Chemical synthesis and evaluation of a backbone‐cyclized minimized 2‐helix Z‐domain

The Z‐molecule is a small, engineered IgG‐binding affinity protein derived from the immunoglobulin‐binding domain B of Staphylococcus aureus protein A. The Z‐domain consists of 58 amino acids forming a well‐defined antiparallel three‐helix structure. Two of the three helices are involved in ligand binding, whereas the third helix provides structural support to the three‐helix bundle. The small size and the stable three‐helix structure are two attractive properties comprised in the Z‐domain, but a further reduction in size of the protein is valuable for several reasons. Reduction in size facilitates synthetic production of any protein‐based molecule, which is beneficial from an economical viewpoint. In addition, a smaller protein is easier to manipulate through chemical modifications. By omitting the third stabilizing helix from the Z‐domain and joining the N‐ and C‐termini by a native peptide bond, the affinity protein obtains the advantageous properties of a smaller scaffold and in addition becomes resistant to exoproteases. We here demonstrate the synthesis and evaluation of a novel cyclic two‐helix Z‐domain. The molecule has retained affinity for its target protein, is resistant to heat treatment, and lacks both N‐ and C‐termini. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

g‐C3N4‐Based Heterostructured Photocatalysts

Photocatalysis is considered as one of the promising routes to solve the energy and environmental crises by utilizing solar energy. Graphitic carbon nitride (g‐C₃N₄) has attracted worldwide attention due to its visible‐light activity, facile synthesis from low‐cost materials, chemical stability, and unique layered structure. However, the pure g‐C₃N₄ photocatalyst still suffers from its low separation efficiency of photogenerated charge carriers, which results in unsatisfactory photocatalytic activity. Recently, g‐C₃N₄‐based heterostructures have become research hotspots for their greatly enhanced charge carrier separation efficiency and photocatalytic performance. According to the different transfer mechanisms of photogenerated charge carriers between g‐C₃N₄ and the coupled components, the g‐C₃N₄‐based heterostructured photocatalysts can be divided into the following categories: g‐C₃N₄‐based conventional type II heterojunction, g‐C₃N₄‐based Z‐scheme heterojunction, g‐C₃N₄‐based p–n heterojunction, g‐C₃N₄/metal heterostructure, and g‐C₃N₄/carbon heterostructure. This review summarizes the recent significant progress on the design of g‐C₃N₄‐based heterostructured photocatalysts and their special separation/transfer mechanisms of photogenerated charge carriers. Moreover, their applications in environmental and energy fields, e.g., water splitting, carbon dioxide reduction, and degradation of pollutants, are also reviewed. Finally, some concluding remarks and perspectives on the challenges and opportunities for exploring advanced g‐C₃N₄‐based heterostructured photocatalysts are presented.     

Differential response of male and female emerald ash borers (Col., Buprestidae) to (Z)‐3‐hexenol and manuka oil

We conducted two trapping experiments in green ash plantations in Ontario, Canada to compare the response of the emerald ash borer (EAB), Agrilus planipennis, to (Z)‐3‐hexenol (Z3‐6:OH) and manuka oil. In the first experiment, Z3‐6:OH (7.6 mg/day) in purple prism traps hung 1.5 m above ground caught significantly more EAB than the unbaited controls, with male catches significantly greater than female catches at two locations. Manuka oil (50 mg/day) attracted equal numbers of males and females but they were significantly greater than the controls at only one location. Adding (Z)‐3‐hexenal or (Z)‐3‐hexenyl acetate in binary or ternary combinations with Z3‐6:OH did not enhance trap catch. In the second experiment, Z3‐6:OH released at two rates (7.6 or 80 mg/day) in light green prism traps placed in the ash canopy also caught significantly more males than females and more males than the unbaited controls or manuka oil‐baited traps. Manuka oil had no significant effect on catches relative to the controls. Combining Z3‐6:OH with manuka oil did not enhance catches of EAB. We conclude that there was a strong male‐biased EAB response to Z3‐6:OH lures, whereas manuka oil, when effective, attracted both sexes equally. Z3‐6:OH in light green prism traps in the canopy is an effective lure for EAB, particularly for males.     

