Pitaya Virus X Coat Protein Acts as an RNA Silencing Suppressor and Can Be Used as a Specific Target for Detection Using RT-LAMP.

Selenicereus undatus (Haworth) D.R. Hunt (pitaya) is a tropical fruit that has been commonly cultivated in Guizhou Province, China, in recent years due to its good taste and high nutritional value. This planting area currently ranks third in China. Viral diseases have increasingly emerged in pitaya cultivation because of the expansion of the pitaya planting area and the characteristics of asexual propagation. The spread of pitaya virus X (PiVX; a Potexvirus) is among the most severe viruses threatening the quality and yield of pitaya fruit. To investigate the occurrence of PiVX in pitaya cultivations in Guizhou Province, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method that can detect PiVX with high sensitivity and specificity at a low cost and produce a visualized result. Our best RT-LAMP system was significantly more sensitive than RT-PCR and was highly specific to PiVX. Furthermore, PiVX coat protein (CP) can form a homodimer, and PiVX may use its CP as a plant RNA silencing suppressor to enhance infection. To the best of our knowledge, this is the first report of fast detection of PiVX and functional exploration of CP in a Potexvirus. These findings will provide an opportunity for early diagnosis and prevention of viruses in pitaya.

Molecular Oxygen Activation by Citric Acid Boosted Pyrite-Photo-Fenton Process for Degradation of PPCPs in Water.

Pyrite has been used in photo-Fenton reactions for the degradation of pollutants, but the application of photo-Fenton processes with extra H2O2 in real water/wastewater treatment has still been limited by the economic cost of H2O2 and artificial light sources. Herein, citric acid (CA) and simulated/natural sunlight are used to develop a pyrite-based photo-Fenton system (pyrite-CA-light) in situ generating H2O2 through the enhanced activation of molecular oxygen. The degradation of pharmaceuticals and personal care products (PPCPs), especially acetaminophen (APAP) as the main target pollutant, in the pyrite-CA-light system was investigated. The effects of influencing factors such as various organic acids, APAP concentration, pH, pyrite dosage, CA concentration and co-existing anions (HCO3-, Cl-, NO3-, SO42- and H2PO4-) were examined. At a pyrite dosage of 0.1 g L-1, CA concentration of 0.6 mM and an initial pH of 6.0, the degradation efficiency of APAP (30 µM) was 99.1% within 30 min under the irradiation of xenon lamp (70 W, λ ≥ 350 nm). Almost the same high efficiency of APAP degradation (93.9%) in the system was achieved under natural sunlight irradiation (ca. 650 W m-2). The scavenging experiments revealed that the dominant active species for degrading APAP was hydroxyl radical (HO•). Moreover, a quantitative structural-activity relationship (QSAR) model for pseudo-first-order rate constants (kobs) was established with a high significance (R2 = 0.932, p = 0.001) by using three descriptors: octanol-water partition coefficient (logKow), dissociation constant (pKa) and highest occupied molecular orbital (HOMO). This work provides an innovative strategy of the photo-Fenton process for the degradation of PPCPs using natural minerals and ordinary carboxylic acid under sunlight.

Characterization of histone deacetylases and their roles in response to abiotic and PAMPs stresses in Sorghum bicolor.

BACKGROUND: Histone deacetylases (HDACs) play an important role in the regulation of gene expression, which is indispensable in plant growth, development, and responses to environmental stresses. In Arabidopsis and rice, the molecular functions of HDACs have been well-described. However, systematic analysis of the HDAC gene family and gene expression in response to biotic and abiotic stresses has not been reported for sorghum. RESULTS: We conducted a systematic analysis of the sorghum HDAC gene family and identified 19 SbHDACs mainly distributed on eight chromosomes. Phylogenetic tree analysis of SbHDACs showed that the gene family was divided into three subfamilies: RPD3/HDA1, SIR2, and HD2. Tissue-specific expression results showed that SbHDACs displayed different expression patterns in different tissues, indicating that these genes may perform different functions in growth and development. The expression pattern of SbHDACs under different stresses (high and low temperature, drought, osmotic and salt) and pathogen-associated molecular model (PAMPs) elf18, chitin, and flg22) indicated that SbHDAC genes may participate in adversity responses and biological stress defenses. Overexpression of SbHDA1, SbHDA3, SbHDT2 and SbSRT2 in Escherichia coli promoted the growth of recombinant cells under abiotic stress. Interestingly, we also showed that the sorghum acetylation level was enhanced when plants were under cold, heat, drought, osmotic and salt stresses. The findings will help us to understand the HDAC gene family in sorghum, and illuminate the molecular mechanism of the responses to abiotic and biotic stresses. CONCLUSION: We have identified and classified 19 HDAC genes in sorghum. Our data provides insights into the evolution of the HDAC gene family and further support the hypothesis that these genes are important for the plant responses to abiotic and biotic stresses.

