Membrane Stress Research Articles

Quinones contained in wastewater as redox mediators for the synergistic removal of azo dye in microbial fuel cells

The present paper aimed to investigate the roles of quinones contained in wastewater and the enhanced effects on microbial fuel cells (MFCs) under different redox conditions. The feasibility of using wastewater rich in quinones to act as co-substrate and redox mediators (RMs) library to strengthen the synergistic removal of azo dye in MFCs was evaluated. The results demonstrated that quinones achieved enhanced effects on electricity generation and COD removal of MFC better at higher current intensity. The addition of pure quinone decreased electron transfer resistance (Rct) of MFCs from 4.76 Ω to 2.13 Ω under 1000 Ω resistance and 1.16 Ω–0.75 Ω under 50 Ω resistance. Meanwhile, higher coulombic efficiency was achieved. Compared with sodium acetate, using quinone-rich traditional Chinese medicine (TCM) wastewater as the co-substrate enhanced the synergistic removal of reactive red 2 (RR2) in MFCs from 79.58% to 92.45% during 24 h. RR2 was also degraded more thoroughly due to the accelerated electron transfer process mediated by RMs. Microbial community analysis demonstrated that the presence of quinone in TCM wastewater can enrich different exoelectrogens under varied redox conditions and thus influenced the enhanced effects on MFC. Metagenomic functional prediction results further indicated that the abundance of functional genes involved in carbohydrate metabolism, membrane transport metabolism, biofilm formation, and stress tolerance increased significantly in presence of RMs. Redundancy analyses revealed that RMs addition was the more important factor driving the variation of the microorganism community. This study revealed the potential effect of quinones as redox mediators on the bioelectrochemical system for pollutants removal.

Design of stiffened panels for stress and buckling via topology optimization

This paper investigates the weight minimization of stiffened panels simultaneously optimizing sizing, layout, and topology under stress and buckling constraints. An effective topology optimization parameterization is presented using multiple level-set functions. Plate elements are employed to model the stiffened panels. The stiffeners are parametrized by implicit level-set functions. The internal topologies of the stiffeners are optimized as well as their layout. A free-form mesh deformation approach is improved to adjust the finite element mesh. Sizing optimization is also included. The thicknesses of the skin and stiffeners are optimized. Bending, shear, and membrane stresses are evaluated at the bottom, middle, and top surfaces of the elements. A p-norm function is used to aggregate these stresses in a single constraint. To solve the optimization problem, a semi-analytical sensitivity analysis is performed, and the optimization algorithm is outlined. Numerical investigations demonstrate and validate the proposed method.

Tomato phytochromes B1 and B2 are part of the responses to the nutritional stress induced by NPK deficiency.

Phytochromes are red-light photoreceptors that play an important role in regulating many responses of plants, including its nutritional control. Nutrient deficiency in plants has become a constraint for agricultural production; thus, we investigated the role of phytochromes B1 and B2 in the nutritional, physiological, and growth changes of the control genotype (WT) and both phyB1 and phyB2 tomato mutants (deficient in phyB1 and phyB2) under nutritional sufficiency and individual deficiency of N, P, and K. Under complete solution, the plants of phyB1 and phyB2 had a decreased N, P, and K accumulation compared with WT and consequently a reduced content of chlorophyll and carotenoids, and dry weight production. In the condition of N deficiency, phyB1 had decreased N absorption, pigments concentration, and plant dry weight, while increased oxidative stress of membranes (MDA content). Similarly, phyB2 also had reduced N absorption. The deficiency of phyB1 mitigated the effects of P deficiency as phyB1 mutant had improved nutritional and physiological responses, increasing plant dry weight production. In contrast, phyB2 reduced N accumulation, quantum efficiency of photosystem II (Fv/Fm), and the concentration of pigments, while it increased MDA. Under K deficiency, phyB1 displayed a reduced P accumulation, as well as the total concentration of chlorophylls and carotenoids and K use efficiency. An increased concentration of MDA was found in phyB2 plants, as well as a reduction in chlorophylls concentration and in the use efficiency of K. Together, these results indicate a new perspective on the control of phytochromes in the nutrition of tomato plants under nutritional stress.

