Microarray and pathway analysis reveals decreased CDC25A and increased CDC42 associated with slow growth of BCL2 overexpressing immortalized breast cell line

Bcl-2 is an anti-apoptotic protein that is frequently overex-pressed in cancer cells but its role in carcinogenesis is not clear. We are interested in how Bcl-2 expression affects non-cancerous breast cells and its role in the cell cycle. We prepared an MCF10A breast epithelial cell line that stably overexpressed Bcl-2. We analyzed the cells by flow cytometry after synchronization, and used cDNA microarrays with quantitative reverse-transcription PCR (qRTPCR) to determine differences in gene expression. The microarray data was subjected to two pathway analysis tools, parametric analysis of gene set enrichment (PAGE) and ingenuity pathway analysis (IPA), and western analysis was carried out to determine the correlation between mRNA and protein levels. The MCF10A/Bcl-2 cells exhibited a slow-growth phenotype compared to control MCF10A/Neo cells that we attributed to a slowing of the G1-S cell cycle transition. A total of 363 genes were differentially expressed by at least two-fold, 307 upregulated and 56 downregulated. PAGE identified 22 significantly changed gene sets. The highest ranked network of genes identified by IPA contained 24 genes. Genes that were chosen for further analysis were confirmed by qRT-PCR, however, the western analysis did not always confirm differential expression of the proteins. Downregulation of the phosphatase CDC25A could solely be responsible for the slow growth pheno-type in MCF10A/Bcl-2 cells. Increased levels of GTPase Cdc42 could be adding to this effect. PAGE and IPA are valuable tools for microarray analysis, but protein expression results do not always follow mRNA expression results. Originally published Cell Cycle, Vol. 7, No. 19, Oct. 200

Label-free classification of cultured cells through diffraction imaging

Automated classification of biological cells according to their 3D morphology is highly desired in a flow cytometer setting. We have investigated this possibility experimentally and numerically using a diffraction imaging approach. A fast image analysis software based on the gray level co-occurrence matrix (GLCM) algorithm has been developed to extract feature parameters from measured diffraction images. The results of GLCM analysis and subsequent classification demonstrate the potential for rapid classification among six types of cultured cells. Combined with numerical results we show that the method of diffraction imaging flow cytometry has the capacity as a platform for high-throughput and label-free classification of biological cells

An online survey of hypothyroid patients demonstrates prominent dissatisfaction

Background: Approximately 15% more patients taking levothyroxine (LT4) report impaired quality of life compared to controls. This could be explained by additional diagnoses independently affecting quality of life and complicating assignment of causation. This study sought to investigate the underpinnings of reduced quality of life in hypothyroid patients and to provide data for discussion at a symposium addressing hypothyroidism. Methods: An online survey for hypothyroid patients was posted on the American Thyroid Association Web site and forwarded to multiple groups. Respondents were asked to rank satisfaction with their treatment for hypothyroidism and their treating physician. They also ranked their perception regarding physician knowledge about hypothyroidism treatments, need for new treatments, and life impact of hypothyroidism on a scale of 1–10. Respondents reported the therapy they were taking, categorized as LT4, LT4 and liothyronine (LT4 + LT3), or desiccated thyroid extract (DTE). They also reported sex, age, cause of hypothyroidism, duration of treatment, additional diagnoses, and prevalence of symptoms. Results: A total of 12,146 individuals completed the survey. The overall degree of satisfaction was 5 (interquartile range [IQR] = 3–8). Among respondents without self-reported depression, stressors, or medical conditions (n = 3670), individuals taking DTE reported a higher median treatment satisfaction of 7 (IQR = 5–9) compared to other treatments. At the same time, the LT4 treatment group exhibited the lowest satisfaction of 5 (IQR = 3–7), and for the LT4 + LT3 treatment group, satisfaction was 6 (IQR = 3–8). Respondents taking DTE were also less likely to report problems with weight management, fatigue/energy levels, mood, and memory compared to those taking LT4 or LT4 + LT3. Conclusions: A subset of patients with hypothyroidism are not satisfied with their current therapy or their physicians. Higher satisfaction with both treatment and physicians is reported by those patients on DTE. While the study design does not provide a mechanistic explanation for this observation, future studies should investigate whether preference for DTE is related to triiodothyronine levels or other unidentified causes

Drug-Dependent Behaviors and Nicotinic Acetylcholine Receptor Expressions in Caenorhabditis elegans Following Chronic Nicotine Exposure

Nicotine, the major psychoactive compound in tobacco, targets nicotinic acetylcholine receptors (nAChRs) and results in drug dependence. The nematode Caenorhabditis elegans’ (C. elegans) genome encodes conserved and extensive nicotinic receptor subunits, representing a useful system to investigate nicotine-induced nAChR expressions in the context of drug dependence. However, the in vivo expression pattern of nAChR genes under chronic nicotine exposure has not been fully investigated. To define the role of nAChR genes involved in nicotine-induced locomotion changes and the development of tolerance to these effects, we characterized the locomotion behavior combining the use of two systems: the Worm Tracker hardware and the WormLab software. Our results indicate that the combined system is an advantageous alternative to define drug-dependent locomotion behavior in C. elegans. Chronic (24-hour dosing) nicotine exposure at 6.17 and 61.7 μM induced nicotine-dependent behaviors, including drug stimulation, tolerance/adaption, and withdrawal responses. Specifically, the movement speed of naïve worms on nicotine-containing environments was significantly higher than on nicotine-free environments, suggesting locomotion stimulation by nicotine. In contrast, the 24-hour 6.17 μM nicotine-treated worms exhibited significantly higher speeds on nicotine-free plates than on nicotine-containing plates. Furthermore significantly increased locomotion behavior during nicotine cessation was observed in worms treated with a higher nicotine concentration of 61.7 μM. The relatively low locomotion speed of nicotine-treated worms on nicotine-containing environments also indicates adaption/tolerance of worms to nicotine following chronic nicotine exposure. In addition, this study provides useful information regarding the comprehensive in vivo expression profile of the 28 “core” nAChRs following different dosages of chronic nicotine treatments. Eleven genes (lev-1, acr-6, acr-7, acr-11, lev-8, acr-14, acr-16, acr-20, acr-21, ric-3, and unc-29) were significantly up-regulated following 61.7 μM nicotine treatment, in which worms showed significantly increased locomotion behavior. This study provides insights into the linkage between nicotine-induced locomotion behavior and the regulation of nicotinic acetylcholine receptors

An Integrated Haplotype Map of the Human Major Histocompatibility Complex

Numerous studies have clearly indicated a role for the major histocompatibility complex (MHC) in susceptibility to autoimmune diseases. Such studies have focused on the genetic variation of a small number of classical human-leukocyte-antigen (HLA) genes in the region. Although these genes represent good candidates, given their immunological roles, linkage disequilibrium (LD) surrounding these genes has made it difficult to rule out neighboring genes, many with immune function, as influencing disease susceptibility. It is likely that a comprehensive analysis of the patterns of LD and variation, by using a high-density map of single-nucleotide polymorphisms (SNPs), would enable a greater understanding of the nature of the observed associations, as well as lead to the identification of causal variation. We present herein an initial analysis of this region, using 201 SNPs, nine classical HLA loci, two TAP genes, and 18 microsatellites. This analysis suggests that LD and variation in the MHC, aside from the classical HLA loci, are essentially no different from those in the rest of the genome. Furthermore, these data show that multi-SNP haplotypes will likely be a valuable means for refining association signals in this region