High-affinity Capture Research Articles

Detection of Dengue Virus 2 with Single Infected Mosquito Resolution Using Yeast Affinity Bionanofragments and Plasmonic SERS Nanoboxes.

Dengue disease is an emerging global threat triggered by dengue virus (DENV) transmission, primarily by the mosquito Aedes aegypti. The accurate surveillance and sensitive detection of DENV in mosquito populations are critical for the protection of human populations worldwide that are in the habitat of these mosquito species. There are four DENV serotypes with DENV2 reported to cause the most severe complications. There are limited ultrasensitive methods to early detect DENV2 mosquito infection and prevent human infection. Herein, we report an innovative nanobased immunoassay platform for early, specific, and ultrasensitive detection of DENV2-secreted nonstructural 1 (NS1) protein biomarker in single infected mosquitoes with the limit of detection of 500 fg of recombinant DENV2 NS1. The high sensitivity and DENV2 serotype specificity of the platform are the result of using nanomixing, plasmonic SERS nanoboxes, and yeast affinity bionanofragments displaying single-chain variable fragments (nanoyeast scFvs). Nanoyeast scFvs used for high affinity capture of DENV2 NS1 provided an innovative and cost-efficient alternative to monoclonal antibodies and differentiated DENV2 NS1 from other DENV serotypes and Zika virus NS1. The platform used electrohydrodynamically driven nanomixing to enhance NS1 capture by the nanoyeast scFvs while reducing nonspecific interactions. High sensitivity detection of captured DENV2 NS1 was achieved using NS1-specific surface-enhanced Raman scattering (SERS) nanotags. These nanotechnologies provide a significant innovation for early DENV2 detection in single infected mosquitoes, improving the accurate surveillance of mosquito habitats and preventing infection and severe disease arising from DENV2 transmission.

Net-Shaped DNA Nanostructures Designed for Rapid/Sensitive Detection and Potential Inhibition of the SARS-CoV-2 Virus.

We present a net-shaped DNA nanostructure (called “DNA Net” herein) design strategy for selective recognition and high-affinity capture of intact SARS-CoV-2 virions through spatial pattern-matching and multivalent interactions between the aptamers (targeting wild-type spike-RBD) positioned on the DNA Net and the trimeric spike glycoproteins displayed on the viral outer surface. Carrying a designer nanoswitch, the DNA Net-aptamers release fluorescence signals upon virus binding that are easily read with a handheld fluorimeter for a rapid (in 10 min), simple (mix-and-read), sensitive (PCR equivalent), room temperature compatible, and inexpensive (∼$1.26/test) COVID-19 test assay. The DNA Net-aptamers also impede authentic wild-type SARS-CoV-2 infection in cell culture with a near 1 × 103-fold enhancement of the monomeric aptamer. Furthermore, our DNA Net design principle and strategy can be customized to tackle other life-threatening and economically influential viruses like influenza and HIV, whose surfaces carry class-I viral envelope glycoproteins like the SARS-CoV-2 spikes in trimeric forms.

Affinity Tag Coating Enables Reliable Detection of Antigen-Specific B Cells in Immunospot Assays.

Assessment of humoral immunity to SARS-CoV-2 and other infectious agents is typically restricted to detecting antigen-specific antibodies in the serum. Rarely does immune monitoring entail assessment of the memory B-cell compartment itself, although it is these cells that engage in secondary antibody responses capable of mediating immune protection when pre-existing antibodies fail to prevent re-infection. There are few techniques that are capable of detecting rare antigen-specific B cells while also providing information regarding their relative abundance, class/subclass usage and functional affinity. In theory, the ELISPOT/FluoroSpot (collectively ImmunoSpot) assay platform is ideally suited for antigen-specific B-cell assessments since it provides this information at single-cell resolution for individual antibody-secreting cells (ASC). Here, we tested the hypothesis that antigen-coating efficiency could be universally improved across a diverse set of viral antigens if the standard direct (non-specific, low affinity) antigen absorption to the membrane was substituted by high-affinity capture. Specifically, we report an enhancement in assay sensitivity and a reduction in required protein concentrations through the capture of recombinant proteins via their encoded hexahistidine (6XHis) affinity tag. Affinity tag antigen coating enabled detection of SARS-CoV-2 Spike receptor binding domain (RBD)-reactive ASC, and also significantly improved assay performance using additional control antigens. Collectively, establishment of a universal antigen-coating approach streamlines characterization of the memory B-cell compartment after SARS-CoV-2 infection or COVID-19 vaccinations, and facilitates high-throughput immune-monitoring efforts of large donor cohorts in general.

