The project will aim to complete the CLASPER system (Cas9-Linked And SNAP-tag Primed Enhancement of Recombination) that will usher in new therapeutics and genetic studies in animals and plants. To build on the CRISPR/Cas9 revolution in genome editing, we are improving the DNA repair activity by engineering a Cas9SNAP hybrid that joins the enzyme to the DNA repair template. Through a 2014 Sprout grant, we successfully engineered and demonstrated that our Cas9SNAP enzyme cuts a human genome site with twice the efficiency at half the dose of the original Cas9 protein. We also uncovered an important discovery that chemical modification of the 5’ terminus of linear DNA completely blocks genome repair. Therefore, we are developing a new scalable chemical labeling system that will place modifications internally into a circular plasmid DNA repair template. This technology will overcome the barrier of linear DNA repair templates and will complete our CLASPER system.
PI - Maria-Eirini Pandelia, Leader - Ben Pomerantz
Nucleotides are not only indispensable constituents of DNA and RNA, but also crucial signaling molecules in all domains of life. Cyclic dinucleotides (CDNs) represent an important and growing family of second messengers, which have been previously recognized as key modulators governing a variety of cellular activities in bacteria, and more recently, in mammalian cells. In 2012, a novel cyclase fromVibrio cholera(Vc), DncV, was shown to produce from ATP and GTP a new hybrid CDN, the 3′,3′-cGAMP. DncV was demonstrated to promote intestinal colonization ofVcby downregulating chemotaxis, previously associated with hyperinfectivity. Predicted homologs of DncV are present only in other bacterial species (several of which are pathogenic), indicating that 3′3′-cGAMP may also regulate a wide range of cellular functions, similar to c-di-GMP and c-di-AMP. Hybrid CDNs have only recently been in the spotlight and their importance though not well understood, has been exemplified by their occurrence in mammalian cells in which pathways involving the non-canonical 2′,3′-cGAMP act to detect cytosolic DNA and induce an immune response. In the present, we propose the employment of DncV as a system for the large scale enzymatic production of the novel 3′,3′-cGAMP in high amounts and purity. 3′3′-cGAMP (as well as 2′,3′-cGAMP) is only commercially available via chemical synthesis in small quantities and high costs. Development of an inexpensive source of 3′,3′-cGAMP (and other CDNs in general) will allow for the broad exploitation of this molecule as well as for functional studies regarding infectivity pathways associated with CDNs and pathogenic bacteria.
Due to the low survival rate of ovarian cancer, we want to focus on the development of a novel drug—using enzyme-instructed assembly (EIA)—to kill ovarian cancer cells. Herein, we provide a design of a novel and cheaper drug that it is pre-installed with an ester bond which can be cleaved by the carboxylesterase (e.g. CES1, CES2) in cancer cells. The remained part will selectively self-assemble in cancer cells instead of normal cells, which can selectively inhibit the ovarian cancer cells. We will do cytotoxicity studies of the precursor we design on high-grade serous ovarian cancer cells as well as drug resistant ovarian cancer cells. We have done some tests on about 6 ovarian cancer cell lines and two normal lines and our future plan is to try more cell lines to make sure the drug is effective enough. Since the mechanism of the death of ovarian cancer cells induced by EIA is still unknown, to further illustrate the mechanism of the inhibition, we will apply ELISA and knock-down technologies with the grant. We will use the funding for the purchase of amino acids, antibodies, chemical reagents, PathScan Sandwich ELISA kits, knock down transfection reagents, cell lines and cell supplements. Our study will be of great commercial significance since there are limited therapies for ovarian cancer and our drug is effective and cheaper. Besides revealing that intracellular EIA can kill the cancer cells, this work will illustrate a new approach to amplify the enzymatic difference between cancer and normal cells and to expand the pool of drug candidates for potentially overcoming drug resistance in ovarian cancer therapy.
