Multiple Protein Analysis of Formalin-fixed and Paraffin-embedded Tissue Samples with Reverse phase Protein Arrays

Reverse-phase protein arrays (RPPAs) have become an important tool for the sensitive and high-throughput detection of proteins from minute amounts of lysates from cell lines and cryopreserved tissue. The current standard method for tissue preservation in almost all hospitals worldwide is formalin fixation and paraffin embedding, and it would be highly desirable if RPPA could also be applied to formalin-fixed and paraffin embedded (FFPE) tissue. We investigated whether the analysis of FFPE tissue lysates with RPPA would result in biologically meaningful data in two independent studies. In the first study on breast cancer samples, we assessed whether a human epidermal growth factor receptor (HER) 2 score based on immunohistochemistry (IHC) could be reproduced with RPPA. The results showed very good concordance between the IHC and RPPA classifications of HER2 expression. In the second study, we profiled FFPE tumor specimens from patients with adenocarcinoma and squamous cell carcinoma in order to find new markers for differentiating these two subtypes of non-small cell lung cancer. p21-activated kinase 2 could be identified as a new differentiation marker for squamous cell carcinoma. Overall, the results demonstrate the technical feasibility and the merits of RPPA for protein expression profiling in FFPE tissue lysates.Many diseases are characterized by the expression of specific proteins and the activation status of distinct signaling pathways (1). Thus, protein expression profiling and activation patterns are instrumental for understanding disease, the development of effective treatments, and the identification of patients who will respond to particular therapies. Traditional ways of analyzing protein expression (e.g. Western blot) can be used for these purposes but often are labor intensive, have low throughput, and consume high sample volumes. Reverse-phase protein array (RPPA)¹ technology is a very promising method that circumvents these issues (2–4). For RPPA, minute amounts of whole protein lysates from a multitude of samples are spotted onto slides, and individual proteins are detected via protein-specific antibodies. This enables medium- to high-throughput analysis of precious low-volume sample material.Lysates for RPPA have so far been generated mainly from cell lines or fresh frozen tissue. However, because of the high amount of effort involved in the use of liquid nitrogen for sample preservation, in almost all hospitals worldwide formalin fixation and paraffin embedding is the preferred method for tissue preservation. Therefore, it would be highly desirable if protein-specific epitopes could be quantitatively extracted and analyzed from formalin-fixed and paraffin embedded (FFPE) tissue, as this would make the majority of clinical specimens accessible for mechanistic protein-based research.In recent years, several research groups have established protocols for protein extraction from FFPE tissue. Common to all of them is the use of high concentrations of ionic detergents, such as sodium dodecyl sulfate, and high temperature. It was shown that these methods even make it possible to extract full-length proteins from FFPE tissue (5–12). The coefficient of variation of the relative extraction efficiency based on Western blot and densitometric assessment of actin typically is below 20% (13). To assess whether the analysis of FFPE tissue lysates would result in biologically meaningful data, we analyzed FFPE breast cancer tissue samples by RPPA for the expression of human epidermal growth factor receptor 2 (HER2) and compared it to HER2 assessment by the gold standard used in clinical practice, which is based on immunohistochemistry (IHC). Successful recovery of HER2 from FFPE tissue should result in concordant HER2 classification between RPPA and IHC.In the second part of the study, FFPE samples of non-small cell lung cancer (NSCLC) were examined via RPPA. Samples from two subtypes of NSCLC, adenocarcinoma (AC) and squamous cell carcinoma (SCC), were analyzed for more than 150 proteins, including two proteins that are known to be differentially expressed between the two subtypes. The objectives of this analysis were to further assess the validity of the approach by confirming the two positive controls and to identify new markers for the differentiation of the two subtypes of NSCLC.     

A DFT study on the sensing behavior of a BC2N nanotube toward formaldehyde

We investigated the viability of using a BC₂N nanotube to detect formaldehyde (H₂CO) molecule by means of B3LYP and M06 density functionals. The results indicate that the molecule is weakly adsorbed on the intrinsic BC₂N nanotube releasing energy of 0.8 kcal mol^-1 (at B3LYP/6-31G(d)) without significant effect on the HOMO-LUMO energy gap and electrical conductivity of the tube. Thus, H₂CO cannot be detected using this intrinsic nanotube. To overcome this problem, a carbon atom of the tube wall was substituted by a Si atom. It was demonstrated that the Si-doped tube cannot only strongly adsorb the H₂CO molecule, but also may effectively detect its presence because of the increase in the electric conductivity of the tube.     