Effect of Cystamine on Sperm and Antioxidant Parameters of Ram Semen Stored at 4 °C for 50 Hours.

Physical and chemical changes caused by oxidative stress in the spermatozoa membrane can reduce spermatozoa function and even lead to death. Cystamine (NH2-CH2-CH2-SH, β-mercaptoethylamine) is a natural substance that modulates the endocrine and metabolic status of animals. This substance has antioxidant and anti-apoptotic effects by inducing intracellular cysteine accumulation. Cystamine is used to treat many diseases despite its many side effects. Sheep semen is sensitive to the stressful condition of chilling storage, which restricts semen storage for artificial insemination in commercial herds. The effect of cystamine on spermatogenesis is not yet fully understood. The present study aimed to investigate the effect of cysteamine addition to the sheep sperm extender during cooling storage on semen quality parameters. Sperm samples were collected from six Edilbayevskaya rams (2 and 3 years old, 70-85 kg). The samples were diluted by extender and supplemented with different concentrations of cysteamine (0, 1, 2, 5, and 10 mM) and cooled to 4ºC for 50 h. Motility parameters, membrane integrity, viability, lipid peroxidation, and mitochondrial activity of cooled semen were evaluated at 0, 25, and 50 h of cooling storage. Although cysteamine failed to affect semen quality at start time (0 hrs), extender supplementation with cysteamine improved sperm total motility, progressive motility, and mitochondrial membrane potential during storage periods (P≤0.01). Moreover, using 1 and 2 mM cysteamine functionally and viably improved (P≤0.01) sperm membrane compared to other treatments. Antioxidant potential (AOP), lipid peroxidation (LPO), and total glutathione (tGSH) (except AOP at 50 h) were significantly different after semen storage at 4 °C. Therefore, levels of AOP and tGSH were significantly increased by using cysteamine. Cysteamine supplementation (1 and 2 mM cysteamine) leads to lower levels of LPO (p<0.01) at 0, 25, and 50 h. Therefore, finding and using the best concentrations of cysteamine in a cooling extender could be effective in saving sheep semen against damages of the cooling storage process.

TMT-based quantitative proteomic analysis of the effects of novel antimicrobial peptide AMP-17 against Candida albicans

Candida albicans is the most common human fungal pathogen in immunocompromised individuals. With the emergence of clinical fungal resistance, there is an urgent need to develop novel antifungal agents. AMP-17, a novel antimicrobial peptide from Musca domestica, has an antifungal effect against C. albicans, but its mechanism of antifungal action remains unclear. In the current study, we performed a proteomics analysis in C. albicans using TMT technique under the treatment of AMP-17. A total of 3931 proteins were identified, of which 3600 included quantitative information. With a 1.5-fold change threshold and a t-test p-value < 0.05 as standard, 423 differentially expressed proteins (DEPs) were up-regulated and 180 DEPs were down-regulated in the AMP-17/control. Notably, GO enrichment revealed that DEPs associated with the cell wall, RNA and oxidative stress were significantly up-regulated, while DEPs involved in ergosterol metabolism and membrane were significantly down-regulated in the AMP-17/control. KEGG pathway enrichment revealed that DEPs involved seven significant metabolic pathways, mainly involved oxidative phosphorylation, RNA degradation, propanoate metabolism and fatty acid metabolism. These results show that AMP-17 induces a complex organism response in C. albicans, indicating that AMP-17 may inhibit growth by affecting multiple targets in C. albicans cells. SignificanceAntimicrobial peptides (AMPs) are an important part of the innate immune system of organisms and having broad range of activity against fungi, bacteria and viruses. These AMPs are considered as probable candidate for forthcoming drugs, due to their broad range of activity, lesser toxicity and decreased resistance development by target cells. AMP-17, a novel antimicrobial peptide from M. domestica, has significant antifungal activity against C. albicans. It has been confirmed that AMP-17 can play an antifungal effect by destroying the cell wall and cell membrane of C. albicans in previous studies, but its mechanism of action at the protein level is currently unclear. In the current study, using the TMT-based quantitative proteomics method, 603 differentially expressed proteins were identified in the cells of C. albicans treated with AMP-17 for 12 h, and these DEPs were closely related to cell wall, cell membrane, RNA degradation and oxidative stress. The results provide new insights into the potential mechanism of action of AMP- 17 against C. albicans. Meanwhile, it provides certain technical support and theoretical basis for the research and development of novel peptide drugs.

