Saturation Fluorescence Labeling of Proteins for Proteomic Analyses

PubMed Central

Pretzer, Elizabeth; Wiktorowicz, John E.

2008-01-01

We present here an optimized and cost-effective approach to saturation fluorescence labeling of protein thiols for proteomic analysis. We investigated a number of conditions and reagent concentrations including a disulfide reducing agent (TCEP), pH, incubation time, linearity of labeling, and saturating dye: protein thiol ratio with protein standards to gauge specific and non-specific labeling. Efficacy of labeling under these conditions was quantified using specific fluorescence estimation, defined as the ratio of fluorescence pixel intensities and Coomassie-stained pixel intensities of bands after digital imaging. Factors leading to specific vs. non-specific labeling in the presence of thiourea are also discussed. We have found that reproducible saturation of available Cys residues of the proteins used as labeling standards (human carbonic anhydrase I, enolase, α-lactalbumin) is achieved at 50-100-fold excess of the uncharged maleimide-functionalized BODIPY™ dyes over Cys. We confirm our previous findings and those of others that the maleimide dyes are not impacted by the presence of 2M thiourea. Moreover, we establish that 2 mM TCEP used as reductant is optimal. We also establish further that labeling is optimal at pH 7.5 and complete after 30 min. Low non-specific labeling was gauged by the inclusion of non-Cys containing proteins (horse myoglobin, bovine carbonic anhydrase) to the labeling mixture. We also show that the dye exhibits little to no effect on the two dimensional mobilities of labeled proteins derived from cells. PMID:18191033

Cryo-Imaging and Software Platform for Analysis of Molecular MR Imaging of Micrometastases

PubMed Central

Qutaish, Mohammed Q.; Zhou, Zhuxian; Prabhu, David; Liu, Yiqiao; Busso, Mallory R.; Izadnegahdar, Donna; Gargesha, Madhusudhana; Lu, Hong; Lu, Zheng-Rong

2018-01-01

We created and evaluated a preclinical, multimodality imaging, and software platform to assess molecular imaging of small metastases. This included experimental methods (e.g., GFP-labeled tumor and high resolution multispectral cryo-imaging), nonrigid image registration, and interactive visualization of imaging agent targeting. We describe technological details earlier applied to GFP-labeled metastatic tumor targeting by molecular MR (CREKA-Gd) and red fluorescent (CREKA-Cy5) imaging agents. Optimized nonrigid cryo-MRI registration enabled nonambiguous association of MR signals to GFP tumors. Interactive visualization of out-of-RAM volumetric image data allowed one to zoom to a GFP-labeled micrometastasis, determine its anatomical location from color cryo-images, and establish the presence/absence of targeted CREKA-Gd and CREKA-Cy5. In a mouse with >160 GFP-labeled tumors, we determined that in the MR images every tumor in the lung >0.3 mm2 had visible signal and that some metastases as small as 0.1 mm2 were also visible. More tumors were visible in CREKA-Cy5 than in CREKA-Gd MRI. Tape transfer method and nonrigid registration allowed accurate (<11 μm error) registration of whole mouse histology to corresponding cryo-images. Histology showed inflammation and necrotic regions not labeled by imaging agents. This mouse-to-cells multiscale and multimodality platform should uniquely enable more informative and accurate studies of metastatic cancer imaging and therapy. PMID:29805438

Octasaccharide is the minimal length unit required for efficient binding of cyclophilin B to heparin and cell surface heparan sulphate.

PubMed

Vanpouille, Christophe; Denys, Agnès; Carpentier, Mathieu; Pakula, Rachel; Mazurier, Joël; Allain, Fabrice

2004-09-01

Cyclophilin B (CyPB) is a heparin-binding protein first identified as a receptor for cyclosporin A. In previous studies, we reported that CyPB triggers chemotaxis and integrin-mediated adhesion of T-lymphocytes by way of interaction with two types of binding sites. The first site corresponds to a signalling receptor; the second site has been identified as heparan sulphate (HS) and appears crucial to induce cell adhesion. Characterization of the HS-binding unit is critical to understand the requirement of HS in pro-adhesive activity of CyPB. By using a strategy based on gel mobility shift assays with fluorophore-labelled oligosaccharides, we demonstrated that the minimal heparin unit required for efficient binding of CyPB is an octasaccharide. The mutants CyPB(KKK-) [where KKK- refers to the substitutions K3A(Lys3-->Ala)/K4A/K5A] and CyPB(DeltaYFD) (where Tyr14-Phe-Asp16 has been deleted) failed to interact with octasaccharides, confirming that the Y14FD16 and K3KK5 clusters are required for CyPB binding. Molecular modelling revealed that both clusters are spatially arranged so that they may act synergistically to form a binding site for the octasaccharide. We then demonstrated that heparin-derived octasaccharides and higher degree of polymerization oligosaccharides inhibited the interaction between CyPB and fluorophore-labelled HS chains purified from T-lymphocytes, and strongly reduced the HS-dependent pro-adhesive activity of CyPB. However, oligosaccharides or heparin were unable to restore adhesion of heparinase-treated T-lymphocytes, indicating that HS has to be present on the cell membrane to support the pro-adhesive activity of CyPB. Altogether, these results demonstrate that the octasaccharide is likely to be the minimal length unit required for efficient binding of CyPB to cell surface HS and consequent HS-dependent cell responses.

Octasaccharide is the minimal length unit required for efficient binding of cyclophilin B to heparin and cell surface heparan sulphate

PubMed Central

2004-01-01

Cyclophilin B (CyPB) is a heparin-binding protein first identified as a receptor for cyclosporin A. In previous studies, we reported that CyPB triggers chemotaxis and integrin-mediated adhesion of T-lymphocytes by way of interaction with two types of binding sites. The first site corresponds to a signalling receptor; the second site has been identified as heparan sulphate (HS) and appears crucial to induce cell adhesion. Characterization of the HS-binding unit is critical to understand the requirement of HS in pro-adhesive activity of CyPB. By using a strategy based on gel mobility shift assays with fluorophore-labelled oligosaccharides, we demonstrated that the minimal heparin unit required for efficient binding of CyPB is an octasaccharide. The mutants CyPBKKK− [where KKK− refers to the substitutions K3A(Lys3→Ala)/K4A/K5A] and CyPBΔYFD (where Tyr14-Phe-Asp16 has been deleted) failed to interact with octasaccharides, confirming that the Y14FD16 and K3KK5 clusters are required for CyPB binding. Molecular modelling revealed that both clusters are spatially arranged so that they may act synergistically to form a binding site for the octasaccharide. We then demonstrated that heparin-derived octasaccharides and higher degree of polymerization oligosaccharides inhibited the interaction between CyPB and fluorophore-labelled HS chains purified from T-lymphocytes, and strongly reduced the HS-dependent pro-adhesive activity of CyPB. However, oligosaccharides or heparin were unable to restore adhesion of heparinase-treated T-lymphocytes, indicating that HS has to be present on the cell membrane to support the pro-adhesive activity of CyPB. Altogether, these results demonstrate that the octasaccharide is likely to be the minimal length unit required for efficient binding of CyPB to cell surface HS and consequent HS-dependent cell responses. PMID:15109301

Proteomic detection of oxidized and reduced thiol proteins in cultured cells.

PubMed

Cuddihy, Sarah L; Baty, James W; Brown, Kristin K; Winterbourn, Christine C; Hampton, Mark B

2009-01-01

The oxidation and reduction of cysteine residues is emerging as an important post-translational control of protein function. We describe a method for fluorescent labelling of either reduced or oxidized thiols in combination with two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (2DE) to detect changes in the redox proteome of cultured cells. Reduced thiols are labelled with the fluorescent compound 5-iodoacetamidofluorescein. To monitor oxidized thiols, the reduced thiols are first blocked with N-ethyl-maleimide, then the oxidized thiols reduced with dithiothreitol and labelled with 5-iodoacetamidofluorescein. The method is illustrated by treating Jurkat T-lymphoma cells with hydrogen peroxide and monitoring increased labelling of oxidized thiol proteins. A decrease in labelling can also be detected, and this is attributed to the formation of higher oxidation states of cysteine that are not reduced by dithiothreitol.

Estrogen Receptor Driven Inhibitor Synthesis

DTIC Science & Technology

2006-09-01

labeling thiols in cellular pro- tein extracts [6,7]. But because these probes are based on nonspecific electrophiles , they are inherently less...selective electrophiles such as maleimide, alkyl halides, and iodoacetamide [5,25,26]. Conclusions DSSA probes composed of fluorescein tethered to rho...Haugland, F. Mao, Fluorinated xanthene derivatives, US patent. 6 (162), (2000) 931. [21] T. Nguyen, M.B. Francis, Practical route to functionalized rhoda

Miniaturization of a homogeneous fluorescence immunoassay based on energy transfer using nanotiter plates as high-density sample carriers.

PubMed

Schobel, U; Coille, I; Brecht, A; Steinwand, G M; Gauglitz, G

2001-11-01

The miniaturization of a homogeneous competitive immunoassay to a final assay volume of 70 nL is described. As the sample carrier, disposable plastic nanotiter plates (NTP) with dimensions of 2 x 2 cm2 containing 25 x 25 wells, corresponding to approximately 15,000 wells on a traditional 96-well microtiter plate footprint, were used. Sample handling was accomplished by a piezoelectrically actuated micropipet. To reduce evaporation while pipetting the assays, the NTP was handled in a closed humid chamber and cooled to the point of condensation. To avoid washing steps, a homogeneous assay was developed that was based on energy-transfer (ET). As a model system, an antibody-based assay for the detection of the environmentally relevant compound, simazine, in drinking water was chosen. Antibodies were labeled with the long-wavelength-excitable sulfoindocyanine dye Cy5 (donor), and a tracer was synthesized by labeling BSA with a triazine derivative and the acceptor dye Cy5.5. At low analyte concentrations, the tracer was preferably bound to the antibody binding sites. As a result of the close proximity of Cy5.5 and Cy5, an efficient quenching of the Cy5 fluorescence occurred. Higher analyte concentrations led to a progressive binding of the analyte to the antibody binding sites. The increased Cy5 fluorescence was determined by using a scanning laser-induced fluorescence detector. The limit of detection (LOD), using an antibody concentration of 20 nM, was 0.32 microg/L, or 1.11 x 10(-16) mol of simazine. In comparison, the LOD of the 96-well microtiter-plate-based ET immunoassay (micro-ETIA) was 0.15 microg/L, or 1.87 x 10(-13) mol. The LOD of the optimized micro-ETIA at 1 nM IgG, was 0.01 microg/L.

Preliminary study on the inhibition of nuclear internalization of Tat peptides by conjugation with a receptor-specific peptide and fluorescent dyes

NASA Astrophysics Data System (ADS)

Shen, Duanwen; Liang, Kexiang; Ye, Yunpeng; Tetteh, Elizabeth; Achilefu, Samuel

2006-02-01

Numerous studies have shown that basic Tat peptide (48-57) internalized non-specifically in cells and localized in the nucleus. However, localization of imaging agents in cellular nucleus is not desirable because of the potential mutagenesis. When conjugated to the peptides that undergo receptor-mediated endocytosis, Tat peptide could target specific cells or pathologic tissue. We tested this hypothesis by incorporating a somatostatin receptor-avid peptide (octreotate, Oct) and two different fluorescent dyes, Cypate 2 (Cy2) and fluorescein 5'-carboxlic acid (5-FAM), into the Tat-peptide sequence. In addition to the Cy2 or 5-FAM-labeled Oct conjugated to Tat peptide (Tat) to produce Tat-Oct-Cypate2 or Tat-Oct-5-FAM, we also labeled the Tat the Tat peptide with these dyes (Tat-Cy2 and Tat-5-FAM) to serve as positive control. A somatostatin receptor-positive pancreatic tumor cell line, AR42J, was used to assess cell internalization. The results show that Tat-5-FAM and Tat-Cypate2 localized in both nucleus and cytoplasm of the cells. In contrast to Tat-Oct-Cypate2, which localized in both the cytoplasm and nucleus, Tat-Oct-5-FAM internalized in the cytoplasm but not in the nucleus of AR42J cells. The internalizations were inhibited by adding non-labeled corresponding peptides, suggesting that the endocytoses of each group of labeled and the corresponding unlabeled compounds occurred through a common pathway. Thus, fluorescent probes and endocytosis complex between octreotate and somatostatin receptors in cytoplasm could control nuclear internalization of Tat peptides.

Proximity hybridization-regulated chemiluminescence resonance energy transfer for homogeneous immunoassay.

PubMed

Liu, Mengmeng; Wu, Jie; Yang, Kaili; Zong, Chen; Lei, Jianping; Ju, Huangxian

2016-07-01

Chemiluminescence resonance energy transfer (CRET) and the proximity ligation assay have been widely used in design of sensors for the bioanalysis. Here, a wash-free and homogeneous strategy was proposed to detect carcino-embryonic antigen (CEA) based on proximity hybridization-regulated CRET. The Cy5 demonstrated strong chemiluminescence (CL) via the oxidation of TCPO in the presence of H2O2 and energy transfer between excited TCPO and Cy5. Graphene oxide (GO) as an excellent quencher was used to produce the "Signal off" mode that little CL emission was observed through CRET between GO and the Cy5-labelled DNA3. Once CEA was introduced, the target-induced proximity hybridization occurred to form a proximate complex, which inhibited the CRET by preventing GO from absorbing Cy5-labelled DNA3. Furthermore, taking advantage of nicking endonuclease Nt.BbvCI for in situ recycling, the signal could be further amplified for highly sensitive CL detection. Our results showed that this strategy enabled a specific response to CEA with a detection range of 5 orders of magnitude, along with a detection limit of 3.2pg mL(-1). Apart from its easy operation, high sensitivity and acceptable accuracy, the proposed method needed only 0.3μL of sample, indicating its great opportunity for commercial application. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a general methodology for labelling peptide-morpholino oligonucleotide conjugates using alkyne-azide click chemistry.

PubMed

Shabanpoor, Fazel; Gait, Michael J

2013-11-11

We describe a general methodology for fluorescent labelling of peptide conjugates of phosphorodiamidate morpholino oligonucleotides (PMOs) by alkyne functionalization of peptides, subsequent conjugation to PMOs and labelling with a fluorescent compound (Cy5-azide). Two peptide-PMO (PPMO) examples are shown. No detrimental effect of such labelled PMOs was seen in a biological assay.

Bioimaging of Nucleolin Aptamer-Containing 5-(N-benzylcarboxyamide)-2′-deoxyuridine More Capable of Specific Binding to Targets in Cancer Cells

PubMed Central

Lee, Kyue Yim; Kang, Hyungu; Ryu, Sung Ho; Lee, Dong Soo; Lee, Jung Hwan; Kim, Soonhag

2010-01-01

Chemically modified nucleotides have been developed and applied into SELEX procedure to find a novel type of aptamers to fit with targets of interest. In this study, we directly performed chemical modification of 5-(N-benzylcarboxyamide)-2′-deoxyuridine (called 5-BzdU) in the AS1411 aptamer, which binds to the nucleolin protein expressed in cancer cells. Forty-seven compounds of AS1411-containing Cy3-labeled 5-BzdU (called Cy3-(5-BzdU)-modified-AS1411) were synthesized by randomly substituting thymidines one to twelve in AS1411 with Cy3-labeled 5-BzdU. Both statistically quantified fluorescence measurements and confocal imaging analysis demonstrated at least three potential compounds of interest: number 12, 29 and 41 that significantly increased the targeting affinity to cancer cells but no significant activity from normal healthy cells. These results suggest that the position and number of substituents in AS1411 are critical parameters to improve the aptamer function. In this study, we demonstrated that chemical modification of the existing aptamers enhanced the binding and targeting affinity to targets of interest without additional SELEX procedures. PMID:20204158

Agonist-induced internalization of the substance P (NK1) receptor expressed in epithelial cells.

PubMed

Garland, A M; Grady, E F; Payan, D G; Vigna, S R; Bunnett, N W

1994-10-01

Internalization of the NK1 receptor (NK1R) and substance P was observed in cells transfected with cDNA encoding the rat NK1R by using anti-receptor antibodies and cyanine 3-labelled substance P (cy3-substance P). After incubation at 4 degrees C, NK1R immunoreactivity and cy3-substance P were confined to the plasma membrane. Within 3 min of incubation at 37 degrees C, NK1R immunoreactivity and cy3-substance P were internalized into small intracellular vesicles located beneath the plasma membrane. Fluorescein isothiocyanate-labelled transferrin and cy3-substance P were internalized into the same vesicles, identifying them as early endosomes. After 60 min at 37 degrees C, NK1R immunoreactivity was detected in larger, perinuclear vesicles. Internalization of 125I-labelled substance P was studied by using an acid wash to dissociate cell-surface label from that which has been internalized. Binding reached equilibrium after incubation for 60 min at 4 degrees C with no detectable internalization. After 10 min incubation at 37 degrees C, 83.5 +/- 1.0% of specifically bound counts were internalized. Hyperosmolar sucrose and phenylarsine oxide, which are inhibitors of endocytosis, prevented internalization of 125I-labelled substance P and accumulation of NK1R immunoreactivity into endosomes. Acidotropic agents caused retention of 125I-labelled substance P within the cell and inhibited degradation of the internalized peptide. Continuous incubation of cells with substance P at 37 degrees C reduced 125I-substance P binding at the cell surface. Therefore, substance P and its receptor are internalized into early endosomes within minutes of binding, and internalized substance P is degraded. Internalization depletes NK1Rs from the cell surface and may down-regulate the response of a cell to substance P.

In vivo imaging of the inflammatory receptor CD40 after cerebral ischemia using a fluorescent antibody.

PubMed

Klohs, Jan; Gräfe, Michael; Graf, Kristof; Steinbrink, Jens; Dietrich, Thore; Stibenz, Dietger; Bahmani, Peyman; Kronenberg, Golo; Harms, Christoph; Endres, Matthias; Lindauer, Ute; Greger, Klaus; Stelzer, Ernst H K; Dirnagl, Ulrich; Wunder, Andreas

2008-10-01

Brain inflammation is a hallmark of stroke, where it has been implicated in tissue damage as well as in repair. Imaging technologies that specifically visualize these processes are highly desirable. In this study, we explored whether the inflammatory receptor CD40 can be noninvasively and specifically visualized in mice after cerebral ischemia using a fluorescent monoclonal antibody, which we labeled with the near-infrared fluorescence dye Cy5.5 (Cy5.5-CD40MAb). Wild-type and CD40-deficient mice were subjected to transient middle cerebral artery occlusion. Mice were either intravenously injected with Cy5.5-CD40MAb or control Cy5.5-IgGMAb. Noninvasive and ex vivo near-infrared fluorescence imaging was performed after injection of the compounds. Probe distribution and specificity was further assessed with single-plane illumination microscopy, immunohistochemistry, and confocal microscopy. Significantly higher fluorescence intensities over the stroke-affected hemisphere, compared to the contralateral side, were only detected noninvasively in wild-type mice that received Cy5.5-CD40MAb, but not in CD40-deficient mice injected with Cy5.5-CD40MAb or in wild-type mice that were injected with Cy5.5-IgGMAb. Ex vivo near-infrared fluorescence showed an intense fluorescence within the ischemic territory only in wild-type mice injected with Cy5.5-CD40MAb. In the brains of these mice, single-plane illumination microscopy demonstrated vascular and parenchymal distribution, and confocal microscopy revealed a partial colocalization of parenchymal fluorescence from the injected Cy5.5-CD40MAb with activated microglia and blood-derived cells in the ischemic region. The study demonstrates that a CD40-targeted fluorescent antibody enables specific noninvasive detection of the inflammatory receptor CD40 after cerebral ischemia using optical techniques.

Tumor Targeting and Pharmacokinetics of a Near-Infrared Fluorescent-Labeled δ-Opioid Receptor Antagonist Agent, Dmt-Tic-Cy5.

PubMed

Huynh, Amanda Shanks; Estrella, Veronica; Stark, Valerie E; Cohen, Allison S; Chen, Tingan; Casagni, Todd J; Josan, Jatinder S; Lloyd, Mark C; Johnson, Joseph; Kim, Jongphil; Hruby, Victor J; Vagner, Josef; Morse, David L

2016-02-01

Fluorescence molecular imaging can be employed for the development of novel cancer targeting agents. Herein, we investigated the pharmacokinetics (PK) and cellular uptake of Dmt-Tic-Cy5, a delta-opioid receptor (δOR) antagonist-fluorescent dye conjugate, as a tumor-targeting molecular imaging agent. δOR expression is observed normally in the CNS, and pathologically in some tumors, including lung liver and breast cancers. In vitro, in vivo, and ex vivo experiments were conducted to image and quantify the fluorescence signal associated with Dmt-Tic-Cy5 over time using in vitro and intravital fluorescence microscopy and small animal fluorescence imaging of tumor-bearing mice. We observed specific retention of Dmt-Tic-Cy5 in tumors with maximum uptake in δOR-expressing positive tumors at 3 h and observable persistence for >96 h; clearance from δOR nonexpressing negative tumors by 6 h; and systemic clearance from normal organs by 24 h. Live-cell and intravital fluorescence microscopy demonstrated that Dmt-Tic-Cy5 had sustained cell-surface binding lasting at least 24 h with gradual internalization over the initial 6 h following administration. Dmt-Tic-Cy5 is a δOR-targeted agent that exhibits long-lasting and specific signal in δOR-expressing tumors, is rapidly cleared from systemic circulation, and is not retained in non-δOR-expressing tissues. Hence, Dmt-Tic-Cy5 has potential as a fluorescent tumor imaging agent.

Hybrid Imaging Labels: Providing the Link Between Mass Spectrometry-Based Molecular Pathology and Theranostics

PubMed Central

Buckle, Tessa; van der Wal, Steffen; van Malderen, Stijn J.M.; Müller, Larissa; Kuil, Joeri; van Unen, Vincent; Peters, Ruud J.B.; van Bemmel, Margaretha E.M.; McDonnell, Liam A.; Velders, Aldrik H.; Koning, Frits; Vanhaeke, Frank; van Leeuwen, Fijs W. B.

2017-01-01

Background: Development of theranostic concepts that include inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS (LA-ICP-MS) imaging can be hindered by the lack of a direct comparison to more standardly used methods for in vitro and in vivo evaluation; e.g. fluorescence or nuclear medicine. In this study a bimodal (or rather, hybrid) tracer that contains both a fluorescent dye and a chelate was used to evaluate the existence of a direct link between mass spectrometry (MS) and in vitro and in vivo molecular imaging findings using fluorescence and radioisotopes. At the same time, the hybrid label was used to determine whether the use of a single isotope label would allow for MS-based diagnostics. Methods: A hybrid label that contained both a DTPA chelate (that was coordinated with either 165Ho or 111In) and a Cy5 fluorescent dye was coupled to the chemokine receptor 4 (CXCR4) targeting peptide Ac-TZ14011 (hybrid-Cy5-Ac-TZ4011). This receptor targeting tracer was used to 1) validate the efficacy of (165Ho-based) mass-cytometry in determining the receptor affinity via comparison with fluorescence-based flow cytometry (Cy5), 2) evaluate the microscopic binding pattern of the tracer in tumor cells using both fluorescence confocal imaging (Cy5) and LA-ICP-MS-imaging (165Ho), 3) compare in vivo biodistribution patterns obtained with ICP-MS (165Ho) and radiodetection (111In) after intravenous administration of hybrid-Cy5-Ac-TZ4011 in tumor-bearing mice. Finally, LA-ICP-MS-imaging (165Ho) was linked to fluorescence-based analysis of excised tissue samples (Cy5). Results: Analysis with both mass-cytometry and flow cytometry revealed a similar receptor affinity, respectively 352 ± 141 nM and 245 ± 65 nM (p = 0.08), but with a much lower detection sensitivity for the first modality. In vitro LA-ICP-MS imaging (165Ho) enabled clear discrimination between CXCR4 positive and negative cells, but fluorescence microscopy was required to determine the intracellular distribution. In vivo biodistribution patterns obtained with ICP-MS (165Ho) and radiodetection (111In) of the hybrid peptide were shown to be similar. Assessment of tracer distribution in excised tissues revealed the location of tracer uptake with both LA-ICP-MS-imaging and fluorescence imaging. Conclusion: Lanthanide-isotope chelation expands the scope of fluorescent/radioactive hybrid tracers to include MS-based analytical tools such as mass-cytometry, ICP-MS and LA-ICP-MS imaging in molecular pathology. In contradiction to common expectations, MS detection using a single chelate imaging agent was shown to be feasible, enabling a direct link between nuclear medicine-based imaging and theranostic methods. PMID:28255355

Incorporation of isotopic, fluorescent, and heavy-atom-modified nucleotides into RNAs by position-selective labeling of RNA.

PubMed

Liu, Yu; Holmstrom, Erik; Yu, Ping; Tan, Kemin; Zuo, Xiaobing; Nesbitt, David J; Sousa, Rui; Stagno, Jason R; Wang, Yun-Xing

2018-05-01

Site-specific incorporation of labeled nucleotides is an extremely useful synthetic tool for many structural studies (e.g., NMR, electron paramagnetic resonance (EPR), fluorescence resonance energy transfer (FRET), and X-ray crystallography) of RNA. However, specific-position-labeled RNAs >60 nt are not commercially available on a milligram scale. Position-selective labeling of RNA (PLOR) has been applied to prepare large RNAs labeled at desired positions, and all the required reagents are commercially available. Here, we present a step-by-step protocol for the solid-liquid hybrid phase method PLOR to synthesize 71-nt RNA samples with three different modification applications, containing (i) a 13 C 15 N-labeled segment; (ii) discrete residues modified with Cy3, Cy5, or biotin; or (iii) two iodo-U residues. The flexible procedure enables a wide range of downstream biophysical analyses using precisely localized functionalized nucleotides. All three RNAs were obtained in <2 d, excluding time for preparing reagents and optimizing experimental conditions. With optimization, the protocol can be applied to other RNAs with various labeling schemes, such as ligation of segmentally labeled fragments.

Fluorescent 6-amino-6-deoxyglycoconjugates for glucose transporter mediated bioimaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Xiangyin; Liu, Shengnan; Liu, Xinyu

Two novel fluorescent bioprobes, namely, 6N-Gly-Cy3 and 6N-Gly-Cy5, were designed and synthesized for real-time glucose transport imaging as well as potentially useful tracer for galactokinase metabolism. The structure of the bioprobes was fully characterized by {sup 1}H NMR, {sup 13}C NMR, IR, and HRMS. The fluorescence properties, glucose transporter (GLUT) specificity, and the quenching and safety profiles were studied. The cellular uptake of both bioprobes was competitively diminished by D-glucose, 2-deoxy-D-glucose and GLUT specific inhibitor in a dose-dependent manner in human colon cancer cells (HT29). Comparison study results revealed that the 6N-derived bioprobes are more useful for real-time imaging ofmore » cell-based glucose uptake than the structurally similar fluorescent tracer 6-NBDG which was not applicable under physiological conditions. The up to 96 h long-lasting quenching property of 6N-Gly-Cy5 in HT29 suggested the potential applcability of the probe for cell labeling in xenograft transplantation as well as in vivo animal imaging studies. - Highlights: • Cy-3 and Cy-5 derived fluorescent 6-amino-6-deoxyglycoconjugates were prepared for glucose transporter mediated bioimaging. • The cellular uptake of the probes was inhibited by natural GLUT substrates and inhibitor. • The probes are useful for real-time imaging of cell-based glucose uptake under physiological conditions. • The probes showed up to 96 h long-lasting quenching profile in labeled cancer cells.« less

Detection of Alicyclobacillus species in fruit juice using a random genomic DNA microarray chip.

PubMed

Jang, Jun Hyeong; Kim, Sun-Joong; Yoon, Bo Hyun; Ryu, Jee-Hoon; Gu, Man Bock; Chang, Hyo-Ihl

2011-06-01

This study describes a method using a DNA microarray chip to rapidly and simultaneously detect Alicyclobacillus species in orange juice based on the hybridization of genomic DNA with random probes. Three food spoilage bacteria were used in this study: Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, and Alicyclobacillus cycloheptanicus. The three Alicyclobacillus species were adjusted to 2 × 10(3) CFU/ml and inoculated into pasteurized 100% pure orange juice. Cy5-dCTP labeling was used for reference signals, and Cy3-dCTP was labeled for target genomic DNA. The molar ratio of 1:1 of Cy3-dCTP and Cy5-dCTP was used. DNA microarray chips were fabricated using randomly fragmented DNA of Alicyclobacillus spp. and were hybridized with genomic DNA extracted from Bacillus spp. Genomic DNA extracted from Alicyclobacillus spp. showed a significantly higher hybridization rate compared with DNA of Bacillus spp., thereby distinguishing Alicyclobacillus spp. from Bacillus spp. The results showed that the microarray DNA chip containing randomly fragmented genomic DNA was specific and clearly identified specific food spoilage bacteria. This microarray system is a good tool for rapid and specific detection of thermophilic spoilage bacteria, mainly Alicyclobacillus spp., and is useful and applicable to the fruit juice industry.

Optical imaging of articular cartilage degeneration using near-infrared dipicolylamine probes.

PubMed

Hu, Xiang; Wang, Qian; Liu, Yang; Liu, Hongguang; Qin, Chunxia; Cheng, Kai; Robinson, William; Gray, Brian D; Pak, Koon Y; Yu, Aixi; Cheng, Zhen

2014-08-01

Articular cartilage is the hydrated tissue that lines the ends of long bones in load bearing joints and provides joints with a smooth, nearly frictionless gliding surface. However, the deterioration of articular cartilage occurs in the early stages of osteoarthritis (OA) and is clinically and radiographically silent. Here two cationic near infrared fluorescent (NIRF) dipicolylamine (DPA) probes, Cy5-DPA-Zn and Cy7-DPA-Zn, were prepared for cartilage degeneration imaging and OA early detection through binding to the anionic glycosaminoglycans (GAGs). The feasibility of NIRF dye labeled DPA-Zn probes for cartilage degeneration imaging was examined ex vivo and in vivo. The ex vivo studies showed that Cy5-DPA-Zn and Cy7-DPA-Zn not only showed the high uptake and electrostatic attractive binding to cartilage, but also sensitively reflected the change of GAGs contents. In vivo imaging study further indicated that Cy5-DPA-Zn demonstrated higher uptake and retention in young mice (high GAGs) than old mice (low GAGs) when administrated via local injection in mouse knee joints. More importantly, Cy5-DPA-Zn showed dramatic higher signals in sham joint (high GAGs) than OA side (low GAGs), through sensitive reflecting the change of GAGs in the surgical induced OA models. In summary, Cy5-DPA-Zn provides promising visual detection for early cartilage pathological degeneration in living subjects. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cu-Au alloy nanostructures coated with aptamers: a simple, stable and highly effective platform for in vivo cancer theranostics

NASA Astrophysics Data System (ADS)

Ye, Xiaosheng; Shi, Hui; He, Xiaoxiao; Yu, Yanru; He, Dinggeng; Tang, Jinlu; Lei, Yanli; Wang, Kemin

2016-01-01

As a star material in cancer theranostics, photoresponsive gold (Au) nanostructures may still have drawbacks, such as low thermal conductivity, irradiation-induced melting effect and high cost. To solve the problem, copper (Cu) with a much higher thermal conductivity and lower cost was introduced to generate a novel Cu-Au alloy nanostructure produced by a simple, gentle and one-pot synthetic method. Having the good qualities of both Cu and Au, the irregularly-shaped Cu-Au alloy nanostructures showed several advantages over traditional Au nanorods, including a broad and intense near-infrared (NIR) absorption band from 400 to 1100 nm, an excellent heating performance under laser irradiation at different wavelengths and even a notable photostability against melting. Then, via a simple conjugation of fluorophore-labeled aptamers on the Cu-Au alloy nanostructures, active targeting and signal output were simultaneously introduced, thus constructing a theranostic platform based on fluorophore-labeled, aptamer-coated Cu-Au alloy nanostructures. By using human leukemia CCRF-CEM cancer and Cy5-labeled aptamer Sgc8c (Cy5-Sgc8c) as the model, a selective fluorescence imaging and NIR photothermal therapy was successfully realized for both in vitro cancer cells and in vivo tumor tissues. It was revealed that Cy5-Sgc8c-coated Cu-Au alloy nanostructures were not only capable of robust target recognition and stable signal output for molecular imaging in complex biological systems, but also killed target cancer cells in mice with only five minutes of 980 nm irradiation. The platform was found to be simple, stable, biocompatible and highly effective, and shows great potential as a versatile tool for cancer theranostics.As a star material in cancer theranostics, photoresponsive gold (Au) nanostructures may still have drawbacks, such as low thermal conductivity, irradiation-induced melting effect and high cost. To solve the problem, copper (Cu) with a much higher thermal conductivity and lower cost was introduced to generate a novel Cu-Au alloy nanostructure produced by a simple, gentle and one-pot synthetic method. Having the good qualities of both Cu and Au, the irregularly-shaped Cu-Au alloy nanostructures showed several advantages over traditional Au nanorods, including a broad and intense near-infrared (NIR) absorption band from 400 to 1100 nm, an excellent heating performance under laser irradiation at different wavelengths and even a notable photostability against melting. Then, via a simple conjugation of fluorophore-labeled aptamers on the Cu-Au alloy nanostructures, active targeting and signal output were simultaneously introduced, thus constructing a theranostic platform based on fluorophore-labeled, aptamer-coated Cu-Au alloy nanostructures. By using human leukemia CCRF-CEM cancer and Cy5-labeled aptamer Sgc8c (Cy5-Sgc8c) as the model, a selective fluorescence imaging and NIR photothermal therapy was successfully realized for both in vitro cancer cells and in vivo tumor tissues. It was revealed that Cy5-Sgc8c-coated Cu-Au alloy nanostructures were not only capable of robust target recognition and stable signal output for molecular imaging in complex biological systems, but also killed target cancer cells in mice with only five minutes of 980 nm irradiation. The platform was found to be simple, stable, biocompatible and highly effective, and shows great potential as a versatile tool for cancer theranostics. Electronic supplementary information (ESI) available: Fig. S1, S2 and Table S1. See DOI: 10.1039/c5nr07017a

Healable Composites

DTIC Science & Technology

2012-03-28

composites: determine mechanical and crack healing properties (4, 5) Composite (3) Prepreg (2) Polymer (1) Furan (1) Maleimide Healable Composites...5. Characterize the composites: determine mechanical and crack healing properties (4, 5) Composite (3) Prepreg (1) Furan (1) Maleimide (2...Furan (1) Maleimide (4, 5) Composite (3) Prepreg Healable Composites AFOSR FA9550-08-1-0314 Christian Nielsen Challenges • At room temperature

Hydrogen sulfide deactivates common nitrobenzofurazan-based fluorescent thiol labeling reagents.

PubMed

Montoya, Leticia A; Pluth, Michael D

2014-06-17

Sulfhydryl-containing compounds, including thiols and hydrogen sulfide (H2S), play important but differential roles in biological structure and function. One major challenge in separating the biological roles of thiols and H2S is developing tools to effectively separate the reactivity of these sulfhydryl-containing compounds. To address this challenge, we report the differential responses of common electrophilic fluorescent thiol labeling reagents, including nitrobenzofurazan-based scaffolds, maleimides, alkylating agents, and electrophilic aldehydes, toward cysteine and H2S. Although H2S reacted with all of the investigated scaffolds, the photophysical response to each scaffold was significantly different. Maleimide-based, alkylating, and aldehydic thiol labeling reagents provided a diminished fluorescence response when treated with H2S. By contrast, nitrobenzofurazan-based labeling reagents were deactivated by H2S addition. Furthermore, the addition of H2S to thiol-activated nitrobenzofurazan-based reagents reduced the fluorescence signal, thus establishing the incompatibility of nitrobenzofurazan-based thiol labeling reagents in the presence of H2S. Taken together, these studies highlight the differential reactivity of thiols and H2S toward common thiol-labeling reagents and suggest that sufficient care must be taken when labeling or measuring thiols in cellular environments that produce H2S due to the potential for both false-positive and eroded responses.

Dual-modality micro-positron emission tomography/computed tomography and near-infrared fluorescence imaging of EphB4 in orthotopic glioblastoma xenograft models.

PubMed

Huang, Miao; Xiong, Chiyi; Lu, Wei; Zhang, Rui; Zhou, Min; Huang, Qian; Weinberg, Jeffrey; Li, Chun

2014-02-01

In glioblastoma, EphB4 receptors, a member of the largest family of receptor tyrosine kinases, are overexpressed in both tumor cells and angiogenic blood vessels. The purpose of this study was to examine whether the EphB4-binding peptide TNYL-RAW labeled with both (64)Cu and near-infrared fluorescence dye Cy5.5 could be used as a molecular imaging agent for dual-modality positron emission tomography/computed tomography [PET/CT] and optical imaging of human glioblastoma in orthotopic brain tumor models. TNYL-RAW was conjugated to Cy5.5 and the radiometal chelator 1,4,7,10-tetraazadodecane-N,N',N″,N‴-tetraacetic acid. The conjugate was then labeled with (64)Cu for in vitro binding and in vivo dual μPET/CT and optical imaging studies in nude mice implanted with EphB4-expressing U251 and EphB4-negative U87 human glioblastoma cells. Tumors and brains were removed at the end of the imaging sessions for immunohistochemical staining and fluorescence microscopic examinations. μPET/CT and near-infrared optical imaging clearly showed specific uptake of the dual-labeled TNYL-RAW peptide in both U251 and U87 tumors in the brains of the nude mice after intravenous injection of the peptide. In U251 tumors, the Cy5.5-labeled peptide colocalized with both tumor blood vessels and tumor cells; in U87 tumors, the tracer colocalized only with tumor blood vessels, not with tumor cells. Dual-labeled EphB4-specific peptide could be used as a noninvasive molecular imaging agent for PET/CT and optical imaging of glioblastoma owing to its ability to bind to both EphB4-expressing angiogenic blood vessels and EphB4-expressing tumor cells.

Dual-Modality Micro-Positron Emission Tomography/Computed Tomography and Near-Infrared Fluorescence Imaging of EphB4 in Orthotopic Glioblastoma Xenograft Models

PubMed Central

Huang, Miao; Xiong, Chiyi; Lu, Wei; Zhang, Rui; Zhou, Min; Huang, Qian; Weinberg, Jeffrey; Li, Chun

2013-01-01

Purpose In glioblastoma, EphB4 receptors, a member of the largest family of receptor tyrosine kinases, are overexpressed in both tumor cells and angiogenic blood vessels. The purpose of this study was to examine whether the EphB4-binding peptide TNYL-RAW labeled with both 64Cu and near-infrared fluorescence dye Cy5.5 could be used as a molecular imaging agent for dual-modality positron emission tomography/computed tomography [PET/CT] and optical imaging of human glioblastoma in orthotopic brain tumor models. Materials and Methods TNYL-RAW was conjugated to Cy5.5 and the radiometal chelator 1,4,7,10-tetraazadodecane-N,N′,N″,N‴ -tetraacetic acid. The conjugate was then labeled with 64Cu for in vitro binding and in vivo dual μPET/CT and optical imaging studies in nude mice implanted with EphB4-expressing U251 and EphB4-negative U87 human glioblastoma cells. Tumors and brains were removed at the end of the imaging sessions for immunohistochemical staining and fluorescence microscopic examinations. Results μPET/CT and near-infrared optical imaging clearly showed specific uptake of the dual-labeled TNYL-RAW peptide in both U251 and U87 tumors in the brains of the nude mice after intravenous injection of the peptide. In U251 tumors, the Cy5.5-labeled peptide colocalized with both tumor blood vessels and tumor cells; in U87 tumors, the tracer colocalized only with tumor blood vessels, not with tumor cells. Conclusions Dual-labeled EphB4-specific peptide could be used as a noninvasive molecular imaging agent for PET/CT and optical imaging of glioblastoma owing to its ability to bind to both EphB4-expressing angiogenic blood vessels and EphB4-expressing tumor cells. PMID:23918654

Oligonucleotide assisted light-emitting Alq3 microrods: energy transfer effect with fluorescent dyes.

PubMed

Cui, Chunzhi; Park, Dong Hyuk; Kim, Jeongyong; Joo, Jinsoo; Ahn, Dong June

2013-06-14

Oligonucleotide assisted tri(8-hydroxyquinoline) aluminium (Alq3) microrods were prepared for the first time. When hybridized with oligonucleotide labeled by Cy3 fluorescent dye, a significant photoluminescence variation of the Alq3 microrods was observed due to Förster resonance energy transfer, unlike when Cy5-oligonucleotide was used. Versatile nucleotide manipulation would open up wider applications of Alq3-based materials, based on this fundamental observation.

A Receptor-targeted Fluorescent Radiopharmaceutical for Multireporter Sentinel Lymph Node Imaging

PubMed Central

Emerson, Derek K.; Limmer, Karl K.; Hall, David J.; Han, Sung-Ho; Eckelman, William C.; Kane, Christopher J.; Wallace, Anne M.

2012-01-01

Purpose: To determine the imaging and receptor-binding properties of a multireporter probe designed for sentinel lymph node (SLN) mapping via nuclear and fluorescence detection. Materials and Methods: The animal experiments were approved by the institutional animal care and use committee. A multireporter probe was synthesized by covalently attaching cyanine 7 (Cy7), a near-infrared cyanine dye, to tilmanocept, a radiopharmaceutical that binds to a receptor specific to recticuloendothelial cells. In vitro binding assays of technetium 99m (99mTc) -labeled Cy7 tilmanocept were conducted at 4°C by using receptor-bearing macrophages. Optical SLN imaging after foot pad administration was performed by using two molar doses of Cy7 tilmanocept. Six mice were injected with 0.11 nmol of 99mTc-labeled Cy7 tilmanocept (low-dose group); an additional six mice were injected with 31 nmol of 99mTc-labeled Cy7 tilmanocept (high-dose group) to saturate the receptor sites within the SLN. After 2.5 hours of imaging, the mice were euthanized, and the sentinel and distal lymph nodes were excised and assayed for radioactivity for calculation of SLN percentage of injected dose and extraction. Four mice were used as controls for autofluorescence. Standard optical imaging software was used to plot integrated fluorescence intensity against time for calculation of the SLN uptake rate constant and scaled peak intensity. Significance was calculated by using the Student t test. Results: In vitro binding assays showed subnanomolar affinity (mean dissociation constant, 0.25 nmol/L ± 0.10 [standard deviation]). Fluorescence imaging showed a detection sensitivity of 1.6 × 103 counts · sec−1 · μW−1 per picomole of Cy7. All four imaging metrics (percentage of injected dose, SLN extraction, SLN uptake rate constant, and expected peak fluorescence intensity) exhibited higher values (P = .005 to P = .042) in the low-dose group than in the high-dose group; this finding was consistent with receptor-mediated image formation. Conclusion: The multireporter probe 99mTc-labeled Cy7 tilmanocept exhibits in vitro and in vivo receptor-binding properties for successful receptor-targeted SLN mapping with nuclear and optical imaging. © RSNA, 2012 Supplemental material: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12120638/-/DC1 PMID:22753678

Synthesis, characterization, and application of cy-dye- and alexa-dye-labeled hongotoxin(1) analogues. The first high affinity fluorescence probes for voltage-gated K+ channels.

