The clinicopathological and gene expression patterns associated with ulceration of primary melanoma

Ulceration of primary melanomas is associated with poor prognosis yet is reported to predict benefit from adjuvant interferon. To better understand the biological processes involved, clinicopathological factors associated with ulceration were determined in 1804 patients. From this cohort, 348 primary tumor blocks were sampled to generate gene expression data using a 502‐gene cancer panel and 195 blocks were used for immunohistochemistry to detect macrophage infiltration and vessel density. Gene expression results were validated using a whole genome array in two independent sample sets. Ulceration of primary melanomas was associated with more proliferative tumors, tumor vessel invasion, and increased microvessel density. Infiltration of tumors with greater number of macrophages and gene expression pathways associated with wound healing and up‐regulation of pro‐inflammatory cytokines suggests that ulceration is associated with tumor‐related inflammation. The relative benefit from interferon reported in patients with ulcerated tumors may reflect modification of signaling pathways involved in inflammation.     

Nanomicellar Topical Aqueous Drop Formulation of Rapamycin for Back-of-the-Eye Delivery

The objective of this study was to develop a clear, aqueous rapamycin-loaded mixed nanomicellar formulations (MNFs) for the back-of-the-eye delivery. MNF of rapamycin (0.2%) was prepared with vitamin E tocopherol polyethylene glycol succinate (TPGS) (Vit E TPGS) and octoxynol-40 (Oc-40) as polymeric matrix. MNF was characterized by various parameters such as size, charge, shape, and viscosity. Proton nuclear magnetic resonance (¹H NMR) was used to identify unentrapped rapamycin in MNF. Cytotoxicity was evaluated in human retinal pigment epithelial (D407) and rabbit primary corneal epithelial cells (rPCECs). In vivo posterior ocular rapamycin distribution studies were conducted in male New Zealand white rabbits. The optimized MNF has excellent rapamycin entrapment and loading efficiency. The average size of MNF was 10.98 ± 0.089 and 10.84 ± 0.11 nm for blank and rapamycin-loaded MNF, respectively. TEM analysis revealed that nanomicelles are spherical in shape. Absence of free rapamycin in the MNF was confirmed by ¹H NMR studies. Neither placebo nor rapamycin-loaded MNF produced cytotoxicity on D407 and rPCECs indicating formulations are tolerable. In vivo studies demonstrated a very high rapamycin concentration in retina-choroid (362.35 ± 56.17 ng/g tissue). No drug was identified in the vitreous humor indicating the sequestration of rapamycin in lipoidal retinal tissues. In summary, a clear, aqueous MNF comprising of Vit E TPGS and Oc-40 loaded with rapamycin was successfully developed. Back-of-the-eye tissue distribution studies demonstrated a very high rapamycin levels in retina-choroid (place of drug action) with a negligible drug partitioning into vitreous humor.KEY WORDS: back-of-the-eye, drug delivery, formulation, mixed nanomicelles, posterior, rabbits, rapamycin/sirolimus, retina/choroid, sclera, topical eye drops     

Novel Pentablock Copolymer-Based Nanoparticulate Systems for Sustained Protein Delivery

The design, synthesis, and application of novel biodegradable and biocompatible pentablock (PB) copolymers, i.e., polyglycolic acid-polycaprolactone-polyethylene glycol-polycaprolactone-polyglycolic acid (PGA-PCL-PEG-PCL-PGA) and polylactic acid-polycaprolactone-polyethylene glycol-polycaprolactone-polylactic acid (PLA-PCL-PEG-PCL-PLA) for sustained protein delivery, are reported. The PB copolymers can be engineered to generate sustained delivery of protein therapeutics to the posterior segment of the eye. PB copolymers with different block arrangements and molecular weights were synthesized by ring-opening polymerization and characterized by proton nuclear magnetic resonance (¹H-NMR), gel permeation chromatography (GPC), and X-ray diffraction (XRD) spectroscopy. Immunoglobulin G (IgG) was selected as a model protein due to its structural similarity to bevacizumab. The influence of polymer molecular weight, composition, and isomerism on formulation parameters such as entrapment efficiency, drug loading, and in vitro release profile was delineated. Crystallinity and molecular weight of copolymers exhibited a substantial effect on formulation parameters. A secondary structure of released IgG was confirmed by circular dichroism (CD) spectroscopy. In vitro cytotoxicity, cell viability, and biocompatibility studies performed on human retinal pigment epithelial cells (ARPE-19) and/or macrophage cell line (RAW 264.7) demonstrated PB copolymers to be excellent biomaterials. Novel PB polymers may be the answer to the unmet need of a sustained release protein formulation.KEY WORDS: block copolymers, controlled drug delivery, IgG, intravitreal, nanoparticles, ocular delivery, pentablock copolymers, posterior segment, protein therapeutics, sustained drug delivery     