Influence of Iron on Physiological Parameters and Intracellular Microcystin in Microcystis Panniformis Strain Isolated from a Reservoir in the Amazon.

In the Amazon, the leaching from soil left unprotected by deforestation increases the entry of iron, among other elements, in aquatic ecosystems, which can cause cyanobacterial blooms. This study aimed to investigate the physiological response of a strain of Microcystis panniformis to iron variation. The strain was isolated from a reservoir located in the Western Amazon and produces microcystin-LR. After a period of iron deprivation, the cultures were submitted to three conditions: control (223 μgFe.L-1), treatment with 23 μgFe.L-1, and absence of iron. At regular intervals for eight days, the cell density, levels of chlorophyll a and microcystins were determined. On the second and fourth day, transcription of genes responsive to iron limitation was quantified. Starting on the fourth day of the experiment, the different iron concentrations affected growth, and on the eighth day in the iron-free condition cell density was 90% lower than in control. Chlorophyll cell quota in 23 μgFe.L-1 and control presented similar values, while without iron the cells became chlorotic as of the fourth day Toxin concentration in cells grow in 0 μgFe.L-1 in relation to the control. Higher transcription levels of the feo and fut genes were observed in the 0 μgFe.L-1 and 23 μgFe.L-1 treatments, indicating that the cells were activating high-affinity capture systems to reestablish an adequate concentration of intracellular iron. The increasing deforestation in the Jamari River Basin (Amazon region), can contribute to the occurrence of toxic cyanobacterial blooms due to the greater entrance of iron in water bodies.

Selective Enrichment of A-to-I Edited Transcripts from Cellular RNA Using Endonuclease V.

Creating accurate maps of A-to-I RNA editing activity is vital to improving our understanding of the biological role of this process and harnessing it as a signal for disease diagnosis. Current RNA sequencing techniques are susceptible to random sampling limitations due to the complexity of the transcriptome and require large amounts of RNA material, specialized instrumentation, and high read counts to accurately interrogate A-to-I editing sites. To address these challenges, we show that Escherichia coli Endonuclease V (eEndoV), an inosine-cleaving enzyme, can be repurposed to bind and isolate A-to-I edited transcripts from cellular RNA. While Mg2+ enables eEndoV to catalyze RNA cleavage, we show that similar levels of Ca2+ instead promote binding of inosine without cleavage and thus enable high affinity capture of inosine in RNA. We leverage this capability to demonstrate EndoVIPER-seq (Endonuclease V inosine precipitation enrichment sequencing) as a facile and effective method to enrich A-to-I edited transcripts prior to RNA-seq, producing significant increases in the coverage and detection of identified editing sites. We envision the use of this approach as a straightforward and cost-effective strategy to improve the epitranscriptomic informational density of RNA samples, facilitating a deeper understanding of the functional roles of A-to-I editing.

A Bio-inspired Nano-pocket Spatial Structure for Targeting Uranyl Capture.

Biology has evolved excellent spatial structures for high-selectivity and high-affinity capture of heavy metals. Inspired by the spatial structure of the superb-uranyl binding protein SUP, we mimic the spatial structure of SUP in metal-organic frameworks (MOFs). The MOF UiO-66-3C4N fabricated by introducing 4-aminoisophthalic acid into UiO-66 shows high uranyl adsorption capacity both in simulated seawater and in natural seawater. In natural seawater, UiO-66-3C4N exhibits 17.03 times higher uranium extraction capacity than that of vanadium, indicating the high selectivity of the adsorbent. The EXAFS analysis and DFT calculation reveal that UiO-66-3C4N forms smaller nano-pocket for uranyl capture than that of SUP protein, which can both restrict the entrance of the other interfering ions with larger size and reinforce the binding by increasing the coordination interaction, and therefore qualify the nano-pocket with high affinity and high selectivity to uranyl.

DNA Framework-Programmed Cell Capture via Topology-Engineered Receptor-Ligand Interactions.

