Genetically-encoded fluorescent proteins have revolutionized cell biology and gene expression studies. Biologists utilize a rainbow of fluorescent proteins, with colors extending across most of the visible spectrum. However, fluorescence imaging in live animals using these proteins has been impeded by the inability of visible light to penetrate the body. Imaging deep into biological tissue is a challenge because proteins in the blood and skin absorb the light wavelengths typically used to excite and visualize fluorescent proteins. Mammalian tissues are penetrable by near-infrared wavelengths but existing infrared technologies for live animal imaging are not optimal; they often consist of non-specific dyes or bulky, multimeric proteins that require the addition of exogenous cofactors. Thus a major limitation in the field of fluorescent imaging is the availability of a genetically-encoded fluorescent protein that is suitable for live animal research.     (more...)

Non-covalent macromolecular interactions of proteins with lipids, nucleic acids, small ligands, and other proteins underlie a vast majority of biological processes. The transient nature of these interactions makes it difficult to use traditional methods to detect specific non-covalent macromolecular interactions. Ubiquitination is one such macromolecular interaction cascade that results in the addition of ubiquitin to a wide variety of substrate proteins. The addition of ubiquitin represents an important regulatory mechanism in the cell to modulate global protein levels and specific signal transduction cascades. Ubiquitination of a substrate protein occurs as a result of a pyramidal cascade involving the sequential action of three classes of E1, E2, and E3 proteins. In general, a small number of E1-activating enzymes transfer ubiquitin to a limited number of E2-conjugating enzymes that in turn function together with a large number of E3-ubiquitin ligases to ubiquitinate a variety of substrate proteins. In humans for example, only two E1 enzymes can transfer ubiquitin to more than three-dozen E2-ubiquitin conjugating enzymes, which in turn can partner with several hundred E3-ligases to ubiquitinate thousands of target substrates. The pervasive use of ubiquitination as a regulatory mechanism in the cell, coupled with the transient nature of the interaction between E3-ligases and their respective substrates, presents the unique challenge of accurately identifying the appropriate E3-ligase/substrate pairs to better understand normal and pathological cellular processes.   (more...)

Multiplex assays are extremely useful in biomedical research for producing genomic and proteomic data.  The ability to translate novel biomarkers for various diseases into new diagnostic multiplex assays is highly attractive from a drug discovery point of view.  However, the actual execution of creating such high-throughput multiplex assays remains challenging, as they require the ability to reliably track the identity and location of individual probes throughout an experiment.  One way of accomplishing this is by using encoded beads, where uniquely identifiable beads are attached to each individual probe.  Spectral encoding is a popular method of encoding beads and involves mixtures of luminescent materials that emit light at different wavelengths in order to generate distinguishable output signatures.  Typically, however, this approach is limited by low photostability and small numbers of usable unique codes.  In order to accelerate the discovery of new biomarkers for drug discovery purposes, there is a need for a more efficient and cost-effective method of creating encoded beads for high-throughput multiplex assays.      (more...)

Optical trapping systems are commercially available through several companies. In these systems, the optical trap precision relies on the passive stability of the instrument itself, and therefore demands costly engineering solutions to limit environmental noise that can be coupled into the optomechanical components. Consequently, high-resolution measurements are not possible in common biological laboratory settings that typically lack appropriate vibration isolation and temperature stability.  Researchers at the University of California, Berkeley have developed an invention that addresses a critical problem currently limiting the performance of high-resolution optical traps: that the mechanical drift of optical components often results in physical drift in the location of an optical trap that obscures the displacement-of-interest. The motion of biological motor proteins that are specific to interacting with DNA often take steps along the double helix that is on the order of 0.3 nanometers in size. Accurate measurement of displacements on this scale requires that drift of the trap positions be limited to no more than a few angstroms. However, the current best-performing optical traps suffer from instrumental drift that is almost twice what can be tolerated. Owing to the critical role of these components in all optical trapping systems, and the previously undetectable levels of mechanical drift they undergo, we sought to measure the trap drift with angstrom-level precision using a new approach. This new approach has successfully measured for and corrected for the mechanical drift of these components and demonstrated that this novel invention is capable of consistently reducing the noise floor to levels that have not previously been accomplished.        (more...)

