Development of intron targeted amplified polymorphic markers of metal homeostasis genes for monitoring their introgression from <Emphasis Type="Italic">Aegilops</Emphasis> species to wheat

The identification of transfers of useful alien genes for metal homeostasis from non-progenitor Aegilops species using the widely available anchored wheat SSR markers is difficult due to their lower polymorphism with the distant related wild species and the lack of locus specificity further restricts their application. The present study deals with the development of intron targeted amplified polymorphic (ITAP) markers for the metal homeostasis genes present on chromosomes of groups 2 and 7 of Triticeae. The mRNA sequences of 27 metal homeostasis genes were retrieved from different plant species using NCBI database and their BLASTn was performed against the wheat draft genome sequences in Ensemblplants to get exonic and intronic sequences of the corresponding metal homeostasis genes in wheat. The ITAP primers were developed in such a way that they would anneal to the conserved flanking exonic regions of the genes and amplify across highly variable introns within the PCR limits. The primers led to the amplification of variable intronic sequences of genes with polymorphism between non-progenitor Aegilops species and the recipient wheat cultivars. Further, the polymorphic ITAP markers were used to characterize the transfers of metal homeostasis genes from the non-progenitor Aegilops species to the BC₂F₅ wheat-Aegilops derivatives, developed through induced homoeologous pairing. The derivatives with significant percent increase in grain Fe and Zn content over the elite cultivar PBW343 LrP showed the introgression of some of the useful Aegilops alleles of the metal homeostasis genes. The use of different metal homeostasis genes using this approach is the first report of the direct contribution of the genes for increasing the grain micronutrient content for developing biofortified wheat lines with reduced linkage drag.     

Aptamer-based multiplexed proteomic technology for biomarker discovery

Background

The interrogation of proteomes (“proteomics”) in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine.

Methodology/Principal Findings

We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (∼100 fM–1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states.

Conclusions/Significance

We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.     

Screening,statistical optimized production,and application of β‐mannanase from some newly isolated fungi

Eighty‐eight fungi isolated from soil and decaying organic matter were screened for mannanolytic activity. Twenty‐eight fungi produced extracellular mannanase on locust bean gum as evidenced by zone of hydrolysis produced on mannan agar gel. Six prominent producers, including four Fusarium species namely Fusarium fusarioides NFCCI 3282, Fusarium solani NFCCI 3283, Fusarium equiseti NFCCI 3284, Fusarium moniliforme NFCCI 3287 with Cladosporium cladosporioides NFCCI 3285 and Acrophialophora levis NFCCI 3286 produced the β‐mannanase in the range of 84–140 nkat/mL. All these grew well on particulate substrates in solid‐state fermentation (SSF), producing relatively higher titers on mannan‐rich palm kernel cake (PKC) and copra meal. Two high yielding strains, F. equiseti (1747 nkat/gds) and A. levis (897 nkat/gds) were selected for statistical optimization of mannanase on PKC. Interaction of two critical solid state fermentation parameters, pH and moisture on mannanase production by these two molds was studied by response surface method. Optimized production on PKC resulted in three‐ to fourfold enhancement in enzyme yield was observed in case of F. equiseti (5945 nkat/gds) and A. levis (4726 nkat/gds). HPLC analysis of mannan hydrolysate indicated that F. equiseti and A. levis mannanase performed efficient hydrolysis of konjac gum (up to 99%) with exclusive mannooligosaccahride (DP of 4) production. A seminative SDS‐PAGE revealed that A. levis and F. solani produced three isoforms, F. moniliforme produced two isoforms while F. fusarioides, F. equiseti, and C. cladosporioides produced a single enzyme.     

Two chimeric regulators of G-protein signaling (RGS) proteins differentially modulate soybean heterotrimeric G-protein cycle

Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gβ, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1-4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1-4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks.     