Postbuckling mechanics in slender steel plates under pure shear: A focus on boundary conditions and load path

Slender steel plates used in buildings, bridges, aircraft, and ships have postbuckling shear strength that is mobilized in design. Despite a century of research on this topic, the mechanics (load path and failure mechanisms) of the postbuckling behavior are still not fully understood. Proper boundary conditions are vital for studying the mechanics in finite element (FE) models, yet several different boundary conditions have been proposed by researchers. This paper uses FE models to examine various boundary conditions on a slender plate under pure shear loading. The models are benchmarked against 16 experimental tests, with a range of web depths (D), panel aspect ratios, and web slenderness ratios. The effects of boundary conditions on the normal stresses on the web plate edges and principal stresses in the web are examined for panel aspect ratios of 1, 2, and 3. The flanges, stiffeners, and “panel extensions” (which extend the web and flange a distance D/2 beyond the stiffener on either side of the panel) are used as boundary conditions (with axially free vertical edges at the ends of the panel). The analysis results show negligible participation from the stiffeners and flanges in the load path up to the ultimate postbuckling shear load, while compressive membrane stresses in the web are observed to redistribute and continue to increase after elastic buckling, in some regions at a greater rate than before buckling. This study advances the understanding of postbuckling shear load path that can be used in code development and performance enhancements for shear buckling in slender plates.

Stability and Microbial Toxicity of Silver Nanoparticles under Denitrifying Conditions

While the antibacterial effect of silver nanoparticles (AgNPs) on environmentally beneficial microbes has drawn considerable attention, the stability and microbial toxicity of AgNPs in a system where nitrate reduction is the dominant terminal electron-accepting process remain understudied. Here, we explore the impact of citrate-coated AgNPs (cit-AgNPs) on the growth and metabolism of two metal-sensitive and one nonsensitive bacterial strains under denitrifying conditions. Dose-response analysis revealed that in contrast to the bacteriostatic effect exhibited at 1 ppm, 5 ppm cit-AgNPs were bactericidal to the metal-sensitive strains. It was observed that the growth of the cells initiated Ag(I) formation, and the supplement of chloride (2.7 mM) to the cultures substantially mitigated the bactericidal capacity of cit-AgNPs, indicating that AgNP dissolution to ionic Ag(I) played a key role in AgNP toxicity. Abiotic experiments confirmed that nitrite, not nitrate, had the capacity to oxidize cit-AgNPs. Transcriptomic analysis revealed that (i) the gene encoding for membrane stress was upregulated proportionally to cit-AgNP concentrations; (ii) cit-AgNPs and Ag(I) at higher levels upregulated genes involved in oxidative stress and iron-sulfur clusters, whereas expressions of the genes responsible for electron transport, ATP synthesis, and denitrification were substantially repressed; (iii) the addition of chloride significantly altered the level of transcriptional profiles of all of the genes. These results not only provide evidence of abiotic AgNP oxidation by metabolic intermediate nitrogen species but also suggest that AgNPs and Ag(I) may induce differential toxicity modes to prokaryotes. Our findings reinforce the importance of evaluating the potential ecological toxicity and risks associated with the transformation of nanomaterials.

