Publications by authors named "Balaji Rao"

76 Publications

Quantitative Yeast-Yeast Two Hybrid for the Discovery and Binding Affinity Estimation of Protein-Protein Interactions.

ACS Synth Biol 2021 03 15;10(3):505-514. Epub 2021 Feb 15.

Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695, United States.

Quantifying the binding affinity of protein-protein interactions is important for elucidating connections within biochemical signaling pathways, as well as characterization of binding proteins isolated from combinatorial libraries. We describe a quantitative yeast-yeast two-hybrid (qYY2H) system that not only enables the discovery of specific protein-protein interactions but also efficient, quantitative estimation of their binding affinities (). In qYY2H, the bait and prey proteins are expressed as yeast cell surface fusions using yeast surface display. We developed a semiempirical framework for estimating the of monovalent bait-prey interactions, using measurements of bait-prey yeast-yeast binding, which is mediated by multivalent interactions between yeast-displayed bait and prey. Using qYY2H, we identified interaction partners of SMAD3 and the tandem WW domains of YAP from a cDNA library and characterized their binding affinities. Finally, we showed that qYY2H could also quantitatively evaluate binding interactions mediated by post-translational modifications on the bait protein.
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http://dx.doi.org/10.1021/acssynbio.0c00472DOI Listing
March 2021

Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands.

Int J Mol Sci 2021 Feb 5;22(4). Epub 2021 Feb 5.

Department of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way, Raleigh, NC 27695, USA.

We present the construction and screening of yeast display libraries of post-translationally modified peptides wherein site-selective enzymatic treatment of linear peptides is achieved using bacterial transglutaminase. To this end, we developed two alternative routes, namely (i) yeast display of linear peptides followed by treatment with recombinant transglutaminase in solution; or (ii) intracellular co-expression of linear peptides and transglutaminase to achieve peptide modification in the endoplasmic reticulum prior to yeast surface display. The efficiency of peptide modification was evaluated via orthogonal detection of epitope tags integrated in the yeast-displayed peptides by flow cytometry, and via comparative cleavage of putative cyclic vs. linear peptides by tobacco etch virus (TEV) protease. Subsequently, yeast display libraries of transglutaminase-treated peptides were screened to isolate binders to the N-terminal region of the Yes-Associated Protein (YAP) and its WW domains using magnetic selection and fluorescence activated cell sorting (FACS). The identified peptide cyclo[LYLAYPAH] featured a K of 1.75 μM for YAP and 0.68 μM for the WW domains of YAP as well as high binding selectivity against albumin and lysozyme. These results demonstrate the usefulness of enzyme-mediated cyclization in screening combinatorial libraries to identify cyclic peptide binders.
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http://dx.doi.org/10.3390/ijms22041634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915732PMC
February 2021

Two distinct trophectoderm lineage stem cells from human pluripotent stem cells.

J Biol Chem 2021 Feb 5:100386. Epub 2021 Feb 5.

Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, 27695, USA; Golden LEAF Biomanufacturing Training and Education Center (BTEC), North Carolina State University, Raleigh, North Carolina, 27695, USA. Electronic address:

The trophectoderm layer of the blastocyst-stage embryo is the precursor for all trophoblast cells in the placenta. Human trophoblast stem (TS) cells have emerged as an attractive tool for studies on early trophoblast development. However, the use of TS cell models is constrained by the limited genetic diversity of existing TS cell lines, and restrictions on using human fetal tissue or embryos needed to generate additional lines. Here we report the derivation of two distinct stem cell types of the trophectoderm lineage from human pluripotent stem cells. Analogous to villous cytotrophoblasts in vivo, the first is a CDX2 stem cell comparable to placenta-derived TS cells - they both exhibit identical expression of key markers, are maintained in culture and differentiate under similar conditions, and share high transcriptome similarity. The second is a CDX2 stem cell with distinct cell culture requirements, and differences in gene expression and differentiation, relative to CDX2 stem cells. Derivation of TS cells from pluripotent stem cells will significantly enable construction of in vitro models for normal and pathological placental development.
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http://dx.doi.org/10.1016/j.jbc.2021.100386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948510PMC
February 2021

Machine Learning and Improved Quality Metrics in Acute Intracranial Hemorrhage by Noncontrast Computed Tomography.

Curr Probl Diagn Radiol 2020 Nov 15. Epub 2020 Nov 15.

Department of Radiology and Biomedical Imaging ,Yale School of Medicine, Yale University, New Haven, CT 06520.. Electronic address:

Objective: The timely reporting of critical results in radiology is paramount to improved patient outcomes. Artificial intelligence has the ability to improve quality by optimizing clinical radiology workflows. We sought to determine the impact of a United States Food and Drug Administration-approved machine learning (ML) algorithm, meant to mark computed tomography (CT) head examinations pending interpretation as higher probability for intracranial hemorrhage (ICH), on metrics across our healthcare system. We hypothesized that ML is associated with a reduction in report turnaround time (RTAT) and length of stay (LOS) in emergency department (ED) and inpatient populations.

Materials And Methods: An ML algorithm was incorporated across CT scanners at imaging sites in January 2018. RTAT and LOS were derived for reports and patients between July 2017 and December 2017 prior to implementation of ML and compared to those between January 2018 and June 2018 after implementation of ML. A total of 25,658 and 24,996 ED and inpatient cases were evaluated across the entire healthcare system before and after ML, respectively.

Results: RTAT decreased from 75 to 69 minutes (P <0.001) at all facilities in the healthcare system. At the level 1 trauma center specifically, RTAT decreased from 67 to 59 minutes (P <0.001). ED LOS decreased from 471 to 425 minutes (P <0.001) for patients without ICH, and from 527 to 491 minutes for those with ICH (P = 0.456). Inpatient LOS decreased from 18.4 to 15.8 days for those without ICH (P = 0.001) and 18.1 to 15.8 days for those with ICH (P = 0.02).