Two frontiers of human medicine are gene therapy and stem cell-derived cellular therapies. However in both cases, effectiveness is limited by cellular diversity. Human organs and most stem cell cultures consist of multiple cell types, yet typically, only one cell type is the desired target for modifying a gene, or for purifying as a cellular therapy. Targeting cell types for research purposes is also at the heart of understanding disease mechanisms that affect all organs, including our most complex organ, the brain. Cell types differ in the transcripts they express, but this is typically only useful for targeting in genetic model organisms.
Here we propose to develop a platform for targetingany specific cell type in any speciesbased on its unique pattern of RNA expression. Our strategy combines designable RNA binding proteins with powerful gene activators. To enhance specificity, nuclear translocation of the transcriptional activator is tightly regulated by the same cell type-specific RNA signal used to turn it on.
Although the individual molecular components already exist, their combined use in such a system is unprecedented. Therefore, we will have to optimize the functions of each of the required components and determine how to combine them optimally. Awarding of this grant will enable us to make a large number of DNA constructs and to test them in cell culture. Once the most efficient combinations of components have been identified, we will produce adeno-associated viruses encoding these components and inject them into mice to demonstrate feasibility forin vivoapplications.
The incidence of insect-borne diseases in New Englanders, including many diseases caused by RNA viruses carried by mosquitoes and by bacteria and protozoa carried ticks, are one the rise due to global warming, increased travel, globalization of economies, and changes in populations of deer and other host animals. Monitoring mosquito and tick populations for these pathogens is the daunting task of Environmental control officers who work with small budgets. Using LATE-PCR and other technologies invented in our laboratory, we are developing several closed-tube assays that will make it convenient and affordable to determine the percentages of infected ticks and mosquitoes in wild populations, as well as which pathogens are present.
Our Comprehensive Tick-Test (CTT) has the capacity to simultaneously identify fifteen species of ticks, as well as nineteen possible pathogens that cause Lyme Disease, Babesiosis, Ehrilichiosis, Anaplasmosis, Relapsing Fever, and Rocky Mountain Spotted Fever. The CTT will be marketed over the internet to health authorities and homeowners who want to know about ticks in the leaf litter, as well as to individuals who remove a tick from their skin and want it analyzed within 24 hrs.
Our Mosquito Pathogen-Test I (MPT-I) will screen batches of mosquitoes for the presence of two species ofAedesmosquitoes,Ae aegyptiandAe albopictus,as well as for several types of viruses: West Nile, EEE, Chikungunya, Yellow Fever, and Zika that may be present. Our Mosquito Pathogen-Test II (MPT-II) will distinguishCulex pipiens and Culex resturansfrom each other (not possible on the basis of morphology) and will also screen for several types of viruses that may be present, including Jamestown Canyon Virus.
With this award, we will purchase reagents to build and optimize these assays. We will also become more familiar with the U.S. and global needs and markets for environmental testing of mosquito and tick borne pathogens.
Cancer and neurodegeneration are arguably the most prevalent and devastating diseases associated with aging. An epidemic of these diseases is looming as the baby boomer generation reaches its twilight years. Up-regulation of the mammalian Target of Rapamycin (mTOR) signaling pathways is common feature of these diseases and inhibition of mTOR holds great promise for the development of treatments.
Unfortunately, down-regulation of mTOR signaling can induce diabetes, and this is a prevalent side effect of currently available drugs targeting mTOR. We have discovered a small molecule (CB3A) that inhibits mTOR signaling via a novel mechanism that may not have this liability. We seek funds to elucidate the mechanism of CB3A action. This information is critical to determine if there will be a therapeutic advantage for CB3A- based treatment and also to identify which diseases are most likely to respond.
