Publications by authors named "Anup K Singh"

87 Publications

Specialized Plant Growth Chamber Designs to Study Complex Rhizosphere Interactions.

Front Microbiol 2021 26;12:625752. Epub 2021 Mar 26.

Climate and Ecosystem Sciences, Earth and Environmental Sciences Area, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.

The rhizosphere is a dynamic ecosystem shaped by complex interactions between plant roots, soil, microbial communities and other micro- and macro-fauna. Although studied for decades, critical gaps exist in the study of plant roots, the rhizosphere microbiome and the soil system surrounding roots, partly due to the challenges associated with measuring and parsing these spatiotemporal interactions in complex heterogeneous systems such as soil. To overcome the challenges associated with study of rhizosphere interactions, specialized plant growth chamber systems have been developed that mimic the natural growth environment. This review discusses the currently available lab-based systems ranging from widely known rhizotrons to other emerging devices designed to allow continuous monitoring and non-destructive sampling of the rhizosphere ecosystems in real-time throughout the developmental stages of a plant. We categorize them based on the major rhizosphere processes it addresses and identify their unique challenges as well as advantages. We find that while some design elements are shared among different systems (e.g., size exclusion membranes), most of the systems are bespoke and speaks to the intricacies and specialization involved in unraveling the details of rhizosphere processes. We also discuss what we describe as the next generation of growth chamber employing the latest technology as well as the current barriers they face. We conclude with a perspective on the current knowledge gaps in the rhizosphere which can be filled by innovative chamber designs.
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http://dx.doi.org/10.3389/fmicb.2021.625752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8032546PMC
March 2021

Induction of APOBEC3B expression by chemotherapy drugs is mediated by DNA-PK-directed activation of NF-κB.

Oncogene 2021 Feb 15;40(6):1077-1090. Epub 2020 Dec 15.

Department of Surgery & Cancer, Imperial College London, London, W12 0NN, UK.

The mutagenic APOBEC3B (A3B) cytosine deaminase is frequently over-expressed in cancer and promotes tumour heterogeneity and therapy resistance. Hence, understanding the mechanisms that underlie A3B over-expression is important, especially for developing therapeutic approaches to reducing A3B levels, and consequently limiting cancer mutagenesis. We previously demonstrated that A3B is repressed by p53 and p53 mutation increases A3B expression. Here, we investigate A3B expression upon treatment with chemotherapeutic drugs that activate p53, including 5-fluorouracil, etoposide and cisplatin. Contrary to expectation, these drugs induced A3B expression and concomitant cellular cytosine deaminase activity. A3B induction was p53-independent, as chemotherapy drugs stimulated A3B expression in p53 mutant cells. These drugs commonly activate ATM, ATR and DNA-PKcs. Using specific inhibitors and gene knockdowns, we show that activation of DNA-PKcs and ATM by chemotherapeutic drugs promotes NF-κB activity, with consequent recruitment of NF-κB to the A3B gene promoter to drive A3B expression. Further, we find that A3B knockdown re-sensitises resistant cells to cisplatin, and A3B knockout enhances sensitivity to chemotherapy drugs. Our data highlight a role for A3B in resistance to chemotherapy and indicate that stimulation of A3B expression by activation of DNA repair and NF-κB pathways could promote cancer mutations and expedite chemoresistance.
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http://dx.doi.org/10.1038/s41388-020-01583-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116738PMC
February 2021

AI26 inhibits the ADP-ribosylhydrolase ARH3 and suppresses DNA damage repair.

J Biol Chem 2020 10 4;295(40):13838-13849. Epub 2020 Aug 4.

School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China. Electronic address:

The ADP-ribosylhydrolase ARH3 plays a key role in DNA damage repair, digesting poly(ADP-ribose) and removing ADP-ribose from serine residues of the substrates. Specific inhibitors that selectively target ARH3 would be a useful tool to examine DNA damage repair, as well as a possible strategy for tumor suppression. However, efforts to date have not identified any suitable compounds. Here, we used and biochemistry screening to search for ARH3 inhibitors. We discovered a small molecule compound named ARH3 inhibitor 26 (AI26) as, to our knowledge, the first ARH3 inhibitor. AI26 binds to the catalytic pocket of ARH3 and inhibits the enzymatic activity of ARH3 with an estimated IC of ∼2.41 μm Moreover, hydrolysis of DNA damage-induced ADP-ribosylation was clearly inhibited when cells were pretreated with AI26, leading to defects in DNA damage repair. In addition, tumor cells with DNA damage repair defects were hypersensitive to AI26 treatment, as well as combinations of AI26 and other DNA-damaging agents such as camptothecin and doxorubicin. Collectively, these results reveal not only a chemical probe to study ARH3-mediated DNA damage repair but also a chemotherapeutic strategy for tumor suppression.
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http://dx.doi.org/10.1074/jbc.RA120.012801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7535916PMC
October 2020

Structural basis of specific DNA binding by the transcription factor ZBTB24.

