fungi defense responses. Notably, ochratoxin A (OTA) is a nephrotoxic, hepatotoxic, teratogenic, immunotoxic and phytotoxic mycotoxin. Herein, defense responses of model system Arabidopsis thaliana detached leaves to infection of Aspergillus ochraceus 3.4412, an OTA high-producing strain, were studied from physiological, proteomic and transcriptional perspectives. During the first 72 h after inoculation (hai), the newly formed hypersensitive responses-like lesions, decreased chlorophyll content, accumulated reactive oxygen species and upregulated defense genes expressions indicated the defense response was induced in the leaves with the possible earlier motivated jasmonic acid/ethylene signaling pathways and the later salicylic acid-related pathway. Moreover, proteomics using two-dimensional gel electrophoresis 72 hai showed 16 spots with significantly changed abundance and 13 spots corresponding to 12 unique proteins were successfully identified by MALDI-TOF/TOF MS/MS. Of these, six proteins were involved in basic metabolism and four in defense-related processes, which included glutathione-S-transferase F7, voltage-dependent anion-selective channel protein 3 (VDAC-3), osmotin-like protein OSM34 and blue copper-binding protein. Verified from proteomic and/or transcriptional perspectives, it is concluded that the primary metabolic pathways were suppressed with the ascorbate-glutathione cycle fluctuated in response to A. ochraceus and the modulation of VDACs suggested the possibility of structural damage and dysfunction of mitochondria in the process. Taken together, these findings exhibited a dynamic overview of the defense responses of A. thaliana to A. ochraceus and provided a better insight into the pathogen-resistance mechanisms in plants.

A-T linker adapter polymerase chain reaction for determining flanking sequences by rescuing inverse PCR or thermal asymmetric interlaced PCR products.

The polymerase chain reaction (PCR)-based genome walking method has been extensively used to isolate unknown flanking sequences, whereas nonspecific products are always inevitable. To resolve these problems, we developed a new strategy to isolate the unknown flanking sequences by combining A-T linker adapter PCR with inverse PCR (I-PCR) or thermal asymmetric interlaced PCR (TAIL-PCR). The result showed that this method can be efficiently achieved with the flanking sequence from the Arabidopsis mutant and papain gene. Our study provides researchers with an additional method for determining genomic DNA flanking sequences to identify the target band from bulk of bands and to eliminate the cloning step for sequencing.

Changes in biosynthesis and metabolism of glutathione upon ochratoxin A stress in Arabidopsis thaliana.

Ochratoxin A (OTA) is one of the most toxic mycotoxins, which is toxic to plants and simulates oxidative stress. Glutathione is an important antioxidant in plants and is closely associated with detoxification in cells. We have previously shown that OTA exposure induces obvious expression differences in genes associated with glutathione metabolism. To characterize glutathione metabolism and understand its role in OTA phytotoxicity, we observed the accumulation of GSH in the detached leaves of Arabidopsis thaliana under OTA treatment. OTA stimulated a defense response through enhancing glutathione-S-transferase, glutathione peroxidase, glutathione reductase activities, and the transcript levels of these enzymes were increased to maintain the total glutathione content. Moreover, the level of oxidized glutathione (GSSG) was increased and the ascorbate-glutathione cycle fluctuated in response to OTA. The depletion of glutathione using buthionine sulfoximine (BSO, inhibitor of glutamate-cysteine ligase) had no profound effect on OTA toxicity, as glutathione was regenerated through the ascorbate-glutathione cycle to maintain the total glutathione content. The ROS, MDA and GSH accumulation was significantly affected in the mutant gsh1, gr1 and gpx2 after treatment with OTA, which indicated that glutathione metabolism is directly involved in the oxidative stress response of Arabidopsis thaliana subjected to OTA. In conclusion, date demonstrate that glutathione-associated metabolism is closely related with OTA stress and glutathione play a role in resistance of Arabidopsis subjected to OTA.

Ochratoxin A biocontrol and biodegradation by Bacillus subtilis CW 14.

BACKGROUND: Ochratoxin A (OTA) is a mycotoxin produced by some Aspergillus and Penicillium species. In this study a strain of Bacillus subtilis was tested for its effects on OTA-producing Aspergillus and OTA degradation. The mechanisms of the effects were also investigated. RESULTS: A strain of Bacillus spp. isolated from fresh elk droppings was screened out using the methods described by Guan et al. (Int J Mol Sci 9:1489-1503 (2008)). The 16S rRNA gene sequence suggested that it was B. subtilis CW 14. It could inhibit the growth of the OTA-producing species Aspergillus ochraceus 3.4412 and Aspergillus carbonarius, with inhibition rates of 33.0 and 33.3% respectively. At 6 µg mL(-1) OTA, both viable and autoclaved (121 °C, 20 min) cells of CW 14 bound more than 60% of OTA. In addition, OTA was degraded by the cell-free supernatant of CW 14. By high-performance liquid chromatography, the cell-free supernatant degraded 97.6% of OTA after 24 h of incubation at 30 °C, and no degradation products were produced. The fastest degradation occurred during the first 2 h. In 3 g samples of contaminated maize, 47.1% of OTA was degraded by 50 mL inocula of overnight cultures of CW 14. CONCLUSION: These findings indicated that B. subtilis CW 14 could both prevent OTA contamination and degrade OTA in crops.

Comparative proteomics and physiological characterization of Arabidopsis thaliana seedlings in responses to Ochratoxin A.