High‐Affinity Antibody Detection with a Bivalent Circularized Peptide Containing Antibody‐Binding Domains

Direct chemical labeling of antibody produces molecules with poorly defined modifications. Use of a small antibody‐binding protein as an adapter can simplify antibody functionalization by forming a specific antibody‐bound complex and introducing site‐specific modifications. To stabilize a noncovalent antibody complex that may be used without chemical crosslinking, a bivalent antibody‐binding protein is engineered with an improved affinity of interaction by joining two Z domains with a conformationally flexible linker. The linker is essential for the increase in affinity because it allows simultaneous binding of both domains. The molecule is further circularized using a split intein, creating a novel adapter protein (“lasso”), which binds human immunoglobulin G1 (IgG1) with K _D = 0.53 n m and a dissociation rate that is 55‐ to 84‐fold slower than Z. The lasso contains a unique cysteine for conjugation with a reporter and may be engineered to introduce other functional groups, including a biotin tag and protease recognition sequences. When used in enzyme‐linked immunosorbent assay (ELISA), the lasso generates a stronger reporter signal compared to a secondary antibody and lowers the limit of detection by 12‐fold. The small size of the lasso and a long half‐life of dissociation make the peptide a useful tool in antibody detection and immobilization.     

Plasma and liver proteomic analysis of 3Z‐3‐[(1H‐pyrrol‐2‐yl)‐methylidene]‐1‐(1‐piperidinylmethyl)‐1,3‐2H‐indol‐2‐one‐induced hepatotoxicity in Wistar rats

3Z‐3‐[(1H‐pyrrol‐2‐yl)‐methylidene]‐1‐(1‐piperidinylmethyl)‐1,3‐2H‐indol‐2‐one (Z24), a synthetic anti‐angiogenic compound, inhibits the growth and metastasis of certain tumors. Previous works have shown that Z24 induces hepatotoxicity in rodents. We examined the hepatotoxic mechanism of Z24 at the protein level and looked for potential biomarkers. We used 2‐DE and MALDI‐TOF/TOF MS to analyze alternatively expressed proteins in rat liver and plasma after Z24 administration. We also examined apoptosis in rat liver and measured levels of intramitochondrial ROS and NAD(P)H redox in liver cells. We found that 22 nonredundant proteins in the liver and 11 in the plasma were differentially expressed. These proteins were involved in several important metabolic pathways, including carbohydrate, lipid, amino acid, and energy metabolism, biotransformation, apoptosis, etc. Apoptosis in rat liver was confirmed with the terminal deoxynucleotidyl transferase dUTP‐nick end labeling assay. In mitochondria, Z24 increased the ROS and decreased the NAD(P)H levels. Thus, inhibition of carbohydrate aerobic oxidation, fatty acid β‐oxidation, and oxidative phosphorylation is a potential mechanism of Z24‐induced hepatotoxicity, resulting in mitochondrial dysfunction and apoptosis‐mediated cell death. In addition, fetub protein and argininosuccinate synthase in plasma may be potential biomarkers of Z24‐induced hepatotoxicity.     

Identification of multi‐component trail pheromones in the most evolutionarily derived termites,the Nasutitermitinae (Termitidae)

In the present study, trail pheromone blends are identified for the first time in termites. In the phylogenetically complex Nasutitermitinae, trail‐following pheromones are composed of dodecatrienol and neocembrene, the proportions of which vary according to species, although neocembrene is always more abundant than dodecatrienol (by 25–250‐fold). Depending on species, termites were more sensitive to dodecatrienol or to neocembrene but the association of both components always elicited significantly higher trail following, with a clear synergistic effect in most of the studied species. A third component, trinervitatriene, was identified in the sternal gland secretion of several species, but its function remains unknown. The secretion of trail pheromone blends appears to be an important step in the evolution of chemical communication in termites. The pheromone optimizes foraging, and promotes their ecological success. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 20–27.     

High Efficiency Photocatalytic Water Splitting Using 2D α‐Fe2O3/g‐C3N4 Z‐Scheme Catalysts

Photocatalysis is the most promising method for achieving artificial photosynthesis, but a bottleneck is encountered in finding materials that could efficiently promote the water splitting reaction. The nontoxicity, low cost, and versatility of photocatalysts make them especially attractive for this application. This study demonstrates that small amounts of α‐Fe₂O₃ nanosheets can actively promote exfoliation of g‐C₃N₄, producing 2D hybrid that exhibits tight interfaces and an all‐solid‐state Z‐scheme junction. These nanostructured hybrids present a high H₂ evolution rate >3 × 10⁴ µmol g^‐1 h^‐1 and external quantum efficiency of 44.35% at λ = 420 nm, the highest value so far reported among the family of g‐C₃N₄ photocatalysts. Besides effectively suppressing the recombination of electron–hole pairs, this Z‐scheme junction also exhibits activity toward overall water splitting without any sacrificial donor. The proposed synthetic route for controlled production of 2D g‐C₃N₄‐based structures provides a scalable alternative toward the development of highly efficient and active photocatalysts.     