Nucleic Acids Res 2019 09;47(16):8388-8398

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

ZBTB24, encoding a protein of the ZBTB family of transcriptional regulators, is one of four known genes-the other three being DNMT3B, CDCA7 and HELLS-that are mutated in immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome, a genetic disorder characterized by DNA hypomethylation and antibody deficiency. The molecular mechanisms by which ZBTB24 regulates gene expression and the biological functions of ZBTB24 are poorly understood. Here, we identified a 12-bp consensus sequence [CT(G/T)CCAGGACCT] occupied by ZBTB24 in the mouse genome. The sequence is present at multiple loci, including the Cdca7 promoter region, and ZBTB24 binding is mostly associated with gene activation. Crystallography and DNA-binding data revealed that the last four of the eight zinc fingers (ZFs) (i.e. ZF5-8) in ZBTB24 confer specificity of DNA binding. Two ICF missense mutations have been identified in the ZBTB24 ZF domain, which alter zinc-binding cysteine residues. We demonstrated that the corresponding C382Y and C407G mutations in mouse ZBTB24 abolish specific DNA binding and fail to induce Cdca7 expression. Our analyses indicate and suggest a structural basis for the sequence specific recognition by a transcription factor centrally important for the pathogenesis of ICF syndrome.
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http://dx.doi.org/10.1093/nar/gkz557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895263PMC
September 2019

Rapid characterization of the activities of lignin-modifying enzymes based on nanostructure-initiator mass spectrometry (NIMS).

Biotechnol Biofuels 2018 27;11:266. Epub 2018 Sep 27.

1Joint BioEnergy Institute, Emeryville, CA 94608 USA.

Background: Producing valuable fuels and chemicals from lignin is a key factor for making lignocellulosic biomass economically feasible; however, significant roadblocks exist due to our lack of detailed understanding of how lignin is enzymatically depolymerized and of the range of possible lignin fragments that can be produced. Development of suitable enzymatic assays for characterization of putative lignin active enzymes is an important step towards improving our understanding of the catalytic activities of relevant enzymes. Previously, we have successfully built an assay platform based on glycan substrates containing a charged perfluorinated tag and nanostructure-initiator mass spectrometry to study carbohydrate active enzymes, especially various glycosyl hydrolyses. Here, we extend this approach to develop a reliable and rapid assay to study lignin-modifying enzymes.

Results: Two β-aryl ether bond containing model lignin dimer substrates, designed to be suitable for studying the activities of lignin-modifying enzymes (LMEs) by nanostructure-initiator mass spectrometry (NIMS), were successful synthesized. Small-angle neutron scattering experiments showed that these substrates form micelles in solution. Two LMEs, laccase from the polypore mushroom , and manganese peroxidase (MnP) from white rot fungus , were tested for catalytic activity against the two model substrates. We show that the reaction of laccase and MnP with phenolic substrate yields products that arise from the cleavage of the carbon-carbon single bond between the α-carbon and the adjacent aryl carbon, consistent with the mechanism for producing phenoxy radical as reaction intermediates. Reactions of the nonphenolic substrate with laccase, on the other hand, adopt a different pathway by producing an α-oxidation product; as well as the cleavage of the β-aryl ether bond. No cleavage of the carbon-carbon bond between the α-carbon and the aryl carbon was observed. To facilitate understanding of reaction kinetics, the reaction time course for laccase activity on the phenolic substrate (I) was generated by the simultaneous measurement of all products at different time points of the reaction. Withdrawal of only a small sample aliquot (0.2 μL at each time point) ensured minimum perturbation of the reaction. The time course can help us to understand the enzyme kinetics.

Conclusions: A new assay procedure has been developed for studying lignin-modifying enzymes by nanostructure-initiator mass spectrometry. Enzyme assays of a laccase and a MnP on phenolic and nonphenolic β-aryl ether substrates revealed different primary reaction pathways due to the availability of the phenoxy radical intermediates. Our assay provides a wealth of information on bond cleavage events not available using conventional colorimetric assays and can easily be carried out in microliter volumes and the quantitative analysis of product formation and kinetics is rapidly achieved by NIMS. This is the first time that NIMS technology was applied to study the activities of lignin-modifying enzymes. Unlike other previous works, our use of amphiphilic guaiacylglycerol β--4 substrate (I) enables the formation of micelles. This approach helps avoid the re-polymerization of the resulting monomeric product. As a result, our assay can clearly demonstrate the degradation pathways of phenolic guaiacylglycerol β--4 type of molecules with laccase and MnP.
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http://dx.doi.org/10.1186/s13068-018-1261-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158898PMC
September 2018

Integrated LAMP and immunoassay platform for diarrheal disease detection.

Biosens Bioelectron 2018 Nov 10;120:93-101. Epub 2018 Aug 10.

Biotechnology and Bioengineering, Sandia National Laboratories, 7011 East Avenue, Livermore, CA 94550, United States. Electronic address:

The challenges of diagnosing infectious disease, especially in the developing world, and the shortcomings of available instrumentation have exposed the need for portable, easy-to-use diagnostic tools capable of detecting the wide range of causative microbes while operating in low resource settings. We present a centrifugal microfluidic platform that combines ultrasensitive immunoassay and isothermal amplification-based screening for the orthogonal detection of both protein and nucleic acid targets at the point-of-care. A disposable disc with automatic aliquoting inlets is paired with a non-contact heating system and precise rotary control system to yield an easy-to-use, field-deployable platform with versatile screening capabilities. The detection of three enterotoxins (cholera toxin, Staphylococcal enterotoxin B, and Shiga-like toxin 1) and three enteric bacteria (C. jejuni, E. coli, and S. typhimurium) were performed independently and shown to be highly sensitive (limit of detection = 1.35-5.50 ng/mL for immunoassays and 1-30 cells for isothermal amplification), highly exclusive in the presence of non-specific targets, and capable of handling a complex sample matrix like stool. The full panel of toxins and bacteria were reliably detected simultaneously on a single disc at clinically relevant sample concentrations in less than an hour. The ability of our technology to detect multiple analyte types in parallel at the point-of-care can serve a variety of needs, from routine patient care to outbreak triage, in a variety of settings to reduce disease impact and expedite effective treatment.
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http://dx.doi.org/10.1016/j.bios.2018.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6145809PMC
November 2018

Insulator Nanostructure Desorption Ionization Mass Spectrometry.