Atg8ylation as a general membrane stress and remodeling response.

The yeast Atg8 protein and its paralogs in mammals, mammalian Atg8s (mAtg8s), have been primarily appreciated for their participation in autophagy. However, lipidated mAtg8s, including the most frequently used autophagosomal membrane marker LC3B, are found on cellular membranes other than autophagosomes. Here we put forward a hypothesis that the lipidation of mAtg8s, termed ‘Atg8ylation', is a general membrane stress and remodeling response analogous to the role that ubiquitylation plays in tagging proteins. Ubiquitin and mAtg8s are related in sequence and structure, and the lipidation of mAtg8s occurs on its C-terminal glycine, akin to the C-terminal glycine of ubiquitin. Conceptually, we propose that mAtg8s and Atg8ylation are to membranes what ubiquitin and ubiquitylation are to proteins, and that, like ubiquitylation, Atg8ylation has a multitude of downstream effector outputs, one of which is autophagy.

Discovery of bactericides as an acute mitochondrial membrane damage inducer.

Mitochondria evolved from endosymbiotic bacteria to become essential organelles of eukaryotic cells. The unique lipid composition and structure of mitochondrial membranes are critical for the proper functioning of mitochondria. However, stress responses that help maintain the mitochondrial membrane integrity are not well understood. One reason for this lack of insight is the absence of efficient tools to specifically damage mitochondrial membranes. Here, through a compound screen, we found that two bis-biguanide compounds, chlorhexidine and alexidine, modified the activity of the inner mitochondrial membrane (IMM)-resident protease OMA1 by altering the integrity of the IMM. These compounds are well-known bactericides whose mechanism of action has centered on their damage-inducing activity on bacterial membranes. We found alexidine binds to the IMM likely through the electrostatic interaction driven by the membrane potential as well as an affinity for anionic phospholipids. Electron microscopic analysis revealed that alexidine severely perturbated the cristae structure. Notably, alexidine evoked a specific transcriptional/proteostasis signature that was not induced by other typical mitochondrial stressors, highlighting the unique property of alexidine as a novel mitochondrial membrane stressor. Our findings provide a chemical-biological tool that should enable the delineation of mitochondrial stress-signaling pathways required to maintain the mitochondrial membrane homeostasis.

Synergistic antibacterial effect of graphene-coated titanium loaded with levofloxacin

In this study, graphene coating was introduced to the modified titanium surface to prevent bacterial infection in oral implants. We modified the titanium surface through SLA and silanization treatment and then coated the surface with graphene. The structure and surface properties were characterized by XPS and SEM. Graphene-coated titanium sheet was incubated with bacteria to test the antibacterial property, which was enhanced by adsorption and release of levofloxacin. We further implanted the graphene-coated titanium sheet loaded with levofloxacin into rabbits to test the antibacterial properties in vivo. The graphene coating exhibited inherent antibacterial properties through membrane stress and the generation of reactive oxygen species (ROS). When loaded with levofloxacin, the graphene coating exhibited a synergistic antibacterial effect and effectively prevented bacterial infections following the implantation. The graphene coating is promising to improve the antibacterial functions of oral implant surfaces to prevent bacterial infection.

Experimental study on residual stresses of dual phase high‐strength cold‐formed steel angles

AbstractThe rapid advance in advanced high‐strength cold‐formed steel makes this material promising for the construction industry to use as structural framing, but necessitates an accurate understanding of mechanical properties, including residual stresses. An experimental study was conducted to measure the residual stress which is generated during the member manufacturing process of a press‐braked angle member composed of advanced high‐strength cold‐formed sheet steel. The amplitude and distribution of membrane and flexural residual stresses around the member cross‐section is presented and the magnitude of residual stresses relative to the yield strength for the test specimen and other previous studies are compared. Additionally, the cutoff length typically recommended for residual stress measurements is investigated by comparing the residual stress distribution around the cross‐section for a section taken from the center and near the end of the member.