Trifluoromethylated amines have improved lipophilicity and metabolic stability over those by the corresponding methyl amines. With such superior properties for drug development, trifluoromethyl amines have received wide attention in pharmaceutical industry. Organic molecules bearing trifluoromethylated amines have been developed into commercially successful therapeutics. The progress in medicinal chemistry in turn greatly increased the demand for chiral trifluoromethylated amines. The isomerization of trifluoromethyl imines represents one of the most general and attractive methods for the preparation of chiral trifluoromethylated amines. Our group was the first in developing an efficient chiral organic catalyst for highly enantioselective isomerizations of trifluoromethyl imines to form chiral trifluoromethylated amines. However, several drawbacks, such as high catalyst loadings, long reaction time and the limitation of providing useful access to only one of the two enantiomers of the trifluoromethylated amines, greatly hampered the wide applications of these new asymmetric reactions in the synthesis of chiral trifluoromethylated amines. In this application, we outlined a new approach for the development of powerful new organic catalysts that will successfully address all the drawbacks of existing catalysts, thereby translating the newly discovered asymmetric isomerization of trifluoromethyl imines into a widely applicable synthetic method for the synthesis of chiral trifluoromethylated amines in the context of drug discovery and environmentally sustainable manufacturing. Moreover, these new catalysts were designed to efficiently promote a series of other mechanistically related isomerizations to form additional chiral building blocks of pharmaceutical importance, such as butenolides, -amino acids, allenes and cyclohexenones.
Genetically tractable organisms like mice, fruit flies (Drosophila) and worms are frequently used to investigate the molecular and cellular mechanisms that underlie basic features of human physiology. This is because the molecules and pathways in these model organisms are similar if not identical to those of humans. These organisms are also used for practical studies, for example to help prevent disease or to develop various treatment strategies.
Drosophilais often the animal of choice. In addition to its abundant genetics tools and resources as well as the large community that has studied it for more than a century,Drosophilahas a short generation time and is cheap to grow and maintain the laboratory. Surprisingly perhaps,Drosophilais also an important organism for studies focused on the brain, cognition and behavior. A good example of aDrosophilabehavior that is studied all over the world is locomotor activity (movement or walking) and its accompanying sleep-wake cycles. Here too there are many similarities with humans.
The current tool to measureDrosophilalocomotor activity is called theDrosophilaActivity Monitor (DAM), which was invented more than 25 years ago. It is a basic “beam break device.” Single infrared beams are located in the middle of glass tubes. The beams detect the movements of individual flies whenever they cross the beam. Although this system is widely used for studying circadian rhythms and sleep in flies, it has many drawbacks and has not been updated since it was invented.
First, the DAM system is quite expensive, especially for young scientists who want to set up a new lab. Each DAM unit costs 800 dollars and only can record the activity of 32 flies. Second, the DAM system has many blind spots and is insufficiently sensitive to characterize or even detect small movements. Third, the size of the DAM system makes it inconvenient, probably impossible, to incorporate additional features such as LEDs for optogenetics or devices for sleep deprivation. In short, these drawbacks of the DAM system are a bottleneck for the broader study ofDrosophilabehavior.
In order to overcome these issues, my team has engineered a totally new, all-in-one system. It utilizes a high sensitivity video camera to record individual fly behavior in four 96-well plates. Our new system has therefore increased the sensitivity of the detection system as well as the throughput, in the latter case by 12 times (96X4 flies vs 32 flies). In addition, we have incorporated UV LEDs for entrainment, red LEDs for optogenetics as well as a solenoid device for arousal. All of this is built into one box, FlyBox, which sits on a bench-top. Importantly, this new system therefore bypasses the need for the expensive and space-hungry incubators into which the DAM systems normally reside.
Dietary modulation is a primary consideration in the prevention and management of Metabolic Syndrome (MetS) and Type 2 Diabetes Mellitus (T2DM). Dietary fiber, especially soluble fiber such as inulin from Chicory root, has benefited humans in such circumstances. The Nile rat is a novel model for T2DM and MetS that, like humans, responds favorably to increased fiber consumption. In a preliminary experiment three different types of fiber (10% cellulose-insoluble, 10% inulin-soluble, 10% carrot pomace, a mixture of soluble-insoluble fiber) were compared to 0% fiber. The carrot pomace powdered fiber (CPP) uniquely appeared to prevent, delay, or reduce T2DM in the male Nile rat model. In a follow-up study, 60 male Nile rats again were fed for 10 weeks these original four diets, plus two additional fibers as the CPP finely ground to 120-mesh, or HydrobindTM, primarily an insoluble form of carrot pomace. Necropsy data was collected to assess organ damage along with other terminal assays to ascertain the degree of diabetes following the consumption of these various fibers.
