Targeting oncogenic transcription factors by polyphenols: A novel approach for cancer therapy.

Inflammation is one of the major causative factor of cancer and chronic inflammation is involved in all the major steps of cancer initiation, progression metastasis and drug resistance. The molecular mechanism of inflammation driven cancer is the complex interplay between oncogenic and tumor suppressive transcription factors which include FOXM1, NF-kB, STAT3, Wnt/ß- Catenin, HIF-1α, NRF2, androgen and estrogen receptors. Several products derived from natural sources modulate the expression and activity of multiple transcription factors in various tumor models as evident from studies conducted in cell lines, pre-clinical models and clinical samples. Further combination of these natural products along with currently approved cancer therapies added an additional advantage and they considered as promising targets for prevention and treatment of inflammation and cancer. In this review we discuss the application of multi-targeting natural products by analyzing the literature and future directions for their plausible applications in drug discovery.

Signaling from the secretory granule to the nucleus.

Neurons and endocrine cells use a complex array of signaling molecules to communicate with each other and with various targets. The majority of these signaling molecules are stored in specialized organelles awaiting release on demand: 40-60 nm vesicles carry conventional or small molecule neurotransmitters, and 200-400 nm granules contain bioactive peptides. The supply of small molecule neurotransmitters is tightly regulated by local feedback of synthetic rates and transport processes at sites of release. The larger granules that contain bioactive peptides present the secretory cell with special challenges, as the peptide precursors are inserted into the lumen of the secretory pathway in the cell soma and undergo biosynthetic processing while being transported to distant sites for eventual secretion. One solution to this dilemma in information handling has been to employ proteolytic cleavage of secretory granule membrane proteins to produce cytosolic fragments that can signal to the nucleus, affecting gene expression. The use of regulated intramembrane proteolysis to signal from secretory granules to the nucleus is compared to its much better understood role in relaying information from the endoplasmic reticulum by SREBP and ATF6 and from the plasma membrane by cadherins, Notch and ErbB4.

Secretory granule membrane protein recycles through multivesicular bodies.

The recycling of secretory granule membrane proteins that reach the plasma membrane following exocytosis is poorly understood. As a model, peptidylglycine alpha-amidating monooxygenase (PAM), a granule membrane protein that catalyzes a final step in peptide processing was examined. Ultrastructural analysis of antibody internalized by PAM and surface biotinylation showed efficient return of plasma membrane PAM to secretory granules. Electron microscopy revealed the rapid movement of PAM from early endosomes to the limiting membranes of multivesicular bodies and then into intralumenal vesicles. Wheat germ agglutinin and PAM antibody internalized simultaneously were largely segregated when they reached multivesicular bodies. Mutation of basally phosphorylated residues (Thr(946), Ser(949)) in the cytoplasmic domain of PAM to Asp (TS/DD) substantially slowed its entry into intralumenal vesicles. Mutation of the same sites to Ala (TS/AA) facilitated the entry of internalized PAM into intralumenal vesicles and its subsequent return to secretory granules. Entry of PAM into intralumenal vesicles is also associated with a juxtamembrane endoproteolytic cleavage that releases a 100-kDa soluble PAM fragment that can be returned to secretory granules. Controlled entry into the intralumenal vesicles of multivesicular bodies plays a key role in the recycling of secretory granule membrane proteins.

Secretion stimulates intramembrane proteolysis of a secretory granule membrane enzyme.

