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Development of Highly Sensitive Excitation Ratiometric Indicators of Cellular Phosphorylation

Protein phosphorylation is one of the most common forms of post-translational modification and is involved in the regulation of key signaling pathways in the cell. Dysfunctional phosphorylation plays a key role in various diseases, especially cancer and neurodegenerative disorders. Protein kinases have been the focus of intense recent interest by the pharmaceutical industry. Indeed, most new cancer drugs approved by the FDA in the last several years target kinases, and there are hundreds of new kinase inhibitors under development.  To this end, it is very important to have quantitative methods for measuring changes in kinase activities.  In vitro kinase activity assays take the target molecules out of cellular contexts. Fluorescent protein-based kinase biosensors have enabled the real-time monitoring of kinase activities within the native context of living cells, yet most commonly used biosensors exhibit poor sensitivity (e.g., dynamic range) for imaging physiological signaling activities in situ.

Assay for Oligonucleotides in Serum Without Extraction or RT-PCR

Prof. Ameae Walker’s laboratory at the University of California, Riverside (UCR) has developed an assay to quantify oligos in sub-picomole amounts without the need for sample purification and amplification. This new competitive assay is called an ELOHA (Enzyme-Linked Oligonucleotide Hybridization Assay). The method is illustrated in Fig. 1, below.  Capture Oligos that are to hybridize with an oligo to be measured are covalently linked to a plate (1), a Detection Oligo, with the same sequence as the oligo to be measured, has a conjugated label, such as horseradish peroxidase or biotin.  The Detection Oligo then competes with the oligo of interest for binding to the Capture Oligo (2).  Once the hybridization is complete, the unbound oligos are washed away (3).  A colorimetric readout is produced (4) to inversely quantify the oligo of interest.  Fig. 1 Schematic of the ELOHA assay Fig. 2 shows the use of an ELOHA for amounts of Antimaia in mouse serum. Antimaia is a splice modulating oligomer therapy for breast cancer developed in the UCR lab of Prof. Walker.  

Methods For High-Throughput Screening and Sorting of Hyperproducing Single Cells

UCLA researchers from the Department of Bioengineering have developed a novel approach to screen and sort single cells and colonies while preserving them in viable culture environments. This innovative technology employs a liquid to gel solution that changes phase under mild conditions to compartmentalize biological components.

“PROTAC-SPARK”: A Genetically-encoded Fluorescent Reporter Platform for Studying PROTAC Interaction Kinetics in Living Cells

This novel class of genetically-encoded fluorescent reporters can be used as powerful tools to study kinetics of protein targeting chimeras (PROTACs) in living cells. These bright, reversible reporters have a large dynamic range and fast kinetics, demonstrating significant advantages over traditional FRET-based fluorescent reporters. This highly-customizable reporter system is ideal for designing novel cell assays to quickly test protein-protein interactions with quantitative results.

A Micro-Bubble Plate For Patterning Biological and Non-Biological Materials

A method for creating a 3D micro-bubble plate for patterning biological and non-biological materials. Because each sample is at a known location, large numbers of samples may be studied and allow for significant statistical data sets, which will aid in diagnosing unknown agents or diseases inexpensively.

A Micro-Patterned Plate Composed Of An Array Of Releaseable Elements Surrounded With Solid Or Gel Walls

This technology is a micro-patterned plate made of an array of releasable elements surrounded by a gel or solid wall, and a process for manufacturing the micro-patterned plate. This is an efficient way of studying samples for statistically significant data sets of cells or biological materials for important scientific research and medicines.

A Novel Method and Protocol to Induce Pluripotent Stem Cells Toward Astrocyte Differentiation

Rett syndrome (RTT) is a devastating disease that affects 1 in every 10,000 children born in the United States, primarily females. RTT patients undergo apparently normal development until 6-18 months of age, followed by impaired motor function, stagnation and then regression of developmental skills, hypotonia, seizures and a spectrum of autistic behaviors. Rett syndrome is a rare disease that shares certain pathways with major developmental disorders such as autism and schizophrenia, increasing the potential impact. There is no cure for Rett syndrome and the animal model does not entirely recapitulate the human disease. Thus, having the possibility to screen drugs directly in human neurons is a major milestone.