Measures of diabetes % incidence and severity (fasting blood glucose [FBG], random blood glucose [RBG], and the oral glucose tolerance test [OGTT]) were pooled with data from the initial study for n=27-28 on each of the first four diets. After 10 weeks on diet, CPP was the only fiber to significantly lower T2DM (as % incidence based on RBG >75 mg/dl, which is the best predictor of organ damage) compared to control. Furthermore, CPP was the only diet to have 0% incidence of T2DM at termination (compared to 43%, 43%, and 30% incidence for control, cellulose, and inulin, respectively). The RBG and 30 min OGTT were significantly lower following CPP than after control or cellulose. In the second study, HydrobindTMand finely ground 120-mesh CPP produced more diabetes and greater incidence and severity of T2DM than the 60-mesh CPP.
In summary, soluble fiber favorably impacts T2DM in the young male Nile rat model, and 60-mesh CPP appeared uniquely more effective at diabetes prevention than cellulose, inulin, or the two other modified versions of CPP. These data suggest that CPP is potentially superior to the most commonly used dietary fibers in the marketplace, and should enjoy a highly beneficial presence in many foods that incorporate extra fiber for health reasons.
Consumption of cruciferous vegetables such as kale and cauliflower, can help prevent cancer and many other diseases. The ‘active ingredients’ in these vegetables are called isothiocyanates (ITCs). Recently, our group discovered the long-sought-after molecular basis for these health effects: ITCs block the action of deubiquitinating enzymes (DUBs). With this finding, a novel DUB inhibitor chemotype has been uncovered. As part of the ubiquitin-proteasome system, DUBs are critical regulators of most cellular processes and many are recognized as attractive therapeutic targets for the treatment of illnesses such as cancer, chronic inflammation, and neurodegenerative diseases. Here we report our evaluation of synthetic isothiocyanate-based DUB inhibitors and describe newly discovered and therapeutically significant ITC DUB targets. Utilizing a SILAC-assisted quantitative proteomics approach, other ITC-DUB targets have been identified. These are DUBs involved in critical pathways such as DNA repair, inflammation, and cell cycle progression which underscores the important role dietary isothiocyanates have on cell health. Using nature as a template has been a successful strategy in drug development and one which should benefit us as we strive to develop potent and selective ITC-based DUB inhibitors.
Taxol® (generic name paclitaxel) is one of the most commercially successful anti-cancer drugs with an estimated annual profit of over 1 billion dollars. Currently, Taxol® is approved for the treatment of a broad range of cancer sub-types and is being investigated for its effectiveness in the treatment of neurological and cardiac diseases. Naturally obtained from the leaves and bark of yew trees, Taxol® was initially difficult to produce on the scale required to match the medical need. In order to increase production, companies have begun favoring cultured plant cells for the generation of Taxol®. However, industrial-scale plant cell cultures have severe drawbacks not least of which are low yields and variable production levels between different cell lines as well as within the same line with time.
We propose to address the increasing demand for Taxol® through the development of a bacterial cell expression line in which we will engineer the biosynthetic pathway. Not only do bacterial cell cultures lack the aforementioned drawbacks of plant cultures, they also have the added benefit of being more cost effective. Since receiving the SPROUT award in July, we have successfully engineered the first two steps of the biosynthetic pathway intoE. colicells and have shown that the first three steps can be catalyzed in anin vitroreaction. The engineering of this cell line will also enable the creation of a platform for the generalized production of terpenes, the family of compounds to which Taxol® belongs. Terpenes are the largest class of naturally derived products and have numerous potential commercial applications ranging from therapeutics to potent and renewable biofuels.