PubMed

Pragl, Bernt; Koschak, Alexandra; Trieb, Maria; Obermair, Gerald; Kaufmann, Walter A; Gerster, Uli; Blanc, Eric; Hahn, Christoph; Prinz, Heino; Schütz, Gerhard; Darbon, Herve; Gruber, Hermann J; Knaus, Hans-Günther

2002-01-01

Hongotoxin(1) (HgTX(1)), a 39-residue peptide recently isolated from the venom of Centruroides limbatus, blocks the voltage-gated K+ channels K(v)1.1, K(v)1.2, and K(v)1.3 at picomolar toxin concentrations (Koschak, A., Bugianesi, R. M., Mitterdorfer, J., Kaczorowski, G. J., Garcia, M. L., and Knaus, H. G. (1998) J. Biol. Chem. 273, 2639-2644). In this report, we determine the three-dimensional structure of HgTX(1) using NMR spectroscopy (PDB-code: 1HLY). HgTX(1) was found to possess a structure similar to previously characterized K+ channel toxins (e.g. margatoxin) consisting of a three-stranded antiparallel beta-sheet (residues 2-4, 26-30, and 33-37) and a helical conformation (part 3(10) helix and part alpha helix; residues 10-20). Due to the importance of residue Lys-28 for high-affinity interaction with the respective channels, lysine-reactive fluorescence dyes cannot be used to label wild-type HgTX(1). On the basis of previous studies (see above) and our NMR data, a HgTX(1) mutant (HgTX(1)-A19C) was engineered, expressed, and purified. HgTX(1)-A19C-SH was labeled using sulfhydryl-reactive Cy3-, Cy5-, and Alexa-dyes. Pharmacological characterization of fluorescently labeled HgTX(1)-A19C in radioligand binding studies indicated that these hongotoxin(1) analogues retain high-affinity for voltage-gated K+ channels and a respective pharmacological profile. Cy3- and Alexa-dye-labeled hongotoxin(1) analogues were used to investigate the localization of K+ channels in brain sections. The distribution of toxin binding closely follows the distribution of K(v)1.2 immunoreactivity with the highest expression levels in the cerebellar Purkinje cell layer. Taken together, these results demonstrate that fluorescently labeled HgTX(1) analogues comprise novel probes to characterize a subset of voltage-gated K+ channels.

Enhancement of Single Molecule Fluorescence Signals by Colloidal Silver Nanoparticles in Studies of Protein Translation

PubMed Central

Bharill, Shashank; Chen, Chunlai; Stevens, Benjamin; Kaur, Jaskiran; Smilansky, Zeev; Mandecki, Wlodek; Gryczynski, Ignacy; Gryczynski, Zygmunt; Cooperman, Barry S.; Goldman, Yale E.

2011-01-01

Metal enhanced fluorescence (MEF) increased total photon emission of Cy3- and Cy5-labeled ribosomal initiation complexes near 50 nm silver particles 4- and 5.5-fold respectively. Fluorescence intensity fluctuations above shot noise, at 0.1 – 5 Hz, were greater on silver particles. Overall signal to noise ratio was similar or slightly improved near the particles. Proximity to silver particles did not compromise ribosome function, as measured by codon-dependent binding of fluorescent tRNA, dynamics of fluorescence resonance energy transfer between adjacent tRNAs in the ribosome, and tRNA translocation induced by elongation factor G. PMID:21158483

Enhancement of single-molecule fluorescence signals by colloidal silver nanoparticles in studies of protein translation.

PubMed

Bharill, Shashank; Chen, Chunlai; Stevens, Benjamin; Kaur, Jaskiran; Smilansky, Zeev; Mandecki, Wlodek; Gryczynski, Ignacy; Gryczynski, Zygmunt; Cooperman, Barry S; Goldman, Yale E

2011-01-25

Metal-enhanced fluorescence (MEF) increased total photon emission of Cy3- and Cy5-labeled ribosomal initiation complexes near 50 nm silver particles 4- and 5.5-fold, respectively. Fluorescence intensity fluctuations above shot noise, at 0.1-5 Hz, were greater on silver particles. Overall signal-to-noise ratio was similar or slightly improved near the particles. Proximity to silver particles did not compromise ribosome function, as measured by codon-dependent binding of fluorescent tRNA, dynamics of fluorescence resonance energy transfer between adjacent tRNAs in the ribosome, and tRNA translocation induced by elongation factor G.

Sensitive Detection of Polynucleotide Kinase Activity by Paper-Based Fluorescence Assay with λ Exonuclease Assistance.

PubMed

Zhang, Hua; Zhao, Zhen; Lei, Zhen; Wang, Zhenxin

2016-12-06

The phosphorylation of nucleic acid with 5'-OH termini catalyzed by polynucleotide kinase (PNK) involves several significant cellular events. Here a paper-based fluorescence assay with λ exonuclease assistance was reported for facile detection of PNK activity through monitoring the change of fluorescence intensity on paper surface. Cy5-labeled ssDNA was first immobilized on the surface of aldehyde group modified paper, and BHQ-labeled ssDNA was then employed to quench the fluorescence of the immobilized Cy5-labeled ssDNA with the help of an adaptor ssDNA. When PNK and λ exonuclease cleavage reaction were introduced, the fluorescence quenching effect on the paper surface was blocked because of the digestion of phosphorylated dsDNA by the coupled enzymes. By using this paper-based assay, PNK activity both in pure reaction buffer and in practical biosample have been successfully measured. Highly sensitive detection of PNK activity down to 0.0001 U mL -1 and lysate of about 50 cells is achieved. The inhibition of PNK activity has also been investigated and a satisfactory result is obtained.

Simultaneous LC/MS/MS determination of thiols and disulfides in urine samples based on differential labeling with ferrocene-based maleimides.

PubMed

Seiwert, Bettina; Karst, Uwe

2007-09-15

A method for the simultaneous determination of a series of thiols and disulfides in urine samples has been developed based on the sequential labeling of free and bound thiol functionalities with two ferrocene-based maleimide reagents. The sample is first exposed to N-(2-ferroceneethyl)maleimide, thus leading to the derivatization of free thiol groups in the sample. After quantitative reaction and subsequent reduction of the disulfide-bound thiols by tris(2-carboxyethyl)phosphine, the newly formed thiol functionalities are reacted with ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide. The reaction products are determined by LC/MS/MS in the multiple reaction mode, and precursor ion scan as well as neutral loss scan is applied to detect unknown further thiols. The method was successfully applied to the analysis of free and disulfide-bound thiols in urine samples. Limits of detection are 30 to 110 nM, and the linear range comprises two decades of concentration, thus covering the relevant concentration range of thiols in urine samples. The thiol and disulfide concentrations were referred to the creatinine content to compensate for different sample volumes. As some calibration standards for the disulfides are not commercially available, they were synthesized in an electrochemical flow-through cell. This allowed the synthesis of hetero- and homodimeric disulfides.

In vivo NIRF imaging-guided delivery of a novel NGR-VEGI fusion protein for targeting tumor vasculature.

PubMed

Ma, Wenhui; Li, Guoquan; Wang, Jing; Yang, Weidong; Zhang, Yingqi; Conti, Peter S; Chen, Kai

2014-12-01

Pathological angiogenesis is crucial in tumor growth, invasion and metastasis. Previous studies demonstrated that the vascular endothelial growth inhibitor (VEGI), a member of the tumor necrosis factor superfamily, can be used as a potent endogenous inhibitor of tumor angiogenesis. Molecular probes containing the asparagine-glycine-arginine (NGR) sequence can specifically bind to CD13 receptor which is overexpressed on neovasculature and several tumor cells. Near-infrared fluorescence (NIRF) optical imaging for targeting tumor vasculature offers a noninvasive method for early detection of tumor angiogenesis and efficient monitoring of response to anti-tumor vasculature therapy. The aim of this study was to develop a new NIRF imaging probe on the basis of an NGR-VEGI protein for the visualization of tumor vasculature. The NGR-VEGI fusion protein was prepared from prokaryotic expression, and its function was characterized in vitro. The NGR-VEGI protein was then labeled with a Cy5.5 fluorophore to afford Cy5.5-NGR-VEGI probe. Using the NIRF imaging technique, we visualized and quantified the specific delivery of Cy5.5-NGR-VEGI protein to subcutaneous HT-1080 fibrosarcoma tumors in mouse xenografts. The Cy5.5-NGR-VEGI probe exhibited rapid HT-1080 tumor targeting, and highest tumor-to-background contrast at 8 h post-injection (pi). Tumor specificity of Cy5.5-NGR-VEGI was confirmed by effective blocking of tumor uptake in the presence of unlabeled NGR-VEGI (20 mg/kg). Ex vivo NIRF imaging further confirmed in vivo imaging findings, demonstrating that Cy5.5-NGR-VEGI displayed an excellent tumor-to-muscle ratio (18.93 ± 2.88) at 8 h pi for the non-blocking group and significantly reduced ratio (4.92 ± 0.75) for the blocking group. In conclusion, Cy5.5-NGR-VEGI provided highly sensitive, target-specific, and longitudinal imaging of HT-1080 tumors. As a novel theranostic protein, Cy5.5-NGR-VEGI has the potential to improve cancer treatment by targeting tumor vasculature.

Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation

PubMed Central

Pleiner, Tino; Bates, Mark; Trakhanov, Sergei; Lee, Chung-Tien; Schliep, Jan Erik; Chug, Hema; Böhning, Marc; Stark, Holger; Urlaub, Henning; Görlich, Dirk

2015-01-01

Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with <2 nm epitope-label displacement. For this, we introduced cysteines at specific positions in the nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks – each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target. DOI: http://dx.doi.org/10.7554/eLife.11349.001 PMID:26633879

Size-matched alkyne-conjugated cyanine fluorophores to identify differences in protein glycosylation.

PubMed

Burnham-Marusich, Amanda R; Plechaty, Anna M; Berninsone, Patricia M

2014-09-01

Currently, there are few methods to detect differences in posttranslational modifications (PTMs) in a specific manner from complex mixtures. Thus, we developed an approach that combines the sensitivity and specificity of click chemistry with the resolution capabilities of 2D-DIGE. In "Click-DIGE", posttranslationally modified proteins are metabolically labeled with azido-substrate analogs, then size- and charge-matched alkyne-Cy3 or alkyne-Cy5 dyes are covalently attached to the azide of the PTM by click chemistry. The fluorescently-tagged protein samples are then multiplexed for 2DE analysis. Whereas standard DIGE labels all proteins, Click-DIGE focuses the analysis of protein differences to a targeted subset of posttranslationally modified proteins within a complex sample (i.e. specific labeling and analysis of azido glycoproteins within a cell lysate). Our data indicate that (i) Click-DIGE specifically labels azido proteins, (ii) the resulting Cy-protein conjugates are spectrally distinct, and (iii) the conjugates are size- and charge-matched at the level of 2DE. We demonstrate the utility of this approach by detecting multiple differentially expressed glycoproteins between a mutant cell line defective in UDP-galactose transport and the parental cell line. We anticipate that the diversity of azido substrates already available will enable Click-DIGE to be compatible with analysis of a wide range of PTMs. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Static micromixer-coaxial electrospray synthesis of theranostic lipoplexes.

PubMed

Wu, Yun; Li, Lei; Mao, Yicheng; Lee, Ly James

2012-03-27

Theranostic lipoplexes are an integrated nanotherapeutic system with diagnostic imaging capability and therapeutic functions. They hold great promise to improve current cancer treatments; however, producing uniform theranostic lipoplexes with multiple components in a reproducible manner is a highly challenging task. Conventional methods, such as bulk mixing, are not able to achieve this goal because of their macroscale and random nature. Here we report a novel technique, called the static micromixer-coaxial electrospray (MCE), to synthesize theranostic lipoplexes in a single step with high reproducibility. In this work, quantum dots (QD605) and Cy5-labeled antisense oligodeoxynucleotides (Cy5-G3139) were chosen as the model imaging reagent and therapeutic drug, respectively. Compared with bulk mixing, QD605/Cy5-G3139-loaded lipoplexes produced by MCE were highly uniform with polydispersity of 0.024 ± 0.006 and mean diameter by volume of 194 ± 15 nm. MCE also showed higher encapsulation efficiency of QD605 and Cy5-G3139. QD605 and Cy5 also formed the Förster resonance energy transfer pair, and thus the cellular uptake and intracellular fate of theranostic lipoplexes could be visualized by flow cytometry and confocal microscopy. The lipoplexes were efficiently delivered to A549 cells (non-small cell lung cancer cell line) and down-regulated the Bcl-2 gene expression by 48 ± 6%. © 2012 American Chemical Society

A method for tracing exogenous DNA uptake in live spermatozoa and embryos.

PubMed

Mu, Y; Jiao, M; Zhao, Y; Lv, J; Wang, J; Hao, J; Zhang, X; Kong, Q; Liu, Z

2018-03-01

Sperm-mediated gene transfer(SMGT) is a simple method for producing transgenic animals. Due to the lack of repeatability in spermatozoa binding and internalization of exogenous DNA, the efficiency of SMGT is still low. Considering this point, the present work aims to develop a method for evaluating the spermatozoa capacity of binding exogenous DNA after co-incubation with DNA. The main approach is using a Cy5-labelled DNA to trace the exogenous DNA and assess the ability of spermatozoa to take up exogenous DNA. Using this technique, we found that the percentage of spermatozoa that are binding and uptaking DNA is higher at concentration of 10 μg/mL and 100 μg/mL than 5 μg/mL, 1 μg/mL and 0 μg/mL after incubation with Cy5-DNA for 30min at 37oC. After fertilization, the DNA fluorescence signal was also detected in zygotes in groups where spermatozoa were incubated with 10 μg/mL and 100 μg/mL of Cy5-DNA. These results showed a simple and convenient method to trace the exogenous DNA in spermatozoa and zygote when compared to conventional methods of labeling DNA during fertilization, resulting in a real-time observation of the exogenous DNA in spermatozoa and zygote. Copyright© by the Polish Academy of Sciences.

Long-term direct visualization of passively transferred fluorophore-conjugated antibodies.

PubMed

Schneider, Jeffrey R; Carias, Ann M; Bastian, Arangaserry R; Cianci, Gianguido C; Kiser, Patrick F; Veazey, Ronald S; Hope, Thomas J

2017-11-01

The use of therapeutic antibodies, delivered by intravenous (IV) instillation, is a rapidly expanding area of biomedical treatment for a variety of conditions. However, little is known about how the antibodies are anatomically distributed following infusion and the underlying mechanism mediating therapeutic antibody distribution to specific anatomical sites remains to be elucidated. Current efforts utilize low resolution and sensitivity methods such as ELISA and indirect labeling imaging techniques, which often leads to high background and difficulty in assessing biodistribution. Here, using the in vivo non-human primate model, we demonstrate that it is possible to utilize the fluorophores Cy5 and Cy3 directly conjugated to antibodies for direct visualization and quantification of passively transferred antibodies in plasma, tissue, and in mucosal secretions. Antibodies were formulated with 1-2 fluorophores per antibody to minimally influence antibody function. Fluorophore conjugated Gamunex-C (pooled human IgG) were tested for binding to protein A, via surface plasmon resonance, and showed similar levels of binding when compared to unlabeled Gamunex-C. In order to assess the effect fluorophore labeling has on turnover and localization, rhesus macaques were IV infused with either labeled or unlabeled Gamunex-C. Plasma, vaginal Weck-Cel fluid, cervicovaginal mucus, and vaginal/rectal tissue biopsies were collected up to 8weeks. Similar turnover and biodistribution was observed between labeled and unlabeled antibodies, showing that the labeling process did not have an obvious deleterious effect on localization or turnover. Cy5 and Cy3 labeled antibodies were readily detected in the same pattern regardless of fluorophore. Tissue distribution was measured in macaque vaginal and rectal biopsies. The labeled antibody in macaque biopsies was found to have similar biodistribution pattern to endogenous antibodies in macaque and human tissues. In the vaginal and rectal mucosa, endogenous and infused antibodies were found primarily within the lamina propria. In the mucosal squamous epithelium of the vaginal vault, significant antibody was also observed in a striated pattern in the superficial, nonviable, stratum corneum. Endogenous antibody distribution in both human and macaque squamous tissues exhibited a similar pattern as seen with the labeled and unlabeled antibodies. This proof-of-principle study reveals that the labeled antibody is stable and physiologically similar relative to endogenous antibody setting the stage for future work to better understand the mechanisms of how antibodies reach unique anatomical sites. Direct visualization of fluorophore-conjugated antibodies following passive infusion can be utilized to assess the kinetics of biodistribution of infused antibodies and may be a useful approach to monitor and predict efficacy of therapeutic antibodies. Copyright © 2017 Elsevier B.V. All rights reserved.

Dual combination therapy targeting DR5 and EMMPRIN in pancreatic adenocarcinoma.

PubMed

Kim, Hyunki; Zhai, Guihua; Samuel, Sharon L; Rigell, Christopher J; Umphrey, Heidi R; Rana, Samir; Stockard, Cecil R; Fineberg, Naomi S; Zinn, Kurt R

2012-02-01

The goal of the study was to assess the efficacy of combined extracellular matrix metalloprotease inducer (EMMPRIN)- and death receptor 5 (DR5)-targeted therapy for pancreatic adenocarcinoma in orthotopic mouse models with multimodal imaging. Cytotoxicity of anti-EMMPRIN antibody and anti-DR5 antibody (TRA-8) in MIA PaCa-2 and PANC-1 cell lines was measured by ATPlite assay in vitro. The distributions of Cy5.5-labeled TRA-8 and Cy3-labeled anti-EMMPRIN antibody in the 2 cell lines were analyzed by fluorescence imaging in vitro. Groups 1 to 12 of severe combined immunodeficient mice bearing orthotopic MIA PaCa-2 (groups 1-8) or PANC-1 (groups 9-12) tumors were used for in vivo studies. Dynamic contrast-enhanced-MRI was applied in group 1 (untreated) or group 2 (anti-EMMPRIN antibody). The tumor uptake of Tc-99m-labeled TRA-8 was measured in group 3 (untreated) and group 4 (anti-EMMPRIN antibody). Positron emission tomography/computed tomography imaging with (18)F-FDG was applied in groups 5 to 12. Groups 5 to 8 (or groups 9 to 12) were untreated or treated with anti-EMMPRIN antibody, TRA-8, and combination, respectively. TRA-8 showed high killing efficacy for both MIA PaCa-2 and PANC-1 cells in vitro, but additional anti-EMMPRIN treatment did not improve the cytotoxicity. Cy5.5-TRA-8 formed cellular caps in both the cell lines, whereas the maximum signal intensity was correlated with TRA-8 cytotoxicity. Anti-EMMPRIN therapy significantly enhanced the tumor delivery of the MR contrast agent, but not Tc-99m-TRA-8. Tumor growth was significantly suppressed by the combination therapy, and the additive effect of the combination was shown in both MIA PaCa-2 and PANC-1 tumor models.

A novel application of maleimide for advanced drug delivery: in vitro and in vivo evaluation of maleimide-modified pH-sensitive liposomes.

PubMed

Li, Tianshu; Takeoka, Shinji

2013-01-01

Maleimide is a stable and easy-to-handle moiety that rapidly and covalently conjugates thiol groups of cysteine residues in proteins or peptides. Herein, we use maleimide to modify the surface of liposomes in order to obtain an advanced drug delivery system. Employing a small amount (0.3 mol%) of maleimide-polyethylene glycol (PEG) to modify the surface of the liposomes M-GGLG-liposomes, composed of 1,5-dihexadecyl N,N-diglutamyl-lysyl-L-glutamate (GGLG)/cholesterol/poly(ethylene glycol) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG5000-DSPE)/maleimide-PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03, drug delivery efficiency was remarkably improved both in vitro and in vivo compared to unmodified liposomes (GGLG-liposomes, composed of GGLG/cholesterol/PEG5000-DSPE/PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03). Moreover, this modification did not elicit any detectable increase in cytotoxicity. The maleimide-modification did not alter the physical characteristics of the liposomes such as size, zeta potential, pH sensitivity, dispersibility and drug encapsulation efficiency. However, M-GGLG-liposomes were more rapidly (≥2-fold) internalized into HeLa, HCC1954, and MDA-MB-468 cells compared to GGLG-liposomes. In vivo, M-GGLG-liposomes encapsulating doxorubicin (M-GGLG-DOX-liposomes) also showed a more potent antitumor effect than GGLG-DOX-liposomes and the widely used 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-DOX-liposomes after two subcutaneous injections around breast cancer tissue in mice. The biodistribution of liposomes in this model was observed using an in vivo imaging system, which showed that M-GGLG-liposomes were present for significantly longer at the injection site compared to GGLG-liposomes. The outstanding biological functions of the maleimide-modified liposomes as a novel drug delivery system make them ideally suited to a wide range of applications.

A novel application of maleimide for advanced drug delivery: in vitro and in vivo evaluation of maleimide-modified pH-sensitive liposomes

PubMed Central

Li, Tianshu; Takeoka, Shinji

2013-01-01

Maleimide is a stable and easy-to-handle moiety that rapidly and covalently conjugates thiol groups of cysteine residues in proteins or peptides. Herein, we use maleimide to modify the surface of liposomes in order to obtain an advanced drug delivery system. Employing a small amount (0.3 mol%) of maleimide-polyethylene glycol (PEG) to modify the surface of the liposomes M-GGLG-liposomes, composed of 1,5-dihexadecyl N,N-diglutamyl-lysyl-L-glutamate (GGLG)/cholesterol/poly(ethylene glycol) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG5000-DSPE)/maleimide-PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03, drug delivery efficiency was remarkably improved both in vitro and in vivo compared to unmodified liposomes (GGLG-liposomes, composed of GGLG/cholesterol/PEG5000-DSPE/PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03). Moreover, this modification did not elicit any detectable increase in cytotoxicity. The maleimide-modification did not alter the physical characteristics of the liposomes such as size, zeta potential, pH sensitivity, dispersibility and drug encapsulation efficiency. However, M-GGLG-liposomes were more rapidly (≥2-fold) internalized into HeLa, HCC1954, and MDA-MB-468 cells compared to GGLG-liposomes. In vivo, M-GGLG-liposomes encapsulating doxorubicin (M-GGLG-DOX-liposomes) also showed a more potent antitumor effect than GGLG-DOX-liposomes and the widely used 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-DOX-liposomes after two subcutaneous injections around breast cancer tissue in mice. The biodistribution of liposomes in this model was observed using an in vivo imaging system, which showed that M-GGLG-liposomes were present for significantly longer at the injection site compared to GGLG-liposomes. The outstanding biological functions of the maleimide-modified liposomes as a novel drug delivery system make them ideally suited to a wide range of applications. PMID:24143089

Excitation of Cy5 in self-assembled lipid bilayers using optical microresonators

NASA Astrophysics Data System (ADS)

Freeman, Lindsay M.; Li, Su; Dayani, Yasaman; Choi, Hong-Seok; Malmstadt, Noah; Armani, Andrea M.

2011-04-01

Due to their sensitivity and temporal response, optical microresonators are used extensively in the biosensor arena, particularly in the development of label-free diagnostics and measurement of protein kinetics. In the present letter, we investigate using microcavities to probe molecules within biomimetic membranes. Specifically, a method for self-assembling lipid bilayers on spherical microresonators is developed and the bilayer-nature is verified. Subsequently, the microcavity is used to excite a Cy5-conjugated lipid located within the bilayer while the optical performance of the microcavity is characterized. The emission wavelength of the dye and the optical behavior of the microcavity agree with theoretical predictions.

Development of a Hybrid Tracer for SPECT and Optical Imaging of Bacterial Infections.

PubMed

Welling, Mick M; Bunschoten, Anton; Kuil, Joeri; Nelissen, Rob G H H; Beekman, Freek J; Buckle, Tessa; van Leeuwen, Fijs W B

2015-05-20

In trauma and orthopedic surgery, infection of implants has a major impact on the outcome for patients. Infections may develop either during the initial implantation or during the lifetime of an implant. Both infections, as well as aseptic loosening of the implant, are reasons for revision of the implants. Therefore, discrimination between aseptic-mechanical-loosening and septic-bacterial-loosening of implants is critical during selection of a patient-tailored treatment policy. Specific detection and visualization of infections is a challenge because it is difficult to discriminate infections from inflammation. An imaging tracer that facilitates bacterial identification in a pre- and intraoperative setting may aid the workup for patients suspicious of bacterial infections. In this study we evaluated an antimicrobial peptide conjugated to a hybrid label, which contains both a radioisotope and a fluorescent dye. After synthesis of DTPA-Cy5-UBI29-41 and-when necessary-radiolabeling with (111)In (yield 96.3 ± 2.7%), in vitro binding to various bacterial strains was evaluated using radioactivity counting and confocal fluorescence microscopy. Intramuscular bacterial infections (S. aureus or K. pneumoniae) were also visualized in vivo using a combined nuclear and fluorescence imaging system. The indium-111 was chosen as label as it has a well-defined coordination chemistry, and in pilot studies labeling DTPA-Cy5-UBI29-41 with technetium-99m, we encountered damage to the Cy5 dye after the reduction with SnCl2. As a reference, we used the validated tracer (99m)Tc-UBI29-41. Fast renal excretion of (111)In-DTPA-Cy5-UBI29-41 was observed. Target to nontarget (T/NT) ratios were highest at 2 h post injection: radioactivity counting yielded T/NT ratios of 2.82 ± 0.32 for S. aureus and 2.37 ± 0.05 for K. pneumoniae. Comparable T/NT ratios with fluorescence imaging of 2.38 ± 0.09 for S. aureus and 3.55 ± 0.31 for K. pneumoniae were calculated. Ex vivo confocal microscopy of excised infected tissues showed specific binding of the tracer to bacteria. Using a combination of nuclear and fluorescence imaging techniques, the hybrid antimicrobial peptide conjugate DTPA-Cy5-UBI29-41 was shown to specifically accumulate in bacterial infections. This hybrid tracer may facilitate integration of noninvasive identification of infections and their extent as well as real-time fluorescence guidance during surgical resection of infected areas.

Imaging vascular endothelial growth factor (VEGF) receptors in turpentine-induced sterile thigh abscesses with radiolabeled single-chain VEGF.

PubMed

Levashova, Zoia; Backer, Marina; Backer, Joseph M; Blankenberg, Francis G

2009-12-01

Angiogenesis plays a central role in the pathogenesis of chronic inflammatory disorders. Vascular endothelial growth factor (VEGF) and its receptors are the most important regulators of angiogenesis. We wished to determine whether labeled forms of single-chain VEGF (scVEGF) could be used to image VEGF receptors in a well-characterized model of sterile soft-tissue inflammation induced by intramuscular injection of turpentine. Anesthetized adult male Swiss-Webster mice received a 20-microL intramuscular injection of turpentine into the right thigh. At 4, 7, or 10 d later, groups of 3-5 mice were injected via the tail vein with 50 microg of either scVEGF that had been site specifically labeled with Cy5.5 (scVEGF/Cy) or inactivated scVEGF/Cy (inVEGF/Cy) and then examined by fluorescence imaging. At 3, 4, 6, 7, 9, 10, or 12 d, additional groups of 3-5 mice were injected via the tail vein with 74-111 MBq of (99m)Tc-scVEGF (or (99m)Tc-inVEGF) and then examined by SPECT imaging. On days 3 through 10, both forms of scVEGF (scVEGF/Cy and (99m)Tc-scVEGF) showed significantly higher uptake (P < 0.05) in the right (abscessed) thigh than in the contralateral thigh (and higher uptake than the inactivated tracer). Peak uptake occurred on day 7 (3.67 +/- 1.79 [ratio of uptake in abscessed thigh to uptake in normal thigh, mean +/- SD] and 0.72 +/- 0.01 for scVEGF/Cy and inVEGF/Cy, respectively, and 3.49 +/- 1.22 and 1.04 +/- 0.41 for (99m)Tc-scVEGF and (99m)Tc-inVEGF, respectively) and slowly decreased thereafter. Autoradiography revealed peak tracer uptake in the thick irregular angiogenic rim of the abscess cavity on day 9 (5.83 x 10(-7) +/- 9.22 x 10(-8) and 5.85 x 10(-8) +/- 5.95 x 10(-8) percentage injected dose per pixel for (99m)Tc-scVEGF and (99m)Tc-inVEGF, respectively); in comparison, a thin circumscribed rim of uptake was seen with (99m)Tc-inVEGF. Immunostaining revealed that VEGFR-2 (VEGF receptor) colocalized with CD31 (endothelial cell marker) at all time points in the abscess rim, whereas F4/80 (macrophage) immunostaining reached a maximum at day 7 and decreased by day 10. The uptake of scVEGF in turpentine-induced abscesses was specific and directly related to VEGFR-2 expression in the neovasculature of the angiogenic rim. Peak tracer uptake coincided with maximum macrophage infiltration, suggesting that scVEGF imaging may be useful for the detection, localization, and monitoring of chronic inflammation in bone, joints, or soft tissues.

Novel water-soluble near-infrared cyanine dyes: synthesis, spectral properties, and use in the preparation of internally quenched fluorescent probes.

PubMed

Bouteiller, Cédric; Clavé, Guillaume; Bernardin, Aude; Chipon, Bertrand; Massonneau, Marc; Renard, Pierre-Yves; Romieu, Anthony

2007-01-01

In this paper, we describe the synthesis and the photophysical properties of two novel near-infrared (NIR) cyanine dyes (NIR5.5-2 and NIR7.0-2) which are water soluble potential substitutes of the commercially available Cy 5.5 and Cy 7.0 fluorescent labels respectively. For each one of these cyanine dyes, the synthetic strategy relies on the postsynthetic derivatization of a cyanine precursor in order to introduce the key functionalities required for bioconjugation of these NIR fluorophores. For NIR5.5-2, a reactive amino group was acylated with an original trisulfonated linker for water solubility. For NIR7.0-2, a vinylic chlorine atom was derivatized through a SRN1 reaction for the introduction of a monoreactive carboxyl group for labeling purposes. Unexpectedly, when these two fluorophores were closely associated within a peptidic architecture, mutual fluorescence quenching between NIR5.5-2 and NIR7.0-2 was observed both at 705 (NIR5.5-2) and 798 nm (NIR7.0-2). On the basis of this property, a novel internally quenched caspase-3-sensitive NIR fluorescent probe was prepared.

A python module to normalize microarray data by the quantile adjustment method.

PubMed

Baber, Ibrahima; Tamby, Jean Philippe; Manoukis, Nicholas C; Sangaré, Djibril; Doumbia, Seydou; Traoré, Sekou F; Maiga, Mohamed S; Dembélé, Doulaye

2011-06-01

Microarray technology is widely used for gene expression research targeting the development of new drug treatments. In the case of a two-color microarray, the process starts with labeling DNA samples with fluorescent markers (cyanine 635 or Cy5 and cyanine 532 or Cy3), then mixing and hybridizing them on a chemically treated glass printed with probes, or fragments of genes. The level of hybridization between a strand of labeled DNA and a probe present on the array is measured by scanning the fluorescence of spots in order to quantify the expression based on the quality and number of pixels for each spot. The intensity data generated from these scans are subject to errors due to differences in fluorescence efficiency between Cy5 and Cy3, as well as variation in human handling and quality of the sample. Consequently, data have to be normalized to correct for variations which are not related to the biological phenomena under investigation. Among many existing normalization procedures, we have implemented the quantile adjustment method using the python computer language, and produced a module which can be run via an HTML dynamic form. This module is composed of different functions for data files reading, intensity and ratio computations and visualization. The current version of the HTML form allows the user to visualize the data before and after normalization. It also gives the option to subtract background noise before normalizing the data. The output results of this module are in agreement with the results of other normalization tools. Published by Elsevier B.V.

New fluorescent labels with tunable hydrophilicity for the rational design of bright optical probes for molecular imaging.

PubMed

Pauli, Jutta; Licha, Kai; Berkemeyer, Janis; Grabolle, Markus; Spieles, Monika; Wegner, Nicole; Welker, Pia; Resch-Genger, Ute

2013-07-17

The rational design of bright optical probes and dye-biomolecule conjugates in the NIR-region requires fluorescent labels that retain their high fluorescence quantum yields when bound to a recognition unit or upon interaction with a target. Because hydrophilicity-controlled dye aggregation in conjunction with homo-FRET presents one of the major fluorescence deactivation pathways in dye-protein conjugates, fluorescent labels are required that enable higher labeling degrees with minimum dye aggregation. Aiming at a better understanding of the factors governing dye-dye interactions, we systematically studied the signal-relevant spectroscopic properties, hydrophilicity, and aggregation behavior of the novel xS-IDCC series of symmetric pentamethines equipped with two, four, and six sulfonic acid groups and selected conjugates of these dyes with IgG and the antibody cetuximab (ctx) directed against the cancer-related epidermal growth factor (EGF) receptor in comparison to the gold standard Cy5.5. With 6S-IDCC, which displays a molar absorption coefficient of 190 000 M(-1) cm(-1) and a fluorescence quantum yield (Φf) of 0.18 in aqueous media like PBS and nearly no aggregation, we could identify a fluorophore with a similarly good performance as Cy5.5. Bioconjugation of 6S-IDCC and Cy5.5 yielded highly emissive targeted probes with comparable Φf values of 0.29 for a dye-to-protein (D/P) ratio <1 and a reduced number of protein-bound dye aggregates in the case of 6S-IDCC. Binding studies of the ctx conjugates of both dyes performed by fluorescence microscopy and FACS revealed that the binding strength between the targeted probes and the EGF receptor at the cell membrane is independent of D/P ratio. These results underline the importance of an application-specific tuning of dye hydrophilicity for the design of bright fluorescent reporters and efficient optical probes. Moreover, we could demonstrate the potential of fluorescence spectroscopy to predict the size of fluorescence signals resulting for other fluorescence techniques such as FACS.

Comparison of Hydrazone Heterobifunctional Crosslinking Agents for Reversible Conjugation of Thiol-Containing Chemistry

PubMed Central

Christie, R. James; Anderson, Diana J.; Grainger, David W.

2010-01-01

Reversible covalent conjugation chemistries that allow site- and condition-specific coupling and uncoupling reactions are attractive components in nanotechnologies, bioconjugation methods, imaging and drug delivery systems. Here, we compare three heterobifunctional crosslinkers, containing both thiol- and amine- reactive chemistry, to form pH-labile hydrazones with hydrazide derivatives of the known and often published water-soluble polymer, poly[N-(2-hydroxypropyl methacrylamide)] (pHPMA), while subsequently coupling thiol-containing molecules to the crosslinker via maleimide addition. Two novel crosslinkers were prepared from the popular heterobifunctional crosslinking agent, succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), modified to contain either terminal aldehyde groups (i.e., 1-(N-3-propanal)-4-(N-maleimidomethyl) cyclohexane carboxamide, PMCA) or methylketone groups (i.e., 1-(N-3-butanone)-4-(N-maleimidomethyl) cyclohexane carboxamide, BMCA). A third crosslinking agent was the commercially available N-4-acetylphenyl maleimide (APM). PMCA and BMCA exhibited excellent reactivity towards hydrazide-derivatized pHPMA with essentially complete hydrazone conjugation to polymer reactive sites, while APM coupled only ~ 60% of available reactive sites on the polymer despite a 3-fold molar excess relative to polymer hydrazide groups. All polymer hydrazone conjugates bearing these bifunctional agents were then further reacted with thiol-modified tetramethylrhodamine dye, confirming crosslinker maleimide reactivity after initial hydrazone polymer conjugation. Incubation of dye-labeled polymer conjugates in phosphate buffered saline at 37°C showed that hydrazone coupling resulting from APM exhibited the greatest difference in stability between pH 7.4 and 5.0, with hydrolysis and dye release increased at pH 5.0 over a 24hr incubation period. Polymer conjugates bearing hydrazones formed from crosslinker BMCA exhibited intermediate stability with hydrolysis much greater at pH 5.0 at early time points, but hydrolysis at pH 7.4 was significant after 5 hrs. Hydrazones formed with the PMCA crosslinker showed no difference in release rates at pH 7.4 and 5.0. PMID:20695431

Development of fluorescent glucose bioprobes and their application on real-time and quantitative monitoring of glucose uptake in living cells.

PubMed

Lee, Hyang Yeon; Lee, Jae Jeong; Park, Jongmin; Park, Seung Bum

2011-01-03

We developed a novel fluorescent glucose bioprobe, GB2-Cy3, for the real-time and quantitative monitoring of glucose uptake in living cells. We synthesized a series of fluorescent glucose analogues by adding Cy3 fluorophores to the α-anomeric position of D-glucose through various linkers. Systematic and quantitative analysis of these Cy3-labeled glucose analogues revealed that GB2-Cy3 was the ideal fluorescent glucose bioprobe. The cellular uptake of this probe competed with the cellular uptake of D-glucose in the media and was mediated by a glucose-specific transport system, and not by passive diffusion. Flow cytometry and fluorescence microscopy analyses revealed that GB2-Cy3 is ten times more sensitive than 2-NBDG, a leading fluorescent glucose bioprobe. GB2-Cy3 can also be utilized for the quantitative flow cytometry monitoring of glucose uptake in metabolically active C2C12 myocytes under various treatment conditions. As opposed to a glucose uptake assay performed by using radioisotope-labeled deoxy-D-glucose and a scintillation counter, GB2-Cy3 allows the real-time monitoring of glucose uptake in living cells under various experimental conditions by using fluorescence microscopy or confocal laser scanning microscopy (CLSM). Therefore, we believe that GB2-Cy3 can be utilized in high-content screening (HCS) for the discovery of novel therapeutic agents and for making significant advances in biomedical studies and diagnosis of various diseases, especially metabolic diseases. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Transcription profiling of a recently colonised pyrethroid resistant Anopheles gambiae strain from Ghana

PubMed Central

Müller, Pie; Donnelly, Martin J; Ranson, Hilary

2007-01-01

Background Mosquito resistance to the pyrethroid insecticides used to treat bednets threatens the sustainability of malaria control in sub-Saharan Africa. While the impact of target site insensitivity alleles is being widely discussed the implications of insecticide detoxification – though equally important – remains elusive. The successful development of new tools for malaria intervention and management requires a comprehensive understanding of insecticide resistance, including metabolic resistance mechanisms. Although three enzyme families (cytochrome P450s, glutathione S-transferases and carboxylesterases) have been widely associated with insecticide detoxification the role of individual enzymes is largely unknown. Results Here, constitutive expression patterns of genes putatively involved in conferring pyrethroid resistance was investigated in a recently colonised pyrethroid resistant Anopheles gambiae strain from Odumasy, Southern Ghana. RNA from the resistant strain and a standard laboratory susceptible strain, of both sexes was extracted, reverse transcribed and labelled with either Cy3- or Cy5-dye. Labelled cDNA was co-hybridised to the detox chip, a custom-made microarray containing over 230 A. gambiae gene fragments predominantly from enzyme families associated with insecticide resistance. After hybridisation, Cy3- and Cy5-signal intensities were measured and compared gene by gene. In both females and males of the resistant strain the cytochrome P450s CYP6Z2 and CYP6M2 are highly over-expressed along with a member of the superoxide dismutase (SOD) gene family. Conclusion These genes differ from those found up-regulated in East African strains of pyrethroid resistant A. gambiae and constitute a novel set of candidate genes implicated in insecticide detoxification. These data suggest that metabolic resistance may have multiple origins in A. gambiae, which has strong implications for the management of resistance. PMID:17261191

Luminescent probes for optical in vivo imaging

NASA Astrophysics Data System (ADS)

Texier, Isabelle; Josserand, Veronique; Garanger, Elisabeth; Razkin, Jesus; Jin, Zhaohui; Dumy, Pascal; Favrot, Marie; Boturyn, Didier; Coll, Jean-Luc

2005-04-01

Going along with instrumental development for small animal fluorescence in vivo imaging, we are developing molecular fluorescent probes, especially for tumor targeting. Several criteria have to be taken into account for the optimization of the luminescent label. It should be adapted to the in vivo imaging optical conditions : red-shifted absorption and emission, limited overlap between absorption and emission for a good signal filtering, optimized luminescence quantum yield, limited photo-bleaching. Moreover, the whole probe should fulfill the biological requirements for in vivo labeling : adapted blood-time circulation, biological conditions compatibility, low toxicity. We here demonstrate the ability of the imaging fluorescence set-up developed in LETI to image the bio-distribution of molecular probes on short times after injection. Targeting with Cy5 labeled holo-transferrin of subcutaneous TS/Apc (angiogenic murine breast carcinoma model) or IGROV1 (human ovarian cancer) tumors was achieved. Differences in the kinetics of the protein uptake by the tumors were evidenced. IGROV1 internal metastatic nodes implanted in the peritoneal cavity could be detected in nude mice. However, targeted metastatic nodes in lung cancer could only be imaged after dissection of the mouse. These results validate our fluorescence imaging set-up and the use of Cy5 as a luminescent label. New fluorescent probes based on this dye and a molecular delivery template (the RAFT molecule) can thus be envisioned.

Enhanced cellular uptake of maleimide-modified liposomes via thiol-mediated transport

PubMed Central

Li, Tianshu; Takeoka, Shinji

2014-01-01

With a small amount of maleimide modification on the liposome surface, enhanced cellular uptake of liposomes and drug-delivery efficiency can be obtained both in vitro and in vivo. Herein, we describe the mechanisms underlying this enhanced cellular uptake. Suppression of the cellular uptake of maleimide-modified liposomes (M-GGLG, composed of 1,5-dihexadecyl N,N-diglutamyl-lysyl-L-glutamate [GGLG]/cholesterol/poly(ethylene glycol) – 1,2-distearoyl-sn-glycero-3-phosphoethanolamine [PEG5000-DSPE]/maleimide [M]-PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03) caused by temperature block and addition of serum was alleviated compared with that of liposomes without maleimide modification (GGLG liposomes, composed of GGLG/cholesterol/PEG5000-DSPE/PEG5000-Glu2C18 at a molar ratio of 5:5:0.03:0.03). When 0.01 nM N-ethylmaleimide was used to pre-block cellular thiols, the cellular uptake of M-GGLG liposomes was decreased to approximately 70% in HeLa, HCC1954, MDA-MB-468, and COS-7 cell lines. Moreover, inhibition of a thiol-related reductase such as protein disulfide isomerase resulted in a 15%–45% inhibition of the cellular uptake of M-GGLG liposomes, whereas GGLG liposomes were not influenced. Further, single and mixed inhibitors of clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis did not efficiently inhibit the cellular uptake of M-GGLG liposomes. Using confocal microscopy, we verified that M-GGLG liposomes were localized partially in lysosomes after inhibition of the mentioned conventional endocytic pathways. Therefore, it was hypothesized that the mechanisms underlying the enhanced cellular uptake of liposomes by maleimide modification was thiol-mediated membrane trafficking, including endocytosis and energy-independent transport. PMID:24940060

Enhanced cellular uptake of maleimide-modified liposomes via thiol-mediated transport.

PubMed

Li, Tianshu; Takeoka, Shinji

2014-01-01

With a small amount of maleimide modification on the liposome surface, enhanced cellular uptake of liposomes and drug-delivery efficiency can be obtained both in vitro and in vivo. Herein, we describe the mechanisms underlying this enhanced cellular uptake. Suppression of the cellular uptake of maleimide-modified liposomes (M-GGLG, composed of 1,5-dihexadecyl N,N-diglutamyl-lysyl-L-glutamate [GGLG]/cholesterol/poly(ethylene glycol) - 1,2-distearoyl-sn-glycero-3-phosphoethanolamine [PEG₅₀₀₀-DSPE]/maleimide [M]-PEG₅₀₀₀-Glu2C18 at a molar ratio of 5:5:0.03:0.03) caused by temperature block and addition of serum was alleviated compared with that of liposomes without maleimide modification (GGLG liposomes, composed of GGLG/cholesterol/PEG₅₀₀₀-DSPE/PEG₅₀₀₀-Glu2C₁₈ at a molar ratio of 5:5:0.03:0.03). When 0.01 nM N-ethylmaleimide was used to pre-block cellular thiols, the cellular uptake of M-GGLG liposomes was decreased to approximately 70% in HeLa, HCC1954, MDA-MB-468, and COS-7 cell lines. Moreover, inhibition of a thiol-related reductase such as protein disulfide isomerase resulted in a 15%-45% inhibition of the cellular uptake of M-GGLG liposomes, whereas GGLG liposomes were not influenced. Further, single and mixed inhibitors of clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis did not efficiently inhibit the cellular uptake of M-GGLG liposomes. Using confocal microscopy, we verified that M-GGLG liposomes were localized partially in lysosomes after inhibition of the mentioned conventional endocytic pathways. Therefore, it was hypothesized that the mechanisms underlying the enhanced cellular uptake of liposomes by maleimide modification was thiol-mediated membrane trafficking, including endocytosis and energy-independent transport.

