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Colorectal cancer stem cell (CSC)‐enriched cultures were obtained efficiently from primary tumor specimens by optimizing a CSC‐isolation protocol. Based on in vitro and in vivo validation, genetic characterization, and drug sensitivity analysis, panels of CSC lines were generated with defined patterns of genetic mutations and therapy sensitivity. An analysis of CSC response to EGFR‐targeted therapy in vivo and an overview of factors implicated in therapy response/resistance are presented. Colorectal cancer (CRC) therapy mainly relies on the use of conventional chemotherapeutic drugs combined, in a subset of patients, with epidermal growth factor receptor [EGFR]‐targeting agents. Although CRC is considered a prototype of a cancer stem cell (CSC)‐driven tumor, the effects of both conventional and targeted therapies on the CSC compartment are largely unknown. We have optimized a protocol for colorectal CSC isolation that allowed us to obtain CSC‐enriched cultures from primary tumor specimens, with high efficiency. CSC isolation was followed by in vitro and in vivo validation, genetic characterization, and drug sensitivity analysis, thus generating panels of CSC lines with defined patterns of genetic mutations and therapy sensitivity. Colorectal CSC lines were polyclonal and maintained intratumor heterogeneity in terms of somatically acquired mutations and differentiation state. Such CSC‐enriched cultures were used to investigate the effects of both conventional and targeted therapies on the CSC compartment in vivo and to generate a proteomic picture of signaling pathways implicated in sensitivity/resistance to anti‐EGFR agents. We propose CSC lines as a sound preclinical framework to test the effects of therapies in vitro and in vivo and to identify novel determinants of therapy resistance. Significance Colorectal cancer stem cells (CSCs) have been shown to be responsible for tumor propagation, metastatic dissemination, and relapse. However, molecular pathways present in CSCs, as well as mechanisms of therapy resistance, are mostly unknown. Taking advantage of genetically characterized CSC lines derived from colorectal tumors, this study provides an extensive analysis of CSC response to EGFR‐targeted therapy in vivo and an overview of factors implicated in therapy response or resistance. Furthermore, the implementation of a biobank of molecularly annotated CSC lines provides an innovative resource for future investigations in colorectal cancer.

P2Y 1 purinergic receptors (P2Y 1 Rs) mediate rises in intracellular Ca 2+ in response to ATP, but the duration and characteristics of this Ca 2+ response are known to vary markedly in distinct cell types. We screened the P2Y 1 R carboxyl terminus against a recently created proteomic array of PDZ (PSD-95/ Drosophila Discs large/ZO-1 homology) domains and identified a previously unrecognized, specific interaction with the second PDZ domain of the scaffold NHERF-2 (Na + /H + exchanger regulatory factor type 2). Furthermore, we found that P2Y 1 R and NHERF-2 associate in cells, allowing NHERF-2-mediated tethering of P2Y 1 R to key downstream effectors such as phospholipase Cβ. Finally, we found that coexpression of P2Y 1 R with NHERF-2 in glial cells prolongs P2Y 1 R-mediated Ca 2+ signaling, whereas disruption of the P2Y 1 R–NHERF-2 interaction by point mutations attenuates the duration of P2Y 1 R-mediated Ca 2+ responses. These findings reveal that NHERF-2 is a key regulator of the cellular activity of P2Y 1 R and may therefore determine cell-specific differences in P2Y 1 R-mediated signaling. G protein-coupled receptor purinergic ATP proteomic array

Abstract The purpose of this article is to integrate the transcriptomic analysis and the proteomic profiles and to reveal and compare the different molecular mechanisms of PC12 cell growth on the surface of chitosan films and collagen/chitosan films. First, the chitosan films and the collagen/chitosan films were prepared. Subsequently, the cell viability assay was performed; the cell viability of the PC12 cells cultured on the collagen/chitosan films for 24 h was significantly higher than that on the chitosan films. Then, with cDNA microarray, the numbers of differentially expressed genes of PC12 cells on the surface of chitosan and collagen/chitosan films were 13349 and 5165, respectively. Next, the biological pathway analysis indicated that the differentially expressed genes were involved in 40 pathways directly related to cell adhesion and growth. The integrated transcriptomic and our previous proteomic analysis revealed that three biological pathways—extracellular matrix–receptor interaction, focal adhesion and regulation of actin cytoskeleton—were regulated in the processes of protein adsorption, cell adhesion and growth. The adsorbed proteins on the material surfaces further influenced the expression of important downstream genes by regulating the expression of related receptor genes in these three pathways. In comparison, chitosan films had a strong inhibitory effect on PC12 cell adhesion and growth, resulting in the significantly lower cell viability on its surface; on the contrary, collagen/chitosan films were more conducive to promoting PC12 cell adhesion and growth, resulting in higher cell viability.

