Strategies for controlling afucosylation in monoclonal antibodies during upstream manufacturing.

Core fucosylation is a highly prevalent and significant feature of N-glycosylation in therapeutic monoclonal antibodies produced by mammalian cells where its absence (afucosylation) plays a key role in treatment safety and efficacy. Notably, even slight changes in the level of afucosylation can have a considerable impact on the antibody-dependent cell-mediated cytotoxicity. Therefore, implementing control over afucosylation levels is important in upstream manufacturing to maintain consistent quality across batches of product, since standard downstream processing does not change afucosylation.

Repurposing antimalarial aminoquinolines and related compounds for treatment of retinal neovascularization.

Neovascularization is the pathological driver of blinding eye diseases such as retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. The loss of vision resulting from these diseases significantly impacts the productivity and quality of life of patients, and represents a substantial burden on the health care system. Current standard of care includes biologics that target vascular endothelial growth factor (VEGF), a key mediator of neovascularization.

A Systematic Approach to Identify Biased Agonists of the Apelin Receptor through High-Throughput Screening.

Biased agonists are defined by their ability to selectively activate distinct signaling pathways of a receptor, and they hold enormous promise for the development of novel drugs that specifically elicit only the desired therapeutic response and avoid potential adverse effects. Unfortunately, most high-throughput screening (HTS) assays are designed to detect signaling of G protein-coupled receptors (GPCRs) downstream of either G protein or β-arrestin-mediated signaling but not both. A comprehensive drug discovery program seeking biased agonists must employ assays that report on the activity of each compound at multiple discrete pathways, particularly for HTS campaigns.

In Vivo Rapid Assessment of Compound Exposure (RACE) for Profiling the Pharmacokinetics of Novel Chemical Probes.

The Rapid Assessment of Compound Exposure (RACE) assay is an easy and efficient method for estimating the pharmacokinetic parameter of exposure (AUC: area under the curve) of novel chemical probe compounds in mice. RACE is a truncated and compressed version of a traditional comprehensive in vivo pharmacokinetics study. The method uses a single standard formulation, dose, route of administration, and a small cohort of mice (n = 4).

Metabolic regulation of an fnr gene knockout Escherichia coli under oxygen limitation.

In addition to our previous study on the effect of fnr gene knockout on the metabolism in Escherichia coli under aerobic conditions (Kumar and Shimizu, Microb Cell Fact 2011), here we further investigated the effect of fnr gene knockout on the metabolism under micro-aerobic condition based on gene expressions, enzyme activities and intracellular metabolic fluxes. The objective of the present research is to clarify the metabolic regulation mechanism on how the culture environment, such as oxygen level, affects the cell metabolism in relation to gene expressions, enzyme activities and fluxes via global regulators such as Fnr and ArcA/B systems. Under micro-aerobic condition, the flux through Pfl and Frd were reduced for the mutant, which are due to fnr gene knockout.

Apelin protects against angiotensin II-induced cardiovascular fibrosis and decreases plasminogen activator inhibitor type-1 production.

Objective: To test the hypothesis that apelin protects against angiotensin II (Ang II)-induced cardiovascular fibrosis and vascular remodeling.

Methods And Results: Wild-type mice administered apelin or apelin along with Ang II exhibited less cardiovascular fibrosis and decreased plasminogen activator inhibitor type-1 (PAI-1) gene expression than mice receiving Ang II, N-nitro-L-arginine methyl ester (L-NAME), apelin plus L-NAME or apelin plus Ang II plus L-NAME. In-vitro analysis using a luciferase construct driven by 3.

Effect of cra gene knockout together with edd and iclR genes knockout on the metabolism in Escherichia coli.

To elucidate the physiological adaptation of Escherichia coli due to cra gene knockout, a total of 3,911 gene expressions were investigated by DNA microarray for continuous culture. About 50 genes were differentially regulated for the cra mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose phosphate (PP) pathway and Entner Doudoroff (ED) pathway were up-regulated in the cra mutant.

An oxazole-based small-molecule Stat3 inhibitor modulates Stat3 stability and processing and induces antitumor cell effects.

Stat3 is hyperactivated in many human tumors and represents a valid target for anticancer drug design. We present a novel small-molecule Stat3 inhibitor, S3I-M2001, and describe the dynamics of intracellular processing of activated Stat3 within the context of the biochemical and biological effects of the Stat3 inhibitor. S3I-M2001 is an oxazole-based peptidomimetic of the Stat3 Src homology (SH) 2 domain-binding phosphotyrosine peptide that selectively disrupts active Stat3:Stat3 dimers.

Selective chemical probe inhibitor of Stat3, identified through structure-based virtual screening, induces antitumor activity.

S3I-201 (NSC 74859) is a chemical probe inhibitor of Stat3 activity, which was identified from the National Cancer Institute chemical libraries by using structure-based virtual screening with a computer model of the Stat3 SH2 domain bound to its Stat3 phosphotyrosine peptide derived from the x-ray crystal structure of the Stat3beta homodimer. S3I-201 inhibits Stat3.Stat3 complex formation and Stat3 DNA-binding and transcriptional activities.

Isoform selective inhibition of STAT1 or STAT3 homo-dimerization via peptidomimetic probes: structural recognition of STAT SH2 domains.

The identification of constitutively activated STAT (Signal Transducers and Activators of Transcription) proteins in aberrant cell signaling pathways has led to investigations targeting the selective disruption of specific STAT isoforms directly associated with oncogenisis. We have identified, through the design of a library of peptidomimetic inhibitors, agents that selectively disrupt STAT1 or STAT3 homo-dimerization at low micromolar concentrations. ISS840 has 20-fold higher inhibition of STAT1 homo-dimerization (IC(50) value of 31 microM) relative to STAT3 (IC(50) value of 560 microM).

Resveratrol inhibits Src and Stat3 signaling and induces the apoptosis of malignant cells containing activated Stat3 protein.

Resveratrol is a naturally occurring phytoalexin with antioxidant and antiinflammatory properties. Recent studies suggest that resveratrol possesses anticancer effects, although its mechanism of action is not well understood. We now show that resveratrol inhibits Src tyrosine kinase activity and thereby blocks constitutive signal transducer and activator of transcription 3 (Stat3) protein activation in malignant cells.

Effect of a pyruvate kinase (pykF-gene) knockout mutation on the control of gene expression and metabolic fluxes in Escherichia coli.

The metabolic regulation of Escherichia coli lacking a functional pykF gene was investigated based on gene expressions, enzyme activities, intracellular metabolite concentrations and the metabolic flux distribution obtained based on (13)C-labeling experiments. RT-PCR revealed that the glycolytic genes such as glk, pgi, pfkA and tpiA were down regulated, that ppc, pckA, maeB and mdh genes were strongly up-regulated, and that the oxidative pentose phosphate pathway genes such as zwf and gnd were significantly up-regulated in the pykF mutant. The catabolite repressor/activator gene fruR was up-regulated in the pykF mutant, but the adenylate cyclase gene cyaA was down-regulated indicating a decreased rate of glucose uptake.