Characterization of TCP-1 probes for molecular imaging of colon cancer.

PubMed

Liu, Zhonglin; Gray, Brian D; Barber, Christy; Bernas, Michael; Cai, Minying; Furenlid, Lars R; Rouse, Andrew; Patel, Charmi; Banerjee, Bhaskar; Liang, Rongguang; Gmitro, Arthur F; Witte, Marlys H; Pak, Koon Y; Woolfenden, James M

2016-10-10

Molecular probes capable of detecting colorectal cancer (CRC) are needed for early CRC diagnosis. The objective of this study was to characterize c[CTPSPFSHC]OH (TCP-1), a small peptide derived from phage display selection, for targeting human CRC xenografts using technetium-99m ((99m)Tc)-labeled TCP-1 and fluorescent cyanine-7 (Cy7)-labeled form of the peptide (Cy7-TCP-1). (99m)Tc-TCP-1 was generated by modifying TCP-1 with succinimidyl-6-hydrazino-nicotinamide (S-HYNIC) followed by radiolabeling. In vitro saturation binding experiments were performed for (99m)Tc-TCP-1 in human HCT116 colon cancer cells. SCID mice with human HCT116 cancer xenografts were imaged with (99m)Tc-TCP-1 or control peptide using a small-animal SPECT imager: Group I (n=5) received no blockade; Group II (n=5) received a blocking dose of non-radiolabeled TCP-1. Group III (n=5) were imaged with (99m)Tc-labeled control peptide (inactive peptide). SCID mice with human PC3 prostate cancer xenografts (Group IV, n=5) were also imaged with (99m)Tc-TCP-1. Eight additional SCID mice bearing HCT116 xenografts in dorsal skinfold window chambers (DSWC) were imaged by direct positron imaging of (18)F-fluorodeoxyglucose ((18)F-FDG) and fluorescence microscopy of Cy7-TCP-1. In vitro(99m)Tc-HYNIC-TCP-1 binding assays on HCT 116 cells indicated a mean Kd of 3.04±0.52nM. In cancer xenografts, (99m)Tc-TCP-1 radioactivity (%ID/g) was 1.01±0.15 in the absence of blockade and was reduced to 0.26±0.04 (P<0.01) with blockade. No radioactive uptake was observed in the PC3 tumors with (99m)Tc-TCP-1 or HCT116 tumors with inactive peptide. Cy7-TCP-1 activity localized not only in metabolically active tumors, as defined by (18)F-FDG imaging, but also in peritumoral microvasculature. In conclusion, TCP-1 probes may have a distinct targeting mechanism with high selectivity for CRC and tumor-associated vasculature. Molecular imaging with TCP-1 probes appears promising to detect malignant colorectal lesions. Copyright © 2016 Elsevier B.V. All rights reserved.

MUB40 Binds to Lactoferrin and Stands as a Specific Neutrophil Marker.

PubMed

Anderson, Mark C; Chaze, Thibault; Coïc, Yves-Marie; Injarabian, Louise; Jonsson, Friederike; Lombion, Naelle; Selimoglu-Buet, Dorothée; Souphron, Judith; Ridley, Caroline; Vonaesch, Pascale; Baron, Bruno; Arena, Ellen T; Tinevez, Jean-Yves; Nigro, Giulia; Nothelfer, Katharina; Solary, Eric; Lapierre, Valérie; Lazure, Thierry; Matondo, Mariette; Thornton, David; Sansonetti, Philippe J; Baleux, Françoise; Marteyn, Benoit S

2018-04-19

Neutrophils represent the most abundant immune cells recruited to inflamed tissues. A lack of dedicated tools has hampered their detection and study. We show that a synthesized peptide, MUB 40 , binds to lactoferrin, the most abundant protein stored in neutrophil-specific and tertiary granules. Lactoferrin is specifically produced by neutrophils among other leukocytes, making MUB 40 a specific neutrophil marker. Naive mammalian neutrophils (human, guinea pig, mouse, rabbit) were labeled by fluorescent MUB 40 conjugates (-Cy5, Dylight405). A peptidase-resistant retro-inverso MUB 40 (RI-MUB 40 ) was synthesized and its lactoferrin-binding property validated. Neutrophil lactoferrin secretion during in vitro Shigella infection was assessed with RI-MUB 40 -Cy5 using live cell microscopy. Systemically administered RI-MUB 40 -Cy5 accumulated at sites of inflammation in a mouse arthritis inflammation model in vivo and showed usefulness as a potential tool for inflammation detection using non-invasive imaging. Improving neutrophil detection with the universal and specific MUB 40 marker will aid the study of broad ranges of inflammatory diseases. Copyright © 2018 Elsevier Ltd. All rights reserved.

Fluorescent tag is not a reliable marker for small RNA transfection in the presence of serum.

PubMed

Han, Jing; Wang, Qi-Wei; Wang, Shi-Qiang

2013-09-01

Chemically synthetic siRNA and miRNA have become powerful tools to study gene function in the past decade. Fluorescent dyes covalently attached to the 5' or 3' ends of synthetic small RNAs are widely used for fluorescently imaging and detection of these RNAs. However, the reliability of fluorescent tags as small RNA markers in different conditions has not attracted enough attention. We used Cy3-labelled small RNAs to explore the reliability of fluorescent tags as small RNA markers in cell cultures involving serum. A strong Cy3-fluorescence signal was observed in the cytoplasm of the cells transfected with Cy3-miR24 in the culture medium containing fetal bovine serum (FBS), but qRT-PCR results showed that little miR24 were detected in these cells. Further study demonstrated that small RNAs were degraded in the presence of FBS, suggesting that it was Cy3-RNA fragments, rather than the original Cy3-miR24, diffused into cells. These phenomena disappeared when FBS was replaced by boiled-FBS, further supporting that the Cy3-fluorescence we observed in cells in the presence of FBS could not represent the presence of intact small RNAs. These findings addressed that fluorescent tags are not reliable for small RNA transfection in the presence of serum in culture.

Binding of the immunomodulatory drug Bz-423 to mitochondrial FoF1-ATP synthase in living cells by FRET acceptor photobleaching

NASA Astrophysics Data System (ADS)

Starke, Ilka; Johnson, Kathryn M.; Petersen, Jan; Gräber, Peter; Opipari, Anthony W.; Glick, Gary D.; Börsch, Michael

2016-03-01

Bz-423 is a promising new drug for treatment of autoimmune diseases. This small molecule binds to subunit OSCP of the mitochondrial enzyme FoF1-ATP synthase and modulates its catalytic activities. We investigate the binding of Bz-423 to mitochondria in living cells and how subunit rotation in FoF1-ATP synthase, i.e. the mechanochemical mechanism of this enzyme, is affected by Bz-423. Therefore, the enzyme was marked selectively by genetic fusion with the fluorescent protein EGFP to the C terminus of subunit γ. Imaging the threedimensional arrangement of mitochondria in living yeast cells was possible at superresolution using structured illumination microscopy, SIM. We measured uptake and binding of a Cy5-labeled Bz-423 derivative to mitochondrial FoF1-ATP synthase in living yeast cells using FRET acceptor photobleaching microscopy. Our data confirmed the binding of Cy5-labeled Bz-423 to the top of the F1 domain of the enzyme in mitochondria of living Saccharomyces cerevisiae cells.

Diagnostic power of laboratory tests for hereditary spherocytosis: a comparison study in 150 patients grouped according to molecular and clinical characteristics

PubMed Central

Bianchi, Paola; Fermo, Elisa; Vercellati, Cristina; Marcello, Anna P.; Porretti, Laura; Cortelezzi, Agostino; Barcellini, Wilma; Zanella, Alberto

2012-01-01

Background The laboratory diagnosis of hereditary spherocytosis commonly relies on NaCl-based or glycerol-based red cell osmotic fragility tests; more recently, an assay directly targeting the hereditary spherocytosis molecular defect (eosin-5′-maleimide-binding test) has been proposed. None of the available tests identifies all cases of hereditary spherocytosis. Design and Methods We compared the performances of the eosin-5′-maleimide-binding test, NaCl-osmotic fragility studies on fresh and incubated blood, the glycerol lysis test, the acidified glycerol lysis test, and the Pink test on a series of 150 patients with hereditary spherocytosis grouped according to clinical phenotype and the defective protein, with the final aim of finding the combination of tests associated with the highest diagnostic power, even in the mildest cases of hereditary spherocytosis. Results The eosin-5′-maleimide-binding test had a sensitivity of 93% and a specificity of 98% for detecting hereditary spherocytosis: the sensitivity was independent of the type and amount of molecular defect and of the clinical phenotype. The acidified glycerol lysis test and Pink test showed comparable sensitivity (95% and 91%). The sensitivity of NaCl osmotic fragility tests, commonly considered the gold standard for the diagnosis of hereditary spherocytosis, was 68% on fresh blood and 81% on incubated blood, and further decreased in compensated cases (53% and 64%, respectively). The combination of the eosin-5′-maleimide-binding test and acidified glycerol lysis test enabled all patients with hereditary spherocytosis to be identified. The eosin-5′-maleimide-binding test showed the greatest disease specificity. Conclusions Each type of test fails to diagnose some cases of hereditary spherocytosis. The association of an eosin-5′-maleimide-binding test and an acidified glycerol lysis test enabled identification of all patients with hereditary spherocytosis in this series and, therefore, represents a currently effective diagnostic strategy for hereditary spherocytosis including mild/compensated cases. PMID:22058213

Cyclen-based lipidic oligomers as potential gene delivery vehicles.

PubMed

Yi, Wen-Jing; Zhang, Qin-Fang; Zhang, Ji; Liu, Qiang; Ren, Laifeng; Chen, Qian-Ming; Guo, Liandi; Yu, Xiao-Qi

2014-03-01

A series of cyclen-based linear oligomers bearing hydrophobic long chains (lipopolymers Cy-LC, where Cy and LC represent cyclen-based linear backbone and hydrophobic long chain substituents, respectively) were designed and synthesized. The effects of type and degree of substitution (DS) of hydrophobic long chains on the transfection efficiency were systematically studied. The nitrogen atoms with relatively strong basicity on the cyclen ensure their good DNA binding ability, which was confirmed by gel retardation and ethidium bromide exclusion assays. Lipopolyplexes could be formed as nanoparticles with suitable sizes and zeta potentials for gene transfection. In vitro gene delivery experiments revealed that the linoleic acid (LIN) substituted material Cy-LIN has better transfection efficiency than 25 kDa polyethylenimine in the absence or in the presence of serum. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and hemolysis assays showed low cytotoxicity and good biocompatibility of the lipopolyplexes. Fluorescent labeled DNA was used to study the cellular uptake and intracellular distribution of transfected DNA. Flow cytometry results suggested that a long chain is necessary for efficient cellular uptake, and images from confocal laser scanning microscopy showed that after 4h transfection, most of the fluorescent labeled DNA accumulated in the perinuclear region, which was required for efficient gene expression. Moreover, it was also found that the DS of the hydrophobic moiety can adjust the balance between DNA binding ability and dissociation of polyplexes, significantly affecting the transfection efficiency. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Peptide-Appended Permethylated β-Cyclodextrins with Hydrophilic and Hydrophobic Spacers

PubMed Central

2017-01-01

A novel synthetic methodology, employing a combination of the strain-promoted azide–alkyne cycloaddition and maleimide–thiol reactions, for the preparation of permethylated β-cyclodextrin-linker-peptidyl conjugates is reported. Two different bifunctional maleimide cross-linking probes, the polyethylene glycol containing hydrophilic linker bicyclo[6.1.0] nonyne-maleimide and the hydrophobic 5′-dibenzoazacyclooctyne-maleimide, were attached to azide-appended permethylated β-cyclodextrin. The successfully introduced maleimide function was exploited to covalently graft a cysteine-containing peptide (Ac-Tyr-Arg-Cys-Amide) to produce the target conjugates. The final target compounds were isolated in high purity after purification by isocratic preparative reverse-phase high-performance liquid chromatography. This novel synthetic approach is expected to give access to many different cyclodextrin–linker peptides. PMID:28697600

Ligase Detection Reaction Generation of Reverse Molecular Beacons for Near Real-Time Analysis of Bacterial Pathogens Using Single-Pair Fluorescence Resonance Energy Transfer and a Cyclic Olefin Copolymer Microfluidic Chip

PubMed Central

Peng, Zhiyong; Soper, Steven A.; Pingle, Maneesh R.; Barany, Francis; Davis, Lloyd M.

2015-01-01

Detection of pathogenic bacteria and viruses require strategies that can signal the presence of these targets in near real-time due to the potential threats created by rapid dissemination into water and/or food supplies. In this paper, we report an innovative strategy that can rapidly detect bacterial pathogens using reporter sequences found in their genome without requiring polymerase chain reaction (PCR). A pair of strain-specific primers was designed based on the 16S rRNA gene and were end-labeled with a donor (Cy5) or acceptor (Cy5.5) dye. In the presence of the target bacterium, the primers were joined using a ligase detection reaction (LDR) only when the primers were completely complementary to the target sequence to form a reverse molecular beacon (rMB), thus bringing Cy5 (donor) and Cy5.5 (acceptor) into close proximity to allow fluorescence resonance energy transfer (FRET) to occur. These rMBs were subsequently analyzed using single-molecule detection of the FRET pairs (single-pair FRET; spFRET). The LDR was performed using a continuous flow thermal cycling process configured in a cyclic olefin copolymer (COC) microfluidic device using either 2 or 20 thermal cycles. Single-molecule photon bursts from the resulting rMBs were detected on-chip and registered using a simple laser-induced fluorescence (LIF) instrument. The spFRET signatures from the target pathogens were reported in as little as 2.6 min using spFRET. PMID:21047095

In vivo stem cell tracking with imageable nanoparticles that bind bioorthogonal chemical receptors on the stem cell surface.

PubMed

Lee, Sangmin; Yoon, Hwa In; Na, Jin Hee; Jeon, Sangmin; Lim, Seungho; Koo, Heebeom; Han, Sang-Soo; Kang, Sun-Woong; Park, Soon-Jung; Moon, Sung-Hwan; Park, Jae Hyung; Cho, Yong Woo; Kim, Byung-Soo; Kim, Sang Kyoon; Lee, Taekwan; Kim, Dongkyu; Lee, Seulki; Pomper, Martin G; Kwon, Ick Chan; Kim, Kwangmeyung

2017-09-01

It is urgently necessary to develop reliable non-invasive stem cell imaging technology for tracking the in vivo fate of transplanted stem cells in living subjects. Herein, we developed a simple and well controlled stem cell imaging method through a combination of metabolic glycoengineering and bioorthogonal copper-free click chemistry. Firstly, the exogenous chemical receptors containing azide (-N 3 ) groups were generated on the surfaces of stem cells through metabolic glycoengineering using metabolic precursor, tetra-acetylated N-azidoacetyl-d-mannosamine(Ac 4 ManNAz). Next, bicyclo[6.1.0]nonyne-modified glycol chitosan nanoparticles (BCN-CNPs) were prepared as imageable nanoparticles to deliver different imaging agents. Cy5.5, iron oxide nanoparticles and gold nanoparticles were conjugated or encapsulated to BCN-CNPs for optical, MR and CT imaging, respectively. These imageable nanoparticles bound chemical receptors on the Ac 4 ManNAz-treated stem cell surface specifically via bioorthogonal copper-free click chemistry. Then they were rapidly taken up by the cell membrane turn-over mechanism resulting in higher endocytic capacity compared non-specific uptake of nanoparticles. During in vivo animal test, BCN-CNP-Cy5.5-labeled stem cells could be continuously tracked by non-invasive optical imaging over 15 days. Furthermore, BCN-CNP-IRON- and BCN-CNP-GOLD-labeled stem cells could be efficiently visualized using in vivo MR and CT imaging demonstrating utility of our stem cell labeling method using chemical receptors. These results conclude that our method based on metabolic glycoengineering and bioorthogonal copper-free click chemistry can stably label stem cells with diverse imageable nanoparticles representing great potential as new stem cell imaging technology. Copyright © 2017 Elsevier Ltd. All rights reserved.

Supramolecular latching system based on ultrastable synthetic binding pairs as versatile tools for protein imaging.

PubMed

Kim, Kyung Lock; Sung, Gihyun; Sim, Jaehwan; Murray, James; Li, Meng; Lee, Ara; Shrinidhi, Annadka; Park, Kyeng Min; Kim, Kimoon

2018-04-27

Here we report ultrastable synthetic binding pairs between cucurbit[7]uril (CB[7]) and adamantyl- (AdA) or ferrocenyl-ammonium (FcA) as a supramolecular latching system for protein imaging, overcoming the limitations of protein-based binding pairs. Cyanine 3-conjugated CB[7] (Cy3-CB[7]) can visualize AdA- or FcA-labeled proteins to provide clear fluorescence images for accurate and precise analysis of proteins. Furthermore, controllability of the system is demonstrated by treating with a stronger competitor guest. At low temperature, this allows us to selectively detach Cy3-CB[7] from guest-labeled proteins on the cell surface, while leaving Cy3-CB[7] latched to the cytosolic proteins for spatially conditional visualization of target proteins. This work represents a non-protein-based bioimaging tool which has inherent advantages over the widely used protein-based techniques, thereby demonstrating the great potential of this synthetic system.

Electronic tuning of self-healing fluorophores for live-cell and single-molecule imaging† †Electronic supplementary information (ESI) available: Chart S1, Fig. S1 to S13, Scheme S1, Tables S1 and S2, note on calculating the effective concentration of protective agents, methods of cellular imaging and details the synthesis and characterization of compounds. Supplementary Movie 1: Single-molecule TIRF movies of living SNAPf-D2s-expressing CHO cells labeled with AF647 (left) or Cy5(4S)-AC(4) (right). Supplementary Movie 2: Single-molecule TIRF movies of living SNAPf-D2s-expressing CHO cells labeled with Dy549 (left) or Cy3(4S)-AC(4) (right). See DOI: 10.1039/c6sc02976k Click here for additional data file. Click here for additional data file. Click here for additional data file.

PubMed Central

Zheng, Qinsi; Jockusch, Steffen; Zhou, Zhou; Altman, Roger B.; Zhao, Hong; Asher, Wesley; Holsey, Michael; Mathiasen, Signe; Geggier, Peter; Javitch, Jonathan A.

2017-01-01

Bright, long-lasting organic fluorophores enable a broad range of imaging applications. “Self-healing” fluorophores, in which intra-molecularly linked protective agents quench photo-induced reactive species, exhibit both enhanced photostability and biological compatibility. However, the self-healing strategy has yet to achieve its predicted potential, particularly in the presence of ambient oxygen where live-cell imaging studies must often be performed. To identify key bottlenecks in this technology that can be used to guide further engineering developments, we synthesized a series of Cy5 derivatives linked to the protective agent cyclooctatetraene (COT) and examined the photophysical mechanisms curtailing their performance. The data obtained reveal that the photostability of self-healing fluorophores is limited by reactivity of the COT protective agent. The addition of electron withdrawing substituents to COT reduced its susceptibility to reactions with molecular oxygen and the fluorophore to which it is attached and increased its capacity to participate in triplet energy transfer. Exploiting these insights, we designed and synthesized a suite of modified COT-fluorophores spanning the visible spectrum that exhibited markedly increased intra-molecular photostabilization. Under ambient oxygen conditions, the photostability of Cy3 and Cy5 fluorophore derivatives increased by 3- and 9-fold in vitro and by 2- and 6-fold in living cells, respectively. We further show that this approach can improve a silicon rhodamine fluorophore. These findings offer a clear strategy for achieving the full potential of the self-healing approach and its application to the gamut of fluorophore species commonly used for biomedical imaging. PMID:28377799

Repetitive Immunosensor with a Fiber-Optic Device and Antibody-Coated Magnetic Beads for Semi-Continuous Monitoring of Escherichia coli O157:H7

PubMed Central

Taniguchi, Midori; Saito, Hirokazu; Mitsubayashi, Kohji

2017-01-01

A rapid and reproducible fiber-optic immunosensor for Escherichia coli O157:H7 (E. coli O157:H7) was described. The biosensor consisted of a flow cell, an optical fiber with a thin Ni layer, and a PC linked fluorometer. First, the samples with E. coli O157:H7 were incubated with magnetic beads coated with anti-E. coli O157:H7 antibodies and anti-E. coli O157:H7 antibodies labeled cyanine 5 (Cy5) to make sandwich complexes. Then the Cy5-(E. coli O157:H7)-beads were injected into a flow cell and pulled to the magnetized Ni layer on the optical fiber set in the flow cell. An excitation light (λ = 635 nm) was used to illuminate the optical fiber, and the Cy5 florescent molecules facing the optical fiber were exposed to an evanescent wave from the optical fiber. The 670 nm fluorescent light was measured using a photodiode. Finally, the magnetic intensity of the Ni layer was removed and the Cy5-E. coli O157:H7-beads were washed out for the next immunoassay. E. coli O157:H7, diluted with phosphate buffer (PB), was measured from 1 × 105 to 1 × 107 cells/mL. The total time required for an assay was less than 15 min (except for the pretreatment process) and repeating immunoassay on one optical fiber was made possible. PMID:28925937

Repetitive Immunosensor with a Fiber-Optic Device and Antibody-Coated Magnetic Beads for Semi-Continuous Monitoring of Escherichia coli O157:H7.

PubMed

Taniguchi, Midori; Saito, Hirokazu; Mitsubayashi, Kohji

2017-09-19

A rapid and reproducible fiber-optic immunosensor for Escherichia coli O157:H7 ( E. coli O157:H7) was described. The biosensor consisted of a flow cell, an optical fiber with a thin Ni layer, and a PC linked fluorometer. First, the samples with E. coli O157:H7 were incubated with magnetic beads coated with anti- E. coli O157:H7 antibodies and anti- E. coli O157:H7 antibodies labeled cyanine 5 (Cy5) to make sandwich complexes. Then the Cy5-( E. coli O157:H7)-beads were injected into a flow cell and pulled to the magnetized Ni layer on the optical fiber set in the flow cell. An excitation light (λ = 635 nm) was used to illuminate the optical fiber, and the Cy5 florescent molecules facing the optical fiber were exposed to an evanescent wave from the optical fiber. The 670 nm fluorescent light was measured using a photodiode. Finally, the magnetic intensity of the Ni layer was removed and the Cy5- E. coli O157:H7-beads were washed out for the next immunoassay. E. coli O157:H7, diluted with phosphate buffer (PB), was measured from 1 × 10⁵ to 1 × 10⁷ cells/mL. The total time required for an assay was less than 15 min (except for the pretreatment process) and repeating immunoassay on one optical fiber was made possible.

T1-Weighted MR imaging of liver tumor by gadolinium-encapsulated glycol chitosan nanoparticles without non-specific toxicity in normal tissues

NASA Astrophysics Data System (ADS)

Na, Jin Hee; Lee, Sangmin; Koo, Heebeom; Han, Hyounkoo; Lee, Kyung Eun; Han, Seung Jin; Choi, Seung Hong; Kim, Hyuncheol; Lee, Seulki; Kwon, Ick Chan; Choi, Kuiwon; Kim, Kwangmeyung

2016-05-01

Herein, we have synthesized Gd(iii)-encapsulated glycol chitosan nanoparticles (Gd(iii)-CNPs) for tumor-targeted T1-weighted magnetic resonance (MR) imaging. The T1 contrast agent, Gd(iii), was successfully encapsulated into 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-modified CNPs to form stable Gd(iii)-encapsulated CNPs (Gd(iii)-CNPs) with an average particle size of approximately 280 nm. The stable nanoparticle structure of Gd(iii)-CNPs is beneficial for liver tumor accumulation by the enhanced permeation and retention (EPR) effect. Moreover, the amine groups on the surface of Gd(iii)-CNPs could be protonated and could induce fast cellular uptake at acidic pH in tumor tissue. To assay the tumor-targeting ability of Cy5.5-labeled Gd(iii)-CNPs, near-infrared fluorescence (NIRF) imaging and MR imaging were used in a liver tumor model as well as a subcutaneous tumor model. Cy5.5-labeled Gd(iii)-CNPs generated highly intense fluorescence and T1 MR signals in tumor tissues after intravenous injection, while DOTAREM®, the commercialized control MR contrast agent, showed very low tumor-targeting efficiency on MR images. Furthermore, damaged tissues were found in the livers and kidneys of mice injected with DOTAREM®, but there were no obvious adverse effects with Gd(iii)-CNPs. Taken together, these results demonstrate the superiority of Gd(iii)-CNPs as a tumor-targeting T1 MR agent.Herein, we have synthesized Gd(iii)-encapsulated glycol chitosan nanoparticles (Gd(iii)-CNPs) for tumor-targeted T1-weighted magnetic resonance (MR) imaging. The T1 contrast agent, Gd(iii), was successfully encapsulated into 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-modified CNPs to form stable Gd(iii)-encapsulated CNPs (Gd(iii)-CNPs) with an average particle size of approximately 280 nm. The stable nanoparticle structure of Gd(iii)-CNPs is beneficial for liver tumor accumulation by the enhanced permeation and retention (EPR) effect. Moreover, the amine groups on the surface of Gd(iii)-CNPs could be protonated and could induce fast cellular uptake at acidic pH in tumor tissue. To assay the tumor-targeting ability of Cy5.5-labeled Gd(iii)-CNPs, near-infrared fluorescence (NIRF) imaging and MR imaging were used in a liver tumor model as well as a subcutaneous tumor model. Cy5.5-labeled Gd(iii)-CNPs generated highly intense fluorescence and T1 MR signals in tumor tissues after intravenous injection, while DOTAREM®, the commercialized control MR contrast agent, showed very low tumor-targeting efficiency on MR images. Furthermore, damaged tissues were found in the livers and kidneys of mice injected with DOTAREM®, but there were no obvious adverse effects with Gd(iii)-CNPs. Taken together, these results demonstrate the superiority of Gd(iii)-CNPs as a tumor-targeting T1 MR agent. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06673e

Reversible optical control of cyanine fluorescence in fixed and living cells: optical lock-in detection immunofluorescence imaging microscopy

PubMed Central

Yan, Yuling; Petchprayoon, Chutima; Mao, Shu; Marriott, Gerard

2013-01-01

Optical switch probes undergo rapid and reversible transitions between two distinct states, one of which may fluoresce. This class of probe is used in various super-resolution imaging techniques and in the high-contrast imaging technique of optical lock-in detection (OLID) microscopy. Here, we introduce optimized optical switches for studies in living cells under standard conditions of cell culture. In particular, a highly fluorescent cyanine probe (Cy or Cy3) is directly or indirectly linked to naphthoxazine (NISO), a highly efficient optical switch that undergoes robust, 405/532 nm-driven transitions between a colourless spiro (SP) state and a colourful merocyanine (MC) state. The intensity of Cy fluorescence in these Cy/Cy3-NISO probes is reversibly modulated between a low and high value in SP and MC states, respectively, as a result of Förster resonance energy transfer. Cy/Cy3-NISO probes are targeted to specific proteins in living cells where defined waveforms of Cy3 fluorescence are generated by optical switching of the SP and MC states. Finally, we introduce a new imaging technique (called OLID-immunofluorescence microscopy) that combines optical modulation of Cy3 fluorescence from Cy3/NISO co-labelled antibodies within fixed cells and OLID analysis to significantly improve image contrast in samples having high background or rare antigens. PMID:23267183

Pyrene maleimide as a probe of microenvironmental and dynamics properties of protein binding sites

NASA Astrophysics Data System (ADS)

Benci, S.; Vaccari, S.; Schianchi, G.; Locatelli, Donata; Vaghi, P.; Bottiroli, Giovanni F.

1995-01-01

N-(1-Pyrene)maleimide is highly fluorescent upon covalent binding with sulfhydryl and amino groups of the proteins. Multiexponential fluorescence decays were observed for the dye bound to different proteins even when a single binding site is involved. The lack of information about the fluorescence decay of free dye does not allow to define the variations of fluorescence parameter following the conjugation and their correlation with the binding properties of the fluorophore. In this work, a study of the fluorescence of the probe, free in solution, bound to different antibodies and to the antigen-antibody complex both in solution and in cell, has been performed. The experimental results showed that chemico-physical properties of the medium influence the fluorescence decay of the probe in both the free and bound forms, although to a different extent. The variations of fluorescence decay and anisotropy of the bound probe are related to the electronic characteristics of microenvironment and show an increased stabilization of the probe binding site with the increasing complexity of the substrate. The sensitivity of the fluorescence properties of the probe to the binding site environment opens interesting perspectives concerning the application of Py- maleimide fluorochromization to assess the degree of specificity of immunocytochemical labelling.

Combined Optical Coherence and Fluorescence Microscopy to assess dynamics and specificity of pancreatic beta-cell tracers

PubMed Central

Berclaz, Corinne; Pache, Christophe; Bouwens, Arno; Szlag, Daniel; Lopez, Antonio; Joosten, Lieke; Ekim, Selen; Brom, Maarten; Gotthardt, Martin; Grapin-Botton, Anne; Lasser, Theo

2015-01-01

The identification of a beta-cell tracer is a major quest in diabetes research. However, since MRI, PET and SPECT cannot resolve individual islets, optical techniques are required to assess the specificity of these tracers. We propose to combine Optical Coherence Microscopy (OCM) with fluorescence detection in a single optical platform to facilitate these initial screening steps from cell culture up to living rodents. OCM can image islets and vascularization without any labeling. Thereby, it alleviates the need of both genetically modified mice to detect islets and injection of external dye to reveal vascularization. We characterized Cy5.5-exendin-3, an agonist of glucagon-like peptide 1 receptor (GLP1R), for which other imaging modalities have been used and can serve as a reference. Cultured cells transfected with GLP1R and incubated with Cy5.5-exendin-3 show full tracer internalization. We determined that a dose of 1 μg of Cy5.5-exendin-3 is sufficient to optically detect in vivo the tracer in islets with a high specificity. In a next step, time-lapse OCM imaging was used to monitor the rapid and specific tracer accumulation in murine islets and its persistence over hours. This optical platform represents a versatile toolbox for selecting beta-cell specific markers for diabetes research and future clinical diagnosis. PMID:25988507

Chemo-mechanical pushing of proteins along single-stranded DNA.

PubMed

Sokoloski, Joshua E; Kozlov, Alexander G; Galletto, Roberto; Lohman, Timothy M

2016-05-31

Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3'-Cy3-labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5' to 3' ssDNA translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from translocase-induced directional (5' to 3') pushing of the SSB toward the 3' ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3' to 5') direction, are observed with EcRep and EcUvrD (both 3' to 5' ssDNA translocases). Simulations indicate that these events reflect active pushing by the translocase. The ability of translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA.

Use of Submicron Vaterite Particles Serves as an Effective Delivery Vehicle to the Respiratory Portion of the Lung

PubMed Central

Gusliakova, Olga; Atochina-Vasserman, Elena N.; Sindeeva, Olga; Sindeev, Sergey; Pinyaev, Sergey; Pyataev, Nikolay; Revin, Viktor; Sukhorukov, Gleb B.; Gorin, Dmitry; Gow, Andrew J.

2018-01-01

Nano- and microencapsulation has proven to be a useful technique for the construction of drug delivery vehicles for use in vascular medicine. However, the possibility of using these techniques within the lung as an inhalation delivery mechanism has not been previously considered. A critical element of particle delivery to the lung is the degree of penetrance that can be achieved with respect to the airway tree. In this study we examined the effectiveness of near infrared (NIR) dye (Cy7) labeled calcium carbonate (vaterite) particles of 3.15, 1.35, and 0.65 μm diameter in reaching the respiratory portion of the lung. First of all, it was shown that, interaction vaterite particles and the components of the pulmonary surfactant occurs a very strong retardation of the recrystallization and dissolution of the particles, which can subsequently be used to create systems with a prolonging release of bioactive substances after the particles penetrate the distal sections of the lungs. Submicro- and microparticles, coated with Cy7 labeled albumin as a model compound, were delivered to mouse lungs via tracheostomy with subsequent imaging performed 24, 48, and 72 h after delivery by in vivo fluorescence. 20 min post administration particles of all three sizes were visible in the lung, with the deepest penetrance observed with 0.65 μm particles. In vivo biodistribution was confirmed by fluorescence tomography imaging of excised organs post 72 h. Laser scanning confocal microscopy shows 0.65 μm particles reaching the alveolar space. The delivery of fluorophore to the blood was assessed using Cy7 labeled 0.65 μm particles. Cy7 labeled 0.65 μm particles efficiently delivered fluorescent material to the blood with a peak 3 h after particle administration. The pharmacokinetics of NIR fluorescence dye will be shown. These studies establish that by using 0.65 μm particles loaded with Cy7 we can efficiently access the respiratory portion of the lung, which represents a potentially efficient delivery mechanism for both the lung and the vasculature.

Use of Submicron Vaterite Particles Serves as an Effective Delivery Vehicle to the Respiratory Portion of the Lung.

PubMed

Gusliakova, Olga; Atochina-Vasserman, Elena N; Sindeeva, Olga; Sindeev, Sergey; Pinyaev, Sergey; Pyataev, Nikolay; Revin, Viktor; Sukhorukov, Gleb B; Gorin, Dmitry; Gow, Andrew J

2018-01-01

Nano- and microencapsulation has proven to be a useful technique for the construction of drug delivery vehicles for use in vascular medicine. However, the possibility of using these techniques within the lung as an inhalation delivery mechanism has not been previously considered. A critical element of particle delivery to the lung is the degree of penetrance that can be achieved with respect to the airway tree. In this study we examined the effectiveness of near infrared (NIR) dye (Cy7) labeled calcium carbonate (vaterite) particles of 3.15, 1.35, and 0.65 μm diameter in reaching the respiratory portion of the lung. First of all, it was shown that, interaction vaterite particles and the components of the pulmonary surfactant occurs a very strong retardation of the recrystallization and dissolution of the particles, which can subsequently be used to create systems with a prolonging release of bioactive substances after the particles penetrate the distal sections of the lungs. Submicro- and microparticles, coated with Cy7 labeled albumin as a model compound, were delivered to mouse lungs via tracheostomy with subsequent imaging performed 24, 48, and 72 h after delivery by in vivo fluorescence. 20 min post administration particles of all three sizes were visible in the lung, with the deepest penetrance observed with 0.65 μm particles. In vivo biodistribution was confirmed by fluorescence tomography imaging of excised organs post 72 h. Laser scanning confocal microscopy shows 0.65 μm particles reaching the alveolar space. The delivery of fluorophore to the blood was assessed using Cy7 labeled 0.65 μm particles. Cy7 labeled 0.65 μm particles efficiently delivered fluorescent material to the blood with a peak 3 h after particle administration. The pharmacokinetics of NIR fluorescence dye will be shown. These studies establish that by using 0.65 μm particles loaded with Cy7 we can efficiently access the respiratory portion of the lung, which represents a potentially efficient delivery mechanism for both the lung and the vasculature.

Identification of potential serum markers for nasopharyngeal carcinoma from a xenografted mouse model using Cy-dye labeling combined with three-dimensional fractionation.

PubMed

Wu, Chih-Ching; Peng, Pei-Hua; Chang, Ya-Ting; Huang, Yu-Shan; Chang, Kai-Ping; Hao, Sheng-Po; Tsang, Ngan-Ming; Yeh, Chau-Ting; Chang, Yu-Sun; Yu, Jau-Song

2008-09-01

Nasopharyngeal carcinoma (NPC), one of the most common cancers in Southeast Asia, is commonly diagnosed late due to its deep location and vague symptoms. To identify biomarkers for improving NPC diagnosis, we established a proteomic platform for detecting aberrant serum proteins in nude mice bearing NPC xenografts. We first removed the three most abundant proteins from serum samples of tumor-bearing and control mice, and then labeled the samples with different fluorescent cyanine (Cy) dyes. The labeled serum proteins were then mixed equally and fractionated with ion-exchange chromatography followed by SDS-PAGE. Differentially expressed proteins were identified by in-gel tryptic digestion and MALDI-TOF MS. We identified peroxiredoxin 2 (Prx-II) and carbonic anhydrase 2 (CA-II) as being elevated in the xenograft mouse model compared to controls. Western blot analysis confirmed up-regulation of Prx-II and CA-II in plasma from five NPC patients, and ELISA showed that plasma Prx-II levels were significantly higher in NPC patients (n = 84) versus healthy controls (n = 90) (3.03 +/- 4.47 versus 1.90 +/- 2.74 microg/mL, p = 0.047). In conclusion, Cy dye labeling combined with three-dimensional fractionation is a feasible strategy for identifying differentially expressed serum proteins in an NPC xenograft model, and Prx-II may represent a potential NPC biomarker.

Sensitive and background-free determination of thiols from wastewater samples by MOF-5 extraction coupled with high-performance liquid chromatography with fluorescence detection using a novel fluorescence probe of carbazole-9-ethyl-2-maleimide.

PubMed

Lv, Zhengxian; Sun, Zhiwei; Song, Cuihua; Lu, Shuaimin; Chen, Guang; You, Jinmao

2016-12-01

A sensitive and background-free pre-column derivatization method for the determination of thiol compounds using metal-organic framework material (MOF-5) as dispersive solid-phase extraction (DSPE) adsorbent followed by high-performance liquid chromatography fluorescence detection (HPLC-FLD) has been developed. In this paper, a novel labeling reagent, carbazole-9-ethyl-2-maleimide(CAEM), was synthesized and reacted with thiols at 40°C for 10min in the presence of PBS buffer (0.02mol/L, pH 7.5). Interestingly, CAEM itself had no fluorescence, while its derivatives exhibited intense fluorescence with an excitation maximum at λ ex 274nm and an emission maximum at λ em 363nm, which greatly reduced the background interference and improved the sensitivity of the method. Furthermore, the MOF-5 was prepared and used as DSPE adsorbent for the selective adsorption of thiols from wastewater sample. Under the optimized experimental conditions, an excellent linearity for all analytes over their concentration ranges of 0.01-1.0μmol/L (R 2 >0.9986)were obtained with the limit of detection (LOD) ranging from 8 to 17.1pmol/L for nine tested thiols. The feasibility of this method for the determination of thiols in wastewater samples had been evaluated and satisfactory average recoveries (n=3) were achieved with the range of 86.6-98.5%. Copyright © 2016 Elsevier B.V. All rights reserved.

Pharmacokinetics of heparin and related polysaccharides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boneu, B.; Dol, F.; Caranobe, C.

1989-01-01

The pharmacodynamic profile of standard heparin (SH), a low molecular weight derivative (CY 216) and of dermatan sulfate (DS), a new potential antithrombotic drug, was investigated in the rabbit over a large range of doses. After bolus i.v. injection of low doses, the biological activity of SH disappeared exponentially; however, its half-life was prolonged when the dose injected increased, and over 158 micrograms/kg (100 anti-factor Xa U/kg) the biological activity disappeared as a concave-convex curve. CY 216 disappeared more slowly than SH at low doses but faster than SH at higher doses. More than 90% of the DS biological activitymore » present 1 minute after the i.v. injection disappeared exponentially without dose-dependent effects. Increasing doses of the three drugs were then delivered for 5 h under continuous infusions. Below 500 micrograms/kg/h the DS and CY 216 plateau concentrations were higher than that of SH while above this dose the SH concentration was higher than that of DS and CY 216. These observations may be explained by the results of pharmacokinetics experiments where /sup 125/I-labeled compounds were delivered by bolus i.v. injection in association with increasing doses of their unlabeled counterparts. For SH there was a 10-fold difference between the half-life of the lower dose (32 micrograms/kg or 5 anti-factor Xa U/kg) and that of the higher dose (3200 micrograms/kg); it was demonstrated that the half-life of SH continuously shortened as its plasma concentration decreased. In contrast the CY 216 and DS half-lives were very close, independent of the dose delivered, and therefore longer than that of SH at low doses and shorter than that of SH at higher doses.« less

In vivo fluorescence imaging of hepatocellular carcinoma xenograft using near-infrared labeled epidermal growth factor receptor (EGFR) peptide

PubMed Central

Li, Z.; Zhou, Q.; Zhou, J.; Duan, X.; Zhu, J.; Wang, T. D.

2016-01-01

Minimally-invasive surgery of hepatocellular carcinoma (HCC) can be limited by poor tumor visualization with white light. We demonstrate systemic administration of a Cy5.5-labeled peptide specific for epidermal growth factor receptor (EGFR) to target HCC in vivo in a mouse xenograft model. We attached a compact imaging module to the proximal end of a medical laparoscope to collect near-infrared fluorescence and reflectance images concurrently at 15 frames/sec. We measured a mean target-to-background ratio of 2.99 ± 0.22 from 13 surgically exposed subcutaneous human HCC tumors in vivo in 5 mice. This integrated imaging methodology is promising to guide laparoscopic resection of HCC. PMID:27699089

Dendrimer probes for enhanced photostability and localization in fluorescence imaging.

PubMed

Kim, Younghoon; Kim, Sung Hoon; Tanyeri, Melikhan; Katzenellenbogen, John A; Schroeder, Charles M

2013-04-02

Recent advances in fluorescence microscopy have enabled high-resolution imaging and tracking of single proteins and biomolecules in cells. To achieve high spatial resolutions in the nanometer range, bright and photostable fluorescent probes are critically required. From this view, there is a strong need for development of advanced fluorescent probes with molecular-scale dimensions for fluorescence imaging. Polymer-based dendrimer nanoconjugates hold strong potential to serve as versatile fluorescent probes due to an intrinsic capacity for tailored spectral properties such as brightness and emission wavelength. In this work, we report a new, to our knowledge, class of molecular probes based on dye-conjugated dendrimers for fluorescence imaging and single-molecule fluorescence microscopy. We engineered fluorescent dendritic nanoprobes (FDNs) to contain multiple organic dyes and reactive groups for target-specific biomolecule labeling. The photophysical properties of dye-conjugated FDNs (Cy5-FDNs and Cy3-FDNs) were characterized using single-molecule fluorescence microscopy, which revealed greatly enhanced photostability, increased probe brightness, and improved localization precision in high-resolution fluorescence imaging compared to single organic dyes. As proof-of-principle demonstration, Cy5-FDNs were used to assay single-molecule nucleic acid hybridization and for immunofluorescence imaging of microtubules in cytoskeletal networks. In addition, Cy5-FDNs were used as reporter probes in a single-molecule protein pull-down assay to characterize antibody binding and target protein capture. In all cases, the photophysical properties of FDNs resulted in enhanced fluorescence imaging via improved brightness and/or photostability. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Hsp47 and cyclophilin B traverse the endoplasmic reticulum with procollagen into pre-Golgi intermediate vesicles. A role for Hsp47 and cyclophilin B in the export of procollagen from the endoplasmic reticulum.