Receptor-ligand interactions (RLIs) that play pivotal roles in living organisms are often depicted with the classic keys-and-locks model. Nevertheless, RLIs on the cell surface are generally highly complex and nonlinear, partially due to the noncontinuous and dynamic distribution of receptors on extracellular membranes. Here, we develop a tetrahedral DNA framework (TDF)-programmed approach to topologically engineer RLIs on the cell membrane, which enables active recruitment-binding of clustered receptors for high-affinity capture of circulating tumor cells (CTCs). The four vertices of a TDF afford orthogonal anchoring of ligands with spatial organization, based on which we synthesized n-simplexes harboring 1-3 aptamers targeting epithelial cell adhesion molecule (EpCAM) that are overexpressed on the membrane of tumor cells. The 2-simplex with three aptamers not only shows increased binding affinity (∼19-fold) but prevents endocytosis by cells. By using 2-simplex as the capture probe, we demonstrate the high-efficiency CTC capture, which is challenged in real clinical breast cancer patient samples. This TDF-programmed platform thus provides a powerful means for studying RLIs in physiological settings and for cancer diagnosis.

Profiling Interactome Networks with the HaloTag-NAPPA In Situ Protein Array.

Physical interactions between proteins and other molecules can be evaluated at a proteome scale using protein arrays, a type of high-throughput pulldown assay. We developed a modified in situ protein array known as the nucleic acid programmable protein assay (NAPPA) that allows the screening of thousands of open reading frames (ORFs) at a lower cost, with less labor, and in less time than conventional protein arrays. The HaloTag-NAPPA protein array can efficiently capture proteins expressed in situ on a glass slide using the Halo high-affinity capture tag. Here, we describe the fabrication of the array using publicly available resources and detection of protein-protein interactions (PPIs) that can be used to generate a protein interactome map. The Basic Protocol includes procedures for preparing the plasmid DNA spotted on glass slides, in situ protein expression, and PPI detection. The supporting protocols outline the construction of vectors and preparation of ORF clones. © 2018 by John Wiley & Sons, Inc.

Proteoliposome-based full-length ZnT8 self-antigen for type 1 diabetes diagnosis on a plasmonic platform

Identified as a major biomarker for type 1 diabetes (T1D) diagnosis, zinc transporter 8 autoantibody (ZnT8A) has shown promise for staging disease risk and disease diagnosis. However, existing assays for ZnT8 autoantibody (ZnT8A) are limited to detection by soluble domains of ZnT8, owing to difficulties in maintaining proper folding of a full-length ZnT8 protein outside its native membrane environment. Through a combined bioengineering and nanotechnology approach, we have developed a proteoliposome-based full-length ZnT8 self-antigen (full-length ZnT8 proteoliposomes; PLR-ZnT8) for efficient detection of ZnT8A on a plasmonic gold chip (pGOLD). The protective lipid matrix of proteoliposomes improved the proper folding and structural stability of full-length ZnT8, helping PLR-ZnT8 immobilized on pGOLD (PLR-ZnT8/pGOLD) achieve high-affinity capture of ZnT8A from T1D sera. Our PLR-ZnT8/pGOLD exhibited efficient ZnT8A detection for T1D diagnosis with ∼76% sensitivity and ∼97% specificity (n = 307), superior to assays based on detergent-solubilized full-length ZnT8 and the C-terminal domain of ZnT8. Multiplexed assays using pGOLD were also developed for simultaneous detection of ZnT8A, islet antigen 2 autoantibody, and glutamic acid decarboxylase autoantibody for diagnosing T1D.

Inhibition of Hsp90 Suppresses PI3K/AKT/mTOR Signaling and Has Antitumor Activity in Burkitt Lymphoma.

Hsp90 is a molecular chaperone that protects proteins, including oncogenic signaling complexes, from proteolytic degradation. PU-H71 is a next-generation Hsp90 inhibitor that preferentially targets the functionally distinct pool of Hsp90 present in tumor cells. Tumors that are driven by the MYC oncoprotein may be particularly sensitive to PU-H71 due to the essential role of Hsp90 in the epichaperome, which maintains the malignant phenotype in the setting of MYC. Burkitt lymphoma (BL) is an aggressive B-cell lymphoma characterized by MYC dysregulation. In this study, we evaluated Hsp90 as a potential therapeutic target in BL. We found that primary BL tumors overexpress Hsp90 and that Hsp90 inhibition has antitumor activity in vitro and in vivo, including potent activity in a patient-derived xenograft model of BL. To evaluate the targets of PU-H71 in BL, we performed high-affinity capture followed by proteomic analysis using mass spectrometry. We found that Hsp90 inhibition targets multiple components of PI3K/AKT/mTOR signaling, highlighting the importance of this pathway in BL. Finally, we found that the anti-lymphoma activity of PU-H71 is synergistic with dual PI3K/mTOR inhibition in vitro and in vivo Overall, this work provides support for Hsp90 as a therapeutic target in BL and suggests the potential for combination therapy with PU-H71 and inhibitors of PI3K/mTOR. Mol Cancer Ther; 16(9); 1779-90. ©2017 AACR.