Morphology of Osteogenesis Imperfecta Collagen Mimetic Peptide Assemblies Correlates with the Identity of Glycine-Substituting Residue.

Osteogenesis imperfecta (OI) is a hereditary bone disorder with various phenotypes ranging from mild multiple fractures to perinatal lethal cases, and it mainly results from the substitution of Gly by a bulkier residue in type I collagen. Triple-helical peptide models of Gly mutations have been widely utilized to decipher the etiology of OI, although these studies are mainly limited to characterizing the peptide features, such as stability and conformation in the solution state. Herein, we have constructed a new series of triple-helical peptides DD(GPO)5 ZPO(GPO)4 DD (Z=Ala, Arg, Asp, Cys, Glu, Ser, and Val) mimicking the most common types of observed OI cases. The inclusion of special terminal aspartic acids enables these collagen mimetic peptides to self-assemble to form nanomaterials upon the trigger of lanthanide ions. We have for the first time systematically evaluated the effect of different OI mutations on the aggregated state of collagen mimetic peptides. We have revealed that the identity of the Gly-substituting residue plays a determinant role in the morphology and secondary structure of the collagen peptide assemblies, showing that bulkier residues tend to result in a disruptive secondary structure and defective morphology, which lead to more severe OI phenotypes. These findings of osteogenesis imperfecta collagen mimetic peptides in the aggregation state provide novel perspectives on the molecular mechanism of osteogenesis imperfecta, and may aid the development of new therapeutic strategies.

Effects of naturally occurring charged mutations on the structure, stability, and binding of the Pin1 WW domain.

Pin1 is a peptidyl-prolyl cis-trans isomerase, whose WW domain specifically recognizes the pSer/Thr-Pro motif. Pin1 is involved in multiple phosphorylation events that regulate the activities of various substrates, and Pin1 deregulation has been reported in various diseases, including cancer and Alzheimer's disease. The WW domain of Pin1 has been used as a small model protein to investigate the folding mechanisms of the ß-sheet structure by studying the effect of mutations or its naturally occurring variants. However, only a few studies have investigated the structure and binding of Pin1 WW mutants. In the present work, two naturally occurring Pin1 WW variants, namely, G20D and S16R, derived from the cynomolgus monkey and African green monkey, respectively, were selected to investigate the influence of charge mutation on the structure, stability, and binding properties of the Pin1 WW domain. Analysis using a combination of nuclear magnetic resonance (NMR) and chemical shift-based calculations revealed that the G20D and S16R mutants had high structural similarity to the wild-type Pin1 WW domain. However, the presence of a charge mutation significantly decreased the stability of the Pin1 WW domain. Both the wild-type and G20D forms of the Pin1 WW domain utilized a three-site mode to bind to a phosphorylated Tau peptide, pT231, whereas the S16R mutant binds to the pT231 peptide either in a non-specific manner or through a totally different binding mechanism. Correspondingly, the wild-type and two mutant Pin1 WW domains showed different binding affinities to the Tau phosphopeptide. Considering that the WW domain participates in the catalytic activity of the Pin1 isomerase, our study represents a novel approach for studying Pin1 function through the analysis of its naturally occurring mutants.

L-F001, a Multifunction ROCK Inhibitor Prevents 6-OHDA Induced Cell Death Through Activating Akt/GSK-3beta and Nrf2/HO-1 Signaling Pathway in PC12 Cells and Attenuates MPTP-Induced Dopamine Neuron Toxicity in Mice.