Regulated intramembrane proteolysis, a highly conserved process employed by diverse regulatory pathways, can release soluble fragments that directly or indirectly modulate gene expression. In this study we used pharmacological tools to identify peptidylglycine α-amidating monooxygenase (PAM), a type I secretory granule membrane protein, as a γ-secretase substrate. PAM, an essential enzyme, catalyzes the final step in the synthesis of the majority of neuropeptides that control metabolic homeostasis. Mass spectroscopy was most consistent with the presence of multiple closely spaced NH(2) termini, suggesting that cleavage occurred near the middle of the PAM transmembrane domain. The luminal domains of PAM must undergo a series of prohormone convertase or α-secretase-mediated cleavages before the remaining transmembrane domain/cytosolic domain fragment can undergo a γ-secretase-like cleavage. Cleavage by γ-secretase generates a soluble fragment of the cytosolic domain (sf-CD) that is known to localize to the nucleus. Although PAM sf-CD is unstable in AtT-20 corticotroph tumor cells, it is readily detected in primary rat anterior pituitary cells. PAM isoform expression, which is tissue-specific and developmentally regulated, affects the efficiency with which sf-CD is produced. sf-CD levels are also modulated by the phosphorylation status of the cytosolic domain and by the ability of the cytosolic domain to interact with cytosolic proteins. sf-CD is produced by primary rat anterior pituitary cells in response to secretogogue, suggesting that sf-CD acts as a signaling molecule relaying information about secretion from the secretory granule to the nucleus.

Secretory granule to the nucleus: role of a multiply phosphorylated intrinsically unstructured domain.

Intrinsically unstructured domains occur in one-third of all proteins and are characterized by conformational flexibility, protease sensitivity, and the occurrence of multiple phosphorylation. They provide large interfaces for diverse protein-protein interactions. Peptidylglycine alpha-amidating monooxygenase (PAM), an enzyme essential for neuropeptide biosynthesis, is a secretory granule membrane protein. As one of the few proteins spanning the granule membrane, PAM is a candidate to relay information about the status of the granule pool and conditions in the granule lumen. Here, we show that the PAM cytosolic domain is unstructured. Mass spectroscopy and two-dimensional gel electrophoresis demonstrated phosphorylation at 10-12 sites in the cytosolic domain. Stimulation of exocytosis resulted in coupled phosphorylation and dephosphorylation of specific sites and in the endoproteolytic release of a soluble, proteasome-sensitive cytosolic domain fragment. Analysis of granule-rich tissues, such as pituitary and heart, showed that a similar fragment was generated endogenously and translocated to the nucleus. This multiply phosphorylated unstructured domain may act as a signaling molecule that relays information from secretory granules to both cytosol and nucleus.

Adsorptive removal of heavy metals from aqueous solution by treated sawdust (Acacia arabica).

The removal of Cr(VI), Pb(II), Hg(II) and Cu(II), by treated sawdust has been found to be concentration, pH, contact time, adsorbent dose and temperature dependent. The adsorption parameters were determined using both Langmuir and Freundlich isotherm models. Adsorption capacity for treated sawdust, i.e. Cr(VI) (111.61 mg/g), Pb(II) (52.38 mg/g), Hg(II) (20.62 mg/g), and Cu(II) (5.64 mg/g), respectively. Surface complexation and ion exchange are the major removal mechanisms involved. The adsorption isotherm studies clearly indicated that the adsorptive behaviour of metal ions on treated sawdust satisfies not only the Langmuir assumptions but also the Freundlich assumptions. The applicability of Lagergren kinetic model has also been investigated. The adsorption follows first-order kinetics. Thermodynamic constant (k(ad)), standard free energy (DeltaG degrees ), enthalpy (DeltaH degrees ) and entropy (DeltaS degrees ) were calculated for predicting the nature of adsorption. The percentage adsorption increases with pH to attain a maximum at pH 6 and thereafter it decreases with further increase in pH. The results indicate the potential application of this method for effluent treatment in industries and also provide strong evidence to support the adsorption mechanism proposed.

Adsorption of Pb(II) and Cd(II) metal ions from aqueous solutions by mustard husk.

The adsorption of Pb(II) and Cd(II) metal ions on mustard husk has been found to be concentration, pH, contact time, adsorbent dose and temperature dependent. The adsorption parameters were determined using Langmuir and Freundlich isotherm models. The adsorption isotherm studies clearly indicated that the adsorptive behavior of Pb(II) and Cd(II) metal ions on mustard husk satisfies not only the Langmuir assumptions but also the Freundlich assumptions, i.e. multilayer formation on the surface of the adsorbent with an exponential distribution of site energy. Ion exchange and surface complexation are the major adsorption mechanisms involved. The applicability of Lagergren kinetic model has also been investigated. Thermodynamic constant (k(ad)), free energy change (DeltaG), enthalpy change (DeltaH) and entropy change (DeltaS) were calculated for predicting the nature of adsorption. The results indicate the potential application of this method for effluent treatment in industries and also provide strong evidence to support the adsorption mechanism proposed.

