The specificity and patterns of staining in human cells and tissues of p16INK4a antibodies demonstrate variant antigen binding

The validity of the identification and classification of human cancer using antibodies to detect biomarker proteins depends upon antibody specificity. Antibodies that bind to the tumour-suppressor protein p16INK4a are widely used for cancer diagnosis and research. In this study we examined the specificity of four commercially available anti-p16INK4a antibodies in four immunological applications. The antibodies H-156 and JC8 detected the same 16 kDa protein in western blot and immunoprecipitation tests, whereas the antibody F-12 did not detect any protein in western blot analysis or capture a protein that could be recognised by the H-156 antibody. In immunocytochemistry tests, the antibodies JC8 and H-156 detected a predominately cytoplasmic localised antigen, whose signal was depleted in p16INK4a siRNA experiments. F-12, in contrast, detected a predominately nuclear located antigen and there was no noticeable reduction in this signal after siRNA knockdown. Furthermore in immunohistochemistry tests, F-12 generated a different pattern of staining compared to the JC8 and E6H4 antibodies. These results demonstrate that three out of four commercially available p16INK4a antibodies are specific to, and indicate a mainly cytoplasmic localisation for, the p16INK4a protein. The F-12 antibody, which has been widely used in previous studies, gave different results to the other antibodies and did not demonstrate specificity to human p16INK4a. This work emphasizes the importance of the validation of commercial antibodies, aside to the previously reported use, for the full verification of immunoreaction specificity

HMGA1 drives stem cell, inflammatory pathway, and cell cycle progression genes during lymphoid tumorigenesis

<p>Abstract</p> <p>Background</p> <p>Although the <it>high mobility group A1 </it>(<it>HMGA1</it>) gene is widely overexpressed in diverse cancers and portends a poor prognosis in some tumors, the molecular mechanisms that mediate its role in transformation have remained elusive. <it>HMGA1 </it>functions as a potent oncogene in cultured cells and induces aggressive lymphoid tumors in transgenic mice. Because HMGA1 chromatin remodeling proteins regulate transcription, <it>HMGA1 </it>is thought to drive malignant transformation by modulating expression of specific genes. Genome-wide studies to define HMGA1 transcriptional networks during tumorigenesis, however, are lacking. To define the HMGA1 transcriptome, we analyzed gene expression profiles in lymphoid cells from <it>HMGA1a </it>transgenic mice at different stages in tumorigenesis.</p> <p>Results</p> <p>RNA from lymphoid samples at 2 months (before tumors develop) and 12 months (after tumors are well-established) was screened for differential expression of > 20,000 unique genes by microarray analysis (Affymetrix) using a parametric and nonparametric approach. Differential expression was confirmed by quantitative RT-PCR in a subset of genes. Differentially expressed genes were analyzed for cellular pathways and functions using Ingenuity Pathway Analysis. Early in tumorigenesis, HMGA1 induced inflammatory pathways with NFkappaB identified as a major node. In established tumors, HMGA1 induced pathways involved in cell cycle progression, cell-mediated immune response, and cancer. At both stages in tumorigenesis, HMGA1 induced pathways involved in cellular development, hematopoiesis, and hematologic development. Gene set enrichment analysis showed that stem cell and immature T cell genes are enriched in the established tumors. To determine if these results are relevant to human tumors, we knocked-down HMGA1 in human T-cell leukemia cells and identified a subset of genes dysregulated in both the transgenic and human lymphoid tumors.</p> <p>Conclusions</p> <p>We found that <it>HMGA1 </it>induces inflammatory pathways early in lymphoid tumorigenesis and pathways involved in stem cells, cell cycle progression, and cancer in established tumors. <it>HMGA1 </it>also dyregulates genes and pathways involved in stem cells, cellular development and hematopoiesis at both early and late stages of tumorigenesis. These results provide insight into <it>HMGA1 </it>function during tumor development and point to cellular pathways that could serve as therapeutic targets in lymphoid and other human cancers with aberrant <it>HMGA1 </it>expression.</p