Embryonic stem cells (ESCs) offer attractive therapeutic alternatives for many diseases and conditions such as diabetes, Parkinson's disease, cancer therapy, and many others. However, teratoma formation of transplanted ESCs is a major concern that needs to be overcome before translating this technology into clinical reality. (more...)

Proteins have found utility for numerous commercial and clinical purposes, including use in biochemical and chemical processes, and as agents for the treatment and prevention of human and veterinary disease. A major challenge associated with the use of proteins is their inherent instability. Many proteins rapidly degrade in response to "environmental stresses," such as changes in temperature, pH, light, and desiccation, which has implications for their production, transport, use and storage. Attachment of poly(ethylene glycol) to therapeutic proteins, a process commonly referred to as PEGylation, has been used successfully to increase their stability in vivo by reducing both protease degradation and renal clearance. However, PEGylation does not necessarily increase protein stability in response to environmental stresses. The development of a technology that enhances the stability of proteins to such stresses would dramatically increase the number of proteins that could be used commercially, reduce costs associated with protein production, storage and transportation, and increase protein shelf-life. (more...)

One barrier to research productivity in assaying cells in culture is the disorganized distribution and random arrangement of cells that can make precise single cell analyses difficult to perform. The challenge is to locate and distinguish individual cells and for some cell types, this includes a specific and consistent orientation, shape and position of the cell. Furthermore, current micropatterning techniques do not produce reliable patterns and often these patterns can be maintained in culture for only a few days.   This platform technology includes methods and compositions that allow for a low cost and user-friendly cell culture product with distinct micropatterns to organize cells into specific orientations for a wide variety of cell-based applications. The robust materials used in this method allow for both increased cell compliance and stability of the micropatterns under cell culture conditions. The material can sit at room temperature for over one month with no noticeable degradation in biological activity or cellular compliance to the micropatterns.   For detailed information on the method and proof of concept studies using hippocampal neurons, please refer to the following publications:   WC Chang and DW Sretavan. Novel High-Resolution Micropatterning for Neuron Culture Using Polylysine Adsorption on a Cell Repellant, Plasma-Polymerized Background. 2008. Langmuir. 24:13048-13057.   US Patent Application No. 13/119,693 (more...)

Although every cell contains the entire genome of the organism, only some of the genes are transcribed and translated in a particular cell type. Cellular functions can, therefore, only be understood once each cellular proteome is known. One of the most important tools in identifying all of the proteins and their post-translational modifications in a particular cell involves digesting the entire mixture of proteins all at the same time, and then piecing the sequence information back together at the end. Nearly all proteomics studies are carried out with a single protease digestion step using trypsin. Sequence coverage of abundant proteins using currently available proteases may approach 50 percent but sequence coverage of most proteins is less than 10 percent. In order to increase the sequence coverage, proteases of alternative specificity are needed. (more...)

Researchers at the University of California, Davis have developed a new method of obtaining a library of sialyl Lewis x and other sialosides containing different sialic acid forms. This method utilizes engineered mutants of sialyltransferase PmST1. These novel mutants show lower donor hydrolysis activity and/or sialidase activity without compromising the sialyltransferase activity. (more...)

Use of synthetic nucleic acids to manipulate gene function has become a powerful tool for both basic research and therapeutics.  Silencing disease targets by RNA interference is a promising approach to drug development, and various experimental RNA therapies are currently in clinical development by both small and large biotechnology companies.  miRNAs are also being developed for disease treatment and diagnosis.  However, lack of specifically targeted, efficient and safe vehicles for systemic delivery of small RNA payloads in vivo is a serious challenge.  Synthetic nucleic acids face a number of physiological barriers in the bloodstream, and their intracellular uptake is hampered by the fact that they are highly charged and have much larger molecular wieght than small-molecule drugs.  Current strategies to circumvent these problems includes local administration, chemical modifications of nucleic acids, viral delivery vectors, lipid-based delivery systems, polymer-based delivery systems and nanoparticle encapsulation.  These methods have serious flaws including toxicity, inummue effects, non-selectively and high cost of manufacturing.  Therefore, novel ways to deliver synthetic nucleic acids for use in humans and experimental animal models are sorely needed. (more...)

