New in vitro cellular model for human Lipodystrophy

Lipodystrophies are disorders characterized by complete or selective loss of adipose tissue from various regions of the body. They might lead to severe metabolic disorders. The development of reliable cellular experimental models mimicking such diseases in vitro is extremely challenging (1). One of the main hurdle in the design of such in vitro cellular models is the access to reliable sources of well-qualified primary cells and the identification of optimal cell culture conditions.

[Read more…]

Thiostrepton: a novel lead compound against Dengue?

Dengue Virus (DenV) is transmitted by mosquito vectors. It infects 50-100 million people each year and is at the origin of Dengue Fever and the more lethal Dengue Hemorrhagic Fever (DHF) and Shock Syndrome (DSS) leading to an estimated 500,000 cases of DHF and 22 000 deaths. The World Health Organization (WHO) estimates that 40% of the world’s population is at risk of infection.

In the June issue of Journal of Biomolecular Screening, investigators at San Diego State University (Dept of Biology) and Institute Pasteur Korea (Seoul, South Korea) developed a multiplexed cell-based assay for the identification of modulators of pre-membrane processing as a target for the discovery of DenV inhibitors. (1)

The DenV pre-membrane protein (prM) is an essential chaperone for the viral envelope protein which prevents premature fusion with vesicles during viral export. Inhibition of pre-membrane protein cleavage restricts fusion and represents, thus, a novel druggable target.

The new in vitro assay developed in this study, is the first described cell-based assay that monitors DenVprM processing within the classical secretory pathway. In a pilot screen of 1,280 small molecules on that assay, Thiostrepton, a known cyclopeptide Antibiotic and FOXM1 inhibitor,  was identified as a novel positive hit in this assay (IC50=4.94 µM).

The utility of this novel assay has been proven by the identification of Thiostrepton (available at Focus Biomolecules cat. nr 10-2108) which may be a novel lead compound for the discovery of new drugs effective against Dengue Virus.

Thiostrepton Focus Biomolecules tebu-bio Catalog # 10-2108

Thiostrepton – Antibiotic and FOXM1 inhibitor. C72H85N19O18S5 – CAS No: 1393-48-2, 98% by TLC and HPLC at Focus Biomolecules.

 

1- Stolp Z.D. et al. “A Multiplexed Cell-Based Assay for the Identification of Modulators of Pre-Membrane Processing as a Target against Dengue Virus” (2015) J. Biomol. Screen. 20:616-626. DOI: 10.1177/1087057115571247.

 

How to easily detect luciferase activity in cell based assays?

Luciferase is the general term given to a class of oxidative enzymes which catalyze reactions that give off light, a process known as bioluminescence (Fig. 1).

structure and mechanism

Fig. 1: Mechanisms of the assay

In biology, researchers take advantage of this reaction to use it as a readout for various biological processes. This has perhaps been exploited most in luciferase reporter cell lines where a promoter region from a gene of interest is placed immediately upstream of the coding sequence for luciferase. In this system, transcriptional activation of the gene of interest leads to a level of luciferase expression that is proportional to the level of gene activation.

The level of activation is then assessed by lysing the cells and adding the luciferase substrate, D-luciferin. Especially in screening processes it is crucial that the assay be accomplished in a single step, meaning the lysis buffer has to be formulated with D-luciferin. [Read more…]

Tumor growth and metastasis monitored in vivo

In vivo monitoring of tumor growth and metastasis provides a powerful means for studying cancer properties and development of effective therapies. Mouse models created with tumor xenografts, resulting from subcutaneous or tail vein injection of cells, have long been used for such purposes. However, without a convenient means to visualize cancer progression in these animals, invasive surgical procedures are required in order to estimate the size and weight of primary and metastatic tumors, and cannot be used for early stages in tumor development. Surgery often requires sacrificing the animals, leading to the need for large numbers of individuals and increasing the cost and risk of sampling errors.

GeneCopoeia has recently announced the introduction of pre-made cancer cell lines labeled with GFP, and pre-made cancer cell lines dual-labeled with luciferase and GFP. A new powerful and sensitive mathods to follow tumor growth & metastasis in vivo! [Read more…]

A 96-well Invasion Assay Compatible with High Content Screening

The transition from non-invasive phenotype to invasive phenotype of tumor cells marks the switch from a benign tumor to a more malignant form of cancer. Understanding the mechanisms underlying this hallmark event, which enables tumor cells to invade through Extracellular matrix, is critical for discovering pathways and new targets to develop anti-metastatic strategies. The discovery of anti-metastatic agents that inhibit cancer cell motility has been hindered by a dearth of cell motility assays that are compatible with high–throughput screening. [Read more…]

Stem cell-derivative Motor Neurons / Muscle fiber co-culture functional bio-assay

Chipman et al. (Dalhousie University, Halifax, Nova Scotia, Canada) have developed a new cell-based system “mimicking” in vitro Mature Neuromuscular Junction (NMJs). (1)

Chipman et al. PLOSOne (2014) DOI: 10.1371/journal.pone.0091643

Chipman et al. PLOSOne (2014) DOI: 10.1371/journal.pone.0091643

NMJs play an important role in the functionality of Motor neurons.

Their dysfunction is seen in the early stages of various Motor Neuron Diseases (MNDs) like the Amyotrophic Lateral Sclerosis (ALS), Spinal Muscular Atrophy (SMA), Primary Lateral Sclerosis (PLS), Progressive Muscular Atrophy (PMA)…

To meet these needs, Chipman et al. have created a clever co-culture system using Embryonic Stem Cell-derived Motor Neurons (ESCMNs) with primary myotubes. They also reported that NCAM-/- ESCMN/myofiber co-cultures exhibit the same phenotypes observed in NCAM-/- mice.

This work give hopes to study MNDs in vitro by using motor neurons differentiated from induced Pluripotent Stem cells (iPS) derived from individuals with ALS and SMA.

Want to know more about cell-based assays and co-culture systems?

(1) Chipman et al. “A Stem-Cell Based Bioassay to Critically Assess the Pathology of Dysfunctional Neuromuscular Junctions” (2014) PLOSone, March 13. DOI: 10.1371/journal.pone.0091643

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