Conclusion: We demonstrated that utilization of ML was associated with a statistically significant decrease in RTAT. There was also a significant decrease in LOS for ED patients without ICH, but not for ED patients with ICH. Further evaluation of the impact of such tools on patient care and outcomes is needed.
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http://dx.doi.org/10.1067/j.cpradiol.2020.10.007DOI Listing
November 2020

Use of Target-Displaying Magnetized Yeast in Screening mRNA-Display Peptide Libraries to Identify Ligands.

ACS Comb Sci 2020 12 22;22(12):738-744. Epub 2020 Oct 22.

Department of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way, Room 2-009, Raleigh, North Carolina 27606, United States.

This work presents the first use of yeast-displayed protein targets for screening mRNA-display libraries of cyclic and linear peptides. The WW domains of Yes-Associated Protein 1 (WW-YAP) and mitochondrial import receptor subunit TOM22 were adopted as protein targets. Yeast cells displaying WW-YAP or TOM22 were magnetized with iron oxide nanoparticles to enable the isolation of target-binding mRNA-peptide fusions. Equilibrium adsorption studies were conducted to estimate the binding affinity () of select WW-YAP-binding peptides: values of 37 and 4 μM were obtained for cyclo[M-AFRLC-K] and its linear cognate, and 40 and 3 μM for cyclo[M-LDFVNHRSRG-K] and its linear cognate, respectively. TOM22-binding peptide cyclo[M-PELNRAI-K] was conjugated to magnetic beads and incubated with yeast cells expressing TOM22 and luciferase. A luciferase-based assay showed a 4.5-fold higher binding of TOM22 yeast compared to control cells. This work demonstrates that integrating mRNA- and yeast-display accelerates the discovery of peptide ligands.
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http://dx.doi.org/10.1021/acscombsci.0c00171DOI Listing
December 2020

Genetically encoded live-cell sensor for tyrosinated microtubules.

J Cell Biol 2020 10;219(10)

Centre for Cardiovascular Biology and Diseases, Institute for Stem Cell Science and Regenerative Medicine, Gandhi Krishi Vigyan Kendra Campus, Bangalore, India.

Microtubule cytoskeleton exists in various biochemical forms in different cells due to tubulin posttranslational modifications (PTMs). Tubulin PTMs are known to affect microtubule stability, dynamics, and interaction with MAPs and motors in a specific manner, widely known as tubulin code hypothesis. At present, there exists no tool that can specifically mark tubulin PTMs in living cells, thus severely limiting our understanding of their dynamics and cellular functions. Using a yeast display library, we identified a binder against terminal tyrosine of α-tubulin, a unique PTM site. Extensive characterization validates the robustness and nonperturbing nature of our binder as tyrosination sensor, a live-cell tubulin nanobody specific towards tyrosinated microtubules. Using this sensor, we followed nocodazole-, colchicine-, and vincristine-induced depolymerization events of tyrosinated microtubules in real time and found each distinctly perturbs the microtubule polymer. Together, our work describes a novel tyrosination sensor and its potential applications to study the dynamics of microtubule and their PTM processes in living cells.
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http://dx.doi.org/10.1083/jcb.201912107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659708PMC
October 2020

Assessing Biota Accumulation Due to Contamination of Sediments by Storm Water Heavy Metals.

Environ Toxicol Chem 2020 12 8;39(12):2475-2484. Epub 2020 Oct 8.

Naval Information Warfare Center Pacific, San Diego, California, USA.

Evaluating sediment recontamination due to storm water discharges is important when evaluating the long-term effectiveness of sediment remediation efforts at reducing biological impacts. The bioaccumulation of the heavy metals zinc, nickel, copper, cadmium, mercury, and lead and the metalloid arsenic in a clam (Macoma nasuta) was studied in surficial sediments before and after storm water inputs from Paleta Creek, California, USA, during wet seasons in 2015 to 2016 and 2016 to 2017. The bioaccumulation was compared with bulk sediment concentrations and porewater concentrations measured by diffusion gradient in thin film devices. Significant reductions in biota accumulation and porewater concentrations were observed in samples collected after storm seasons compared with before storm seasons despite bulk sediment concentrations remaining the same or increasing. This was apparently the result of the deposition of storm water contaminants in low bioavailable forms. The bioaccumulation of all the measured contaminants showed a positive significant correlation with porewater concentrations (p < 0.1, α = 0.1) and weak or no correlations with bulk sediment concentration. In conclusion, observed bulk sediment recontamination due to storm water should not be assumed to lead directly to greater biota accumulation without bioavailability assessment. Environ Toxicol Chem 2020;39:2475-2484. © 2020 SETAC.
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http://dx.doi.org/10.1002/etc.4862DOI Listing
December 2020

Evaluating the transport of Hg(II) in the presence of natural organic matter through a diffusive gradient in a thin-film passive sampler.

Sci Total Environ 2020 Dec 3;749:141217. Epub 2020 Aug 3.

Department of Civil, Environmental, and Construction Engineering, Texas Tech University, Lubbock, TX 79409, United States. Electronic address:

The effects of a model natural organic matter (NOM) on the transport of Hg(II) into diffusive gradient in thin-film devices (DGTs) was evaluated in order to better understand their ability to measure colloidal Hg species in porewater. The presence of NOM significantly reduced the diffusivity of the Hg(II) species and the reduction was dependent upon NOM to Hg(II) ratio. This relationship was modeled by determining the Hg(II) partition coefficients (K) of size fractionated NOM obtained by ultrafiltration and estimating the Hg diffusivity through the DGT for the different NOM size fractions across a range of Hg-NOM ratios. The estimated diffusivities were consistent with experimental observations of uptake into the DGT. Overall, this study indicated that Hg(II) associated with NOM passes into a DGT, however the transport is slowed in accordance with the diffusivity of the NOM to which the Hg(II) is associated. Thus, the Hg-NOM association and complex diffusivities need to be considered when relating DGT uptake to Hg porewater concentration. The results also suggest that Hg(II) associated with colloidal or larger particles of negligible diffusivity are unlikely to contribute significantly to DGT measurements.
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http://dx.doi.org/10.1016/j.scitotenv.2020.141217DOI Listing
December 2020

Discovery of Membrane-Permeating Cyclic Peptides via mRNA Display.