Researchers at the University of California, Irvine have developed methods to enable greater multiplexing abilities for digital polymerase chain reaction (PCR) so that up to 100 genetic targets may be analyzed. In the past multiplexing of digital PCR samples has been limited to only one probe per color. However multiple probes may be labeled by using combinatorial encoding of color, exploiting reaction rates of PCR cycles and modulating the intensity of Taqman and/or intercalating dyes therefore allowing a greater number of probes to be labeled. (more...)

Researchers at the University of California, Irvine have developed a plasmid that expresses recombinant GST-RILP protein. RILP is a Rab7 effector protein and therefore selectively binds the GTP-bound form of Rab7. (more...)

UC San Diego researchers have discovered an enhancer function of an approximately 30 bp DNA fragment that had been reported to function as a protein transduction domain in microbial cells. (more...)

UCLA investigators have determined that Receptor Interacting Protein 2 (RIP2) has novel functions in regulating Th1 helper T cells and the IL-1/IL-18 Toll-like receptor responses of NK cells and presents a target for therapeutic approaches to the treatment of disorders mediated by these cells, including toxic shock and certain autoimmune diseases. Their results suggest that RIP2 plays a pivotal role in Th1 and NK cell-mediated immune responses and that its regulation should provide a therapeutic approach to the treatment of disorders mediated by these cells, such as toxic shock and of certain autoimmune diseases. (more...)

Systemic lupus erythematosus (SLE or lupus) is a disease characterized by the production of autoantibodies that react with native cells and tissues, causing inflammation, pain, and damage throughout the body. Increased production of IgG in SLE causes the precipitation of immune complexes in the kidney, resulting in irreversible renal damage and failure. There is no known definitive cure for SLE, and treatment is relegated to symptomatic relief of inflammation flare-ups and to non-antigen specific immunosuppression. Non-steroidal anti-inflammatory drugs and anti-malarials are used to treat milder forms of SLE, though corticosteroids and immunosuppressants are used in more severe cases. However, the efficacy of immunomodulating drugs is limited by the increased risk of infection in lupus patients, while corticosteroid therapy is also limited by its side effects, such as obesity, diabetes, and osteoporosis. There is an estimated 1.5 to 2 million Americans with lupus who will benefit from therapeutics that intervene at the gene product level and interrupt the pathogenic process. (more...)

Mycobacterium tuberculosis is a disease that infects millions of people each year; in addition, the related bacterium, Mycobacterium bovis, infects domesticated animals, resulting in substantial economic losses. Currently, humans are administered Bacille Calmette-Guerin (BCG) vaccine to prevent tuberculosis. However, BCG vaccines have variable efficacy - on average about 50%. Recombinant BCG vaccines have been developed that express a key antigen of M. tuberculosis and are more potent than BCG. However, these recombinant BCG vaccines contain antibiotic resistance markers; regulatory authorities want vaccines to be free of such antibiotic resistance markers to diminish their dissemination to other pathogens in the environment. Unmarked vaccine vectors (i.e. those lacking an antibiotic resistance marker) have been produced by various means, but these methods have resulted in low levels of expression of recombinant proteins. Preferably, unmarked strains would not only express large amounts of the recombinant proteins, but express them from genes integrated into the chromosome because such constructs tend to be more stable than when the genes are expressed from a plasmid. Due to safety, potency, regulatory, and stability issues, there is a need for a better vaccine that can prevent and treat tuberculosis in humans and animals. (more...)

The use of antibodies to target tumor cell-associated antigens for diagnostic and therapeutic purposes has been a critical step forward in cancer research. As protein engineering capabilities grow, researchers modify antibodies to alter inherent characteristics, such as affinity and immunogenicity, for enhanced imaging and tumor response. One example of this is in the conjugation of various radionuclides to small recombinant antibody fragments (i.e. diabodies and minibodies) for in vivo tumor cell targeting applications. However, it is not always advantageous to use radioactivity, and thus alternative detection systems are necessary. To that end, the search for high-sensitivity and high-specificity probes that circumvent the limitations of organic dyes and fluorescent proteins has led to the discovery and utilization of quantum dots, nanometer-sized semiconductor particles. Quantum dots are brighter than traditional chromophores, have greater stability, and can be used in multiplex imaging due to size-tunable emission wavelengths. To date, bioconjugates with quantum dots are coupled to intact antibodies whose large size makes it difficult to penetrate tissues and tumors. Therefore, it would be advantageous to monitor tumors with a robust, but small, bioconjugate for tandem in vivo monitoring and treatment. (more...)