PubMed

Smith, T; Ferreira, L R; Hebert, C; Norris, K; Sauk, J J

1995-08-04

Hsp47 and cyclophilin B (CyPB) are residents of the endoplasmic reticulum (ER). Both of these proteins are closely associated with polysome-associated alpha 1(I) procollagen chains. Hsp47 possesses chaperone properties early during the translation of procollagen while the cis/trans-isomerase properties of CyPB facilitate procollagen folding. In this report, we further investigate the interaction of these proteins with procollagen I during export from the ER. To inhibit vesicular budding and retain procollagen within the ER, cells were treated with the heterotrimeric G protein inhibitor mastoparan or calphostin C, a specific inhibitor of diacylglycerol/phorbol ester binding proteins. To arrest procollagen in pre-Golgi intermediate vesicles, cells were treated with guanosine 5'-3-O-(thio)triphosphate. Pulse-chase experiments of cells labeled with [35S]methionine followed by immunoprecipitation during the chase period with anti-procollagen, anti-Hsp47, and anti-CyPB antibodies were performed to reveal the relationship between Hsp47/CyPB/procollagen I. The distribution of procollagen, Hsp47, and CyPB to the ER and/or pre-Golgi vesicles was verified by immunofluorescence. Hsp47 and CyPB remained associated with procollagen retained within the ER. Hsp47 and CyPB were also associated with procollagen exported from the ER into pre-Golgi intermediate vesicles. Treatment of cells with cyclosporin A diminished the levels of CyPB bound to procollagen and diminished the rate of Hsp47 released from procollagen and the rate of procollagen secretion, suggesting that Hsp47 release from procollagen may be driven by helix formation. Also, these studies suggest that Hsp47 may resemble protein disulfide isomerase and possess both chaperone and anti-chaperone properties. During translation, high levels of Hsp47 are seen to limit protein aggregation and facilitate chain registration. Later, Hsp47 and/or CyPB and protein disulfide isomerase act as anti-chaperones and provide the basis for concentration of procollagen for ER export.

A novel BRET-based binding assay for interaction studies of relaxin family peptide receptor 3 with its ligands.

PubMed

Wang, Jia-Hui; Shao, Xiao-Xia; Hu, Meng-Jun; Wei, Dian; Liu, Ya-Li; Xu, Zeng-Guang; Guo, Zhan-Yun

2017-05-01

Relaxin family peptide receptor 3 (RXFP3) is an A-class G protein-coupled receptor that is implicated in the regulation of food intake and stress response upon activation by its cognate agonist relaxin-3. To study its interaction with various ligands, we developed a novel bioluminescence resonance energy transfer (BRET)-based binding assay using the brightest NanoLuc as an energy donor and a newly developed cyan-excitable orange fluorescent protein (CyOFP) as an energy acceptor. An engineered CyOFP without intrinsic cysteine residues but with an introduced cysteine at the C-terminus was overexpressed in Escherichia coli and chemically conjugated to the A-chain N-terminus of an easily labeled chimeric R3/I5 peptide via an intermolecular disulfide linkage. After the CyOFP-conjugated R3/I5 bound to a shortened human RXFP3 (removal of 33 N-terminal residues) fused with the NanoLuc reporter at the N-terminus, high BRET signals were detected. Saturation binding and real-time binding assays demonstrated that this BRET pair retained high binding affinity with fast association/dissociation. Using this BRET pair, binding potencies of various ligands with RXFP3 were conveniently measured through competition binding assays. Thus, the novel BRET-based binding assay facilitates interaction studies of RXFP3 with various ligands. The engineered CyOFP without intrinsic cysteine residues may also be applied to other BRET-based binding assays in future studies.

Spin-labelling study of interactions of ovalbumin with multilamellar liposomes and specific anti-ovalbumin antibodies.

PubMed

Brgles, Marija; Mirosavljević, Krunoslav; Noethig-Laslo, Vesna; Frkanec, Ruza; Tomasić, Jelka

2007-03-10

Ovalbumin (OVA) has been used continuously as the model antigen in numerous studies of immune reactions and antigen processing, very often encapsulated into liposomes. The purpose of this work was to study the possible interactions of spin-labelled OVA and lipids in liposomal membranes using electron spin resonance (ESR) spectroscopy. OVA was covalently spin-labelled with 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO-maleimide), characterized and encapsulated into multilamellar, negatively charged liposomes. ESR spectra of this liposomal preparation gave evidence for the interaction of OVA with the lipid bilayers. Such an interaction was also evidenced by the ESR spectra of liposomal preparation containing OVA, where liposomes were spin-labelled with n-doxyl stearic acids. The spin-labelled OVA retains its property to bind specific anti-OVA antibodies, as shown by ESR spectroscopy, but also in ELISA for specific anti-OVA IgG.

Statistical methodology for the analysis of dye-switch microarray experiments

PubMed Central

Mary-Huard, Tristan; Aubert, Julie; Mansouri-Attia, Nadera; Sandra, Olivier; Daudin, Jean-Jacques

2008-01-01

Background In individually dye-balanced microarray designs, each biological sample is hybridized on two different slides, once with Cy3 and once with Cy5. While this strategy ensures an automatic correction of the gene-specific labelling bias, it also induces dependencies between log-ratio measurements that must be taken into account in the statistical analysis. Results We present two original statistical procedures for the statistical analysis of individually balanced designs. These procedures are compared with the usual ML and REML mixed model procedures proposed in most statistical toolboxes, on both simulated and real data. Conclusion The UP procedure we propose as an alternative to usual mixed model procedures is more efficient and significantly faster to compute. This result provides some useful guidelines for the analysis of complex designs. PMID:18271965

Evaluation of a 99mTc-labeled AnnexinA5 variant for non-invasive SPECT imaging of cell death in liver, spleen and prostate.

PubMed

Greupink, Rick; Sio, Charles F; Ederveen, Antwan; Orsel, Joke

2009-12-01

We investigate radio-labeling and pharmacokinetics of a new AnnexinA5 variant (HYNIC-cys-AnxA5) and then assess its utility for the non-invasive detection of cell death in liver, spleen and prostate. AnnexinA5 binds to phosphatidylserine expressed on the surface of apoptotic and necrotic cells. Contrary to other AnnexinA5 variants, the new cys-AnxA5 allows for site-specific conjugation of a hydrazinonicotinamide-maleimide moiety and subsequent radio-labeling with (99m)Tc at a position not involved in the AnxA5-phosphatidylserine interaction. Distribution of (99m)Tc-HYNIC-cys-AnxA5 was studied in rats, both invasively and via SPECT/CT. Cycloheximide was used to induce cell death in liver and spleen, whereas apoptosis in the prostate was induced by castration. HYNIC-cys-AnxA5 was efficiently and reproducibly labeled with (99m)Tc. Blood clearance of radioactivity after iv-injection was adequately described by a two-compartment model, the renal cortex representing the main site of accumulation. Cycloheximide treatment resulted in increased accumulation of intravenous-injected (99m)Tc-HYNIC-cys-AnxA5 in liver and spleen over controls, which correlated well with TUNEL staining for cell death in corresponding tissue sections. However, the increase in TUNEL-positive prostate epithelial cells observed following castration was not paralleled by greater (99m)Tc-HYNIC-cys-AnxA5 accumulation. (99m)Tc-HYNIC-cys-AnxA5 appears a suitable tracer for assessment of cell death in liver and spleen, but not prostate.

Optical Fluorescent Imaging to Monitor Temporal Effects of Microbubble-Mediated Ultrasound Therapy

PubMed Central

Sorace, Anna G.; Saini, Reshu; Rosenthal, Eben; Warram, Jason M.; Zinn, Kurt R.; Hoyt, Kenneth

2013-01-01

Microbubble-mediated ultrasound therapy can noninvasively enhance drug delivery to localized regions in the body. This technique can be beneficial in cancer therapy, but currently there are limitations to tracking the therapeutic effects. The purpose of this experiment was to investigate the potential of fluorescent imaging for monitoring the temporal effects of microbubble-mediated ultrasound therapy. Mice were implanted with 2LMP breast cancer cells. The animals underwent microbubble-mediated ultrasound therapy in the presence of Cy5.5 fluorescent-labeled IgG antibody (large molecule) or Cy5.5 dye (small molecule) and microbubble contrast agents. Control animals were administered fluorescent molecules only. Animals were transiently imaged in vivo at 1, 10, 30, and 60 min post therapy using a small animal optical imaging system. Tumors were excised and analyzed ex vivo. Tumors were homogenized and emulsion imaged for Cy5.5 fluorescence. Monitoring in vivo results showed significant influx of dye into the tumor (p < 0.05) using the small molecule, but not in the large molecule group (p > 0.05). However, after tumor emulsion, significantly higher dye concentration was detected in therapy group tumors for both small and large molecule groups in comparison to their control counterparts (p < 0.01). This paper explores a noninvasive optical imaging method for monitoring the effects of microbubble-mediated ultrasound therapy in a cancer model. It provides temporal information following the process of increasing extravasation of molecules into target tumors. PMID:23357902

Optical fluorescent imaging to monitor temporal effects of microbubble-mediated ultrasound therapy.

PubMed

Sorace, Anna G; Saini, Reshu; Rosenthal, Eben; Warram, Jason M; Zinn, Kurt R; Hoyt, Kenneth

2013-02-01

Microbubble-mediated ultrasound therapy can noninvasively enhance drug delivery to localized regions in the body. This technique can be beneficial in cancer therapy, but currently there are limitations to tracking the therapeutic effects. The purpose of this experiment was to investigate the potential of fluorescent imaging for monitoring the temporal effects of microbubble-mediated ultrasound therapy. Mice were implanted with 2LMP breast cancer cells. The animals underwent microbubble-mediated ultrasound therapy in the presence of Cy5.5 fluorescent-labeled IgG antibody (large molecule) or Cy5.5 dye (small molecule) and microbubble contrast agents. Control animals were administered fluorescent molecules only. Animals were transiently imaged in vivo at 1, 10, 30, and 60 min post therapy using a small animal optical imaging system. Tumors were excised and analyzed ex vivo. Tumors were homogenized and emulsion imaged for Cy5.5 fluorescence. Monitoring in vivo results showed significant influx of dye into the tumor (p < 0.05) using the small molecule, but not in the large molecule group (p > 0.05). However, after tumor emulsion, significantly higher dye concentration was detected in therapy group tumors for both small and large molecule groups in comparison to their control counterparts (p <0.01). This paper explores a noninvasive optical imaging method for monitoring the effects of microbubble-mediated ultrasound therapy in a cancer model. It provides temporal information following the process of increasing extravasation of molecules into target tumors.

Scanning confocal fluorescence microscopy for single molecule analysis of nucleotide excision repair complexes.

PubMed

Segers-Nolten, G M J; Wyman, C; Wijgers, N; Vermeulen, W; Lenferink, A T M; Hoeijmakers, J H J; Greve, J; Otto, C

2002-11-01

We used scanning confocal fluorescence microscopy to observe and analyze individual DNA- protein complexes formed between human nucleotide excision repair (NER) proteins and model DNA substrates. For this purpose human XPA protein was fused to EGFP, purified and shown to be functional. Binding of EGFP-labeled XPA protein to a Cy3.5-labeled DNA substrate, in the presence and absence of RPA, was assessed quantitatively by simultaneous excitation and emission detection of both fluorophores. Co-localization of Cy3.5 and EGFP signals within one diffraction limited spot indicated complexes of XPA with DNA. Measurements were performed on samples in a 1% agarose matrix in conditions that are compatible with protein activity and where reactions can be studied under equilibrium conditions. In these samples DNA alone was freely diffusing and protein-bound DNA was immobile, whereby they could be discriminated resulting in quantitative data on DNA binding. On the single molecule level approximately 10% of XPA co-localized with DNA; this increased to 32% in the presence of RPA. These results, especially the enhanced binding of XPA in the presence of RPA, are similar to those obtained in bulk experiments, validating the utility of scanning confocal fluorescence microscopy for investigating functional interactions at the single molecule level.

Chemo-mechanical pushing of proteins along single-stranded DNA

PubMed Central

Sokoloski, Joshua E.; Kozlov, Alexander G.; Galletto, Roberto; Lohman, Timothy M.

2016-01-01

Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3′-Cy3–labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5′ to 3′ ssDNA translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from translocase-induced directional (5′ to 3′) pushing of the SSB toward the 3′ ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3′ to 5′) direction, are observed with EcRep and EcUvrD (both 3′ to 5′ ssDNA translocases). Simulations indicate that these events reflect active pushing by the translocase. The ability of translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA. PMID:27185951

Comment on "An unexpected formation of the novel 7-oxa-2-azabicyclo[2.2.1]hept-5-ene skeleton during the reaction of furfurylamine with maleimides and their bioprospection using a zebrafish embryo model" by C. E. Puerto Galvis and V. V. Kouznetsov, Org. Biomol. Chem., 2013, 11, 407.

PubMed

Zubkov, F I; Kvyatkovskaya, E A; Nikitina, E V; Amoyaw, P N-A; Kouznetsov, V V; Lazarenko, V A; Khrustalev, V N

2017-08-02

It has been proved that the reaction between furfuryl amines and N-R-maleimides leads to the formation of aza-Michael addition products - 3-(furylmethylamino)-N-R-pyrrolidine-2,5-diones, instead of 7-oxa-2-azabicyclo[2.2.1]hept-5-enes, as this journal reported previously.

Highly sensitive detection of human IgG using a novel bio-barcode assay combined with DNA chip technology

NASA Astrophysics Data System (ADS)

Liu, Zhenbao; Zhou, Bo; Wang, Haiqing; Lu, Feng; Liu, Tianjun; Song, Cunxian; Leng, Xigang

2013-09-01

A simple and ultrasensitive detection of human IgG based on signal amplification using a novel bio-barcode assay and DNA chip technology was developed. The sensing platform was a sandwich system made up of antibody-modified magnetic microparticles (Ab-MMPs)/human IgG/Cy3-labeled single-stranded DNA and antibody-modified gold nanoparticles (Cy3-ssDNA-Ab-AuNPs). The MMPs (2.5 μm in diameter) modified with mouse anti-human IgG monoclonal-antibodies could capture human IgG and further be separated and enriched via a magnetic field. The AuNPs (13 nm in diameter) conjugated with goat anti-human IgG polyclonal-antibodies and Cy3-ssDNA could further combine with the human IgG/Ab-MMP complex. The Cy3-ssDNA on AuNPs was then released by TCEP to hybridize with the DNA chip, thus generating a detectable signal by the fluorescence intensity of Cy3. In order to improve detection sensitivity, a three-level cascaded signal amplification was developed: (1) The MMP enrichment as the first-level; (2) Large quantities of Cy3-ssDNA on AuNPs as the second-level; (3) The Cy3-ssDNA conjugate with DNA chip as the third-level. The highly sensitive technique showed an increased response of the fluorescence intensity to the increased concentration of human IgG through a detection range from 1 pg mL-1 to 10 ng mL-1. This sensing technique could not only improve the detection sensitivity for the low concentration of human IgG but also present a robust and efficient signal amplification model. The detection method has good stability, specificity, and reproducibility and could be applied in the detection of human IgG in the real samples.

Evaluating the efficacy of subcellular fractionation of blast cells using live cell labeling and 2D DIGE.

PubMed

Ho, Yin Ying; Penno, Megan; Perugini, Michelle; Lewis, Ian; Hoffmann, Peter

2012-01-01

Labeling of exposed cell surface proteins of live cells using CyDye DIGE fluor minimal dyes is an efficient strategy for cell surface proteome profiling and quantifying differentially expressed proteins in diseases. Here we describe a strategy to evaluate a two-step detergent-based protein fractionation method using live cell labeling followed by visualization of the fluorescently labeled cell surface proteins and fractionated proteins within a single 2D gel.

Cy5.5 conjugated MnO nanoparticles for magnetic resonance/near-infrared fluorescence dual-modal imaging of brain gliomas.

PubMed

Chen, Ning; Shao, Chen; Li, Shuai; Wang, Zihao; Qu, Yanming; Gu, Wei; Yu, Chunjiang; Ye, Ling

2015-11-01

The fusion of molecular and anatomical modalities facilitates more reliable and accurate detection of tumors. Herein, we prepared the PEG-Cy5.5 conjugated MnO nanoparticles (MnO-PEG-Cy5.5 NPs) with magnetic resonance (MR) and near-infrared fluorescence (NIRF) imaging modalities. The applicability of MnO-PEG-Cy5.5 NPs as a dual-modal (MR/NIRF) imaging nanoprobe for the detection of brain gliomas was investigated. In vivo MR contrast enhancement of the MnO-PEG-Cy5.5 nanoprobe in the tumor region was demonstrated. Meanwhile, whole-body NIRF imaging of glioma bearing nude mouse exhibited distinct tumor localization upon injection of MnO-PEG-Cy5.5 NPs. Moreover, ex vivo CLSM imaging of the brain slice hosting glioma indicated the preferential accumulation of MnO-PEG-Cy5.5 NPs in the glioma region. Our results therefore demonstrated the potential of MnO-PEG-Cy5.5 NPs as a dual-modal (MR/NIRF) imaging nanoprobe in improving the diagnostic efficacy by simultaneously providing anatomical information from deep inside the body and more sensitive information at the cellular level. Copyright © 2015 Elsevier Inc. All rights reserved.

mTORC1/2 and rapamycin in female Han:SPRD rats with polycystic kidney disease.

PubMed

Belibi, Franck; Ravichandran, Kameswaran; Zafar, Iram; He, Zhibin; Edelstein, Charles L

2011-01-01

Rapamycin slows disease progression in the male Han:SPRD (Cy/+) rat with polycystic kidney disease (PKD). The aim of this study was to determine the effect of rapamycin on PKD and the relative contributions of the proproliferative mammalian target of rapamycin complexes 1 and 2 (mTORC1 and mTORC2) in female Cy/+ rats. Female Cy/+ rats were treated with rapamycin from 4 to 12 wk of age. In vehicle-treated Cy/+ rats, kidney volume increased by 40% and cyst volume density (CVD) was 19%. Phosphorylated S6 (p-S6) ribosomal protein, a marker of mTORC1 activity, was increased in Cy/+ rats compared with normal littermate controls (+/+) and decreased by rapamycin. Despite activation of mTORC1 in female Cy/+ rats, rapamycin had no effect on kidney size, CVD, number of PCNA-positive cystic tubular cells, caspase-3 activity, or the number of terminal deoxynucleotidyl transferase dUTP-mediated nick-end label-positive apoptotic cells. To determine a reason for the lack of effect of rapamycin, we studied the mTORC2 signaling pathway. On immunoblot of kidney, phosphorylated (Ser473) Akt (p-Akt), a marker of mTORC2 activity, was increased in female Cy/+ rats treated with rapamycin. Phosphorylated (Ser657) PKCα, a substrate of mTORC2, was unaffected by rapamycin in females. In contrast, in male rats, where rapamycin significantly decreases PKD, p-Akt (Ser473) was decreased by rapamcyin. PKCα (Ser657) was increased in male Cy/+ rats but was unaffected by rapamycin. In summary, in female Cy/+ rats, rapamycin had no effect on PKD and proproliferative p-Akt (Ser473) activity was increased by rapamycin. There were differential effects of rapamycin on mTORC2 signaling in female vs. male Cy/+ rats.

Near-Infrared Fluorescent Deoxyglucose Analog for Tumor Optical Imaging in Cell Culture and in Living Mice

PubMed Central

Cheng, Zhen; Levi, Jelena; Xiong, Zhengming; Gheysens, Olivier; Keren, Shay; Chen, Xiaoyuan; Gambhir, Sanjiv Sam

2011-01-01

2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG) has extensively been used for clinical diagnosis, staging and therapy monitoring of cancer and other diseases. Non-radioactive glucose analogs enabling the screening of the glucose metabolic rate of tumors are of particular interest for anticancer drug development. A non-radioactive fluorescent deoxyglucose analog may have many applications for both imaging of tumors and monitoring therapeutic efficacy of drugs in living animals and may eventually translate to clinical applications. We found that a fluorescent 2-deoxyglucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) can be delivered in several tumor cells via the glucose transporters (GLUTs). We therefore conjugated d-glucosamine with a near-infrared (NIR) fluorphor Cy5.5 and tested the feasibility of Cy5.5-d-glucosamine conjugate (Cy5.5-2DG) for NIR fluorescence imaging of tumors in a pre-clinical xenograft animal model. Cy5.5-2DG was prepared by conjugating Cy5.5 monofunctional N-hydroxysuccinimide ester (Cy5.5-NHS) and d-glucosamine followed by high-performance liquid chromatography purification. The accumulation of Cy5.5-2DG and Cy5.5-NHS in different tumor cell lines at 37 °C and 4 °C were imaged using a fluorescence microscope. Tumor targeting and retention of Cy5.5-2DG and Cy5.5-NHS in a subcutaneous U87MG glioma and A375M melanoma tumor model were evaluated and quantified by a Xenogen IVIS 200 optical cooled charged-coupled device system. Fluorescence microscopy imaging shows that Cy5.5-2DG and Cy5.5-NHS are taken up and trapped by a variety of tumor cell lines at 37 °C incubation, while they exhibit marginal uptake at 4 °C. The tumor cell uptake of Cy5.5-2DG can not be blocked by the 50 mM d-glucose, suggesting that Cy5.5-2DG may not be delivered in tumor cells by GLUTs. U87MG and A375M tumor localization were clearly visualized in living mice with both NIR fluorescent probes. Tumor/muscle contrast was clearly visible as early as 30 min post-injection, and the highest U87MG tumor/muscle ratio of 2.81 ± 0.10, 3.34 ± 0.23 were achieved 24 hours post-injection for Cy5.5-2DG and Cy5.5-NHS, respectively. While as a comparison, the micro-positron emission tomography imaging study shows that [18F]FDG preferentially localize to the U87MG tumor, with resulting tumor/muscle ratios ranging from 3.89 to 4.08 after 30 min to 2 h post-administration of the probe. In conclusion, the NIR fluorescent glucose analog, Cy5.5-2DG and Cy5.5-NHS both demonstrate tumor targeting abilities in cell culture and in living mice. More studies are warranted to further explore their application for optical tumor imaging. In order to develop NIR glucose analog with ability to targeting GLUTs/hexokinase, it is highly important to select NIR dyes with reasonable molecular size. PMID:16704203

Preparation and evaluation of APTES-PEG coated iron oxide nanoparticles conjugated to rhenium-188 labeled rituximab.

PubMed

Azadbakht, Bakhtiar; Afarideh, Hossein; Ghannadi-Maragheh, Mohammad; Bahrami-Samani, Ali; Asgari, Mehdi

2017-05-01

Radioimmuno-conjugated (Rhenium-188 labeled Rituximab), 3-aminopropyltriethoxysilane (APTES)-polyethylene glycol (PEG) coated iron oxide nanoparticles were synthesized and then characterized. Therapeutic effect and targeting efficacy of complex were evaluated in CD20 express B cell lines and tumor bearing Balb/c mice respectively. To reach these purposes, superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized using coprecipitation method and then their surface was treated with APTES for increasing retention time of SPIONs in blood circulation and amine group creation. In the next step, N-hydroxysuccinimide (NHS) ester of polyethylene glycol maleimide (NHS-PEG-Mal) was conjugated to the APTES-treated SPIONs. After radiolabeling of Rituximab antibody with Rhenium-188 (T 1/2 =16.9h) using synthesized N 2 S 4 chelator, it was attached to the APTES-PEG-MAL-SPIONs surface through thiol-maleimide coupling reaction. In vitro evaluation of the 188 ReN 2 S 4 -Rituximab-SPION-complex thus obtained revealed that at 24 and 48h post-treatment effective cancer cell killing had been achieved. Bio-distribution study in tumor bearing mice showed capability of this complex for targeted cancer therapy. Active and passive tumor targeting strategies were applied through incorporated anti-CD20 (Rituximab) antibody and also enhanced permeability and retention (EPR) effect of solid tumors for nanoparticles respectively. Copyright © 2016 Elsevier Inc. All rights reserved.

Combined image guided monitoring the pharmacokinetics of rapamycin loaded human serum albumin nanoparticles with a split luciferase reporter

NASA Astrophysics Data System (ADS)

Wang, Fu; Yang, Kai; Wang, Zhe; Ma, Ying; Gutkind, J. Silvio; Hida, Naoki; Niu, Gang; Tian, Jie

2016-02-01

Imaging guided techniques have been increasingly employed to investigate the pharmacokinetics (PK) and biodistribution of nanoparticle based drug delivery systems. In most cases, however, the PK profiles of drugs could vary significantly from those of drug delivery carriers upon administration in the blood circulation, which complicates the interpretation of image findings. Herein we applied a genetically encoded luciferase reporter in conjunction with near infrared (NIR) fluorophores to investigate the respective PK profiles of a drug and its carrier in a biodegradable drug delivery system. In this system, a prototype hydrophobic agent, rapamycin (Rapa), was encapsulated into human serum albumin (HSA) to form HSA Rapa nanoparticles, which were then labeled with Cy5 fluorophore to facilitate the fluorescence imaging of HSA carrier. Meanwhile, we employed transgenetic HN12 cells that were modified with a split luciferase reporter, whose bioluminescence function is regulated by Rapa, to reflect the PK profile of the encapsulated agent. It was interesting to discover that there existed an obvious inconsistency of PK behaviors between HSA carrier and rapamycin in vitro and in vivo through near infrared fluorescence imaging (NIFRI) and bioluminescence imaging (BLI) after treatment with Cy5 labeled HSA Rapa. Nevertheless, HSA Rapa nanoparticles manifested favorable in vivo PK and tumor suppression efficacy in a follow-up therapeutic study. The developed strategy of combining a molecular reporter and a fluorophore in this study could be extended to other drug delivery systems to provide profound insights for non-invasive real-time evaluation of PK profiles of drug-loaded nanoparticles in pre-clinical studies.Imaging guided techniques have been increasingly employed to investigate the pharmacokinetics (PK) and biodistribution of nanoparticle based drug delivery systems. In most cases, however, the PK profiles of drugs could vary significantly from those of drug delivery carriers upon administration in the blood circulation, which complicates the interpretation of image findings. Herein we applied a genetically encoded luciferase reporter in conjunction with near infrared (NIR) fluorophores to investigate the respective PK profiles of a drug and its carrier in a biodegradable drug delivery system. In this system, a prototype hydrophobic agent, rapamycin (Rapa), was encapsulated into human serum albumin (HSA) to form HSA Rapa nanoparticles, which were then labeled with Cy5 fluorophore to facilitate the fluorescence imaging of HSA carrier. Meanwhile, we employed transgenetic HN12 cells that were modified with a split luciferase reporter, whose bioluminescence function is regulated by Rapa, to reflect the PK profile of the encapsulated agent. It was interesting to discover that there existed an obvious inconsistency of PK behaviors between HSA carrier and rapamycin in vitro and in vivo through near infrared fluorescence imaging (NIFRI) and bioluminescence imaging (BLI) after treatment with Cy5 labeled HSA Rapa. Nevertheless, HSA Rapa nanoparticles manifested favorable in vivo PK and tumor suppression efficacy in a follow-up therapeutic study. The developed strategy of combining a molecular reporter and a fluorophore in this study could be extended to other drug delivery systems to provide profound insights for non-invasive real-time evaluation of PK profiles of drug-loaded nanoparticles in pre-clinical studies. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07308a

A new approach to the synthesis of monomers and polymers incorporating furan/maleimide Diels-Alder adducts

NASA Astrophysics Data System (ADS)

Banella, Maria Barbara; Gioia, Claudio; Vannini, Micaela; Colonna, Martino; Celli, Annamaria; Gandini, Alessandro

2016-05-01

The Diels-Alder reaction between furan and maleimide moieties is a well-known and widely used strategy to build bio-based macromolecular structures with peculiar properties. The furan-maleimide adducts are thermally reversible because they can be broken above about 120°C and recombined at lower temperatures. At the moment only the monomers exhibiting the furan or the maleimide moieties on their extremity are used in order to get linear or cross-linked polymeric structures. The innovative idea described here consists in using a monomer bearing two carboxylic acidic groups on its extremities and a furan-maleimide Diels-Alder adduct within its structure. This monomer can give rise to classical polycondensation reactions leading to polymers. These polymers (which are polyesters in the present case) can be broken at high temperatures in correspondence of the furane-maleimide Diels-Alder adduct leading to segments exhibiting furan or maleimide moieties at their extremities, which at lower temperature recombine leading to random or block copolymers.

Molecular recognition of a model globular protein apomyoglobin by synthetic receptor cyclodextrin: effect of fluorescence modification of the protein and cavity size of the receptor in the interaction.

PubMed

Saha, Ranajay; Rakshit, Surajit; Pal, Samir Kumar

2013-11-01

Labelling of proteins with some extrinsic probe is unavoidable in molecular biology research. Particularly, spectroscopic studies in the optical region require fluorescence modification of native proteins by attaching polycyclic aromatic fluoroprobe with the proteins under investigation. Our present study aims to address the consequence of the attachment of a fluoroprobe at the protein surface in the molecular recognition of the protein by selectively small model receptor. A spectroscopic study involving apomyoglobin (Apo-Mb) and cyclodextrin (CyD) of various cavity sizes as model globular protein and synthetic receptors, respectively, using steady-state and picosecond-resolved techniques, is detailed here. A study involving Förster resonance energy transfer, between intrinsic amino acid tryptophan (donor) and N, N-dimethyl naphthalene moiety of the extrinsic dansyl probes at the surface of Apo-Mb, precisely monitor changes in donor acceptor distance as a consequence of interaction of the protein with CyD having different cavity sizes (β and γ variety). Molecular modelling studies on the interaction of tryptophan and dansyl probe with β-CyD is reported here and found to be consistent with the experimental observations. In order to investigate structural aspects of the interacting protein, we have used circular dichroism spectroscopy. Temperature-dependent circular dichroism studies explore the change in the secondary structure of Apo-Mb in association with CyD, before and after fluorescence modification of the protein. Overall, the study well exemplifies approaches to protein recognition by CyD as a synthetic receptor and offers a cautionary note on the use of hydrophobic fluorescent labels for proteins in biochemical studies involving recognition of molecules. Copyright © 2013 John Wiley & Sons, Ltd.

An Integrin-Targeting RGDK-Tagged Nanocarrier: Anticancer Efficacy of Loaded Curcumin.

PubMed

Das, Krishnendu; Nimushakavi, Sahithi; Chaudhuri, Arabinda; Das, Prasanta Kumar

2017-05-22

Herein we report the design and development of α 5 β 1 integrin-specific noncovalent RGDK-lipopeptide-functionalized single-walled carbon nanotubes (SWNTs) that selectively deliver the anticancer drug curcumin to tumor cells. RGDK tetrapeptide-tagged amphiphiles were synthesized that efficiently disperse SWNTs with a suspension stability index of >80 % in cell culture media. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)- and lactate dehydrogenase (LDH)-based cell viability assays in tumor (B16F10 melanoma) and noncancerous (NIH3T3 mouse fibroblast) cells revealed the non-cytotoxic nature of these RGDK-lipopeptide-SWNT conjugates. Cellular uptake experiments with monoclonal antibodies against α v β 3 , α v β 5 , and α 5 β 1 integrins showed that these SWNT nanovectors deliver their cargo (Cy3-labeled oligonucleotides, Cy3-oligo) to B16F10 cells selectively via α 5 β 1 integrin. Notably, the nanovectors failed to deliver the Cy3-oligo to NIH3T3 cells. The RGDK-SWNT is capable of delivering the anticancer drug curcumin to B16F10 cells more efficiently than NIH3T3 cells, leading to selective killing of B16F10 cells. Results of Annexin V binding based flow cytometry experiments are consistent with selective killing of tumor cells through the late apoptotic pathway. Biodistribution studies in melanoma (B16F10)-bearing C57BL/6J mice showed tumor-selective accumulation of curcumin intravenously administered via RGDK-lipopeptide-SWNT nanovectors. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nucleotide-Protectable Labeling of Sulfhydryl Groups in Subunit I of the ATPhase from Halobacterium Saccharovorum

NASA Technical Reports Server (NTRS)

Sulzner, Michael; Stan-Lotter, Helga; Hochstein, Lawrence I.

1992-01-01

A membrane-bound ATPase from the archaebacterium Halobacterium saccharovorum is inhibited by N-ethyl-maleimide in a nucleotide-protectable manner. When the enzyme was incubated with N-[C-14]jethylmaleimide, the bulk of radioactivity was as- sociated with the 87,000-Da subunit (subunit 1). ATP, ADP, or AMP reduced incorporation of the inhibitor. No charge shift of subunit I was detected following labeling with N-ethylmaleimide, indicating an electroneutral reaction. The results are consistent with the selective modification of sulfhydryl groups in subunit I at or near the catalytic site and are further evidence of a resemblance between this archaebacterial ATPase and the vacuolar-type ATPases.

Heat Shock Protein 27-Targeted Heptapeptide of the PKC{Delta} Catalytic V5 Region Sensitizes Tumors With Radio- and Chemoresistance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Hae-June; Kim, Eun-Ho; School of Life Sciences and Biotechnology, Korea University, Seoul

Purpose: Previous data suggest that the PKC{delta} catalytic V5 (PKC{delta}-V5) heptapeptide (HEPT) (FEQFLDI) binds HSP27 and blocks HSP27-mediated radio- or chemoresistance. Here we investigated further the in vivo function of the PKC{delta}-V5 HEPT. Methods and Materials: Labeling of HEPT with Cy5.5 or fluorescein isothiocyanate was performed to evaluate in vitro or in vivo distribution of HEPT. A clonogenic survival assay, flow cytometry, and Western blotting of cleaved caspase-3 were performed to determine in vitro sensitization effects of HEPT plus ionizing radiation (IR) versus IR alone or those of HEPT plus cisplatin(Cis) versus Cis alone. A nude mouse xenografting system wasmore » also applied to detect in vivo sensitizing effects of HEPT. Results: HEPT efficiently bound to HSP27 and showed sensitization after combined treatment with IR versus treatment with Cis alone in NCI-H1299 lung carcinoma cells, with higher HSP27 expression, which was similar to that of combined treatment with IR or with Cis alone in NCI-H460 lung carcinoma cells with lower HSP27 expression. In vivo image analysis using Cy5.5-labeled HEPT showed that HEPT was retained in HSP27-overexpressing cancer cells after xenografting to nude mice. Combined treatment of HEPT with IR versus that with Cis alone in xenografted mice showed that HEPT increased radio- or chemosensitization in NCI-H1299 cells compared to that in mice xenografted with NCI-H460 cells. Conclusions: The novel PKC{delta}-V5 HEPT may help overcome HSP27-mediated radio- or chemoresistance.« less

Synthesis and characterization of maleimide-functionalized polystyrene-SiO2/TiO2 hybrid nanocomposites by sol-gel process

NASA Astrophysics Data System (ADS)

Ramesh, Sivalingam; Sivasamy, Arumugam; Kim, Joo-Hyung

2012-06-01

Maleimide-functionalized polystyrene (PSMA-SiO2/TiO2) hybrid nanocomposites were prepared by sol-gel reaction starting from tratraethoxysilane (TEOS) and titanium isopropoxide in the solution of polystyrene maleimide in 1,4-dioxane. The hybrid films were obtained by the hydrolysis and polycondensation of TEOS and titanium isopropoxide in maleimide-functionalized polystyrene solution followed by the Michael addition reaction. The transparency of polymer (PSMA-SiO2/TiO2) hybrid was prepared from polystyrene titanium isopropoxide using the γ-aminopropyltriethoxy silane as crosslinking agent by in situ sol-gel process via covalent bonding between the organic-inorganic hybrid nanocomposites. The maleimide-functionalized polystyrene was synthesized by Friedel-Crafts reaction from N-choloromethyl maleimide. The FTIR spectroscopy data conformed the occurrence of Michael addition reaction between the pendant maleimide moieties of the styrene and γ-aminopropyltriethoxysilane. The chemical structure and morphology of PSMA-SiO2/TiO2 hybrid nanocomposites were characterized by FTIR, nuclear magnetic resonance (NMR), 13 C NMR, SEM, XRD, and TEM analyses. The results also indicate that the inorganic particles are much smaller in the ternary systems than in the binary systems; the shape of the inorganic particles and compatibility for maleimide-functionalized polystrene and inorganic moieties are varied with the ratio of the inorganic moieties in the hybrids. Furthermore, TGA and DSC results indicate that the thermal stability of maleimide-functionalized polystyrene was enhanced through the incorporation of the inorganic moieties in the hybrid materials.

Gating mass cytometry data by deep learning.

PubMed

Li, Huamin; Shaham, Uri; Stanton, Kelly P; Yao, Yi; Montgomery, Ruth R; Kluger, Yuval

2017-11-01

Mass cytometry or CyTOF is an emerging technology for high-dimensional multiparameter single cell analysis that overcomes many limitations of fluorescence-based flow cytometry. New methods for analyzing CyTOF data attempt to improve automation, scalability, performance and interpretation of data generated in large studies. Assigning individual cells into discrete groups of cell types (gating) involves time-consuming sequential manual steps, untenable for larger studies. We introduce DeepCyTOF, a standardization approach for gating, based on deep learning techniques. DeepCyTOF requires labeled cells from only a single sample. It is based on domain adaptation principles and is a generalization of previous work that allows us to calibrate between a target distribution and a source distribution in an unsupervised manner. We show that DeepCyTOF is highly concordant (98%) with cell classification obtained by individual manual gating of each sample when applied to a collection of 16 biological replicates of primary immune blood cells, even when measured across several instruments. Further, DeepCyTOF achieves very high accuracy on the semi-automated gating challenge of the FlowCAP-I competition as well as two CyTOF datasets generated from primary immune blood cells: (i) 14 subjects with a history of infection with West Nile virus (WNV), (ii) 34 healthy subjects of different ages. We conclude that deep learning in general, and DeepCyTOF specifically, offers a powerful computational approach for semi-automated gating of CyTOF and flow cytometry data. Our codes and data are publicly available at https://github.com/KlugerLab/deepcytof.git. yuval.kluger@yale.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Long-Term Efficacy and Safety of Cyclosporine in a Cohort of Steroid-Refractory Acute Severe Ulcerative Colitis Patients from the ENEIDA Registry (1989-2013): A Nationwide Multicenter Study.

PubMed

Ordás, I; Domènech, E; Mañosa, M; García-Sánchez, V; Iglesias-Flores, E; Peñalva, M; Cañas-Ventura, A; Merino, O; Fernández-Bañares, F; Gomollón, F; Vera, M; Gutiérrez, A; Garcia-Planella, E; Chaparro, M; Aguas, M; Gento, E; Muñoz, F; Aguirresarobe, M; Muñoz, C; Fernández, L; Calvet, X; Jiménez, C E; Montoro, M A; Mir, A; De Castro, M L; García-Sepulcre, M F; Bermejo, F; Panés, J; Esteve, M

2017-11-01

To determine the efficacy and safety of cyclosporine (CyA) in a large national registry-based population of patients with steroid-refractory (SR) acute severe ulcerative colitis (ASUC) and to establish predictors of efficacy and adverse events. Multicenter study of SR-ASUC treated with CyA, based on data from the ENEIDA registry. SR-ASUC patients treated with infliximab (IFX) or sequential rescue therapy (CyA-IFX or IFX-CyA) were used as comparators. Of 740 SR-ASUC patients, 377 received CyA, 131 IFX and 63 sequential rescue therapy. The cumulative colectomy rate was higher in the CyA (24.1%) and sequential therapy (32.7%) than in the IFX group (14.5%; P=0.01) at 3 months and 5 years. There were no differences in early and late colectomy between CyA and IFX in patients treated after 2005. 62% of patients receiving CyA remained colectomy-free in the long term (median 71 months). There were no differences in mortality between CyA (2.4%), IFX (1.5%) and sequential therapy (0%; P=0.771). The proportion of patients with serious adverse events (SAEs) was lower in CyA (15.4%) than in IFX treated patients (26.5%) or sequential therapy (33.4%; P<0.001). This difference in favor of CyA was maintained when only patients treated after 2005 were analyzed. Treatment with CyA showed a lower rate of SAE and a similar efficacy to that of IFX thereby supporting the use of either CyA or IFX in SR-ASUC. In addition, the risk-benefit of sequential CyA-IFX for CyA non-responders is acceptable.

A low-cost method for visible fluorescence imaging.

PubMed

Tarver, Crissy L; Pusey, Marc

2017-12-01

A wide variety of crystallization solutions are screened to establish conditions that promote the growth of a diffraction-quality crystal. Screening these conditions requires the assessment of many crystallization plates for the presence of crystals. Automated systems for screening and imaging are very expensive. A simple approach to imaging trace fluorescently labeled protein crystals in crystallization plates has been devised, and can be implemented at a cost as low as $50. The proteins β-lactoglobulin B, trypsin and purified concanavalin A (ConA) were trace fluorescently labeled using three different fluorescent probes: Cascade Yellow (CY), Carboxyrhodamine 6G (CR) and Pacific Blue (PB). A crystallization screening plate was set up using β-lactoglobulin B labeled with CR, trypsin labeled with CY, ConA labeled with each probe, and a mixture consisting of 50% PB-labeled ConA and 50% CR-labeled ConA. The wells of these plates were imaged using a commercially available macro-imaging lens attachment for smart devices that have a camera. Several types of macro lens attachments were tested with smartphones and tablets. Images with the highest quality were obtained with an iPhone 6S and an AUKEY Ora 10× macro lens. Depending upon the fluorescent probe employed and its Stokes shift, a light-emitting diode or a laser diode was used for excitation. An emission filter was used for the imaging of protein crystals labeled with CR and crystals with two-color fluorescence. This approach can also be used with microscopy systems commonly used to observe crystallization plates.

Construction of a cDNA microarray derived from the ascidian Ciona intestinalis.

PubMed

Azumi, Kaoru; Takahashi, Hiroki; Miki, Yasufumi; Fujie, Manabu; Usami, Takeshi; Ishikawa, Hisayoshi; Kitayama, Atsusi; Satou, Yutaka; Ueno, Naoto; Satoh, Nori

2003-10-01

A cDNA microarray was constructed from a basal chordate, the ascidian Ciona intestinalis. The draft genome of Ciona has been read and inferred to contain approximately 16,000 protein-coding genes, and cDNAs for transcripts of 13,464 genes have been characterized and compiled as the "Ciona intestinalis Gene Collection Release I". In the present study, we constructed a cDNA microarray of these 13,464 Ciona genes. A preliminary experiment with Cy3- and Cy5-labeled probes showed extensive differential gene expression between fertilized eggs and larvae. In addition, there was a good correlation between results obtained by the present microarray analysis and those from previous EST analyses. This first microarray of a large collection of Ciona intestinalis cDNA clones should facilitate the analysis of global gene expression and gene networks during the embryogenesis of basal chordates.

Fluorescence Resonance Energy Transfer Glucose Sensor from Site-Specific Dual Labeling of Glucose/Galactose Binding Protein Using Ligand Protection

PubMed Central

Hsieh, Helen V.; Sherman, Douglas B.; Andaluz, Sandra A.; Amiss, Terry J.; Pitner, J. Bruce

2012-01-01

Background Site-selective modification of proteins at two separate locations using two different reagents is highly desirable for biosensor applications employing fluorescence resonance energy transfer (FRET), but few strategies are available for such modification. To address this challenge, sequential selective modification of two cysteines in glucose/galactose binding protein (GGBP) was demonstrated using a technique we call “ligand protection.” Method In this technique, two cysteines were introduced in GGBP and one cysteine is rendered inaccessible by the presence of glucose, thus allowing sequential attachment of two different thiol-reactive reagents. The mutant E149C/A213C/L238S was first labeled at E149C in the presence of the ligand glucose. Following dialysis and removal of glucose, the protein was labeled with a second dye, either Texas Red (TR) C5 bromoacetamide or TR C2 maleimide, at the second site, A213C. Results Changes in glucose-dependent fluorescence were observed that were consistent with FRET between the nitrobenzoxadiazole and TR fluorophores. Comparison of models and spectroscopic properties of the C2 and C5 TR FRET constructs suggests the greater rigidity of the C2 linker provides more efficient FRET. Conclusions The ligand protection strategy provides a simple method for labeling GGBP with two different fluorophores to construct FRET-based glucose sensors with glucose affinity within the human physiological glucose range (1–30 mM). This general strategy may also have broad utility for other protein-labeling applications. PMID:23294773

Fully automated synthesis of 4-[18F]fluorobenzylamine based on borohydride/NiCl2 reduction.