Lipidomic metabolism associated with acetic acid priming-induced salt tolerance in Carex rigescens

Acetic acid priming may mitigate salt stress to plants by modulating lipid metabolism. Carex rigescens is a stress-tolerant turfgrass species with a widespread distribution in north China. The objective of this study was to figure out whether modification of lipid profiles, including the contents, compositions and saturation levels of leaf lipids, may contribute to acetic acid modulated salt tolerance in C. rigescens. Plants of C. rigescens were primed with or without acetic acid (30 mM) and subsequently exposed to salt stress (300 mM NaCl) for 15 days. Salt stress affected the physiological performance of C. rigescens, while acetic acid-primed plants showed significantly lower malondialdehyde content, proline content, and electrolyte leakage than non-primed plants under salt stress. Acetic acid priming enhanced the contents of phospholipids and glycolipids involved in membrane stabilization and stress signaling (phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, digalactosyl diacylglycerol, monogalactosyl diacylglycerol, and sulfoquinovosyldiacylglycerol), reduced the content of toxic lipid intermediates (free fatty acids) during subsequent exposure to salt stress. Furthermore, expression levels of genes involved in lipid metabolism such as CK and PLDα changed due to acetic acid priming. These results demonstrated that acetic acid priming could enhance salt tolerance of C. rigescens by regulating lipid metabolism. The lipids could be used as biomarkers to select for salt-tolerant grass germplasm.

TMEM16F and dynamins control expansive plasma membrane reservoirs

Cells can expand their plasma membrane laterally by unfolding membrane undulations and by exocytosis. Here, we describe a third mechanism involving invaginations held shut by the membrane adapter, dynamin. Compartments open when Ca activates the lipid scramblase, TMEM16F, anionic phospholipids escape from the cytoplasmic monolayer in exchange for neutral lipids, and dynamins relax. Deletion of TMEM16F or dynamins blocks expansion, with loss of dynamin expression generating a maximally expanded basal plasma membrane state. Re-expression of dynamin2 or its GTPase-inactivated mutant, but not a lipid binding mutant, regenerates reserve compartments and rescues expansion. Dynamin2-GFP fusion proteins form punctae that rapidly dissipate from these compartments during TMEM16F activation. Newly exposed compartments extend deeply into the cytoplasm, lack numerous organellar markers, and remain closure-competent for many seconds. Without Ca, compartments open slowly when dynamins are sequestered by cytoplasmic dynamin antibodies or when scrambling is mimicked by neutralizing anionic phospholipids and supplementing neutral lipids. Activation of Ca-permeable mechanosensitive channels via cell swelling or channel agonists opens the compartments in parallel with phospholipid scrambling. Thus, dynamins and TMEM16F control large plasma membrane reserves that open in response to lateral membrane stress and Ca influx.

High levels of Tryptophan reduce cell wall or membrane stress tolerance in Saccharomyces cerevisiae.

Tryptophan (Trp) is a proteinogenic aromatic amino acid; however, high levels of Trp are toxic in animals and yeast with unknown mechanisms. Previously, we suggested that aromatic aminotransferase Aro9 is important for excess Trp degradation. Besides, Schroeder and Ikui showed that aro9Δ is sensitive to membrane stress by sodium dodecyl sulfate. Therefore, Trp accumulation may reduce the cell wall or membrane (CW/M) stress tolerance through participation of cell wall integrity (CWI) pathway, which detects and responds to CW/M perturbations. In this study, we found that yeast mutants of the CWI mitogen-activated protein kinase cascade were susceptible to excess Trp. Also, the Trp degradation deficient mutant aro8Δ aro9Δ cells, in which Trp accumulation was confirmed, were sensitive to several CW/M stresses. These results indicated that accumulation of Trp is adverse for the CW/M stress resistance and may disturb appropriate signal transduction responding to the stress.