The present invention involves a method of artificially introducing an allosteric site into a protein whereby an addition of an artificially designed or a natural modulator of that allosteric site leads to a mechanical tension onto the protein. This mechanical tension alters the proteins affinity for its substrate, and, in the case of an enzyme, the catalysis rate.In addition to the above-described method, the invention involves, but is not limited to, the following: 1. An allosteric protein, selected from a group of well-studied proteins, artificially engineered with an allosteric site, herein referred to as the chimera); 2. A modulator engineered to affect the specific allosteric site on the chimera to effectively affect the chimeras substrate affinity; 3. A well-known substrate for the chimera that can be implemented in an in vitro or an in vivo reporting system.This differential change in substrate affinity or catalysis rate caused by modulator binding can be used in multiple applications. One is the use of such chimera system as an amplified molecular probe, to detect the presence of a natural biological modulator specific to the chimera in an in vitro or in vivo assay. In addition, the chimera-modulator system can be used as a method to study protein conformation and the effect of modulating such conformation on a proteins function. (more...)

An efficient chemical synthesis has been developed for a new low molecular weight compound, Fluoro Nissl Green, which selectively binds RNA in vitro and in vivo in the presence of DNA. The compound is highly fluorescent with an emission at 510 nm and it has been purified and well characterized. Unlike other known RNA-binding compounds, it does not significantly bind to DNA. Unlike antibodies or oligonucleotide sequences targeted for detection of nucleic acids, it is inexpensive to prepare, stable tohydrolysis and is not limited by sequence specificity. Potential uses for Fluoro Nissl Green and related derivatives are in the areas of cancer diagnosis (specifically astrocytic neoplasia), fluorescent activated cell sorting as well as RNA labeling, targeting, separation and quantization. (more...)

Prior to translation, transcription generates a precursor molecule (pre-mRNA) that contains both introns (intervening sequences) and exons (protein coding regions). Alternative splicing pathways vary the production of a mature mRNA strand by modifying the introns removed and the exons joined. Depending on the splice sites, these mRNA variances give rise to proteome diversity by changing the encoded protein structure, which in turn can affect ligand binding, allosteric regulation, protein localization, etc. Although mutations in splice signals account for 15% of genetic diseases caused by point mutations indicating a pressing need for research into the mechanisms controlling alternative splicing, experimental efforts to discover compounds targeting splicing are hampered by a lack of reliable, reproducible, and high-throughput techniques. (more...)

G2A, initially an orphan GPCR, was identified in a search for downstream transcriptional targets of the oncogene BCR-ABL tyrosine kinase. G2A belongs to a family of sequence related GPCRs that were initially thought to bind to proinflammatory lipids. However it was recently discovered that G2A and its related GPCRs act as proton sensors that increase the acid-induced production of secondary messengers such as inositol phosphates and cyclic AMP.G2A is expressed mainly in immune cells including T and B-lymphocytes, monocytes, macrophages and dendritic cells. Currently, G2A and its related GPCRs are implicated in a variety of diseases including autoimmune disorders, inflammation and cancer. However the exact biological functions of these GPCRs have not been elucidated. Therefore, readily available research tools such as those generated by the UCLA investigators could significantly accelerate the research to better understand the role of these GPCRs under physiological and pathological conditions. (more...)

Brutons tyrosine kinase (Btk) is a kinase enzyme that plays an important role in B cell stimulation and maturation. B cell maturation and function is impaired when there is a Btk mutation, leading to X-linked agammaglobulinemia (XLA). It is known that B cell stimulation occurs after the activation of Btk, which is correlated with an increase in the phosphorylation of the regulatory Btk tyrosine site, Btk Y223. Currently, the only method for distinguishing the phosphorylation site is through the process of metabolic radiolabeling of phosphates, enzymatic digestion of the Btk protein, and then the separation of the resulting phosphopeptide fragments (phosphopeptide mapping). Phosphopeptide mapping is a difficult and time-consuming technique where only a few samples can be processed. There is a commercially available anti-phosphotyrosine antibody that recognizes the phosphorylated site through immunoblot and immunoprecipitation assays. However, there is not an antibody that has more specificity to the regulatory Btk 223 tyrosine site. There is a need for a more specific and easier technique of distinguishing the Btk tyrosine site, and consequently for the detection of B cell stimulation and maturation. (more...)