Interface engineering of metal organic framework on graphene oxide with enhanced adsorption capacity for organophosphorus pesticide

A simple solvothermal synthetic method was proposed for the fabrication of metal organic framework/grapheme oxide hybrid nanocomposite (UiO-67/GO), achieving dense and ordered UiO-67 on the surface of GO for the high-affinity capture of organophosphorus pesticides (OPPs). The composites were characterized by SEM, XRD, FTIR and TGA. The adsorption process and adsorption capacity of glyphosate was evaluated by kinetics and equilibrium adsorption isotherm study, respectively. The results indicate that the pseudo-second-order kinetic model and the Langmuir model correlated satisfactorily to the experimental data. The maximum adsorption capacity of glyphosate on UiO-67/GO is ∼2.855mmol (482.69mg)g−1 at pH 4, which catches up with and even surpasses that of most GO-based adsorbents. The excellent adsorption performances of UiO-67/GO toward OPPs are also presented in a wide range of pH 2–8. Moreover, the adsorption mechanism was further revealed using XPS and FTIR. Taking advantage of the abundant ZrOH groups on the surface of GO, as well as the large surface of available GO for enhancing the sequestration of target organophosphorus-pollutants, the as-prepared UiO-67/GO composites exhibit enhancement of adsorption capacity toward glyphosate. The experimental results suggest that the UiO-67/GO is a promising adsorbent for efficient removal of OPPs for the contaminated water purification.

Conformational Plasticity in the Transsynaptic Neurexin-Cerebellin-Glutamate Receptor Adhesion Complex

Synaptic specificity is a defining property of neural networks. In the cerebellum, synapses between parallel fiber neurons and Purkinje cells are specified by the simultaneous interactions of secreted protein cerebellin with pre-synaptic neurexin and post-synaptic delta-type glutamate receptors (GluD). Here, we determined the crystal structures of the trimeric C1q-like domain of rat cerebellin-1, and the first complete ectodomain of a GluD, rat GluD2. Cerebellin binds to the LNS6 domain of α- and β-neurexin-1 through a high-affinity interaction that involves its highly flexible N-terminal domain. In contrast, we show that the interaction of cerebellin with isolated GluD2 ectodomain is low affinity, which is not simply an outcome of lost avidity when compared with binding with a tetrameric full-length receptor. Rather, high-affinity capture of cerebellin by post-synaptic terminals is likely controlled by long-distance regulation within this transsynaptic complex. Altogether, our results suggest unusual conformational flexibility within all components of the complex.

Ixekizumab, a novel anti–IL-17A antibody, exhibits low immunogenicity during long-term treatment in patients with psoriasis

