High-throughput ligand profile characterization in novel cell lines expressing seven heterologous insect olfactory receptors for the detection of volatile plant biomarkers.

Agriculturally important crop plants emit a multitude of volatile organic compounds (VOCs), which are excellent indicators of their health status and their interactions with pathogens and pests. In this study, we have developed a novel cellular olfactory panel for detecting fungal pathogen-related VOCs we had identified in the field, as well as during controlled inoculations of several crop plants. The olfactory panel consists of seven stable HEK293 cell lines each expressing a functional Drosophila olfactory receptor as a biosensing element along with GCaMP6, a fluorescent calcium indicator protein.

An Advanced Automated Patch Clamp Protocol Design to Investigate Drug-Ion Channel Binding Dynamics.

Standard high throughput screening projects using automated patch-clamp instruments often fail to grasp essential details of the mechanism of action, such as binding/unbinding dynamics and modulation of gating. In this study, we aim to demonstrate that depth of analysis can be combined with acceptable throughput on such instruments. Using the microfluidics-based automated patch clamp, IonFlux Mercury, we developed a method for a rapid assessment of the mechanism of action of sodium channel inhibitors, including their state-dependent association and dissociation kinetics.

Characterization of Compound-Specific, Concentration-Independent Biophysical Properties of Sodium Channel Inhibitor Mechanism of Action Using Automated Patch-Clamp Electrophysiology.

We have developed an automated patch-clamp protocol that allows high information content screening of sodium channel inhibitor compounds. We have observed that individual compounds had their specific signature patterns of inhibition, which were manifested irrespective of the concentration. Our aim in this study was to quantify these properties.

The mechanism of non-blocking inhibition of sodium channels revealed by conformation-selective photolabeling.

Background And Purpose: Sodium channel inhibitors can be used to treat hyperexcitability-related diseases, including epilepsies, pain syndromes, neuromuscular disorders and cardiac arrhythmias. The applicability of these drugs is limited by their nonspecific effect on physiological function. They act mainly by sodium channel block and in addition by modulation of channel kinetics.

A Mouse Model to Assess STAT3 and STAT5A/B Combined Inhibition in Health and Disease Conditions.

Genetically-engineered mouse models (GEMMs) lacking diseased-associated gene(s) globally or in a tissue-specific manner represent an attractive tool with which to assess the efficacy and toxicity of targeted pharmacological inhibitors. and transcription factors have been implicated in several pathophysiological conditions, and pharmacological inhibition of both transcription factors has been proposed to treat certain diseases, such as malignancies. To model combined inhibition of and we have developed a GEMM harboring a flox - allele ( mice) and generated mice lacking and in hepatocytes ().

Directed Evolution of Canonical Loops and Their Swapping between Unrelated Serine Proteinase Inhibitors Disprove the Interscaffolding Additivity Model.

Reversible serine proteinase inhibitors comprise 18 unrelated families. Each family has a distinct representative structure but contains a surface loop that adopts the same, canonical conformation in the enzyme-inhibitor complex. The Laskowski mechanism universally applies for the action of all canonical inhibitors independent of their scaffold, but it has two nontrivial extrapolations.

AKT3 drives adenoid cystic carcinoma development in salivary glands.

Salivary gland cancer is an aggressive and painful cancer, but a rare tumor type accounting for only ~0.5% of cancer cases. Tumors of the salivary gland exhibit heterogeneous histologic and genetic features and they are subdivided into different subtypes, with adenoid cystic carcinomas (ACC) being one of the most abundant.

Overlapping Specificity of Duplicated Human Pancreatic Elastase 3 Isoforms and Archetypal Porcine Elastase 1 Provides Clues to Evolution of Digestive Enzymes.

Chymotrypsin-like elastases (CELAs) are pancreatic serine proteinases that digest dietary proteins. CELAs are typically expressed in multiple isoforms that can vary among different species. The human pancreas does not express CELA1 but secretes two CELA3 isoforms, CELA3A and CELA3B.

Heterologous protein production using euchromatin-containing expression vectors in mammalian cells.

Upon stable cell line generation, chromosomal integration site of the vector DNA has a major impact on transgene expression. Here we apply an active gene environment, rather than specified genetic elements, in expression vectors used for random integration. We generated a set of Bacterial Artificial Chromosome (BAC) vectors with different open chromatin regions, promoters and gene regulatory elements and tested their impact on recombinant protein expression in CHO cells.

Disruption of STAT3 signalling promotes KRAS-induced lung tumorigenesis.

STAT3 is considered to play an oncogenic role in several malignancies including lung cancer; consequently, targeting STAT3 is currently proposed as therapeutic intervention. Here we demonstrate that STAT3 plays an unexpected tumour-suppressive role in KRAS mutant lung adenocarcinoma (AC). Indeed, lung tissue-specific inactivation of Stat3 in mice results in increased Kras(G12D)-driven AC initiation and malignant progression leading to markedly reduced survival.

Modeling cancer using genetically engineered mice.

Genetically engineered mouse (GEM) models have proven to be a powerful tool to study tumorigenesis. The mouse is the preferred complex organism used in cancer studies due to the high number and versatility of genetic tools available for this species. GEM models can mimic point mutations, gene amplifications, short and large deletions, translocations, etc.

Exploration of BAC versus plasmid expression vectors in recombinant CHO cells.

Vector engineering approaches are commonly used to increase recombinant protein production in mammalian cells, and among various concepts, bacterial artificial chromosomes (BAC) have been proposed to serve as open chromatin regions to omit chromosome positional effects. For proof of concept, we developed stable recombinant Chinese hamster ovary (CHO) cell lines using different expression vector systems: the plasmid vectors contained the identical expression cassette as the BAC constructs. Two anti-HIV1 antibody derivates served as model proteins (3D6scFc and 2F5scFc) for generation of four stable recombinant CHO cell lines.

High affinity small protein inhibitors of human chymotrypsin C (CTRC) selected by phage display reveal unusual preference for P4' acidic residues.

Human chymotrypsin C (CTRC) is a pancreatic protease that participates in the regulation of intestinal digestive enzyme activity. Other chymotrypsins and elastases are inactive on the regulatory sites cleaved by CTRC, suggesting that CTRC recognizes unique sequence patterns. To characterize the molecular determinants underlying CTRC specificity, we selected high affinity substrate-like small protein inhibitors against CTRC from a phage library displaying variants of SGPI-2, a natural chymotrypsin inhibitor from Schistocerca gregaria.