Bioconjug Chem 2020 10 8;31(10):2325-2338. Epub 2020 Sep 8.

Department of Chemical and Biomolecular Engineering, North Carolina State University, 911 Partners Way room 2-009, Raleigh, North Carolina 27606, United States.

Small synthetic peptides capable of crossing biological membranes represent valuable tools in cell biology and drug delivery. While several cell-penetrating peptides (CPPs) of natural or synthetic origin have been reported, no peptide is currently known to cross both cytoplasmic and outer embryonic membranes. Here, we describe a method to engineer membrane-permeating cyclic peptides (MPPs) with broad permeation activity by screening mRNA display libraries of cyclic peptides against embryos at different developmental stages. The proposed method was demonstrated by identifying peptides capable of permeating (fruit fly) embryos and mammalian cells. The selected peptide cyclo[-MRKRHASRRE-*] showed a strong permeation activity of embryos exposed to minimal permeabilization pretreatment, as well as human embryonic stem cells and a murine fibroblast cell line. Notably, in both embryos and mammalian cells, the cyclic peptide outperformed its linear counterpart and the control MPPs. Confocal microscopy and single cell flow cytometry analysis were utilized to assess the degree of permeation both qualitatively and quantitatively. These MPPs have potential application in studying and nondisruptively controlling intracellular or intraembryonic processes.
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http://dx.doi.org/10.1021/acs.bioconjchem.0c00413DOI Listing
October 2020

Isolation of Chemically Cyclized Peptide Binders Using Yeast Surface Display.

ACS Comb Sci 2020 10 26;22(10):519-532. Epub 2020 Aug 26.

Department of Chemical and Biomolecular Engineering, North Carolina State University, Box 7905, Engineering Building I, Raleigh, North Carolina 27695, United States.

Cyclic peptides with engineered protein-binding activity have gained increasing attention for use in therapeutic and biotechnology applications. We describe the efficient isolation and characterization of cyclic peptide binders from genetically encoded combinatorial libraries using yeast surface display. Here, peptide cyclization is achieved by disuccinimidyl glutarate-mediated cross-linking of amine groups within a linear peptide sequence that is expressed as a yeast cell surface fusion. Using this approach, we first screened a library of cyclic heptapeptides using magnetic selection, followed by fluorescence activated cell sorting (FACS) to isolate binders for a model target (lysozyme) with low micromolar binding affinity ( ∼ 1.2-3.7 μM). The isolated peptides bind lysozyme selectively and only when cyclized. Importantly, we showed that yeast surface displayed cyclic peptides can be used to efficiently obtain quantitative estimates of binding affinity, circumventing the need for chemical synthesis of the selected peptides. Subsequently, to demonstrate broader applicability of our approach, we isolated cyclic heptapeptides that bind human interleukin-17 (IL-17) using yeast-displayed IL-17 as a target for magnetic selection, followed by FACS using recombinant IL-17. Molecular docking simulations and follow-up experimental analyses identified a candidate cyclic peptide that likely binds IL-17 in its receptor binding region with moderate apparent affinity ( ∼ 300 nM). Taken together, our results show that yeast surface display can be used to efficiently isolate and characterize cyclic peptides generated by chemical modification from combinatorial libraries.
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http://dx.doi.org/10.1021/acscombsci.0c00076DOI Listing
October 2020

Assessing sediment recontamination from metals in stormwater.

Sci Total Environ 2020 Oct 27;737:139726. Epub 2020 May 27.

Geosyntec Consultants, 924 Anacapa St #4a, Santa Barbara, CA 93101, USA.

Recontamination of sediments by stormwater is a major concern when evaluating the potential effectiveness of sediment remediation. Stormwater and sediment sampling were conducted in a mixed-use watershed at Paleta Creek in San Diego, CA to evaluate methods for assessing sediment recontamination by metals. Size-segregated stormwater contaminant loads with simultaneous receiving water and sediment measurements were used to identify dominant sources and contaminants with respect to their impact on sediment recontamination. Most of the stormwater contaminant loads of Cd, Cu, Pb, and Zn were associated with residential and highway sources from the upstream portions of the watershed and As, Ni and Hg were more significantly influenced by the downstream area of the watershed. Cd was strongly associated with large particles (>63 μm) and observed to settle in near shore areas with some attenuation due to mixing and dilution. Cu, in contrast, was associated more with the filtered fraction (<0.45 μm) and clay fraction (0.45-5 μm), resulting in less near shore sediment recontamination. Depositing sediment and other metals, particularly Cu and Hg, exhibited greater accumulation in settling traps than could be attributed to stormwater loads indicating the importance of other sources or resuspension of bay sediments on surficial sediment concentrations. Pb, Zn, Ni, and As showed influences of both stormwater and other sources. The study showed that measurement of size-segregated stormwater contaminant mass and concentrations combined with simultaneous measurements of deposition in sediment traps could differentiate between recontamination by stormwater and that of other sources.
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http://dx.doi.org/10.1016/j.scitotenv.2020.139726DOI Listing
October 2020

Past, Present, and Future of Affinity-based Cell Separation Technologies.

Acta Biomater 2020 08 19;112:29-51. Epub 2020 May 19.

Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695-7905, USA; Biomanufacturing Training and Education Center (BTEC), North Carolina State University, Raleigh, NC 27695-7928, USA. Electronic address:

Progress in cell purification technology is critical to increase the availability of viable cells for therapeutic, diagnostic, and research applications. A variety of techniques are now available for cell separation, ranging from non-affinity methods such as density gradient centrifugation, dielectrophoresis, and filtration, to affinity methods such as chromatography, two-phase partitioning, and magnetic-/fluorescence-assisted cell sorting. For clinical and analytical procedures that require highly purified cells, the choice of cell purification method is crucial, since every method offers a different balance between yield, purity, and bioactivity of the cell product. For most applications, the requisite purity is only achievable through affinity methods, owing to the high target specificity that they grant. In this review, we discuss past and current methods for developing cell-targeting affinity ligands and their application in cell purification, along with the benefits and challenges associated with different purification formats. We further present new technologies, like stimuli-responsive ligands and parallelized microfluidic devices, towards improving the viability and throughput of cell products for tissue engineering and regenerative medicine. Our comparative analysis provides guidance in the multifarious landscape of cell separation techniques and highlights new technologies that are poised to play a key role in the future of cell purification in clinical settings and the biotech industry. STATEMENT OF SIGNIFICANCE: Technologies for cell purification have served science, medicine, and industrial biotechnology and biomanufacturing for decades. This review presents a comprehensive survey of this field by highlighting the scope and relevance of all known methods for cell isolation, old and new alike. The first section covers the main classes of target cells and compares traditional non-affinity and affinity-based purification techniques, focusing on established ligands and chromatographic formats. The second section presents an excursus of affinity-based pseudo-chromatographic and non-chromatographic technologies, especially focusing on magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Finally, the third section presents an overview of new technologies and emerging trends, highlighting how the progress in chemical, material, and microfluidic sciences has opened new exciting avenues towards high-throughput and high-purity cell isolation processes. This review is designed to guide scientists and engineers in their choice of suitable cell purification techniques for research or bioprocessing needs.
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http://dx.doi.org/10.1016/j.actbio.2020.05.004DOI Listing
August 2020

Utility of Artificial Intelligence Tool as a Prospective Radiology Peer Reviewer - Detection of Unreported Intracranial Hemorrhage.

Acad Radiol 2021 01 24;28(1):85-93. Epub 2020 Feb 24.

Department of Radiology and Biomedical Imaging, Yale University School of Medicine, 330 Cedar St. Tompkins East TE-2, New Haven, CT 06520.

Rationale And Objectives: Misdiagnosis of intracranial hemorrhage (ICH) can adversely impact patient outcomes. The increasing workload on the radiologists may increase the chance of error and compromise the quality of care provided by the radiologists.

Materials And Methods: We used an FDA approved artificial intelligence (AI) solution based on a convolutional neural network to assess the prevalence of ICH in scans, which were reported as negative for ICH. We retrospectively applied the AI solution to all consecutive noncontrast computed tomography (CT) head scans performed at eight imaging sites affiliated to our institution.

Results: In the 6565 noncontrast CT head scans, which met the inclusion criteria, 5585 scans were reported to have no ICH ("negative-by-report" cases). We applied AI solution to these "negative-by-report" cases. AI solution suggested there were ICH in 28 of these scans ("negative-by-report" and "positive-by-AI solution"). After consensus review by three neuroradiologists, 16 of these scans were found to have ICH, which was not reported (missed diagnosis by radiologists), with a false-negative rate of radiologists for ICH detection at 1.6%. Most commonly missed ICH was overlying the cerebral convexity and in the parafalcine regions.

Conclusion: Our study demonstrates that an AI solution can help radiologists to diagnose ICH and thus decrease the error rate. AI solution can serve as a prospective peer review tool for non-contrast head CT scans to identify ICH and thus minimize false negatives.
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http://dx.doi.org/10.1016/j.acra.2020.01.035DOI Listing
January 2021

A Fyn biosensor reveals pulsatile, spatially localized kinase activity and signaling crosstalk in live mammalian cells.

Elife 2020 02 4;9. Epub 2020 Feb 4.

Institute for Stem Cell Science and Regenerative Medicine, Bangalore, India.

Cell behavior is controlled through spatio-temporally localized protein activity. Despite unique and often contradictory roles played by Src-family-kinases (SFKs) in regulating cell physiology, activity patterns of individual SFKs have remained elusive. Here, we report a biosensor for specifically visualizing active conformation of SFK-Fyn in live cells. We deployed combinatorial library screening to isolate a binding-protein (F29) targeting activated Fyn. Nuclear-magnetic-resonance (NMR) analysis provides the structural basis of F29 specificity for Fyn over homologous SFKs. Using F29, we engineered a sensitive, minimally-perturbing fluorescence-resonance-energy-transfer (FRET) biosensor () that reveals cellular Fyn activity to be spatially localized, pulsatile and sensitive to adhesion/integrin signaling. Strikingly, growth factor stimulation further enhanced Fyn activity in pre-activated intracellular zones. However, inhibition of focal-adhesion-kinase activity not only attenuates Fyn activity, but abolishes growth-factor modulation. imaging uncovers spatially organized, sensitized signaling clusters, direct crosstalk between integrin and growth-factor-signaling, and clarifies how compartmentalized Src-kinase activity may drive cell fate.
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http://dx.doi.org/10.7554/eLife.50571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000222PMC
February 2020

Screening Yeast Display Libraries against Magnetized Yeast Cell Targets Enables Efficient Isolation of Membrane Protein Binders.

ACS Comb Sci 2019 12 20;21(12):817-832. Epub 2019 Nov 20.

When isolating binders from yeast displayed combinatorial libraries, a soluble, recombinantly expressed form of the target protein is typically utilized. As an alternative, we describe the use of target proteins displayed as surface fusions on magnetized yeast cells. In our strategy, the target protein is coexpressed on the yeast surface with an iron oxide binding protein; incubation of these yeast cells with iron oxide nanoparticles results in their magnetization. Subsequently, binder cells that interact with the magnetized target cells can be isolated using a magnet. Using a known binder-target pair with modest binding affinity ( ≈ 400 nM), we showed that a binder present at low frequency (1 in 10) could be enriched more than 100-fold, in a single round of screening, suggesting feasibility of screening combinatorial libraries. Subsequently, we screened yeast display libraries of Sso7d and nanobody variants against yeast displayed targets to isolate binders specific to the cytosolic domain of the mitochondrial membrane protein TOM22 ( ≈ 272-1934 nM) and the extracellular domain of the c-Kit receptor ( ≈ 93 to > 2000 nM). Additional studies showed that the TOM22 binders identified using this approach could be used for the enrichment of mitochondria from cell lysates, thereby confirming binding to the native mitochondrial protein. The ease of expressing a membrane protein or a domain thereof as a yeast cell surface fusion-in contrast to recombinant soluble expression-makes the use of yeast-displayed targets particularly attractive. Therefore, we expect the use of magnetized yeast cell targets will enable efficient isolation of binders to membrane proteins.
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http://dx.doi.org/10.1021/acscombsci.9b00147DOI Listing
December 2019

Simultaneous Soluble Secretion and Surface Display of Proteins in Saccharomyces cerevisiae Using Inefficient Ribosomal Skipping.