SELMA (SELection with Modified Aptamers) is a directed evolution technique used to discover DNA-scaffolded multivalent presentations of a ligand to bind specifically to a protein target with multiple ligand sites. We have applied this technique to the selection of multivalent carbohydrate structures of interest as HIV vaccines. A library of DNAs containing unnatural, alkynyl bases is glycosylated with a carbohydrate azide using click chemistry. The carbohydrate modified DNA library can then undergo selection in the presence of the carbohydrate-binding protein of interest to obtain carbohydrate presentations which are matched to the spacing of binding sites in the target protein. Using SELMA, we have discovered carbohydrate clusters which bind to HIV broadly-neutralizing antibody 2G12 with low-nanomolar affinities as strong as the recognition of the HIV protein gp120. Our current efforts are directed toward developing an optimal formulation of these carbohydrate clusters for vaccine testing in animals, and toward selection of multivalent ligands for other targets, including additional HIV neutralizing antibodies.
The ultimate goal of cancer therapy is to kill cancer cells selectively without harming normal cells. We are using the SPROUT funding to aid our research in the development of a novel cancer therapy: the use of enzymatic reactions to recruit chiral nanoparticles (i.e., nanoparticles decorated with D-phosphotyrosine) to selectively inhibit cancer cells in their co-culture with normal cells. Specifically, alkaline phosphatase (ALP), a membrane enzyme overexpressed on the cancer cells, catalytically dephosphorylates the D-phosphotyrosines on the nanoparticles to enable them to adhere selectively on the cancer cells for inhibiting cancer cells via extrinsic death pathways. Without phosphate groups or being prematurely dephosphorylated, the nanoparticles are innocuous to cells. Illustrating the use of a multistep process, but not the dogmatic receptor-ligand (or “lock-key”) interaction, to amplify the generic difference between cancer and normal cells for selectively killing cancer cells, this work promises a paradigm-shifting strategy, which engages “undruggable” enzymes such as phosphatase, for future cancer nanomedicine.
PI - Sacha Nelson, Leader - Yasmin Escobedo Lozoya
Organotypic brain slice cultures (BSCs) have been used successfully in neuroscience research since the pioneering studies of Gahwiler in the 1980s and gained popularity after the refinements introduced by the Muller lab in the early 1990s. Many basic research studies have found slice cultures to be a more accurate model for the brain than other alternatives (7), however their adoption as a model system has been limited by the low viability and poor throughput of standard methods for production of brain slices. To eliminate these barriers, we have modified the mechanical design of brain slicers and produce an instrument that achieves the reliability and cutting precision of slicers such as the industry leader VT1000S™ vibratome while producing higher quality slice cultures with greater throughput.
PI - Christine Thomas, Leader - Deirdra Evers-McGregor
Commodity chemicals that directly impact our daily lives often synthesized in chemical industrial processes that are not environmentally responsible or economical. A few examples include polymerization reactions required for plastic manufacturing, petroleum cracking and emissions processing in automobiles in which the catalytic converters often use expensive starting materials and expensive transition metals. Researchers around the world are always looking for new catalysts that can lead to a reduction in both reaction costs and the environmental impact. To address this need, we have used the SPROUT funding to develop a potential catalyst capable of supplementing the current technologies using greener, less expensive, and more efficient processes.
We have established that by coordinating our novel class of ligands to platinum and palladium, these non-innocent chelators are capable of undergoing multi-electron catalytic transformations. We are now focused on harnessing this unique redox chemistry to promote analogous functionality in nickel-containing compounds. Because nickel is Earth abundant and significantly less expensive than platinum and palladium, this can be considered a more green catalyst. The nickel compounds synthesized could be used for the activation and functionalization of small molecules that would be applicable to both organic chemists as well as the industrial production of bulk commodity and fine chemicals.
Lack of inhibition in the nervous system is an underlying cause of epilepsy, a disease characterized by runaway excitation in the brain. Millions of Americans suffer from epilepsy, and 1/3 of patients do not respond to currently available treatments. Further, all currently available anti-epilepsy drugs (AEDs) only treat the symptoms of epilepsy (i.e. seizure) without addressing the underlying cause of the seizures: decreased inhibition.