Amounting evidences demonstrated that Rho/Rho-associated kinase (ROCK) might be a novel target for the therapy of Parkinson's disease (PD). Recently, we synthesized L-F001 and revealed it was a potent ROCK inhibitor with multifunctional effects. Here we investigated the effects of L-F001 in PD models. We found that L-F001 potently attenuated 6-OHDA-induced cytotoxicity in PC12 cells and significantly decreased intracellular reactive oxygen species (ROS), prevented the 6-OHDA-induced decline of mitochondrial membrane potential and intracellular GSH levels. In addition, L-F001 increased Akt and GSK-3beta phosphorylation and induced the nuclear Nrf2 and HO-1 expression in a time- and concentration-dependent manner. Moreover, L-F001 restored the levels of p-Akt and p-GSK-3beta (Ser9) as well as HO-1 expression reduced by 6-OHDA. Those effects were blocked by the specific PI3K inhibitor, LY294002, indicating the involvement of Akt/GSK-3beta pathway in the neuroprotective effect of L-F001. In addition, L-F001 significantly attenuated the tyrosinehydroxylase immunoreactive cell loss in 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP)-induced mice PD model. Together, our findings suggest that L-F001 prevents 6-OHDA-induced cell death through activating Akt/GSK-3beta and Nrf2/HO-1 signaling pathway and attenuates MPTP-induced dopaminergic neuron toxicity in mice. L-F001 might be a promising drug candidate for PD.

Degradation mechanism of Bisphenol S via hydrogen peroxide/persulfate activated by sulfidated nanoscale zero valent iron.

Fenton-like oxidation processes are widely used to degrade recalcitrant organic pollutants, but are limited by narrow application pH and low reaction efficiency. This study investigated the synchronous activation of H2O2 and persulfate (PDS) by sulfidated zero valent iron (S-nZVI) in ambient conditions for Fenton-like oxidation of bisphenol S (BPS), an estrogenic endocrine-disrupting chemical. The activation of S-nZVI induced H2O2 or PDS could be greatly enhanced with the assistance of PDS and H2O2, respectively, even across a wide range of pH value (3-11). The first-order rate constant of S-nZVI/H2O2/PDS, S-nZVI/PDS and S-nZVI/H2O2 systems was found to be 0.2766 min-1, 0.0436 min-1, and 0.0113 min-1, respectively. A significant synergy between H2O2 and PDS was achieved when the PDS-H2O2 molar ratio was above 1:1, and where sulfidation promoted iron corrosion and decreased solution pH were observed in the S-nZVI/H2O2/PDS system. Radical scavenging experiments and electron paramagnetic resonance (EPR) investigations suggest that both SO4•- and •OH were generated and that •OH played a crucial role in BPS removal. Furthermore, four BPS degradation intermediates were detected and three degradation pathways were proposed in line with the HPLC-Q-TOF-MS analysis. This study demonstrated that compared to the traditional Fenton-like system, the S-nZVI/H2O2/PDS system could be a more efficient, advanced oxidation technology capable of being used across a broad pH range for emerging pollutants' degradation.

Quantitative structure-activity relationship for the photooxidation of aromatic micro-pollutants induced by graphene oxide in water.

The photocatalytic degradation behavior of aromatic micro-pollutants (AMPs) exhibits complexity and uncertainty, which mainly depends on the properties of different substituents on benzene. And with similar catalytic reaction substrates, the reaction rate constant could reveal the influence of different characteristics of molecular structure in a specific system. Therefore, the photooxidation pseudo first-order kinetic rate constants (kobs) of 30 AMPs were experimentally determined in Photo-GO system. A quantitative structure-activity relationship (QSAR) model for predicting the photooxidation reaction of AMPs has been developed by stepwise multiple linear regression (MLR) method, based on the lg kobs and representative molecule descriptors (20 in total) including physicochemical, quantum chemical and electrostatic descriptors. Afterwards, Radj2, QLOO2, and Qext2 were calculated as 0.870, 0.841, and 0.732 respectively, which exhibited the excellent goodness-of-fit, robustness, and predictability of the QSAR model, indicating its great prediction ability for photooxidation behavior of AMPs. Meanwhile, during the photooxidation process of AMPs with GO, the model revealed that the one-electron oxidation potential (Eox), molecular dipole moment (µ), and number of hydrogen bond donors (#HD) were the most important molecular structural parameters, which showed that the single electron transfer pathway and adsorption were as the significant steps. Additionally, the Hammett correlation showed that photooxidation of AMPs in Photo-GO system is of typical electrophilic reactions, which demonstrated that the electron-donating substituents could promote the photooxidation of AMPs. The QSAR model was constructed and evaluated to perform the prediction of AMPs reaction kinetics, which provided a guidance for the study of the mechanism and selective oxidation of AOPs photooxidation system based on GO.