Fluorescent adducts formed by reaction of oxidized unsaturated fatty acids with amines increase macrophage viability

Macrophages are prominent components of human atherosclerotic lesions and they are believed to accelerate the progression and/or complications of both early and advanced atherosclerotic lesions. We and others have shown that oxidized low-density lipoprotein (oxLDL) induces growth and inhibits apoptosis in murine bone marrow-derived macrophages. In this study, we sought to characterize the oxidative modification of LDL that is responsible for this prosurvival effect. We found that both the modified lipid and the modified protein components of oxLDL can increase the viability of macrophages. The key modification appeared to involve derivatization of amino groups in apoB or in phosphatidylethanolamine by lipid peroxidation products. These reactive oxidation products were primarily unfragmented hydroperoxide- or endoperoxide-containing oxidation products of linoleic acid or arachidonic acid. LC-MS/MS studies showed that some of the arachidonic acid-derived lysine adducts were isolevuglandins that contain lactam and hydroxylactam rings. MS/MS analysis of linoleic acid autoxidation adducts was consistent with 5- or 6-membered nitrogen-containing heterocycles derived from unfragmented oxidation products. The amine modification by oxidation products generated a fluorescence pattern with an excitation maximum at 350nm and emission maximum at 430nm. This is very similar to the fluorescence spectrum of copper-oxidized LDL.     

The antimicrobial potential of a new derivative of cathelicidin from <Emphasis Type="Italic">Bungarus fasciatus</Emphasis> against methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Cathelicidins are a family of antimicrobial peptides which exhibit broad antimicrobial activities against antibiotic-resistant bacteria. Considering the progressive antibiotic resistance, cathelicidin is a candidate for use as an alternative approach to treat and overcome the challenge of antimicrobial resistance. Cathelicidin-BF (Cath-BF) is a short antimicrobial peptide, which was originally extracted from the venom of Bungarus fasciatus. Recent studies have reported that Cath-BF and some related derivatives exert strong antimicrobial and weak hemolytic properties. This study investigates the bactericidal and cytotoxic effects of Cath-BF and its analogs (Cath-A and Cath-B). Cath-A and Cath-B were designed to increase their net positive charge, to have more activity against methicillin resistant S. aureus (MRSA). The results of this study show that Cath-A, with a +17-net charge, has the most noteworthy antimicrobial activity against MRSA strains, with minimum inhibitory concentration (MIC) ranging between 32–128 μg/ml. The bacterial kinetic analysis by 1 × MIC concentration of each peptide shows that Cath-A neutralizes the clinical MRSA isolate for 60 min. The present data support the notion that increasing the positive net charge of antimicrobial peptides can increase their potential antimicrobial activity. Cath-A also displayed the weakest cytotoxicity effect against human umbilical vein endothelial and H9c2 rat cardiomyoblast cell lines. Analysis of the hemolytic activity reveals that all three peptides exhibit minor hemolytic activity against human erythrocytes at concentrations up to 250 μg/ml. Altogether, these results suggest that Cath-A and Cath-B are competent candidates as novel antimicrobial compounds against MRSA and possibly other multidrug resistant bacteria.     

Therapeutic gene transfer to dystrophic diaphragm by an adenoviral vector deleted of all viral genes

Duchenne muscular dystrophy is caused by defects in the dystrophin gene, and the mdx mouse is the most frequently employed genetic model of this disease. It is well known that different muscle groups do not respond in the same way to dystrophin deficiency. In particular, the mdx mouse diaphragm exhibits severe morphological and functional changes not found in other mdx muscles. Use of early generation adenoviral vectors to deliver genes to the diaphragm in immunocompetent mdx mice has been associated with substantial functional toxicity and a rapid loss of transgene expression. Here we determined the response to dystrophin gene replacement in the mdx diaphragm using a "gutted" adenoviral vector that contains the coding sequence of two full-length dystrophin genes and is deleted of most viral DNA sequences. At 1 wk postdelivery of the vector, 23.6 +/- 4% of total fibers in the injected diaphragm bundle expressed dystrophin at the sarcolemma, which remained stable over the study duration of 30 days without the need for continuous immunosuppression. Treated diaphragms showed a significantly improved resistance to the abnormal force deficits induced by high-stress muscle contractions, the latter being a functional hallmark of dystrophin-deficient muscle. This functional amelioration was achieved despite the presence of mildly increased inflammation (CD4+ and CD8+ lymphocytes) within the vector-treated diaphragms. To our knowledge, this is the first demonstration that a viral vector can achieve reversal of functional abnormalities in the dystrophic diaphragm via therapeutic dystrophin gene transfer without the need for sustained immunosuppressive therapy.     

