BSKs are partially redundant positive regulators of brassinosteroid signaling in Arabidopsis

Arabidopsis thaliana brassinosteroid signaling kinases (BSKs) constitute a receptor‐like cytoplasmic kinase sub‐family (RLCK‐XII) with 12 members. Previous analysis demonstrated a positive role for BSK1 and BSK3 in the initial steps of brassinosteroid (BR) signal transduction. To investigate the function of BSKs in plant growth and BR signaling, we characterized T‐DNA insertion lines for eight BSK genes (BSK1–BSK8) and multiple mutant combinations. Simultaneous elimination of three BSK genes caused alterations in growth and the BR response, and the most severe phenotypes were observed in the bsk3,4,7,8 quadruple and bsk3,4,6,7,8 pentuple mutants, which displayed reduced rosette size, leaf curling and enhanced leaf inclination. In addition, upon treatment with 24‐epibrassinolide, these mutants showed reduced hypocotyl elongation, enhanced root growth and alteration in the expression of BR‐responsive genes. Some mutant combinations also showed antagonistic interactions. In support of a redundant function in BR signaling, multiple BSKs interacted in vivo with the BR receptor BRI1, and served as its phosphorylation substrates in vitro. The BIN2 and BIL2 GSK3‐like kinases, which are negative regulators of BR signaling, interacted in vivo with BSKs and phosphorylated them in vitro, probably at different sites to BRI1. This study demonstrates redundant biological functions for BSKs, and suggests the existence of a regulatory link between BSKs and GSK3‐like kinases.     

A Vaccine That Co-Targets Tumor Cells and Cancer Associated Fibroblasts Results in Enhanced Antitumor Activity by Inducing Antigen Spreading

Dendritic cell (DC) vaccines targeting only cancer cells have produced limited antitumor activity in most clinical studies. Targeting cancer-associated fibroblasts (CAFs) in addition to cancer cells may enhance antitumor effects, since CAFs, the central component of the tumor stroma, directly support tumor growth and contribute to the immunosuppressive tumor microenvironment. To co-target CAFs and tumor cells we developed a new compound DC vaccine that encodes an A20-specific shRNA to enhance DC function, and targets fibroblast activation protein (FAP) expressed in CAFs and the tumor antigen tyrosine-related protein (TRP)2 (DC-shA20-FAP-TRP2). DC-shA20-FAP-TRP2 vaccination induced robust FAP- and TRP2-specific T-cell responses, resulting in greater antitumor activity in the B16 melanoma model in comparison to monovalent vaccines or a vaccine encoding antigens and a control shRNA. DC-shA20-FAP-TRP2 vaccination enhanced tumor infiltration of CD8-positive T cells, and induced antigen-spreading resulting in potent antitumor activity. Thus, co-targeting of tumor cells and CAFs results in the induction of broad-based tumor-specific T-cell responses and has the potential to improve current vaccine approaches for cancer.     

Xanthomonas euvesicatoria type III effector XopQ interacts with tomato and pepper 14–3–3 isoforms to suppress effector‐triggered immunity

Effector‐triggered immunity (ETI) to host‐adapted pathogens is associated with rapid cell death at the infection site. The plant‐pathogenic bacterium Xanthomonas euvesicatoria (Xcv) interferes with plant cellular processes by injecting effector proteins into host cells through the type III secretion system. Here, we show that the Xcv effector XopQ suppresses cell death induced by components of the ETI‐associated MAP kinase cascade MAPKKKα MEK2/SIPK and by several R/avr gene pairs. Inactivation of xopQ by insertional mutagenesis revealed that this effector inhibits ETI‐associated cell death induced by avirulent Xcv in resistant pepper (Capsicum annuum), and enhances bacterial growth in resistant pepper and tomato (Solanum lycopersicum). Using protein–protein interaction studies in yeast (Saccharomyces cerevisiae) and in planta, we identified the tomato 14–3–3 isoform SlTFT4 and homologs from other plant species as XopQ interactors. A mutation in the putative 14–3–3 binding site of XopQ impaired interaction of the effector with CaTFT4 in yeast and its virulence function in planta. Consistent with a role in ETI, TFT4 mRNA abundance increased during the incompatible interaction of tomato and pepper with Xcv. Silencing of NbTFT4 in Nicotiana benthamiana significantly reduced cell death induced by MAPKKKα. In addition, silencing of CaTFT4 in pepper delayed the appearance of ETI‐associated cell death and enhanced growth of virulent and avirulent Xcv, demonstrating the requirement of TFT4 for plant immunity to Xcv. Our results suggest that the XopQ virulence function is to suppress ETI and immunity‐associated cell death by interacting with TFT4, which is an important component of ETI and a bona fide target of XopQ.     

Extraction of Y2O3:Cr3+ nanophosphor by eco‐friendly approach and its suitability for white light‐emitting diode applications

Cr³⁺‐doped Y₂O₃ (0.5–9 mol%) was synthesized by a simple solution combustion method using Aloe vera gel as a fuel/surfactant. The final obtained product was calcined at 750°C for 3 h, which is the lowest temperature reported so far for the synthesis of this compound. The calcined product was confirmed for its crystallinity and purity by powder X‐ray diffraction (PXRD) studies which showed a single‐phase nano cubic phosphor. The particles size estimated by Scherrer formula was in the range of 6–19 nm. The UV–vis spectra showed absorption bands at 198, 272 and 372 nm having band gap energy in the range 4.00–4.26 eV. In order to investigate the possibility of its use in white light emitting display applications, the photoluminescence properties of Cr³⁺‐doped Y₂O₃ nanophosphors were studied at an excitation wavelength in the near ultraviolet (UV) light region (361 nm). The emission spectra consisted of emission peaks in the blue (⁴F_9/2 → ⁶H_15/2), orange (⁴F_9/2 → ⁶H_13/2) and red (⁴F_9/2 → ⁶H_11/2) regions. The CIE coordinates (0.33, 0.33) lie in the white light region. Hence Y₂O₃:Cr³⁺ can be used for white light‐emitting diode (LED) applications.     