PubMed

Way, Jenilee; Wuest, Frank

2013-04-01

4-[(18)F]Fluorobenzylamine ([(18)F]FBA) is an important building block for the synthesis of (18)F-labeled compounds. Synthesis of [(18)F]FBA usually involves application of strong reducing agents like LiAlH4 which is challenging to handle in automated synthesis units (ASUs). Therefore, alternative methods for the preparation of [(18)F]FBA compatible with remotely-controlled syntheses in ASUs are needed. (18)F]FBA was prepared in a remotely-controlled synthesis unit (GE TRACERlab™ FX) based on Ni(II)-mediated borohydride exchange resin (BER) reduction of 4-[(18)F]fluorobenzonitrile ([(18)F]FBN). [(18)F]FBA was used for the synthesis of novel thiol-reactive prosthetic group 4-[(18)F]fluorobenzyl)maleimide [(18)F]FBM and Hsp90 inhibitor 17-(4-[(18)F]fluorobenzylamino)-17-demethoxy-geldanamycin [(18)F] GA. [(18)F]FBA could be prepared in high radiochemical yield greater than 80% (decay-corrected) within 60min. In a typical experiment, 7.4GBq of [(18)F]FBA could be obtained in high radiochemical purity of greater than 95% starting from 10GBq of cyclotron-produced n.c.a. [(18)F]fluoride. [(18)F]FBA was used for the preparation of 4-[(18)F]fluorobenzyl)maleimide as a novel prosthetic group for labeling of thiol groups as demonstrated with tripeptide glutathione. [(18)F]FBA was also used as building block for the syntheses of small molecules as exemplified by the preparation of Hsp90 inhibitor 17-(4-[(18)F]fluorobenzylamino)-17-demethoxy-geldanamycin. The described remotely-controlled synthesis of [(18)F]FBA will significantly improve the availability of [(18)F]FBA as an important and versatile building block for the development of novel (18)F-labeled compounds containing a fluorobenzylamine moiety. Copyright © 2013 Elsevier Inc. All rights reserved.

The unexpected product of Diels-Alder reaction between "indanocyclon" and maleimide

NASA Astrophysics Data System (ADS)

Dobrowolski, Michał A.; Roszkowski, Piotr; Struga, Marta; Szulczyk, Daniel

2017-02-01

A heterocyclic compound commonly known as "indanocyclon" undergoes an unexpected Diels-Alder addition with maleimide. The resulting product has been isolated and characterized in order to get an information about its structure and possible mechanism of the reaction. Extensive comparison of single crystal properties of 3-(2,8-dioxo-1,3-diphenyl-2,8-dihydrocyclopenta[a]inden-8a(1H)-yl)pyrrolidine-2,5-dione and favorable product of the reaction has been also performed.

Simultaneous monitoring of insulin and islet amyloid polypeptide secretion from islets of Langerhans on a microfluidic device.

PubMed

Lomasney, Anna R; Yi, Lian; Roper, Michael G

2013-08-20

A method was developed that allowed simultaneous monitoring of the acute secretory dynamics of insulin and islet amyloid polypeptide (IAPP) from islets of Langerhans using a microfluidic system with two-color detection. A flow-switching feature enabled changes in the perfusion media within 5 s, allowing rapid exchange of the glucose concentrations delivered to groups of islets. The perfusate was continuously sampled by electroosmotic flow and mixed online with Cy5-labeled insulin, fluorescein isothiocyanate (FITC)-labeled IAPP, anti-insulin, and anti-IAPP antibodies in an 8.15 cm mixing channel maintained at 37 °C. The immunoassay mixture was injected for 0.3 s onto a 1.5 cm separation channel at 11.75 s intervals and immunoassay reagents detected using 488 and 635 nm lasers with two independent photomultiplier tubes for detection of the FITC and Cy5 signal. RSD of the bound-to-free immunoassay ratios ranged from 2 to 7% with LODs of 20 nM for insulin and 1 nM for IAPP. Simultaneous secretion profiles of the two peptides were monitored from groups of 4-10 islets during multiple step changes in glucose concentration. Insulin and IAPP were secreted in an approximately 10:1 ratio and displayed similar responses to step changes from 3 to 11 or 20 mM glucose. The ability to monitor the secretory dynamics of multiple peptides from islets of Langerhans in a highly automated fashion is expected to be a useful tool for investigating hormonal regulation of glucose homeostasis.

Novel Micro/Nano Approaches for Glucose Measurement Using pH-Sensitive Hydrogels

DTIC Science & Technology

2005-06-01

hydrochloride ) (PAH) (polycation). A total of three bilayers of GOx/PAH were assembled, and enzyme activity was confirmed by colorimetric assay...D- glucosamine residues in chitosan can be conjugated with amine-reactive dyes, such as succinimidyl esters (Alexa Fluor 647TM, CY5®), isothiocyanates...were found to be: A 4 94 nm=0.153276 MW=161 [D- glucosamine ]=0.77g/L Sdye=68000cm-M-1 DOL=4.71 x 10-’ Therefore, the labeling ratio of FITC:chitosan

An Unsymmetric Ligand Framework for Noncoupled Homo- and Heterobimetallic Complexes.

PubMed

Haas, Ruth M; Hern, Zachary; Sproules, Stephen; Hess, Corinna R

2017-12-18

We introduce a new unsymmetric ligand, PDIpCy (PDI = pyridyldiimine; Cy = cyclam), that offers two distinct, noncoupled coordination sites. A series of homo- and heterobimetallic complexes, [Zn 2 (PDIpCy)(THF)(OTf) 4 ] (1; THF = tetrahydrofuran and OTf = triflate), [Ni 2 (PDIpCy)(THF)(OTf) 2 ](OTf) 2 (2), and [NiZn(PDIpCy)(THF)(OTf) 4 ] (3), are described. The one-electron-reduced compounds, [Zn 2 (PDIpCy)(OTF) 3 ] (4), [Ni 2 (PDIpCy)(OTf)](OTf) 2 (5), and [NiZn(PDIpCy)(OTf) 3 ] (6), were isolated, and their electronic structures were characterized. The reduced compounds are charge-separated species, with electron storage at either the PDI ligand (4) or at the PDI-bound metal ion (5 and 6).

131I-Anti-CD45 Antibody Plus Busulfan and Cyclophosphamide before Allogeneic Hematophoietic Cell Transplantation for Treatment of Acute Myeloid Leukemia in First Remission

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pagel, John M.; Appelbaum, Frederick R.; Eary, Janet F.

2006-03-01

In an attempt to improve outcomes for patients with acute myeloid leukemia (AML) after allogeneic hematopoietic cell transplantation (HCT), we conducted a Phase I/II study in which targeted irradiation delivered by 131I-anti-CD45 antibody was combined with targeted busulfan (BU; area-under-curve, 600-900 ng/ml) and cyclophosphamide (CY; 120 mg/kg). Fifty-two of 59 patients (88%) receiving a trace 131I-labeled dose of 0.5 mg/kg anti-CD45 murine antibody had higher estimated absorbed radiation in bone marrow and spleen than in any other organ. Forty-six patients were treated with 102-298 mCi 131I delivering an estimated 5.3-19 (mean 11.3) Gy to marrow, 17-72 (mean 29.7) Gy tomore » spleen, and 3.5 Gy (n=4) to 5.25 Gy (n=42) to the liver. The estimated 3-year non-relapse mortality and disease-free survival (DFS) were 21% and 61%, respectively. These results were compared to those from 509 similar International Bone Marrow Transplant Registry patients transplanted using BU/CY alone. After adjusting for differences in age and cytogenetics-risk, the hazard of mortality among all antibody-treated patients was 0.65 times that of the Registry patients (95% CI 0.39-1.08; p=.09). The addition of targeted hematopoietic irradiation to conventional BU/CY is feasible and well tolerated, and Phase II results are sufficiently encouraging to warrant further study.« less

Monohalogenated maleimides as potential agents for the inhibition of Pseudomonas aeruginosa biofilm.

PubMed

Carteau, David; Soum-Soutéra, Emmanuelle; Faÿ, Fabienne; Dufau, Chrystèle; Cérantola, Stéphane; Vallée-Réhel, Karine

2010-01-01

New monohalogenated maleimide derivatives (with bromine, chlorine or iodine) were synthesized to test the effect of halogen atoms in inhibiting the formation of Pseudomonas aeruginosa biofilm. The evaluation of their biological activities clearly defines a structure-activity relationship. In this study, the bactericidal action of the three compounds was observed at the concentration range 0.3-5.0 mM on Luria-Bertani agar plates. The halogen atom of these molecules was critical in modulating the antibacterial activity, with a slightly higher effectiveness for chlorine. Confocal laser scanning microscopy was used to examine P. aeruginosa biofilms cultivated in flow cells. At concentration as low as 40 microM, the bromine and iodine compounds displayed a total inhibition towards the formation of bacterial biofilm. At this concentration, the bacterial attachment to glass surfaces was strongly affected by the presence of bromine and iodine whereas the chlorine derivative behaved as a bactericidal compound. A bioluminescent reporter strain was then used to detect the effect of the chemically synthesized maleimides on quorum sensing (QS) in P. aeruginosa. At the concentration range 10-100 microM, bioluminescence assays reveal that halogenated maleimides were able to interfere with the QS of the bacterium. Although the relationship between the weak inhibition of cell-to-cell communication (15-55% of the signal) and the high inhibition of biofilm formation has not been elucidated clearly, the results demonstrate that bromo- and iodo-N-substituted maleimides bromine and iodine may be used as new potent inhibitors that control bacterial biofilms.

Synthesis and spectral properties of polymethine-cyanine dye-nitroxide radical hybrid compounds for use as fluorescence probes to monitor reducing species and radicals

NASA Astrophysics Data System (ADS)

Sato, Shingo; Tsunoda, Minoru; Suzuki, Minoru; Kutsuna, Masahiro; Takido-uchi, Kiyomi; Shindo, Mitsuru; Mizuguchi, Hitoshi; Obara, Heitaro; Ohya, Hiroaki

2009-01-01

Various hybrid compounds comprised of two types of nitroxide radicals and either a pentamethine (Cy5) or trimethine cyanine (Cy3) were synthesized. The nitroxide radicals were linked either via an ester-bond to one or two N-alkyl carboxyl-terminated groups of Cy5, or via two amido-bonds (aminocarbonyl or carbonylamino group) to the 5-position of the indolenine moieties of Cy5 and Cy3. Changes in fluorescence and ESR intensities of the hybrid compounds were measured before and after addition of Na ascorbate in PBS (pH 7.0) to reduce the radicals. Among the hybrid compounds synthesized, those that linked the nitroxide radicals via an aminocarbonyl residue at the 5-position of the indolenine moieties on Cy5 and Cy3 exhibited a 1.8- and 5.1-fold increase in fluorescence intensity with the reduction of the nitroxide segment by the addition of Na ascorbate, respectively. In contrast, fluorescence intensity was not enhanced in the other hybrid compounds. Thus, the hybrid compounds which exhibited an increase in fluorescent intensity with radical reduction can be used in the quantitative measurement of reducing species such as Fe 2+ and ascorbic acid, and hydroxyl radicals. Because these hybrid compounds have the advantage of fluorescing at longer wavelengths—661 (Cy5) or 568 (Cy3) nm, respectively, they can be used to measure radical-reducing species or radicals either in solution or in vivo.

NIR fluorescent chitosan-based nanoparticles for tracking and delivery of cancer therapeutic molecule in living systems

NASA Astrophysics Data System (ADS)

Suarato, Giulia; Chin, Amanda; Meng, Yizhi

2013-03-01

Tumor metastasis is associated with the epithelial-to-mesenchymal transition (EMT), in which cells lose their polarized phenotype to acquire the asymmetry and motility of mesenchymal cells. Among the many molecular determinants for EMT is bone morphogenetic protein-7 (BMP-7), a critical regulator of skeletal tissue formation and kidney development. Current treatments for metastatic cancer primarily involve surgery and chemotherapy, both with considerable side effects. Therefore the goal of our research is to evaluate the ability of BMP-7 to reverse EMT using a delivery system based on glycol chitosan nanoparticles (GCNP), naturally biodegradable. The GCNP are labeled with Cy5.5, a near infrared (NIR) excitable dye that enables non-invasive imaging in living systems. The chitosan shell provides affinity for the cell surface and protection from intracellular enzymes during transport. Preliminary data show that Cy5.5-GCNP vehicles were successfully delivered to murine preosteoblast (MC3T3-E1), rat osteosarcoma (ROS) 17/2.8 and human embryonic kidney (HEK293) cells. Release kinetics using a model protein (BSA) and BMP-7, and the stability of the protein nano-cargo are currently being evaluated. Cell morphology will be examined with immunofluorescence microscopy.

Development of a Multiplexed Microsphere PCR for Culture-Free Detection and Gram-Typing of Bacteria in Human Blood Samples.

PubMed

Liang, Fang; Browne, Daniel J; Gray, Megan J; Gartlan, Kate H; Smith, David D; Barnard, Ross T; Hill, Geoffrey R; Corrie, Simon R; Markey, Kate A

2018-05-11

Bloodstream infection is a significant clinical problem, particularly in vulnerable patient groups such as those undergoing chemotherapy and bone marrow transplantation. Clinical diagnostics for suspected bloodstream infection remain centered around blood culture (highly variable timing, in the order of hours to days to become positive), and empiric use of broad-spectrum antibiotics is therefore employed for patients presenting with febrile neutropenia. Gram-typing provides the first opportunity to target therapy (e.g., combinations containing vancomycin or teicoplanin for Gram-positives; piperacillin-tazobactam or a carbapenem for Gram-negatives); however, current approaches require blood culture. In this study, we describe a multiplexed microsphere-PCR assay with flow cytometry readout, which can distinguish Gram-positive from Gram-negative bacterial DNA in a 3.5 h time period. The combination of a simple assay design (amplicon-dependent release of Gram-type specific Cy3-labeled oligonucleotides) and the Luminex-based readout (for quantifying each specific Cy3-labeled sequence) opens opportunities for further multiplexing. We demonstrate the feasibility of detecting common Gram-positive and Gram-negative organisms after spiking whole bacteria into healthy human blood prior to DNA extraction. Further development of DNA extraction methods is required to reach detection limits comparable to blood culture.

Development of species-specific rDNA probes for Giardia by multiple fluorescent in situ hybridization combined with immunocytochemical identification of cyst wall antigens.

PubMed

Erlandsen, Stanley L; Jarroll, Edward; Wallis, Peter; van Keulen, Harry

2005-08-01

In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17-22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts. Specificity of fluorescent in situ hybridization (FISH) was shown using RNase digestion and high stringency to diminish the hybridization signal, and oligonucleotide probes for rRNA in Giardia lamblia, Giardia muris, and Giardia ardeae were shown to specifically stain rRNA only within cysts or trophozoites of those species. The fluorescent oligonucleotide specific for rRNA in human isolates of Giardia was positive for ten different strains. A method for simultaneous FISH detection of cysts using fluorescent antibody (genotype marker) and two oligonucleotide probes (species marker) permitted visualization of G. lamblia and G. muris cysts in the same preparation. Testing of an environmental water sample revealed the presence of FISH-positive G. lamblia cysts with a specific rDNA probe for rRNA, while negative cysts were presumed to be of animal or bird origin.

[DNA microarray reveals changes in gene expression of endothelial cells under shear stress].

PubMed

Cheng, Min; Zhang, Wensheng; Chen, Huaiqing; Wu, Wenchao; Huang, Hua

2004-04-01

cDNA microarray technology is used as a powerful tool for rapid, comprehensive, and quantitative analysis of gene profiles of cultured human umbilical vein endothelial cells(HUVECs) in the normal static group and the shear stressed (4.20 dyne/cm2, 2 h) group. The total RNA from normal static cultured HUVECs was labeled by Cy3-dCTP, and total RNA of HUVECs from the paired shear stressed experiment was labeled by Cy5-dCTP. The expression ratios reported are the average from the two separate experiments. After bioinformatics analysis, we identified a total of 108 genes (approximately 0.026%) revealing differential expression. Of these 53 genes expressions were up-regulated, the most enhanced ones being human homolog of yeast IPP isomerase, human low density lipoprotein receptor gene, Squalene epoxidase gene, 7-dehydrocholesterol reductase, and 55 were down-regulated, the most decreased ones being heat shock 70 kD protein 1, TCB gene encoding cytosolic thyroid hormone-binding protein in HUVECs exposed to low shear stress. These results indicate that the cDNA microarray technique is effective in screening the differentially expressed genes in endothelial cells induced by various experimental conditions and the data may serve as stimuli to further researches.

Monte Carlo N-Particle (MCNP) Modeling of the Cellular Dosimetry of 64Cu: Comparison with MIRDcell S Values and Implications for Studies of Its Cytotoxic Effects.

PubMed

Cai, Zhongli; Kwon, Yongkyu Luke; Reilly, Raymond M

2017-02-01

64 Cu emits positrons as well as β - particles and Auger and internal conversion electrons useful for radiotherapy. Our objective was to model the cellular dosimetry of 64 Cu under different geometries commonly used to study the cytotoxic effects of 64 Cu. Monte Carlo N-Particle (MCNP) was used to simulate the transport of all particles emitted by 64 Cu from the cell surface (CS), cytoplasm (Cy), or nucleus (N) of a single cell; monolayer in a well (radius = 0.32-1.74 cm); or a sphere (radius = 50-6,000 μm) of cells to calculate S values. The radius of the cell and N ranged from 5 to 12 μm and 2 to 11 μm, respectively. S values were obtained by MIRDcell for comparison. MCF7/HER2-18 cells were exposed in vitro to 64 Cu-labeled trastuzumab. The subcellular distribution of 64 Cu was measured by cell fractionation. The surviving fraction was determined in a clonogenic assay. The relative differences of MCNP versus MIRDcell self-dose S values (S self ) for 64 Cu ranged from -0.2% to 3.6% for N to N (S N←N ), 2.3% to 8.6% for Cy to N (S N←Cy ), and -12.0% to 7.3% for CS to N (S N←CS ). The relative differences of MCNP versus MIRDcell cross-dose S values were 25.8%-30.6% for a monolayer and 30%-34% for a sphere, respectively. The ratios of S N←N versus S N←Cy and S N←Cy versus S N←CS decreased with increasing ratio of the N of the cell versus radius of the cell and the size of the monolayer or sphere. The surviving fraction of MCF7 /: HER2-18 cells treated with 64 Cu-labeled trastuzumab (0.016-0.368 MBq/μg, 67 nM) for 18 h versus the absorbed dose followed a linear survival curve with α = 0.51 ± 0.05 Gy -1 and R 2 = 0.8838. This is significantly different from the linear quadratic survival curve of MCF7 /: HER2-18 cells exposed to γ-rays. MCNP- and MIRDcell-calculated S values agreed well. 64 Cu in the N increases the dose to the N in isolated single cells but has less effect in a cell monolayer or small cluster of cells simulating a micrometastasis, and little effect in a sphere analogous to a tumor xenograft compared with 64 Cu in the Cy or on the CS. The dose deposited by 64 Cu is less effective for cell killing than γ-rays. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Topology of OxlT, the oxalate transporter of Oxalobacter formigenes, determined by site-directed fluorescence labeling.

PubMed

Ye, L; Jia, Z; Jung, T; Maloney, P C

2001-04-01

The topology of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes, was established by site-directed fluorescence labeling, a simple strategy that generates topological information in the context of the intact protein. Accessibility of cysteine to the fluorescent thiol-directed probe Oregon green maleimide (OGM) was examined for a panel of 34 single-cysteine variants, each generated in a His(9)-tagged cysteine-less host. The reaction with OGM was readily scored by examining the fluorescence profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of material purified by Ni2+ linked affinity chromatography. A position was assigned an external location if its single-cysteine derivative reacted with OGM added to intact cells; a position was designated internal if OGM labeling required cell lysis. We also showed that labeling of external, but not internal, positions was blocked by prior exposure of cells to the impermeable and nonfluorescent thiol-specific agent ethyltrimethylammonium methanethiosulfonate. Of the 34 positions examined in this way, 29 were assigned unambiguously to either an internal or external location; 5 positions could not be assigned, since the target cysteine failed to react with OGM. There was no evidence of false-positive assignment. Our findings document a simple and rapid method for establishing the topology of a membrane protein and show that OxlT has 12 transmembrane segments, confirming inferences from hydropathy analysis.

Topology of OxlT, the Oxalate Transporter of Oxalobacter formigenes, Determined by Site-Directed Fluorescence Labeling

PubMed Central

Ye, Liwen; Jia, Zhenzhen; Jung, Thomas; Maloney, Peter C.

2001-01-01

The topology of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes, was established by site-directed fluorescence labeling, a simple strategy that generates topological information in the context of the intact protein. Accessibility of cysteine to the fluorescent thiol-directed probe Oregon green maleimide (OGM) was examined for a panel of 34 single-cysteine variants, each generated in a His9-tagged cysteine-less host. The reaction with OGM was readily scored by examining the fluorescence profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of material purified by Ni2+-linked affinity chromatography. A position was assigned an external location if its single-cysteine derivative reacted with OGM added to intact cells; a position was designated internal if OGM labeling required cell lysis. We also showed that labeling of external, but not internal, positions was blocked by prior exposure of cells to the impermeable and nonfluorescent thiol-specific agent ethyltrimethylammonium methanethiosulfonate. Of the 34 positions examined in this way, 29 were assigned unambiguously to either an internal or external location; 5 positions could not be assigned, since the target cysteine failed to react with OGM. There was no evidence of false-positive assignment. Our findings document a simple and rapid method for establishing the topology of a membrane protein and show that OxlT has 12 transmembrane segments, confirming inferences from hydropathy analysis. PMID:11274108

Molecular MRI of Cardiomyocyte Apoptosis with Simultaneous Delayed Enhancement MRI Distinguishes Apoptotic and Necrotic Myocytes In Vivo: Potential for Midmyocardial Salvage in Acute Ischemia

PubMed Central

Sosnovik, David E.; Garanger, Elisabeth; Aikawa, Elena; Nahrendorf, Matthias; Figuiredo, Jose-Luiz; Dai, Guangping; Reynolds, Fred; Rosenzweig, Anthony; Weissleder, Ralph; Josephson, Lee

2009-01-01

Background A novel dual contrast molecular MRI technique to image both cardiomyocyte (CM) apoptosis and necrosis in-vivo within 4-6 hours of ischemia is presented. The technique utilizes the annexin-based nanoparticle AnxCLIO-Cy5.5 (apoptosis) and simultaneous delayed enhancement (DE) imaging with a novel gadolinium chelate, Gd-DTPA-NBD (necrosis). Methods and Results Mice with transient coronary ligation were injected intravenously at the onset of reperfusion with AnxCLIO-Cy5.5 (n=7) or the control probe Inact_CLIO-Cy5.5 (n=6). T2* weighted MR images (9.4 Tesla) were acquired within 4-6 hours of reperfusion. The contrast-to-noise ratio (CNR) between injured and uninjured myocardium was measured. The mice were then injected with Gd-DTPA-NBD and DE imaging was performed within 10-30 minutes. Uptake of AnxCLIO-Cy5.5 was most prominent in the midmyocardium and was significantly greater than that of Inact_CLIO-Cy5.5 (CNR 8.82 +/− 1.5 versus 3.78 +/− 1.1, p < 0.05). Only 21 +/− 3% of the myocardium with accumulation of AnxCLIO-Cy5.5 showed DE of Gd-DTPA-NBD. Wall thickening was significantly reduced in segments with DE and/or transmural accumulation of AnxCLIO-Cy5.5 (p < 0.001). Fluorescence microscopy of AnxCLIO-Cy5.5 and immunohistochemistry of Gd-DTPA-NBD confirmed the presence of large numbers of apoptotic but potentially viable CMs (AnxCLIO-Cy5.5 positive, Gd-DTPA-NBD negative) in the midmyocardium. Conclusions A novel technique to image CM apoptosis and necrosis in-vivo within 4-6 hours of injury is presented, and reveals large areas of apoptotic but viable myocardium in the midmyocardium. Strategies to salvage the numerous apoptotic but potentially viable CMs in the midmyocardium in acute ischemia should be investigated. PMID:19920044

Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase.

PubMed

Watashi, Koichi; Ishii, Naoto; Hijikata, Makoto; Inoue, Daisuke; Murata, Takayuki; Miyanari, Yusuke; Shimotohno, Kunitada

2005-07-01

Viruses depend on host-derived factors for their efficient genome replication. Here, we demonstrate that a cellular peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin B (CyPB), is critical for the efficient replication of the hepatitis C virus (HCV) genome. CyPB interacted with the HCV RNA polymerase NS5B to directly stimulate its RNA binding activity. Both the RNA interference (RNAi)-mediated reduction of endogenous CyPB expression and the induced loss of NS5B binding to CyPB decreased the levels of HCV replication. Thus, CyPB functions as a stimulatory regulator of NS5B in HCV replication machinery. This regulation mechanism for viral replication identifies CyPB as a target for antiviral therapeutic strategies.

Treating hepatitis C: can you teach old dogs new tricks?

PubMed

Rice, Charles M; You, Shihyun

2005-12-01

Viruses depend on host-derived factors for their efficient genome replication. Here, we demonstrate that a cellular peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin B (CyPB), is critical for the efficient replication of the hepatitis C virus genome. CyPB interacted with the HCV RNA polymerase NS5B to directly stimulate its RNA binding activity. Both the RNA interference (RNAi)-mediated reduction of endogenous CyPB expression and the induced loss of NS5B binding to CyPB decreased the levels of HCV replication. Thus, CyPB functions as a stimulatory regulator of NS5B in HCV replication machinery. This regulation mechanism for viral replication identifies CyPB as a target for antiviral therapeutic strategies.

Simplified Microarray Technique for Identifying mRNA in Rare Samples

NASA Technical Reports Server (NTRS)

Almeida, Eduardo; Kadambi, Geeta

2007-01-01

Two simplified methods of identifying messenger ribonucleic acid (mRNA), and compact, low-power apparatuses to implement the methods, are at the proof-of-concept stage of development. These methods are related to traditional methods based on hybridization of nucleic acid, but whereas the traditional methods must be practiced in laboratory settings, these methods could be practiced in field settings. Hybridization of nucleic acid is a powerful technique for detection of specific complementary nucleic acid sequences, and is increasingly being used for detection of changes in gene expression in microarrays containing thousands of gene probes. A traditional microarray study entails at least the following six steps: 1. Purification of cellular RNA, 2. Amplification of complementary deoxyribonucleic acid [cDNA] by polymerase chain reaction (PCR), 3. Labeling of cDNA with fluorophores of Cy3 (a green cyanine dye) and Cy5 (a red cyanine dye), 4. Hybridization to a microarray chip, 5. Fluorescence scanning the array(s) with dual excitation wavelengths, and 6. Analysis of the resulting images. This six-step procedure must be performed in a laboratory because it requires bulky equipment.

Detailed analysis of complex single molecule FRET data with the software MASH

NASA Astrophysics Data System (ADS)

Hadzic, Mélodie C. A. S.; Kowerko, Danny; Börner, Richard; Zelger-Paulus, Susann; Sigel, Roland K. O.

2016-04-01

The processing and analysis of surface-immobilized single molecule FRET (Förster resonance energy transfer) data follows systematic steps (e.g. single molecule localization, clearance of different sources of noise, selection of the conformational and kinetic model, etc.) that require a solid knowledge in optics, photophysics, signal processing and statistics. The present proceeding aims at standardizing and facilitating procedures for single molecule detection by guiding the reader through an optimization protocol for a particular experimental data set. Relevant features were determined from single molecule movies (SMM) imaging Cy3- and Cy5-labeled Sc.ai5γ group II intron molecules synthetically recreated, to test the performances of four different detection algorithms. Up to 120 different parameterizations per method were routinely evaluated to finally establish an optimum detection procedure. The present protocol is adaptable to any movie displaying surface-immobilized molecules, and can be easily reproduced with our home-written software MASH (multifunctional analysis software for heterogeneous data) and script routines (both available in the download section of www.chem.uzh.ch/rna).

Site-Specific Three-Color Labeling of α-Synuclein via Conjugation to Uniquely Reactive Cysteines during Assembly by Native Chemical Ligation.

PubMed

Lee, Taehyung C; Moran, Crystal R; Cistrone, Philip A; Dawson, Philip E; Deniz, Ashok A

2018-04-12

Single-molecule fluorescence is widely used to study conformational complexity in proteins, and has proven especially valuable with intrinsically disordered proteins (IDPs). Protein studies using dual-color single-molecule Förster resonance energy transfer (smFRET) are now quite common, but many could benefit from simultaneous measurement of multiple distances through multi-color labeling. Such studies, however, have suffered from limitations in site-specific incorporation of more than two dyes per polypeptide. Here we present a fully site-specific three-color labeling scheme for α-synuclein, an IDP with important putative functions and links to Parkinson disease. The convergent synthesis combines native chemical ligation with regiospecific cysteine protection of expressed protein fragments to permit highly controlled labeling via standard cysteine-maleimide chemistry, enabling more global smFRET studies. Furthermore, this modular approach is generally compatible with recombinant proteins and expandable to accommodate even more complex experiments, such as by labeling with additional colors. Copyright © 2018 Elsevier Ltd. All rights reserved.

(67/68)Ga-labeling agent that liberates (67/68)Ga-NOTA-methionine by lysosomal proteolysis of parental low molecular weight polypeptides to reduce renal radioactivity levels.

PubMed

Uehara, Tomoya; Rokugawa, Takemi; Kinoshita, Mai; Nemoto, Souki; Fransisco Lazaro, Guerra Gomez; Hanaoka, Hirofumi; Arano, Yasushi

2014-11-19

The renal localization of gallium-67 or gallium-68 ((67/68)Ga)-labeled low molecular weight (LMW) probes such as peptides and antibody fragments constitutes a problem in targeted imaging. Wu et al. previously showed that (67)Ga-labeled S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bz-NOTA)-conjugated methionine ((67)Ga-NOTA-Met) was rapidly excreted from the kidney in urine following lysosomal proteolysis of the parental (67)Ga-NOTA-Bz-SCN-disulfide-stabilized Fv fragment (Bioconjugate Chem., (1997) 8, 365-369). In the present study, a new (67/68)Ga-labeling reagent for LMW probes that liberates (67/68)Ga-NOTA-Met was designed, synthesized, and evaluated using longer-lived (67)Ga in order to reduce renal radioactivity levels. We employed a methionine-isoleucine (MI) dipeptide bond as the cleavable linkage. The amine residue of MI was coupled with SCN-Bz-NOTA for (67)Ga-labeling, while the carboxylic acid residue of MI was derivatized to maleimide for antibody conjugation in order to synthesize NOTA-MI-Mal. A Fab fragment of the anti-Her2 antibody was thiolated with iminothiolane, and NOTA-MI-Mal was conjugated with the antibody fragment by maleimide-thiol chemistry. The Fab fragment was also conjugated with SCN-Bz-NOTA (NOTA-Fab) for comparison. (67)Ga-NOTA-MI-Fab was obtained at radiochemical yields of over 95% and was stable in murine serum for 24 h. In the biodistribution study using normal mice, (67)Ga-NOTA-MI-Fab registered significantly lower renal radioactivity levels from 1 to 6 h postinjection than those of (67)Ga-NOTA-Fab. An analysis of urine samples obtained 6 h after the injection of (67)Ga-NOTA-MI-Fab showed that the majority of radioactivity was excreted as (67)Ga-NOTA-Met. In the biodistribution study using tumor-bearing mice, the tumor to kidney ratios of (67)Ga-NOTA-MI-Fab were 4 times higher (6 h postinjection) than those of (67)Ga-NOTA-Fab. Although further studies including the structure of radiometabolites and/or cleavable linkages are required, the results of the present study indicate that the current chemical design is applicable to the development of (67)Ga-labeled Fabs for low renal radioactivity levels.

Automation of sample preparation for mass cytometry barcoding in support of clinical research: protocol optimization.

PubMed

Nassar, Ala F; Wisnewski, Adam V; Raddassi, Khadir

2017-03-01

Analysis of multiplexed assays is highly important for clinical diagnostics and other analytical applications. Mass cytometry enables multi-dimensional, single-cell analysis of cell type and state. In mass cytometry, the rare earth metals used as reporters on antibodies allow determination of marker expression in individual cells. Barcode-based bioassays for CyTOF are able to encode and decode for different experimental conditions or samples within the same experiment, facilitating progress in producing straightforward and consistent results. Herein, an integrated protocol for automated sample preparation for barcoding used in conjunction with mass cytometry for clinical bioanalysis samples is described; we offer results of our work with barcoding protocol optimization. In addition, we present some points to be considered in order to minimize the variability of quantitative mass cytometry measurements. For example, we discuss the importance of having multiple populations during titration of the antibodies and effect of storage and shipping of labelled samples on the stability of staining for purposes of CyTOF analysis. Data quality is not affected when labelled samples are stored either frozen or at 4 °C and used within 10 days; we observed that cell loss is greater if cells are washed with deionized water prior to shipment or are shipped in lower concentration. Once the labelled samples for CyTOF are suspended in deionized water, the analysis should be performed expeditiously, preferably within the first hour. Damage can be minimized if the cells are resuspended in phosphate-buffered saline (PBS) rather than deionized water while waiting for data acquisition.

Different mechanisms of hepatitis C virus RNA polymerase activation by cyclophilin A and B in vitro.

PubMed

Weng, Leiyun; Tian, Xiao; Gao, Yayi; Watashi, Koichi; Shimotohno, Kunitada; Wakita, Takaji; Kohara, Michinori; Toyoda, Tetsuya

2012-12-01

Cyclophilins (CyPs) are cellular proteins that are essential to hepatitis C virus (HCV) replication. Since cyclosporine A was discovered to inhibit HCV infection, the CyP pathway contributing to HCV replication is a potential attractive stratagem for controlling HCV infection. Among them, CyPA is accepted to interact with HCV nonstructural protein (NS) 5A, although interaction of CyPB and NS5B, an RNA-dependent RNA polymerase (RdRp), was proposed first. CyPA, CyPB, and HCV RdRp were expressed in bacteria and purified using combination column chromatography. HCV RdRp activity was analyzed in vitro with purified CyPA and CyPB. CyPA at a high concentration (50× higher than that of RdRp) but not at low concentration activated HCV RdRp. CyPB had an allosteric effect on genotype 1b RdRp activation. CyPB showed genotype specificity and activated genotype 1b and J6CF (2a) RdRps but not genotype 1a or JFH1 (2a) RdRps. CyPA activated RdRps of genotypes 1a, 1b, and 2a. CyPB may also support HCV genotype 1b replication within the infected cells, although its knockdown effect on HCV 1b replicon activity was controversial in earlier reports. CyPA activated HCV RdRp at the early stages of transcription, including template RNA binding. CyPB also activated genotype 1b RdRp. However, their activation mechanisms are different. These data suggest that both CyPA and CyPB are excellent targets for the treatment of HCV 1b, which shows the greatest resistance to interferon and ribavirin combination therapy. Copyright © 2012 Elsevier B.V. All rights reserved.

Antibody persistence for up to 5 years after a fourth dose of Haemophilus influenzae type b and Neisseria meningitidis serogroups C and Y-tetanus toxoid conjugate vaccine (HibMenCY-TT) given at 12-15 months of age.

PubMed

Marshall, Gary S; Blatter, Mark; Marchant, Colin; Aris, Emmanuel; Mesaros, Narcisa; Miller, Jacqueline M

2013-06-01

A 4-dose series of recently licensed Haemophilus influenzae type b-meningococcal serogroups C and Y-tetanus toxoid conjugate vaccine (HibMenCY-TT) was immunogenic with a clinically acceptable safety profile in infants, with antibodies persisting in most participants for 1 year following dose 4. This study assessed antibody persistence up to 5 years after vaccination. Participants had received HibMenCY-TT or Hib-TT at 2, 4 and 6 months of age. At age 12-15 months, HibMenCY-TT vaccinees received a fourth HibMenCY-TT dose (HibMenCY x 4 group), whereas those who received Hib-TT received a fourth dose of either Hib-TT (Hib) or HibMenCY-TT (HibMenCY x 1). Blood samples were collected 1 month and 1, 3 and 5 years after the last dose for measurement of antipolyribosylribitol phosphate (the Hib capsular polysaccharide) antibodies and serum bactericidal activity (human complement source) against meningococcal serogroups C and Y. Five years after the fourth dose, the percentages of children with antipolyribosylribitol phosphate ≥0.15 μg/mL in HibMenCY x 4, HibMenCY x 1 and Hib groups were 98.8% (95% confidence interval: 93.5%-100%), 97.3% (85.8%-99.9%) and 92.3% (79.1%-98.4%), respectively. The percentages with human complement serum bactericidal activity ≥1:8 for meningococcal serogroup C were 82.9% (72.5%-90.6%), 73.5% (55.6%-87.1%) and 21.1% (9.6%-37.3%), respectively. The percentages with human complement serum bactericidal activity ≥1:8 for serogroup Y were 69.5% (58.4%-79.2%), 54.3% (36.6%-71.2%) and 18.4% (7.7%-34.3%), respectively. HibMenCY-TT given as a 4-dose series or as a single dose at 12-15 months of age induced immune responses for all 3 antigens that lasted for up to 5 years after vaccination in more than half of recipients.

Super-resolution microscopy reveals structural diversity in molecular exchange among peptide amphiphile nanofibres

DOE PAGES

da Silva, Ricardo M. P.; van der Zwaag, Daan; Albertazzi, Lorenzo; ...

2016-05-19

The dynamic behaviour of supramolecular systems is an important dimension of their potential functions. Here, we report on the use of stochastic optical reconstruction microscopy to study the molecular exchange of peptide amphiphile nanofibres, supramolecular systems known to have important biomedical functions. Solutions of nanofibres labelled with different dyes (Cy3 and Cy5) were mixed, and the distribution of dyes inserting into initially single-colour nanofibres was quantified using correlative image analysis. Our observations are consistent with an exchange mechanism involving monomers or small clusters of molecules inserting randomly into a fibre. Different exchange rates are observed within the same fibre, suggestingmore » that local cohesive structures exist on the basis of beta-sheet discontinuous domains. The results reported here show that peptide amphiphile supramolecular systems can be dynamic and that their intermolecular interactions affect exchange patterns. Lastly, this information can be used to generate useful aggregate morphologies for improved biomedical function.« less

High ordered biomineralization induced by carbon nanoparticles in the sea urchin Paracentrotus lividus.

PubMed

Manno, Daniela; Carata, Elisabetta; Tenuzzo, Bernadetta A; Panzarini, Elisa; Buccolieri, Alessandro; Filippo, Emanuela; Rossi, Marco; Serra, Antonio; Dini, Luciana

2012-12-14

A surprising and unexpected biomineralization process was observed during toxicological assessment of carbon nanoparticles on Paracentrotus lividus (sea urchin) pluteus larvae. The larvae activate a process of defense against external material, by incorporating the nanoparticles into microstructures of aragonite similarly to pearl oysters. Aiming at a better understanding of this phenomenon, the larvae were exposed to increasing concentrations of carbon nanoparticles and the biomineralization products were analyzed by electron microscopy, x-ray diffraction and Raman spectroscopy. In order to evaluate the possible influence of Sp-CyP-1 expression on this biomineralization process by larvae, analyses of gene expression (Sp-CyP-1) and calcein labeling were performed. Overall, we report experimental evidence about the capability of carbon nanoparticles to induce an increment of Sp-CyP-1 expression with the consequent activation of a biomineralization process leading to the production of a new pearl-like biomaterial never previously observed in sea urchins.

High ordered biomineralization induced by carbon nanoparticles in the sea urchin Paracentrotus lividus

NASA Astrophysics Data System (ADS)

Manno, Daniela; Carata, Elisabetta; Tenuzzo, Bernadetta A.; Panzarini, Elisa; Buccolieri, Alessandro; Filippo, Emanuela; Rossi, Marco; Serra, Antonio; Dini, Luciana

2012-12-01

A surprising and unexpected biomineralization process was observed during toxicological assessment of carbon nanoparticles on Paracentrotus lividus (sea urchin) pluteus larvae. The larvae activate a process of defense against external material, by incorporating the nanoparticles into microstructures of aragonite similarly to pearl oysters. Aiming at a better understanding of this phenomenon, the larvae were exposed to increasing concentrations of carbon nanoparticles and the biomineralization products were analyzed by electron microscopy, x-ray diffraction and Raman spectroscopy. In order to evaluate the possible influence of Sp-CyP-1 expression on this biomineralization process by larvae, analyses of gene expression (Sp-CyP-1) and calcein labeling were performed. Overall, we report experimental evidence about the capability of carbon nanoparticles to induce an increment of Sp-CyP-1 expression with the consequent activation of a biomineralization process leading to the production of a new pearl-like biomaterial never previously observed in sea urchins.

Cyanine 5.5 Conjugated Nanobubbles as a Tumor Selective Contrast Agent for Dual Ultrasound-Fluorescence Imaging in a Mouse Model

PubMed Central

Li, Jing; Wei, Qiong; Yuchi, Ming; He, Xiaoling; Ding, Mingyue; Zhou, Qibing

2013-01-01

Nanobubbles and microbubbles are non-invasive ultrasound imaging contrast agents that may potentially enhance diagnosis of tumors. However, to date, both nanobubbles and microbubbles display poor in vivo tumor-selectivity over non-targeted organs such as liver. We report here cyanine 5.5 conjugated nanobubbles (cy5.5-nanobubbles) of a biocompatible chitosan–vitamin C lipid system as a dual ultrasound-fluorescence contrast agent that achieved tumor-selective imaging in a mouse tumor model. Cy5.5-nanobubble suspension contained single bubble spheres and clusters of bubble spheres with the size ranging between 400–800 nm. In the in vivo mouse study, enhancement of ultrasound signals at tumor site was found to persist over 2 h while tumor-selective fluorescence emission was persistently observed over 24 h with intravenous injection of cy5.5-nanobubbles. In vitro cell study indicated that cy5.5-flurescence dye was able to accumulate in cancer cells due to the unique conjugated nanobubble structure. Further in vivo fluorescence study suggested that cy5.5-nanobubbles were mainly located at tumor site and in the bladder of mice. Subsequent analysis confirmed that accumulation of high fluorescence was present at the intact subcutaneous tumor site and in isolated tumor tissue but not in liver tissue post intravenous injection of cy5.5-nanobubbles. All these results led to the conclusion that cy5.5-nanobubbles with unique crosslinked chitosan–vitamin C lipid system have achieved tumor-selective imaging in vivo. PMID:23637799

Cyanine 5.5 conjugated nanobubbles as a tumor selective contrast agent for dual ultrasound-fluorescence imaging in a mouse model.

