Physicochemical and biological stability of diluted vedolizumab in intravenous infusion bags.

Introduction: Intravenous 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.

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.

How tech-savvy employees make the difference in core facilities: Recognizing core facility expertise with dedicated career tracks: Recognizing core facility expertise with dedicated career tracks.

Core facilities have a different mission than academic research labs. Accordingly, they require different career paths and structures.

Non-cell autonomous and spatiotemporal signalling from a tissue organizer orchestrates root vascular development.

During plant development, a precise balance of cytokinin is crucial for correct growth and patterning, but it remains unclear how this is achieved across different cell types and in the context of a growing organ. Here we show that in the root apical meristem, the TMO5/LHW complex increases active cytokinin levels via two cooperatively acting enzymes. By profiling the transcriptomic changes of increased cytokinin at single-cell level, we further show that this effect is counteracted by a tissue-specific increase in CYTOKININ OXIDASE 3 expression via direct activation of the mobile transcription factor SHORTROOT.

Off-target glycans encountered along the synthetic biology route toward humanized N-glycans in Pichia pastoris.

The glycosylation pathways of several eukaryotic protein expression hosts are being engineered to enable the production of therapeutic glycoproteins with humanized application-customized glycan structures. In several expression hosts, this has been quite successful, but one caveat is that the new N-glycan structures inadvertently might be substrates for one or more of the multitude of endogenous glycosyltransferases in such heterologous background. This then results in the formation of novel, undesired glycan structures, which often remain insufficiently characterized.

COSY catalyses trans-cis isomerization and lactonization in the biosynthesis of coumarins.

Coumarins, also known as 1,2-benzopyrones, comprise a large class of secondary metabolites that are ubiquitously found throughout the plant kingdom. In many plant species, coumarins are particularly important for iron acquisition and plant defence. Here, we show that COUMARIN SYNTHASE (COSY) is a key enzyme in the biosynthesis of coumarins.

IL-33trap is a novel IL-33-neutralizing biologic that inhibits allergic airway inflammation.

Background: The emergence of IL-33 as a key molecular player in the development and propagation of widespread inflammatory diseases, including asthma and atopic dermatitis, has established the need for effective IL-33-neutralizing biologics.

Objective: Here we describe the development and validation of a new antagonist of IL-33, termed IL-33trap, which combines the extracellular domains of the IL-33 receptor (ST2) and its coreceptor, IL-1 receptor accessory protein, into a single fusion protein.

Methods: We produced and purified recombinant IL-33trap from human cells and analyzed its IL-33-binding affinity and IL-33 antagonistic activity in cultured cells and mice.

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.

Identification and characterization of Nanobodies targeting the EphA4 receptor.

The ephrin receptor A4 (EphA4) is one of the receptors in the ephrin system that plays a pivotal role in a variety of cell-cell interactions, mostly studied during development. In addition, EphA4 has been found to play a role in cancer biology as well as in the pathogenesis of several neurological disorders such as stroke, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease. Pharmacological blocking of EphA4 has been suggested to be a therapeutic strategy for these disorders.

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.

Generation of a new Gateway-compatible inducible lentiviral vector platform allowing easy derivation of co-transduced cells.

In contrast to most common gene delivery techniques, lentiviral vectors allow targeting of almost any mammalian cell type, even non-dividing cells, and they stably integrate in the genome. Therefore, these vectors are a very powerful tool for biomedical research. Here we report the generation of a versatile new set of 22 lentiviral vectors with broad applicability in multiple research areas.

Development and Validation of a Small Single-domain Antibody That Effectively Inhibits Matrix Metalloproteinase 8.

A detrimental role for matrix metalloproteinase 8 (MMP8) has been identified in several pathological conditions, e.g., lethal hepatitis and the systemic inflammatory response syndrome.

Generation and characterization of small single domain antibodies inhibiting human tumor necrosis factor receptor 1.

The cytokine TNF is a well known drug target for several inflammatory diseases such as Crohn disease. Despite the great success of TNF blockers, therapy could be improved because of high costs and side effects. Selective inhibition of TNF receptor (TNFR) 1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, thereby preserving the advantageous immunomodulatory signals mediated by TNFR2.

Phenylcoumaran benzylic ether reductase prevents accumulation of compounds formed under oxidative conditions in poplar xylem.