Transcriptional Profiling of TGF-ß Superfamily Members in Lumbar DRGs of Rats Following Sciatic Nerve Axotomy and Activin C Inhibits Neuropathic Pain.

BACKGROUND: Neuroinflammation and cytokines play critical roles in neuropathic pain and axon degeneration/regeneration. Cytokines of transforming growth factor-ß superfamily have implications in pain and injured nerve repair processing. However, the transcriptional profiles of the transforming growth factor-ß superfamily members in dorsal root ganglia under neuropathic pain and axon degeneration/regeneration conditions remain elusive. OBJECTIVE: We aimed to plot the transcriptional profiles of transforming growth factor-ß superfamily components in lumbar dorsal root ganglia of sciatic nerve-axotomized rats and to further verify the profiles by testing the analgesic effect of activin C, a representative cytokine, on neuropathic pain. METHODS: Adult male rats were axotomized in sciatic nerves, and lumbar dorsal root ganglia were isolated for total RNA extraction or section. A custom microarray was developed and employed to plot the gene expression profiles of transforming growth factor-ß superfamily components. Realtime RT-PCR was used to confirm changes in the expression of activin/inhibin family genes, and then in situ hybridization was performed to determine the cellular locations of inhibin α, activin ßC, BMP-5 and GDF-9 mRNAs. The rat spared nerve injury model was performed, and a pain test was employed to determine the effect of activin C on neuropathic pain. RESULTS: The expression of transforming growth factor-ß superfamily cytokines and their signaling, including some receptors and signaling adaptors, were robustly upregulated. Activin ßC subunit mRNAs were expressed in the small-diameter dorsal root ganglion neurons and upregulated after axotomy. Single intrathecal injection of activin C inhibited neuropathic pain in spared nerve injury model. CONCLUSION: This is the first report to investigate the transcriptional profiles of members of transforming growth factor-ß superfamily in axotomized dorsal root ganglia. The distinct cytokine profiles observed here might provide clues toward further study of the role of transforming growth factor-ß superfamily in the pathogenesis of neuropathic pain and axon degeneration/regeneration after peripheral nerve injury.

[Determination of multi-veterinary drug residues in complementary foods for infants and young children by ultra performance liquid chromatography-tandem mass spectrometry].

Special attention must be paid to children and infants because health issues stemming from a poor immunologic system and low weight make them a vulnerable risk group. Complementary foods for infants and young children, which are a class of special dietary foods, are essential transitional foods for infants from lactation to adaptation to ordinary food. Some complementary foods for infants and young children are of animal origin such as fish, meat, and the liver, which may contain veterinary drug residues. Veterinary drugs are usually small-molecular-weight chemicals that are essential for treating infections, increasing production, and improving animal husbandry. However, abuse of these substances can provoke transfer to the food chain, leading to negative consequences for humans, especially infants and young children. For a more comprehensive safety supervision of infant supplementary foods, a method based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed and used for the determination of 50 antibiotics and antivirals (grouped into six categories: fluoroquinolones, sulfonamides, macrolides, nitroimidazoles, chloramphenicols, and antivirals) in complementary foods for infants and young children. The matrix of complementary foods for infants and young children is complex and contains a large number of proteins and lipids, which poses a serious challenge for sample pretreatment. The Captiva EMR-Lipid solid-phase extraction (SPE) column is a new type of product that allows for selective and efficient lipid/matrix removal without negatively affecting the recovery of the analyte. In this study, samples were extracted with acidified acetonitrile and then purified using a Captiva EMR-Lipid SPE column. The target analytes were separated on a BEH C18 column by gradient elution using acetonitrile and water containing 0.1% (v/v) formic acid as the mobile phases. MS detection was performed with an electrospray source in the positive and negative modes in the multireaction monitoring (MRM) mode. The ion spray voltages were set at 5500 V and -4500 V in the positive and negative modes, respectively. The source temperature for both the ionization modes was set to 500 ℃. Instrumental parameters such as collision energy and declustering potential were optimized. The samples were quantified using the external standard method with matrix calibration curves to reduce the influence of the matrix effect on the quantitative results.The results showed that the 50 veterinary drug residues had good linear relationships in the range of 0.5 to 50 µg/L, with correlation coefficients higher than 0.995. The limits of detection (LODs) and quantification (LOQs) were in the range of 0.03-0.70 µg/kg and 0.09-2.33 µg/kg, respectively. The average recoveries for all the compounds under different matrices ranged from 64.37% to 119.3% at spiked levels of 5 µg/kg and 50 µg/kg, with relative standard deviations of less than 15%. Compared to QuEChERS, this method has a better purification effect. The recoveries of the 50 veterinary drugs extracted by this method were also much higher than those in the case of QuEChERS. This method was applied to the detection of 14 domestic and six imported infant supplementary foods. Sulfaquinoxaline, sulfamethazine, and tilmicosin were detected in one imported meat-based baby food. With its simple operation, high sensitivity and accuracy, and low sample quantity consumption, this method is suitable for the determination of multiveterinary drug residues in complementary foods for infants and young children. This study provides an effective analysis method for risk monitoring and troubleshooting of complementary foods for infants and young children, which is of great significance in ensuring the healthy growth of the next generation.