The Origin and Functions of Exosomes in Cancer.

Exosomes are nanovesicles having a maximum size of 150 nm and is a newly emerging focus in various fields of research. Its role in cargo trafficking along with its differential expression is associated with the disrupted homeostasis and provides an opportunity to defend against different diseases like cancer. Furthermore, exosomes are rich in cargos, which contain proteins and nucleic acids that directly reflect the metabolic state of the cells from which it originates. This review summarizes recent studies on tumor-derived exosomes with an overview about biogenesis, their functions and potential of using as diagnostic and prognostic markers. We also discussed the current challenges and microfluidic-based detection approaches that might improve the detection of exosomes in different settings. More intricate studies of the molecular mechanisms in angiogenesis, pre-metastatic niche formation, and metastasis can give more promising insights and novel strategies in oncotherapeutics.

Rational library design by functional CDR resampling.

Successful antibody discovery relies on diversified libraries, where two aspects are implied, namely the absolute number of unique clones and the percentage of functional clones. Instead of pursuing the absolute quantity thresholded by current display technology, we have sought to maximize the effective diversity by improving functional clone percentage. With the combined effort of bioinformatics, structural biology, molecular immunology and phage display technology, we devised a bioinformatic pipeline to construct and validate libraries via combinatorial assembly of sequences from a database of experimentally validated antibodies. Furthermore, we showed that the libraries constructed as such yielded a significantly increased success rate against different antigen types and generated over 20-fold more unique hits per targets compared with libraries based on traditional degenerate nucleotide methods. Our study indicated that predefined CDR sequences with optimized CDR-framework compatibility could be a productive direction of functional library construction for in vitro antibody development.

Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells.

In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL.

Removal of lead(II) by adsorption using treated granular activated carbon: batch and column studies.

In the present study, a deeper understanding of adsorption behavior of Pb(II) from aqueous systems onto activated carbon and treated activated carbon has been attempted via static and column mode studies under various conditions. It probes mainly two adsorbents that is, activated carbon (AC) and modified activated carbon (AC-S). Characterization of both the adsorbents was one of the key focal areas of the present study. This has shown a clear change or demarcation in the various physical and chemical properties of the modified adsorbent from its precursor activated carbon. Both the adsorbents are subjected to static mode adsorption studies and then after a comparison based on isotherm analysis; more efficient adsorbent is screened for column mode adsorption studies. The lead removal increased for sample of treated carbon. The extent of Pb(II) removal was found to be higher in the treated activated carbon. The aim of carrying out the continuous-flow studies was to assess the effect of various process variables, viz., of bed height, hydraulic loading rate and initial feed concentration on breakthrough time and adsorption capacity. This has helped in ascertaining the practical applicability of the adsorbent. Breakthrough curves were plotted for the adsorption of lead on the adsorbent using continuous-flow column operation by varying different operating parameters like hydraulic loading rate (3.0-10.5 m3/(hm2)), bed height (0.3-0.5 m) and feed concentrations (2.0-6.0 mg/l). At the end, an attempt has also been made to model the data generated from column studies using the empirical relationship based on Bohart-Adams model. This model has provided an objective framework to the subjective interpretation of the adsorption system and the model constant obtained here can be used to achieve the ultimate objective of our study that is, up scaling and designing of adsorption process at the pilot plant scale level. AC-S column regeneration using 0.5 and 1.0M concentration of HNO3 has been investigated. It has shown a regeneration efficiency of 52.0% with 0.5 M HNO3.

Removal of heavy metal ions from aqueous solutions using carbon aerogel as an adsorbent.

