Measurement of gene-expression profiles using microarray technology is becoming increasingly popular among the biomedical research community. Although there has been great progress in this field, investigators are still confronted with a difficult question after completing their experiments: how to validate the large data sets that are generated? This review summarizes current approaches to verifying global expression results, discusses the caveats that must be considered, and describes some methods that are being developed to address outstanding problems.

The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE) Proteomics (1) includes the systematic cataloging of protein expression on a large scale, providing complementary information to that obtained from mRNA profiling by microarray (2, 3). Such studies could lead to the molecular characterization of cellular events associated with cancer progression, cellular signaling, and developmental stages (4 -7). Proteomics studies of clinical tumor samples have led to the identification of cancer-specific protein markers, which provide a basis for developing new methods for early diagnosis and early detection and clues to understand the molecular characterization of cancer progression (5, 8 -10).,A mainstay of conventional proteomics is high resolution 2D 1 gel electrophoresis (11, 12) followed by protein identification using mass spectrometry (13-15). The state of the art 2D gel system can be loaded with a few milligrams of protein and separates thousands of protein spots (5, 16). Although the technique has been widely used and successfully applied in a variety of biological systems, several technical limitations exist. Because of subtle changes in experimental conditions, the protein expression patterns on a single 2D gel usually cannot be fully duplicated, which makes it difficult to find the proteins changed between gels and to quantify the changes in protein expression. Although a comparison of protein expression profiles from regular 2D gel electrophoresis can be carried out with the assistance of various software programs, it typically requires some computerized justification of 2D gel images so that two images can be superimposed and compared. These difficulties limit the speed and accuracy of quantitation of protein spots in 2D gel electrophoresis.The differential in-gel electrophoresis (DIGE) technique recently introduced by Amersham Biosciences, Inc. is aimed at improving reproducibility. The concept of DIGE was originally developed by Minden and colleagues (17). To analyze the samples in DIGE, two pools of protein extracts are labeled covalently with fluorescent cyanine dyes, Cy3 and Cy5, re-

Using a general strategy for evaluating clinical tissue specimens, we found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies. Human prostate and kidney were used as test tissues. The protein content of the samples was analyzed by one-dimensional gel electrophoresis, immunoblot, two-dimensional gel electrophoresis, and layered expression scanning. In each case, the fixed and embedded tissues produced results similar to that obtained from snap-frozen specimens, although the protein quantity was somewhat decreased. Recovery of mRNA was reduced in both quantity and quality in the ethanol-fixed samples, but was superior to that obtained from formalin-fixed samples and sufficient to perform reverse transcription polymerase chain reactions. Recovery of DNA from ethanol-fixed specimens was superior to formalin-fixed samples as determined by one-dimensional gel electrophoresis and polymerase chain reaction. In conclusion, specimens fixed in 70% ethanol and embedded in paraffin produce good histology and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses. Complete protocols and additional discussion of relevant issues are available on an accompanying website (http://cgap-mf.nih.gov/).

Background-The cathelicidin family of anti-microbial peptides is an integral component of the innate immune response that exhibits activity against bacterial, fungal and viral pathogens. Eczema herpeticum (ADEH) develops in a subset of AD patients due to disseminated infection with herpes simplex virus (HSV).

A single-point mutation in exon 15 of the BRAF gene has recently been reported in a high percentage in cultured melanoma cells and in 6 of 9 primary melanomas examined. To evaluate the impact of the T1796A BRAF mutation, we screened primary melanomas, various types of nevi and lesions where a melanoma developed in an underlying nevus. We could detect the mutation in 28 of 97 (29%) melanomas and in 39 of 187 (21%) nevi, including blue nevi (0/20) and Spitz nevi (0/69), which did not carry the mutation. In melanomas with an underlying nevus, either the mutation was present in both the laser-microdissected nevus cells and the laser-microdissected melanoma cells (3/14) or both lesions were negative for the BRAF mutation except one case. In conclusion, mutations in exon 15 of the BRAF gene are nonspecific for progression of a nevus to a melanoma. Other so far unknown cofactors seem to be of importance.

We previously reported that ethanol fixation and paraffin embedding of tissues produce excellent histomorphology and good preservation of macromolecules. Here, we present a detailed evaluation of ethanol-fixed tissues for proteomic initiatives. When proteins were extracted from ethanol-fixed, paraffin-embedded prostate tissue, resolved by two-dimensional gel electrophoresis (2-DE), and stained by standard methods, several hundred protein molecules could be detected and successfully analyzed by mass spectrometry. Protein profiles obtained from ethanol-fixed tissues were highly similar to those observed from frozen tissues, in contrast to the poor protein recovery from formalin-fixed material. The protein content of specific cells that were microdissected from ethanol-fixed tissue sections using laser capture microdissection could also be successfully analyzed by 2-DE. We observed that eosin staining of tissue sections had a detrimental effect on protein separation, whereas hematoxylin staining had minimal consequence. In order to illustrate the applicability of ethanol-fixed tissues for proteomic discovery studies, we compared the protein profiles of patient-matched, normal prostatic epithelial cells and invasive adenocarcinoma cells obtained from ethanol-fixed, paraffin-embedded tissues. A number of differentially expressed proteins was discovered and identified by mass spectrometry. Immunohistochemical analyses performed on ethanol-fixed tissue sections were in agreement with the proteomic discovery findings. In light of these results, we conclude that ethanol-fixed tissues can be successfully utilized for proteomic analyses.

Background: The diagnosis of most cancers is made by a board-certified pathologist based on a tissue biopsy under the microscope. Recent research reveals a high discordance between individual pathologists. For melanoma, the literature reports on 25e26% of discordance for classifying a benign nevus versus malignant melanoma. A recent study indicated the potential of deep learning to lower these discordances. However, the performance of deep learning in classifying histopathologic melanoma images was never compared directly to human experts. The aim of this study is to perform such a first direct comparison. Methods: A total of 695 lesions were classified by an expert histopathologist in accordance with current guidelines (350 nevi/345 melanoma). Only the haematoxylin & eosin (H&E) slides

The magnitude of therapeutic success correlates with type of venom, duration of therapy, and venom dose. Adult-onset MIS and/or a BTC > 20 μg/L is a significant, albeit not the strongest determinant for VIT failure. According to its odds ratio, ACE inhibitor therapy appears to be associated with the highest risk for VIT failure.