Chemiluminescence-Assisted Cell Endocytosis Kinetics Assay

UCLA researchers in the Department of Chemical and Biomolecular Engineering have developed a novel method to monitor the intracellular kinetics using the bioluminescent reaction of nanoparticles.

Insect Repellents and Assay

Prof. Anandasankar Ray and his colleagues at the University of California, Riverside (UCR) have developed insect repellants to deter insects from detecting and biting humans. The repellants are comprised of a group of compounds consisting of an aldehyde, mono- or  diketone and an alcohol. Repellants mask the insect’s ability to detect CO2. These repellants can be delivered in a variety of forms and can be used in much smaller concentrations and remain effective for much longer when compared to traditional repellants like DEET. The UCR lab also developed and patented a computational assay to screen and identify mosquito repellents. This assay was used to identify the patented compounds that disrupt CO2 sensing in mosquitos.   Fig. 1 Effect of inhibitory odor, 1-hexanol, on mosquito neuronal CO2 response. The small black bar indicates an 0.5 second exposure to inhibitory odor overlayed with a 3 second response to CO2. The second chart shows how CO2 response is mitigated by the odor   Fig. 2 Effect of pre-exposure to inhibitory odors on long-term reduction to CO2 response. The response to a 0.3% CO2 impulse over 6 minutes was measured every 30 seconds after an initial 3-second exposure to an ‘odor’ mixture (black bars). The odor mixture consisted of 1-hexanolo, pentanal, butanal, and 2,3-butanedione at 10-2 concentration. Paraffin oil (white bars) was used as a control      

A Cell-Based Seeding Assay for Huntingtin Aggregation

UCLA researchers from the Department of Psychiatry has created a novel cell-based seeding assay for sensitive, specific and high throughput detection of mutant Huntingtin proteins in biological samples.

Hydrostatic pressure-driven passive micropumps

Researchers at UCI have developed an inexpensive and entirely passive pump for microfluidic devices, which yields steady, controllable, and long-lived fluid flow through the device.

“SPARK (Separation of Phases-based Activity Reporter of Kinase)”_A Genetically-encoded Fluorescent Reporter Platform for Studying Cell Signaling in Living Cells

This novel class of genetically-encoded fluorescent reporters can be used as powerful tools to study protein-protein interactions (PPIs) in living cells. These bright, reversible reporters have a large dynamic range and fast kinetics, demonstrating significant advantages over traditional FRET-based fluorescent reporters.

A Method for Making a Human Alzheimer’s Disease Neuronal Model Using Purified ApoE-HDL

Alzheimer's disease (AD) is a common neurodegenerative disease and the most common cause of dementia. Alzheimer’s disease is defined post-mortem by the increased presence of amyloid plaques and neurofibrillary tangles (NFTs) in the brain. Amyloid plaques are extracellular deposits consisting primarily of amyloid-ß (Aß) peptides, and NFTs are intraneuronal aggregations of hyperphosphorylated tau, a microtubule-associated protein involved in microtubule stabilization. The discovery of new drugs for treating Alzheimer’s disease is currently limited by difficulties in obtaining live neurons from patients and the inability to accurately model Alzheimer’s disease. Animal models of Alzheimer’s disease have been developed, however, these animal models do not completely mimic true human disease, and none of these animal models are neuronal models of the disease. There is a need to develop a human neuronal model that more accurately mimics true human Alzheimer’s disease, and then use such a model for Alzheimer’s disease drug discovery and research.

A Highly Error-Prone Orthogonal Replication System For Targeted Continuous Evolution In Vivo

Inventors at UC Irvine have engineered an orthogonal DNA replication system capable of rapid, accelerated continuous evolution. This system enables the directed evolution of specific biomolecules towards user-defined functions and is applicable to problems of protein, enzyme, and metabolic pathway engineering.

