Protein extraction from the stem of Panax ginseng C. A. Meyer: A tissue of lower protein extraction efficiency for proteomic analysis

Ginseng stem, the aerial part of the ginseng is more susceptible to diseases and environmental damages in its long process of growth. Since the molecular mechanism of why ginseng stems are vulnerable remains unclear, the comparison between healthy and pathogen suspicious tissues via proteomics approaches, such as 2-DE, could facilitate the deciphering of the pathogenesis of ginseng and improve ginseng planting industry. A major obstacle for the proteomics study of ginseng stem is the low extraction efficacy of protein due to the properties of its interfering compounds. Here, we tested six different protocols of protein extraction, and identified a protocol that gave us satisfactory yield for 2-DE analysis. The protein extraction was further optimized by chloroform/isoamylol and Tris-saturated phenol extraction that reached the standard of protein purity for 2-DE. Then, using the new extraction protocol, we can efficiently analyze the protein expression patterns of ginseng stem which might provide important information for our understanding of the disease mechanism. Also, our study would lay a foundation for the systematic analysis of the proteomics of ginseng and provide a methodological reference for other similar plant tissues.Key words: Protein extraction, lower protein extraction efficiency, ginsengstem, two-dimensional electrophoresis

A new fault diagnosis and fault-tolerant control method for mechanical and aeronautical systems with neural estimators

A new method of fault detection and fault tolerant control is proposed in this paper for mechanical systems and aeronautical systems. The faults to be estimated and diagnosed are malfunctions occurred within the control loops of the systems, rather than some static faults, such as gearbox fault, component cracks, etc. In the proposed method two neural networks are used as on-line estimators, the fault will be accurately estimated when the estimators are adapted on-line with the post fault dynamic information. Furthermore, the estimated value of faults are used to compensate for the impact of the faults, so that the stability and performance of the system with the faults are maintained until the faulty components to be repaired. The sliding mode control is used to maintain system stability under the post fault dynamics. The control law and the neural network learning algorithms are derived using the Lyapunov method, so that the neural estimators are guaranteed to converge to the fault to be diagnosed, while the entire closed-loop system stability is guaranteed with all variables bounded. The main contribution of this paper to the knowledge in this field is that the proposed method cannot only diagnose and tolerant with constant fault, also diagnose and tolerant with the time-varying faults. This is very important because most faults occurred in industrial systems are time-varying in nature. A simulation example is used to demonstrate the design procedure and the effectiveness of the method. The simulation results are compared with two existing methods that can cope with constant faults only, and the superiority is demonstrated

Effect of bainite layer by LSMCIT on wear resistance of medium-carbon bainite steel at different temperatures

In this work, bainite layer was prepared by Laser surface melting combined with isothermal treatment (LSMCIT) at 250ºC. The microstructures of the samples were analyzed by scanning electron microscopy (SEM), X-ray Diffraction (XRD) and transmission electron microscopy (TEM). Their wear resistances at 20ºC, 100ºC and 200ºC were measured using reciprocating tribometer. After the wear test, the confocal laser scanning microscope and SEM were used to characterize the topography of all abrasion surfaces, and the phase transformations occurred on the contact surfaces were analyzed by XRD. The results show that the microstructure of the LSMCIT sample has been refined to nanoscale. The wear volume reduction ratio of LSMCIT sample is 40.9% at 20ºC. The wear resistances of the samples are decreased with increasing of the temperature, however, the decrease in amplitude of the bainite is relatively small. The harder surface of the LSMCIT sample can provides higher mechanical support, and the white-etching layer on surface are difficult to remove by the reciprocating friction. The wear resistances of the LSMCIT samples at 20ºC, 100ºC and 200ºC are excellent, which shows the wide temperature ranges in wear applications

AGO6 Functions in RNA-Mediated Transcriptional Gene Silencing in Shoot and Root Meristems in Arabidopsis thaliana

RNA-directed DNA methylation (RdDM) is a small interfering RNA (siRNA)-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery that includes specialized RNA polymerases, named Pol IV and Pol V, as well as chromatin remodelling proteins, transcription factors, RNA binding proteins, and other plant-specific proteins whose functions are not yet clarified. In Arabidopsis thaliana, DICER-LIKE3 and members of the ARGONAUTE4 group of ARGONAUTE (AGO) proteins are involved, respectively, in generating and using 24-nt siRNAs that trigger methylation and transcriptional gene silencing of homologous promoter sequences. AGO4 is the main AGO protein implicated in the RdDM pathway. Here we report the identification of the related AGO6 in a forward genetic screen for mutants defective in RdDM and transcriptional gene silencing in shoot and root apical meristems in Arabidopsis thaliana. The identification of AGO6, and not AGO4, in our screen is consistent with the primary expression of AGO6 in shoot and root growing points

IDN2 and Its Paralogs Form a Complex Required for RNA–Directed DNA Methylation

IDN2/RDM12 has been previously identified as a component of the RNA–directed DNA methylation (RdDM) machinery in Arabidopsis thaliana, but how it functions in RdDM remains unknown. By affinity purification of IDN2, we co-purified two IDN2 paralogs IDP1 and IDP2 (IDN2 PARALOG 1 and 2). The coiled-coil domain between the XS and XH domains of IDN2 is essential for IDN2 homodimerization, whereas the IDN2 C-terminal XH domain but not the coiled-coil domain is required for IDN2 interaction with IDP1 and IDP2. By introducing the wild-type IDN2 sequence and its mutated derivatives into the idn2 mutant for complementation testing, we demonstrated that the previously uncharacterized IDN2 XH domain is required for the IDN2-IDP1/IDP2 complex formation as well as for IDN2 function. IDP1 is required for de novo DNA methylation, siRNA accumulation, and transcriptional gene silencing, whereas IDP2 has partially overlapping roles with IDP1. Unlike IDN2, IDP1 and IDP2 are incapable of binding double-stranded RNA, suggesting that the roles of IDP1 and IDP2 are different from those of IDN2 in the IDN2-IDP1/IDP2 complex and that IDP1 and IDP2 are essential for the functioning of the complex in RdDM