Ochratoxin A (OTA) is a mycotoxin that is primarily produced by Aspergillus ochraceus and Penicillium verrucosum. This mycotoxin is a contaminant of food and feedstock worldwide and may induce cell death in plants. To investigate the dynamic growth process of Arabidopsis seedlings in response to OTA stress and to obtain a better understanding of the mechanism of OTA toxicity towards Arabidopsis, a comparative proteomics study using 2-DE and MALDI-TOF/TOF MS/MS was performed. Mass spectrometry analysis identified 59 and 51 differentially expressed proteins in seedlings exposed to 25 and 45 µM OTA for 7 days, respectively. OTA treatment decreased root elongation and leaf area, increased anthocyanin accumulation, damaged the photosynthetic apparatus and inhibited photosynthesis. Treatment of the seedlings with 25 µM OTA enhanced energy metabolism, whereas higher concentration of OTA (45 µM) inhibited energy metabolism in the seedlings. OTA treatment caused an increase of ROS, an enhancement of antioxidant enzyme defense responses, disturbance of redox homeostasis and activation of lipid oxidation. Glutamine and S-adenosylmethionine metabolism may also play important roles in the response to OTA. In conclusion, our study provided novel insights regarding the response of Arabidopsis to OTA at the level of the proteome. These results are expected to be highly useful for understanding the physiological responses and dissecting the OTA response pathways in higher plants.

Zinc protects HepG2 cells against the oxidative damage and DNA damage induced by ochratoxin A.

Oxidative stress and DNA damage are the most studied mechanisms by which ochratoxin A (OTA) induces its toxic effects, which include nephrotoxicity, hepatotoxicity, immunotoxicity and genotoxicity. Zinc, which is an essential trace element, is considered a potential antioxidant. The aim of this paper was to investigate whether zinc supplement could inhibit OTA-induced oxidative damage and DNA damage in HepG2 cells and the mechanism of inhibition. The results indicated that that exposure of OTA decreased the intracellular zinc concentration; zinc supplement significantly reduced the OTA-induced production of reactive oxygen species (ROS) and decrease in superoxide dismutase (SOD) activity but did not affect the OTA-induced decrease in the mitochondrial membrane potential (Δψm). Meanwhile, the addition of the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) strongly aggravated the OTA-induced oxidative damage. This study also demonstrated that zinc helped to maintain the integrity of DNA through the reduction of OTA-induced DNA strand breaks, 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation and DNA hypomethylation. OTA increased the mRNA expression of metallothionein1-A (MT1A), metallothionein2-A (MT2A) and Cu/Zn superoxide dismutase (SOD1). Zinc supplement further enhanced the mRNA expression of MT1A and MT2A, but it had no effect on the mRNA expression of SOD1 and catalase (CAT). Zinc was for the first time proven to reduce the cytotoxicity of OTA through inhibiting the oxidative damage and DNA damage, and regulating the expression of zinc-associated genes. Thus, the addition of zinc can potentially be used to reduce the OTA toxicity of contaminated feeds.

Loop-linker PCR: an advanced PCR technique for genome walking.

In this article, we developed a novel PCR method, termed loop-linker PCR, to isolate flanking sequences in transgenic crops. The novelty of this approach is its use of a stem-loop structure to design a loop-linker adapter. The adapter is designed to form a nick site when ligated with restricted DNA. This modification not only can prevent the self-ligation of adapters but also promotes the elongation of the 3' end of the loop-linker adapter to generate a stem-loop structure in the ligation products. Moreover, the suppressive effect of the stem-loop structure decreases nonspecific amplification and increases the success rate of the approach; all extension products will suppress exponential amplification except from the ligation product that contains the specific primer binding site. Using this method, 442, 1830, 107, and 512 bp left border flanking sequences were obtained from the transgenic maizes LY038, DAS-59122-7, Event 3272, and the transgenic soybean MON89788, respectively. The experimental results demonstrated that loop-linker PCR is an efficient, reliable, and cost-effective method for identifying flanking sequences in transgenic crops and could be applied for other genome walking applications.

Transcript and protein profiling analysis of OTA-induced cell death reveals the regulation of the toxicity response process in Arabidopsis thaliana.

Ochratoxin A (OTA) is a toxic isocoumarin derivative produced by various species of mould which mainly grow on grain, coffee, and nuts. Recent studies have suggested that OTA induces cell death in plants. To investigate possible mechanisms of OTA phytotoxicity, both digital gene expression (DGE) transcriptomic and two-dimensional electrophoresis proteomic analyses were used, through which 3118 genes and 23 proteins were identified as being up- or down-regulated at least 2-fold in Arabidopsis leaf in response to OTA treatment. First, exposure of excised Arabidopsis thaliana leaves to OTA rapidly causes the hypersensitive reponse, significantly accelerates the increase of reactive oxygen species and malondialdehyde, and enhances antioxidant enzyme defence responses and xenobiotic detoxification. Secondly, OTA stimulation causes dynamic changes in transcription factors and activates the membrane transport system dramatically. Thirdly, a concomitant persistence of compromised photosynthesis and photorespiration is indicative of a metabolic shift from a highly active to a weak state. Finally, the data revealed that ethylene, salicylic acid, jasmonic acid, and mitogen-activated protein kinase signalling molecules mediate the process of toxicity caused by OTA. Profiling analyses on Arabidopsis in response to OTA will provide new insights into signalling transduction that modulates the OTA phytotoxicity mechanism, facilitate mapping of regulatory networks, and extend the ability to improve OTA tolerance in Arabidopsis.