Phenylcoumaran benzylic ether reductase (PCBER) is one of the most abundant proteins in poplar (Populus spp) xylem, but its biological role has remained obscure. In this work, metabolite profiling of transgenic poplar trees downregulated in PCBER revealed both the in vivo substrate and product of PCBER. Based on mass spectrometry and NMR data, the substrate was identified as a hexosylated 8-5-coupling product between sinapyl alcohol and guaiacylglycerol, and the product was identified as its benzyl-reduced form.

Caffeoyl shikimate esterase (CSE) is an enzyme in the lignin biosynthetic pathway in Arabidopsis.

Lignin is a major component of plant secondary cell walls. Here we describe caffeoyl shikimate esterase (CSE) as an enzyme central to the lignin biosynthetic pathway. Arabidopsis thaliana cse mutants deposit less lignin than do wild-type plants, and the remaining lignin is enriched in p-hydroxyphenyl units.

Generation and in vivo characterization of a chimeric αvβ5-targeting antibody 14C5 and its derivatives.

Background: Previous studies showed that radiolabeled murine monoclonal antibody (mAb) 14C5 and its Fab and F(ab')2 fragments, targeting αvβ5 integrin, have promising properties for diagnostic and therapeutic applications in cancer. To diminish the risk of generating a human anti-mouse antibody response in patients, chimeric variants were created. The purpose of this study was to recombinantly produce chimeric antibody (chAb) derivatives of the murine mAb 14C5 and to evaluate the in vitro and in vivo characteristics.

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.

Fed-batch fermentation of GM-CSF-producing glycoengineered Pichia pastoris under controlled specific growth rate.

Background: Yeast expression systems with altered N-glycosylation are now available to produce glycoproteins with homogenous, defined N-glycans. However, data on the behaviour of these strains in high cell density cultivation are scarce.

Results: Here, we report on cultivations under controlled specific growth rate of a GlycoSwitch-Man5 Pichia pastoris strain producing Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) at high levels (hundreds of milligrams per liter).

Further pharmacological and genetic evidence for the efficacy of PlGF inhibition in cancer and eye disease.

Our findings that PlGF is a cancer target and anti-PlGF is useful for anticancer treatment have been challenged by Bais et al. Here we take advantage of carcinogen-induced and transgenic tumor models as well as ocular neovascularization to report further evidence in support of our original findings of PlGF as a promising target for anticancer therapies. We present evidence for the efficacy of additional anti-PlGF antibodies and their ability to phenocopy genetic deficiency or silencing of PlGF in cancer and ocular disease but also show that not all anti-PlGF antibodies are effective.

Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris.

Background: Tumour associated antigens on the surface of tumour cells, such as MUC1, are being used as specific antibody targets for immunotherapy of human malignancies. In order to address the poor penetration of full sized monoclonal antibodies in tumours, intermediate sized antibodies are being developed. The cost-effective and efficient production of these molecules is however crucial for their further success as anti-cancer therapeutics.

Role of magnesium in the failure of rhDNase therapy in patients with cystic fibrosis.

Background: In the management of cystic fibrosis (CF), rhDNase-I inhalation is widely used to facilitate the removal of the highly viscous and elastic mucus (often called sputum) from the lungs. However, an important group of CF patients does not benefit from rhDNase-I treatment. A study was undertaken to elucidate the reason for the failure of rhDNase-I in these patients and to evaluate strategies to overcome this.

Application of the restricted-access precolumn packing material alkyl-diol silica in a column-switching system for the determination of ketoprofen enantiomers in horse plasma.

The group of LiChrospher ADS (alkyl-diol silica) sorbents that make part of a unique family of restricted-access materials, have been developed as special packings for precolumns used in the LC-integrated sample processing of biofluids. The advantage of these sorbents lies in the direct injection of untreated biological fluids, that is without sample clean-up, the elimination of the protein matrix with a quantitative recovery together with an on-column enrichment. The present method is based on previous work applying UV detection at 260 nm for ketoprofen determinations.

Direct HPLC analysis of ketoprofen in horse plasma applying an ADS-restricted access-phase.

Making up part of the unique family of restricted access materials (RAM) the Lichrospher ADS (alkyl-diol silica) sorbents have been developed as special packing materials for precolumns used for LC-integrated sample processing of biofluids. The advantage of such phases consists of direct injection of untreated biological fluids without sample clean-up and elimination of the protein matrix together with an on-column enrichment. The plasma samples, with internal standard phenacetin added (not essential), were brought onto the precolumn (C-18 ADS, 25 micron, 25 x 4 mm i.