388 Ixekizumab, a novel anti-IL-17A antibody, exhibits low immunogenicity during long-term treatment in patients with psoriasis A Blauvelt, R Langley, C Leonardi, K Gordon, T Luger, M Ohtsuki, B Nickoloff, L Kerr, L Mallbris and K Reich 1 Oregon Medical Research Center, Portland, OR, 2 Dalhousie University, Halifax, NS, Canada, 3 Saint Louis University, St. Louis, MO, 4 Northwestern University Feinberg School of Medicine, St. Louis, MO, 5 University of Munster, Munster, Germany, 6 Jichi Medical University, Shimotsuke, Japan, 7 Eli Lilly and Company, Indianapolis, IN and 8 Dermatologikum Hamburg and SCIderm Research Institute, Hamburg, Germany The potential association of treatment-emergent anti-drug antibodies (TE-ADAs) with efficacy and safety in patients treated with ixekizumab (IXE), a high binding affinity monoclonal antibody that selectively targets interleukin-17A, was evaluated. TE-ADAs were evaluated in patients randomized to 80 mg IXE either every 2 weeks (IXE Q2W; N1⁄41150) or 4 weeks (IXE Q4W; N1⁄41143) for up to 12 weeks following a 160-mg starting dose. IXE responders at Week 12 were re-randomized to IXE Q4W (N1⁄4397) or IXE Q12W (N1⁄4382) until Week 60. A highaffinity capture elution screening assay was used to assess ADA serum trough levels. Serum TE-ADAs were divided into four titer groups (negative, low, moderate, and high). At 12 weeks, the majority of patients were TE-ADA negative (IXE Q2W: 91.0%; IXE Q4W: 86.6%). TE-ADE titers at 12 weeks for IXE Q2W and IXE Q4W patients were, respectively, 5.7% and 8.0% (low), 1.6% and 3.0% (moderate), and 1.7% and 2.4% (high). IXE Q2W patients with high TEADA titers had reduced PASI responses versus TE-ADA negative patients; 12-week mean percent PASI improvement was 91.7% (negative), 85.7% (low), 88.7% (moderate), and 53.5% (high); 12-week PASI 75 response rates were 90.7% (negative), 84.8% (low), 83.3% (moderate), and 36.8% (high). Mean percent improvement from baseline to 60 weeks PASI was 94.3% (negative), 97.0% (low), and 100% (moderate) for TE-ADA titer patients. IXE Q4W data were similar. No safety findings were correlated to TE-ADAs. TE-ADA levels in IXE patients did not affect maintenance of response and safety up to 60 weeks among initial IXE responders. 389 The extravesicular miRNome of senescent human fibroblasts and its impact on keratinocyte functionality L Terlecki-Zaniewicz, V Pils, J Schwestka, J Latreille, I Lammermann, R Weinmullner, I Berlin, M Hackl, F Morizot and J Grillari 1 Dept. of Biotechnology, BOKU University Vienna, Austria, Christian Doppler Laboratory for Biotechnology of Skin Aging, Vienna, Austria, 2 CE.R.I.E.S. (Research Centre on Human Skin of CHANEL), Neuilly sur Seine, France and 3 TAmiRNA GmbH, Vienna, Austria In aged skin up to 25% of senescent cells are present and supposed to impair tissue homeostasis and regeneration resulting in a gradual loss of juvenile appearance and functionality, while transiently the presence of senescent cells might be beneficial for cutaneous wound healing. The senescence associated secretory phenotype (SASP) is one pivotal hallmark of senescent cells, whereby proinflammtory cytokines, chemokines and metalloproteases are secreted and alter the tissue microenvironment. Recently, micro RNAs (miRNAs) packaged into extracellular vesicles (EV) have been found as part of SASP (miRSASP). Here, we report the identification of EV-miRNAs as part of the human dermal skin fibroblasts’ SASP. For this purpose, stress induced premature senescence was triggered by repeated low doses of H2O2 in cells of 3 donors and EV of senescent and control cells were harvested by ultracentrifugation for miRNomics and functional biological studies. We identified differentially secreted miRNAs by qPCR arrays and correlated their extracellular and intracellular abundance. Based on that, we have confirmed selected prominent, highly secreted miRNAs to be more abundant in EVs of senescent cells. Furthermore, by nanoparticle tracking analysis we observed that senescent fibroblasts secrete more vesicles than controls. In order to test if EV-miRNAs might be part of a paracrine cross-talk between dermal fibroblasts and epidermal keratinocytes, we confirmed uptake of cel-miR-39 enclosed in fibroblast derived EV by keratinocytes in monolayers and in full thickness skin-equivalents. To summarize, our data indicate that extravesicular miRNAs of senescent fibroblasts are bona fide members of the SASP and that they contribute to the communication between fibroblasts and keratinocytes in 2D and 3D human skin models.