A β‐ketoacyl carrier protein reductase confers heat tolerance via the regulation of fatty acid biosynthesis and stress signaling in rice

Summary Heat stress is a major environmental threat affecting crop growth and productivity. However, the molecular mechanisms associated with plant responses to heat stress are poorly understood.Here, we identified a heat stress‐sensitive mutant, hts1, in rice. HTS1 encodes a thylakoid membrane‐localized β‐ketoacyl carrier protein reductase (KAR) involved in de novo fatty acid biosynthesis. Phylogenetic and bioinformatic analysis showed that HTS1 probably originated from streptophyte algae and is evolutionarily conserved in land plants.Thermostable HTS1 is predominantly expressed in green tissues and strongly induced by heat stress, but is less responsive to salinity, cold and drought treatments. An amino acid substitution at A254T in HTS1 causes a significant decrease in KAR enzymatic activity and, consequently, impairs fatty acid synthesis and lipid metabolism in the hts1 mutant, especially under heat stress. Compared to the wild‐type, the hts1 mutant exhibited heat‐induced higher H2O2 accumulation, a larger Ca2+ influx to mesophyll cells, and more damage to membranes and chloroplasts. Also, disrupted heat stress signaling in the hts1 mutant depresses the transcriptional activation of HsfA2s and the downstream target genes.We suggest that HTS1 is critical for underpinning membrane stability, chloroplast integrity and stress signaling for heat tolerance in rice.

Phenothiazines alter plasma membrane properties and sensitize cancer cells to injury by inhibiting annexin-mediated repair

Repair of damaged plasma membrane in eukaryotic cells is largely dependent on the binding of annexin repair proteins to phospholipids. Changing the biophysical properties of the plasma membrane may provide means to compromise annexin-mediated repair and sensitize cells to injury. Since, cancer cells experience heightened membrane stress and are more dependent on efficient plasma membrane repair, inhibiting repair may provide approaches to sensitize cancer cells to plasma membrane damage and cell death. Here, we show that derivatives of phenothiazines, which have widespread use in the fields of psychiatry and allergy treatment, strongly sensitize cancer cells to mechanical-, chemical-, and heat-induced injury by inhibiting annexin-mediated plasma membrane repair. Using a combination of cell biology, biophysics, and computer simulations, we show that trifluoperazine acts by thinning the membrane bilayer, making it more fragile and prone to ruptures. Secondly, it decreases annexin binding by compromising the lateral diffusion of phosphatidylserine, inhibiting the ability of annexins to curve and shape membranes, which is essential for their function in plasma membrane repair. Our results reveal a novel avenue to target cancer cells by compromising plasma membrane repair in combination with noninvasive approaches that induce membrane injuries.

Continuous production of PVA-based hydrogel nanoparticles by membrane nanoprecipitation

The use of membranes as dispersing tools opens new development perspectives to the traditional nanoprecipitation technique by overcoming one of its major limitations, related to its exclusive use in batch processes and for small scale formulations. In this work the suitability of membrane nanoprecipitation for the formulation of uniform hydrogel nanoparticles (H-NPs) based on polyvinyl alcohol (PVA) in a continuous process has been demonstrated. This is a challenging formulation even for the traditional nanoprecipitation process, which has been well studied mainly for polymers of a hydrophobic nature. A systematic study of the phase composition and operating conditions of membrane nanoprecipitation allowed us to clarify the influence of the components involved in PVA nanoprecipitation promoted by non-solvent-induced phase separation and to identify the conditions to ensure the continuous production of uniform PVA hydrogel NPs. The size of PVA-H-NPs was tailored by tuning the solvent, non-solvent (dispersing medium), PVA concentration and volumetric ratio between solvent and non-solvent. The use of a porous membrane to assist nanoprecipitation in the batch allowed us to demonstrate that nanoprecipitation was not influenced by flux through the membrane or wall shear stress, but mainly by the solvent/non-solvent volumetric ratio. The obtained knowledge permitted to set up the membrane system for the continuous preparation of PVA-H-NPs, which was carried out in single-pass cross-flow mode. To the best of our knowledge, this is the first preparation of PVA-H-NPs in a continuous membrane nanoprecipitation process. Compared to batch nanoprecipitation, an order of magnitude higher mass productivity was achieved.