Methods Mol Biol 2020 ;2070:321-334

Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, USA.

Combinatorial library screening platforms, such as yeast surface display, typically identify several candidate proteins that need further characterization and validation using soluble recombinant protein. However, recombinant production of these candidate proteins involves tedious and time-consuming subcloning steps. This, in turn, limits the number of candidate proteins that can be characterized. To address this bottleneck, we have developed a platform that exploits inefficient ribosomal skipping by the F2A peptide for simultaneous soluble secretion and cell surface display of protein in the yeast Saccharomyces cerevisiae. Here we provide detailed protocols utilizing this F2A-based yeast display system. We discuss specific recommendations for the purification of the secreted protein. Additionally, we provide suggestions for testing the functionality and binding specificity of the soluble secreted proteins using flow cytometry analysis.
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http://dx.doi.org/10.1007/978-1-4939-9853-1_18DOI Listing
December 2020

Seasonal Toxicity Observed with Amphipods (Eohaustorius estuarius) at Paleta Creek, San Diego Bay, USA.

Environ Toxicol Chem 2020 01 18;39(1):229-239. Epub 2019 Dec 18.

Texas Tech University, Lubbock, Texas, USA.

To assess potential impacts on receiving systems, associated with storm water contaminants, laboratory 10-d amphipod (Eohaustorius estuarius) survival toxicity tests were performed using intact sediment cores collected from Paleta Creek (San Diego Bay, CA, USA) on 5 occasions between 2015 and 2017. The approach included deposition-associated sediment particles collected from sediment traps placed at each of 4 locations during the 2015 to 2016 wet seasons. The bioassays demonstrated wet season toxicity, especially closest to the creek mouth, and greater mortality associated with particles deposited in the wet season compared with dry season samples. Grain size analysis of sediment trap material indicated coarser sediment at the mouth of the creek and finer sediment in the outer depositional areas. Contaminant concentrations of metals (Cd, Cu, Hg, Ni, Pb, and Zn) and organic compounds (polycyclic aromatic hydrocarbons [PAHs], polychlorinated biphenyls [PCBs], and pesticides) were quantified to assess possible causes of toxicity. Contaminant concentrations were determined in the top 5 cm of sediment and porewater (using passive samplers). Whereas metals, PAHs, and PCBs were rarely detected at sufficient concentrations to elicit a response, pyrethroid pesticides were highly correlated with amphipod toxicity. Summing individual pyrethroid constituents using a toxic unit approach suggested that toxicity to E. estuarius could be associated with pyrethroids. This unique test design allowed delineation of spatial and temporal differences in toxicity, suggesting that storm water discharge from Paleta Creek may be the source of seasonal toxicity. Environ Toxicol Chem 2019;39:229-239. © 2019 SETAC.
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http://dx.doi.org/10.1002/etc.4619DOI Listing
January 2020

Design and evaluation of engineered protein biosensors for live-cell imaging of EGFR phosphorylation.

Sci Signal 2019 06 4;12(584). Epub 2019 Jun 4.

Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA.

Live-cell fluorescence microscopy is broadly applied to study the dynamics of receptor-mediated cell signaling, but the availability of intracellular biosensors is limited. A biosensor based on the tandem SH2 domains from phospholipase C-γ1 (PLCγ1), tSH2-WT, has been used to measure phosphorylation of the epidermal growth factor receptor (EGFR). Here, we found that tSH2-WT lacked specificity for phosphorylated EGFR, consistent with the known promiscuity of SH2 domains. Further, EGF-stimulated membrane recruitment of tSH2-WT differed qualitatively from the expected kinetics of EGFR phosphorylation. Analysis of a mathematical model suggested, and experiments confirmed, that the high avidity of tSH2-WT resulted in saturation of its target and interference with EGFR endocytosis. To overcome the apparent target specificity and saturation issues, we implemented two protein engineering strategies. In the first approach, we screened a combinatorial library generated by random mutagenesis of the C-terminal SH2 domain (cSH2) of PLCγ1 and isolated a mutant form (mSH2) with enhanced specificity for phosphorylated Tyr (pTyr) of EGFR. A biosensor based on mSH2 closely reported the kinetics of EGFR phosphorylation but retained cross-reactivity similar to tSH2-WT. In the second approach, we isolated a pTyr-binding protein (SPY992) from a combinatorial library generated by mutagenesis of the Sso7d protein scaffold. Compared to tSH2-WT and mSH2, SPY992 exhibited superior performance as a specific, moderate-affinity biosensor. We extended this approach to isolate a biosensor for EGFR pTyr (SPY1148). This approach of integrating theoretical considerations with protein engineering strategies can be generalized to design and evaluate suitable biosensors for various phospho-specific targets.
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http://dx.doi.org/10.1126/scisignal.aap7584DOI Listing
June 2019

An Engineered Sso7d Variant Enables Efficient Magnetization of Yeast Cells.

ACS Comb Sci 2018 10 11;20(10):579-584. Epub 2018 Sep 11.

Department of Chemical and Biomolecular Engineering , North Carolina State University , Box 7905, Engineering Building I , Raleigh , North Carolina 27695 , United States.