Our laboratory discovered a previously unknown role for the protein Semaphorin4D (Sema4D) as an important regulator of inhibitory synapse development. Using anin vitromodel of epileptiform activity, we demonstrated that 2 hours of Sema4D treatment rapidly and dramatically reduces the hyperexcitability of this tissue. Preliminary data indicates that this effect persists for at least 24 hours post-Sema4D treatment, suggesting that these new synapses are stabilized and outlast the activation of the signaling pathway. Further, we find that Sema4D treatment reduces mortality in anin vivomodel of epilepsy. Taken together, these results suggest that Sema4D is a promising antiepileptic drug (AED) candidate.
In ongoing experiments, we are examining thein vivoeffects of Sema4D treatment on seizure severity using electroencephalography (EEG). Seizures are characterized by abnormal EEG activity, and seizure severity is correlated with EEG frequency and amplitude. Using EEG, we may identify effects of Sema4D treatment that are not observable by behavioral measures. Further, we are exploring a newin vivoseizure model, intravenous pentylenetetrazol (PTZ). PTZ infusion produces highly stereotyped seizure behaviors and EEG waveforms. Thus, effects of Sema4D treatment will be more apparent when compared to these consistent baselines. Using these new paradigms, we will further evaluate Sema4D’s potential as an AED.
We have set up a plasmid based CRISPR system with a modified Cas9-SNAP protein. We have tested a variety of transgenes to set a baseline for integration efficiency. We have modified the 5’ and 3’ ends of the transgene with benzylguanine (BG) to test if directly binding the transgene to Cas9 to directly target it to the genomic locus of interest improves integration efficiency. We can correctly target transgenes into the desired genomic locus when all parts of the CRISPR system are present. 5’ and 3’ end modifications decrease homologous recombination efficiency. We are working on adding BG to the transgene internally to work around problems with end modifications. Additionally we are working on cloning and expressing a recombinant Cas9-SNAP to bind the BG coupled transgene in vitro before introduction into the cell.
Aurora A kinase is an oncoprotein, whose upregulation stimulates uncontrolled cell proliferation in a multitude of cancers. It has been the target of several drugs currently in clinical trials, but these compounds are found to be highly toxic, because they bind nonspecifically to the ATP site conserved in all kinases. To circumvent this problem, we propose an innovative idea that the TPX2- interacting pocket in Aurora A kinase is a novel docking site for which new drugs with high specificity and potency could be designed to suppress cancer development. Binding of the TPX2 protein is absolutely necessary for Aurora A kinase to promote cell cycle progression, and therefore a drug that disrupts TPX2-Aurora A interaction could in principle arrest cancer cells growth. Our in vitro work has successfully identified a small-molecule compound PS48 that inhibits the enzymatic
activity of purified Aurora A kinase by perturbing TPX2 binding. Moreover, we found that PS48 triggers death of cultured human cancer cell lines. The Sprout grant supports our research endeavor to further understand how PS48 exerts its pharmacological effect in vivo, and this knowledge will be used to develop a cell-based assay compatible with high throughput drug screening facilities in major government agencies or private-sectors.
Industrial insulin production, relying on the expression of insulin from either yeast or E. coli, supports adequate healthcare delivery for millions of diabetic patients. Currently insulin is made by two methods: Eli Lilly refolds recombinant insulin expressed in bacteria; Novo Nordisk purifies insulin that has been secreted by yeast. Both methods are environmentally damaging and costly compared to the large-scale production of many other biologics, largely due to insulin misfolding and aggregation. To improve the efficiency of industrial insulin secretion from yeast, we will systematically identify genes that inhibit or promote the secretion of insulin from the yeast. An optimal insulin expression system will be developed for yeast in which the identified inhibitory genes are suppressed, while the enhancing genes are overexpressed. Insulin will then fold and be secreted more efficiently by yeast, resulting in an improved secretion system for industrial applications. Sprout Grant funding will support the identification of these genes so that the optimal expression system can be designed. This technology can then be licensed into a healthy and rapidly expanding market of not only insulin related products, but as a platform technology for any biological pharmaceutical products secreted by yeast.