The removal of Cd(II), Pb(II), Hg(II), Cu(II), Ni(II), Mn(II) and Zn(II) by carbon aerogel has been found to be concentration, pH, contact time, adsorbent dose and temperature dependent. The adsorption parameters were determined using both Langmuir and Freundlich isotherm models. Surface complexation and ion exchange are the major removal mechanisms involved. The adsorption isotherm studies clearly indicated that the adsorptive behaviour of metal ions on carbon aerogel satisfies not only the Langmuir assumptions but also the Freundlich assumptions, i.e. multilayer formation on the surface of the adsorbent with an exponential distribution of site energy. The applicability of the Lagergren kinetic model has also been investigated. Thermodynamic constant (K(ad)), standard free energy (DeltaG(0)), enthalpy (DeltaH(0)) and entropy (DeltaS(0)) were calculated for predicting the nature of adsorption. The results indicate the potential application of this method for effluent treatment in industries and also provide strong evidence to support the adsorption mechanism proposed.

Quantitative risk assessment for accidental release of titanium tetrachloride in a titanium sponge production plant.

This paper outlines the quantitative risk assessment for storage and purification section of a titanium sponge production facility. Based on qualitative HAZAN technique, which involves a detailed FETI and HAZOP study of the entire plant, the storage and the purification section were found to be the most hazardous sections. Titanium tetrachloride (TiCl(4)) is the major reactant used in this plant. TiCl(4) is a toxic, corrosive water reactive chemical and on spillage from containment creates a liquid pool that can either boil or evaporate leading to the evolution of toxic hydrogen chloride (HCl). Fault tree analysis technique has been used to identify the basic events responsible for the top event occurrence and calculate their probabilities. Consequence analysis of the probable scenarios has been carried out and the risk has been estimated in terms of fatality and injuries. These results form the basic inputs for the risk management decisions.

Signaling from the secretory granule to the nucleus: Uhmk1 and PAM.

Neurons and endocrine cells package peptides in secretory granules (large dense-core vesicles) for storage and stimulated release. Studies of peptidylglycine alpha-amidating monooxygenase (PAM), an essential secretory granule membrane enzyme, revealed a pathway that can relay information from secretory granules to the nucleus, resulting in alterations in gene expression. The cytosolic domain (CD) of PAM, a type 1 membrane enzyme essential for the production of amidated peptides, is basally phosphorylated by U2AF homology motif kinase 1 (Uhmk1) and other Ser/Thr kinases. Proopiomelanocortin processing in AtT-20 corticotrope tumor cells was increased when Uhmk1 expression was reduced. Uhmk1 was concentrated in the nucleus, but cycled rapidly between nucleus and cytosol. Endoproteolytic cleavage of PAM releases a soluble CD fragment that localizes to the nucleus. Localization of PAM-CD to the nucleus was decreased when PAM-CD with phosphomimetic mutations was examined and when active Uhmk1 was simultaneously overexpressed. Membrane-tethering Uhmk1 did not eliminate its ability to exclude PAM-CD from the nucleus, suggesting that cytosolic Uhmk1 could cause this response. Microarray analysis demonstrated the ability of PAM to increase expression of a small subset of genes, including aquaporin 1 (Aqp1) in AtT-20 cells. Aqp1 mRNA levels were higher in wild-type mice than in mice heterozygous for PAM, indicating that a similar relationship occurs in vivo. Expression of PAM-CD also increased Aqp1 levels whereas expression of Uhmk1 diminished Aqp1 expression. The outlines of a pathway that ties secretory granule metabolism to the transcriptome are thus apparent.

Functional characterization of beta-ketoacyl-ACP reductase (FabG) from Plasmodium falciparum.

The malaria parasite, Plasmodium falciparum, unlike its human host, utilizes type II fatty acid synthesis, in which steps of fatty acid biosynthesis are catalyzed by independent enzymes. Due to this difference, the enzymes of this pathway are a potential target of newer antimalarials. Here we report the functional characterization of Plasmodium FabG expressed in Escherichia coli. The purified recombinant FabG from P. falciparum is soluble and active. The K(m) of the enzyme for acetoacetyl-CoA was estimated to be 75 microM with a V(max) of 0.0054 micromol/min/ml and a k(cat) value of 0.014s(-1). NADPH exhibited negative cooperativity for its interaction with FabG. We have also modeled P. falciparum FabG using Brassica napus FabG as the template. This model provides a structural rationale for the specificity of FabG towards its cofactor, NADPH.