PubMed

Mai, Liyi; Yao, Anna; Li, Jing; Wei, Qiong; Yuchi, Ming; He, Xiaoling; Ding, Mingyue; Zhou, Qibing

2013-01-01

Nanobubbles and microbubbles are non-invasive ultrasound imaging contrast agents that may potentially enhance diagnosis of tumors. However, to date, both nanobubbles and microbubbles display poor in vivo tumor-selectivity over non-targeted organs such as liver. We report here cyanine 5.5 conjugated nanobubbles (cy5.5-nanobubbles) of a biocompatible chitosan-vitamin C lipid system as a dual ultrasound-fluorescence contrast agent that achieved tumor-selective imaging in a mouse tumor model. Cy5.5-nanobubble suspension contained single bubble spheres and clusters of bubble spheres with the size ranging between 400-800 nm. In the in vivo mouse study, enhancement of ultrasound signals at tumor site was found to persist over 2 h while tumor-selective fluorescence emission was persistently observed over 24 h with intravenous injection of cy5.5-nanobubbles. In vitro cell study indicated that cy5.5-flurescence dye was able to accumulate in cancer cells due to the unique conjugated nanobubble structure. Further in vivo fluorescence study suggested that cy5.5-nanobubbles were mainly located at tumor site and in the bladder of mice. Subsequent analysis confirmed that accumulation of high fluorescence was present at the intact subcutaneous tumor site and in isolated tumor tissue but not in liver tissue post intravenous injection of cy5.5-nanobubbles. All these results led to the conclusion that cy5.5-nanobubbles with unique crosslinked chitosan-vitamin C lipid system have achieved tumor-selective imaging in vivo.

Cy3 and Cy5 dyes attached to oligonucleotide terminus stabilize DNA duplexes: predictive thermodynamic model.

PubMed

Moreira, Bernardo G; You, Yong; Owczarzy, Richard

2015-03-01

Cyanine dyes are important chemical modifications of oligonucleotides exhibiting intensive and stable fluorescence at visible light wavelengths. When Cy3 or Cy5 dye is attached to 5' end of a DNA duplex, the dye stacks on the terminal base pair and stabilizes the duplex. Using optical melting experiments, we have determined thermodynamic parameters that can predict the effects of the dyes on duplex stability quantitatively (ΔG°, Tm). Both Cy dyes enhance duplex formation by 1.2 kcal/mol on average, however, this Gibbs energy contribution is sequence-dependent. If the Cy5 is attached to a pyrimidine nucleotide of pyrimidine-purine base pair, the stabilization is larger compared to the attachment to a purine nucleotide. This is likely due to increased stacking interactions of the dye to the purine of the complementary strand. Dangling (unpaired) nucleotides at duplex terminus are also known to enhance duplex stability. Stabilization originated from the Cy dyes is significantly larger than the stabilization due to the presence of dangling nucleotides. If both the dangling base and Cy3 are present, their thermodynamic contributions are approximately additive. New thermodynamic parameters improve predictions of duplex folding, which will help design oligonucleotide sequences for biophysical, biological, engineering, and nanotechnology applications. Copyright © 2015. Published by Elsevier B.V.

Sequence-Independent Cloning and Post-Translational Modification of Repetitive Protein Polymers through Sortase and Sfp-Mediated Enzymatic Ligation.

PubMed

Ott, Wolfgang; Nicolaus, Thomas; Gaub, Hermann E; Nash, Michael A

2016-04-11

Repetitive protein-based polymers are important for many applications in biotechnology and biomaterials development. Here we describe the sequential additive ligation of highly repetitive DNA sequences, their assembly into genes encoding protein-polymers with precisely tunable lengths and compositions, and their end-specific post-translational modification with organic dyes and fluorescent protein domains. Our new Golden Gate-based cloning approach relies on incorporation of only type IIS BsaI restriction enzyme recognition sites using PCR, which allowed us to install ybbR-peptide tags, Sortase c-tags, and cysteine residues onto either end of the repetitive gene polymers without leaving residual cloning scars. The assembled genes were expressed in Escherichia coli and purified using inverse transition cycling (ITC). Characterization by cloud point spectrophotometry, and denaturing polyacrylamide gel electrophoresis with fluorescence detection confirmed successful phosphopantetheinyl transferase (Sfp)-mediated post-translational N-terminal labeling of the protein-polymers with a coenzyme A-647 dye (CoA-647) and simultaneous sortase-mediated C-terminal labeling with a GFP domain containing an N-terminal GG-motif in a one-pot reaction. In a further demonstration, we installed an N-terminal cysteine residue into an elastin-like polypeptide (ELP) that was subsequently conjugated to a single chain poly(ethylene glycol)-maleimide (PEG-maleimide) synthetic polymer, noticeably shifting the ELP cloud point. The ability to straightforwardly assemble repetitive DNA sequences encoding ELPs of precisely tunable length and to post-translationally modify them specifically at the N- and C- termini provides a versatile platform for the design and production of multifunctional smart protein-polymeric materials.

Site Specific Discrete PEGylation of 124I-Labeled mCC49 Fab′ Fragments Improves Tumor MicroPET/CT Imaging in Mice

PubMed Central

Ding, Haiming; Carlton, Michelle M.; Povoski, Stephen P.; Milum, Keisha; Kumar, Krishan; Kothandaraman, Shankaran; Hinkle, George H.; Colcher, David; Brody, Rich; Davis, Paul D.; Pokora, Alex; Phelps, Mitchell; Martin, Edward W.; Tweedle, Michael F.

2014-01-01

The tumor-associated glycoprotein-72 (TAG-72) antigen is highly overexpressed in various human adenocarcinomas and anti-TAG-72 monoclonal antibodies, and fragments are therefore useful as pharmaceutical targeting vectors. In this study, we investigated the effects of site-specific PEGylation with MW 2–4 kDa discrete, branched PEGylation reagents on mCC49 Fab′ (MW 50 kDa) via in vitro TAG72 binding, and in vivo blood clearance kinetics, biodistribution, and mouse tumor microPET/CT imaging. mCC49Fab′ (Fab′-NEM) was conjugated at a hinge region cysteine with maleimide-dPEG12-(dPEG24COOH)3 acid (Mal-dPEG-A), maleimide-dPEG12-(dPEG12COOH)3 acid (Mal-dPEG-B), or maleimide-dPEG12-(m-dPEG24)3 (Mal-dPEG-C), and then radiolabeled with iodine-124 (124I) in vitro radioligand binding assays and in vivo studies used TAG-72 expressing LS174T human colon carcinoma cells and xenograft mouse tumors. Conjugation of mCC49Fab′ with Mal-dPEG-A (Fab′-A) reduced the binding affinity of the non PEGylated Fab′ by 30%; however, in vivo, Fab′-A significantly lengthened the blood retention vs Fab′-NEM (47.5 vs 28.1%/ID at 1 h, 25.1 vs 8.4%/ID at 5 h, p < 0.01), showed excellent tumor to background, better microPET/CT images due to higher tumor accumulation, and increased tumor concentration in excised tissues at 72 h by 130% (5.09 ± 0.83 vs 3.83 ± 1.50%ID/g, p < 0.05). Despite the strong similarity of the three PEGylation reagents, PEGylation with Mal-dPEG-B or -C reduced the in vitro binding affinity of Fab′-NEM by 70%, blood retention, microPET/CT imaging tumor signal intensity, and residual 72 h tumor concentration by 49% (3.83 ± 1.50 vs 1.97 ± 0.29%ID/g, p < 0.05) and 63% (3.83 ± 1.50 vs 1.42 ± 0.35%ID/g, p < 0.05), respectively. We conclude that remarkably subtle changes in the structure of the PEGylation reagent can create significantly altered biologic behavior. Further study is warranted of conjugates of the triple branched, negatively charged Mal-dPEG-A. PMID:24175669

Concerted loop motion triggers induced fit of FepA to ferric enterobactin

PubMed Central

Smallwood, Chuck R.; Jordan, Lorne; Trinh, Vy; Schuerch, Daniel W.; Gala, Amparo; Hanson, Mathew; Shipelskiy, Yan; Majumdar, Aritri; Newton, Salete M.C.

2014-01-01

Spectroscopic analyses of fluorophore-labeled Escherichia coli FepA described dynamic actions of its surface loops during binding and transport of ferric enterobactin (FeEnt). When FeEnt bound to fluoresceinated FepA, in living cells or outer membrane fragments, quenching of fluorophore emissions reflected conformational motion of the external vestibular loops. We reacted Cys sulfhydryls in seven surface loops (L2, L3, L4, L5, L7 L8, and L11) with fluorophore maleimides. The target residues had different accessibilities, and the labeled loops themselves showed variable extents of quenching and rates of motion during ligand binding. The vestibular loops closed around FeEnt in about a second, in the order L3 > L11 > L7 > L2 > L5 > L8 > L4. This sequence suggested that the loops bind the metal complex like the fingers of two hands closing on an object, by individually adsorbing to the iron chelate. Fluorescence from L3 followed a biphasic exponential decay as FeEnt bound, but fluorescence from all the other loops followed single exponential decay processes. After binding, the restoration of fluorescence intensity (from any of the labeled loops) mirrored cellular uptake that depleted FeEnt from solution. Fluorescence microscopic images also showed FeEnt transport, and demonstrated that ferric siderophore uptake uniformly occurs throughout outer membrane, including at the poles of the cells, despite the fact that TonB, its inner membrane transport partner, was not detectable at the poles. PMID:24981231

Concerted loop motion triggers induced fit of FepA to ferric enterobactin.

PubMed

Smallwood, Chuck R; Jordan, Lorne; Trinh, Vy; Schuerch, Daniel W; Gala, Amparo; Hanson, Mathew; Hanson, Matthew; Shipelskiy, Yan; Majumdar, Aritri; Newton, Salete M C; Klebba, Phillip E

2014-07-01

Spectroscopic analyses of fluorophore-labeled Escherichia coli FepA described dynamic actions of its surface loops during binding and transport of ferric enterobactin (FeEnt). When FeEnt bound to fluoresceinated FepA, in living cells or outer membrane fragments, quenching of fluorophore emissions reflected conformational motion of the external vestibular loops. We reacted Cys sulfhydryls in seven surface loops (L2, L3, L4, L5, L7 L8, and L11) with fluorophore maleimides. The target residues had different accessibilities, and the labeled loops themselves showed variable extents of quenching and rates of motion during ligand binding. The vestibular loops closed around FeEnt in about a second, in the order L3 > L11 > L7 > L2 > L5 > L8 > L4. This sequence suggested that the loops bind the metal complex like the fingers of two hands closing on an object, by individually adsorbing to the iron chelate. Fluorescence from L3 followed a biphasic exponential decay as FeEnt bound, but fluorescence from all the other loops followed single exponential decay processes. After binding, the restoration of fluorescence intensity (from any of the labeled loops) mirrored cellular uptake that depleted FeEnt from solution. Fluorescence microscopic images also showed FeEnt transport, and demonstrated that ferric siderophore uptake uniformly occurs throughout outer membrane, including at the poles of the cells, despite the fact that TonB, its inner membrane transport partner, was not detectable at the poles. © 2014 Smallwood et al.

Differential reactivity of maleimide and bromoacetyl functions with thiols: application to the preparation of liposomal diepitope constructs.

PubMed

Schelté, P; Boeckler, C; Frisch, B; Schuber, F

2000-01-01

The comparative reactivity of maleimide and bromoacetyl groups with thiols (2-mercaptoethanol, free cysteine, and cysteine residues present at the N-terminus of peptides) was investigated in aqueous media. These studies were performed (i) with water-soluble functionalized model molecules, i.e., polyoxyethylene-based spacer arms that could also be coupled to lipophilic anchors destined to be incorporated into liposomes, and (ii) with small unilamellar liposomes carrying at their surface these thiol-reactive functions. Our results indicate that an important kinetic discrimination (2-3 orders of magnitude in terms of rate constants) can be achieved between the maleimide and bromoacetyl functions when the reactions with thiols are performed at pH 6.5. The bromoacetyl function which reacts at higher pH values (e.g., pH 9.0) retained a high chemoselectivity; i.e., under conditions where it reacted appreciably with the thiols of, e.g., HS-peptides, it did react with other nucleophilic functions such as alpha- and epsilon-amino groups or imidazole, which could also be present in peptides. This differential reactivity was applied to design chemically defined and highly immunogenic liposomal diepitope constructs as synthetic vaccines, i.e., vesicles carrying at their surface two different peptides conjugated each to a specific amphiphilic anchor. This was realized by coupling sequentially at pH 6.5 and 9.0 two HS-peptides to preformed vesicles containing lipophilic anchors functionalized with maleimide and bromoacetyl groups [Boeckler, C., et al. (1999) Eur. J. Immunol. 29, 2297-2308].

Identification and Characterization of Cell Wall Proteins of a Toxic Dinoflagellate Alexandrium catenella Using 2-D DIGE and MALDI TOF-TOF Mass Spectrometry

PubMed Central

Wang, Da-Zhi; Dong, Hong-Po; Li, Cheng; Xie, Zhang-Xian; Lin, Lin; Hong, Hua-Sheng

2011-01-01

The cell wall is an important subcellular component of dinoflagellate cells with regard to various aspects of cell surface-associated ecophysiology, but the full range of cell wall proteins (CWPs) and their functions remain to be elucidated. This study identified and characterized CWPs of a toxic dinoflagellate, Alexandrium catenella, using a combination of 2D fluorescence difference gel electrophoresis (DIGE) and MALDI TOF-TOF mass spectrometry approaches. Using sequential extraction and temperature shock methods, sequentially extracted CWPs and protoplast proteins, respectively, were separated from A. catenella. From the comparison between sequentially extracted CWPs labeled with Cy3 and protoplast proteins labeled with Cy5, 120 CWPs were confidently identified in the 2D DIGE gel. These proteins gave positive identification of protein orthologues in the protein database using de novo sequence analysis and homology-based search. The majority of the prominent CWPs identified were hypothetical or putative proteins with unknown function or no annotation, while cell wall modification enzymes, cell wall structural proteins, transporter/binding proteins, and signaling and defense proteins were tentatively identified in agreement with the expected role of the extracellular matrix in cell physiology. This work represents the first attempt to investigate dinoflagellate CWPs and provides a potential tool for future comprehensive characterization of dinoflagellate CWPs and elucidation of their physiological functions. PMID:21904561

Randomized, Controlled Trial of Intravenous Immunoglobulin for Pediatric Autoimmune Neuropsychiatric Disorders Associated With Streptococcal Infections.

PubMed

Williams, Kyle A; Swedo, Susan E; Farmer, Cristan A; Grantz, Heidi; Grant, Paul J; D'Souza, Precilla; Hommer, Rebecca; Katsovich, Liliya; King, Robert A; Leckman, James F

2016-10-01

Pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS) are hypothesized to occur as a result of cross-reactive antibodies produced in response to group A streptococcal infections. Previous research suggests that immunomodulatory therapies, such as intravenous immunoglobulin (IVIG), may lead to rapid and sustained symptom improvement in patients with PANDAS. A total of 35 children meeting criteria for PANDAS and moderate to severe obsessive-compulsive disorder (OCD) were enrolled in a randomized-entry, double-blind, placebo-controlled, 6-week trial of IVIG (1 g/kg/day on 2 consecutive days), followed by optional open-label treatment for nonresponders, with follow-up at 12 and 24 weeks. Primary outcome measures were the Children's Yale-Brown Obsessive Compulsive Scale (CY-BOCS) and the Clinical Global Impressions-Improvement (CGI-I) rating. "Responders" were defined, a priori, by a ≥ 30% decrease in CY-BOCS total score, and a "much" or "very much" improved rating on CGI-I. During the double-blind phase, the mean decrease in CY-BOCS score was 24% ± 31% in the IVIG group (n = 17) and 12% ± 27% in the placebo group (n = 18), with six responders in the IVIG group (35%) versus four (22%) in the placebo group; these differences were not statistically significant. Twenty-four participants met criteria for nonresponse to double-blind infusion and received open-label IVIG at week 6. Among all participants, the mean CY-BOCS improvement from baseline was 55% ± 33% at week 12 and 62% ± 33% at week 24. IVIG was safe and well tolerated. Between-group differences were smaller than anticipated, and the double-blind comparison failed to demonstrate superiority of IVIG over placebo. The observed open-label improvements indicate that future trials would benefit from larger sample sizes designed in part to aid in the identification of biomarkers predictive of a positive response to immunotherapy. Future investigations focused on the natural history of PANDAS are also warranted. Clinical trial registration information-Intravenous Immunoglobulin for PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated With Streptococcal Infections); http://clinicaltrials.gov/; NCT01281969ZIAMH002666. Published by Elsevier Inc.

Electron paramagnetic resonance study of lipid and protein membrane components of erythrocytes oxidized with hydrogen peroxide

PubMed Central

Mendanha, S.A.; Anjos, J.L.V.; Silva, A.H.M.; Alonso, A.

2012-01-01

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to monitor membrane dynamic changes in erythrocytes subjected to oxidative stress with hydrogen peroxide (H2O2). The lipid spin label, 5-doxyl stearic acid, responded to dramatic reductions in membrane fluidity, which was correlated with increases in the protein content of the membrane. Membrane rigidity, associated with the binding of hemoglobin (Hb) to the erythrocyte membrane, was also indicated by a spin-labeled maleimide, 5-MSL, covalently bound to the sulfhydryl groups of membrane proteins. At 2% hematocrit, these alterations in membrane occurred at very low concentrations of H2O2 (50 µM) after only 5 min of incubation at 37°C in azide phosphate buffer, pH 7.4. Lipid peroxidation, suggested by oxidative hemolysis and malondialdehyde formation, started at 300 µM H2O2 (for incubation of 3 h), which is a concentration about six times higher than those detected with the probes. Ascorbic acid and α-tocopherol protected the membrane against lipoperoxidation, but did not prevent the binding of proteins to the erythrocyte membrane. Moreover, the antioxidant (+)-catechin, which also failed to prevent the cross-linking of cytoskeletal proteins with Hb, was very effective in protecting erythrocyte ghosts from lipid peroxidation induced by the Fenton reaction. This study also showed that EPR spectroscopy can be useful to assess the molecular dynamics of red blood cell membranes in both the lipid and protein domains and examine oxidation processes in a system that is so vulnerable to oxidation. PMID:22473321

The modifier effects of chymotrypsin and trypsin enzymes on fluorescence lifetime distribution of "N-(1-pyrenyl)maleimide-bovine serum albumin" complex

NASA Astrophysics Data System (ADS)

Özyiğit, İbrahim Ethem; Karakuş, Emine; Pekcan, Önder

2016-02-01

Chymotrypsin and trypsin are the well known proteolytic enzymes, both of which are synthesized in the pancreas as their precursors - the inactive forms; chymotrypsinogen and trypsinogen - and then are released into the duodenum to cut proteins into smaller peptides. In this paper, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat bovine serum albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. In the labeling study of BSA with PM, it is aimed to attach PM to the single free thiol (Cys34) and to all the free amine groups in accessible positions in order to produce excimers of pyrene planes of the possible highest amount to form the lifetime distributions in the widest range, that may show specifically distinguishing changes resulting from the activities of the proteases. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analyzed by using the exponential series method (ESM) to obtain the changes of lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the enzymes, the fluorescence lifetime distributions exhibited different structures which were attributed to the different activities of the proteases.

A pretreatment method for HPLC analysis of cypermethrin in microbial degradation systems.

PubMed

Liu, Shuliang; Yao, Kai; Jia, Dongying; Zhao, Nan; Lai, Wen; Yuan, Huaiyu

2012-07-01

In this paper, a pretreatment method for high-performance liquid chromatography (HPLC) determination of cypermethrin (CY) in microbial degradation systems was systemically studied, primarily to solve the problem of inaccurate determination of CY concentration caused by its uneven distribution in the systems. A suitable pretreatment method was established, including sampling, extraction and dehydration of CY. Partial sampling could be taken for bacterial and yeast systems in which CY was uniformly dispersed by an emulsifying agent, while total sampling was only suitable for mold systems with or without an emulsifying agent. CY could be fully extracted from the samples in which microbial cells were disrupted by ultrasonic treatment with acetonitrile under ultrasonic condition. The extract could be effectively dehydrated and purified by passing it through an anhydrous Na(2)SO(4) column followed by an elution with acetonitrile. The determination of CY in the pretreated sample by HPLC showed a high precision [relative standard deviation (RSD) = 1.14%, n = 5] and a good stability over a period of five days (RSD = 1.57%, n = 5). The recoveries of CY in microbial degradation systems at three different spiked levels ranged from 95.68 to 108.09% (RSD = 0.50-5.87%, n = 5).

Multicolor fluorescence "click"-chemistry as a means to select membrane targets for pre-targeting approaches by function of their receptor kinetics.

PubMed

van der Wal, Steffen; de Korne, Clarize M; Sand, Laurens L G; van Willigen, Danny M; Hogendoorn, Pancras C W; Szuhai, Karoly; van Leeuwen, Fijs W B; Buckle, Tessa

2018-06-04

Availability of a receptor for theranostic pre-targeting approaches was assessed using a novel "click" chemistry-based de-activatable fluorescence-quenching concept. Efficacy was evaluated in a cell-based model system that exhibits both membranous (available) and internalized (unavailable) receptor-fractions of the clinically relevant receptor chemokine receptor 4 (CXCR4). Proof of concept was based on a de-activatable tracer consisting out of a CXCR4 specific peptide functionalized with a Cy5 dye comprising a chemo-selective azide handle (N3-Cy5-AcTZ14011). Reaction with a Cy7 quencher dye (Cy7-DBCO) resulted in optically silent Cy7-["click"]-Cy5-AcTZ14011. In situ a >90% FRET-based reduction of signal intensity of N3-Cy5-AcTZ14011 (KD 222.4 ± 25.2 nM) was seen within minutes after quencher addition. In cells, discrimination between the membranous and internalized receptor-fraction could be made through quantitative assessment of quenching/internalization kinetics. As such, using this approach screening of membrane receptors and their applicability in receptor-(pre-)targeted theranostics can become straightforward. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Investigation of energy transfer between semiconducting polymer dot donors and hydrophilic and hydrophobic Cy5 acceptors

NASA Astrophysics Data System (ADS)

Lix, Kelsi; Algar, W. Russ

2016-09-01

Semiconducting polymer dots (Pdots) are rapidly emerging fluorescent probes for bioanalysis. Pdots have extraordinarily strong absorption and bright emission compared to other commonly used fluorescent probes, making them very attractive for applications involving Förster resonance energy transfer (FRET). Here, we investigated two FRET systems with green-emitting poly(9,9-dioctylfluorene-alt-benzothiadiazole) (F8BT) Pdots as donors and two different Cyanine 5 (Cy5) dyes as acceptors. A hydrophilic sulfo-Cy5 dye was directly conjugated to the Pdot surface using carbodiimide chemistry, and a hydrophobic Cy5 dye was observed to spontaneously partition into the core of the Pdot. FRET was observed to depend on the acceptor dye concentration with both systems, and was characterized using a combination of fluorescence emission spectra, excitation spectra, and lifetime measurements. Much stronger quenching of Pdot emission and FRET-sensitized acceptor dye emission were observed for the hydrophobic Cy5 system, and these trends were attributed to reduced donor-acceptor distances in comparison to the hydrophilic sulfo-Cy5 system. Current limitations in the experimental format are discussed. The results show that Pdots are effective FRET donors for acceptor dyes located both within and at the surface of Pdots.

An excited state intramolecular proton transfer dye based fluorescence turn-on probe for fast detection of thiols and its applications in bioimaging

NASA Astrophysics Data System (ADS)

Zhao, Yun; Xue, Yuanyuan; Li, Haoyang; Zhu, Ruitao; Ren, Yuehong; Shi, Qinghua; Wang, Song; Guo, Wei

2017-03-01

In this study, a new fluorescent probe 2-(2‧-hydroxy-5‧-N-maleimide phenyl)-benzothiazole (probe 1), was designed and synthesized by linking the excited state intramolecular proton transfer (ESIPT) fluorophore to the maleimide group for selective detection of thiols in aqueous solution. The fluorescence of probe 1 is strongly quenched by maleimide group through the photo-induced electron transfer (PET) mechanism, but after reaction with thiol, the fluorescence of ESIPT fluorophore is restored, affording a large Stokes shifts. Upon addition of cysteine (Cys), probe 1 exhibited a fast response time (complete within 30 s) and a high signal-to-noise ratio (up to 23-fold). It showed a high selectivity and excellent sensitivity to thiols over other relevant biological species, with a detection limit of 3.78 × 10- 8 M (S/N = 3). Moreover, the probe was successfully applied to the imaging of thiols in living cells.

Design, Synthesis of Novel Lipids as Chemical Permeation Enhancers and Development of Nanoparticle System for Transdermal Drug Delivery

PubMed Central

Shah, Punit P.; Etukala, Jagan Reddy; Vemuri, Adithi; Singh, Mandip

2013-01-01

In the present study, we designed and developed novel lipids that include (Z)-1-(Octadec-9-en-1-yl)-pyrrolidine (Cy5T), 1, 1-Di-((Z)-octadec-9-en-1-yl)pyrrolidin-1-ium iodide (Cy5), (Z)-1-(Octadec-9-en-1-yl)-piperidine (Cy6T), and 1, 1-Di-((Z)-octadec-9-en-1-yl) piperidin-1-ium iodide (Cy6) to enhance the transdermal permeation of some selected drugs. Firstly, we evaluated the transdermal permeation efficacies of these lipids as chemical permeation enhancers in vehicle formulations for melatonin, ß-estradiol, caffeine, α-MSH, and spantide using franz diffusion cells. Among them Cy5 lipid was determined to be the most efficient by increasing the transdermal permeation of melatonin, ß-estradiol, caffeine, α-MSH, and spantide by 1.5 to 3.26-fold more at the epidermal layer and 1.3 to 2.5-fold more at the dermal layer, in comparison to either NMP or OA. Hence we developed a nanoparticle system (cy5 lipid ethanol drug nanoparticles) to evaluate any further improvement in the drug penetration. Cy5 lipid formed uniformly sized nanoparticles ranging from 150–200 nm depending on the type of drug. Further, Cy5 based nanoparticle system significantly (p<0.05) increased the permeation of all the drugs in comparison to the lipid solution and standard permeation enhancers. There were about 1.54 to 22-fold more of drug retained in the dermis for the Cy5 based nanoparticles compared to OA/NMP standard enhancers and 3.87 to 66.67-fold more than lipid solution. In addition, epifluorescent microscopic analysis in rhodamine-PE permeation studies confirmed the superior permeation enhancement of LEDs (detection of fluorescence up to skin depth of 340 μm) more than lipid solution, which revealed fluorescence up to skin depth of only 260 μm. In summary the present findings demonstrate that i) cationic lipid with 5 membered amine heterocyclic ring has higher permeating efficacy than the 6 membered amine hertocyclic ring. ii) The nanoparticle system prepared with Cy5 showed significant (p<0.05) increase in the permeation of the drugs than the control penetration enhancers, oleic acid and NMP. PMID:24349315

Rapid detection of Cyprinid herpesvirus-3 (CyHV-3) using a gold nanoparticle-based hybridization assay.

PubMed

Saleh, Mona; El-Matbouli, Mansour

2015-06-01

Cyprinid herpesvirus-3 (CyHV-3) is a highly infectious pathogen that causes fatal disease in common and koi carp Cyprinus carpio L. CyHV-3 detection is usually based on virus propagation or amplification of the viral DNA using the PCR or LAMP techniques. However, due to the limited susceptibility of cells used for propagation, it is not always possible to successfully isolate CyHV-3 even from tissue samples that have high virus titres. All previously described detection methods including PCR-based assays are time consuming, laborious and require specialized equipment. To overcome these limitations, gold nanoparticles (AuNPs) have been explored for direct and sensitive detection of DNA. In this study, a label-free colorimetric nanodiagnostic method for direct detection of unamplified CyHV-3 DNA using gold nanoparticles is introduced. Under appropriate conditions, DNA probes hybridize with their complementary target sequences in the sample DNA, which results in aggregation of the gold nanoparticles and a concomitant colour change from red to blue, whereas test samples with non complementary DNA sequences remain red. In this study, gold nanoparticles were used to develop and evaluate a specific and sensitive hybridization assay for direct and rapid detection of the highly infectious pathogen termed Cyprinid herpesvirus-3. Copyright © 2015 Elsevier B.V. All rights reserved.

Fluorescent TEM-1 β-lactamase with wild-type activity as a rapid drug sensor for in vitro drug screening

PubMed Central

Cheong, Wing-Lam; Tsang, Ming-San; So, Pui-Kin; Chung, Wai-Hong; Leung, Yun-Chung; Chan, Pak-Ho

2014-01-01

We report the development of a novel fluorescent drug sensor from the bacterial drug target TEM-1 β-lactamase through the combined strategy of Val216→Cys216 mutation and fluorophore labelling for in vitro drug screening. The Val216 residue in TEM-1 is replaced with a cysteine residue, and the environment-sensitive fluorophore fluorescein-5-maleimide is specifically attached to the Cys216 residue in the V216C mutant for sensing drug binding at the active site. The labelled V216C mutant has wild-type catalytic activity and gives stronger fluorescence when β-lactam antibiotics bind to the active site. The labelled V216C mutant can differentiate between potent and impotent β-lactam antibiotics and can distinguish active-site binders from non-binders (including aggregates formed by small molecules in aqueous solution) by giving characteristic time-course fluorescence profiles. Mass spectrometric, molecular modelling and trypsin digestion results indicate that drug binding at the active site is likely to cause the fluorescein label to stay away from the active site and experience weaker fluorescence quenching by the residues around the active site, thus making the labelled V216C mutant to give stronger fluorescence in the drug-bound state. Given the ancestor's role of TEM-1 in the TEM family, the fluorescent TEM-1 drug sensor represents a good model to demonstrate the general combined strategy of Val216→Cys216 mutation and fluorophore labelling for fabricating tailor-made fluorescent drug sensors from other clinically significant TEM-type β-lactamase variants for in vitro drug screening. PMID:25074398

Nanobarcoding: detecting nanoparticles in biological samples using in situ polymerase chain reaction

PubMed Central

Eustaquio, Trisha; Leary, James F

2012-01-01

Background Determination of the fate of nanoparticles (NPs) in a biological system, or NP biodistribution, is critical in evaluating an NP formulation for nanomedicine. Current methods to determine NP biodistribution are greatly inadequate, due to their limited detection thresholds. Herein, proof of concept of a novel method for improved NP detection based on in situ polymerase chain reaction (ISPCR), coined “nanobarcoding,” is demonstrated. Methods Nanobarcoded superparamagnetic iron oxide nanoparticles (NB-SPIONs) were characterized by dynamic light scattering, zeta potential, and hyperspectral imaging measurements. Cellular uptake of Cy5-labeled NB-SPIONs (Cy5-NB-SPIONs) was imaged by confocal microscopy. The feasibility of the nanobarcoding method was first validated by solution-phase PCR and “pseudo”-ISPCR before implementation in the model in vitro system of HeLa human cervical adenocarcinoma cells, a cell line commonly used for ISPCR-mediated detection of human papilloma virus (HPV). Results Dynamic light-scattering measurements showed that NB conjugation stabilized SPION size in different dispersion media compared to that of its precursor, carboxylated SPIONs (COOH-SPIONs), while the zeta potential became more positive after NB conjugation. Hyperspectral imaging confirmed NB conjugation and showed that the NB completely covered the SPION surface. Solution-phase PCR and pseudo-ISPCR showed that the expected amplicons were exclusively generated from the NB-SPIONs in a dose-dependent manner. Although confocal microscopy revealed minimal cellular uptake of Cy5-NB-SPIONs at 50 nM over 24 hours in individual cells, ISPCR detected definitive NB-SPION signals inside HeLa cells over large sample areas. Conclusion Proof of concept of the nanobarcoding method has been demonstrated in in vitro systems, but the technique needs further development before its widespread use as a standardized assay. PMID:23144562

Maleimide-functionalized closo-dodecaborate albumin conjugates (MID-AC): Unique ligation at cysteine and lysine residues enables efficient boron delivery to tumor for neutron capture therapy.

PubMed

Kikuchi, Shunsuke; Kanoh, Daisuke; Sato, Shinichi; Sakurai, Yoshinori; Suzuki, Minoru; Nakamura, Hiroyuki

2016-09-10

Maleimide-conjugating closo-dodecaborate sodium form 5c (MID) synthesized by the nucleophilic ring-opening reaction of closo-dodecaborate-1,4-dioxane complex 2 with tetrabutylammonium (TBA) azide was found to conjugate to free SH of cysteine and lysine residues in BSA under physiological conditions, forming highly boronated BSA that showed high and selective accumulation in tumor and significant tumor growth inhibition in colon 26 tumor-bearing mice subjected to thermal neutron irradiation. Copyright © 2016 Elsevier B.V. All rights reserved.

Visualization of cell death in mice with focal cerebral ischemia using fluorescent annexin A5, propidium iodide, and TUNEL staining.

PubMed

Bahmani, Peyman; Schellenberger, Eyk; Klohs, Jan; Steinbrink, Jens; Cordell, Ryan; Zille, Marietta; Müller, Jochen; Harhausen, Denise; Hofstra, Leo; Reutelingsperger, Chris; Farr, Tracy Deanne; Dirnagl, Ulrich; Wunder, Andreas

2011-05-01

To monitor stroke-induced brain damage and assess neuroprotective therapies, specific imaging of cell death after cerebral ischemia in a noninvasive manner is highly desirable. Annexin A5 has been suggested as a marker for imaging cell death under various disease conditions including stroke. In this study, C57BL6/N mice received middle cerebral artery occlusion (MCAO) and were injected intravenously with either active or inactive Cy5.5-annexin A5 48 hours after reperfusion. Some mice also received propidium iodide (PI), a cell integrity marker. Only in mice receiving active Cy5.5-annexin A5 were fluorescence intensities significantly higher over the hemisphere ipsilateral to MCAO than on the contralateral side. This was detected noninvasively and ex vivo 4 and 8 hours after injection. The majority of cells positive for fluorescent annexin A5 were also positive for PI and fragmented DNA as detected by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining. This study demonstrates the high specificity of annexin A5 for visualization of cell death in a mouse model of stroke. To our knowledge, this is the first study to compare the distribution of injected active and inactive annexin A5, PI, and TUNEL staining. It provides important information on the experimental and potential clinical applications of annexin A5-based imaging agents in stroke.

Visualization of cell death in mice with focal cerebral ischemia using fluorescent annexin A5, propidium iodide, and TUNEL staining

PubMed Central

Bahmani, Peyman; Schellenberger, Eyk; Klohs, Jan; Steinbrink, Jens; Cordell, Ryan; Zille, Marietta; Müller, Jochen; Harhausen, Denise; Hofstra, Leo; Reutelingsperger, Chris; Farr, Tracy Deanne; Dirnagl, Ulrich; Wunder, Andreas

2011-01-01

To monitor stroke-induced brain damage and assess neuroprotective therapies, specific imaging of cell death after cerebral ischemia in a noninvasive manner is highly desirable. Annexin A5 has been suggested as a marker for imaging cell death under various disease conditions including stroke. In this study, C57BL6/N mice received middle cerebral artery occlusion (MCAO) and were injected intravenously with either active or inactive Cy5.5-annexin A5 48 hours after reperfusion. Some mice also received propidium iodide (PI), a cell integrity marker. Only in mice receiving active Cy5.5-annexin A5 were fluorescence intensities significantly higher over the hemisphere ipsilateral to MCAO than on the contralateral side. This was detected noninvasively and ex vivo 4 and 8 hours after injection. The majority of cells positive for fluorescent annexin A5 were also positive for PI and fragmented DNA as detected by terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling (TUNEL) staining. This study demonstrates the high specificity of annexin A5 for visualization of cell death in a mouse model of stroke. To our knowledge, this is the first study to compare the distribution of injected active and inactive annexin A5, PI, and TUNEL staining. It provides important information on the experimental and potential clinical applications of annexin A5-based imaging agents in stroke. PMID:21245871

Timed sequential chemotherapy of cytoxan-refractory multiple myeloma with cytoxan and adriamycin based on induced tumor proliferation.

PubMed

Karp, J E; Humphrey, R L; Burke, P J

1981-03-01

Malignant plasma cell proliferation and induced humoral stimulatory activity (HSA) occur in vivo at a predictable time following drug administration. Sequential sera from 11 patients with poor-risk multiple myeloma (MM) undergoing treatment with Cytoxan (CY) 2400 mq/sq m were assayed for their in vitro effects on malignant bone marrow plasma cell tritiated thymidine (3HTdR) incorporation. Peak HSA was detected day 9 following CY. Sequential changes in marrow malignant plasma cell 3HTdR-labeling indices (LI) paralleled changes in serum activity, with peak LI occurring at the time of peak HS. An in vitro model of chemotherapy demonstrated that malignant plasma cell proliferation was enhanced by HSA, as determined by 3HTdR incorporation assay, 3HTdR LI, and tumor cells counts, and that stimulated plasma cells were more sensitive to cytotoxic effects of adriamycin (ADR) than were cells cultured in autologous pretreatment serum. Based on these studies, we designed a clinical trial to treat 12 CY-refractory poor-risk patients with MM in which ADR (60 mg/sq m) was administered at the time of peak HSA and residual tumor cell LI (day 9) following initial CY, 2400 mg/m (CY1ADR9). Eight of 12 (67%) responded to timed sequential chemotherapy with a greater than 50% decrement in monoclonal protein marker and a median survival projected to be greater than 8 mo duration (range 4-21+ mo). These clinical results using timed sequential CY1ADR9 compare favorably with results obtained using ADR in nonsequential chemotherapeutic regimens.

Development of poly(lactic-co-glycolic) acid nanoparticles-embedded hyaluronic acid-ceramide-based nanostructure for tumor-targeted drug delivery.

PubMed

Park, Ju-Hwan; Lee, Jae-Young; Termsarasab, Ubonvan; Yoon, In-Soo; Ko, Seung-Hak; Shim, Jae-Seong; Cho, Hyun-Jong; Kim, Dae-Duk

2014-10-01

A hyaluronic acid-ceramide (HACE) nanostructure embedded with docetaxel (DCT)-loaded poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) was fabricated for tumor-targeted drug delivery. NPs with a narrow size distribution and negative zeta potential were prepared by embedding DCT-loaded PLGA NPs into a HACE nanostructure (DCT/PLGA/HACE). DCT-loaded PLGA and DCT/PLGA/HACE NPs were characterized by solid-state techniques, including Fourier-transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), and powder X-ray diffraction (PXRD). A sustained drug release pattern from the NPs developed was observed and negligible cytotoxicity was seen in NIH3T3 cells (normal fibroblast, CD44 receptor negative) and MDA-MB-231 cells (breast cancer cells, CD44 receptor positive). PLGA/HACE NPs containing coumarin 6, used as a fluorescent dye, exhibited improved cellular uptake efficiency, based on the HA-CD44 receptor interaction, compared to plain PLGA NPs. Cyanine 5.5 (Cy5.5)-labeled PLGA/HACE NPs were injected intravenously into a MDA-MB-231 tumor xenograft mouse model and demonstrated enhanced tumor targetability, compared with Cy5.5-PLGA NPs, according to a near-infrared fluorescence (NIRF) imaging study. Considering these experimental results, the DCT/PLGA/HACE NPs developed may be useful as a tumor-targeted drug delivery system. Copyright © 2014 Elsevier B.V. All rights reserved.

[Construction of lentiviral mediated CyPA siRNA and its functions in non-small cell lung cancer].

PubMed

FENG, Yan-ming; WU, Yi-ming; TU, Xin-ming; XU, Zheng-shun; WU, Wei-dong

2010-02-01

To construct a lentiviral-vector-mediated CyPA small interference RNA (siRNA) and study its function in non-small cell lung cancer. First, four target sequences were selected according to CyPA mRNA sequence, the complementary DNA contained both sense and antisense oligonucleotides were designed, synthesized and cloned into the pGCL-GFP vector, which contained U6 promoter and green fluorescent protein (GFP). The resulting lentiviral vector containing CyPA shRNA was named Lv-shCyPA, and it was confirmed by PCR and sequencing. Next, it was cotransfected by Lipofectamine 2000 along with pHelper1.0 and pHelper 2.0 into 293T cells to package lentivirus particles. At the same time, the packed virus infected non-small cell lung cancer cell (A549), the level of CyPA protein at 5 d after infection was detected by Western Blot to screen the target of CyPA. A549 were infected with Lv-shCyPA and grown as xenografts in severe combined immunodeficient mice. Cell cycle and apoptosis were measured by FCM. It was confirmed by PCR and DNA sequencing that lentiviral-vector-mediated CyPA siRNA (Lv-shCyPA) producing CyPA shRNA was constructed successfully. The titer of concentrated virus were 1 x 10(7) TU/ml. Flow cytometric analysis demonstrated G2-M phase (11.40% +/- 0.68%) was decreased relatively in A549/LvshCyPA compared with control groups (14.52% +/- 1.19%) (P<0.05). The apoptosis rate of A549/Lv-shCyPA (5.01% +/- 0.5%) was higher than control groups (0.35% +/- 0.17%) (P<0.05). Visible tumors were only detectable at 6th day after inoculated by A549/Lv-shCyPA. The xenograft tumors of A549/Lv-shCyPA remarkably delayed tumor growth and remained at a similarly small average size at 38th days after inoculation compared with the control group (P < 0.05). Lentiviral-vector-mediated siRNA technique effectively inhibits the expression of CyPA, induces the NSCLC cell apoptosis, inhibits the tumor growth. Elucidation of the precise role of CypA in these pathways may lead to new targeted therapies for non-small cell lung cancer.

Development of cyclosporine A microemulsion for parenteral delivery.

PubMed

Yuan, Yue; Che, Xin; Zhao, Mingyi; Wang, Yan; Liu, Yajun; Schwendeman, Anna; Li, Sanming

2015-01-01

The goal of this study was to develop a parenteral microemulsion formulation of cyclosporine A (CyA). The CyA solubility in caprylic capric triglyceride (GTCC), ethyl oleate and soybean oil were determined. The pseudo-ternary diagrams of oil (GTCC), surfactant (Solutol® HS-15), cosurfactants (ethanol/polyethylene glycol 400 [PEG 400] mixture) and water were constructed to identify boundaries for microemulsion existence. The CyA was added at 3, 6 and 9% w/w to the optimal microemulsion composition. Microemulsion particle size, solution viscosity and conductivity were examined. The microemulsion stability and haemolytic potential were examined after dilution in 5% dextrose solution for injection to 1 mg/mL CyA. Microemulsion stability was examined after a three-month storage at 4 and 25 °C. The GTCC was selected as an oil phase for CyA microemulsion based on solubility results. The optimum CyA microemulsion formulation consisted of 2.5% CyA, 9% GTCC, 24% Solutol® HS 15, 8% PEG 400, 4% ethanol and 52.5% water based on weight percent. The average particle sizes of the optimized blank and drug-loaded microemulsions were 68.7 nm and 71.6 nm, respectively and remained unchanged upon 25-fold dextrose dilution. The results of microemulsion physical and CyA chemical were confirmed by a three-month stability study at 4 and 25 °C. In vitro haemolysis studies indicated that CyA microemulsions were well tolerated by erythrocytes. The novel microemulsion formulation of CyA was developed that is suitable for parenteral administration. This new formulation could potentially have less vehicle-associated side effects that current commercial formulation of CyA based on Cremophor® EL and ethanol solution.

Fibrin-binding, peptide amphiphile micelles for targeting glioblastoma☆

PubMed Central

Chung, Eun Ji; Cheng, Yu; Morshed, Ramin; Nord, Kathryn; Han, Yu; Wegscheid, Michelle L.; Auffinger, Brenda; Wainwright, Derek A.; Lesniak, Maciej S.; Tirrell, Matthew V.

