Immunohistochemical assessment of protein phosphorylation state: the dream and the reality

The development of phosphorylation state-specific antibodies (PSSAs) in the 1980s, and their subsequent proliferation promised to enable in situ analysis of the activation states of complex intracellular signaling networks. The extent to which this promise has been fulfilled is the topic of this review. I review some applications of PSSAs primarily in the assessment of solid tumor signaling pathway activation status. PSSAs have received considerable attention for their potential to reveal cell type-specific activation status, provide added prognostic information, aid in the prediction of response to therapy, and most recently, demonstrate the efficacy of kinase-targeted chemotherapies. However, despite some successes, many studies have failed to demonstrate added value of PSSAs over general antibody immunohistochemistry. Moreover, there is still a large degree of uncertainty about the interpretation of complex and heterogeneous staining patterns in tissue samples and their relationship to the actual phosphorylation states in vivo. The next phase of translational research in applications of PSSAs will entail the hard work of antibody validation, gathering of detailed information about epitope-specific lability, and implementation of methods for standardization

YK-4-279 Inhibits ERG and ETV1 Mediated Prostate Cancer Cell Invasion

Background: Genomic rearrangements involving the ETS family of transcription factors occur in 40–70 % of prostate cancer cases. ERG and ETV1 are the most common ETS members observed in these genetic alterations. The high prevalence of these rearrangements and their biological significance represents a novel therapeutic target for the treatment of prostate cancer. Methods and Findings: We recently reported the development of YK-4-279, a small molecule inhibitor of EWS-FLI1 oncoprotein in Ewing’s Sarcoma. Since ERG and ETV1 belong to the same class of ETS factors as FLI1, we tested the ability of YK-4-279 to inhibit biological functions of ERG and ETV1 proteins in prostate cancer. YK-4-279 inhibited ERG and ETV1 mediated transcriptional activity in a luciferase assay. YK-4-279 also decreased ERG and ETV1 downstream target mRNA and protein expression in ETV1-fusion positive LNCaP and ERG fusion positive VCaP cells. YK-4-279 reduced the motility of LNCaP cells in a scratch assay and the invasive phenotype of both LNCaP and VCaP cells in a HUVEC invasion assay. Fusion-negative PC3 cells were unresponsive to YK-4-279. SiRNA mediated ERG knockdown in VCaP cells resulted in a loss of drug responsiveness. Concurrently, transient ERG expression in PC-3 cells resulted in increased invasive potential, which was reduced by YK-4-279. Conclusion: These data demonstrate that YK-4-279 inhibits ERG and ETV1 biological activity in fusion-positive prostat

Rapid Transient Production in Plants by Replicating and Non-Replicating Vectors Yields High Quality Functional Anti-HIV Antibody

Background: The capacity of plants and plant cells to produce large amounts of recombinant protein has been well established. Due to advantages in terms of speed and yield, attention has recently turned towards the use of transient expression systems, including viral vectors, to produce proteins of pharmaceutical interest in plants. However, the effects of such high level expression from viral vectors and concomitant effects on host cells may affect the quality of the recombinant product. Methodology/Principal Findings: To assess the quality of antibodies transiently expressed to high levels in plants, we have expressed and characterised the human anti-HIV monoclonal antibody, 2G12, using both replicating and non-replicating systems based on deleted versions of Cowpea mosaic virus (CPMV) RNA-2. The highest yield (approximately 100 mg/kg wet weight leaf tissue) of affinity purified 2G12 was obtained when the non-replicating CPMV-HT system was used and the antibody was retained in the endoplasmic reticulum (ER). Glycan analysis by mass-spectrometry showed that the glycosylation pattern was determined exclusively by whether the antibody was retained in the ER and did not depend on whether a replicating or non-replicating system was used. Characterisation of the binding and neutralisation properties of all the purified 2G12 variants from plants showed that these were generally similar to those of the Chinese hamster ovary (CHO) cell-produced 2G12. Conclusions: Overall, the results demonstrate that replicating and non-replicating CPMV-based vectors are able to direct the production of a recombinant IgG similar in activity to the CHO-produced control. Thus, a complex recombinant protein was produced with no apparent effect on its biochemical properties using either high-level expression or viral replication. The speed with which a recombinant pharmaceutical with excellent biochemical characteristics can be produced transiently in plants makes CPMV-based expression vectors an attractive option for biopharmaceutical development and production

A Method for the Simultaneous Estimation of Selection Intensities in Overlapping Genes

Inferring the intensity of positive selection in protein-coding genes is important since it is used to shed light on the process of adaptation. Recently, it has been reported that overlapping genes, which are ubiquitous in all domains of life, seem to exhibit inordinate degrees of positive selection. Here, we present a new method for the simultaneous estimation of selection intensities in overlapping genes. We show that the appearance of positive selection is caused by assuming that selection operates independently on each gene in an overlapping pair, thereby ignoring the unique evolutionary constraints on overlapping coding regions. Our method uses an exact evolutionary model, thereby voiding the need for approximation or intensive computation. We test the method by simulating the evolution of overlapping genes of different types as well as under diverse evolutionary scenarios. Our results indicate that the independent estimation approach leads to the false appearance of positive selection even though the gene is in reality subject to negative selection. Finally, we use our method to estimate selection in two influenza A genes for which positive selection was previously inferred. We find no evidence for positive selection in both cases

Cell-to-Cell Transformation in Escherichia coli: A Novel Type of Natural Transformation Involving Cell-Derived DNA and a Putative Promoting Pheromone

