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.

Bi-allelic GOT2 Mutations Cause a Treatable Malate-Aspartate Shuttle-Related Encephalopathy.

Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate diagnosis to guide proper management. Whole-exome sequencing was used to investigate the disease etiology in four children from independent families with intellectual disability and epilepsy, revealing bi-allelic GOT2 mutations. In-depth metabolic studies in individual 1 showed low plasma serine, hypercitrullinemia, hyperlactatemia, and hyperammonemia. The epilepsy was serine and pyridoxine responsive. Functional consequences of observed mutations were tested by measuring enzyme activity and by cell and animal models. Zebrafish and mouse models were used to validate brain developmental and functional defects and to test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate transaminase. GOT2 enzyme activity was deficient in fibroblasts with bi-allelic mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role in the intracellular NAD(H) redox balance. De novo serine biosynthesis was impaired in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the highly oxidized cytosolic NAD-redox state by pyruvate supplementation restored serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted in a brain developmental defect associated with seizure-like electroencephalography spikes, which could be rescued by supplying pyridoxine in embryo water. Both pyridoxine and serine synergistically rescued embryonic developmental defects in zebrafish got2a morphants. The two treated individuals reacted favorably to their treatment. Our data provide a mechanistic basis for the biochemical abnormalities in GOT2 deficiency that may also hold for other MAS defects.

Preparation and characterization of Ca(II) cross-linking modified pectin microspheres for Pb(II) adsorption.

A novel adsorbent, composed of cross-linked de-esterified pectin microspheres, was prepared via cross-linking with Ca(II) and modification by de-esterified pectin, low-methoxyl pectin (LMP) and pectic acid (PA). Fourier transform infrared (FTIR), energy dispersive spectrometry (EDS), scanning electron microscopy (SEM) and atomic absorption spectroscopy (AAS) were applied too, exhibiting a successful fabrication, good adsorption ability, and well-defined surface microstructure beneficial to Pb(II) adsorption. The adsorption ability of pectin microspheres (PMs), low-methoxyl pectin microspheres (LMPMs) and pectic acid microspheres (PAMs) for Pb(II) in aqueous solution were explored. The maximum adsorption capacity of PMs, LMPMs and PAMs was 127 mg·g-1, 292 mg·g-1 and 325 mg·g-1 at pH 5.0 respectively, indicating a great improvement of LMPMs and PAMs in the adsorption ability for Pb(II) compared with PMs. Furthermore, the adsorption mechanism was proposed. The experimental data were well fitted with pseudo-second-order kinetic and Langmuir isotherm models. Five-cycle reusability tests demonstrated that microspheres could be used repeatedly. All the results confirmed that LMPMs and PAMs, which presented outstanding adsorption capability and reusability, could be a good candidate for wastewater purification.

Effect of auricular points treatment combined with acupoints application in patients with constipation after lung cancer surgery.

OBJECT: To assess the effect of auricular points treatment combined with acupoints application on patient with constipation after lung cancer surgery. METHODS: Design and participants: This is a single-center randomized controlled trial. Totally 341 after lung cancer surgery patients were randomly assigned into the experimental group (n = 174) and the control group (n = 167). The control group received routine nursing care, which was included psychological support, diet instruction, and post-operative activities guidance. The experimental group received auricular points treatment combined with acupoints application therapy in addition to the routine nursing care. All the patients had defecation within 3 days after operation. The characteristics of the stool were recorded, and the progress and performance of the incidence of constipation was recorded in two groups. RESULTS: The incidence of constipation in the control group was higher than that in the experimental group (P < 0.001). Moreover, the stool characteristics of experimental group were better than it in the control group by rank-sum test (P = 0.047). CONCLUSION: On the basis of routine measures to prevent constipation after lung cancer surgery, auricular point sticking combined with acupoint application therapy can effectively decrease the incidence of postoperative constipation.

Neuroleptics as therapeutic compounds stabilizing neuromuscular transmission in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, fatal disorder with no effective treatment. We used simple genetic models of ALS to screen phenotypically for potential therapeutic compounds. We screened libraries of compounds in C. elegans, validated hits in zebrafish, and tested the most potent molecule in mice and in a small clinical trial. We identified a class of neuroleptics that restored motility in C. elegans and in zebrafish, and the most potent was pimozide, which blocked T-type Ca2+ channels in these simple models and stabilized neuromuscular transmission in zebrafish and enhanced it in mice. Finally, a short randomized controlled trial of sporadic ALS subjects demonstrated stabilization of motility and evidence of target engagement at the neuromuscular junction. Simple genetic models are, thus, useful in identifying promising compounds for the treatment of ALS, such as neuroleptics, which may stabilize neuromuscular transmission and prolong survival in this disease.

