An evaluation of the PowerSeq™ Auto System: A multiplex short tandem repeat marker kit compatible with massively parallel sequencing.

Capillary electrophoresis (CE) and multiplex amplification with fluorescent tagging have been routinely used for STR typing in forensic genetics. However, CE-based methods restrict the number of markers that can be multiplexed simultaneously and cannot detect any intra-repeat variations within STRs. Several studies already have indicated that massively parallel sequencing (MPS) may be another potential technology for STR typing.

Identification of known drugs targeting the endoplasmic reticulum stress response.

The endoplasmic reticulum (ER), a multifunctional organelle, plays a central role in cellular signaling, development, and stress response. Dysregulation of ER homeostasis has been associated with human diseases, such as cancer, inflammation, and diabetes. A broad spectrum of stressful stimuli including hypoxia as well as a variety of pharmacological agents can lead to the ER stress response.

Pharmacological inhibition of LRRK2 cellular phosphorylation sites provides insight into LRRK2 biology.

Mutations in LRRK2 (leucine-rich repeat kinase 2) have been linked to inherited forms of PD (Parkinson's disease). Substantial pre-clinical research and drug discovery efforts have focused on LRRK2 with the hope that small-molecule inhibitors of the enzyme may be valuable for the treatment or prevention of the onset of PD. The pathway to develop therapeutic or neuroprotective agents based on LRRK2 function (i.

Screening for novel LRRK2 inhibitors using a high-throughput TR-FRET cellular assay for LRRK2 Ser935 phosphorylation.

Background: Mutations in the leucine-rich repeat kinase-2 (LRRK2) have been linked to Parkinson's disease. Recent studies show that inhibition of LRRK2 kinase activity decreased the level of phosphorylation at its own Ser910 and Ser935, indicating that these sites are prime targets for cellular readouts of LRRK2 inhibition.

Methodology/principal Findings: Using Time-Resolved Förster Resonance Energy Transfer (TR-FRET) technology, we developed a high-throughput cellular assay for monitoring LRRK2 phosphorylation at Ser935.

A quantitative TR-FRET plate reader immunoassay for measuring autophagy.

Autophagy involves the isolation and targeting of unwanted cellular components to lysosomes for their digestion and reuse. Autophagic dysregulation has recently been implicated in a wide range of disease processes, yet facile methods for quantifying autophagy have been lacking in the field. Here we describe the generation of a quantitative plate reader assay for measuring the autophagic activity of cells.

TR-FRET cellular assays for interrogating posttranslational modifications of histone H3.

Posttranslational modifications such as phosphorylation, acetylation, and methylation play important roles in regulating the structures and functions of histones, which in turn regulate gene expression and DNA repair and replication. Histone-modifying enzymes, such as deacetylases, methyltransferases and demethylases, have been pursued as therapeutic targets for various diseases. However, detection of the activities of these enzymes in high-throughput cell-based formats has remained challenging.

Hemicellulases and auxiliary enzymes for improved conversion of lignocellulosic biomass to monosaccharides.

Background: High enzyme loading is a major economic bottleneck for the commercial processing of pretreated lignocellulosic biomass to produce fermentable sugars. Optimizing the enzyme cocktail for specific types of pretreated biomass allows for a significant reduction in enzyme loading without sacrificing hydrolysis yield. This is especially important for alkaline pretreatments such as Ammonia fiber expansion (AFEX) pretreated corn stover.

Development and validation of a cell-based assay for the nuclear receptor retinoid-related orphan receptor gamma.

The nuclear receptor retinoid-related orphan receptor gamma (RORγ) has become an attractive target for drug discovery due to its important role in the development and differentiation of Th17 cells, a subset of T cells that produce interleukin-17 and are involved in the pathogenesis of human inflammatory and autoimmune diseases. To facilitate the drug discovery efforts in this area, we have developed a cellular assay for screening for RORγ inverse agonists. We stably engineered a tetracycline-inducible Gal4 DNA-binding domain/RORγ ligand-binding domain fusion protein into an upstream activation sequence driven-beta-lactamase reporter gene cell line.