Anal Chem 2018 08 6;90(16):9657-9661. Epub 2018 Aug 6.

DOE Joint BioEnergy Institute , 5885 Hollis Street , Emeryville , California 94608 , United States.

Surface-assisted laser desorption ionization (SALDI) is an approach for gas-phase ion generation for mass spectrometry using laser excitation on typically conductive or semiconductive nanostructures. Here, we introduce insulator nanostructure desorption ionization mass spectrometry (INDI-MS), a nanostructured polymer substrate for SALDI-MS analysis of small molecules and peptides. INDI-MS surfaces are produced through the self-assembly of a perfluoroalkyl silsesquioxane nanostructures in a single chemical vapor deposition silanization-step. We find that surfaces formed from the perfluorooctyltrichlorosilane monomer assemble semielliptical features with a 10 nm height, diameters between 10 and 50 nm, and have attomole-femtomole sensitivities for selected analytes. Surfaces prepared with silanes that either lack the trichloro or perfluoro groups, lack sensitivity. Further, we demonstrate that hydrophobic INDI regions can be micropatterned onto hydrophilic surfaces to perform on-chip self-desalting in an array format.
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http://dx.doi.org/10.1021/acs.analchem.8b01989DOI Listing
August 2018

De novo DNA synthesis using polymerase-nucleotide conjugates.

Nat Biotechnol 2018 08 18;36(7):645-650. Epub 2018 Jun 18.

Joint BioEnergy Institute, Emeryville, California, USA.

Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of ∼200 mers and produces hazardous waste. Here, we describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the tethered dNTP, the 3' end of the primer remains covalently bound to TdT and is inaccessible to other TdT-dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. We demonstrate that TdT-dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10-20 s, and that the scheme can be iterated to write a defined sequence. This approach may form the basis of an enzymatic oligonucleotide synthesizer.
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http://dx.doi.org/10.1038/nbt.4173DOI Listing
August 2018

Nanoporous Hydrogels for the Observation of Anthrax Exotoxin Translocation Dynamics.

ACS Appl Mater Interfaces 2018 Apr 4;10(16):13342-13349. Epub 2018 Apr 4.

Center for Biological and Engineering Sciences , Sandia National Laboratories , Albuquerque , New Mexico 87185 , United States.

The ability to observe lethal anthrax exotoxins translocating through size-constricting nanopores in vitro, combined with detailed sequence and structural data, has aided in elucidated mechanisms of exotoxin cell entry and toxicity. However, due to limited observations of anthrax exotoxins translocating through protective antigen nanopores in vitro and the instability of protective antigen-functionalized suspended lipid bilayers, questions remain regarding the native mechanisms of cell entry. Nanoporous hydrogel membranes offer a robust tool for studying protein translocation with ensemble measurements that complement conventional single-molecule translocation measurements. Here, we utilize nanoporous hydrogel membranes to assess the translocation of full-length anthrax lethal and edema factors through nanopores similar in diameter to protective antigen translocons. We find that, relative to globular serum and other proteins that do not translocate natively through nanopores, anthrax exotoxins demonstrate significantly reduced barriers to pore entry. Computed free-energy barriers to the unfolding of proteins and the dissociation of macromolecular complexes are generally found to coincide with translocation. Finally, a nanopore-blocking strategy is developed that utilizes nonspecific synthetic peptide constructs and effectively prevents LF translocation within the nanoporous hydrogel.
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http://dx.doi.org/10.1021/acsami.8b01871DOI Listing
April 2018

Timescale Separation of Positive and Negative Signaling Creates History-Dependent Responses to IgE Receptor Stimulation.

Sci Rep 2017 Nov 14;7(1):15586. Epub 2017 Nov 14.

Biological and Material Sciences, Sandia National Laboratories, Livermore, CA, USA.

The high-affinity receptor for IgE expressed on the surface of mast cells and basophils interacts with antigens, via bound IgE antibody, and triggers secretion of inflammatory mediators that contribute to allergic reactions. To understand how past inputs (memory) influence future inflammatory responses in mast cells, a microfluidic device was used to precisely control exposure of cells to alternating stimulatory and non-stimulatory inputs. We determined that the response to subsequent stimulation depends on the interval of signaling quiescence. For shorter intervals of signaling quiescence, the second response is blunted relative to the first response, whereas longer intervals of quiescence induce an enhanced second response. Through an iterative process of computational modeling and experimental tests, we found that these memory-like phenomena arise from a confluence of rapid, short-lived positive signals driven by the protein tyrosine kinase Syk; slow, long-lived negative signals driven by the lipid phosphatase Ship1; and slower degradation of Ship1 co-factors. This work advances our understanding of mast cell signaling and represents a generalizable approach for investigating the dynamics of signaling systems.
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http://dx.doi.org/10.1038/s41598-017-15568-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5686181PMC
November 2017

The Use of M-Mode Ultrasonography to Differentiate the Causes of B Lines.