Phosphorylation of Puma modulates its apoptotic function by regulating protein stability

Puma is a potent BH3-only protein that antagonises anti-apoptotic Bcl-2 proteins, promotes Bax/Bak activation and has an essential role in multiple apoptotic models. Puma expression is normally kept very low, but can be induced by several transcription factors including p53, p73, E2F1 and FOXO3a, whereby it can induce an apoptotic response. As Puma can to bind and inactivate all anti-apoptotic members of the Bcl-2 family, its activity must be tightly controlled. We report here, for the first time, evidence that Puma is subject to post-translational control through phosphorylation. We show that Puma is phosphorylated at multiple sites, with the major site of phosphorylation being serine 10. Replacing serine 10 with alanine causes reduced Puma turnover and enhanced cell death. Interestingly, Puma turnover occurs through the proteasome, and substitution of serine 10 causes elevated Puma levels independently of macroautophagy, Bcl-2 family member binding, caspase activity and apoptotic death. We conclude, therefore, that phosphorylation of Puma at serine 10 promotes Puma turnover, represses Puma's cell death potential and promotes cell survival. Owing to the highly pro-apoptotic nature of Puma, these studies highlight an important additional regulatory step in the determination of cellular life or death

Loss of ubiquitin E2 Ube2w rescues hypersensitivity of Rnf4 mutant cells to DNA damage

SUMO and ubiquitin play important roles in the response of cells to DNA damage. These pathways are linked by the SUMO Targeted ubiquitin Ligase Rnf4 that catalyses transfer of ubiquitin from a ubiquitin loaded E2 conjugating enzyme to a polySUMO modified substrate. Rnf4 can functionally interact with multiple E2s, including Ube2w, in vitro. Chicken cells lacking Rnf4 are hypersensitive to hyroxyurea, DNA alkylating drugs and DNA crosslinking agents, but this sensitivity is suppressed by simultaneous depletion of Ube2w. Cells depleted of Ube2w alone are not hypersensitive to the same DNA damaging agents. Similar results were also obtained in human cells. These data indicate that Ube2w does not have an essential role in the DNA damage response, but is deleterious in the absence of Rnf4. Thus, although Rnf4 and Ube2w functionally interact in vitro, our genetic experiments indicate that in response to DNA damage Ube2w and Rnf4 function in distinct pathways

Bim Nuclear Translocation and Inactivation by Viral Interferon Regulatory Factor

Viral replication efficiency is in large part governed by the ability of viruses to counteract pro-apoptotic signals induced by infection of the host cell. Human herpesvirus 8 (HHV-8) uses several strategies to block the host's innate antiviral defenses via interference with interferon and apoptotic signaling. Contributors include the four viral interferon regulatory factors (vIRFs 1–4), which function in dominant negative fashion to block cellular IRF activities in addition to targeting IRF signaling-induced proteins such as p53 and inhibiting other inducers of apoptosis such as TGFβ receptor-activated Smad transcription factors. Here we identify direct targeting by vIRF-1 of BH3-only pro-apoptotic Bcl-2 family member Bim, a key negative regulator of HHV-8 replication, to effect its inactivation via nuclear translocation. vIRF-1-mediated relocalization of Bim was identified in transfected cells, by both immunofluorescence assay and western analysis of fractionated cell extracts. Also, co-localization of vIRF-1 and Bim was detected in nuclei of lytically infected endothelial cells. In vitro co-precipitation assays using purified vIRF-1 and Bim revealed direct interaction between the proteins, and Bim-binding residues of vIRF-1 were mapped by deletion and point mutagenesis. Generation and experimental utilization of Bim-refractory vIRF-1 variants revealed the importance of vIRF-1:Bim interaction, specifically, in pro-replication and anti-apoptotic activity of vIRF-1. Furthermore, blocking of the interaction with cell-permeable peptide corresponding to the Bim-binding region of vIRF-1 confirmed the relevance of vIRF-1:Bim association to vIRF-1 pro-replication activity. To our knowledge, this is the first report of an IRF protein that interacts with a Bcl-2 family member and of nuclear sequestration of Bim or any other member of the family as a means of inactivation. The data presented reveal a novel mechanism utilized by a virus to control replication-induced apoptosis and suggest that inhibitory targeting of vIRF-1:Bim interaction may provide an effective antiviral strategy

Reemerging superconductivity at 48 K across quantum criticality in iron chalcogenides

Pressure plays an essential role in the induction1 and control2,3 of superconductivity in iron-based superconductors. Substitution of a smaller rare-earth ion for the bigger one to simulate the pressure effects has surprisingly raised the superconducting transition temperature Tc to the record high 55 K in these materials4,5. However, Tc always goes down after passing through a maximum at some pressure and the superconductivity eventually tends to disappear at sufficiently high pressures1-3. Here we show that the superconductivity can reemerge with a much higher Tc after its destruction upon compression from the ambient-condition value of around 31 K in newly discovered iron chalcogenide superconductors. We find that in the second superconducting phase the maximum Tc is as high as 48.7 K for K0.8Fe1.70Se2 and 48 K for (Tl0.6Rb0.4)Fe1.67Se2, setting the new Tc record in chalcogenide superconductors. The presence of the second superconducting phase is proposed to be related to pressure-induced quantum criticality. Our findings point to the potential route to the further achievement of high-Tc superconductivity in iron-based and other superconductors.Comment: 20 pages and 7 figure