Transforming growth factor ␤ (TGF␤) signals primarily through the Smad proteins to regulate cell growth, differentiation, and extracellular matrix production. Post-translational modifications, such as phosphorylation, play an important role in the regulation of the Smad proteins. TGF␤ signaling results in the phosphorylation of Smad2 and Smad3 that then oligomerize with Smad4 and translocate into the nucleus to initiate transcription of TGF␤ target genes. The initiation of transcription is significantly enhanced by the direct interaction of the Smad complex with p300/CBP (CREB-binding protein), a co-activator with intrinsic acetyltransferase activity. However, how p300/ CBP enhances transcription through this interaction is not entirely understood. In this report, we show that Smad2, but not the highly homologous Smad3, can be acetylated by p300/CBP in a ligand-dependent manner. At least three lysine residues, Lys 19 , Lys 20 , and Lys 39 , are required for efficient acetylation of Smad2, as mutations altering these lysines abolished Smad2 acetylation in vivo. This acetylation event is required for the ability of Smad2 to mediate activin and TGF␤ signaling. Mutation of the three key lysine residues did not alter the stability of Smad2 or the ability of Smad2 to form a complex with Smad4 on promoter DNA, but it prevented nuclear accumulation of Smad2 and subsequent TGF␤ and activin responses. Thus, our studies reveal a novel mechanism of modulating Smad2 activity and localization through protein acetylation.

Key Points• BCL-2 homology domain 3 mimetic inhibitor ABT-737 targets leukemia initiating cells and progenitors.• Dephosphorylates RAS signaling proteins and regulates proliferation and differentiation genes detected by gene expression profiling.Myelodysplastic syndrome (MDS) transforms into an acute myelogenous leukemia (AML) with associated increased bone marrow (BM) blast infiltration. Using a transgenic mouse model, MRP8[NRASD12/hBCL-2], in which the NRAS:BCL-2 complex at the mitochondria induces MDS progressing to AML with dysplastic features, we studied the therapeutic potential of a BCL-2 homology domain 3 mimetic inhibitor, ABT-737. Treatment significantly extended lifespan, increased survival of lethally irradiated secondary recipients transplanted with cells from treated mice compared with cells from untreated mice, with a reduction of BM blasts, Lin-/Sca-1 1 /c-Kit 1 , and progenitor populations by increased apoptosis of infiltrating blasts of diseased mice assessed in vivo by technicium-labeled annexin V single photon emission computed tomography and ex vivo by annexin V/7-amino actinomycin D flow cytometry, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, caspase 3 cleavage, and re-localization of the NRAS:BCL-2 complex from mitochondria to plasma membrane. Phosphoprotein analysis showed restoration of wild-type (WT) AKT or protein kinase B, extracellular signal-regulated kinase 1/2 and mitogen-activated protein kinase patterns in spleen cells after treatment, which showed reduced mitochondrial membrane potential. Exon specific gene expression profiling corroborates the reduction of leukemic cells, with an increase in expression of genes coding for stem cell development and maintenance, myeloid differentiation, and apoptosis. Myelodysplastic features persist underscoring targeting of BCL-2-mediated effects on MDS-AML transformation and survival of leukemic cells. (Blood. 2013; 122(16):2864-2876

Characterization of charge heterogeneity of recombinant monoclonal antibodies (mAbs) requires high throughput analytical methods to support clone selection and formulation screens. We applied the NanoPro technology to rapidly measure relative charge distribution of mAbs in early stage process development. The NanoPro is a multiplexed capillary-based isoelectric immunoassay with whole-column imaging detection. This assay offers specificity, speed and sensitivity advantages over conventional capillary isoelectric focusing (CIEF) platforms. After CIEF, charge variants are photochemically immobilized to the wall of a short coated capillary. Once immobilized, mAbs are probed using a secondary anti-IgG conjugated with horseradish peroxidase. After flushing away excess reagents, secondary antibodies bound to their targets are then detected by chemiluminescence upon incubation with peroxidase reactive substrates. Charge heterogeneity as determined by chemiluminescence was similar to that measured by conventional CIEF technology with absorbance detection for purified mAbs and contaminated mAbs derived directly from host cellular extract. Upon method optimization, the automated CIEF immunoassay was applied to several mAbs of varying isoelectric points, demonstrating the suitability of NanoPro as a rugged high-throughput product characterization tool. Furthermore, qualification of detection sensitivity, precision, and dynamic range are reported with discussion of its advantages as an alternative approach to rapidly characterize charge variants during process development of mAbs.

Microarrays can be used to simultaneously measure the differential expression states of many mRNA's in two samples. Such measurements are limited by systematic and random errors. Systematic errors include labeling bias, imperfect feature morphologies, mismatched sample concentrations, and cross-hybridization. Random errors arise from chemical and scanning noise, particularly for low signals. We have used a combination of fluor-exchanged two-color labeling and improved normalization methods to minimize systematic errors from labeling bias, imperfect features, and mismatched sample concentrations. On-array specificity control probes and experimentally proven probe design algorithms were used to correct for cross-hybridization. Random errors were reduced via automated non-uniform feature flagging and an advanced scanner design. We have scored feature significance, using established statistical tests. We have then estimated the intrinsic random measurement error as a function of average probe signal via sample self-comparison experiments (human K-562 cell mRNA). Finally, we have combined all of these tools in the analysis of differential expression measurements between K-562 cells and HeLa cells. The results establish the importance of the elimination of systematic errors and the objective assessment of the effects of random errors in producing reliable estimates of differential expression.

Abstract-We seek to understand the current state of equity, scalability, and sustainability of data science education infrastructure in both the U.S. and Canada. Our analysis of the technological, funding, and organizational structure of four types of institutions shows an increasing divergence in the ability of universities across the United States to provide students with accessible data science education infrastructure, primarily JupyterHub. We observe that generally liberal arts colleges, community colleges, and other institutions with limited IT staff and experience have greater difficulty setting up and maintaining JupyterHub, compared to well-funded private institutions or large public research universities with a deep technical bench of IT staff. However, by leveraging existing public-private partnerships and the experience of Canada's national JupyterHub (Syzygy), the U.S. has an opportunity to provide a wider range of institutions and students access to JupyterHub.