Chest 2018 03 26;153(3):689-696. Epub 2017 Oct 26.

Department of Pulmonary and Critical Care Medicine, Long Island Jewish Medical Center, New Hyde Park, NY.

Background: The presence of B lines on lung ultrasonography is a characteristic feature of both cardiogenic pulmonary edema (CPE) and noncardiogenic alveolar interstitial syndrome (NCAIS), so their presence does not allow the clinician to differentiate between the two entities. Our study used M-mode ultrasonography of the pleura to differentiate CPE from NCAIS.

Methods: A total of 43 subjects were enrolled in the study, and based on history, physical examination, and chart review, the patients were divided into three groups: an NCAIS group, a CPE group, and a control group. Three distinct pleural line morphologic categories were identified: a continuous pleural line, a fragmented pleural line, and a sinusoidal pleural line. In addition, two separate subpleural patterns were independently identified by the investigators: a horizontal pattern and a vertical pattern. These pleural and subpleural patterns were correlated with subject diagnoses.

Results: A fragmented pleural line and a vertical subpleural pattern on M-mode ultrasonography is associated with patients who have NCAIS. Most patients with CPE have a continuous pleural line and a vertical subpleural pattern on M-mode ultrasonography. A sinusoidal pleural line on M-mode ultrasonography is suggestive of the presence of a pleural effusion.

Conclusions: Our results indicate that M-mode ultrasonography is useful to distinguish CPE from NCAIS based on the pleural and the subpleural morphologic features.
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http://dx.doi.org/10.1016/j.chest.2017.10.019DOI Listing
March 2018

p53 controls expression of the DNA deaminase APOBEC3B to limit its potential mutagenic activity in cancer cells.

Nucleic Acids Res 2017 Nov;45(19):11056-11069

Department of Surgery & Cancer, Imperial College London, Hammersmith Hospital Campus, London W12 0NN, UK.

Cancer genome sequencing has implicated the cytosine deaminase activity of apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) genes as an important source of mutations in diverse cancers, with APOBEC3B (A3B) expression especially correlated with such cancer mutations. To better understand the processes directing A3B over-expression in cancer, and possible therapeutic avenues for targeting A3B, we have investigated the regulation of A3B gene expression. Here, we show that A3B expression is inversely related to p53 status in different cancer types and demonstrate that this is due to a direct and pivotal role for p53 in repressing A3B expression. This occurs through the induction of p21 (CDKN1A) and the recruitment of the repressive DREAM complex to the A3B gene promoter, such that loss of p53 through mutation, or human papilloma virus-mediated inhibition, prevents recruitment of the complex, thereby causing elevated A3B expression and cytosine deaminase activity in cancer cells. As p53 is frequently mutated in cancer, our findings provide a mechanism by which p53 loss can promote cancer mutagenesis.
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http://dx.doi.org/10.1093/nar/gkx721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737468PMC
November 2017

The role of poly ADP-ribosylation in the first wave of DNA damage response.

Nucleic Acids Res 2017 Aug;45(14):8129-8141

Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA 91010, USA.

Poly ADP-ribose polymerases (PARPs) catalyze massive protein poly ADP-ribosylation (PARylation) within seconds after the induction of DNA single- or double-strand breaks. PARylation occurs at or near the sites of DNA damage and promotes the recruitment of DNA repair factors via their poly ADP-ribose (PAR) binding domains. Several novel PAR-binding domains have been recently identified. Here, we summarize these and other recent findings suggesting that PARylation may be the critical event that mediates the first wave of the DNA damage response. We also discuss the potential for functional crosstalk with other DNA damage-induced post-translational modifications.
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http://dx.doi.org/10.1093/nar/gkx565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737498PMC
August 2017

Droplet microfluidics for synthetic biology.

Lab Chip 2017 10;17(20):3388-3400

Technology Division, DOE Joint BioEnergy Institute, Emeryville, California 94608, USA.

Synthetic biology is an interdisciplinary field that aims to engineer biological systems for useful purposes. Organism engineering often requires the optimization of individual genes and/or entire biological pathways (consisting of multiple genes). Advances in DNA sequencing and synthesis have recently begun to enable the possibility of evaluating thousands of gene variants and hundreds of thousands of gene combinations. However, such large-scale optimization experiments remain cost-prohibitive to researchers following traditional molecular biology practices, which are frequently labor-intensive and suffer from poor reproducibility. Liquid handling robotics may reduce labor and improve reproducibility, but are themselves expensive and thus inaccessible to most researchers. Microfluidic platforms offer a lower entry price point alternative to robotics, and maintain high throughput and reproducibility while further reducing operating costs through diminished reagent volume requirements. Droplet microfluidics have shown exceptional promise for synthetic biology experiments, including DNA assembly, transformation/transfection, culturing, cell sorting, phenotypic assays, artificial cells and genetic circuits.
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http://dx.doi.org/10.1039/c7lc00576hDOI Listing
October 2017

Discovery of a Novel Small-Molecule Inhibitor that Targets PP2A-β-Catenin Signaling and Restricts Tumor Growth and Metastasis.

Mol Cancer Ther 2017 09 12;16(9):1791-1805. Epub 2017 May 12.