Targeting the Hsp90 Oncoproteome in Burkitt Lymphoma

Introduction: Novel therapies are urgently needed in pediatric Burkitt lymphoma (pBL) where the survival for relapsed disease is less than 20%. Heat shock protein 90 (Hsp90) is molecular chaperone that protects proteins from proteolytic degradation including oncogenic signaling complexes. The clinical development of broad-spectrum Hsp90 inhibitors has previously been limited by suboptimal target inhibition and off-target toxicities. PU-H71 is a next-generation Hsp90 inhibitor that preferentially targets tumor enriched (te-Hsp90), the functionally distinct pool of Hsp90 present in tumor cells. PU-H71 is not toxic to normal B-cells and has demonstrated pre-clinical efficacy in diffuse large B-cell lymphoma, but has not been studied in Burkitt lymphoma. In the current study, we evaluated te-Hsp90 as a potential therapeutic target in pediatric Burkitt lymphoma.Methods and Results: To evaluate overall Hsp90 protein expression in primary pBL tumors we performed immunohistochemistry on a tissue microarray. Fifty-three of 59 cases (90%) demonstrated high levels of Hsp90 expression defined as >90% tumor cell positivity (Fig. 1A). To evaluate the sensitivity of pBL to te-Hsp90 inhibition we performed in-vitro viability assays with the ATP-based CellTiter-Glo¨ in a panel of pBL cell lines (Ramos, DG-75, Raji, Namalwa, Daudi, Jiyoye, CA-46, Raji 2R, Raji 4RH) and in-vivo studies with a Ramos xenograft model of pBL. pBL cells were sensitive to inhibition by PU-H71 with IC50s in the low nanomolar range (151-337nM, Fig 1B). The Raji 2R and Raji 4RH pBL cell lines with acquired resistance to chemotherapy (Czuczman et al, Clin Cancer Res 2008) were also sensitive to PU-H71 (IC50 175-181nM). In contrast, normal peripheral blood lymphocytes were resistant (IC50 >37,000nM). In pBL xenograft studies, PU-H71 decreased tumor volume (p<0.01, Fig. 1C) and prolonged survival (p<0.01, Fig. 1D).To evaluate the targets of PU-H71 in pBL we performed high affinity capture followed by proteomic analysis using mass spectrometry. Cellular lysates from pBL cell lines were incubated with PU-H71 conjugated agarose beads and the cargo was subject to tandem mass spectrometry. Data was analyzed using Ingenuity Pathway Analysis¨. PI3K and mTOR signaling were among the top networks identified. Given the data supporting a crucial role for PI3K in BL (Sanders et al, Cancer Cell 2012), we further investigated protein targets in this pathway. We found a significant number of proteins in the mTOR (p<0.0001) and PI3K (p<0.0001) signaling pathways including ERK, mTOR, and multiple isoforms of PI3K (p110-β; p110-γ, p110-δ). PI3K targets were validated by pull-downs with PU-H71 conjugated beads (Fig 2A).To investigate the activity of PI3K inhibition in pBL we evaluated a panel of PI3K inhibitors in pBL. Agents with a narrow spectrum of PI3K isoform inhibition such as idelalisib (p110δ inhibition) and IPI-145 (p110 δ and γ inhibition) did not have activity in pBL. In contrast, agents that broadly target PI3K/mTOR such as BEZ235 (pan PI3K and mTOR inhibition) and BKM120 (pan PI3K inhibition) demonstrated activity (IC50 35nM-2602nM). Based on this we concluded that PI3K inhibition is effective in pBL only when multiple components of the pathway are targeted. We hypothesized that PU-H71 will therefore synergize with PI3K inhibitors through dual targeting of PI3K signaling. To test this we evaluated the combination of PU-H71 + BKM-120 and PU-H71 + BEZ235 in four pBL cell lines (Ramos, Namalwa, Daudi, DG-75). The combined response at 48-72 hours was evaluated using the Chou-Talalay method. Both combinations were synergistic in 3 of 4 cell lines with a combinatorial index at the IC50 of <1 (CI 0.44-0.79, Fig. 2B).Conclusion: Our work demonstrates that Hsp90 is over-expressed in primary pBL tumors and that te-Hsp90 inhibition with PU-H71 is toxic to pBL in-vitro and in-vivo. The oncoprotein targets of Hsp90 in pBL include multiple components of the PI3K/mTOR signaling pathway, highlighting the importance of this pathway in pBL. The anti-lymphoma activity of PU-H71 is synergistic with pan-PI3K inhibition as well as dual PI3K/mTOR inhibition. Overall this work provides support for te-Hsp90 as a therapeutic target in BL and suggests the potential for combination therapy with PU-H71 and inhibitors of PI3K/mTOR. [Display omitted] [Display omitted] DisclosuresCesarman:Weill Cornell Medical College: Patents & Royalties: applied for patent for 6-ETI.