2013-01-01

Glioblastoma-targeted drug delivery systems facilitate efficient delivery of chemotherapeutic agents to malignant gliomas, while minimizing systemic toxicity and side effects. Taking advantage of the fibrin deposition that is characteristic of tumors, we constructed spherical, Cy7-labeled, targeting micelles to glioblastoma through the addition of the fibrin-binding pentapeptide, cysteine–arginine–glutamic acid–lysine–alanine, or CREKA. Conjugation of the CREKA peptide to Cy7-micelles increased the average particle size and zeta potential. Upon intravenous administration to GL261 glioma bearing mice, Cy7-micelles passively accumulated at the brain tumor site via the enhanced permeability and retention (EPR) effect, and Cy7-CREKA-micelles displayed enhanced tumor homing via active targeting as early as 1 h after administration, as confirmed via in vivo and ex vivo imaging and immunohistochemistry. Biodistribution of micelles showed an accumulation within the liver and kidneys, leading to micelle elimination via renal clearance and the reticuloendothelial system (RES). Histological evaluation showed no signs of cytotoxicity or tissue damage, confirming the safety and utility of this nanoparticle system for delivery to glioblastoma. Our findings offer strong evidence for the glioblastoma-targeting potential of CREKA-micelles and provide the foundation for CREKA-mediated, targeted therapy of glioma. PMID:24211079

Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms.

PubMed

Ford, Nicole R; Hecht, Karen A; Hu, DeHong; Orr, Galya; Xiong, Yijia; Squier, Thomas C; Rorrer, Gregory L; Roesijadi, Guritno

2016-03-18

The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins comprising either enhanced green fluorescent protein (EGFP) or single chain antibodies engineered with a tetracysteine tagging sequence. Of interest were the site-specific binding of (1) the fluorescent biarsenical probe AsCy3 and AsCy3e to the tetracysteine tagged fusion proteins and (2) high and low molecular mass antigens, the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT), to biosilica-immobilized single chain antibodies. Analysis of biarsenical probe binding using fluorescence and structured illumination microscopy indicated differential colocalization with EGFP in nascent and mature biosilica, supporting the use of either EGFP or bound AsCy3 and AsCy3e in studying biosilica maturation. Large increases in the lifetime of a fluorescent analogue of TNT upon binding single chain antibodies provided a robust signal capable of discriminating binding to immobilized antibodies in the transformed frustule from nonspecific binding to the biosilica matrix. In conclusion, our results demonstrate an ability to engineer diatoms to create antibody-functionalized mesoporous silica able to selectively bind chemical and biological agents for the development of sensing platforms.

A CD45-based barcoding approach to multiplex mass-cytometry (CyTOF).

PubMed

Lai, Liyun; Ong, Raymond; Li, Juntao; Albani, Salvatore

2015-04-01

CyTOF enables the study of the immune system with a complexity, depth, and multidimensionality never achieved before. However, the full potential of using CyTOF can be limited by scarce cell samples. Barcoding strategies developed based on direct labeling of cells using maleimido-monoamide-DOTA (m-DOTA) provide a very useful tool. However, using m-DOTA has some inherent problems, mainly associated with signal intensity. This may be a source of uncertainty when samples are multiplexed. As an alternative or complementary approach to m-DOTA, conjugating an antibody, specific for a membrane protein present on most immune cells, with different isotopes could address the issues of stability and signal intensity needed for effective barcoding. We chose for this purpose CD45, and designed experiments to address different types of cultures and the ability to detect extra- and intra-cellular targets. We show here that our approach provides an useful alternative to m-DOTA in terms of sensitivity, specificity, flexibility, and user-friendliness. Our manuscript provides details to effectively barcode immune cells, overcoming limitations in current technology and enabling the use of CyTOF with scarce samples (for instance precious clinical samples). © 2015 The Authors. Published by Wiley Periodicals, Inc.

Tracers for fluorescence-guided surgery: how elongation of the polymethine chain in cyanine dyes alters the pharmacokinetics of a (bimodal) c[RGDyK] tracer.

PubMed

Buckle, Tessa; van Willigen, Danny M; Spa, Silvia J; Hensbergen, Albertus W; van der Wal, Steffen; de Korne, Clarize M; Welling, Mick M; van der Poel, Henk G; Hardwick, James C H; van Leeuwen, Fijs W B

2018-02-15

Objectives: The potential of (receptor-mediated) fluorescence-based image-guided surgery tracers is generally linked to the near-infrared emission profile and good manufacturing production (GMP) availability of fluorescent dyes. Surprisingly, little is known about the critical interaction between the structural composition of the dye and the pharmacokinetics of the tracers. In this study, a bimodal/hybrid tracer design was used to systematically and quantitatively evaluate the influence of elongation of the polymethine chain in a fluorescent cyanine (Cy) dye on the imaging potential of a targeted (RGD-based) tracer. Methods: As model system, the integrin marker ανβ3 was targeted using c[RGDyK] vectors functionalized with a ( 111 In-)DTPA chelate and a fluorescent dye (Cy3-(SO3)Methyl-COOH (λem 580nm), Cy5-(SO3)Methyl-COOH (λem 680nm), or Cy7-(SO3)Methyl-COOH (λem 780nm)). Tracers were analyzed for differences in (photo-) physical properties, serum protein binding, chemical/optical stability and signal penetration through tissue. Receptor affinities (KD) were evaluated using saturation and competition experiments. In vivo biodistribution (SPECT imaging and percentage injected dose per gram of tissue (%ID/g)) was assessed in tumor-bearing mice and complimented with in- and ex vivo fluorescence images obtained using a clinical grade multispectral fluorescence laparoscope. Results: Two carbon-atom-step variations in the polymethine chain of the fluorescent Cy-dyes were shown to significantly influence the chemical and photophysical characteristics e.g. stability, brightness and tissue penetration of the hybrid RGD-tracers. Herein DTPA-Cy5-(SO3)Methyl-COOH-c[RGDyK] systematically outperformed its Cy3- and Cy7- derivatives. Radioactivity-based evaluation of in vivo tracer pharmacokinetics yielded the lowest non-specific uptake and highest tumor-to-background ratio (T/B) for DTPA-Cy5-(SO3)Methyl-COOH-c[RGDyK] (13.2 ± 1.7), with the Cy3- and Cy7- analogs trailing at a respective T/B of 5.7 ± 0.7 and 4.7 ± 0.7. Fluorescence-based assessment of the tumor visibility revealed a similar trend. Conclusion: These findings underline that variations in the polymethine chain lengths of Cy dyes have a profound influence on the photophysical properties, stability and in vivo targeting capabilities of fluorescent imaging tracers. In a direct comparison the intermediate length dye (Cy5) yielded a superior c[RGDyK] -tracer compared to the shorter (Cy3-) and longer (Cy7-) analogs. Copyright © 2018 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Multimodality Imaging with Silica-Based Targeted Nanoparticle Platforms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jason S. Lewis

2012-04-09

Objectives: To synthesize and characterize a C-Dot silica-based nanoparticle containing 'clickable' groups for the subsequent attachment of targeting moieties (e.g., peptides) and multiple contrast agents (e.g., radionuclides with high specific activity) [1,2]. These new constructs will be tested in suitable tumor models in vitro and in vivo to ensure maintenance of target-specificity and high specific activity. Methods: Cy5 dye molecules are cross-linked to a silica precursor which is reacted to form a dye-rich core particle. This core is then encapsulated in a layer of pure silica to create the core-shell C-Dot (Figure 1) [2]. A 'click' chemistry approach has beenmore » used to functionalize the silica shell with radionuclides conferring high contrast and specific activity (e.g. 64Cu and 89Zr) and peptides for tumor targeting (e.g. cRGD and octreotate) [3]. Based on the selective Diels-Alder reaction between tetrazine and norbornene, the reaction is bioorthogonal, highyielding, rapid, and water-compatible. This radiolabeling approach has already been employed successfully with both short peptides (e.g. octreotate) and antibodies (e.g. trastuzumab) as model systems for the ultimate labeling of the nanoparticles [1]. Results: PEGylated C-Dots with a Cy5 core and labeled with tetrazine have been synthesized (d = 55 nm, zeta potential = -3 mV) reliably and reproducibly and have been shown to be stable under physiological conditions for up to 1 month. Characterization of the nanoparticles revealed that the immobilized Cy5 dye within the C-Dots exhibited fluorescence intensities over twice that of the fluorophore alone. The nanoparticles were successfully radiolabeled with Cu-64. Efforts toward the conjugation of targeting peptides (e.g. cRGD) are underway. In vitro stability, specificity, and uptake studies as well as in vivo imaging and biodistribution investigations will be presented. Conclusions: C-Dot silica-based nanoparticles offer a robust, versatile, and multi-functional platform to enhance in vivo detection sensitivity and non-invasively assay receptor expression/status of tumor cellular targets, including those of low abundance, using nuclear-NIR fluorescence imaging approaches [2]. Improvements in molecular diagnostics, refined by the availability of nanotechnology platforms, will be a key determinant in driving early-stage disease detection and prevention, ultimately leading to decreases in mortality.« less

Onset of carbon-carbon bonding in Ta(5)C(y) (y = 0-6) clusters: a threshold photoionization and density functional theory study.

PubMed

Dryza, Viktoras; Alvino, Jason F; Metha, Gregory F

2010-04-01

We have used photoionization efficiency spectroscopy to determine ionization energies (IEs) of the gas-phase tantalum-carbide clusters Ta(5)C(y) (y = 0-6). The structures of the clusters observed in the experiment are assigned by comparing the experimental IEs with those of candidate isomers, calculated by density functional theory. Two competing geometries of the underlying Ta(5) cluster are found to be present in the assigned Ta(5)C(y) structures; either a "prolate" or "distorted oblate" trigonal bipyramid geometry. The onset of carbon-carbon bonding in the Ta(5)C(y) series is proposed to occur at y = 6, with the structure of Ta(5)C(6) containing two molecular C(2) units.

USAF Hearing Conservation Program, DOEHRS-HC Data Repository Annual Report: CY15

DTIC Science & Technology

2017-05-31

AFRL-SA-WP-SR-2017-0014 USAF Hearing Conservation Program, DOEHRS-HC Data Repository Annual Report: CY15 Daniel A. Williams...Conservation Program, DOEHRS-HC Data Repository Annual Report: CY15 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR...Health Readiness System-Hearing Conservation Data Repository (DOEHRS-HC DR). Major command- and installation-level reports are available quarterly

Double walled POE/PLGA microspheres: encapsulation of water-soluble and water-insoluble proteins and their release properties.

PubMed

Shi, Meng; Yang, Yi-Yan; Chaw, Cheng-Shu; Goh, Suat-Hong; Moochhala, Shabbir M; Ng, Steve; Heller, Jorge

2003-04-29

The poly(orthoester) (POE)-poly(D,L-lactide-co-glycolide) (50:50) (PLGA) double-walled microspheres with 50% POE in weight were loaded with hydrophilic bovine serum albumin (BSA) and hydrophobic cyclosporin A (CyA). Most of the BSA and CyA was entrapped within the shell and core, respectively, because of the difference in their hydrophilicity. The morphologies and release mechanisms of proteins-loaded double-walled POE/PLGA microspheres were investigated. Scanning electron microscope studies revealed that the CyA-BSA-loaded double-walled POE/PLGA microspheres yielded a more porous surface and PLGA shell than those without BSA. The neat POE and PLGA yielded slow and incomplete CyA and BSA release. In contrast, nearly complete BSA and more than 95% CyA were released in a sustained manner from the double-walled POE/PLGA microspheres. Both the BSA- and CyA-BSA-loaded POE/PLGA microspheres yielded a sustained BSA release over 5 days. The CyA release pattern of the CyA-loaded double-walled POE/PLGA microspheres was biphasic, characterized by a slow release over 15 days followed by a sustained release over 27 days. However, the CyA-BSA-loaded double-walled POE/PLGA microspheres provided a more constant and faster CyA release due to their more porous shell. In the CyA-BSA-loaded double-walled POE/PLGA microspheres system, the PLGA layer acted as a carrier for BSA and mild reservoir for CyA. During the first 5 days, most BSA was released from the shell but only 14% CyA was left from the microspheres. Subsequently, more than 80% CyA were released in the next 25 days. The distinct structure of double-walled POE/PLGA microspheres would make an interesting device for controlled delivery of therapeutic agents.

Solid-state molecular organometallic chemistry. Single-crystal to single-crystal reactivity and catalysis with light hydrocarbon substrates.

PubMed

Chadwick, F Mark; McKay, Alasdair I; Martinez-Martinez, Antonio J; Rees, Nicholas H; Krämer, Tobias; Macgregor, Stuart A; Weller, Andrew S

2017-08-01

Single-crystal to single-crystal solid/gas reactivity and catalysis starting from the precursor sigma-alkane complex [Rh(Cy 2 PCH 2 CH 2 PCy 2 )(η 2 η 2 -NBA)][BAr F 4 ] (NBA = norbornane; Ar F = 3,5-(CF 3 ) 2 C 6 H 3 ) is reported. By adding ethene, propene and 1-butene to this precursor in solid/gas reactions the resulting alkene complexes [Rh(Cy 2 PCH 2 CH 2 PCy 2 )(alkene) x ][BAr F 4 ] are formed. The ethene ( x = 2) complex, [Rh(Cy 2 PCH 2 CH 2 PCy 2 )(ethene) 2 ][BAr F 4 ]-Oct , has been characterized in the solid-state (single-crystal X-ray diffraction) and by solution and solid-state NMR spectroscopy. Rapid, low temperature recrystallization using solution methods results in a different crystalline modification, [Rh(Cy 2 PCH 2 CH 2 PCy 2 )(ethene) 2 ][BAr F 4 ]-Hex , that has a hexagonal microporous structure ( P 6 3 22). The propene complex ( x = 1) [Rh(Cy 2 PCH 2 CH 2 PCy 2 )(propene)][BAr F 4 ] is characterized as having a π-bound alkene with a supporting γ-agostic Rh···H 3 C interaction at low temperature by single-crystal X-ray diffraction, variable temperature solution and solid-state NMR spectroscopy, as well as periodic density functional theory (DFT) calculations. A fluxional process occurs in both the solid-state and solution that is proposed to proceed via a tautomeric allyl-hydride. Gas/solid catalytic isomerization of d 3 -propene, H 2 C 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 CHCD 3 , using [Rh(Cy 2 PCH 2 CH 2 PCy 2 )(η 2 η 2 -NBA)][BAr F 4 ] scrambles the D-label into all possible positions of the propene, as shown by isotopic perturbation of equilibrium measurements for the agostic interaction. Periodic DFT calculations show a low barrier to H/D exchange (10.9 kcal mol -1 , PBE-D3 level), and GIPAW chemical shift calculations guide the assignment of the experimental data. When synthesized using solution routes a bis-propene complex, [Rh(Cy 2 PCH 2 CH 2 PCy 2 )(propene) 2 ][BAr F 4 ] , is formed. [Rh(Cy 2 PCH 2 CH 2 PCy 2 )(butene)][BAr F 4 ] ( x = 1) is characterized as having 2-butene bound as the cis -isomer and a single Rh···H 3 C agostic interaction. In the solid-state two low-energy fluxional processes are proposed. The first is a simple libration of the 2-butene that exchanges the agostic interaction, and the second is a butene isomerization process that proceeds via an allyl-hydride intermediate with a low computed barrier of 14.5 kcal mol -1 . [Rh(Cy 2 PCH 2 CH 2 PCy 2 )(η 2 η 2 -NBA)][BAr F 4 ] and the polymorphs of [Rh(Cy 2 PCH 2 CH 2 PCy 2 )(ethene) 2 ][BAr F 4 ] are shown to be effective in solid-state molecular organometallic catalysis (SMOM-Cat) for the isomerization of 1-butene to a mixture of cis - and trans -2-butene at 298 K and 1 atm, and studies suggest that catalysis is likely dominated by surface-active species. [Rh(Cy 2 PCH 2 CH 2 PCy 2 )(η 2 η 2 -NBA)][BAr F 4 ] is also shown to catalyze the transfer dehydrogenation of butane to 2-butene at 298 K using ethene as the sacrificial acceptor.

BioTextQuest: a web-based biomedical text mining suite for concept discovery.

PubMed

Papanikolaou, Nikolas; Pafilis, Evangelos; Nikolaou, Stavros; Ouzounis, Christos A; Iliopoulos, Ioannis; Promponas, Vasilis J

2011-12-01

BioTextQuest combines automated discovery of significant terms in article clusters with structured knowledge annotation, via Named Entity Recognition services, offering interactive user-friendly visualization. A tag-cloud-based illustration of terms labeling each document cluster are semantically annotated according to the biological entity, and a list of document titles enable users to simultaneously compare terms and documents of each cluster, facilitating concept association and hypothesis generation. BioTextQuest allows customization of analysis parameters, e.g. clustering/stemming algorithms, exclusion of documents/significant terms, to better match the biological question addressed. http://biotextquest.biol.ucy.ac.cy vprobon@ucy.ac.cy; iliopj@med.uoc.gr Supplementary data are available at Bioinformatics online.

Simple, mild, one-step labelling of proteins with gallium-68 using a tris(hydroxypyridinone) bifunctional chelator: a 68Ga-THP-scFv targeting the prostate-specific membrane antigen.

PubMed

Nawaz, Saima; Mullen, Gregory E D; Sunassee, Kavitha; Bordoloi, Jayanta; Blower, Philip J; Ballinger, James R

2017-10-25

Labelling proteins with gallium-68 using bifunctional chelators is often problematic because of unsuitably harsh labelling conditions such as low pH or high temperature and may entail post-labelling purification. To determine whether tris(hydroxypyridinone) (THP) bifunctional chelators offer a potential solution to this problem, we have evaluated the labelling and biodistribution of a THP conjugate with a new single-chain antibody against the prostate-specific membrane antigen (PSMA), an attractive target for staging prostate cancer (PCa). A single-chain variable fragment (scFv) of J591, a monoclonal antibody that recognises an external epitope of PSMA, was prepared in order to achieve biokinetics matched to the half-life of gallium-68. The scFv, J591c-scFv, was engineered with a C-terminal cysteine. J591c-scFv was produced in HEK293T cells and purified by size-exclusion chromatography. A maleimide THP derivative (THP-mal) was coupled site-specifically to the C-terminal cysteine residue. The THP-mal-J591c-scFv conjugate was labelled with ammonium acetate-buffered gallium-68 from a 68 Ge/ 68 Ga generator at room temperature and neutral pH. The labelled conjugate was evaluated in the PCa cell line DU145 and its PSMA-overexpressing variant in vitro and xenografted in SCID mice. J591c-scFv was produced in yields of 4-6 mg/l culture supernatant and efficiently coupled with the THP-mal bifunctional chelator. Labelling yields > 95% were achieved at room temperature following incubation of 5 μg conjugate with gallium-68 for 5 min without post-labelling purification. 68 Ga-THP-mal-J591c-scFv was stable in serum and showed selective binding to the DU145-PSMA cell line, allowing an IC50 value of 31.5 nM to be determined for unmodified J591c-scFv. Serial PET/CT imaging showed rapid, specific tumour uptake and clearance via renal elimination. Accumulation in DU145-PSMA xenografts at 90 min post-injection was 5.4 ± 0.5%ID/g compared with 0.5 ± 0.2%ID/g in DU145 tumours (n = 4). The bifunctional chelator THP-mal enabled simple, rapid, quantitative, one-step room temperature radiolabelling of a protein with gallium-68 at neutral pH without a need for post-labelling purification. The resultant gallium-68 complex shows high affinity for PSMA and favourable in vivo targeting properties in a xenograft model of PCa.

Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

PubMed Central

Kishen, Ria E. B.; Kluth, David C.; Bellamy, Christopher O. C.

2016-01-01

The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family). Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705), Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches. PMID:27632367

Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability.

PubMed

Moreira, Bernardo G; You, Yong; Behlke, Mark A; Owczarzy, Richard

2005-02-11

Single and dual-labeled fluorescent oligodeoxynucleotides are used in many molecular biology applications. We investigated the effects of commonly used fluorescent dyes and quenchers on the thermodynamic stability of a model probe-target DNA duplex. We demonstrate that those effects can be significant. Fluorescent dyes and quenchers were attached to the probe ends. In certain combinations, these groups stabilized the duplex up to 1.8kcal/mol and increased T(m) up to 4.3 degrees C. None of the groups tested significantly destabilized the duplex. Rank order of potency was, starting with the most stabilizing group: Iowa Black RQ approximately Black Hole 2>Cy5 approximately Cy3>Black Hole 1>QSY7 approximately Iowa Black FQ>Texas Red approximately TAMRA>FAM approximately HEX approximately Dabcyl>TET. Longer linkers decreased stabilizing effects. Hybridizations to targets with various dangling ends were also studied and were found to have only minor effects on thermodynamic stability. Depending on the dye/quencher combination employed, it can be important to include thermodynamic contributions from fluorophore and quencher when designing oligonucleotide probe assays.

Radiohalogenated half-antibodies and maleimide intermediate therefor

DOEpatents

Kassis, Amin I.; Khawli, Leslie A.

1991-01-01

N-(m-radiohalophenyl) maleimide can be conjugated with a reduced antibody having a mercapto group to provide a radiolabelled half-antibody having immunological specific binding characteristics of whole antibody.

Potentiating action of 5-fluorouracil when used in combination with platinium compounds and cyclophosphamide in treatment of advanced L1210 leukemia.

PubMed

Gale, G R; Atkins, L M; Schwartz, P; Meischen, S J

1978-01-01

Nine new organoplatinum (Pt) compounds, cyclophosphamide (CY), and 5-fluorouracil (FU) were used singly and in combination in treatment of advanced L1210 leukemia in C57BL/6 X DBA/2 hybrid mice. In each experiment the Pt + CY dual combination was minimally supra-additive at the doses chosen. However, eight of the nine Pt + CY + FU combination regimens enhanced markedly the increased life span of treated mice as compared with the corresponding dual Pt + CY combination. Collectively, the cure rate (greater than 60-day survival) was less than 6% with the various Pt + CY combinations, and was increased to over 63% upon inclusion of FU in the regimens.

EphB4-targeted imaging with antibody h131, h131-F(ab′)2 and h131-Fab

PubMed Central

Li, Dan; Liu, Shuanglong; Liu, Ren; Zhou, Yue; Park, Ryan; Naga, Kranthi; Krasnoperov, Valery; Gill, Parkash S.; Li, Zibo; Shan, Hong; Conti, Peter S.

2013-01-01

Accumulating evidence suggests that overexpression of the tyrosine kinase receptor EphB4, a mediator of vascular development, is a novel target for tumor diagnosis, prognosis and therapy. Noninvasive imaging of EphB4 expression could therefore be valuable for evaluating disease course and therapeutic efficacy at the earliest stages of anti-EphB4 treatment. In this study, we systematically investigated the use of anti-EphB4 antibody h131 (150 kD) and its fragments (h131-F(ab′)2, 110 kD; h131-Fab, 50 kD) for near-infrared fluorescence (NIRF) imaging of EphB4 expression in vivo. h131-F(ab′)2 and h131-Fab were produced through pepsin and papain digestion of h131 respectively, whose purity was confirmed by FPLC and SDS-PAGE. After conjugation with Cy5.5, in vivo characteristics of h131, h131-F(ab′)2 and h131-Fab were evaluated in EphB4-positive HT29 tumor model. Although h131-Cy5.5 demonstrated highest tumor uptake among these probes, its optimal tumor uptake level was obtained at 2 d post injection (p.i.). For h131-Fab-Cy5.5, maximum tumor uptake was achieved at 4 h p.i.. However, no significant difference was observed between h131-Fab-Cy5.5 and hIgG-Fab-Cy5.5, indicating the tumor accumulation was mainly caused by passive targeting. In contrast, h131-F(ab′)2-Cy5.5 demonstrated prominent tumor uptake at 6 h p.i. The target specificity was confirmed by hIgG-F(ab′)2-Cy5.5 control and immunofluorescent staining. Collectively, h131-F(ab′)2 exhibited prominent and specific tumor uptake at early time points, which suggests it is a promising agent for EphB4-targeted imaging. PMID:24147882

Maleimido substituted aromatic cyclotriphosphazenes

NASA Technical Reports Server (NTRS)

Kumar, D.; Fohlen, G. M.; Parker, J. A. (Inventor)

1985-01-01

4-Aminophenoxy cyclotriphosphazenes are reacted with maleic anhydride to produce maleamic acids which are converted to the maleimides. The maleimides are polymerized. By selection of starting materials (e.g., hexakis amino or trisaminophenoxy-trisphenoxy-cyclo-triphosphazenes), selection of molar proportions of reactants, use of mixtures of anhydrides and use of dianhydrides as bridging groups a variety of maleimides and polymers are produced. The polymers have high limiting oxygen indices, high char yields and other useful heat and fire resistant properties making them useful as, for example, impregnants of fabrics.

Crosslinked polyimides prepared from N-(3-ethynylphenyl)maleimide

NASA Technical Reports Server (NTRS)

Gerber, Margaret K. (Inventor); St.clair, Terry L. (Inventor)

1993-01-01

The compound N-(3-ethynylphenyl)maleimide (NEPMI) was used to prepare thermally stable, glassy polyimides which did not exhibit glass transition temperatures below 500 C. NEPMI was blended with the maleimide of methylene dianiline (BMI) and heated to form the polyimide. NEPMI was also mixed with Thermid 600 R, a commercially available bisethynyl oligomeric material, and heated to form a thermally stable, glassy polyimide. Lastly, NEPMI was blended with both BMI and Thermid 600 R to form thermally stable, glassy polyimides.

N-(3-ethynylphenyl)maleimide

NASA Technical Reports Server (NTRS)

Hergenrother, Paul M. (Inventor); Connell, John W. (Inventor); Havens, Stephen J. (Inventor)

1990-01-01

Acetylene terminated aspartimides are prepared using two methods. In the first, an amino-substituted aromatic acetylene is reacted with an aromatic bismaleimide in a solvent of glacial acetic acid and/or m-cresol. In the second method, an aromatic diamine is reacted with an ethynyl containing maleimide, such as N-(3-ethynylphenyl) maleimide, in a solvent of glacial acetic acid and/or m-cresol. In addition, acetylene terminated aspartimides are blended with various acetylene terminated oligomers and polymers to yield composite materials exhibiting improved mechanical properties.

Aromatic cyclotriphosphazenes

NASA Technical Reports Server (NTRS)

Kamar, Devendra (Inventor); Fohlen, George M. (Inventor); Parker, John A. (Inventor)

1988-01-01

Four-Aminophenoxy cyclotriphosphazenes are reacted with maleic anhydride to produce maleamic acids which are converted to the maleimides. The maleimides are polymerized. By selection of starting materials (e.g., hexakis amino or trisaminophenoxy trisphenoxy cyclotrisphosphazenes), selection of molar porportions of reactants, use of mixtures of anhydrides and use of dianhydrides as bridging groups a variety of maleimides and polymers are produced. The polymers have high limiting oxygen indices, high char yields and other useful heat and fire resistant properties making them useful as, for example, impregnants of fabrics.

Radiohalogenated half-antibodies and maleimide intermediate therefor

DOEpatents

Kassis, A.I.; Khawli, L.A.

1991-02-19

N-(m-radiohalophenyl) maleimide can be conjugated with a reduced antibody having a mercapto group to provide a radiolabeled half-antibody having immunological specific binding characteristics of whole antibody. No Drawings

Enhanced rectal absorption and reduced local irritation of the anti-inflammatory drug ethyl 4-biphenylylacetate in rats by complexation with water-soluble beta-cyclodextrin derivatives and formulation as oleaginous suppository.

PubMed

Arima, H; Kondo, T; Irie, T; Uekama, K

1992-11-01

To improve the rectal delivery of ethyl 4-biphenylylacetate (EBA), a prodrug of the anti-inflammatory drug 4-biphenylylacetic acid (BPAA), the use of highly water-soluble 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) and heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CyD) was investigated and compared with the use of the parent beta-cyclodextrin (beta-CyD). Among the three beta-CyDs, HP-beta-CyD was best at improving the rectal bioavailability of EBA in rats after single and multiple administrations of oleaginous suppositories (Witepsol H-5) containing the complexes. To gain insight into the enhancing effect of beta-CyDs, the absorption behaviors of EBA (observed by monitoring BPAA as an active metabolite of EBA) and beta-CyDs themselves were examined in vitro, in situ, and in vivo. The in situ recirculation study revealed that the complexed form of EBA was less absorbable from the rectal lumen in the solution state, but this disadvantageous effect of beta-CyDs was compensated in part by the inhibition of the bioconversion of EBA to BPAA. When beta-CyDs were coadministered with EBA in vivo, however, rather high amounts of HP-beta-CyD (approximately 26% of dose) and DM-beta-CyD (approximately 21% of dose), compared with beta-CyD (approximately 5% of dose), were absorbed from the rat rectum. Thus, the enhancement of rectal absorption of EBA in vivo can be explained by the facts that the hydrophilic beta-CyDs increased the release rate of EBA from the vehicle and stabilized EBA in the rectal lumen and that the drug was partly absorbed in the form of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibrin-binding, peptide amphiphile micelles for targeting glioblastoma.

PubMed

Chung, Eun Ji; Cheng, Yu; Morshed, Ramin; Nord, Kathryn; Han, Yu; Wegscheid, Michelle L; Auffinger, Brenda; Wainwright, Derek A; Lesniak, Maciej S; Tirrell, Matthew V

2014-01-01

Glioblastoma-targeted drug delivery systems facilitate efficient delivery of chemotherapeutic agents to malignant gliomas, while minimizing systemic toxicity and side effects. Taking advantage of the fibrin deposition that is characteristic of tumors, we constructed spherical, Cy7-labeled, targeting micelles to glioblastoma through the addition of the fibrin-binding pentapeptide, cysteine-arginine-glutamic acid-lysine-alanine, or CREKA. Conjugation of the CREKA peptide to Cy7-micelles increased the average particle size and zeta potential. Upon intravenous administration to GL261 glioma bearing mice, Cy7-micelles passively accumulated at the brain tumor site via the enhanced permeability and retention (EPR) effect, and Cy7-CREKA-micelles displayed enhanced tumor homing via active targeting as early as 1 h after administration, as confirmed via in vivo and ex vivo imaging and immunohistochemistry. Biodistribution of micelles showed an accumulation within the liver and kidneys, leading to micelle elimination via renal clearance and the reticuloendothelial system (RES). Histological evaluation showed no signs of cytotoxicity or tissue damage, confirming the safety and utility of this nanoparticle system for delivery to glioblastoma. Our findings offer strong evidence for the glioblastoma-targeting potential of CREKA-micelles and provide the foundation for CREKA-mediated, targeted therapy of glioma. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

The modifier effects of chymotrypsin and trypsin enzymes on fluorescence lifetime distribution of "N-(1-pyrenyl)maleimide-bovine serum albumin" complex.

PubMed

Özyiğit, İbrahim Ethem; Karakuş, Emine; Pekcan, Önder

2016-02-05

Chymotrypsin and trypsin are the well known proteolytic enzymes, both of which are synthesized in the pancreas as their precursors - the inactive forms; chymotrypsinogen and trypsinogen - and then are released into the duodenum to cut proteins into smaller peptides. In this paper, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat bovine serum albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. In the labeling study of BSA with PM, it is aimed to attach PM to the single free thiol (Cys34) and to all the free amine groups in accessible positions in order to produce excimers of pyrene planes of the possible highest amount to form the lifetime distributions in the widest range, that may show specifically distinguishing changes resulting from the activities of the proteases. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analyzed by using the exponential series method (ESM) to obtain the changes of lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the enzymes, the fluorescence lifetime distributions exhibited different structures which were attributed to the different activities of the proteases. Copyright © 2015 Elsevier B.V. All rights reserved.

Sensitive determination of thiols in wine samples by a stable isotope-coded derivatization reagent d0/d4-acridone-10-ethyl-N-maleimide coupled with high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis.

PubMed

Lv, Zhengxian; You, Jinmao; Lu, Shuaimin; Sun, Weidi; Ji, Zhongyin; Sun, Zhiwei; Song, Cuihua; Chen, Guang; Li, Guoliang; Hu, Na; Zhou, Wu; Suo, Yourui

2017-03-31

As the key aroma compounds, varietal thiols are the crucial odorants responsible for the flavor of wines. Quantitative analysis of thiols can provide crucial information for the aroma profiles of different wine styles. In this study, a rapid and sensitive method for the simultaneous determination of six thiols in wine using d 0 /d 4 -acridone-10-ethyl-N-maleimide (d 0 /d 4 -AENM) as stable isotope-coded derivatization reagent (SICD) by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) has been developed. Quantification of thiols was performed by using d 4 -AENM labeled thiols as the internal standards (IS), followed by stable isotope dilution HPLC-ESI-MS/MS analysis. The AENM derivatization combined with multiple reactions monitoring (MRM) not only allowed trace analysis of thiols due to the extremely high sensitivity, but also efficiently corrected the matrix effects during HPLC-MS/MS and the fluctuation in MS/MS signal intensity due to instrument. The obtained internal standard calibration curves for six thiols were linear over the range of 25-10,000pmol/L (R 2 ≥0.9961). Detection limits (LODs) for most of analytes were below 6.3pmol/L. The proposed method was successfully applied for the simultaneous determination of six kinds of thiols in wine samples with precisions ≤3.5% and recoveries ≥78.1%. In conclusion, the developed method is expected to be a promising tool for detection of trace thiols in wine and also in other complex matrix. Copyright © 2017 Elsevier B.V. All rights reserved.

Four-coordinate, 14-electron Ru(II) complexes: unusual trigonal pyramidal geometry enforced by bis(phosphino)silyl ligation.

PubMed

MacInnis, Morgan C; McDonald, Robert; Ferguson, Michael J; Tobisch, Sven; Turculet, Laura

2011-08-31

Unprecedented diamagnetic, four-coordinate, formally 14-electron (Cy-PSiP)RuX (Cy-PSiP = [κ(3)-(2-R(2)PC(6)H(4))(2)SiMe](-); X = amido, alkoxo) complexes that do not require agostic stabilization and that adopt a highly unusual trigonal pyramidal coordination geometry are reported. The tertiary silane [(2-Cy(2)PC(6)H(4))(2)SiMe]H ((Cy-PSiP)H) reacted with 0.5 [(p-cymene)RuCl(2)](2) in the presence of Et(3)N and PCy(3) to afford [(Cy-PSiP)RuCl](2) (1) in 74% yield. Treatment of 1 with KO(t)Bu led to the formation of (Cy-PSiP)RuO(t)Bu (2, 97% yield), which was crystallographically characterized and shown to adopt a trigonal pyramidal coordination geometry in the solid state. Treatment of 1 with NaN(SiMe(3))(2) led to the formation of (Cy-PSiP)RuN(SiMe(3))(2) (3, 70% yield), which was also found to adopt a trigonal pyramidal coordination geometry in the solid state. The related anilido complexes (Cy-PSiP)RuNH(2,6-R(2)C(6)H(3)) (4, R = H; 5, R = Me) were also prepared in >90% yields by treating 1 with LiNH(2,6-R(2)C(6)H(3)) (R = H, Me) reagents. The solid state structure of 5 indicates a monomeric trigonal pyramidal complex that features a C-H agostic interaction. Complexes 2 and 3 were found to react readily with 1 equiv of H(2)O to form the dimeric hydroxo-bridged complex [(Cy-PSiP)RuOH](2) (6, 94% yield), which was crystallographically characterized. Complexes 2 and 3 also reacted with 1 equiv of PhOH to form the new 18-electron η(5)-oxocyclohexadienyl complex (Cy-PSiP)Ru(η(5)-C(6)H(5)O) (7, 84% yield). Both amido and alkoxo (Cy-PSiP)RuX complexes reacted with H(3)B·NHRR' reagents to form bis(σ-B-H) complexes of the type (Cy-PSiP)RuH(η(2):η(2)-H(2)BNRR') (8, R = R' = H; 9, R = R' = Me; 10, R = H, R' = (t)Bu), which illustrates that such four-coordinate (Cy-PSiP)RuX (X = amido, alkoxo) complexes are able to undergo multiple E-H (E = main group element) bond activation steps. Computational methods were used to investigate structurally related PCP, PPP, PNP, and PSiP four-coordinate Ru complexes and confirmed the key role of the strongly σ-donating silyl group of the PSiP ligand set in enforcing the unusual trigonal pyramidal coordination geometry featured in complexes 2-5, thus substantiating a new strategy for the synthesis of low-coordinate Ru species. The mechanism of the activation of ammonia-borane by such low-coordinate (R-PSiP)RuX (X = amido, alkoxo) species was also studied computationally and was determined to proceed most likely in a stepwise fashion via intramolecular deprotonation of ammonia and subsequent borane B-H bond oxidative addition steps.

[Characterization of microstructure of ibuprofen-hydroxypropyl-beta-cyclodextrin and ibuprofen-beta-cyclodextrin by atomic force microscope].

PubMed

Wang, Li-juan; Zhu, Zhao-jing; Che, Ke-ke; Ju, Feng-ge

2008-09-01

The microstructures of ibuprofen-hydroxypropyl-bets-cyclodextrin (IBU-HP-beta-CyD) and ibuprofen-beta-cyclodextrin (IBU-beta-CyD) were observed by atomic force microscope (AFM). The high resolving capability of AFM has the tungsten filament probe with the spring constant of 0.06 N x m(-1). Samples were observed in a small scale scanning area of 10.5 nm x 10.5 nm and 800 x 800 pixels. The original scanning images were gained by tapping mode at room temperature. Their three-dimensional reconstruction of microstructure was performed by G3DR software. The outer diameters of HP-beta-CyD and beta-CyD are 1.53 nm. The benzene diameter of IBU is 0.62 nm, fitting to the inner diameters of HP-beta-CyD and beta-CyD. The benzene and hydrophobic chain of IBU enter into the hole of cyclodextrin at 1:1 ratio. The results were evidenced by IR, X-ray diffraction and the phase solubility.

[Pharmaceutical application of cyclodextrins as multi-functional drug carriers].

PubMed

Uekama, Kaneto

2004-12-01

Owing to the increasingly globalized nature of the cyclodextrin (CyD)-related science and technology, development of the CyD-based pharmaceutical formulation is rapidly progressing. The pharmaceutically useful CyDs are classified into hydrophilic, hydrophobic, and ionic derivatives. Because of the multi-functional characteristics and bioadaptability, these CyDs are capable of alleviating the undesirable properties of drug molecules through the formation of inclusion complexes or the form of CyD/drug conjugates. This review outlines the current application of CyDs in drug delivery and pharmaceutical formulation, focusing on the following evidences. 1) The hydrophilic CyDs enhance the rate and extent of bioavailability of poorly water-soluble drugs. 2) The amorphous CyDs such as 2-hydroxypropyl-beta-CyD are useful for inhibition of polymorphic transition and crystallization rates of drugs during storage. 3) The delayed release formulation can be obtained by the use of enteric type CyDs such as O-carboxymethyl-O-ethyl-beta-CyD. 4) The hydrophobic CyDs are useful for modification of the release site and/or time profile of water-soluble drugs with prolonged therapeutic effects. 5) The branched CyDs are particularly effective in inhibiting the adsorption to hydrophobic surface of containers and aggregation of polypeptide and protein drugs. 6) The combined use of different CyDs and/or pharmaceutical additives can serve as more functional drug carriers, improving efficacy and reducing side effects. 7) The CyD/drug conjugates may provide a versatile means for the constructions of not only colonic delivery system but also site-specific drug release system, including gene delivery. On the basis of the above-mentioned knowledge, the advantages and limitations of CyDs in the design of advanced dosage forms will be discussed.

Fire and heat resistant laminating resins based on maleimido substituted aromatic cyclotriphosphazene polymer

NASA Technical Reports Server (NTRS)

Kumar, Devendra (Inventor); Fohlen, George M. (Inventor); Parker, John A. (Inventor)

1987-01-01

4-Aminophenoxy cyclotriphosphazenes are reacted with maleic anhydride to produce maleamic acids which are converted to the maleimides. The maleimides are polymerized. By selection of starting materials (e.g., hexakis amino or trisaminophenoxy trisphenoxy cyclotriphosphazenes), selection of molar proportions of reactants, use of mixtures of anhydrides and use of dianhydrides as bridging groups a variety of maleimides and polymers are produced. The polymers have high limiting oxygen indices, high char yields and other useful heat and fire resistant properties making them useful as, for example, impregnants of fabrics.

Spotting effect in microarray experiments

PubMed Central

Mary-Huard, Tristan; Daudin, Jean-Jacques; Robin, Stéphane; Bitton, Frédérique; Cabannes, Eric; Hilson, Pierre

2004-01-01

Background Microarray data must be normalized because they suffer from multiple biases. We have identified a source of spatial experimental variability that significantly affects data obtained with Cy3/Cy5 spotted glass arrays. It yields a periodic pattern altering both signal (Cy3/Cy5 ratio) and intensity across the array. Results Using the variogram, a geostatistical tool, we characterized the observed variability, called here the spotting effect because it most probably arises during steps in the array printing procedure. Conclusions The spotting effect is not appropriately corrected by current normalization methods, even by those addressing spatial variability. Importantly, the spotting effect may alter differential and clustering analysis. PMID:15151695

Production of the immunosuppressant cyclosporin A by a new soil isolate, Aspergillus fumigatus, in submerged culture.

PubMed

Ismaiel, Ahmed A

2017-04-01

Cyclosporin A (CyA) has received meticulous attention owing to its immunosuppressive and biological activities. In this study, a soil isolate, capable of producing CyA, was named Zag1 strain and identified as Aspergillus fumigatus based on macroscopic and microscopic characteristics, 18S rDNA sequence, and phylogenetic characteristic analysis. To maximize the production of CyA, the fungal culture was grown under various fermentation conditions including selection of the cultivation medium, agitation rate, fermentation time, incubation temperature, pH value, inoculum nature, and medium volume. A simple medium (pH 5.0) containing 5% maltose as a carbon source and 2% potassium nitrate as a nitrogen source favored the highest CyA production when the fermentation process was maintained at 120 rpm for 9 days and at 30 °C using 3% standard inoculum of 5-day-old. The final CyA titer under these conditions was intensified to 2.23-3.31-fold, as compared with the amount obtained with seven types of basal media. A. fumigatus Zag1 appears to possess a good biotechnological potential for CyA production under favorable culture conditions.

G-CSF plus preemptive plerixafor vs hyperfractionated CY plus G-CSF for autologous stem cell mobilization in multiple myeloma: effectiveness, safety and cost analysis.

PubMed

Antar, A; Otrock, Z K; Kharfan-Dabaja, M A; Ghaddara, H A; Kreidieh, N; Mahfouz, R; Bazarbachi, A

2015-06-01

The optimal stem cell mobilization regimen for patients with multiple myeloma (MM) remains undefined. We retrospectively compared our experience in hematopoietic cell mobilization in 83 MM patients using fractionated high-dose CY and G-CSF with G-CSF plus preemptive plerixafor. All patients in the CY group (n=56) received fractionated high-dose CY (5 g/m(2) divided into five doses of 1 g/m(2) every 3 h) with G-CSF. All patients in the plerixafor group (n=27) received G-CSF and plerixafor preemptively based on an established algorithm. Compared with plerixafor, CY use was associated with higher total CD34+ cell yield (7.5 × 10(6) vs 15.5 × 10(6) cells/kg, P=0.005). All patients in both groups yielded ⩾4 × 10(6) CD34+ cells/kg. Conversely, CY use was associated with high frequency of febrile neutropenia, blood and platelet transfusions need and hospitalizations. The average total cost of mobilization in Lebanon was slightly higher in the plerixafor group ($7886 vs $7536; P=0.16). Our data indicate robust stem cell mobilization in MM patients with either fractionated high-dose CY and G-CSF or G-CSF alone with preemptive plerixafor. The chemo-mobilization approach was associated with twofold stem cell yield, slightly lower cost but significantly increased toxicity.