Biochemistry Division, Council of Scientific & Industrial Research (CSIR), Central Drug Research Institute (CDRI), Lucknow, India.

Molecular hybridization of different pharmacophores to tackle both tumor growth and metastasis by a single molecular entity can be very effective and unique if the hybrid product shows drug-like properties. Here, we report synthesis and discovery of a novel small-molecule inhibitor of PP2A-β-catenin signaling that limits both tumor growth and metastasis. Our molecular hybridization approach resulted in cancer cell selectivity and improved drug-like properties of the molecule. Inhibiting PP2A and β-catenin interaction by selectively engaging PR55α-binding site, our most potent small-molecule inhibitor diminished the expression of active β-catenin and its target proteins c-Myc and Cyclin D1. Furthermore, it promotes robust E-cadherin upregulation on the cell surface and increases β-catenin-E-Cadherin association, which may prevent dissemination of metastatic cells. Altogether, we report synthesis and mechanistic insight of a novel drug-like molecule to differentially target β-catenin functionality via interacting with a particular subunit of PP2A. .
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http://dx.doi.org/10.1158/1535-7163.MCT-16-0584DOI Listing
September 2017

On-chip integration of droplet microfluidics and nanostructure-initiator mass spectrometry for enzyme screening.

Lab Chip 2017 01;17(2):323-331

Joint Bioenergy Institute, Emeryville, California 94608, USA and Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA. and Joint Genome Institute, Walnut creek, California, 94598, USA.

Biological assays often require expensive reagents and tedious manipulations. These shortcomings can be overcome using digitally operated microfluidic devices that require reduced sample volumes to automate assays. One particular challenge is integrating bioassays with mass spectrometry based analysis. Towards this goal we have developed μNIMS, a highly sensitive and high throughput technique that integrates droplet microfluidics with nanostructure-initiator mass spectrometry (NIMS). Enzyme reactions are carried out in droplets that can be arrayed on discrete NIMS elements at defined time intervals for subsequent mass spectrometry analysis, enabling time resolved enzyme activity assay. We apply the μNIMS platform for kinetic characterization of a glycoside hydrolase enzyme (CelE-CMB3A), a chimeric enzyme capable of deconstructing plant hemicellulose into monosaccharides for subsequent conversion to biofuel. This study reveals NIMS nanostructures can be fabricated into arrays for microfluidic droplet deposition, NIMS is compatible with droplet and digital microfluidics, and can be used on-chip to assay glycoside hydrolase enzyme in vitro.
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http://dx.doi.org/10.1039/c6lc01182aDOI Listing
January 2017

Rapid, Portable, Multiplexed Detection of Bacterial Pathogens Directly from Clinical Sample Matrices.

Biosensors (Basel) 2016 Sep 23;6(4). Epub 2016 Sep 23.

Biotechnology and Bioengineering, Sandia National Laboratories, Livermore, CA 94551, USA.

Enteric and diarrheal diseases are a major cause of childhood illness and death in countries with developing economies. Each year, more than half of a million children under the age of five die from these diseases. We have developed a portable, microfluidic platform capable of simultaneous, multiplexed detection of several of the bacterial pathogens that cause these diseases. This platform can perform fast, sensitive immunoassays directly from relevant, complex clinical matrices such as stool without extensive sample cleanup or preparation. Using only 1 µL of sample per assay, we demonstrate simultaneous multiplexed detection of four bacterial pathogens implicated in diarrheal and enteric diseases in less than 20 min.
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http://dx.doi.org/10.3390/bios6040049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192369PMC
September 2016

Digital Droplet Multiple Displacement Amplification (ddMDA) for Whole Genome Sequencing of Limited DNA Samples.

PLoS One 2016 4;11(5):e0153699. Epub 2016 May 4.

Sandia National Laboratories, Biotechnology and Bioengineering Department, Livermore, California, United States of America.

Multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet), ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli) compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0153699PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856258PMC
July 2017

A Centrifugal Microfluidic Platform That Separates Whole Blood Samples into Multiple Removable Fractions Due to Several Discrete but Continuous Density Gradient Sections.

PLoS One 2016 7;11(4):e0153137. Epub 2016 Apr 7.

Sandia National Laboratories, Livermore, California, United States of America.

We present a miniaturized centrifugal platform that uses density centrifugation for separation and analysis of biological components in small volume samples (~5 μL). We demonstrate the ability to enrich leukocytes for on-disk visualization via microscopy, as well as recovery of viable cells from each of the gradient partitions. In addition, we simplified the traditional Modified Wright-Giemsa staining by decreasing the time, volume, and expertise involved in the procedure. From a whole blood sample, we were able to extract 95.15% of leukocytes while excluding 99.8% of red blood cells. This platform has great potential in both medical diagnostics and research applications as it offers a simpler, automated, and inexpensive method for biological sample separation, analysis, and downstream culturing.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0153137PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824354PMC
September 2016

Exometabolomics Assisted Design and Validation of Synthetic Obligate Mutualism.

ACS Synth Biol 2016 07 17;5(7):569-76. Epub 2016 Feb 17.

Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.