Studies of the B‐Z transition of DNA: The temperature dependence of the free‐energy difference,the composition of the counterion sheath in mixed salt,and the preparation of a sample of the 5′‐d[T‐(m5C‐G)12‐T] duplex in pure B‐DNA or Z‐DNA form

It is often envisioned that cations might coordinate at specific sites of nucleic acids and play an important structural role, for instance in the transition between B‐DNA and Z‐DNA. However, nucleic acid models explicitly devoid of specific sites may also exhibit features previously considered as evidence for specific binding. Such is the case of the “composite cylinder” (or CC) model which spreads out localized features of DNA structure and charge by cylindrical averaging, while sustaining the main difference between the B and Z structures, namely the better immersion of the B‐DNA phosphodiester charges in the solution. Here, we analyze the non‐electrostatic component of the free‐energy difference between B‐DNA and Z‐DNA. We also compute the composition of the counterion sheath in a wide range of mixed‐salt solutions and of temperatures: in contrast with the large difference of composition between the B‐DNA and Z‐DNA forms, the temperature dependence of sheath composition, previously unknown, is very weak. In order to validate the model, the mixed‐salt predictions should be compared to experiment. We design a procedure for future measurements of the sheath composition based on Anomalous Small‐Angle X‐ray Scattering and complemented by ³¹P NMR. With due consideration for the kinetics of the B‐Z transition and for the capacity of generating at will the B or Z form in a single sample, the 5′‐d[T‐(m⁵C‐G)₁₂‐T] 26‐mer emerges as a most suitable oligonucleotide for this study. Finally, the application of the finite element method to the resolution of the Poisson‐Boltzmann equation is described in detail. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 369–384, 2016.     

Heteromerous Bistricyclic Aromatic Enes: Dynamic Stereochemistry of Xanthylidene‐Anthrones

The heteromerous bistricyclic aromatic ene (BAE) 2,2'‐dimethyl‐10‐(9H‐xanthylidene)‐9(10H)‐anthrone (DMXA) was synthesized by a condensation of 10,10‐dichloro‐2‐methylxanthene with 2‐methylanthrone. X‐ray crystallography of (E)‐DMXA and xanthylidene‐anthrone (XA) indicated that the molecules adopt anti‐folded conformations with folding dihedral angles of 44°/44° and 39°/41°, respectively . The crystal structure of anti‐folded (E)‐DMXA does not indicate any xanthenylium–anthracenolate push–pull effect. E,Z‐diastereomerization of DMXA was studied by ¹H‐NMR coalescence‐temperature measurements at different magnetic field strengths and by kinetic equilibration experiments . Free energy of activation for this process was 81.5 (±1.3) kJ/mol. B3LYP/6‐311+G(d,p) calculations showed that anti‐folded conformers of XA, (E)‐DMXA, bianthrone (AA), and dixanthylene (XX) were global minima. The twisted conformers of XA, AA, and XX were local minima (ΔG₂₉₈ = 16, 18, and 24 kJ/mol) with a substantial dipolar xanthenylium–anthracenolate dipolar contribution for XA. Chirality 27:919–928, 2015. © 2015 Wiley Periodicals, Inc.     

Long‐term evaluation of field‐wide oriental beetle (Col., Scarabaeidae) mating disruption in blueberries using female‐mimic pheromone lures

The oriental beetle, Anomala orientalis (Waterhouse) (Col., Scarabaeidae), is the most important root‐feeding pest of blueberries and turfgrass in New Jersey, USA. Previous studies showed that mating disruption is a feasible option for oriental beetle management; however, assessing its efficiency can be challenging, and little is known on its long‐term effects. Accordingly, we conducted studies to investigate low‐dose pheromone lures equivalent to oriental beetle females (i.e. female mimics) as easy‐to‐use indicators of mating disruption success, determine the distance at which oriental beetle males respond to female‐mimic lures and assess the long‐term (3‐year) effects of mating disruption on oriental beetle populations in entire blueberry fields. Our studies showed that rubber septa baited with 0.3 μg of the oriental beetle sex pheromone (Z)‐7‐tetradecen‐2‐one attract similar numbers of males as compared with virgin females and can thus be used as a female mimic. The range of attraction of this lure was found to be also similar to virgin females and <30 m. In blueberries, mating disruption provided 87% inhibition of oriental beetle populations (trap shutdown) over a 3‐year period. Oriental beetle male captures in disrupted fields were threefold higher along the field edges than in the field interiors, indicating movement of males from nearby areas into the pheromone‐treated fields. In addition, mating disruption reduced male attraction to female‐mimic lures by 93% in all 3 years and reduced the number of larvae in sentinel potted plants in 1 of 2 years. These results show for the first time that mating disruption provides consistent long‐term field‐wide control of oriental beetle populations and that female‐mimic pheromone lures can be used as a new tool to assess oriental beetle mating disruption success.     