Magnetization using cheap and minimally toxic materials, such as iron oxide nanoparticles can enable easy separation of cells from culture medium and is relevant to several industrial applications. Here, we show that cell surface expression of a mutant protein that binds iron oxide can enable efficient magnetization of yeast cells. We screened a combinatorial library of mutants derived from the Sso7d protein scaffold to isolate proteins that exhibit preferential binding to iron oxide. One of the isolated mutants, SsoFe2, was chosen for further characterization. Yeast cells expressing SsoFe2 as fusions to a cell wall protein-but not other Sso7d mutants with similar overall protein charge or amino acid composition-preferentially bind iron oxide when present in a solution with high protein concentration and in the presence of 1000-fold excess of competitor yeast cells. Moreover, coexpression of cell surface SsoFe2 enables efficient magnetic capture and separation of yeast cells expressing an enzyme (glucose oxidase) on the cell surface from yeast culture medium, and solutions with high protein concentration or containing other metal oxides. Therefore, SsoFe2-enabled magnetization can enable a range of industrial and biotechnology applications, where easy separation of cells or organelles from complex media is desirable.
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http://dx.doi.org/10.1021/acscombsci.8b00084DOI Listing
October 2018

Design and Fabrication of Wettability Gradients with Tunable Profiles through Degrafting Organosilane Layers from Silica Surfaces by Tetrabutylammonium Fluoride.

Langmuir 2017 12 11;33(51):14556-14564. Epub 2017 Dec 11.

Department of Chemical and Biomolecular Engineering, North Carolina State University , Raleigh, North Carolina 27695-7905, United States.

Surface-bound wettability gradients allow for a high-throughput approach to evaluate surface interactions for many biological and chemical processes. Here we describe the fabrication of surface wettability gradients on flat surfaces by a simple, two-step procedure that permits precise tuning of the gradient profile. This process involves the deposition of homogeneous silane SAMs followed by the formation of a surface coverage gradient through the selective removal of silanes from the substrate. Removal of silanes from the surface is achieved by using tetrabutylammonium fluoride which selectively cleaves the Si-O bonds at the headgroup of the silane. The kinetics of degrafting has been modeled by using a series of first order rate equations, based on the number of attachment points broken to remove a silane from the surface. Degrafting of monofunctional silanes exhibits a single exponential decay in surface coverage; however, there is a delay in degrafting of trifunctional silanes due to the presence of multiple attachment points. The effects of degrafting temperature and time are examined in detail and demonstrate the ability to reliably and precisely control the gradient profile on the surface. We observe a relatively homogeneous coverage of silane (i.e., without the presence of islands or holes) throughout the degrafting process, providing a much more uniform surface when compared to additive approaches of gradient formation. Linear gradients were formed on the substrates to demonstrate the reproducibility and tuneability of this subtractive approach.
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http://dx.doi.org/10.1021/acs.langmuir.7b02961DOI Listing
December 2017

Identification and characterization of a novel Sso7d scaffold-based binder against Notch1.

Sci Rep 2017 09 20;7(1):12021. Epub 2017 Sep 20.

New York University Langone Medical center, Perlmutter Cancer Center, Deparment of Biochemistry and Molecular Pharmacology, 522 1st Ave., Smilow Research Building, Rm 1206, New York, NY, 10016, USA.

Notch signaling has important functions in regulating cell growth and development, misregulation of which has been implicated in various cancers. Monoclonal antibodies (mAbs) targeting Notch protein activity have already moved into clinical trials. However due to the limitations associated with cost and productivity of mAbs, there has been a surge in the development of complementary approaches that are based on non-antibody scaffolds. Non-antibody scaffolds are small proteins that are stable and can be engineered to develop high-affinity binders against specific targets of interest. Here we describe the isolation and characterization of a novel Notch1-binding protein, N9, obtained by screening of a combinatorial library based on the ultra-stable Sso7d scaffold. N9 targets the extracellular EGF-like repeats (ELR) 11-13 in Notch1, and therefore serves as a competitive inhibitor for Notch ligands to decrease expression of Notch target genes. We demonstrate that N9 recognizes surface expression of Notch1 on the plasma membrane and binds preferentially to cell lines misexpressing Notch1. Although N9 was selected against Notch1, we also observe cross-reactivity against other Notch receptors, including Notch2/3. Finally, we demonstrate that N9 inhibits proliferation and generation of tumorspheres in Notch expressing cancer cell lines, suggesting its potential as a therapeutic agent in Notch-associated malignancies.
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http://dx.doi.org/10.1038/s41598-017-12246-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5607287PMC
September 2017

Inefficient Ribosomal Skipping Enables Simultaneous Secretion and Display of Proteins in Saccharomyces cerevisiae.

ACS Synth Biol 2017 11 14;6(11):2096-2107. Epub 2017 Aug 14.

Department of Chemical and Biomolecular Engineering, North Carolina State University , Raleigh, North Carolina 27695, United States.

The need for recombinant expression of soluble protein slows the validation of engineered proteins isolated from combinatorial libraries and limits the number of protein variants evaluated. To overcome this bottleneck, we describe a system for simultaneous cell surface display and soluble secretion of proteins in Saccharomyces cerevisiae based on inefficient ribosomal skipping. Ribosomal skipping mediated by "self-cleaving" 2A peptides produces two proteins from a single open reading frame. Incorporation of the F2A peptide sequence-with ∼50% efficiency of ribosomal skipping-between the protein of interest and the yeast cell wall protein Aga2 results in simultaneous expression of both the solubly secreted protein and the protein-Aga2 fusion that is tethered to the yeast cell surface. We show that binding proteins derived from the Sso7d scaffold and the homodimeric enzyme glucose oxidase can be simultaneously secreted solubly and expressed as yeast cell surface fusions using the F2A-based system. Furthermore, a combinatorial library of Sso7d mutants can be screened to isolate binders with higher affinity for a model target (lysozyme), and the pool of higher affinity binders can be characterized in soluble form. Significantly, we show that both N- and C-terminal fusions to Aga2 can be simultaneously secreted solubly and displayed on the cell surface; this is particularly advantageous because protein functionality can be affected by the specific position of Aga2 in the protein fusion. We expect that the F2A-based yeast surface display and secretion system will be a useful tool for protein engineering and enable efficient characterization of individual clones isolated from combinatorial libraries.
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http://dx.doi.org/10.1021/acssynbio.7b00144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5905331PMC
November 2017

Stable isotopic composition of perchlorate and nitrate accumulated in plants: Hydroponic experiments and field data.