Expression Analysis Reveals That Sorghum Disease Resistance Protein SbSGT1 Is Regulated by Auxin.

SGT1 (suppressor of the skp1 G2 allele) is an important plant disease resistance-related protein, which plays an important role in plant resistance to pathogens and regulates signal transduction during the process of plant disease resistance. In this study, we analyzed the expression profile of SbSGT1 in sorghum under phytohormones treatment. Quantitative real-time PCR results showed that SbSGT1 was most expressed in sorghum leaves, and could respond to plant hormones such as auxin, abscisic acid, salicylic acid, and brassinolide. Subsequently, we determined the optimal soluble prokaryotic expression conditions for SbSGT1 and purified it using a protein purification system in order to evaluate its potential interactions with plant hormones. Microscale thermophoretic analysis showed that SbSGT1 exhibited significant interactions with indole-3-acetic acid (IAA), with a Kd value of 1.5934. Furthermore, the transient expression of SbSGT1 in Nicotiana benthamiana indicated that treatment with exogenous auxin could inhibit SbSGT1 expression, both at the transcriptional and translational level, demonstrating that there exists an interaction between SbSGT1 and auxin.

Sulfamethoxazole degradation by alpha-MnO2/periodate oxidative system: Role of MnO2 crystalline and reactive oxygen species.

Pollutant degradation via periodate ([Formula: see text]) and transitional metal oxides provides an economical, energy-efficient way for chemical oxidation process in environmental remediation. However, catalytic activation of periodate by manganese dioxide and the associated mechanism were barely investigated. In this study, four MnO2 polymorphs (α-, ß-, γ- and δ-MnO2) were synthesized and tested to activate [Formula: see text] for the degradation of sulfamethoxazole (SMX). The reactivity of different MnO2 structures followed the order of α-MnO2 > ß-MnO2 > γ-MnO2 > δ-MnO2, suggesting that the particular crystalline structure in α-MnO2 would exhibit higher activities via [Formula: see text] activation. Herein, in α-MnO2/[Formula: see text] system, 91.1% of SMX was eliminated within 30 min with degradation rate constant of 0.0649 min-1, and the neutral pH exhibited higher efficiency in SMX degradation compared with acidic and alkaline conditions. Singlet oxygen (1O2) was unveiled to be the dominant ROS according to the results of electron paramagnetic resonance, chemical probes and radical quenching experiments, whereas [Formula: see text] and •OH were mainly acted as a free-radical precursor. Six oxidation products were identified by LC-MS, and the elimination of sulfonamide bond, hydroxylation and direct oxidation were found to be the important oxidation pathways. The study dedicates to the mechanistic study into periodate activation over alpha-MnO2 and provides a novel catalytic activation for selective removal in aqueous contaminants.

Adsorption-enforced Fenton-like process using activated carbon-supported iron oxychloride catalyst for wet scrubbing of airborne dichloroethane.