Epigallocatechin Gallate/Layered Double Hydroxide Nanohybrids: Preparation,Characterization, and In Vitro Anti-Tumor Study

In recent years, nanotechnology in merging with biotechnology has been employed in the area of cancer management to overcome the challenges of chemopreventive strategies in order to gain promising results. Since most biological processes occur in nano scale, nanoparticles can act as carriers of certain drugs or agents to deliver it to specific cells or targets. In this study, we intercalated Epigallocatechin-3-Gallate (EGCG), the most abundant polyphenol in green tea, into Ca/Al-NO₃ Layered double hydroxide (LDH) nanoparticles, and evaluated its efficacy compared to EGCG alone on PC3 cell line. The EGCG loaded LDH nanohybrids were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy (TEM) and nanosizer analyses. The anticancer activity of the EGCG-loaded LDH was investigated in prostate cancer cell line (PC3) while the release behavior of EGCG from LDH was observed at pH 7.45 and 4.25. Besides enhancing of apoptotic activity of EGCG, the results showed that intercalation of EGCG into LDH can improve the anti- tumor activity of EGCG over 5-fold dose advantages in in-vitro system. Subsequently, the in-vitro release data showed that EGCG-loaded LDH had longer release duration compared to physical mixture, and the mechanism of diffusion through the particle was rate-limiting step. Acidic attack was responsible for faster release of EGCG molecules from LDH at pH of 4.25 compared to pH of 7.4. The results showed that Ca/Al-LDH nanoparticles could be considered as an effective inorganic host matrix for the delivery of EGCG to PC3 cells with controlled release properties.     

A kinetic study on the Novozyme 435‐catalyzed esterification of free fatty acids with octanol to produce octyl esters

Octyl esters can serve as an important class of biolubricant components replacing their mineral oil counterparts. The purpose of the current work was to investigate the enzymatic esterification reaction of free fatty acids (FFA, from waste cooking oil) with octanol in a solvent‐free system using a commercial lipase Novozyme 435. It was found that the esterificaton reaction followed the Ping‐pong bi‐bi kinetics with no inhibition by substrates or products within the studied concentration range. The maximum reaction rate was estimated to be 0.041 mol L^?1 g^?1 h^?1. Additionally, the stability of Novozyme 435 in the current reaction system was studied by determining its activity and final conversion of FFA to esters after 12 successive utilizations. Novozyme 435 exhibited almost 100% enzyme activity up to 7 cycles of reaction and gradually decreased (by 5%) thereafter. The kinetic parameters evaluated from the study shall assist in the design of reactors for large‐scale production of octyl esters from a cheap biomass source. The enzyme reusability data can further facilitate mass production by curtailing the cost of expensive enzyme consumption. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1494–1499, 2015     

Proteomic Profiling of SupT1 Cells Reveal Modulation of Host Proteins by HIV-1 Nef Variants

Nef is an accessory viral protein that promotes HIV-1 replication, facilitating alterations in cellular pathways via multiple protein-protein interactions. The advent of proteomics has expanded the focus on better identification of novel molecular pathways regulating disease progression. In this study, nef was sequenced from randomly selected patients, however, sequence variability identified did not elicited any specific mutation that could have segregated HIV-1 patients in different stages of disease progression. To explore the difference in Nef functionality based on sequence variability we used proteomics approach. Proteomic profiling was done to compare the effect of Nef variants in host cell protein expression. 2DGE in control and Nef transfected SupT1 cells demonstrated several differentially expressed proteins. Fourteen protein spots were detected with more than 1.5 fold difference. Significant down regulation was seen in six unique protein spots in the Nef treated cells. Proteins were identified as Cyclophilin A, EIF5A-1 isoform B, Rho GDI 1 isoform a, VDAC1, OTUB1 and α-enolase isoform 1 (ENO1) through LC-MS/MS. The differential expression of the 6 proteins was analyzed by Real time PCR, Western blotting and Immunofluorescence studies with two Nef variants (RP14 and RP01) in SupT1 cells. There was contrasting difference between the effect of these Nef variants upon the expression of these six proteins. Downregulation of α-enolase (ENO1), VDAC1 and OTUB1 was more significant by Nef RP01 whereas Cyclophilin A and RhoGDI were found to be more downregulated by Nef RP14. This difference in Nef variants upon host protein expression was also studied through a site directed mutant of Nef RP01 _{(55AAAAAAA61)} and the effect was found to be reversed. Deciphering the role of these proteins mediated by Nef variants will open a new avenue of research in understanding Nef mediated pathogenesis. Overall study determines modulation of cellular protein expression in T cells by HIV-1 Nef variants.     