Sci Total Environ 2017 Oct 8;595:556-566. Epub 2017 Apr 8.

Department of Civil, Environmental, and Construction Engineering, Texas Tech University, Lubbock, TX 79409-1023, USA. Electronic address:

Natural perchlorate (ClO) in soil and groundwater exhibits a wide range in stable isotopic compositions (δCl, δO, and ΔO), indicating that ClO may be formed through more than one pathway and/or undergoes post-depositional isotopic alteration. Plants are known to accumulate ClO, but little is known about their ability to alter its isotopic composition. We examined the potential for plants to alter the isotopic composition of ClO in hydroponic and field experiments conducted with snap beans (Phaseolus vulgaris L.). In hydroponic studies, anion ratios indicated that ClO was transported from solutions into plants similarly to NO but preferentially to Cl (4-fold). The ClO isotopic compositions of initial ClO reagents, final growth solutions, and aqueous extracts from plant tissues were essentially indistinguishable, indicating no significant isotope effects during ClO uptake or accumulation. The ClO isotopic composition of field-grown snap beans was also consistent with that of ClO in varying proportions from irrigation water and precipitation. NO uptake had little or no effect on NO isotopic compositions in hydroponic solutions. However, a large fractionation effect with an apparent ε (N/O) ratio of 1.05 was observed between NO in hydroponic solutions and leaf extracts, consistent with partial NO reduction during assimilation within plant tissue. We also explored the feasibility of evaluating sources of ClO in commercial produce, as illustrated by spinach, for which the ClO isotopic composition was similar to that of indigenous natural ClO. Our results indicate that some types of plants can accumulate and (presumably) release ClO to soil and groundwater without altering its isotopic characteristics. Concentrations and isotopic compositions of ClO and NO in plants may be useful for determining sources of fertilizers and sources of ClO in their growth environments and consequently in food supplies.
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http://dx.doi.org/10.1016/j.scitotenv.2017.03.223DOI Listing
October 2017

IFPA meeting 2016 workshop report I: Genomic communication, bioinformatics, trophoblast biology and transport systems.

Placenta 2017 12 11;60 Suppl 1:S5-S9. Epub 2017 Jan 11.

Center for Reproductive Health, MetroHealth Medical Center, Case Western Reserve University, Cleveland, OH, USA. Electronic address:

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2016 there were twelve themed workshops, four of which are summarized in this report. These workshops covered innovative technologies applied to new and traditional areas of placental research: 1) genomic communication; 2) bioinformatics; 3) trophoblast biology and pathology; 4) placental transport systems.
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http://dx.doi.org/10.1016/j.placenta.2017.01.103DOI Listing
December 2017

Targeted Mutagenesis and Combinatorial Library Screening Enables Control of Protein Orientation on Surfaces and Increased Activity of Adsorbed Proteins.

Langmuir 2016 08 16;32(34):8660-7. Epub 2016 Aug 16.

Department of Chemical and Biomolecular Engineering, North Carolina State University , Raleigh, North Carolina 27695, United States.

While nonspecific adsorption is widely used for immobilizing proteins on solid surfaces, the random nature of protein adsorption may reduce the activity of immobilized proteins due to occlusion of the active site. We hypothesized that the orientation a protein assumes on a given surface can be controlled by systematically introducing mutations into a region distant from its active site, thereby retaining activity of the immobilized protein. To test this hypothesis, we generated a combinatorial protein library by randomizing six targeted residues in a binding protein derived from highly stable, nonimmunoglobulin Sso7d scaffold; mutations were targeted in a region that is distant from the binding site. This library was screened to isolate binders that retain binding to its cognate target (chicken immunoglobulin Y, cIgY) as well as exhibit adsorption on unmodified silica at pH 7.4 and high ionic strength conditions. A single mutant, Sso7d-2B5, was selected for further characterization. Sso7d-2B5 retained binding to cIgY with an apparent dissociation constant similar to that of the parent protein; both mutant and parent proteins saturated the surface of silica with similar densities. Strikingly, however, silica beads coated with Sso7d-2B5 could achieve up to 7-fold higher capture of cIgY than beads coated with the parent protein. These results strongly suggest that mutations introduced in Sso7d-2B5 alter its orientation relative to the parent protein, when adsorbed on silica surfaces. Our approach also provides a generalizable strategy for introducing mutations in proteins so as to improve their activity upon immobilization, and has direct relevance to development of protein-based biosensors and biocatalysts.
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http://dx.doi.org/10.1021/acs.langmuir.6b01446DOI Listing
August 2016

Identification of Epigenetic Factor Proteins Expressed in Human Embryonic Stem Cell-Derived Trophoblasts and in Human Placental Trophoblasts.

J Proteome Res 2016 08 18;15(8):2433-44. Epub 2016 Jul 18.

Department of Obstetrics and Gynecology, University of North Carolina-Chapel Hill , Chapel Hill, North Carolina 27599, United States.

Human embryonic stem cells (hESCs) have been used to derive trophoblasts through differentiation in vitro. Intriguingly, mouse ESCs are prevented from differentiation to trophoblasts by certain epigenetic factor proteins such as Dnmt1, thus necessitating the study of epigenetic factor proteins during hESC differentiation to trophoblasts. We used stable isotope labeling by amino acids in cell culture and quantitative proteomics to study changes in the nuclear proteome during hESC differentiation to trophoblasts and identified changes in the expression of 30 epigenetic factor proteins. Importantly, the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B were downregulated. Additionally, we hypothesized that nuclear proteomics of hESC-derived trophoblasts may be used for screening epigenetic factor proteins expressed by primary trophoblasts in human placental tissue. Accordingly, we conducted immunohistochemistry analysis of six epigenetic factor proteins identified from hESC-derived trophoblasts-DNMT1, DNMT3B, BAF155, BAF60A, BAF57, and ING5-in 6-9 week human placentas. Indeed, expression of these proteins was largely, though not fully, consistent with that observed in 6-9 week placental trophoblasts. Our results support the use of hESC-derived trophoblasts as a model for placental trophoblasts, which will enable further investigation of epigenetic factors involved in human trophoblast development.
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http://dx.doi.org/10.1021/acs.jproteome.5b01118DOI Listing
August 2016

Combinatorial Pairwise Assembly Efficiently Generates High Affinity Binders and Enables a "Mix-and-Read" Detection Scheme.