The inventors describe three different knock-in mice, each with a specific mutation associated with different disease phenotypes relating to cryopyrin associated periodic syndromes: familial cold autoinflammatory syndrome, Muckle Wells Syndrome, and neonatal onset multisystem inflammatory disease. These mouse models allow genetic studies of the human disease as well as to study pharmacologic compounds that may be useful for treatment. (more...)

Monoclonal Antibodies Against the Prokaryotic Cyclic Nucleotide-Modulated Ion Channel Mlok1 (more...)

Novel fluorescent heterodimeric DNA-staining energy transfer dyes are provided combining asymmetric cyanine azole-indolenine dyes, which provide for strong DNA affinity, large Stokes shifts and emission in the red region of the spectrum. The dyes find particular application in gel electorphoresis and for labels which may be bound to a variety of compositions in a variety of contexts. Kits and individual compounds are provided, where the kits find use for simultaneous detection of a variety of moities, particularly using a single narrow wavelength irradiation source. The individual compounds are characterized by high donor quenching and high affinity to dsbDNA as a result of optimizing the length of the linking group separating the two chromophores. (more...)

Scientists at the University of California, Berkeley have developed a novel method to produce probes with broad analytical applications. This technique uses cationic fluorescent intercalating dyes which have high affinities for double-stranded DNA. One of the fluorescent dyes, ethidium homodimer cation, complexes with double-stranded DNA at a ratio of one homodimer per four or five base pairs (Proc. Nat'l. Acad. Sci. 87, 3851-3855, 1990). This method enables multiple fluorescent molecules to be loaded onto a single molecule of double-stranded DNA, thus enhancing the fluorescent signal. (more...)

Scientific progress is often associated with the invention of a new experimental apparatus. New tools can increase the ease and efficiency of routine experiments as well as provide the means to make new discoveries by making possible novel experiments. The development of Lab on Chip (LOC) devices is playing an important role in the progression of many different areas of research ranging from point of care diagnostics to the search for life on Mars. LOC devices hold promise to replace existing techniques with processes that are not only more automated and consistent but also require less time and valuable reagents. Researchers at the University of California have developed an integrated LOC for cell-based studies/analysis/research. The device has integrated biological fluidic circuits with the capability of culturing cells inside of a microfluidic ?chip?, the ability to lyse the cells on demand, and the ability to perform on chip analysis of the lysate, which contains both genetic and proteinaceous material. The device is essentially a completely integrated cell-based platform capable of performing practically all of the common cell-based studies currently employed in laboratories across the world. (more...)

The invention provides multifunctional fusion constructs which are rapidly incorporated into a macromolecular structure such as a phycobilisome such that the fusion proteins are separated from one another and unable to self-associate. The invention provides methods and compositions for displaying a functional polypeptide domain on an oligomeric phycobiliprotein, including fusion proteins comprising a functional displayed domain and a functional phycobiliprotein domain incorporated in a functional oligomeric phycobiliprotein. The fusion proteins provide novel specific labeling reagents. (more...)

Isolated and sequenced gene encoding a neuro-specific isoform of a K-Cl cotransporter protein. (more...)

System for bioengineering functional phytochrome assemblies in living cells which produces functional phtochromes in nonphotosynthetic organism E.coli (more...)

One-pot Synthesis for Alpha Glycolipids and their Analogs Using Glycosyl Iodide Donors (more...)

Phytochromes as Fluorescent Markers (more...)

Mucin-Specific Monoclonal Antibodies (more...)

Researchers at the University of California, Davis have developed transgenic mouse models that constitutively express B cell Activating Factor (BAFF) in the Tumor Necrosis Factor (TNF) family, and Green Fluorescent Protein (GFP) under the control of Glial Fibrillary Acidic Protein (GFAP) promoter in astrocytes. (more...)