Preclinical Models for Investigation of Herbal Medicines in Liver Diseases: Update and Perspective.

Liver disease results from a dynamic pathological process associated with cellular and genetic alterations, which may progress stepwise to liver dysfunction. Commonly, liver disease begins with hepatocyte injury, followed by persistent episodes of cellular regeneration, inflammation, and hepatocyte death that may ultimately lead to nonreversible liver failure. For centuries, herbal remedies have been used for a variety of liver diseases and recent studies have identified the active compounds that may interact with liver disease-associated targets. Further study on the herbal remedies may lead to the formulation of next generation medicines with hepatoprotective, antifibrotic, and anticancer properties. Still, the pharmacological actions of vast majority of herbal remedies remain unknown; thus, extensive preclinical studies are important. In this review, we summarize progress made over the last five years of the most commonly used preclinical models of liver diseases that are used to screen for curative herbal medicines for nonalcoholic fatty liver disease, liver fibrosis/cirrhosis, and liver. We also summarize the proposed mechanisms associated with the observed liver-protective, antifibrotic, and anticancer actions of several promising herbal medicines and discuss the challenges faced in this research field.

The Wnt signaling pathway effector TCF7L2 controls gut and brain proglucagon gene expression and glucose homeostasis.

The type 2 diabetes risk gene TCF7L2 is the effector of the Wnt signaling pathway. We found previously that in gut endocrine L-cell lines, TCF7L2 controls transcription of the proglucagon gene (gcg), which encodes the incretin hormone glucagon-like peptide-1 (GLP-1). Whereas peripheral GLP-1 stimulates insulin secretion, brain GLP-1 controls energy homeostasis through yet-to-be defined mechanisms. We aim to determine the metabolic effect of a functional knockdown of TCF7L2 by generating transgenic mice that express dominant-negative TCF7L2 (TCF7L2DN) specifically in gcg-expressing cells. The gcg-TCF7L2DN transgenic mice showed reduced gcg expression in their gut and brain, but not in pancreas. Defects in glucose homeostasis were observed in these mice, associated with attenuated plasma insulin levels in response to glucose challenge. The defect in glucose disposal was exacerbated with high-fat diet. Brain Wnt activity and feeding-mediated hypothalamic AMP-activated protein kinase (AMPK) repression in these mice were impaired. Peripheral injection of the cAMP-promoting agent forskolin increased brain ß-cat Ser675 phosphorylation and brain gcg expression and restored feeding-mediated hypothalamic AMPK repression. We conclude that TCF7L2 and Wnt signaling control gut and brain gcg expression and glucose homeostasis and speculate that positive cross-talk between Wnt and GLP-1/cAMP signaling is an underlying mechanism for brain GLP-1 in exerting its metabolic functions.

Effect of surfactant concentration on transdermal lidocaine delivery with linker microemulsions.

A limited number of studies have been conducted to investigate the effect of surfactant concentration on microemulsion-mediated transdermal transport. Some studies suggest that increasing surfactant concentration reduces the partition of the active in the skin, and the overall transport. Other studies suggest that increasing surfactant concentration improves mass transport across membranes by increasing the number of "carriers" available for transport. To decouple these partition and mass transport effects, a three-compartment (donor, skin, receiver) mass balance model was introduced. The model has three permeation parameters, the skin-donor partition coefficient (K(sd)), the donor-skin mass transfer coefficient (k(ds)) and the skin-receiver mass transfer coefficient (k(sr)), also known as skin permeability. The model was used to fit the permeation profile of lidocaine formulated in oil-in-water (Type I) and water-in-oil (Type II) lecithin-linker microemulsions. The results show that surfactant concentration has a relatively minor effect on the mass transfer coefficients, suggesting that permeation enhancement via disruption of the structure of the skin is not a relevant mechanism in these lecithin-linker microemulsions. The most significant effect was the increase in the concentration of lidocaine in the skin with increasing surfactant concentration. For Type I systems such increase in lidocaine concentration in the skin was linked to the increase in lidocaine solubilization in the microemulsion with increasing surfactant concentration. For Type II systems, the increase in lidocaine concentration in the skin was linked to the increase in skin-donor partition. A surfactant-mediated absorption/permeation mechanism was proposed to explain the increase in lidocaine concentration in skin with increasing surfactant concentration. The penetration profiles of hydrophobic and amphiphilic fluorescence probes are consistent with the proposed mechanism.