A new stress-based topology optimization approach for finding flexible structures

This paper presents a new FE-based stress-related topology optimization approach for finding bending governed flexible designs. Thereby, the knowledge about an output displacement or force as well as the detailed mounting position is not necessary for the application. The newly developed objective function makes use of the varying stress distribution in the cross section of flexible structures. Hence, each element of the design space must be evaluated with respect to its stress state. Therefore, the method prefers elements experiencing a bending or shear load over elements which are mainly subjected to membrane stresses. In order to determine the stress state of the elements, we use the principal stresses at the Gauss points. For demonstrating the feasibility of the new topology optimization approach, three academic examples are presented and discussed. As a result, the developed sensitivity-based algorithm is able to find usable flexible design concepts with a nearly discrete 0 − 1 density distribution for these examples.

Sub-10-nm-thick SiN nanopore membranes fabricated using the SiO2 sacrificial layer process

In our previous studies, ultrathin SiN membranes down to 3 nm in thickness were fabricated using the poly-Si sacrificial layer process, and nanopores were formed in those membranes. The region of the SiN membrane fabricated using this process was small, and the poly-Si sacrificial layer remained throughout the other region. On the other hand, to reduce the noise of the current through the nanopore, it is preferable to reduce the capacitance of the nanopore chip by replacing the poly-Si layer with an insulator with low permittivity, such as SiO2. Thus, in this study, the fabrication of SiN membranes with thicknesses of 3–7 nm using the SiO2 sacrificial layer process was examined. SiN membranes with thicknesses of less than 5 nm could not be formed when the thickness of the top SiN layer deposited onto the sacrificial layer was 100 nm. In contrast, SiN membranes down to 3.07 nm in thickness could be formed when the top SiN layer was 40 nm in thickness. This is thought to be due to the difference in membrane stress. Nanopores were then fabricated in the membranes via dielectric breakdown. The current noise of the nanopore membranes was approximately 3/5 that of membranes fabricated using the poly-Si sacrificial layer process. Last, ionic current blockades were measured when poly(dT)60 passed through the nanopores, and the effective thickness of the nanopores was estimated based on those current-blockade values. The effective thickness was approximately 4.8 nm when the deposited thickness of the SiN membrane was 6.03 nm. On the other hand, the effective thickness and the deposited thickness were almost the same when the deposited thickness was 3.07 nm. This suggests it became difficult to form a shape in which the thickness of the nanopore edge was thinner than the deposited membrane thickness as the deposited thickness decreased.

Transcriptome Response to Cadmium Exposure in Barley (Hordeum vulgare L.).

Cadmium is an environmental pollutant with high toxicity that negatively affects plant growth and development. To understand the molecular mechanisms of plant response to cadmium stress, we have performed a genome-wide transcriptome analysis on barley plants treated with an increased concentration of cadmium. Differential gene expression analysis revealed 10,282 deregulated transcripts present in the roots and 7,104 in the shoots. Among them, we identified genes related to reactive oxygen species metabolism, cell wall formation and maintenance, ion membrane transport and stress response. One of the most upregulated genes was PLANT CADMIUM RESISTACE 2 (HvPCR2) known to be responsible for heavy metal detoxification in plants. Surprisingly, in the transcriptomic data we identified four different copies of the HvPCR2 gene with a specific pattern of upregulation in individual tissues. Heterologous expression of all five barley copies in a Cd-sensitive yeast mutant restored cadmium resistance. In addition, four HvPCR2 were located in tandem arrangement in a single genomic region of the barley 5H chromosome. To our knowledge, this is the first example showing multiplication of the PCR2 gene in plants.