ACS Synth Biol 2016 12 15;5(12):1348-1354. Epub 2016 Jun 15.

Department of Chemical and Biomolecular Engineering, North Carolina State University , Raleigh, North Carolina 27695, United States.

We show that a combinatorial library constructed by random pairwise assembly of low affinity binders can efficiently generate binders with increased affinity. Such a library based on the Sso7d scaffold, from a pool of low affinity binders subjected to random mutagenesis, contained putative high affinity clones for a model target (lysozyme) at higher frequency than a library of monovalent mutants generated by random mutagenesis alone. Increased binding affinity was due to intramolecular avidity generated by linking binders targeting nonoverlapping epitopes; individual binders of K ∼ 1.3 μM and 250 nM produced a bivalent binder with apparent K ∼ 2 nM. Furthermore, the bivalent protein retained thermal stability (T = 84.5 °C) and high recombinant expression yields in E. coli. Finally, when binders comprising the bivalent protein are fused to two of the three fragments of tripartite split-green fluorescent protein (GFP), target-dependent reconstitution of fluorescence occurs, thereby enabling a "mix-and-read" assay for target quantification.
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http://dx.doi.org/10.1021/acssynbio.6b00034DOI Listing
December 2016

Activin/nodal signaling switches the terminal fate of human embryonic stem cell-derived trophoblasts.

J Biol Chem 2015 Apr 10;290(14):8834-48. Epub 2015 Feb 10.

From the Department of Chemical and Biomolecular Engineering,

Human embryonic stem cells (hESCs) have been routinely treated with bone morphogenetic protein and/or inhibitors of activin/nodal signaling to obtain cells that express trophoblast markers. Trophoblasts can terminally differentiate to either extravillous trophoblasts or syncytiotrophoblasts. The signaling pathways that govern the terminal fate of these trophoblasts are not understood. We show that activin/nodal signaling switches the terminal fate of these hESC-derived trophoblasts. Inhibition of activin/nodal signaling leads to formation of extravillous trophoblast, whereas loss of activin/nodal inhibition leads to the formation of syncytiotrophoblasts. Also, the ability of hESCs to form bona fide trophoblasts has been intensely debated. We have examined hESC-derived trophoblasts in the light of stringent criteria that were proposed recently, such as hypomethylation of the ELF5-2b promoter region and down-regulation of HLA class I antigens. We report that trophoblasts that possess these properties can indeed be obtained from hESCs.
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http://dx.doi.org/10.1074/jbc.M114.620641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4423675PMC
April 2015

Coupled mercury-cell sorption, reduction, and oxidation on methylmercury production by Geobacter sulfurreducens PCA.

Environ Sci Technol 2014 Oct 7;48(20):11969-76. Epub 2014 Oct 7.

Environmental Sciences Division and ‡Biosciences Division, Oak Ridge National Laboratory , Oak Ridge, Tennessee 37831, United States.

G. sulfurreducens PCA cells have been shown to reduce, sorb, and methylate Hg(II) species, but it is unclear whether this organism can oxidize and methylate dissolved elemental Hg(0) as shown for Desulfovibrio desulfuricans ND132. Using Hg(II) and Hg(0) separately as Hg sources in washed cell assays in phosphate buffered saline (pH 7.4), we report how cell-mediated Hg reduction and oxidation compete or synergize with sorption, thus affecting the production of toxic methylmercury by PCA cells. Methylation is found to be positively correlated to Hg sorption (r = 0.73) but negatively correlated to Hg reduction (r = -0.62). These reactions depend on the Hg and cell concentrations or the ratio of Hg to cellular thiols (-SH). Oxidation and methylation of Hg(0) are favored at relatively low Hg to cell-SH molar ratios (e.g., <1). Increasing Hg to cell ratios from 0.25 × 10(-19) to 25 × 10(-19) moles-Hg/cell (equivalent to Hg/cell-SH of 0.71 to 71) shifts the major reaction from oxidation to reduction. In the absence of five outer membrane c-type cytochromes, mutant ΔomcBESTZ also shows decreases in Hg reduction and increases in methylation. However, the presence of competing thiol-binding ions such as Zn(2+) leads to increased Hg reduction and decreased methylation. These results suggest that the coupled cell-Hg sorption and redox transformations are important in controlling the rates of Hg uptake and methylation by G. sulfurreducens PCA in anoxic environments.
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http://dx.doi.org/10.1021/es502537aDOI Listing
October 2014

Alzheimer disease: An interactome of many diseases.

Ann Indian Acad Neurol 2014 Jan;17(1):48-54

Department of Bioinformatics, NTHRYS Biotech Labs, Hyderabad, Andhra Pradesh, India.

Alzheimer Disease (AD) is an outcome as well as source of many diseases. Alzheimer is linked with many other diseases like Diabetes type 2, cholesterolemia, hypertension and many more. But how each of these diseases affecting other is still unknown to scientific community. Signaling Pathways of one disease is interlinked with other disease. But to what extent healthy brain is affected when any signaling in human body is disturbed is the question that matters. There is a need of Pathway analysis, Protein-Protein interaction (PPI) and the conserved interactome study in AD and linked diseases. It will be helpful in finding the potent drug or vaccine target in conscious manner. In the present research the Protein-Protein interaction of all the proteins involved in Alzheimer Disease is analyzed using ViSANT and osprey tools and pathway analysis further reveals the significant genes/proteins linking AD with other diseases.
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http://dx.doi.org/10.4103/0972-2327.128551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3992769PMC
January 2014