Automated in vivo compound screening with zebrafish and the discovery and validation of PD 81,723 as a novel angiogenesis inhibitor.

Angiogenesis is a critical process in tumor progression. Inhibition of angiogenesis by blocking VEGF signaling can impair existing tumor vessels and halt tumor progression. However, the benefits are transient, and most patients who initially respond to these therapies develop resistance. Accordingly, there is a need for new anti-angiogenesis therapeutics to delay the processes of resistance or eliminate the resistive effects entirely. This manuscript presents the results of a screen of the National Institutes of Health Clinical Collections Libraries I & II (NIHCCLI&II) for novel angiogenesis inhibitors. The 727 compounds of the NIHCCLI&II library were screened with a high-throughput drug discovery platform (HTP) developed previously with angiogenesis-specific protocols utilizing zebrafish. The screen resulted in 14 hit compounds that were subsequently narrowed down to one, with PD 81,723 chosen as the lead compound. PD 81,723 was validated as an inhibitor of angiogenesis in vivo in zebrafish and in vitro in human umbilical vein endothelial cells (HUVECs). Zebrafish exposed to PD 81,723 exhibited several signs of a diminished endothelial network due to the inhibition of angiogenesis. Immunochemical analysis did not reveal any significant apoptotic or mitotic activity in the zebrafish. Assays with cultured HUVECs elucidated the ability of PD 81,723 to inhibit capillary tube formation, migration, and proliferation of endothelial cells. In addition, PD 81,723 did not induce apoptosis while significantly down regulating p21, AKT, VEGFR-2, p-VEGFR-2, eNOS, and p-eNOS, with no notable change in endogenous VEGF-A in cultured HUVECs.

Protocol development for discovery of angiogenesis inhibitors via automated methods using zebrafish.

Their optical clarity as larvae and embryos, small size, and high fecundity make zebrafish ideal for whole animal high throughput screening. A high-throughput drug discovery platform (HTP) has been built to perform fully automated screens of compound libraries with zebrafish embryos. A Tg(kdrl:EGFP) line, marking endothelial cell cytoplasm, was used in this work to help develop protocols and functional algorithms for the system, with the intent of screening for angiogenesis inhibitors. Indirubin 3' Monoxime (I3M), a known angiogenesis inhibitor, was used at various concentrations to validate the protocols. Consistent with previous studies, a dose dependant inhibitory effect of I3M on angiogenesis was confirmed. The methods and protocols developed here could significantly increase the throughput of drug screens, while limiting human errors. These methods are expected to facilitate the discovery of novel anti-angiogenesis compounds and can be adapted for many other applications in which samples have a good fluorescent signal.

Sialic acid catabolism by N-acetylneuraminate pyruvate lyase is essential for muscle function.

Sialic acids are important components of glycoproteins and glycolipids essential for cellular communication, infection, and metastasis. The importance of sialic acid biosynthesis in human physiology is well illustrated by the severe metabolic disorders in this pathway. However, the biological role of sialic acid catabolism in humans remains unclear. Here, we present evidence that sialic acid catabolism is important for heart and skeletal muscle function and development in humans and zebrafish. In two siblings, presenting with sialuria, exercise intolerance/muscle wasting, and cardiac symptoms in the brother, compound heterozygous mutations [chr1:182775324C>T (c.187C>T; p.Arg63Cys) and chr1:182772897A>G (c.133A>G; p.Asn45Asp)] were found in the N-acetylneuraminate pyruvate lyase gene (NPL). In vitro, NPL activity and sialic acid catabolism were affected, with a cell-type-specific reduction of N-acetyl mannosamine (ManNAc). A knockdown of NPL in zebrafish resulted in severe skeletal myopathy and cardiac edema, mimicking the human phenotype. The phenotype was rescued by expression of wild-type human NPL but not by the p.Arg63Cys or p.Asn45Asp mutants. Importantly, the myopathy phenotype in zebrafish embryos was rescued by treatment with the catabolic products of NPL: N-acetyl glucosamine (GlcNAc) and ManNAc; the latter also rescuing the cardiac phenotype. In conclusion, we provide the first report to our knowledge of a human defect in sialic acid catabolism, which implicates an important role of the sialic acid catabolic pathway in mammalian muscle physiology, and suggests opportunities for monosaccharide replacement therapy in human patients.