Measurement of the cellular deacetylase activity of SIRT1 on p53 via LanthaScreen® technology.

Upon genomic insult, the tumor suppressor p53 is phosphorylated and acetylated at specific serine and lysine residues, increasing its stability and transactivation function. Deacetylases, including the type III histone deacetylase SIRT1, remove acetyl groups from p53 and counterbalance acetyltransferase activity during a DNA damage response. This report describes a series of high-throughput LanthaScreen® time-resolved Förster resonance energy transfer (TR-FRET) immunoassays for detection of intracellular p53 phosphorylation of Ser15 and acetylation of Lys382 upon treatment with DNA damage agents, such as etoposide.

Mining Dictyoglomus turgidum for enzymatically active carbohydrases.

The genome of Dictyoglomus turgidum was sequenced and analyzed for carbohydrases. The broad range of carbohydrate substrate utilization is reflected in the high number of glycosyl hydrolases, 54, and the high percentage of CAZymes present in the genome, 3.09% of its total genes.

WNT5A mutations in patients with autosomal dominant Robinow syndrome.

Robinow syndrome is a skeletal dysplasia with both autosomal dominant and autosomal recessive inheritance patterns. It is characterized by short stature, limb shortening, genital hypoplasia, and craniofacial abnormalities. The etiology of dominant Robinow syndrome is unknown; however, the phenotypically more severe autosomal recessive form of Robinow syndrome has been associated with mutations in the orphan tyrosine kinase receptor, ROR2, which has recently been identified as a putative WNT5A receptor.

Gene-breaking transposon mutagenesis reveals an essential role for histone H2afza in zebrafish larval development.

We report a novel gene tagging, identification and mutagenicity ('gene-breaking') method for the zebrafish, Danio rerio. This modular approach consists of two distinct and separable molecular cassettes. The first is a gene-finding cassette.

Enhancer trapping in zebrafish using the Sleeping Beauty transposon.

Background: Among functional elements of a metazoan gene, enhancers are particularly difficult to find and annotate. Pioneering experiments in Drosophila have demonstrated the value of enhancer "trapping" using an invertebrate to address this functional genomics problem.

Results: We modulated a Sleeping Beauty transposon-based transgenesis cassette to establish an enhancer trapping technique for use in a vertebrate model system, zebrafish Danio rerio.

Efficient gene delivery and gene expression in zebrafish using the Sleeping Beauty transposon.

We used the Tc1/mariner family transposable element Sleeping Beauty (SB) for transgenesis and long-term expression studies in the zebrafish (Danio rerio), a popular organism for clinical disease, vertebrate patterning, and cell biology applications. SB transposase enhanced the transgenesis and expression rate sixfold (from 5 to 31%) and more than doubled the total number of tagged chromosomes over standard, plasmid injection-based transgenesis methods. Molecular analysis of these loci demonstrated a precise integration of these elements into recipient chromosomes with genetic footprints diagnostic of transposition.

Syndecan-2 is essential for angiogenic sprouting during zebrafish development.

We used a morpholino-based gene-targeting screen to identify a novel protein essential for vascular development using the zebrafish, Danio rerio. We show that syndecan-2, a cell-surface heparan sulfate proteoglycan, is essential for angiogenic sprouting during embryogenesis. The vascular function of syndecan-2 is likely conserved, as zebrafish and mouse syndecan-2 show similar expression patterns around major trunk vessels, and human syndecan-2 can restore angiogenic sprouting in syndecan-2 morphants.

Lateral line, nervous system, and maternal expression of Frizzled 7a during zebrafish embryogenesis.

We have isolated and mapped a new wnt receptor frizzled family member, zebrafish frizzled 7a. Fz7a and a previously reported zebrafish fz7 (El-Messaoudi and Renucci, 2001) make an orthologous gene pair, however, they display distinct expression patterns. Fz7a shows strong maternal as well as zygotic expression.