Wet scrubbing is a low-cost process for disposing of air pollutants. Nevertheless, this method is rarely used for the treatment of volatile organic compounds (VOCs) because of their poor water solubility. In this study, we used a unique wet scrubbing system containing H2O2 and activated carbon (AC)-supported iron oxychloride (FeOCl) nanoparticles to remove airborne dichloroethane (DCE). The operating conditions of the wet scrubber were optimized, and the mechanism was explored. The results showed that the adsorption of dissolved DCE onto AC promoted its transfer from air to water, while the accumulation of DCE on AC facilitated its oxidation by •OH generated on FeOCl catalyst. The wet scrubber performed well at pH 3 and low H2O2 concentrations. By pulsed or continuous dosing H2O2, the cooperative adsorption-catalytic oxidation allowed long-term DCE removal from air. Benefiting from satisfactory cost-effectiveness, avoidance of toxic byproduct formation, and less corrosion and catalyst poisoning, wet scrubbers coupled with cooperative adsorption and heterogeneous advanced oxidation processes could have broad application potentials in VOC control.

Cytochrome P450 Superfamily: Evolutionary and Functional Divergence in Sorghum (Sorghum bicolor) Stress Resistance.

Cytochrome P450 (CYP) genes encode enzymes that catalyze various growth-, development-, and stress-related reactions. Sorghum (Sorghum bicolor) is a type of C4 plant and an important cash crop. However, systematic identification and analysis of functional differentiation and evolution of CYP genes have not been carried out in this species. In the present study, we revealed that the sorghum genome contains 351 CYP genes, which can be divided into nine classes. These genes are from ancestors and repeated segments, rather than tandem repeats. Based on collinearity results, a large number of CYPs were extended before cotyledon differentiation, during the emergence of Gramineae, suggesting that genomewide duplication events and stress adaptation processes were important for the expansion of CYP genes. Their gene structure and motifs contain conserved regions and include various changes and loci. The expression characteristics and functional annotation of CYP genes indicated tissue specificity and selective expression. Overall, we identified all CYP genes in the sorghum genome and preliminarily explored their naming, structure, evolution, expression, and functional differentiation. The results advanced our understanding of plant gene family evolution and functional differentiation.

Ln3+-Triggered self-assembly of a heterotrimer collagen mimetic peptide into luminescent nanofibers.

Type I collagen, the most abundant and arguably the most complex molecule in the human body, is an ABB heterotrimer that self-assembles to form well-defined nanofibers. We herein for the first time report the construction of peptides that could simultaneously mimic the heterotrimer composition and the self-assembly features of Type I collagen.

High prevalence of Clonorchis sinensis infections and coinfection with hepatitis virus in riverside villages in northeast China.

In China, the prevalence of Clonorchis sinensis (C. sinensis) infections is only evaluated at the provincial level by national sampling surveys, and data from villages and counties are still lacking. In this study, we conducted a cross-sectional survey in 10 villages located along the Lalin River in northeast China. Clonorchiasis was diagnosed using a modified Kato-Katz method that detects the C. sinensis egg in stools. A total of 3,068 persons were screened and 2,911 were recruited for the study. Overall, the prevalence of C. sinensis infection was 29.3%. Among 175 participants who were cured after antiparasitic treatment, 54 (30.86%) were re-infected in this survey. After calibration of potential confounders, male gender, occupation as a farmer, smoking, and occasionally or frequently eating raw fish were independent risk factors for C. sinensis infection. The results of laboratory examinations in the C. sinensis/hepatitis B or C virus co-infection group were similar to those in the hepatitis B or C virus mono-infection groups. In conclusion, C. sinensis is highly endemic in villages along the Lalin River, and the primary route of infection is the consumption of raw freshwater fish. Co-infection with C. sinensis did't aggravate the clinical manifestations of viral hepatitis in this cross-sectional study.

Detection of target collagen peptides with single amino acid mutation using two fluorescent peptide probes.