Supercritical carbon dioxide pretreatment of corn stover and switchgrass for lignocellulosic ethanol production

Supercritical CO₂ (SC-CO₂), a green solvent suitable for a mobile lignocellulosic biomass processor, was used to pretreat corn stover and switchgrass at various temperatures and pressures. The CO₂ pressure was released as quickly as possible by opening a quick release valve during the pretreatment. The biomass was hydrolyzed after pretreatment using cellulase combined with β-glucosidase. The hydrolysate was analyzed for the amount of glucose released. Glucose yields from corn stover samples pretreated with SC-CO₂ were higher than the untreated sample’s 12% glucose yield (12 g/100 g dry biomass) and the highest glucose yield of 30% was achieved with SC-CO₂ pretreatment at 3500 psi and 150 °C for 60 min. The pretreatment method showed very limited improvement (14% vs. 12%) in glucose yield for switchgrass. X-ray diffraction results indicated no change in crystallinity of the SC-CO₂ treated corn stover when compared to the untreated, while SEM images showed an increase in surface area.     

Phosphocaveolin-1 is a mechanotransducer that induces caveola biogenesis via Egr1 transcriptional regulation

Maintenance of epithelial cell adhesion is crucial for epidermal morphogenesis and homeostasis and relies predominantly on the interaction of keratins with desmosomes. Although the importance of desmosomes to epidermal coherence and keratin organization is well established, the significance of keratins in desmosome organization has not been fully resolved. Here, we report that keratinocytes lacking all keratins show elevated, PKC-α–mediated desmoplakin phosphorylation and subsequent destabilization of desmosomes. We find that PKC-α activity is regulated by Rack1–keratin interaction. Without keratins, desmosomes assemble but are endocytosed at accelerated rates, rendering epithelial sheets highly susceptible to mechanical stress. Re-expression of the keratin pair K5/14, inhibition of PKC-α activity, or blocking of endocytosis reconstituted both desmosome localization at the plasma membrane and epithelial adhesion. Our findings identify a hitherto unknown mechanism by which keratins control intercellular adhesion, with potential implications for tumor invasion and keratinopathies, settings in which diminished cell adhesion facilitates tissue fragility and neoplastic growth.     

BRASSINOSTEROID-SIGNALLING KINASES 7 and 8 associate with the FLS2 immune receptor and are required for flg22-induced PTI responses

Pattern-triggered immunity (PTI) is typically initiated in plants by recognition of pathogen- or damage-associated molecular patterns (PAMP/DAMPs) by cell surface-localized pattern recognition receptors (PRRs). Here, we investigated the role in PTI of Arabidopsis thaliana brassinosteroid-signalling kinases 7 and 8 (BSK7 and BSK8), which are members of the receptor-like cytoplasmic kinase subfamily XII. BSK7 and BSK8 localized to the plant cell periphery and interacted in yeast and in planta with FLS2, but not with other PRRs. Consistent with a role in FLS2 signalling, bsk7 and bsk8 single and bsk7,8 double mutant plants were impaired in several immune responses induced by flg22, but not by other PAMP/DAMPs. These included resistance to Pseudomonas syringae and Botrytis cinerea, reactive oxygen species accumulation, callose deposition at the cell wall, and expression of the defence-related gene PR1, but not activation of MAP kinases and expression of the FRK1 and WRKY29 genes. bsk7, bsk8, and bsk7,8 plants also displayed enhanced susceptibility to P. syringae and B. cinerea. Finally, BSK7 and BSK8 variants mutated in their myristoylation site or in the ATP-binding site failed to complement defective phenotypes of the corresponding mutants, suggesting that localization to the cell periphery and kinase activity are critical for BSK7 and BSK8 functions. Together, these findings demonstrate that BSK7 and BSK8 play a role in PTI initiated by recognition of flg22 by interacting with the FLS2 immune receptor.     