The powerful genetics makes fruit flies a major model organism for biological studies. However, the very basic fly genetics is not easy for lab beginners. It takes them several hours, even several days, to learn how to pick virgins or to get familiar with a marker. The aim of the smart phone App “An Introduction to Fly Genetics for Lab Beginners” is to help lab beginners to learn the basic fly genetics during the first few days or weeks in fly labs. Plenty of smart phone Apps have been designed for shopping, books, games, music, movies, elementary educations and so on; but very few for lab researches. Comparing with traditional textbooks, the advantage of this smart phone App will be: 1) “Real” pictures. All pictures will be taken under dissecting microscope so that lab beginners can see the “real” flies rather than cartoons. Lab beginners can adjust the size of the pictures in smart phones. It will be easier for them to compare and to decide whether they choose the right genotypes. 2) Searchable. This App is like an e-book, which can be easily searched and marked. 3) Easy to access. Smart phones are easily accessed at any time and at any places. The heavy textbooks will be left at home, but smart phones will be always on you. 4) Low cost and environmentally friendly. It is paperless. During the year of Sprout Grant, we will develop the first version. It will include five parts: male vs female, virgins vs non-virgins, dominant markers, balancers vs dominant markers, and basic crosses. Pictures and texts will be used for explanation. In the later versions, we will include more comprehensive fly genetic technology. Moreover, animations, voices and simple quizzes will also be used to explain the technology.
The chemoprotective effects of a diet rich in broccoli or kale has been appreciated for several decades. Such cruciferous vegetables are a rich source of isothiocyanates (ITCs) such as benzyl ITC (BITC) and phenethyl ITC (PEITC). Each of these ITCs have antiproliferative activity against various tumors and PEITC is in clinical trials for lung and oral cancers. However, the mechanism by which ITCs suppress carcinogenesis has been the subject of much debate and numerous potential targets have been proposed. Here we show that BITC and PEITC inhibit the deubiquitinating enzyme (DUB) USP9xin vitroand in living cells. Both ITC treatment and USP9x knockdown decrease the levels of the oncogenic proteins MCL1 and Bcr-Abl kinase. BITC and PEITC also inhibit UCH37, a proteasome associated DUB involved in the degradation of many proteins. Competitive activity profiling in cells pre-treated with these ITCs suggests that other DUBs may also be inhibited. Inhibition occurs at physiologically relevant concentrations and time scales, and thus can explain many of the anticancer properties of dietary ITCs.
"Structure determines function" is the mantra of structural biology. X-ray diffraction from protein crystals is the most prevalent method of determining protein structure; therefore crystallizing protein is a critical step in the structure pipeline. The pipeline consists of screening a protein against a wide range of precipitants. In the case that crystallization occurs, crystals are cryo-cooled to minimize radiation damage and mounted in an x-ray beam. Crystals are selected to be large enough to provide diffraction from many different crystal orientations in order to collect enough information to solve the structure. This standard method is challenging when the crystals are small and fragile as thermal stresses induced during cryo-cooling damages the crystals, which in turn degrades the quality of the diffraction data. Fabricating a microfluidic platform that allows growing protein crystals using well-controlled crystallization conditions combined with in-situ x-ray diffraction data collection eliminates the above-mentioned challenges. I will discuss the device we’ve fabricated in which the diffraction data is collected in-situ at room temperature using emulsion method. Diffraction data are measured; one crystal at a time, from a series of room-temperature crystals stored in an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different un-oriented crystals. As proof of concept, the structure of glucose isomerase was solved to 2.1 angstroms demonstrating the feasibility of high-throughput serial x-ray crystallography using synchrotron radiation. This device will be purchased by thousands of researchers, X-ray structure based drug discovery companies which are multi-billion dollar /year businesses.