Synthetic microbial ecology has the potential to enhance the productivity and resiliency of biotechnology processes compared to approaches using single isolates. Engineering microbial consortia is challenging; however, one approach that has attracted significant attention is the creation of synthetic obligate mutualism using auxotrophic mutants that depend on each other for exchange or cross-feeding of metabolites. Here, we describe the integration of mutant library fitness profiling with mass spectrometry based exometabolomics as a method for constructing synthetic mutualism based on cross-feeding. Two industrially important species lacking known ecological interactions, Zymomonas mobilis and Escherichia coli, were selected as the test species. Amino acid exometabolites identified in the spent medium of Z. mobilis were used to select three corresponding E. coli auxotrophs (proA, pheA and IlvA), as potential E. coli counterparts for the coculture. A pooled mutant fitness assay with a Z. mobilis transposon mutant library was used to identify mutants with improved growth in the presence of E. coli. An auxotroph mutant in a gene (ZMO0748) with sequence similarity to cysteine synthase A (cysK), was selected as the Z. mobilis counterpart for the coculture. Exometabolomic analysis of spent E. coli medium identified glutathione related metabolites as potentially available for rescue of the Z. mobilis cysteine synthase mutant. Three sets of cocultures between the Z. mobilis auxotroph and each of the three E. coli auxotrophs were monitored by optical density for growth and analyzed by flow cytometry to confirm high cell counts for each species. Taken together, our methods provide a technological framework for creating synthetic mutualisms combining existing screening based methods and exometabolomics for both the selection of obligate mutualism partners and elucidation of metabolites involved in auxotroph rescue.
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http://dx.doi.org/10.1021/acssynbio.5b00236DOI Listing
July 2016

Centrifugal sedimentation immunoassays for multiplexed detection of enteric bacteria in ground water.

Biomicrofluidics 2016 Jan 12;10(1):014103. Epub 2016 Jan 12.

Biotechnology and Bioengineering Department, Sandia National Laboratories , Livermore, California 94550, USA.

Waterborne pathogens pose significant threat to the global population and early detection plays an important role both in making drinking water safe, as well as in diagnostics and treatment of water-borne diseases. We present an innovative centrifugal sedimentation immunoassay platform for detection of bacterial pathogens in water. Our approach is based on binding of pathogens to antibody-functionalized capture particles followed by sedimentation of the particles through a density-media in a microfluidic disk. Beads at the distal end of the disk are imaged to quantify the fluorescence and determine the bacterial concentration. Our platform is fast (20 min), can detect as few as ∼10 bacteria with minimal sample preparation, and can detect multiple pathogens simultaneously. The platform was used to detect a panel of enteric bacteria (Escherichia coli, Salmonella typhimurium, Shigella, Listeria, and Campylobacter) spiked in tap and ground water samples.
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http://dx.doi.org/10.1063/1.4939099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4714988PMC
January 2016

A Droplet Microfluidic Platform for Automating Genetic Engineering.

ACS Synth Biol 2016 05 22;5(5):426-33. Epub 2016 Feb 22.

Technology Division, Joint BioEnergy Institute (JBEI) , Emeryville, California 94608, United States.

We present a water-in-oil droplet microfluidic platform for transformation, culture and expression of recombinant proteins in multiple host organisms including bacteria, yeast and fungi. The platform consists of a hybrid digital microfluidic/channel-based droplet chip with integrated temperature control to allow complete automation and integration of plasmid addition, heat-shock transformation, addition of selection medium, culture, and protein expression. The microfluidic format permitted significant reduction in consumption (100-fold) of expensive reagents such as DNA and enzymes compared to the benchtop method. The chip contains a channel to continuously replenish oil to the culture chamber to provide a fresh supply of oxygen to the cells for long-term (∼5 days) cell culture. The flow channel also replenished oil lost to evaporation and increased the number of droplets that could be processed and cultured. The platform was validated by transforming several plasmids into Escherichia coli including plasmids containing genes for fluorescent proteins GFP, BFP and RFP; plasmids with selectable markers for ampicillin or kanamycin resistance; and a Golden Gate DNA assembly reaction. We also demonstrate the applicability of this platform for transformation in widely used eukaryotic organisms such as Saccharomyces cerevisiae and Aspergillus niger. Duration and temperatures of the microfluidic heat-shock procedures were optimized to yield transformation efficiencies comparable to those obtained by benchtop methods with a throughput up to 6 droplets/min. The proposed platform offers potential for automation of molecular biology experiments significantly reducing cost, time and variability while improving throughput.
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http://dx.doi.org/10.1021/acssynbio.6b00011DOI Listing
May 2016

Anti-breast tumor activity of Eclipta extract in-vitro and in-vivo: novel evidence of endoplasmic reticulum specific localization of Hsp60 during apoptosis.

Sci Rep 2015 Dec 17;5:18457. Epub 2015 Dec 17.

Biochemistry Division, CSIR-Central Drug Research Institute (CDRI), Lucknow-226031, India.