Defining process design space for monoclonal antibody cell culture

The concept of design space has been taking root as a foundation of in‐process control strategies for biopharmaceutical manufacturing processes. During mapping of the process design space, the multidimensional combination of operational variables is studied to quantify the impact on process performance in terms of productivity and product quality. An efficient methodology to map the design space for a monoclonal antibody cell culture process is described. A failure modes and effects analysis (FMEA) was used as the basis for the process characterization exercise. This was followed by an integrated study of the inoculum stage of the process which includes progressive shake flask and seed bioreactor steps. The operating conditions for the seed bioreactor were studied in an integrated fashion with the production bioreactor using a two stage design of experiments (DOE) methodology to enable optimization of operating conditions. A two level Resolution IV design was followed by a central composite design (CCD). These experiments enabled identification of the edge of failure and classification of the operational parameters as non‐key, key or critical. In addition, the models generated from the data provide further insight into balancing productivity of the cell culture process with product quality considerations. Finally, process and product‐related impurity clearance was evaluated by studies linking the upstream process with downstream purification. Production bioreactor parameters that directly influence antibody charge variants and glycosylation in CHO systems were identified. Biotechnol. Bioeng. 2010;106: 894–905. © 2010 Wiley Periodicals, Inc.     

Cation exchange as a single polishing step for conjugated peptides

Purification of peptides typically includes expensive reverse phase (RP) processes, which utilize high pressure and large volumes of solvent. For two conjugated peptides, chromatography process development targeted a low-pressure aqueous process that could achieve target product purities of ≥95%, comparable to purities seen with traditional RP. A high throughput screening approach of different modalities was used to identify binding and elution conditions on a cation exchange resin and small-scale columns were used in order to assess impurity removal and process yield. The parameters for load and gradient elution were optimized to increase product purity and process productivity with a wide operating window identified where high purity and productivity are achieved. Computational modeling was then used to validate experimental chromatography results and to gain insight on the effect of the chemical modifications on the surface properties of the two peptides. Both modeling and experimental data showed that with optimization, cation exchange could be utilized as a single polishing step for conjugated peptides. Similar purities were achieved as those seen with RP with up to double the productivity.     

Antiarthritic and antiinflammatory propensity of 4-methylesculetin,a coumarin derivative

Coumarins are a group of natural compounds widely distributed in plants. Of late, coumarins and their derivatives have grabbed much attention from the pharmacological and pharmaceutical arena due to their broad range of therapeutical qualities. A coumarin derivative 4-methylesculetin (4-ME) has known to possess effective antioxidant and radical-scavenging properties. Recently they have also shown to down regulate nuclear factor-kappa B (NF-κB) and protein kinase B (Akt) that play a vital role in inflammation and apoptosis. In view of this, the present study investigated the anti-arthritic potentiality of 4-ME by assessing its ability to inhibit cartilage and bone degeneration, inflammation and associated oxidative stress. Arthritis being a debilitating joint disease, results in the deterioration of extracellular matrix (ECM) of cartilage and synovium. Participation of both enzymatic and non-enzymatic factors in disease perpetuation is well documented. The present study demonstrated the mitigation of augmented serum levels of hyaluronidase and matrix metalloproteinases (MMP-13, MMP-3 and MMP-9) responsible for cartilage degeneration by 4-ME. It also protected bone resorption by reducing the elevated levels of bone-joint exoglycosidases, cathepsin-D and tartrate resistant acid phosphatases. Further, 4-ME significantly ameliorated the upregulated non-enzymatic inflammatory markers like TNF-α, IL-1β, IL-6, COX-2 and PGE₂. Besides, 4-ME effectively stabilized the arthritis-induced oxidative stress by restoring the levels of reactive oxygen species, lipid and hydro peroxides and antioxidant enzymes such as superoxide dismutase, catalase and glutathione-S-transferase. Thus, the study suggests that 4-ME could be an effective agent to treat arthritis and associated secondary complications like oxidative stress.     

Clinical applications of breast cancer metabolomics using high-resolution magic angle spinning proton magnetic resonance spectroscopy (HRMAS 1H MRS): systematic scoping review

Metabolomics - Oral microflora is a well-orchestrated and acts as a sequential defense mechanism for any infection related to oral disease. Chronic periodontitis is a disease of a microbial...     

Atypical antidepressants extend lifespan of Caenorhabditis elegans by activation of a non‐cell‐autonomous stress response

Oxidative stress has long been associated with aging and has recently been linked to psychiatric disorders, including psychosis and depression. We identified multiple antipsychotics and antidepressants that extend Caenorhabditis elegans lifespan and protect the animal from oxidative stress. Here, we report that atypical antidepressants activate a neuronal mechanism that regulates the response to oxidative stress throughout the animal. While the activation of the oxidative stress response by atypical antidepressants depends on synaptic transmission, the activation by reactive oxygen species does not. Lifespan extension by atypical antidepressants depends on the neuronal oxidative stress response activation mechanism. Neuronal regulation of the oxidative stress response is likely to have evolved as a survival mechanism to protect the organism from oxidative stress, upon detection of adverse or dangerous conditions by the nervous system.