CRISPR/Cas9 mediated knockout of rb1 and rbl1 leads to rapid and penetrant retinoblastoma development in Xenopus tropicalis

Retinoblastoma is a pediatric eye tumor in which bi-allelic inactivation of the Retinoblastoma 1 (RB1) gene is the initiating genetic lesion. Although recently curative rates of retinoblastoma have increased, there are at this time no molecular targeted therapies available. This is, in part, due to the lack of highly penetrant and rapid retinoblastoma animal models that facilitate rapid identification of targets that allow therapeutic intervention. Different mouse models are available, all based on genetic deactivation of both Rb1 and Retinoblastoma-like 1 (Rbl1), and each showing different kinetics of retinoblastoma development. Here, we show by CRISPR/Cas9 techniques that similar to the mouse, neither rb1 nor rbl1 single mosaic mutant Xenopus tropicalis develop tumors, whereas rb1/rbl1 double mosaic mutant tadpoles rapidly develop retinoblastoma. Moreover, occasionally presence of pinealoblastoma (trilateral retinoblastoma) was detected. We thus present the first CRISPR/Cas9 mediated cancer model in Xenopus tropicalis and the first genuine genetic non-mammalian retinoblastoma model. The rapid kinetics of our model paves the way for use as a pre-clinical model. Additionally, this retinoblastoma model provides unique possibilities for fast elucidation of novel drug targets by triple multiplex CRISPR/Cas9 gRNA injections (rb1 + rbl1 + modifier gene) in order to address the clinically unmet need of targeted retinoblastoma therapy

Maackia Amurensis Seed Lectin (MASL) and soluble human podoplanin (shPDPN) Sequence Analysis and Effects on Human Oral Squamous Cell Carcinoma (OSCC) Cell Migration and Viability

Maackia amurensis lectins serve as research and botanical agents that bind to sialic residues on proteins. For example, M. amurensis seed lectin (MASL) targets the sialic acid modified podoplanin (PDPN) receptor to suppress arthritic chondrocyte inflammation, and inhibit tumor cell growth and motility. However, M. amurensis lectin nomenclature and composition are not clearly defined. Here, we sought to definitively characterize MASL and its effects on tumor cell behavior. We utilized SDS-PAGE and LC-MS/MS to find that M. amurensis lectins can be divided into two groups. MASL is a member of one group which is composed of subunits that form dimers, evidently mediated by a cysteine residue in the carboxy region of the protein. In contrast to MASL, members of the other group do not dimerize under nonreducing conditions. These data also indicate that MASL is composed of 4 isoforms with an identical amino acid sequence, but unique glycosylation sites. We also produced a novel recombinant soluble human PDPN receptor (shPDPN) with 17 threonine residues glycosylated with sialic acid moieties with potential to act as a ligand trap that inhibits OSCC cell growth and motility. In addition, we report here that MASL targets PDPN with very strong binding kinetics in the nanomolar range. Moreover, we confirm that MASL can inhibit the growth and motility of human oral squamous cell carcinoma (OSCC) cells that express the PDPN receptor. Taken together, these data characterize M. amurensis lectins into two major groups based on their intrinsic properties, clarify the composition of MASL and its subunit isoform sequence and glycosylation sites, define sialic acid modifications on the PDPN receptor and its ability to act as a ligand trap, quantitate MASL binding to PDPN with KD in the nanomolar range, and verify the ability of MASL to serve as a potential anticancer agent

A concise guide to choosing suitable gene expression systems for recombinant protein production

This overview guides both novices and experienced researchers facing challenging targets to select the most appropriate gene expression system for producing a particular protein. By answering four key questions, readers can determine the most suitable gene expression system following a decision scheme. This guide addresses the most commonly used and accessible systems and provides brief descriptions of the main gene expression systems' key characteristics to assist decision making. Additionally, information has been included for selected less frequently used "exotic" gene expression systems

Role of glutamine synthetase in angiogenesis beyond glutamine synthesis

Glutamine synthetase, encoded by the gene GLUL, is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible glutamine-synthesizing activity in these cells at physiological glutamine levels. Here we show in mice that genetic deletion of Glul in endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cells GLUL knockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced by GLUL knockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to the known formation of glutamine, the enzyme glutamine synthetase shows unknown activity in endothelial cell migration during pathological angiogenesis through RHOJ palmitoylation

Production of antibody derivatives in the methylotrophic yeast Pichia pastoris

New antibody derivatives are continuously being generated to interact with a range of therapeutic targets. The cost-effective and efficient production of these and other antibody derivatives is crucial for their further success. Here, we describe the construction of the expression vectors needed for heterologous expression of a Fab fragment in the yeast Pichia pastoris. The experimental conditions for lab-scale expressions are discussed, and an overview of an efficient purification strategy is presented

Physicochemical and biological stability of diluted vedolizumab in intravenous infusion bags

IntroductionIntravenous vedolizumab is a widely used monoclonal antibody for outpatients with inflammatory bowel disease. Drug preparation is performed on the day of administration, but is time consuming, causing unnecessary in-hospital patient delay and inefficient logistics for preparation and distribution. Storage of vedolizumab ready-to-administer infusions and distribution via pneumatic air tubes could streamline logistics in the outpatient setting. The aim of this study was to test the shelf life and stability of ready-to-administer intravenous infusion bags containing vedolizumab.MethodsFor assessing in-use shelf life, the reconstituted product (300 mg fixed dose) was diluted to a concentration of 1.2 mg/mL in 0.9% NaCl under aseptic conditions, and stored in polyolefin infusion bags at 2-8 degrees C prior to analysis. On replicate samples, we measured concentration, physical and chemical stability using sodium dodecyl sulphate polyacrylamide gel electrophoresis, size exclusion chromatography, and multi-angle laser light scattering, as well as biological activity using a biolayer interferometry assay to study target engagement, and endotoxin content to assess microbiological stability. Stability of ready-to-use vedolizumab was assessed also after transportation via pneumatic tube system. Samples were taken at different time points over an observation period of 30 days on four replicate samples.ResultsFor all parameters assessed, the ready-to-use solution of vedolizumab remained stable over a period of at least 30 days. There were no signs of protein aggregation, chemical instability, or loss of binding of the antibody to the alpha 4 beta 7 integrin target. There was no increase in endotoxin concentration over time. No significant difference was seen in antibody structural stability and protein aggregation between samples before and after transportation via pneumatic tube system.ConclusionWhen prepared under aseptic conditions, dissolved ready-to-administer vedolizumab infusion bags can be stored long term at 2-8 degrees C and transported via pneumatic air tube, without observable loss of antibody stability or binding activity