Escherichia coli is not assumed to be naturally transformable. However, several recent reports have shown that E. coli can express modest genetic competence in certain conditions that may arise in its environment. We have shown previously that spontaneous lateral transfer of non-conjugative plasmids occurs in a colony biofilm of mixed E. coli strains (a set of a donor strain harbouring a plasmid and a plasmid-free recipient strain). In this study, with high-frequency combinations of strains and a plasmid, we constructed the same lateral plasmid transfer system in liquid culture. Using this system, we demonstrated that this lateral plasmid transfer was DNase-sensitive, indicating that it is a kind of transformation in which DNase-accessible extracellular naked DNA is essential. However, this transformation did not occur with purified plasmid DNA and required a direct supply of plasmid from co-existing donor cells. Based on this feature, we have termed this transformation type as ‘cell-to-cell transformation’. Analyses using medium conditioned with the high-frequency strain revealed that this strain released a certain factor(s) that promoted cell-to-cell transformation and arrested growth of the other strains. This factor is heat-labile and protease-sensitive, and its roughly estimated molecular mass was between ∼9 kDa and ∼30 kDa, indicating that it is a polypeptide factor. Interestingly, this factor was effective even when the conditioned medium was diluted 10–5–10–6, suggesting that it acts like a pheromone with high bioactivity. Based on these results, we propose that cell-to-cell transformation is a novel natural transformation mechanism in E. coli that requires cell-derived DNA and is promoted by a peptide pheromone. This is the first evidence that suggests the existence of a peptide pheromone-regulated transformation mechanism in E. coli and in Gram-negative bacteria

Deoxynivalenol and its toxicity

Deoxynivalenol (DON) is one of several mycotoxins produced by certain Fusarium species that frequently infect corn, wheat, oats, barley, rice, and other grains in the field or during storage. The exposure risk to human is directly through foods of plant origin (cereal grains) or indirectly through foods of animal origin (kidney, liver, milk, eggs). It has been detected in buckwheat, popcorn, sorgum, triticale, and other food products including flour, bread, breakfast cereals, noodles, infant foods, pancakes, malt and beer. DON affects animal and human health causing acute temporary nausea, vomiting, diarrhea, abdominal pain, headache, dizziness, and fever. This review briefly summarizes toxicities of this mycotoxin as well as effects on reproduction and their antagonistic and synergic actions

Insight on genes affecting tuber development in potato upon <i>Potato spindle tuber viroid</i> (PSTVd) infection

Potato (Solanum tuberosum L) is a natural host of Potato spindle tuber viroid (PSTVd) which can cause characteristic symptoms on developing plants including stunting phenotype and distortion of leaves and tubers. PSTVd is the type species of the family Pospiviroidae, and can replicate in the nucleus and move systemically throughout the plant. It is not well understood how the viroid can affect host genes for successful invasion and which genes show altered expression levels upon infection. Our primary focus in this study is the identification of genes which can affect tuber formation since viroid infection can strongly influence tuber development and especially tuber shape. In this study, we used a large-scale method to identify differentially expressed genes in potato. We have identified defence, stress and sugar metabolism related genes having altered expression levels upon infection. Additionally, hormone pathway related genes showed significant up- or down-regulation. DWARF1/DIMINUTO, Gibberellin 7-oxidase and BEL5 transcripts were identified and validated showing differential expression in viroid infected tissues. Our study suggests that gibberellin and brassinosteroid pathways have a possible role in tuber development upon PSTVd infection

YangZheng XiaoJi exerts anti-tumour growth effects by antagonising the effects of HGF and its receptor, cMET, in human lung cancer cells

BACKGROUND: Hepatocyte growth factor (HGF) is a cytokine that has a profound effect on cancer cells by stimulating migration and invasion and acting as an angiogenic factor. In lung cancer, the factor also plays a pivotal role and is linked to a poor outcome in patients. In particular, HGF is known to work in combination with EGF on lung cancer cells. In the present study, we investigated the effect of a traditional Chinese medicine reported in cancer therapies, namely YangZheng XiaoJi (YZXJ) on lung cancer and on HGF mediated migration and invasion of lung cancer cells. METHODS: Human lung cancer cells, SKMES1 and A549 were used in the study. An extract from the medicine was used. Cell migration was investigated using the EVOS and by ECIS. Cell–matrix adhesion and in vitro invasion were assessed. In vivo growth of lung cancer was tested using an in vivo xenograft tumour model and activation of the HGF receptor in lung tumours by an immunofluorescence method. RESULTS: Both lung cancer cells increased their migration in response to HGF and responded to YZXJ by reducing their speed of migration. YZXJ markedly reduced the migration and in vitro invasiveness induced by HGF. It worked synergistically with PHA665752 and SU11274, HGF receptor inhibitors on the lung cancer cells both on HGF receptor activation and on cell functions. A combination of HGF and EGF resulted in a greater increase in cell migration, which was similarly inhibited by YZXJ, and in combination with the HGF receptor and EGF receptor inhibitors. In vivo, YZXJ reduced the rate of tumour growth and potentiated the effects of PHA665752 on tumour growth. It was further revealed that YZXJ significantly reduced the degree of phosphorylation of the HGF receptor in lung tumours. CONCLUSION: YZXJ has a significant role in reducing the migration, invasion and in vivo tumour growth of lung cancer and acts to inhibit the migratory and invasive effects induced by HGF and indeed by HGF/EGF. This effect is likely attributed to the inhibition of the HGF receptor activation. These results indicate that YZXJ has a therapeutic role in lung cancer and that combined strategy with methods to block HGF and EGF should be considered. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12967-015-0639-1) contains supplementary material, which is available to authorized users