Glucosylation of (Z)‐3‐hexenol informs intraspecies interactions in plants: A case study in Camellia sinensis

Plants emit a variety of volatiles in response to herbivore attack, and (Z)‐3‐hexenol and its glycosides have been shown to function as defence compounds. Although the ability to incorporate and convert (Z)‐3‐hexenol to glycosides is widely conserved in plants, the enzymes responsible for the glycosylation of (Z)‐3‐hexenol remained unknown until today. In this study, uridine‐diphosphate‐dependent glycosyltransferase (UGT) candidate genes were selected by correlation analysis and their response to airborne (Z)‐3‐hexenol, which has been shown to be taken up by the tea plant. The allelic proteins UGT85A53‐1 and UGT85A53‐2 showed the highest activity towards (Z)‐3‐hexenol and are distinct from UGT85A53‐3, which displayed a similar catalytic efficiency for (Z)‐3‐hexenol and nerol. A single amino acid exchange E59D enhanced the activity towards (Z)‐3‐hexenol, whereas a L445M mutation reduced the catalytic activity towards all substrates tested. Transient overexpression of CsUGT85A53‐1 in tobacco significantly increased the level of (Z)‐3‐hexenyl glucoside. The functional characterization of CsUGT85A53 as a (Z)‐3‐hexenol UGT not only provides the foundation for the biotechnological production of (Z)‐3‐hexenyl glucoside but also delivers insights for the development of novel insect pest control strategies in tea plant and might be generally applicable to other plants.     

Contractile activity is required for Z‐disc sarcomere maturation in vivo

Sarcomere structure underpins structural integrity, signaling, and force transmission in the muscle. In embryos of the frog Xenopus tropicalis, muscle contraction begins even while sarcomerogenesis is ongoing. To determine whether contractile activity plays a role in sarcomere formation in vivo, chemical tools were used to block acto‐myosin contraction in embryos of the frog X. tropicalis, and Z‐disc assembly was characterized in the paralyzed dicky ticker mutant. Confocal and ultrastructure analysis of paralyzed embryos showed delayed Z‐disc formation and defects in thick filament organization. These results suggest a previously undescribed role for contractility in sarcomere maturation in vivo. genesis 53:299–307, 2015. © 2015 The Authors. Genesis Published by Wiley Periodicals, Inc.     

(+)‐Z‐Bisdehydrodoisynolic Acid Ameliorates Obesity and the Metabolic Syndrome in Female ZDF Rats

Objective: The putative selective estrogen receptor modulator (+)‐Z‐bisdehydrodoisynolic acid (Z‐BDDA) has been found to improve cardiovascular risk in rodents. The objective of this study was to investigate the effectiveness of (+)‐Z‐BDDA compared with the antidiabetic drug, rosiglitazone, in treating obesity and risk factors associated with the metabolic syndrome. Research Methods and Procedures: Female Zucker Diabetic Fatty rats were randomly assigned to three treatment groups for 29 weeks: control (C), 1.8 mg (+)‐Z‐BDDA/kg diet [control diet + (+)‐Z‐BDDA (CB)], or 100 mg rosiglitazone/kg diet [control diet + rosiglitazone (CR)]. At sacrifice, physiological, biochemical, and molecular parameters were examined. Results: CB animals gained less weight and exhibited a decrease in total body lipids (p < 0.05) as compared with C or CR rats. Body weight and total body lipids were the highest in CR rats (p < 0.05). Liver weights in CB and CR rats were lower (p < 0.05) than in C rats, whereas kidney weights were lower in CB (p < 0.05) than in C and CR animals. Fasting plasma glucose was lower (p < 0.05) in the CB and CR animals when compared with C animals. C rats exhibited the highest concentration of total plasma cholesterol, and CR‐treated rats exhibited the lowest concentration. Plasma triglycerides followed the same pattern as plasma cholesterol. Histomorphometry of heart vasculature revealed that CB and CR treatments produced a significant shift from small to large venules and arterioles compared with C (p < 0.05). Liver expression profiles of peroxisome proliferator‐activated receptor (PPAR) α, PPARγ, and PPAR‐regulated genes revealed encouraging CB‐induced effects. Discussion: These results suggest that (+)‐Z‐BDDA may have applications in treating obesity and complications associated with the metabolic syndrome.     