Modular Miniature Microscopy System

UCLA researchers have developed a modular miniature microscopy system for brain imaging in behaving animals.

Platform for predicting a compound’s cardioactivity

The invention is a platform that combines a screening system and machine learning algorithms to investigate and report the cardio-activity related information of a certain compound. Through screening cardiac tissue strips, the platform determines whether a compound is cardio-active or not, as well as the associated cardio-active mechanism based on a drug library that is automatically developed. Such information is crucial for the drug development process, especially for evidence based decisions.

Controlled 'One-Cell-One-Bead' Encapsulation in Droplets

Improving droplet encapsulation of a single-cell and single-bead to increase pharmacological assay throughput.

EpiSort: A Novel Method Using Deep Bisulfite Sequencing to Determine Immune Cell Types in Solid Tissue Samples

EpiSort is a novel method of using DNA methylation patterns to determine the proportion of immune cell populations in solid tissue samples.

SHARPR-MPRA (Systematic High-Resolution Activation And Repression Profiling With Reporter-Tiling Massively Parallel Reporter Assay)

UCLA researchers in the Department of Biological Chemistry have developed a method to screen hundreds to thousands of genes to identify their regulatory functions.

In Vivo and In Vitro Methods for Site-specific Protein Labeling: Leveraging the CoA Biosynthetic and Protein Modification Pathways

Selective chemical control of biochemical processes within a living cell enables the study and modification of natural biological systems in ways that may not be obtained through in vitro experiments. Accordingly, access to promiscuous metabolic pathways has provided a unique chemical entry into small molecule engineering in vivo.  A method for covalent reporter labeling of carrier proteins using permissive phosphopantetheinyltransferase (PPTase) enzymes and reporter-labeled coenzyme A (CoA) has been commonly used but has been limited to in vitro and cell-surface protein labeling, as CoA derivatives have not been shown to penetrate the cell.

Microfluidics Device For Digestion Of Tissues Into Cellular Suspension

A microfluidic device that separates single cells from whole tissue in a rapid and gentle manner using hydrodynamic fluid flow. The separated single cell suspensions can then be used in tissue engineering applications, regenerative medicine and the study of cancer.

Sieve Container For Contactless Media Exchange For Cell Growth

Media that contains nutrients and growth factors is necessary to grow all types of cells, a process that is widely used in many fields of research. Such media should be routinely changed either to different media or a fresh batch of the same media. This change currently involves either using a pipette to transfer cells from their current dish of media to a new dish, or aspirating the media out of the dish and replacing it with new media. Both methods have inherent risks to stressing and damaging the cells. Researchers at UCI have developed a unique dish for growing cells that allows for safer aspiration of the old media, which reduces stress and damage to the cells.

Assay for Inhibitors of Nonsense-Mediated RNA Decay

Prof. Sika Zheng at UCR has developed a new endogenous NMD assay that is both sensitive and quantitative. The assay can be used on its own to assess changes in cellular NMD activity with high specificity and sensitivity. It can facilitate analysis of NMD controls by cellular pathways in response to stimuli or during development and is particularly suitable for unbiased screening of NMD modulators. The assay is designed to distinguish NMD regulation from transcriptional regulation and alternative splicing control.

Compound Library Made Through Phosphine-Catalyzed Annulation/Tebbe/Diels-Alder Reaction

UCLA researchers in the Department of Chemistry and Biochemistry have developed a small molecule library consisting of a large variety stereochemical variants.

In Situ Lipid Synthesis for Protein Reconstitution (INSYRT)

While current methods for membrane protein functional reconstitution in biomimetic membranes approaches are powerful and have uncovered fundamental properties of protein function, they are methodologically cumbersome, requiring chromatography steps to remove detergents. Moreover, structural features normally found in cell membranes such as curvature and polarity are mostly absent. In this regard, an efficient reconstitution methodology that better mimics the native chemical environment of a whole-cell embedded protein would be highly useful.

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