Major challenges for current therapeutic strategies against breast cancer are associated with drug-induced toxicities. Considering the immense potential of bioactive phytochemicals to deliver non-toxic, efficient anti-cancer therapeutics, we performed bio-guided fractionation of Eclipta alba extract and discovered that particularly the chloroform fraction of Eclipta alba (CFEA) is selectively inducing cytotoxicity to breast cancer cells over non-tumorigenic breast epithelial cells. Our unbiased mechanistic hunt revealed that CFEA specifically activates the intrinsic apoptotic pathway by disrupting the mitochondrial membrane potential, upregulating Hsp60 and downregulating the expression of anti-apoptotic protein XIAP. By utilizing Hsp60 specific siRNA, we identified a novel pro-apoptotic role of Hsp60 and uncovered that following CFEA treatment, upregulated Hsp60 is localized in the endoplasmic reticulum (ER). To our knowledge, this is the first evidence of ER specific localization of Hsp60 during cancer cell apoptosis. Further, our LC-MS approach identified that luteolin is mainly attributed for its anti-cancer activities. Moreover, oral administration of CFEA not only offers potential anti-breast cancer effects in-vivo but also mitigates tumor induced hepato-renal toxicity. Together, our studies offer novel mechanistic insight into the CFEA mediated inhibition of breast cancer and may potentially open up new avenues for further translational research.
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http://dx.doi.org/10.1038/srep18457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4682077PMC
December 2015

Microfluidic Flow Cytometry for Single-Cell Protein Analysis.

Methods Mol Biol 2015 ;1346:69-83

Biological Science and Technology, Sandia National Laboratories, 969, MS 9292, 7011 East Avenue, Livermore, CA, 94551-0969, USA.

Flow-cytometric (FC) detection of proteins in single cells is a rapid, quantitative method for single-cell protein analysis. Recent advancements in microfluidic technologies have leveraged miniaturization and automation to adapt flow cytometry for analyzing single cell protein profiles both for cell surface and intracellular proteins. Here, we describe the method for microfluidic FC, along with instructions to build a microfluidic platform capable of automated cell culture, cell surface receptor immunostaining, intracellular phosphoprotein and intracellular cytokine immunostaining, and analysis using micro-flow cytometry. As a demonstration of our platform and protocol, we detail the profiling of TLR4 receptor activation, ERK1/2 phosphorylation, and TNFα production in LPS stimulated macrophages using the microfluidic platform.
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http://dx.doi.org/10.1007/978-1-4939-2987-0_6DOI Listing
August 2016

Development of a High Throughput Platform for Screening Glycoside Hydrolases Based on Oxime-NIMS.

Front Bioeng Biotechnol 2015 13;3:153. Epub 2015 Oct 13.

US Department of Energy Joint BioEnergy Institute , Emeryville, CA , USA ; Lawrence Berkeley National Laboratory , Berkeley, CA , USA.

Cost-effective hydrolysis of biomass into sugars for biofuel production requires high-performance low-cost glycoside hydrolase (GH) cocktails that are active under demanding process conditions. Improving the performance of GH cocktails depends on knowledge of many critical parameters, including individual enzyme stabilities, optimal reaction conditions, kinetics, and specificity of reaction. With this information, rate- and/or yield-limiting reactions can be potentially improved through substitution, synergistic complementation, or protein engineering. Given the wide range of substrates and methods used for GH characterization, it is difficult to compare results across a myriad of approaches to identify high performance and synergistic combinations of enzymes. Here, we describe a platform for systematic screening of GH activities using automatic biomass handling, bioconjugate chemistry, robotic liquid handling, and nanostructure-initiator mass spectrometry (NIMS). Twelve well-characterized substrates spanning the types of glycosidic linkages found in plant cell walls are included in the experimental workflow. To test the application of this platform and substrate panel, we studied the reactivity of three engineered cellulases and their synergy of combination across a range of reaction conditions and enzyme concentrations. We anticipate that large-scale screening using the standardized platform and substrates will generate critical datasets to enable direct comparison of enzyme activities for cocktail design.
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http://dx.doi.org/10.3389/fbioe.2015.00153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4603251PMC
November 2015

A Versatile Microfluidic Device for Automating Synthetic Biology.

ACS Synth Biol 2015 Oct 15;4(10):1151-64. Epub 2015 Jun 15.

Sandia National Laboratories , 7011 East Avenue, Livermore, California 94550, United States.

New microbes are being engineered that contain the genetic circuitry, metabolic pathways, and other cellular functions required for a wide range of applications such as producing biofuels, biobased chemicals, and pharmaceuticals. Although currently available tools are useful in improving the synthetic biology process, further improvements in physical automation would help to lower the barrier of entry into this field. We present an innovative microfluidic platform for assembling DNA fragments with 10× lower volumes (compared to that of current microfluidic platforms) and with integrated region-specific temperature control and on-chip transformation. Integration of these steps minimizes the loss of reagents and products compared to that with conventional methods, which require multiple pipetting steps. For assembling DNA fragments, we implemented three commonly used DNA assembly protocols on our microfluidic device: Golden Gate assembly, Gibson assembly, and yeast assembly (i.e., TAR cloning, DNA Assembler). We demonstrate the utility of these methods by assembling two combinatorial libraries of 16 plasmids each. Each DNA plasmid is transformed into Escherichia coli or Saccharomyces cerevisiae using on-chip electroporation and further sequenced to verify the assembly. We anticipate that this platform will enable new research that can integrate this automated microfluidic platform to generate large combinatorial libraries of plasmids and will help to expedite the overall synthetic biology process.
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http://dx.doi.org/10.1021/acssynbio.5b00062DOI Listing
October 2015

A 75-year-old woman with fever and a right upper lobe pulmonary mass.

Chest 2015 Jan;147(1):e1-e4

Division of Pulmonary, Critical Care, and Sleep Medicine, Hofstra North Shore-LIJ School of Medicine, New Hyde Park, NY.