Cyclooxygenase (COX)-2 inhibitor celecoxib abrogates TNF-induced NF-kappa B activation through inhibition of activation of I kappa B alpha kinase and Akt in human non-small cell lung carcinoma: correlation with suppression of COX-2 synthesis

The cyclooxygenase 2 (COX-2) inhibitor celecoxib (also called celebrex), approved for the treatment of colon carcinogenesis, rheumatoid arthritis, and other inflammatory diseases, has been shown to induce apoptosis and inhibit angiogenesis. Because NF-kappa B plays a major role in regulation of apoptosis, angiogenesis, carcinogenesis, and inflammation, we postulated that celecoxib modulates NF-kappa B. In the present study, we investigated the effect of this drug on the activation of NF-kappa B by a wide variety of agents. We found that celecoxib suppressed NF-kappa B activation induced by various carcinogens, including TNF, phorbol ester, okadaic acid, LPS, and IL-1 beta. Celecoxib inhibited TNF-induced I kappa B alpha kinase activation, leading to suppression of I kappa B alpha phosphorylation and degradation. Celecoxib suppressed both inducible and constitutive NF-kappa B without cell type specificity. Celecoxib also suppressed p65 phosphorylation and nuclear translocation. Akt activation, which is required for TNF-induced NF-kappa B activation, was also suppressed by this drug. Celecoxib also inhibited the TNF-induced interaction of Akt with I kappa B alpha kinase (IKK). Celecoxib abrogated the NF-kappa B-dependent reporter gene expression activated by TNF, TNF receptor, TNF receptor-associated death domain, TNF receptor-associated factor 2, NF-kappa B-inducing kinase, and IKK, but not that activated by p65. The COX-2 promoter, which is regulated by NF-kappa B, was also inhibited by celecoxib, and this inhibition correlated with suppression of TNF-induced COX-2 expression. Besides NF-kappa B, celecoxib also suppressed TNF-induced JNK, p38 MAPK, and ERK activation. Thus, overall, our results indicate that celecoxib inhibits NF-kappa B activation through inhibition of IKK and Akt activation, leading to down-regulation of synthesis of COX-2 and other genes needed for inflammation, proliferation, and carcinogenesis.     

Emerging Insights into Noncanonical Inflammasome Recognition of Microbes

Inflammasomes are cytosolic multi-molecular complexes that sense intracellular microbial danger signals and metabolic perturbations. Inflammasome activation leads to the activation of caspase-1 and the release of pro-inflammatory cytokines IL-1β and IL-18 accompanied by cell death. An inflammasome-based surveillance machinery for Gram-negative bacterial infections has been recently discovered. This noncanonical inflammasome relies on sensing the cytosolic presence of lipopolysaccharide of Gram-negative bacteria via inflammatory caspases such as caspase-4, -5, and -11. This review discusses the recent findings related to the mechanism of activation of the noncanonical inflammasome and its biological functions.     

Pharmacological and Genetic Targeting of the PI4KA Enzyme Reveals Its Important Role in Maintaining Plasma Membrane Phosphatidylinositol 4-Phosphate and Phosphatidylinositol 4,5-Bisphosphate Levels

Phosphatidylinositol 4-kinase type IIIα (PI4KA) is a host factor essential for hepatitis C virus replication and hence is a target for drug development. PI4KA has also been linked to endoplasmic reticulum exit sites and generation of plasma membrane phosphoinositides. Here, we developed highly specific and potent inhibitors of PI4KA and conditional knock-out mice to study the importance of this enzyme in vitro and in vivo. Our studies showed that PI4KA is essential for the maintenance of plasma membrane phosphatidylinositol 4,5-bisphosphate pools but only during strong stimulation of receptors coupled to phospholipase C activation. Pharmacological blockade of PI4KA in adult animals leads to sudden death closely correlating with the drug''s ability to induce phosphatidylinositol 4,5-bisphosphate depletion after agonist stimulation. Genetic inactivation of PI4KA also leads to death; however, the cause in this case is due to severe intestinal necrosis. These studies highlight the risks of targeting PI4KA as an anti-hepatitis C virus strategy and also point to important distinctions between genetic and pharmacological studies when selecting host factors as putative therapeutic targets.