Engineering of CRISPR/Cas9‐mediated potyvirus resistance in transgene‐free Arabidopsis plants

Members of the eukaryotic translation initiation factor (eIF) gene family, including eIF4E and its paralogue eIF(iso)4E, have previously been identified as recessive resistance alleles against various potyviruses in a range of different hosts. However, the identification and introgression of these alleles into important crop species is often limited. In this study, we utilise CRISPR/Cas9 technology to introduce sequence‐specific deleterious point mutations at the eIF(iso)4E locus in Arabidopsis thaliana to successfully engineer complete resistance to Turnip mosaic virus (TuMV), a major pathogen in field‐grown vegetable crops. By segregating the induced mutation from the CRISPR/Cas9 transgene, we outline a framework for the production of heritable, homozygous mutations in the transgene‐free T₂ generation in self‐pollinating species. Analysis of dry weights and flowering times for four independent T₃ lines revealed no differences from wild‐type plants under standard growth conditions, suggesting that homozygous mutations in eIF(iso)4E do not affect plant vigour. Thus, the established CRISPR/Cas9 technology provides a new approach for the generation of Potyvirus resistance alleles in important crops without the use of persistent transgenes.     

Biological and computational evaluation of resveratrol inhibitors against Alzheimer’s disease

It has been reported that beta amyloid induces production of radical oxygen species and oxidative stress in neuronal cells, which in turn upregulates β-secretase (BACE-1) expression and beta amyloid levels, thereby propagating oxidative stress and increasing neuronal injury. A series of resveratrol derivatives, known to be inhibitors of oxidative stress-induced neuronal cell death (oxytosis) were biologically evaluated against BACE-1 using homogeneous time-resolved fluorescence (TRF) assay. Correlation between oxytosis inhibitory and BACE-1 inhibitory activity of resveratrol derivatives was statistically significant, supporting the notion that BACE-1 may act as pivotal mediator of neuronal cell oxytosis. Four of the biologically evaluated resveratrol analogs demonstrated considerably higher activity than resveratrol in either assay. The discovery of some “hits” led us to initiate detailed docking studies associated with Molecular Dynamics in order to provide a plausible explanation for the experimental results and understand their molecular basis of action.     

Exploring the unbinding of Leishmania (L.) amazonensis CPB derived‐epitopes from H2 MHC class I proteins

New strategies to control Leishmania disease demand an extensive knowledge about several aspects of infection including the understanding of its molecular events. In murine models, cysteine proteinase B from Leishmania amazonensis promotes regulation of immune response, and fragments from its C‐terminus extension (cyspep) can play a decisive role in the host‐parasite interaction. The interaction between cyspep‐derived peptides and major histocompatibility complex (MHC) proteins is a crucial factor in Leishmania infections. Seven cyspep‐derived peptides, previously identified as capable of interacting with H‐2 (murine) MHC class I proteins, were studied in this work. We established a protocol to simulate the unbinding of these peptides from the cleft of H‐2 receptors. From the simulations, we estimated the corresponding free energy of dissociation (ΔG_d) and described the molecular events that occur during the exit of peptides from the cleft. To test the reliability of this method, we first applied it to a calibration set of four crystallographic MHC/peptide complexes. Next, we explored the unbinding of the seven complexes mentioned above. Results were consistent with ΔG_d values obtained from surface plasmon resonance (SPR) experiments. We also identified some of the primary interactions between peptides and H‐2 receptors, and we detected three regions of influence for the interaction. This pattern was systematically observed for the peptides and helped determine a minimum distance for the real interaction between peptides and H‐2 proteins occurring at ～25 Å. Proteins 2016; 84:473–487. © 2016 Wiley Periodicals, Inc.     

增温和CO2浓度加倍对川西亚高山针叶林土壤可溶性氮的影响

采用全自动微气候控制的"人工模拟气候实验系统"研究了增温和CO2浓度加倍对川西亚高山针叶林土壤硝态氮(NO_3~--N)、铵态氮(NH_4~+-N)、游离氨基酸(FAA)、可溶性有机氮(DON)和可溶性总氮(TSN)的影响。结果表明:1在种植油松苗木组,增温处理显著降低了土壤NO_3~--N含量,不同处理0—15 cm土层NO_3~--N含量均显著小于15—30 cm层;而在未种树组,增温处理显著增加了土壤NO_3~--N含量,0—15 cm土层NO_3~--N含量显著高于15—30 cm层,这表明增温促进了油松苗对NO_3~--N的吸收。2在种植油松苗木组,增温(ET)、增CO_2(EC)及两者的共同作用(ETC)均显著增加了土壤NH_4~+-N、DON和TSN含量;在未种树组,ET显著增加了土壤NH_4~+-N、FAA、DON和TSN含量,EC和ETC对NH_4~+、FAA、DON和TSN含量具有微弱影响或没有显著影响。不同处理0—15cm层土壤NH_4~+-N、FAA、DON和TSN的含量显著大于15—30 cm层。3种植油松苗木组土壤NO_3~--N、NH_4~+-N、FAA、DON和TSN含量均显著低于未种树组,这是由植物对氮素的吸收消耗造成的。研究结果表明,EC、ETC主要通过植物根系作用促进了NH_4~+-N、DON和TSN含量增加,而ET处理通过影响土壤微生物和植物根系来促进NH_4~+-N、FAA、DON和TSN含量的增加。     