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http://dx.doi.org/10.1378/chest.14-1161DOI Listing
January 2015

Centrifugal microfluidic platform for ultrasensitive detection of botulinum toxin.

Anal Chem 2015 Jan 5;87(2):922-8. Epub 2015 Jan 5.

Sandia National Laboratories, 7011 East Avenue, Livermore, California 94551, United States.

We present an innovative centrifugal microfluidic immunoassay platform (SpinDx) to address the urgent biodefense and public health need for ultrasensitive point-of-care/incident detection of botulinum toxin. The simple, sample-to-answer centrifugal microfluidic immunoassay approach is based on binding of toxins to antibody-laden capture particles followed by sedimentation of the particles through a density-media in a microfluidic disk and quantification by laser-induced fluorescence. A blind, head-to-head comparison study of SpinDx versus the gold-standard mouse bioassay demonstrates 100-fold improvement in sensitivity (limit of detection = 0.09 pg/mL), while achieving total sample-to-answer time of <30 min with 2-μL required volume of the unprocessed sample. We further demonstrate quantification of botulinum toxin in both exogeneous (human blood and serum spiked with toxins) and endogeneous (serum from mice intoxicated via oral, intranasal, and intravenous routes) samples. SpinDx can analyze, without any sample preparation, multiple sample types including whole blood, serum, and food. It is readily expandable to additional analytes as the assay reagents (i.e., the capture beads and detection antibodies) are disconnected from the disk architecture and the reader, facilitating rapid development of new assays. SpinDx can also serve as a general-purpose immunoassay platform applicable to diagnosis of other conditions and diseases.
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http://dx.doi.org/10.1021/ac504054uDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303339PMC
January 2015

Versatile on-demand droplet generation for controlled encapsulation.

Biomicrofluidics 2014 May 12;8(3):034112. Epub 2014 Jun 12.

Sandia National Laboratories , Livermore, California 94550, USA.

We present a droplet-based microfluidic system for performing bioassays requiring controlled analyte encapsulation by employing highly flexible on-demand droplet generation. On-demand droplet generation and encapsulation are achieved pneumatically using a microdispensing pump connected to a constant pressure source. The system generates single droplets to the collection route only when the pump is actuated with a designated pressure level and produces two-phase parallel flow to the waste route during the stand-by state. We analyzed the effect of actuation pressure on the stability and size of droplets and optimized conditions for generation of stable droplets over a wide pressure range. By increasing the duration of pump actuation, we could either trigger a short train of identical size droplets or generate a single larger droplet. We also investigated the methodology to control droplet contents by fine-tuning flow rates or implementing a resistance bridge between the pump and main channels. We demonstrated the integrated chip for on-demand mixing between two aqueous phases in droplets and on-demand encapsulation of Escherichia coli cells. Our unique on-demand feature for selective encapsulation is particularly appropriate for bioassays with extremely dilute samples, such as pathogens in a clinical sample, since it can significantly reduce the number of empty droplets that impede droplet collection and subsequent data analysis.
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http://dx.doi.org/10.1063/1.4874715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4162415PMC
May 2014

A droplet-to-digital (D2D) microfluidic device for single cell assays.

Lab Chip 2015 Jan;15(1):225-36

Sandia National Laboratories, 7011 East Ave, Livermore, CA, USA.

We have developed a new hybrid droplet-to-digital microfluidic platform (D2D) that integrates droplet-in-channel microfluidics with digital microfluidics (DMF) for performing multi-step assays. This D2D platform combines the strengths of the two formats-droplets-in-channel for facile generation of droplets containing single cells, and DMF for on-demand manipulation of droplets including control of different droplet volumes (pL-μL), creation of a dilution series of ionic liquid (IL), and parallel single cell culturing and analysis for IL toxicity screening. This D2D device also allows for automated analysis that includes a feedback-controlled system for merging and splitting of droplets to add reagents, an integrated Peltier element for parallel cell culture at optimum temperature, and an impedance sensing mechanism to control the flow rate for droplet generation and preventing droplet evaporation. Droplet-in-channel is well-suited for encapsulation of single cells as it allows the careful manipulation of flow rates of aqueous phase containing cells and oil to optimize encapsulation. Once single cell containing droplets are generated, they are transferred to a DMF chip via a capillary where they are merged with droplets containing IL and cultured at 30 °C. The DMF chip, in addition to permitting cell culture and reagent (ionic liquid/salt) addition, also allows recovery of individual droplets for off-chip analysis such as further culturing and measurement of ethanol production. The D2D chip was used to evaluate the effect of IL/salt type (four types: NaOAc, NaCl, [C2mim] [OAc], [C2mim] [Cl]) and concentration (four concentrations: 0, 37.5, 75, 150 mM) on the growth kinetics and ethanol production of yeast and as expected, increasing IL concentration led to lower biomass and ethanol production. Specifically, [C2mim] [OAc] had inhibitory effects on yeast growth at concentrations 75 and 150 mM and significantly reduced their ethanol production compared to cells grown in other ILs/salts. The growth curve trends obtained by D2D matched conventional yeast culturing in microtiter wells, validating the D2D platform. We believe that our approach represents a generic platform for multi-step biochemical assays such as drug screening, digital PCR, enzyme assays, immunoassays and cell-based assays.
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http://dx.doi.org/10.1039/c4lc00794hDOI Listing
January 2015