Colorimetric estimation of human glucose level using γ-Fe2O3 nanoparticles: An easily recoverable effective mimic peroxidase

This article reports simple, green and efficient synthesis of γ-Fe₂O₃ nanoparticles (NPs) (maghemite) through single-source precursor approach for colorimetric estimation of human glucose level. The γ-Fe₂O₃ NPs, having cubic morphology with an average particle size of 30 nm, exhibited effective peroxidase-like activity through the catalytic oxidation of peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H₂O₂ producing a blue-colored solution. On the basis of this colored-reaction, we have developed a simple, cheap, highly sensitive and selective colorimetric method for estimation of glucose using γ-Fe₂O₃/TMB/glucose–glucose oxidase (GOx) system in the linear range from 1 to 80 μM with detection limit of 0.21 μM. The proposed glucose sensor displays faster response, good stability, reproducibility and anti-interference ability. Based on this simple reaction process, human blood and urine glucose level can be monitored conveniently.     

Features analysis for identification of date and party hubs in protein interaction network of <Emphasis Type="Italic">Saccharomyces Cerevisiae</Emphasis>

Background  

It has been understood that biological networks have modular organizations which are the sources of their observed complexity. Analysis of networks and motifs has shown that two types of hubs, party hubs and date hubs, are responsible for this complexity. Party hubs are local coordinators because of their high co-expressions with their partners, whereas date hubs display low co-expressions and are assumed as global connectors. However there is no mutual agreement on these concepts in related literature with different studies reporting their results on different data sets. We investigated whether there is a relation between the biological features of Saccharomyces Cerevisiae 's proteins and their roles as non-hubs, intermediately connected, party hubs, and date hubs. We propose a classifier that separates these four classes.     

Accumulation of p-hydroxybenzoic acid in hairy roots of Daucus carota

We report here the accumulation of p-hydroxybenzoic acid in Agrobacterium rhizogenes-induced hairy root cultures of Daucus carota. This phenolic acid finds application in food, pharmaceutical and polymer industries. Metabolic profiling of phenolics by HPLC/ESI-MS from these hairy roots showed a considerable amount of p-hydroxybenzoic acid accumulation both in cytosol and in the cell wall. Analyses of HCl and NaOH treated soluble phenolic fractions resulted in the elution of peaks with same retention time and similar UV-absorption spectra as observed with p-hydroxybenzoic acid standard. This suggests that p-hydroxybenzoic acid is present in the cytosol as free-form (unconjugated). A correlation has been drawn between the accumulation of soluble and wall-bound phenolic acids on a time-course basis. An apparent absence of any p-hydroxybenzoic acid-glucoside supports this observation, which in turn encourages the idea of its incorporation in the cell wall in an alkaline-labile form.     

Tumor necrosis factor-alpha stimulates focal adhesion kinase activity required for mitogen-activated kinase-associated interleukin 6 expression

Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase that promotes cell migration, survival, and gene expression. Here we show that FAK signaling is important for tumor necrosis factor-alpha (TNFalpha)-induced interleukin 6 (IL-6) mRNA and protein expression in breast (4T1), lung (A549), prostate (PC-3), and neural (NB-8) tumor cells by FAK short hairpin RNA knockdown and by comparisons of FAK-null (FAK(-/-)) and FAK(+/+) mouse embryo fibroblasts. FAK promoted TNFalpha-stimulated MAPK activation needed for maximal IL-6 production. FAK was not required for TNFalpha-mediated nuclear factor-kappaB or c-Jun N-terminal kinase activation. TNFalpha-stimulated FAK catalytic activation and IL-6 production were inhibited by FAK N-terminal but not FAK C-terminal domain overexpression. Analysis of FAK(-/-) fibroblasts stably reconstituted with wild type or various FAK point mutants showed that FAK catalytic activity, Tyr-397 phosphorylation, and the Pro-712/713 proline-rich region of FAK were required for TNFalpha-stimulated MAPK activation and IL-6 production. Constitutively activated MAPK kinase-1 (MEK1) expression in FAK(-/-) and A549 FAK short hairpin RNA-expressing cells rescued TNFalpha-stimulated IL-6 production. Inhibition of Src protein-tyrosine kinase activity or mutation of Src phosphorylation sites on FAK (Tyr-861 or Tyr-925) did not affect TNFalpha-stimulated IL-6 expression. Moreover, analyses of Src(-/-), Yes(-/-), and Fyn(-/-) fibroblasts showed that Src expression was inhibitory to TNFalpha-stimulated IL-6 production. These studies provide evidence for a novel Src-independent FAK to MAPK signaling pathway regulating IL-6 expression with potential importance to inflammation and tumor progression.