An inkjet-printed bioactive paper sensor that reports ATP through odour generation.

We describe an inkjet printed paper-based sensor that reports ATP by the enzyme catalysed hydrolysis of S-methyl-L-cysteine generating an odour (methyl mercaptan) that is easily detectable by the human nose.

Tuning cellulose nanocrystal gelation with polysaccharides and surfactants.

Gelation of cellulose nanocrystal (CNC) dispersions was measured as a function of the presence of four nonionic polysaccharides. Addition of hydroxyethyl cellulose (HEC), hydroxypropyl guar (HPG), or locust bean gum (LBG) to CNC dispersions induced the gelation of dilute CNC dispersions, whereas dextran (DEX) did not. These behaviors correlated with adsorption tendencies; HEC, HPG, and LBG adsorbed onto CNC-coated quartz crystal microbalance sensors, whereas DEX did not adsorb. We propose that the adsorbing polysaccharides greatly increased the effective volume fraction of dilute CNC dispersions, driving more of the nanocrystals into anisotropic domains. SDS and Triton X-100 addition disrupted HEC-CNC gels whereas CTAB did not. Surface plasmon resonance measurements with CNC-coated sensors showed that SDS and Triton X-100 partially removed adsorbed HEC, whereas CTAB did not. These behaviors illustrate the complexities associated with including CNC dispersions in formulated products: low CNC contents can induce spectacular changes in rheology; however, surfactants and soluble polymers may promote gel formation or induce CNC coagulation.

Centrifugal seeding of mammalian cells in nonwoven fibrous matrices.

Three-dimensional (3D) cell cultures have many advantages over two-dimensional cultures. However, seeding cells in 3D scaffolds such as nonwoven fibrous polyethylene terephthalate (PET) matrices has been a challenge task in tissue engineering and cell culture bioprocessing. In this study, a centrifugal seeding method was investigated to improve the cell seeding efficiency in PET matrices with two different porosities (93% and 88%). Both the centrifugal force and centrifugation time were found to affect the seeding efficiency. With an appropriate centrifugation speed, a high 80-90% cell seeding efficiency was achieved and the time to reach this high seeding efficiency was less than 5 min. The seeding efficiency was similar for matrices with different porosities, although the optimal seeding time was significantly shorter for the low-porosity scaffold. Post seeding cell viability was demonstrated by culturing colon cancer cells seeded in PET matrices for over 5 days. The centrifugal seeding method developed in this work can be used to efficiently and uniformly seed small fibrous scaffolds for applications in 3D cell-based assays for high-throughput screening.

Long-term culturing of undifferentiated embryonic stem cells in conditioned media and three-dimensional fibrous matrices without extracellular matrix coating.

ESCs have unlimited proliferation potential and capability to differentiate into all tissue types. They are ideal cell sources for tissue engineering and cell therapy, but their supplies are limited. Current in vitro ESC cultures are carried out in tissue flasks with the surface precoated with extracellular matrix (ECM) proteins. T-flask cultures also require frequent subculturing because their limited surface area cannot support long-term growth of ESCs. In this work, ECM coating and frequent subculturing required in two-dimensional (2D) cultures were circumvented by culturing murine ESCs in three-dimensional (3D) polyethylene terephthalate (PET) fibrous matrices. Also, media conditioned with STO fibroblast cells were used to replace leukemia inhibitory factor and to effectively maintain the pluripotency of murine ESCs in a long-term static culture. However, the lactic acid present in the conditioned medium could inhibit ESC growth and induce spontaneous differentiation when its concentration exceeded 1.5 g/l. In addition, the 3D static culture could be limited by oxygen, which was depleted in the long-term culture when cell density in the matrix was high. However, these problems can be alleviated in dynamic culture with improved oxygen transfer and continuous media perfusion. The matrix pore size also had profound effects on ESCs. The smaller-pore (30-60 mum) matrix gave a higher proliferation rate and Oct-4 and stage specific embryonic antigen-1 expressions. Overall, the 3D culturing method is superior to the 2D culture method and can provide an economical way to mass-produce undifferentiated ESCs in uncoated matrices and conditioned media.