Z‐FA.FMK activates duodenal epithelial cell proliferation through oxidative stress,NF‐κB and IL‐1β in d‐GalN/TNF‐α‐administered mice

This study was designed to evaluate the effect of Z‐FA.FMK (benzyloxycarbonyl‐l ‐phenylalanyl‐alanine‐fluoromethylketone), a pharmacological inhibitor of cathepsin B, on the proliferation of duodenal mucosal epithelial cells and the cellular system that controls this mechanism in these cells in vivo. For this investigation, BALB/c male mice were divided into four groups. The first group received physiological saline, the second group was administered Z‐FA.FMK, the third group received d ‐GalN (d ‐galactosamine) and TNF‐α (tumour necrosis factor‐α) and the fourth group was given both d ‐GalN/TNF‐α and Z‐FA.FMK. When d ‐GalN/TNF‐α was administered alone, we observed an increase in IL‐1β‐positive and active NF‐κB‐positive duodenal epithelial cells, a decrease in PCNA (proliferative cell nuclear antigen)‐positive duodenal epithelial cells and an increase in degenerative changes in duodenum. On the other hand, Z‐FA.FMK pretreatment inhibited all of these changes. Furthermore, lipid peroxidation, protein carbonyl and collagen levels were increased, glutathione level and superoxide dismutase activity were decreased, while there was no change in catalase activity by d ‐GalN/TNF‐α injection. On the contrary, the Z‐FA.FMK pretreatment before d ‐GalN/TNF‐α blocked these effects. Based on these findings, we suggest that Z‐FA.FMK might act as a proliferative mediator which is controlled by IL‐1β through NF‐κB and oxidative stress in duodenal epithelial cells of d ‐GalN/TNF‐α‐administered mice.     

Cell‐free synthesis of functional aquaporin Z in synthetic liposomes

The challenges involved in producing sufficient quantities of aquaporins for precise biophysical characterization have limited our knowledge of this important class of molecules. This article describes a cell‐free protein synthesis method for producing high concentrations of the E. coli water transporter, aquaporin Z (AqpZ), in synthetic liposomes. To our knowledge, this is the first report of in vitro synthesis of a membrane protein directly into synthetic liposomes with verified function, (i.e., transport activity and selectivity). Titration of DOPC lipid vesicles added to the cell‐free reaction show that production yields of active AqpZ are dependent on the concentration of DOPC lipid vesicles added to the cell‐free reaction, with 224 ± 24 lipids required per aquaporin monomer. Supplementation of the signal recognition particle receptor (FtsY) to the cell‐free reaction increases production of vesicle‐associated AqpZ but not active AqpZ. Cell‐free reactions using 7 mg/mL lipids that were not supplemented with FtsY produced 507 ± 11 µg/mL of vesicle‐associated AqpZ that exhibited a specific water transport activity of (2.2 ± 0.3) × 10^?14 cm³ s^?1 monomer^?1. Proteinase K protection, activation energy determination, and selectivity against glycerol and urea transport also confirmed the production of correctly folded AqpZ. This technique is capable of producing milligram quantities of aquaporin that can be readily assayed for function, facilitating biophysical characterization and high‐throughput analysis. Biotechnol. Bioeng. 2009; 104: 40–49 © 2009 Wiley Periodicals, Inc.     

Effect of Different Z‐Inducers on the Stabilization of Z Portion in BZ‐DNA Sequence: Correlation Between Experimental and Simulation Data

In this study we show the outstanding agreement between simulation and experimental data concerning the efficient stabilization effect by NaCl of Z conformation. We demonstrate by circular dichroism (CD) experiments that Na⁺ not only is able to induce a B to Z form transition in a short (GC)₃ alternated portion of a sequence having 17 basis, but also is the best stabilizer in comparison with other Z inducers used (spermine and NiCl₂). This result was confirmed by free energy calculations. Chirality 27:773–778, 2015. © 2015 Wiley Periodicals, Inc.