Understanding Plant-Microbe Interactions for Phytoremediation of Petroleum-Polluted Soil

Plant-microbe interactions are considered to be important processes determining the efficiency of phytoremediation of petroleum pollution, however relatively little is known about how these interactions are influenced by petroleum pollution. In this experimental study using a microcosm approach, we examined how plant ecophysiological traits, soil nutrients and microbial activities were influenced by petroleum pollution in Phragmites australis, a phytoremediating species. Generally, petroleum pollution reduced plant performance, especially at early stages of plant growth. Petroleum had negative effects on the net accumulation of inorganic nitrogen from its organic forms (net nitrogen mineralization (NNM)) most likely by decreasing the inorganic nitrogen available to the plants in petroleum-polluted soils. However, abundant dissolved organic nitrogen (DON) was found in petroleum-polluted soil. In order to overcome initial deficiency of inorganic nitrogen, plants by dint of high colonization of arbuscular mycorrhizal fungi might absorb some DON for their growth in petroleum-polluted soils. In addition, through using a real-time polymerase chain reaction method, we quantified hydrocarbon-degrading bacterial traits based on their catabolic genes (i.e. alkB (alkane monooxygenase), nah (naphthalene dioxygenase) and tol (xylene monooxygenase) genes). This enumeration of target genes suggests that different hydrocarbon-degrading bacteria experienced different dynamic changes during phytoremediation and a greater abundance of alkB was detected during vegetative growth stages. Because phytoremediation of different components of petroleum is performed by different hydrocarbon-degrading bacteria, plants’ ability of phytoremediating different components might therefore vary during the plant life cycle. Phytoremediation might be most effective during the vegetative growth stages as greater abundances of hydrocarbon-degrading bacteria containing alkB and tol genes were observed at these stages. The information provided by this study enhances our understanding of the effects of petroleum pollution on plant-microbe interactions and the roles of these interactions in the phytoremediation of petroleum-polluted soil

Inter-Species Complementation of the Translocon Beta Subunit Requires Only Its Transmembrane Domain

In eukaryotes, proteins enter the secretory pathway through the translocon pore of the endoplasmic reticulum. This protein translocation channel is composed of three major subunits, called Sec61α, β and γ in mammals. Unlike the other subunits, the β subunit is dispensable for translocation and cell viability in all organisms studied. Intriguingly, the knockout of the Sec61β encoding genes results in different phenotypes in different species. Nevertheless, the β subunit shows a high level of sequence homology across species, suggesting the conservation of a biological function that remains ill-defined. To address its cellular roles, we characterized the homolog of Sec61β in the fission yeast Schizosaccharomyces pombe (Sbh1p). Here, we show that the knockout of sbh1+ results in severe cold sensitivity, increased sensitivity to cell-wall stress, and reduced protein secretion at 23°C. Sec61β homologs from Saccharomyces cerevisiae and human complement the knockout of sbh1+ in S. pombe. As in S. cerevisiae, the transmembrane domain (TMD) of S. pombe Sec61β is sufficient to complement the phenotypes resulting from the knockout of the entire encoding gene. Remarkably, the TMD of Sec61β from S. cerevisiae and human also complement the gene knockouts in both yeasts. Together, these observations indicate that the TMD of Sec61β exerts a cellular function that is conserved across species

Effects of Heavy Metals and Arbuscular Mycorrhiza on the Leaf Proteome of a Selected Poplar Clone: A Time Course Analysis

Arbuscular mycorrhizal (AM) fungi establish a mutualistic symbiosis with the roots of most plant species. While receiving photosynthates, they improve the mineral nutrition of the plant and can also increase its tolerance towards some pollutants, like heavy metals. Although the fungal symbionts exclusively colonize the plant roots, some plant responses can be systemic. Therefore, in this work a clone of Populus alba L., previously selected for its tolerance to copper and zinc, was used to investigate the effects of the symbiosis with the AM fungus Glomus intraradices on the leaf protein expression. Poplar leaf samples were collected from plants maintained in a glasshouse on polluted (copper and zinc contaminated) or unpolluted soil, after four, six and sixteen months of growth. For each harvest, about 450 proteins were reproducibly separated on 2DE maps. At the first harvest the most relevant effect on protein modulation was exerted by the AM fungi, at the second one by the metals, and at the last one by both treatments. This work demonstrates how importantly the time of sampling affects the proteome responses in perennial plants. In addition, it underlines the ability of a proteomic approach, targeted on protein identification, to depict changes in a specific pattern of protein expression, while being still far from elucidating the biological function of each protein