A Role for Versican in the Development of Leiomyosarcoma

Leiomyosarcoma (LMS) is a mesenchymal cancer that occurs throughout the body. Although LMS is easily recognized histopathologically, the cause of the disease remains unknown. Versican, an extracellular matrix proteoglycan, increases in LMS. Microarray analyses of 80 LMSs and 24 leiomyomas showed a significant elevated expression of versican in human LMS versus benign leiomyomas. To explore the importance of versican in this smooth muscle cell tumor, we used versican-directed siRNA to knock down versican expression in a LMS human cell line, SK-LMS-1. Decreased versican expression was accompanied by slower rates of LMS cell proliferation and migration, increased adhesion, and decreased accumulation of the extracellular matrix macromolecule hyaluronan. Addition of purified versican to cells expressing versican siRNA restored cell proliferation to the level of LMS controls, increased the pericellular coat and the retention of hyaluronan, and decreased cell adhesion in a dose-dependent manner. The presence of versican was not only synergistic with hyaluronan in increasing cell proliferation, but the depletion of versican decreased hyaluronan synthase expression and decreased the retention of hyaluronan. When LMS cells stably expressing versican siRNA were injected into nude mice, the resulting tumors displayed significantly less versican and hyaluronan staining, had lower volumes, and had reduced levels of mitosis as compared with controls. Collectively, these results suggest a role for using versican as a point of control in the management and treatment of LMS.     

LOX-1 augments oxLDL uptake by lysoPC-stimulated murine macrophages but is not required for oxLDL clearance from plasma

Oxidized LDL (oxLDL) promotes lipid accumulation as well as growth and survival signaling in macrophages. OxLDL uptake is mainly due to scavenger receptors SR-AI/II and CD36. However, other scavenger receptors such as lectin-like oxLDL receptor-1 (LOX-1) may also play a role. We used mice with targeted inactivation of the LOX-1 gene to define the role of this receptor in the uptake of oxLDL and in activation of survival pathways. There was no difference in uptake or degradation of ¹²⁵I-oxLDL in unstimulated macrophages from wild-type and LOX-1 knockout mice and no difference in the rate of clearance of oxLDL from plasma in vivo. However, when expression of LOX-1 was induced with lysophosphatidylcholine, oxLDL uptake and degradation increased 2-fold in wild-type macrophages but did not change in LOX-1 knockout macrophages. Macrophages lacking LOX-1 showed the same stimulation of PKB phosphorylation and enhancement of survival by oxLDL as wild-type cells. These data show that LOX-1 does not alter the uptake of oxLDL in unstimulated macrophages and is not essential for the pro-survival effect of oxLDL in these cells. However, LOX-1 expression is highly inducible by lysophosphatidylcholine and pro-inflammatory cytokines, and if that occurred in macrophages within atheromas, LOX-1 could substantially increase oxLDL uptake by lesion macrophages.     

A large gap opening of graphene induced by the adsorption of CO on the Al-doped site

We investigated CO adsorption on the pristine, Stone-Wales (SW) defected, Al- and Si- doped graphenes by using density functional calculations in terms of geometric, energetic and electronic properties. It was found that CO molecule is weakly adsorbed on the pristine and SW defected graphenes and their electronic properties were slightly changed. The Al- and Si- doped graphenes show high reactivity toward CO, so calculated adoption energies are about ?11.40 and ?13.75 kcal mol^?1 in the most favorable states. It was found that, among all the structures, the electronic properties of Al-doped graphene are strongly sensitive to the presence of CO molecule. We demonstrate the existence of a large E_g opening of 0.87 eV in graphene which is induced by Al-doping and CO adsorption.