Introduction Identifying serum biomarkers that reflect the restoration of dystrophin in skeletal muscle is important for evaluating the effect of dystrophin‐restoring therapies in preclinical and clinical trials. Many potential blood biomarkers have been identified in Duchenne muscular dystrophy (DMD) patients, which change with disease progression or respond to pharmacological treatment. In this study, it was suggested that a panel of such blood biomarker candidates could be used to monitor dystrophin rescue in mdx mice treated with microdystrophin based therapies. Methods Plasma samples from mdx mice treated with the microdystrophin therapy SGT‐001 were analysed with an antibody suspension bead array consisting of 87 antibodies. The array targets 83 unique proteins previously identified as biomarker candidates for DMD. Each sample was assayed at two different plasma dilutions to cover a broader concentration range. Protein concentrations estimated as Median fluorescent intensities (MFI) were correlated to dystrophin expression in muscle tissue, as measured by immunohistochemistry and Western blot. Thirteen of the targets were selected and analysed in a DMD and Becker muscular dystrophy (BMD) longitudinal natural history cohort using a suspension bead array. Results Ten proteins were found to be significantly elevated in untreated mdx mice compared with C57 wild‐type mice and to correlate with dystrophin expression (Spearman's correlation, FDR < 0.05) upon gene transfer in mdx mice. Translatability of these biomarkers from animal models to patients was evaluated by exploring abundance trajectories over time in both DMD and BMD patients and association with dystrophin expression in BMD patients. Consistent with the observations in mouse, six of these biomarker candidates were more abundant in DMD patients compared with controls, and six were also differentially abundant between BMD and DMD patients. Among them, serum titin was shown to be associated with dystrophin expression in BMD patients, having a steeper decline over time in patients with low dystrophin expression in tibialis anterior compared with patients with high expression. Myosine light chain 3 had a steeper decline with time in DMD patients compared with BMD patients. Conclusions The 10 biomarker candidates identified in mouse plasma are related to muscle contraction, glycolysis, microtubule formation and protein degradation. Human titin and myosine light chain 3 were the most interesting candidates as explorative biomarkers to monitor microdystrophin expression in gene therapies. If confirmed, these biomarkers could be used to detect restoration of dystrophin expression per se, monitor changes in dystrophin expression over time and potentially confirm disease phenotype changes from severe to mild disease forms.

Information technology (IT) is leading us to reconsider some of the approaches we have been using in both basic research and clinical work in allergology. Resources mainly coming from the advent of the Internet are further amplified by the parallel development of other new tools, such as molecular biology and nanotechnology. These three powerful tools are now available and are cross-linked to a certain degree to express their power when applied to biomedical fields. Bioinformatics applied to allergy simplifies our way of handling an increasing wealth of knowledge. This review assesses the current status of allergen databases that are mainly dedicated to sequence homology collection for computational purposes. Whether or not they integrate features that are now typical of IT in other biomedical fields is analyzed as well. The results of these analyses are discussed with a view to the critical need of integrating biochemical data with clinical, epidemiological information and how this goal can be reached by the use of proteomic microarrays for IgE detection. Future directions for a more comprehensive use of allergen databases are proposed.

Cassava is cultivated for two ends proposals: \"sweet cassava\" as fresh consumes and \"industry cassava\" as source of starch and farina. Landraces were used to discover \"spontaneous mutations\" and to develop evolutionary and breeding perspective of gene function. Genomic and Proteomic resources were obtained. Gene expression by RNA blot and Microarray analysis were performed to identify differentially expressed genes. A new sugary cassava was identified to be related to missing expression of BEI and a nonsense mutation in GBSSI gene leading to amylose free starch. A pink phenotype showed no expression of CasLYB gene, and a yellow phenotype a down regulation of CasHYb. Proteomic analysis of carotenoid-protein complex together with gene expression analysis of CAP4 revealed a heteroduplex double strand cDNA associated with high carotenoid content. GBSSI gene sequencing identified 22 haplotypes and large nucleotide diversity. Segregating populations by crossing differential biochemical phenotypes and parents adapted to Cerrado's Region were obtained.