Inter-Species Complementation of the Translocon Beta Subunit Requires Only Its Transmembrane Domain

In eukaryotes, proteins enter the secretory pathway through the translocon pore of the endoplasmic reticulum. This protein translocation channel is composed of three major subunits, called Sec61α, β and γ in mammals. Unlike the other subunits, the β subunit is dispensable for translocation and cell viability in all organisms studied. Intriguingly, the knockout of the Sec61β encoding genes results in different phenotypes in different species. Nevertheless, the β subunit shows a high level of sequence homology across species, suggesting the conservation of a biological function that remains ill-defined. To address its cellular roles, we characterized the homolog of Sec61β in the fission yeast Schizosaccharomyces pombe (Sbh1p). Here, we show that the knockout of sbh1+ results in severe cold sensitivity, increased sensitivity to cell-wall stress, and reduced protein secretion at 23°C. Sec61β homologs from Saccharomyces cerevisiae and human complement the knockout of sbh1+ in S. pombe. As in S. cerevisiae, the transmembrane domain (TMD) of S. pombe Sec61β is sufficient to complement the phenotypes resulting from the knockout of the entire encoding gene. Remarkably, the TMD of Sec61β from S. cerevisiae and human also complement the gene knockouts in both yeasts. Together, these observations indicate that the TMD of Sec61β exerts a cellular function that is conserved across species

Strategies to Target Tumor Immunosuppression

The tumor microenvironment is currently in the spotlight of cancer immunology research as a key factor impacting tumor development and progression. While antigen-specific immune responses play a crucial role in tumor rejection, the tumor hampers these immune responses by creating an immunosuppressive microenvironment. Recently, major progress has been achieved in the field of cancer immunotherapy, and several groundbreaking clinical trials demonstrated the potency of such therapeutic interventions in patients. Yet, the responses greatly vary among individuals. This calls for the rational design of more efficacious cancer immunotherapeutic interventions that take into consideration the “immune signature” of the tumor. Multimodality treatment regimens that aim to enhance intratumoral homing and activation of antigen-specific immune effector cells, while simultaneously targeting tumor immunosuppression, are pivotal for potent antitumor immunity

Impact of an Interdisciplinary Food, Nutrition and Health Education Program for adolescent Brazilian volleyball players

ABSTRACT Objective: To evaluate the impact of an Interdisciplinary Food, Nutrition and Health Education Program on nutrition knowledge, intention to change eating behavior, and body dissatisfaction of adolescent volleyball players. Methods: The sample consisted of 10 female volleyball players from the juvenile category of the city of Santos, São Paulo, Brazil, who participated in a program with eight monthly meetings (one discussion group followed by six educational activities and one final discussion group for evaluation). Results: Nutrition knowledge, body perception, intention to change eating behavior, eating attitudes and practices were investigated using questionnaires and discussion groups before and after the athletes' participation in ludic activities designed to address nutrition strategies for athletic performance and healthy eating, and how to deal with pressure for results and self-image. Nutrition knowledge improved from 57.0%±9.9 to 63.0%±11.8 (p=0.03) of correct answers. The mean body distortion score did not change (70.0±14.9 versus 76.5±22.4, p=0.235). Six athletes advanced in their intention to change eating behavior. Positive food practices were reported during the program and the identified discourses indicated the intention of changing the daily eating habits in the future. Conclusion: The program had a positive impact on nutrition knowledge and intention of changing eating behavior; however, for other issues, especially involving emotional aspects, further interventions should be planned