Zirconia/Graphene Oxide Hybrid Micromotors for Selective Capture of Nerve Agents

Self-propelled zirconia-graphene/Pt hybrid tubular micromotors, prepared by electrochemical template synthesis, are used for both high-affinity capture and isolation of nerve agents. The simultaneous electrochemical deposition of zirconia and reduced graphene oxide leads to high surface area with a needle-like zirconia microstructure. The attractive surface properties of graphene sheets are used as growth directing template for the electrochemical synthesis of high surface area of zirconia nanostructures for effective and selective binding of organophosphate compounds. Such selective binding is dramatically enhanced by the rapid movement of the motors and the corresponding bubble-induced solution mixing. The greatly increased fluid transport leads to a 15-fold faster remediation compared to the use of the static counterpart structures. The unique material properties allow the convenient alkaline regeneration of the micromotor surface, improving the cost effectiveness of this methodology. The new strategy ...

Abstract 376: Capture and release of circulating tumor cells by temperature-sensitive graphene oxide-polymer composite

Abstract Introduction and Objective: Recently, various microfluidics-based circulating tumor cells (CTC) isolation technologies have been developed to isolate, enumerate, and characterize CTCs, but there are few studies reporting CTC release after selective capture. Existing devices face impediments to the easy release of captured cells due to the conjugation of antibodies to permanent structures within the devices. Here we present a tunable microfluidic polymer graphene oxide (GO) Chip based on a temperature-sensitive graphene oxide-polymer composite for selective capture and efficient subsequent release of CTCs. Methods: A blend film of GO sheets and thermo-responsive polymers serves as a platform enabling cell capture and release in our microfluidic devices. The polymers with a tunable lower critical solution temperature (LCST) allow for stimulated release of the captured cells when the device temperature is below the LCST. The GO sheets serve as a carrier for phosophlipid-PEG-amine, which is used for the immobilization of an antibody against the epithelial-cell-adhesion-molecule (EpCAM). To investigate the capture efficiency, MCF-7 cells labeled with cell tracker dye were spiked into 1 mL of whole blood and flowed through the microfluidic device. After thoroughly washing the device with PBS at 25 °C to remove any non-specifically bound cells, we dissolved the thermo-responsive composite with 1 mL of PBS at 5 °C in a cold room and collected released cells from the device outlet. The released cells were cytospun and stained to identify CTCs as cytokeratin positive, DAPI positive, and CD45 negative cells. We also tested peripheral blood samples obtained from patients with metastatic breast cancer in a similar manner. Results: The graphene oxide-polymer based CTC-chip showed a capture efficiency of 95.21% at 95.21% and a release efficiency of 92.45% for MCF-7 breast cancer cells. This new GO Chip was also able to isolate viable CTCs from blood samples of metastatic breast cancer patients for further molecular characterizations. Conclusion: We present a sensitive and tunable microfluidic chip based on a GO-polymer composite to capture, release, identify, and characterize extremely rare CTCs. The CTCs released from the chip are viable, suggesting that this polymer based GO Chip offers great potential for clinical applications due to its high-affinity cell capture and the ability to enable various downstream analyses of captured CTCs. Citation Format: Hyeun Joong Yoon, Apoorv Shanker, Yang Wang, Molly Kozminsky, Shamileh Fouladdel, Monika L. Burness, Ebrahim Azizi, Max S. Wicha, Jinsang Kim, Sunitha Nagrath. Capture and release of circulating tumor cells by temperature-sensitive graphene oxide-polymer composite. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 376. doi:10.1158/1538-7445.AM2015-376

A robust pipeline for rapid production of versatile nanobody repertoires.

Nanobodies are single domain antibodies derived from the variable regions of Camelidae atypical immunoglobulins. They show great promise as high affinity reagents for research, diagnostics and therapeutics due to their high specificity, small size (~15 kDa) and straightforward bacterial expression. However, identification of repertoires with sufficiently high affinity has proven time consuming and difficult, hampering nanobody implementation. Here, we present a rapid, straightforward approach that generates large repertoires of readily expressible recombinant nanobodies with high affinities and specificities against a given antigen. We demonstrate the efficacy of this approach through the production of large repertoires of nanobodies against two antigens, GFP and mCherry, with Kd values into the sub-nanomolar range. After mapping diverse epitopes on GFP, we were also able to design ultra-high affinity dimeric nanobodies with Kds down to ~30 pM. The approach presented is well-suited for the routine production of high affinity capture reagents for various biomedical applications.