Radiation chemical studies of Gly-Met-Gly in aqueous solution

Abstract

Important biological consequences are related to the reaction of HO^? radicals with methionine (Met). Several fundamental aspects remain to be defined when Met is an amino acid residue incorporated in the interior of peptides and proteins. The present study focuses on Gly-Met-Gly, the simplest peptide where Met is not a terminal residue. The reactions of HO^? with Gly-Met-Gly and its N-acetyl derivative were studied by pulse radiolysis technique. The transient absorption spectra were resolved into contributions from specific components of radical intermediates. Moreover, a detailed product analysis is provided for the first time for Met-containing peptides in radiolytic studies to support the mechanistic proposal. By parallel radiolytical and electrochemical reactions and consequent product identification, the formation of sulfoxide attributed to the direct HO^? radical attack on the sulfide functionality of the Met residue could be excluded, with the in situ generated hydrogen peroxide responsible for this oxidation. LC–MS and high resolution MS/MS were powerful analytical tools to envisage the structures of five products, thus allowing to complete the mechanistic picture of the overall Met-containing peptide reactivity.     

Structure Evolution of Oligomer Fused‐Ring Electron Acceptors toward High Efficiency of As‐Cast Polymer Solar Cells

The structure evolution of oligomer fused‐ring electron acceptors (FREAs) toward high efficiency of as‐cast polymer solar cells (PSCs) is reported. First, a series of FREAs (IC‐(1‐3)IDT‐IC) based on indacenodithiophene (IDT) oligomers as cores are designed and synthesized, effects of IDT number (1–3) on their basic optical and electronic properties are investigated, and more importantly, the relationship between device performance of as‐cast PSCs and donor(D)/acceptor(A) matching (absorption, energy level, morphology, and charge transport) of IC‐(1‐3)IDT‐IC acceptors and two representative polymer donors, PTB7‐Th and PDBT‐T1 is surveyed. Then, the most promising D/A system (PDBT‐T1/IC‐1IDT‐IC) with the best D/A harmony among the six D/A combinations, which yields a power conversion efficiency (PCE) of 7.39%, is found. Finally, changing the side‐chains in IC‐1IDT‐IC from alkylphenyl to alkyl enhances the PCE from 7.39% to 9.20%.     

Identification of glycoproteins associated with HIV latently infected cells using quantitative glycoproteomics

HIV infection is not curable due to viral latency. Compelling reports suggest that there is a distinct profile of surface proteins that can be used for targeting latently infected cells. We have recently reported that glycoproteins were differentially secreted from HIV latently infected ACH‐2 cells compared to the parental A3.01 cells. This finding suggests that glyco‐phenotype might be different in these two cell lines. To determine the difference, the ACH‐2 and A3.01 cell lines were subjected to a glycoproteomic analysis. A total number of 940 unique N‐linked glycosite‐containing peptides from 515 glycoproteins were identified. Among the glycoproteins, 365 and 104 were annotated as cell surface and membrane‐associated proteins, respectively. Quantitative LC‐MS/MS analysis revealed a change of 236 glycosite‐containing peptides from 172 glycoproteins between the two cell lines without reactivation. Bioinformatic analysis suggests that cell adhesion, immune response, glycoprotein metabolic process, cell motion, and cell activation were associated with the changed proteins. After reactivation of latency, changes in glycosite‐containing peptides were observed in both cell lines. The changed proteins suggest that cell migration, response to wounding and immune response might be impaired in reactivated latently infected cells. Glycoproteomics merits future application using primary cells to discover reveal mechanisms in HIV pathogenesis.     

Breakdown of Galactolipids in Senescent Barly Leaves

The changes of galactolipids (MGDG and DGDG, largely 18:3/18:3), free fatty acids (FFA), and phosphatidylcholine (PC) taking place during senescence of primary barley leaves were analysed employing HPLC and GLC. Upon induction of senescence MGDG and, with some delay, DGDG began to disappear and were largely broken down at the end of the senescence period. A concomitant appearance of a pool of FFA could not be observed. However, PC accumulated during the main period of galactolipid breakdown. This change was due to the marked increase of the 18:3/18:3 molecular species of PC. An inverse correlation between the changes of galactolipids and PC could be established. A hypothesis featuring the conversion of galactolipids via diacylglycerol to PC is presented as the principal route of galactolipid breakdown.