Antibodies that recognise the active phosphorylated forms of mitogen-activated protein kinase (MAPK) kinase 5 (MKK5) and extracellular signal-regulated kinase 5 (ERK5) in untransfected cells have been exploited to show that the epidermal growth factor (EGF)-induced activation of MKK5 and ERK5 occurs subsequent to the activation of ERK1 and ERK2 in HeLa cells. The drugs U0126 and PD184352, which prevent the activation of MKK1 (and hence the activation of ERK1/ERK2), also prevent the activation of MKK5, although higher concentrations are required. Our studies define physiological targets of the MKK5/ERK5 pathway as proteins whose phosphorylation is largely prevented by 10 W WM PD184352, but unaffected by 2 W WM PD184352. Surprisingly, 2 W WM PD184352 prolongs the activation of MKK5 and ERK5 induced by EGF or H 2 O 2 , indicating negative control of the MKK5/ERK5 pathway by the classical MAPK cascade. Our results also indicate that ERK5 is not a significant activator of MAPK-activated protein kinase-1/RSK in HeLa cells. ß

Rho kinase is known to control smooth muscle contractility by phosphorylating the 110 kDa myosin-targetting subunit (MYPT1) of the myosin-associated form of protein phosphatase 1 (PP1M). Phosphorylation of MYPT1 at Thr695 has previously been reported to inhibit the catalytic activity of PP1. Here, we show that the phosphorylation of Thr850 by Rho kinase dissociates PP1M from myosin, providing a second mechanism by which myosin phosphatase activity is inhibited. ß

Plants respond to low temperatures by altering the mRNA abundance of thousands of genes contributing to numerous physiological and metabolic processes that allow them to adapt. At the post-transcriptional level, these cold stress-responsive transcripts undergo alternative splicing, microRNA-mediated regulation and alternative polyadenylation, amongst others. Recently, m 6 A, m 5 C and other mRNA modifications that can affect the regulation and stability of RNA were discovered, thus revealing another layer of posttranscriptional regulation that plays an important role in modulating gene expression. The importance of m 6 A in plant growth and development has been appreciated, although its significance under stress conditions is still underexplored. To assess the role of m 6 A modifications during cold stress responses, methylated RNA immunoprecipitation sequencing was performed in Arabidopsis seedlings esposed to low temperature stress (4°C) for 24 h. This transcriptome-wide m 6 A analysis revealed large-scale shifts in this modification in response to low temperature stress. Because m 6 A is known to affect transcript stability/ degradation and translation, we investigated these possibilities. Interestingly, we found that cold-enriched m 6 A-containing transcripts demonstrated the largest increases in transcript abundance coupled with increased ribosome occupancy under cold stress. The significance of the m 6 A epitranscriptome on plant cold tolerance was further assessed using the mta mutant in which the major m 6 A methyltransferase gene was mutated. Compared to the wild-type, along with the differences in CBFs and COR gene expression levels, the mta mutant exhibited hypersensitivity to cold treatment as determined by primary root growth, biomass, and reactive oxygen species accumulation. Furthermore, and most importantly, both non-acclimated and cold-acclimated mta mutant demonstrated hypersensitivity to freezing tolerance. Taken together, these findings suggest a critical role for the epitranscriptome in cold tolerance of Arabidopsis.

Developments in high-throughput sequencing (HTS) result in an exponential increase in the amount of data generated by sequencing experiments, an increase in the complexity of bioinformatics analysis reporting and an increase in the types of data generated. These increases in volume, diversity and complexity of the data generated and their analysis expose the necessity of a structured and standardized reporting template. BioCompute Objects (BCOs) provide the requisite support for communication of HTS data analysis that includes support for workflow, as well as data, curation, accessibility and reproducibility of communication. BCOs standardize how researchers report provenance and the established verification and validation protocols used in workflows while also being robust enough to convey content integration or curation in knowledge bases. BCOs that encapsulate tools, platforms, datasets and workflows are FAIR (findable, accessible, interoperable and reusable) compliant. Providing operational workflow and data information facilitates interoperability between platforms and incorporation of future dataset within an HTS analysis for use within industrial, academic and regulatory settings. Cloud-based platforms, including High-performance Integrated Virtual Environment (HIVE), Cancer Genomics Cloud (CGC) and Galaxy, support BCO generation for users. Given the 100K+ userbase between these platforms, BioCompute can be leveraged for workflow documentation. In this paper, we report the availability of platform-dependent and platform-independent BCO tools: HIVE BCO App, CGC BCO App, Galaxy BCO API Extension and BCO Portal. Community engagement was utilized to evaluate tool efficacy. We demonstrate that these tools further advance BCO creation from text editing approaches used in earlier releases of the standard. Moreover, we demonstrate that integrating BCO generation within existing analysis platforms greatly streamlines BCO creation while capturing granular workflow details. We also demonstrate that the BCO tools described in the paper provide an approach to solve the long-standing challenge of standardizing workflow descriptions that are both human and machine readable while accommodating manual and automated curation with evidence tagging. Database URL:  https://www.biocomputeobject.org/resources

Anthracyclines are a class of highly effective chemotherapy drugs used in the treatment of many cancers, including leukemias, lymphomas, breast, uterine, ovarian, bladder and lung cancers. Approximately 50% of childhood cancer treatment regimens include anthracyclines. Dose dependent cardiotoxicity is a major side effect of anthracyclines. Cancer survivors receiving anthracycline therapy are 15 times more likely to develop heart failure and 8 times more likely to die of cardiovascular disease. Dexrazoxane is the only clinically approved cardio-protectant that has been shown to reduce the risk of cardiotoxicity. However, it is only indicated for metastatic breast cancer and has been linked to secondary malignancies. It is, therefore, critical to develop safer and more effective cardio-protectant drugs to address this major unmet medical need. The recent advancement in generation of human cardiomyocytes from induced pluripotent stem cells (hiPSC-CMs) enables a biologically relevant setting for compound screening because hiPSC-CMs possess many similar functional characteristics as primary human cardiomyocytes and can be generated in significant quantities. We have established a platform to screen small compounds and natural product for efficacy and safety across multiple donor lines using hiPSC-CMs. Compounds to be screened were selected in collaboration with Capella Biosciences who used their SMarTR computational analysis platform to identify potential candidates for further screening in hiPSC-CMs. Using this novel drug discovery engine that combines bioinformatics-driven in-silico screening and hiPSC-CM-based compound screening platforms, we discovered four drugs that protect hiPSC-CMs from doxorubicin-induced cardiotoxicity. We used two cancer cell lines to confirm that these candidates do not interfere with doxorubicin’s anti-cancer activity. We also demonstrated that these candidates do not show acute cardiotoxicity mediated through ion channels. Thus, a unique platform combining SMarTR computational approaches and hiPSC-CMs screening technology allows for rapid identification of highly effective cardio-protective drugs.

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