Preparation and characterization of fluorophenylboronic acid-functionalized monolithic columns for high affinity capture of cis-diol containing compounds

Boronic acids are important ligands for the selective recognition and capture of cis-diol containing compounds, such as nucleosides and glycoproteins. In a recent study, it was found that 2,4-difluoro-3-formyl-phenylboronic acid (DFFPBA) exhibited an ultrahigh boronate affinity for binding with monosaccharides. Herein three DFFPBA-functionalized monolithic columns with varying spacer arms were synthesized and characterized. Different cis-diol containing compounds were used for the evaluation of the boronate affinity of the DFFPBA-functionalized monoliths. The DFFPBA-functionalized monoliths exhibited advantageous characteristics. These monoliths exhibited an ultrahigh boronate affinity toward cis-diol containing compounds. Moreover, the monolith with appropriate spacer arm exhibited a low binding pH (6.0) for cis-diols of small molecular weight. These advantages made DFFPBA-functionalized monoliths suitable for the enrichment of trace cis-diol containing compounds in neutral and weak acidic real samples. In addition, it was interesting that the length of spacer arms strongly influenced the boronate affinity: increasing the spacer arm resulted in apparently reduced boronate affinity, and inappropriate spacer arm length even eliminated the boronate affinity toward glycoprotein. To explain such a phenomenon, a possible mechanism was proposed. Finally, the potential of DFFPBA-functionalized monoliths for real applications was demonstrated with the selective enrichment of modified nucleosides from human urine.

High affinity capture and concentration of quinacrine in polymorphonuclear neutrophils via vacuolar ATPase-mediated ion trapping: Comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs

Many cationic drugs are concentrated in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping), with an ensuing vacuolar and autophagic cytopathology. In solid tissues, there is evidence that phagocytic cells, e.g., histiocytes, preferentially concentrate cationic drugs. We hypothesized that peripheral blood leukocytes could differentially take up a fluorescent model cation, quinacrine, depending on their phagocytic competence. Quinacrine transport parameters were determined in purified or total leukocyte suspensions at 37°C. Purified polymorphonuclear leukocytes (PMNLs, essentially neutrophils) exhibited a quinacrine uptake velocity inferior to that of lymphocytes, but a consistently higher affinity (apparent KM 1.1 vs. 6.3μM, respectively). However, the vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine transport or initiated its release in either cell type. PMNLs capture most of the quinacrine added at low concentrations to fresh peripheral blood leukocytes compared with lymphocytes and monocytes (cytofluorometry). Accumulation of the autophagy marker LC3-II occurred rapidly and at low drug concentrations in quinacrine-treated PMNLs (significant at ≥2.5μM, ≥2h). Lymphocytes contained more LAMP1 than PMNLs, suggesting that the mass of lysosomes and late endosomes is a determinant of quinacrine uptake Vmax. PMNLs, however, exhibited the highest capacity for pinocytosis (uptake of fluorescent dextran into endosomes). The selectivity of quinacrine distribution in peripheral blood leukocytes may be determined by the collaboration of a non-concentrating plasma membrane transport mechanism, tentatively identified as pinocytosis in PMNLs, with V-ATPase-mediated concentration. Intracellular reservoirs of cationic drugs are a potential source of toxicity (e.g., loss of lysosomal function in phagocytes).

Simplified detection of food-borne pathogens: An in situ high affinity capture and staining concept

Food industries need simple, rapid and cost-effective solutions for pathogen detection in food and environmental samples. In this paper, we describe a simple but novel detection concept combining an affinity capture surface and intracellular metabolic marker to visualize the bacterial presence on the affinity surface. The surface of a Solid Phase Support (SPS) is functionalized with specific phage tail proteins targeted to the bacterial pathogen of interest. The SPS is placed directly into the primary food enrichment bag after stomaching. Following incubation, the captured bacteria are visually detected in situ as a result of the bacterial reduction of the colorless soluble substrate triphenyltetrazolium chloride (TTC) (present in the primary culture medium) to an intracellular red insoluble formazan product. Detection on the SPS is observed as an intense red color after 22 to 40hours of enrichment. This is not impaired by the presence of food particles and the natural background microflora. The in situ method significantly simplifies pathogen detection by eliminating any post-enrichment intervention that is necessary in the traditional methods of analysis. We have demonstrated the application of this new approach for the detection of Escherichia coli O157: H7, Listeria spp. and Salmonella spp. in artificially contaminated food samples.