Collagen with a single amino acid substitution is the main cause of a plethora of heritable disorders such as Osteogenesis Imperfecta and Ehlers-Danlos syndrome. Though significant advances have been achieved in the development of protein assays, it remains very challenging to distinguish a protein with a single amino acid mutation from the wild-type protein. A novel fluorescent self-quenching assay has been constructed to detect target collagen peptides with a single amino acid mutation using two probe peptides. The hybridization of the probe peptide and the natural target collagen peptide results in a complete heterotrimer and strong fluorescence, whereas the mixture of the probe peptide and the mutation collagen sequences leads to a partial homotrimer and pronounced fluorescence self-quenching. The extent of fluorescence quenching is dependent on the identity of the residue replacing Gly following the order of Ala < Ser < Arg, while the Gly-Ala mutation causes the mildest fluorescence loss. The probe peptide-based fluorescence self-quenching assay facilitates specific detection of the target collagen sequence with a single Gly mutation at the nM level. The simultaneous utilization of both probe peptides enables efficient discrimination between different mutation peptides. To our knowledge, our work may be the first report of a robust analytical assay that can identify collagen fragments with single amino acid mutation, which will greatly contribute to deciphering the molecular mechanism of Osteogenesis Imperfecta as well as developing novel diagnostic strategies.

Luminescent Biofunctional Collagen Mimetic Nanofibers.

Collagen has long been one of the top targets for biomimetic design due to its superior structural and functional properties. Significant progress has been achieved to construct self-assembling peptides to mimic the fibrous nanostructure of native collagen, while it is still very demanding to fabricate peptide assemblies that can recapitulate both structural and biofunctional features of collagen. Herein, collagen-like peptides have been synthesized to contain negatively charged amino acids as the binding groups of lanthanide ions and the integrin-binding motif GFOGER. The simultaneous inclusion of negatively charged amino acids in the middle as well as at both terminals drives the peptides to self-assemble to form well-ordered nanofibers with distinct periodic banding patterns specifically mediated by lanthanide ions. The aggregation tendency and the morphology of the final assembled materials for the peptides are modulated in a pH-cooperative manner, which well mimics the pH-dependent fibrillogenesis of Type I collagen. The utilization of lanthanide ions in the system not only offers a convenient external stimulus but also functionalizes assembled materials with excellent luminescent features. Most notably, the lanthanide-triggered peptide assembled nanomaterials possess good cell adhesion properties, which resemble the biological function of collagen. This peptide-Ln3+ system provides a facile and potent strategy to generate nanofibers that mimic both the structural and functional properties of natural collagen. These novel pH-responsive, luminescent, and biofunctional collagen mimetic nanofibers open fascinating opportunities in the development of improved functional biomaterials in tissue engineering, drug delivery, and medical diagnostics.

Expression of puroindoline a enhances leaf rust resistance in transgenic tetraploid wheat.

The purouindoline gene (pin) coding for puroindoline proteins (PINs) is located on chromosome 5D, controls grain hardness, and the PINs have in vitro antimicrobial activity against gram-positive (G+) bacteria, gram-negative (G-) bacteria and fungi. Wheat leaf rust caused by Puccinia triticina is one of the most important fungal diseases for common wheat with AABBDD genomes. Tetraploid wheat (AABB genome) varieties Luna and Venusia were transformed with the purouindoline a (pinA) gene by bombardment, express PINA consititutively. Transgenic plants showed enhanced response to leaf rust in greenhouse and field. Comparative study of harvesting parameters showed significant differences between transgenic and control plants. These indexes were significantly lower (P < 0.05) in control plants than that in transgenic plants, which suggests that they are significantly affected by pinA gene and that the puroindoline a protein (PINA) can effectively inhibit in vivo the growth of fungal, and the transgenic tetraploid wheat can grow well in Hubei Province, Central China, where the tetraploid wheat varieties Luna and Venusia have poor yield due to their disease-sensitivity.

An optimal pooling strategy applied to high-throughput screening for rare marker-free transformants.

An optimal pooling system, called Accurate and Fast Target Screening, has been developed for high-throughput identifying the rare marker-free transformants. This system can identify targets between 10- and 100-fold more efficiently than analysis of individual samples. By calculating the efficiency for different proportions of targets and the optimal group size in a worst case scenario, we are able to estimate an upper limit for the number of tests that are required. The application of this system to determine the transgene in an artificially constructed population of transgenic and non-transgenic wheat lines successfully identified the 10 positive samples located randomly with 990 negative samples using only 92 PCR reactions. The same approach was also applied to determine transgene expression by SDS-PAGE of seed proteins. This system gives unambiguous positive or negative results and should facilitate marker-free transformation.