Tissue microarray (TMA) technology is a high-throughput research tool, which has greatly facilitated and accelerated tissue analyses by in-situ technologies. TMAs are amenable to every research method that can be applied on the standard whole sections at enhanced speed. It plays a central role in target verification of results from cDNA arrays, expression profiling of tumors and tissues, and is proving to be a powerful platform for proteomic research. In this review article, primarily meant for students of pathology and oncology, we briefly discuss its basic methodology, applications and merits and limitations.

This chapter contains sections titled: Introduction Concluding Remarks References

Goals of Dissertation Research. Cocaine use and abuse is a burden on society with high treatment costs and high relapse rates despite rehabilitation efforts. Many studies have tried to identify changes in gene expression that are associated cocaine abuse (Chapter 1A); however, the goal of this dissertation research was to identify molecules that may play a role not only in cocaine abuse, but also in relapse-associated behaviors. The central hypothesis of this research was that increases in drug-seeking and drug-taking behavior, which occur during periods of abstinence from cocaine self-administration, produce (or are caused by) differential regulation of the proteomic and gene expression profiles in brain regions associated with drug abuse. Identification of these changes may help to elucidate the molecular mechanisms of abstinence-induced behaviors and provide targets for therapeutic intervention. Specific Aims. Specific Aim 1: Testing the hypothesis that enforced abstinence from cocaine self-administration is accompanied by protein expression changes in the mesolimbic system (Chapter 2). A large-scale discovery experiment was conducted using 2-dimensional differential in-gel electrophoresis (2D-DIGE), to elucidate protein expression changes that occur during abstinence from cocaine self-administration in the medial prefrontal cortex. Specific Aim 2: Modify existing methods to isolate high-quality synaptosomes from brain tissue and examine the idea that changes occur in the expression of proteins associated with exocytosis and endocytosis at the synapse during periods of abstinence from cocaine self-administration (Chapter 3). A directed study of protein expression was conducted on isolated synaptosomes to directly look at the effect of enforced abstinence on this sub-cellular compartment. Specific Aim 3: Testing the hypothesis that enforced abstinence from cocaine self-administration is accompanied by gene expression changes in the mesolimbic system (Chapter 4). A whole-genome microarray analysis of the medial prefrontal cortex and nucleus accumbens was conducted to identify changes in mRNA expression that result from cocaine self-administration followed by increasing periods of abstinence. Original Breakthroughs and Findings. Identified in these studies was a panel of changes that occur following cocaine self-administration and increasing periods of enforced abstinence. Using a systems biology approach, the combined analysis of these changes reveals possible mechanisms by which relapse-associated behaviors incubate over periods of abstinence from cocaine. Such mechanisms include changes in neuronal structure and synaptic plasticity, signal transduction (specifically MAPK, TNF, and calcium signaling), and changes in glial cell function. Additionally, preliminary epigenetic analysis of identified changes in mRNA expression (Appendix) illuminates a possible mechanism by which persistent changes in gene and protein expression may occur. Together, these data open the door for future examination of the functional effect of these changes, as well as providing possible targets for therapeutic intervention.

Proteomics plays a vital role in biomedical research in the post-genomic era. With the technological revolution and emerging computational and statistic models, proteomic methodology has evolved rapidly in the past decade and shed light on solving complicated biomedical problems. Here, we summarize scientific research and clinical practice of existing and emerging high-throughput proteomics approaches, including mass spectrometry, protein pathway array, next-generation tissue microarrays, single-cell proteomics, single-molecule proteomics, Luminex, Simoa and Olink Proteomics. We also discuss important computational methods and statistical algorithms that can maximize the mining of proteomic data with clinical and/or other ‘omics data. Various principles and precautions are provided for better utilization of these tools. In summary, the advances in high-throughput proteomics will not only help better understand the molecular mechanisms of pathogenesis, but also to identify the signature signaling networks of specific diseases. Thus, modern proteomics have a range of potential applications in basic research, prognostic oncology, precision medicine, and drug discovery.