Localization of deformed wing virus infection in queen and drone Apis mellifera L

PubMed Central

Fievet, Julie; Tentcheva, Diana; Gauthier, Laurent; de Miranda, Joachim; Cousserans, François; Colin, Marc Edouard; Bergoin, Max

2006-01-01

The distribution of deformed wing virus infection within the honey bee reproductive castes (queens, drones) was investigated by in situ hybridization and immunohistology from paraffin embedded sections. Digoxygenin or CY5.5 fluorochrome end-labelled nucleotide probes hybridizing to the 3' portion of the DWV genome were used to identify DWV RNA, while a monospecific antibody to the DWV-VP1 structural protein was used to identify viral proteins and particles. The histological data were confirmed by quantitative RT-PCR of dissected organs. Results showed that DWV infection is not restricted to the digestive tract of the bee but spread in the whole body, including queen ovaries, queen fat body and drone seminal vesicles. PMID:16569216

Maleimide-activated aryl diazonium salts for electrode surface functionalization with biological and redox-active molecules.

PubMed

Harper, Jason C; Polsky, Ronen; Wheeler, David R; Brozik, Susan M

2008-03-04

A versatile and simple method is introduced for formation of maleimide-functionalized surfaces using maleimide-activated aryl diazonium salts. We show for the first time electrodeposition of N-(4-diazophenyl)maleimide tetrafluoroborate on gold and carbon electrodes which was characterized via voltammetry, grazing angle FTIR, and ellipsometry. Electrodeposition conditions were used to control film thickness and yielded submonolayer-to-multilayer grafting. The resulting phenylmaleimide surfaces served as effective coupling agents for electrode functionalization with ferrocene and the redox-active protein cytochrome c. The utility of phenylmaleimide diazonium toward formation of a diazonium-activated conjugate, followed by direct electrodeposition of the diazonium-modified DNA onto the electrode surface, was also demonstrated. Effective electron transfer was obtained between immobilized molecules and the electrodes. This novel application of N-phenylmaleimide diazonium may facilitate the development of bioelectronic devices including biofuel cells, biosensors, and DNA and protein microarrays.

[Establishment of myocardial targeted nanoparticles and preliminary evaluation of its effects on prevention and treatment of myocardial injury].

PubMed

Liu, Y Y; Wang, C; Luo, P F; Xia, Z F

2017-11-20

Objective: To establish 3-{4-[2-hydroxyl-(1-methylethylamino) propoxy] phenyl} propionic acid cetylesters (PAC) modified nanoparticles, and preliminarily explore its cardiomyocyte-targeting function and protection effects on myocardium. Methods: (1) HL-1 myocardial cells were divided into cyanidin-3 (Cy3) marked non-targeted small interference RNA (Cy3-siNC) group and Cy3 marked small interference RNA designed for the nuclear factor kappa B (NF-κB)-p65 gene (Cy3-si435) group according to the random number table, with 3 wells in each group. Cells in Cy3-siNC group were transfected with Cy3-siNC, while cells in Cy3-si435 group were transfected with Cy3-si435. At transfection hour 24, the mRNA expression of NF-κB-p65 of cells was determined by real-time fluorescent quantitative polymerase chain reaction. (2) Multiple emulsificating solvent evaporating method was adopted to prepare PAC modified nanoparticles carried with Cy3-siNC (Cy3-siNC-PAC) and PAC modified nanoparticles carried with Cy3-si435 (Cy3-si435-PAC). The morphology of Cy3-si435-PAC nanoparticles was observed with scanning electron microscope, and the size and potential of Cy3-si435-PAC nanoparticles were detected by nanometer particle size and zeta potential analyzer. The entrapment efficiency and drug loadings of Cy3-si435-PAC nanoparticle were determined with ultraviolet spectrophotometer. The release of Cy3-si435 of Cy3-si435-PAC nanoparticles was determined by dialysis method. (3) Another batch of HL-1 cells were divided into 4 groups according to the random number table, with 9 wells in each group. Cells in negative control group were added with 5 μL phosphate buffer. Cells in 25, 50, and 100 mg/mL Cy3-si435-PAC nanoparticles groups were added with 5 μL 25, 50, and 100 mg/mL Cy3-si435-PAC nanoparticles, respectively. At transfection hour 6, 12, and 24, proliferation activity of cells in 3 wells of each group was detected by methyl thiazolyl tetrazolium method, respectively. (4) Another batch of HL-1 cells were cultured for 24 h, and then treated with 100 μL Cy3-si435-PAC nanoparticles. At transfection hour 0, 4, 8, 12, and 24, the percentage of cells uptaking Cy3-si435-PAC nanoparticles in 3 wells were detected by flow cytometry, respectively. (5) Another batch of HL-1 cells were divided into 2 groups according to the random number table, with 3 wells in each group. Cells in Cy3-siNC-PAC group were added with 100 μL Cy3-siNC-PAC nanoparticles, while cells in Cy3-si435-PAC group were added with 100 μL Cy3-si435-PAC nanoparticles. At transfection hour 24, the mRNA expression of NF-κB-p65 of cells was determined by real-time fluorescent quantitative polymerase chain reaction. (6) Six male C57BL/6J mice were divided into 2 groups according to the random number table, with 3 mice in each group. Mice in Cy3-siNC-lipopolysaccharide (LPS) group and Cy3-si435-LPS group were respectively injected with 500 μL Cy3-siNC-PAC nanoparticles and Cy3-si435-PAC nanoparticles (50 mg/mL) in the tail vein. At injection hour 24, mice in the two groups were intraperitoneally injected with 10 mg/kg LPS to induce myocardial injury. At post injury hour 24, the distribution of nanoparticles in mice was detected with small animal imager. (7) Another 9 male C57BL/6J mice were divided into 3 groups according to the random number table, with 3 mice in each group. Mice in Cy3-siNC-normal saline (NS) group and Cy3-siNC-LPS group were injected with 500 μL 50 mg/mL Cy3-siNC-PAC nanoparticles in the tail vein, while mice in Cy3-si435-LPS group were injected with 500 μL 50 mg/mL Cy3-si435-PAC nanoparticles. At injection hour 24, mice in Cy3-siNC-NS group were intraperitoneally injected with NS, while mice in Cy3-siNC-LPS group and Cy3-si435-LPS group were injected with 10 mg/kg LPS to induce myocardial injury. At post injury hour 24, pathological changes of myocardium of mice in each group were observed with HE staining. Data were processed with t test and one-way analysis of variance. Results: (1) The mRNA expression of NF-κB-p65 of cells in Cy3-si435 group was 0.183±0.004, significantly lower than 1.003±0.092 in Cy3-siNC group ( t =15.46, P <0.01). (2) The form of prepared Cy3-si435-PAC nanoparticles was good, with particle size of 146.0 nm, potential of -29.2 mV, entrapment efficiency of (86.9±1.1) %, drug loadings of (25.4±0.9) %, and stable Cy3-si435 release. (3) At transfection hour 6, 12, and 24, there were no statistically significant differences in proliferation activity of cells in the 4 groups (with F values from 0.129 to 2.512, P values above 0.05). (4) At transfection hour 0, 4, 8, 12, and 24, the percentages of cells uptaking Cy3-si435-PAC nanoparticles were (0.79±0.06)%, (31.04±1.59)%, (51.64±2.67)%, (68.15±2.60)%, and (83.68±4.67)%, respectively. (5) The mRNA expression of NF-κB-p65 of cells in Cy3-si435-PAC group was 0.286±0.015, significantly lower than 1.002±0.073 in Cy3-siNC-PAC group ( t =16.62, P <0.01). (6) At post injury hour 24, uniform distribution of nanoparticles could be observed in cardiomyocytes of mice in Cy3-siNC-LPS group and Cy3-si435-LPS group. (7) The structure of myocardial fibers of mice in Cy3-siNC-NS group was dense, with no inflammatory cells infiltration and uniform distribution of cytoplasm. The structure of myocardial fibers of mice in Cy3-siNC-LPS group were loose, with inflammatory cells infiltration and scattered distribution of cytoplasm. The structure of myocardial fibers of mice in Cy3-si435-LPS group was denser, with no obvious inflammatory cells infiltration and uniform distribution of cytoplasm. Conclusions: Cy3-si435-PAC nanoparticles have good morphology, uniform particle size, normal potential distribution, and no cell cytotoxicity. Cy3-si435-PAC nanoparticles can be effectively uptaked by HL-1 cells and suppress NF-κB-p65 mRNA expression. They also can effectively target to mice cardiomyocytes to reduce inflammatory cells infiltration and alleviate the myocardial injury of mice induced by LPS.

Investigation of latent infections caused by cyprinid herpesvirus 3 in koi ( Cyprinus carpio) in southern China.

PubMed

Zheng, Shucheng; Wang, Qing; Bergmann, Sven M; Li, Yingying; Zeng, Weiwei; Wang, Yingying; Liu, Chun; Shi, Cunbin

2017-05-01

Although herpesviruses such as cyprinid herpesvirus 3 (CyHV-3) can establish lifelong latent infections, little is known about latency conditions in farmed koi populations in China. We used nested polymerase chain reaction targeting the TK gene and an indirect antibody ELISA to screen asymptomatic fish obtained from southern China for evidence of CyHV-3 infection. CyHV-3 DNA could be detected either in peripheral blood leukocytes or from gills of asymptomatic koi. Most koi sera did not contain anti-CyHV-3 antibodies; however, 5 samples were ELISA positive, providing evidence of prior CyHV-3 infections. These findings suggest that koi may survive CyHV-3 infections and become virus carriers.

Protein-based photothermal theranostics for imaging-guided cancer therapy

NASA Astrophysics Data System (ADS)

Rong, Pengfei; Huang, Peng; Liu, Zhiguo; Lin, Jing; Jin, Albert; Ma, Ying; Niu, Gang; Yu, Lun; Zeng, Wenbin; Wang, Wei; Chen, Xiaoyuan

2015-10-01

The development of imageable photothermal theranostics has attracted considerable attention for imaging guided photothermal therapy (PTT) with high tumor ablation accuracy. In this study, we strategically constructed a near-infrared (NIR) cyanine dye by introducing a rigid cyclohexenyl ring to the heptamethine chain to obtain a heptamethine dye CySCOOH with high fluorescence intensity and good stability. By covalent conjugation of CySCOOH onto human serum albumin (HSA), the as-prepared HSA@CySCOOH nanoplatform is highly efficient for NIR fluorescence/photoacoustic/thermal multimodality imaging and photothermal tumor ablation. The theranostic capability of HSA@CySCOOH was systematically evaluated both in vitro and in vivo. Most intriguingly, complete tumor elimination was achieved by intravenous injection of HSA@CySCOOH (CySCOOH, 1 mg kg-1 808 nm, 1.0 W cm-2 for 5 min) into 4T1 tumor-bearing mice, with no weight loss, noticeable toxicity, or tumor recurrence being observed. This as-prepared protein-based nanotheranostics exhibits high water dispersibility, no off target cytotoxicity, and good biodegradability and biocompatibility, thus facilitating its clinical translation to cancer photothermal theranostics.The development of imageable photothermal theranostics has attracted considerable attention for imaging guided photothermal therapy (PTT) with high tumor ablation accuracy. In this study, we strategically constructed a near-infrared (NIR) cyanine dye by introducing a rigid cyclohexenyl ring to the heptamethine chain to obtain a heptamethine dye CySCOOH with high fluorescence intensity and good stability. By covalent conjugation of CySCOOH onto human serum albumin (HSA), the as-prepared HSA@CySCOOH nanoplatform is highly efficient for NIR fluorescence/photoacoustic/thermal multimodality imaging and photothermal tumor ablation. The theranostic capability of HSA@CySCOOH was systematically evaluated both in vitro and in vivo. Most intriguingly, complete tumor elimination was achieved by intravenous injection of HSA@CySCOOH (CySCOOH, 1 mg kg-1 808 nm, 1.0 W cm-2 for 5 min) into 4T1 tumor-bearing mice, with no weight loss, noticeable toxicity, or tumor recurrence being observed. This as-prepared protein-based nanotheranostics exhibits high water dispersibility, no off target cytotoxicity, and good biodegradability and biocompatibility, thus facilitating its clinical translation to cancer photothermal theranostics. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr04428f

Maleimide conjugation markedly enhances the immunogenicity of both human and murine idiotype-KLH vaccines

PubMed Central

Kafi, Kamran; Betting, David J.; Yamada, Reiko E.; Bacica, Michael; Steward, Kristopher K.; Timmerman, John M.

2009-01-01

The collection of epitopes present within the variable regions of the tumor-specific clonal immunoglobulin expressed by B cell lymphomas (idiotype, Id) can serve as a target for active immunotherapy. Traditionally, tumor-derived Id protein is chemically-conjugated to the immunogenic foreign carrier protein keyhole limpet hemocyanin (KLH) using glutaraldehyde to serve as a therapeutic vaccine. While this approach offered promising results for some patients treated in early clinical trials, glutaraldehyde Id-KLH vaccines have failed to induce immune and clinical responses in many vaccinated subjects. We recently described an alternative conjugation method employing maleimide-sulfhydryl chemistry that significantly increased the therapeutic efficacy of Id-KLH vaccines in three different murine B cell lymphoma models, with protection mediated by either CD8+ T cells or antibodies. We now define in detail the methods and parameters critical for enhancing the in vivo immunogenicity of human as well as murine Id-KLH conjugate vaccines. Optimal conditions for Id sulfhydryl pre-reduction were determined, and maleimide Id-KLH conjugates maintained stability and potency even after prolonged storage. Field flow fractionation analysis of Id-KLH particle size revealed that maleimide conjugates were far more uniform in size than glutaraldehyde conjugates. Under increasingly stringent conditions, maleimide Id-KLH vaccines maintained superior efficacy over glutaraldehyde Id-KLH in treating established, disseminated murine lymphoma. More importantly, human maleimide Id-KLH conjugates were consistently superior to glutaraldehyde Id-KLH conjugates in inducing Id-specific antibody and T cell responses. The described methods should be easily adaptable to the production of clinical grade vaccines for human trials in B cell malignancies. PMID:19046770

Synthesis and characterization of Her2-NLP peptide conjugates targeting circulating breast cancer cells: cellular uptake and localization by fluorescent microscopic imaging.

PubMed

Cai, Huawei; Singh, Ajay N; Sun, Xiankai; Peng, Fangyu

2015-01-01

To synthesize a fluorescent Her2-NLP peptide conjugate consisting of Her2/neu targeting peptide and nuclear localization sequence peptide (NLP) and assess its cellular uptake and intracellular localization for radionuclide cancer therapy targeting Her2/neu-positive circulating breast cancer cells (CBCC). Fluorescent Cy5.5 Her2-NLP peptide conjugate was synthesized by coupling a bivalent peptide sequence, which consisted of a Her2-binding peptide (NH2-GSGKCCYSL) and an NLP peptide (CGYGPKKKRKVGG) linked by a polyethylene glycol (PEG) chain with 6 repeating units, with an activated Cy5.5 ester. The conjugate was separated and purified by HPLC and then characterized by Maldi-MS. The intracellular localization of fluorescent Cy5.5 Her2-NLP peptide conjugate was assessed by fluorescent microscopic imaging using a confocal microscope after incubation of Cy5.5-Her2-NLP with Her2/neu positive breast cancer cells and Her2/neu negative control breast cancer cells, respectively. Fluorescent signals were detected in cytoplasm of Her2/neu positive breast cancer cells (SKBR-3 and BT474 cell lines), but not or little in cytoplasm of Her2/neu negative breast cancer cells (MDA-MB-231), after incubation of the breast cancer cells with Cy5.5-Her2-NLP conjugates in vitro. No fluorescent signals were detected within the nuclei of Her2/neu positive SKBR-3 and BT474 breast cancer cells, neither Her2/neu negative MDA-MB-231 cells, incubated with the Cy5.5-Her2-NLP peptide conjugates, suggesting poor nuclear localization of the Cy5.5-Her2-NLP conjugates localized within the cytoplasm after their cellular uptake and internalization by the Her2/neu positive breast cancer cells. Her2-binding peptide (KCCYSL) is a promising agent for radionuclide therapy of Her2/neu positive breast cancer using a β(-) or α emitting radionuclide, but poor nuclear localization of the Her2-NLP peptide conjugates may limit its use for eradication of Her2/neu-positive CBCC using I-125 or other Auger electron emitting radionuclide.

Effect of ketoconazole on cyclosporine dose in healthy dogs.

PubMed

Dahlinger, J; Gregory, C; Bea, J

1998-01-01

To determine the degree to which the dose of oral cyclosporine (CyA), in healthy dogs, can be decreased by concurrent oral administration of ketoconazole. Dogs in this study were observed for physical or biochemical side effects that might have been caused by the administration of CyA and ketoconazole. Prospective research study. Five healthy, intact female Beagle dogs. CyA was administered orally twice daily to achieve stable whole blood trough levels of 400 to 600 ng/mL. Ketoconazole was added at a low therapeutic dose (average dose: 13.6 mg/kg/d) then at a subtherapeutic dose (average dose: 4.7 mg/kg/d). CyA whole blood trough levels were monitored every 3 to 4 days and maintained at 400 to 600 ng/mL by adjusting CyA doses accordingly. Physical examination, CBC, biochemical profile, and urinalysis were performed at 2-week intervals throughout the study period. The initial mean dose of CyA required to achieve target blood levels was 14.5 mg/ kg/d. With concurrent ketoconazole (low therapeutic dose, average dose: 13.6 mg/kg/d) and CyA administration, the CyA dose declined to 3.4 mg/kg/day (range: 1.2 to 5.2 mg/kg/d), representing a 75% reduction in CyA dose and monetary savings of 57.8%. At a subtherapeutic dose of ketoconazole (average dose: 4.7 mg/kg/d), combination therapy resulted in a CyA dose of 10.1 mg/kg/day (4.9 to 10.6 mg/kg/d), representing a 38% reduction in CyA dose and monetary savings of 23.8%. Weight loss and transient hypoalbuminemia of unknown clinical significance were observed. Other physical and biochemical evaluations were unremarkable over the 12-week study period. The oral administration of ketoconazole can be used to reduce substantially the oral CyA dose needed to maintain selected blood levels in healthy dogs. The oral administration of ketoconazole can result in substantial cost savings to owners of dogs receiving CyA after renal allograft transplantation or for the treatment of autoimmune disease.

Synthesis and Evaluation of 3-(furo[2,3-b]pyridin-3-yl)-4-(1H-indol-3-yl)-maleimides as Novel GSK-3β Inhibitors and Anti-Ischemic Agents.

PubMed

Ye, Qing; Li, Qiu; Zhou, Yubo; Xu, Lei; Mao, Weili; Gao, Yuanxue; Li, Chenhui; Xu, Yuan; Xu, Yazhou; Liao, Hong; Zhang, Luyong; Gao, Jianrong; Li, Jia; Pang, Tao

2015-10-01

A series of novel 3-(furo[2,3-b]pyridin-3-yl)-4-(1H-indol-3-yl)-maleimides were designed, synthesized, and biologically evaluated for their GSK-3β inhibitory activities. Most compounds showed favorable inhibitory activities against GSK-3β protein. Among them, compounds 5n, 5o, and 5p significantly reduced GSK-3β substrate tau phosphorylation at Ser396 in primary neurons, indicating inhibition of cellular GSK-3β activity. In the in vitro neuronal injury models, compounds 5n, 5o, and 5p prevented neuronal death against glutamate, oxygen-glucose deprivation, and nutrient serum deprivation which are closely associated with cerebral ischemic stroke. In the in vivo cerebral ischemia animal model, compound 5o reduced infarct size by 10% and improved the neurological deficit. The results may provide new insights into the development of novel GSK-3β inhibitors with potential neuroprotective activity against brain ischemic stroke. © 2015 John Wiley & Sons A/S.

Cyclipostins and Cyclophostin analogs as promising compounds in the fight against tuberculosis.

PubMed

Nguyen, Phuong Chi; Delorme, Vincent; Bénarouche, Anaïs; Martin, Benjamin P; Paudel, Rishi; Gnawali, Giri R; Madani, Abdeldjalil; Puppo, Rémy; Landry, Valérie; Kremer, Laurent; Brodin, Priscille; Spilling, Christopher D; Cavalier, Jean-François; Canaan, Stéphane

2017-09-18

A new class of Cyclophostin and Cyclipostins (CyC) analogs have been investigated against Mycobacterium tuberculosis H37Rv (M. tb) grown either in broth medium or inside macrophages. Our compounds displayed a diversity of action by acting either on extracellular M. tb bacterial growth only, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth with very low toxicity towards host macrophages. Among the eight potential CyCs identified, CyC 17 exhibited the best extracellular antitubercular activity (MIC 50  = 500 nM). This compound was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 23 potential candidates, most of them being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA and HsaD, have previously been reported as essential for in vitro growth of M. tb and/or survival and persistence in macrophages. Overall, our findings support the assumption that CyC 17 may thus represent a novel class of multi-target inhibitor leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes participating in important physiological processes.

A Facile One-Pot Construction of Succinimide-Fused Spiro[Pyrrolidine-2,3'-Oxindoles] via 1,3-Dipolar Cycloaddition Involving 3-Amino Oxindoles and Maleimides.

PubMed

Jin, Lunqiang; Liang, Feng

2018-03-05

Increasing interests have been invested in the development of synthetic strategies toward the construction of spiro[pyrrolidine-2,3'-oxindole], which is the core structural skeleton in some compounds with diverse biological activities. In this work, an efficient diastereoselective 1,3-dipolar cycloaddition reaction of azomethine ylides generated in situ from 3-amino oxindoles and aldehydes with maleimides has been described. The protocol provides a facile and efficient access to structurally diverse succinimide-fused spiro[pyrrolidine-2,3'-oxindole] compounds in good to high yields (up to 93%) with moderate to excellent diastereoselectivities (up to >95:5). The relative stereochemistry of cycloaddition products has been assigned by X-ray diffraction analysis.

Paper-based solid-phase nucleic acid hybridization assay using immobilized quantum dots as donors in fluorescence resonance energy transfer.

PubMed

Noor, M Omair; Shahmuradyan, Anna; Krull, Ulrich J

2013-02-05

A paper-based solid-phase assay is presented for transduction of nucleic acid hybridization using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) were FRET-paired with Cy3 acceptor. Hybridization of Cy3-labeled oligonucleotide targets provided the proximity required for FRET-sensitized emission from Cy3, which served as an analytical signal. The assay exhibited rapid transduction of nucleic acid hybridization within minutes. Without any amplification steps, the limit of detection of the assay was found to be 300 fmol with the upper limit of the dynamic range at 5 pmol. The implementation of glutathione-coated QDs for the development of nucleic acid hybridization assay integrated on a paper-based platform exhibited excellent resistance to nonspecific adsorption of oligonucleotides and showed no reduction in the performance of the assay in the presence of large quantities of noncomplementary DNA. The selectivity of nucleic acid hybridization was demonstrated by single-nucleotide polymorphism (SNP) detection at a contrast ratio of 19 to 1. The reuse of paper over multiple cycles of hybridization and dehybridization was possible, with less than 20% reduction in the performance of the assay in five cycles. This work provides an important framework for the development of paper-based solid-phase QD-FRET nucleic acid hybridization assays that make use of a ratiometric approach for detection and analysis.

Structural mapping of divergent regions in the type 1 ryanodine receptor using fluorescence resonance energy transfer

PubMed Central

Mahalingam, Mohana; Girgenrath, Tanya; Svensson, Bengt; Thomas, David D.; Cornea, Razvan L.; Fessenden, James D.

2014-01-01

Summary Ryanodine receptors (RyR) release Ca2+ to initiate striated muscle contraction. Three highly divergent regions in the RyR protein sequence (DR1, DR2, DR3) are proposed to confer isoform-specific functional properties to the RyRs. We used cell-based fluorescence resonance energy transfer (FRET) measurements to localize these DRs to the cryo-electron microscopic (EM) map of the skeletal muscle RyR isoform (RyR1). FRET donors were targeted to RyR1 using five different FKBP12.6 variants labeled with Alexa Fluor 488. FRET was then measured to Cy3NTA or Cy5NTA, FRET acceptors targeted to decahistidine tags introduced within the DRs. DR2 and DR3 were localized to separate positions within the “clamp” region of the RyR1 cryo-EM map, which is presumed to interface with Cav1.1. DR1 was localized to the “handle” region, near the regulatory calmodulin binding site on the RyR. These localizations provide new insights into the roles of DRs in RyR allosteric regulation during excitation-contraction coupling. PMID:25132084

Synthesis and biological evaluation of radio and dye labeled amino functionalized dendritic polyglycerol sulfates as multivalent anti-inflammatory compounds.

PubMed

Gröger, Dominic; Paulus, Florian; Licha, Kai; Welker, Pia; Weinhart, Marie; Holzhausen, Cornelia; Mundhenk, Lars; Gruber, Achim D; Abram, Ulrich; Haag, Rainer

2013-09-18

Herein we describe a platform technology for the synthesis and characterization of partially aminated, (35)S-labeled, dendritic polyglycerol sulfate (dPG(35)S amine) and fluorescent dPGS indocarbocyanine (ICC) dye conjugates. These polymer conjugates, based on a biocompatible dendritic polyglycerol scaffold, exhibit a high affinity to inflamed tissue in vivo and represent promising candidates for therapeutic and diagnostic applications. By utilizing a one-step sequential copolymerization approach, dendritic polyglycerol (Mn ≈ 4.5 kDa) containing 9.4% N-phthalimide protected amine functionalities was prepared on a large scale. Sulfation and simultaneous radio labeling with (35)SO3 pyridine complex, followed by cleavage of the N-phthalimide protecting groups, yielded dPG(35)S amine as a beta emitting, inflammation specific probe with free amino functionalities for conjugation. Furthermore, efficient labeling procedures with ICC via iminothiolane modification and subsequent "Michael" addition of the maleimide functionalized ICC dye, as well as by amide formation via NHS derivatized ICC on a dPGS amine scaffold, are described. The dPGS-ICC conjugates were investigated with respect to their photophysical properties, and both the radiolabeled and fluorescent compounds were comparatively visualized in histological tissue sections (radio detection and fluorescence microscopy) of animals treated with dPGS. Furthermore, cellular uptake of dPGS-ICC was found in endothelial cord blood (HUVEC) and the epithelial lung cells (A549). The presented synthetic routes allow a reproducible, controlled synthesis of dPGS amine on kilogram scale applying a one-pot batch reaction process. dPGS amine can be used for analysis via radioactivity or fluorescence, thereby creating a new platform for inflammation specific, multimodal imaging purposes using other attachable probes or contrast agents.

Solid-state molecular organometallic chemistry. Single-crystal to single-crystal reactivity and catalysis with light hydrocarbon substrates† †Electronic supplementary information (ESI) available: Full details of experimental details, spectroscopic and other analytical data, X-ray crystallography, catalytic conditions, and computational studies. CCDC 1539832–1539836. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c7sc01491k

PubMed Central

Chadwick, F. Mark; McKay, Alasdair I.; Martinez-Martinez, Antonio J.; Rees, Nicholas H.; Krämer, Tobias

2017-01-01

Single-crystal to single-crystal solid/gas reactivity and catalysis starting from the precursor sigma-alkane complex [Rh(Cy2PCH2CH2PCy2)(η2η2-NBA)][BArF4] (NBA = norbornane; ArF = 3,5-(CF3)2C6H3) is reported. By adding ethene, propene and 1-butene to this precursor in solid/gas reactions the resulting alkene complexes [Rh(Cy2PCH2CH2PCy2)(alkene)x][BArF4] are formed. The ethene (x = 2) complex, [Rh(Cy2PCH2CH2PCy2)(ethene)2][BArF4]-Oct, has been characterized in the solid-state (single-crystal X-ray diffraction) and by solution and solid-state NMR spectroscopy. Rapid, low temperature recrystallization using solution methods results in a different crystalline modification, [Rh(Cy2PCH2CH2PCy2)(ethene)2][BArF4]-Hex, that has a hexagonal microporous structure (P6322). The propene complex (x = 1) [Rh(Cy2PCH2CH2PCy2)(propene)][BArF4] is characterized as having a π-bound alkene with a supporting γ-agostic Rh···H3C interaction at low temperature by single-crystal X-ray diffraction, variable temperature solution and solid-state NMR spectroscopy, as well as periodic density functional theory (DFT) calculations. A fluxional process occurs in both the solid-state and solution that is proposed to proceed via a tautomeric allyl-hydride. Gas/solid catalytic isomerization of d3-propene, H2C 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 CHCD3, using [Rh(Cy2PCH2CH2PCy2)(η2η2-NBA)][BArF4] scrambles the D-label into all possible positions of the propene, as shown by isotopic perturbation of equilibrium measurements for the agostic interaction. Periodic DFT calculations show a low barrier to H/D exchange (10.9 kcal mol–1, PBE-D3 level), and GIPAW chemical shift calculations guide the assignment of the experimental data. When synthesized using solution routes a bis-propene complex, [Rh(Cy2PCH2CH2PCy2)(propene)2][BArF4], is formed. [Rh(Cy2PCH2CH2PCy2)(butene)][BArF4] (x = 1) is characterized as having 2-butene bound as the cis-isomer and a single Rh···H3C agostic interaction. In the solid-state two low-energy fluxional processes are proposed. The first is a simple libration of the 2-butene that exchanges the agostic interaction, and the second is a butene isomerization process that proceeds via an allyl-hydride intermediate with a low computed barrier of 14.5 kcal mol–1. [Rh(Cy2PCH2CH2PCy2)(η2η2-NBA)][BArF4] and the polymorphs of [Rh(Cy2PCH2CH2PCy2)(ethene)2][BArF4] are shown to be effective in solid-state molecular organometallic catalysis (SMOM-Cat) for the isomerization of 1-butene to a mixture of cis- and trans-2-butene at 298 K and 1 atm, and studies suggest that catalysis is likely dominated by surface-active species. [Rh(Cy2PCH2CH2PCy2)(η2η2-NBA)][BArF4] is also shown to catalyze the transfer dehydrogenation of butane to 2-butene at 298 K using ethene as the sacrificial acceptor. PMID:28989631

Application of a new convenience gender sorting method for mouse spermatozoa to mouse reproductive engineering technology.

PubMed

Hashimoto, Haruo; Eto, Tomoo; Suemizu, Hiroshi; Ito, Mamoru

2013-02-01

In this study, we attempted to apply new convenience gender sorting methods using sex-determining region Y (SRY) gene expression on Y spermatozoa to mice. Mouse spermatozoa labeled with Cy3-SRY antibody conjugate were used for intracytoplasmic sperm injection (ICSI). In addition, spermatozoa conjugated with SRY antibody were conjugated with magnetic beads (Mag) and were pulled to the bottom of the medium. The supernatant of the medium was used for in vitro fertilization (IVF). The rate of males reproduced by ICSI using the spermatozoa conjugated with Cy3-SRY antibody was 86.1%. The female proportion reproduced by IVF using the spermatozoa separated in the supernatant after Mag-SRY antibody conjugation was 67.3%. These gender sorting methods are effective for the reproduction of transgenic mice.

Resonance energy transfer between sites in rat liver glutathione S-transferase, 1-1, selectively modified at cysteine-17 and cysteine-111.

PubMed

Hu, L; Colman, R F

1997-02-18

Monobromobimane (mBBr) can label both Cys111 and Cys17 of rat liver glutathione S-transferase, 1-1 (GST 1-1). However, selective modification of Cys111 was achieved by the maleimide-based sulfhydryl reagents N-ethylmaleimide (NEM) and fluorescein 5-maleimide (NFM). Incubation of GST 1-1 with 5 mM NEM for 30 min at pH 7.5 and 25 degrees C leads to the formation of modified enzyme with 92% residual activity toward 1-chloro-2,4-dinitrobenzene and completely blocks Cys111 from subsequent reaction with either NFM or mBBr. Reaction of GST 1-1 with 0.2 mM NFM under the same conditions affords a modified enzyme with only 14% residual activity even though NFM and NEM target the same Cys111. The results indicate that when the bulky fluorescein is covalently bound to Cys111, the ligand projects into both the xenobiotic binding site and the glutathione site. After NEM or NFM modification of GST 1-1, the enzyme was further modified by monobromobimane at Cys17 with loss of activity. Together with the only tryptophan (Trp20), fluorescein linked to Cys111 and bimane to Cys17 provide three fluorescent probes to study the solution structure of GST 1-1. Fluorescence spectral analysis suggests that Trp20 and bimane linked to Cys17 are located in a relatively hydrophobic environment, while fluorescein linked to Cys111 is located in a charged environment. These fluorescent probes constitute three sets of donor-acceptor pairs for the measurement of fluorescence energy transfer, and distances calculated from such measurements are 20 A between Trp20 and bimane at Cys17, 19 A between Trp20 and fluorescein at Cys111, and < 22 A between bimane at Cys17 and fluorescein at Cys111. Molecular modeling studies indicate that fluorescein lies between the two subunits, is surrounded by charged residues, and is extended into the xenobiotic binding site. They also suggest that mBBr must approach from the dimer interface in order to reach the reaction site at Cys17.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry.

PubMed

Mason, Alexander F; Thordarson, Pall

2016-07-20

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation. We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Establishment of a novel and highly permissive cell line for the efficient replication of cyprinid herpesvirus 2 (CyHV-2).

PubMed

Ma, Jie; Jiang, Nan; LaPatra, Scott E; Jin, Ling; Xu, Jin; Fan, Yuding; Zhou, Yong; Zeng, Lingbing

2015-06-12

Haematopoietic necrosis of gibel carp (Carassius auratus gibelio) is caused by cyprinid herpesvirus 2 (CyHV-2) and has caused huge economic losses in aquaculture worldwide. Currently the isolation and propagation of CyHV-2 in vitro is very difficult due to the lack of permissive cell lines. Studies on the pathogenesis of CyHV-2 have been hampered because the virus has not been extensively characterized. In this study, a novel cell line from the brain of gibel carp, denoted GiCB, has been established and characterized. Sustainable propagation of CyHV-2 in the GiCB cell line has been confirmed by virus infection and titration, PCR, transmission electron microscopy, immunofluorescence assay and fluorescence in situ hybridization. The GiCB cells showed typical cytopathic effect by day 6 post-infection with CyHV-2 including cell shrinkage, rounding, and cell fusion with cytoplasmic vacuolization. The virus titer reached 10(7.5 ± 0.37)TCID₅₀/ml and has been successfully passaged over 50 times in the GiCB cell line. Electron microscopy analysis revealed the complete replication of CyHV-2 in GiCB cells. CyHV-2-infected GiCB cells reacted strongly with polyclonal antibodies against CyHV-2 and CyHV-2 RNA in cells hybridized specifically with the virus RNA probes. Additionally, an experimental infection demonstrated that CyHV-2 produced in GiCB cells caused 100% mortality in gibel carp. All the results provide solid evidence that the GiCB cell line is highly permissive for the isolation and propagation of CyHV-2. This is a significant advancement that will promote additional research on CyHV-2 infection in fish in the future. Copyright © 2015 Elsevier B.V. All rights reserved.

Acidosis and weight loss are induced by cyclosporin A in uninephrectomized rats.

PubMed

Jaramillo-Juárez, F; Rodríguez-Vázquez, M L; Namorado, M C; Martín, D; Reyes, J L

2000-02-01

The effects of cyclosporin A (CyA, 50 mg/kg body weight) or its commercial vehicle (cremophor) on the acid-base regulation of uninephrectomized rats were assessed for 7 days and in non-nephrectomized rats for 15 days. CyA induced a marked systemic acidosis, accompanied by decreases in blood PCO(2) and plasma bicarbonate. Untreated uninephrectomized rats did not show the acidosis. In CyA-treated rats the urine pH decreased (control 6. 65+/-0.06 vs. CyA 6.18+/-0.08; P<0.01) as well as urinary bicarbonate (non-nephrectomized rats 7.50+/-1.88 mM vs. uninephrectomy plus CyA 0.75+/- 0.06 mM; P<0.01), suggesting partial renal compensation of systemic acidosis. Titratable acidity increased in CyA-treated rats (control 21.6+/-1.2 vs. CyA 63.3+/-12.0 microEq/l; P<0.001). Phosphate, glucose, and osmolar clearances were not significantly altered in non-nephrectomized rats treated with CyA for 15 days. There was a striking decrease in body weight in CyA-treated rats (control 274.0+/-3.8 vs. CyA 225.0+/-5.1 g; P<0. 01), but compensatory growth of the remaining kidney was not prevented by this drug or by its vehicle. In summary, CyA induced a severe metabolic acidosis in uninephrectomized rats that was not compensated by the remaining kidney, in spite of the well-preserved compensatory weight gain of this organ. Loss of body weight was significant in CyA-treated animals.

A novel upconversion@polydopamine core@shell nanoparticle based aptameric biosensor for biosensing and imaging of cytochrome c inside living cells.

PubMed

Ma, Lina; Liu, Fuyao; Lei, Zhen; Wang, Zhenxin

2017-01-15

Herein, a novel upconversion@polydopamine core@shell nanoparticle (termed as UCNP@PDA NP) -based aptameric biosensor has been fabricated for the quantitative analysis of cytochrome c (Cyt c) inside living cells, which comprises an UCNP@PDA NP, acting as an internal reference and fluorescence quenching agent, and Cy3 modified aptamer enabling ratiometric quantitative Cyt c measurement. After the hybridization of Cy3 labeled aptamer with amino-terminated single DNA on the UCNP@PDA NP surface (termed as UCNP@PDA@AP), the fluorescence of Cy3 can be efficiently quenched by the PDA shell. With the spontaneous cellular uptake of UCNP@PDA@AP, the Cyt c aptamer dissociates from UCNP@PDA NP surface through formation of aptamer-Cyt c complex, resulting in concomitant activation of the Cy3 fluorescence. High amount of Cyt c leads to high fluorescence emission, enabling direct visualization/measurement of the Cyt c by fluorescence microscopy/spectroscopy. The steady upconversion luminescent (UCL) signals can be employed not only for intracellular imaging, but also as an internal reference for evaluating intracellular Cyt c amount using the ratio of fluorescence intensity of Cy3 with the UCL intensity of UCNP. The UCNP@PDA@AP shows a reasonable detection limit (20nM) and large dynamic range (50nM to 10μM, which covers the literature reported values (1-10μM) for cytosolic Cyt c in apoptotic cells) for detecting Cyt c in buffer with excellent selectivity. In addition, the UCNP@PDA@AP has been successfully used to monitor etoposide induced intracellular releasing of Cyt c, providing the possibility for cell-based screening of apoptosis-inducing drugs. Copyright © 2016 Elsevier B.V. All rights reserved.

Cytochrome c conformations resolved by the photon counting histogram: Watching the alkaline transition with single-molecule sensitivity

PubMed Central

Perroud, Thomas D.; Bokoch, Michael P.; Zare, Richard N.

2005-01-01

We apply the photon counting histogram (PCH) model, a fluorescence technique with single-molecule sensitivity, to study pH-induced conformational changes of cytochrome c. PCH is able to distinguish different protein conformations based on the brightness of a fluorophore sensitive to its local environment. We label cytochrome c through its single free cysteine with tetramethylrhodamine-5-maleimide (TMR), a fluorophore with specific brightnesses that we associate with specific protein conformations. Ensemble measurements demonstrate two different fluorescence responses with increasing pH: (i) a decrease in fluorescence intensity caused by the alkaline transition of cytochrome c (pH 7.0–9.5), and (ii) an increase in intensity when the protein unfolds (pH 9.5–10.8). The magnitudes of these two responses depend strongly on the molar ratio of TMR used to label cytochrome c. Using PCH we determine that this effect arises from the proportion of a nonfunctional conformation in the sample, which can be differentiated from the functional conformation. We further determine the causes of each ensemble fluorescence response: (i) during the alkaline transition, the fluorophore enters a dark state and discrete conformations are observed, and (ii) as cytochrome c unfolds, the fluorophore incrementally brightens, but discrete conformations are no longer resolved. Moreover, we also show that functional TMR-cytochrome c undergoes a response of identical magnitude regardless of the proportion of nonfunctional protein in the sample. As expected for a technique with single-molecule sensitivity, we demonstrate that PCH can directly observe the most relevant conformation, unlike ensemble fluorometry. PMID:16314563

Inhibition of Glutathione Biosynthesis Alters Compartmental Redox Status and the Thiol Proteome in Organogenesis-Stage Rat Conceptuses

PubMed Central

Harris, Craig; Shuster, Daniel Z.; Gomez, Rosaicela Roman; Sant, Karilyn E.; Reed, Matthew S.; Pohl, Jan; Hansen, Jason M.

2013-01-01

Developmental signals that control growth and differentiation are regulated by environmental factors that generate reactive oxygen species (ROS) and alter steady state redox environments in tissues and fluids. Protein thiols are selectively oxidized and reduced in distinct spatial and temporal patterns in conjunction with changes in glutathione/glutathione disulfide (GSH/GSSG) and cysteine/cystine (Cys/CySS) redox potentials (E0) to regulate developmental signaling. The purpose of this study was to measure compartment specific thiol redox status in cultured organogenesis-stage rat conceptuses and to evaluate the impact of thiol oxidation on the redox proteome. The visceral yolk sac (VYS) has the highest initial (0 hr) total intracellular GSH (GSH + 2GSSG) concentrations (5.5 mM) and the lowest Eh (−223 mV) as determined by HPLC analysis. Total embryo (EMB) GSH concentrations ranged lower (3.2 mM) and were only slightly more oxidized than the VYS. Total GSH concentrations in yolk sac fluid (YSF) and amniotic fluid (AF) are >500-fold lower than in tissues and are highly oxidized (YSF Eh = −121 mV and AF Eh = −49 mV). Steady state total Cys concentrations (Cys + 2CySS) were significantly lower than GSH in tissues but were otherwise equal in VYS and EMB near 0.5 mM. On gestational day 11, total GSH and Cys concentrations in EMB and VYS increase significantly over the 6 hr time course while Eh remains relatively constant. The Eh (GSH/GSSG) in YSF and AF become more reduced over time while Eh (Cys/CySS) become more oxidized. Addition of L-buthionine-S,R-sulfoximine (BS0) to selectively inhibit GSH synthesis and mimic the effects of some GSH-depleting environmental chemicals, significantly decreased VYS and EMB GSH and cys concentrations and increased Eh over the 6 hr exposure period, showing a greater overall oxidation. In the YSF, BSO caused a significant increase in total Cys concentrations to 1.7 mM but did not significantly change the Eh for Cys/CySS. A significant net oxidation was seen in the BSO-treated AF compartment after 6 hr. Biotinylated iodoacetamide (BIAM) labeling of proteins revealed the significant thiol-oxidation of many EMB proteins following BSO treatment. Quantitative changes in the thiol proteome, associated with developmentally-relevant pathways, were detected using isotope coded affinity tag (ICAT) labeling and mass spectroscopy. Adaptive pathways were selectively enriched with increased concentrations of proteins involved in mRNA processing (splicesome) and mRNA stabilization (glycolysis, GAPDH), as well as, protein synthesis (aminoacyl-tRNA) and protein folding (antigen processing, Hsp70, protein disulfide isomerase). These results show the ability of chemical and environmental modulators to selectively alter compartmental intracellular and extracellular GSH and Cys concentrations and change their corresponding Eh within the intact viable conceptus. The altered Eh were also of sufficient magnitude to alter the redox proteome and change relative protein concentrations suggesting that the mechanistic links through which environmental factors inform and regulate developmental signaling pathways may be discovered using systems developmental biology techniques. PMID:23736079

Inhibition of glutathione biosynthesis alters compartmental redox status and the thiol proteome in organogenesis-stage rat conceptuses.

PubMed

Harris, Craig; Shuster, Daniel Z; Roman Gomez, Rosaicela; Sant, Karilyn E; Reed, Matthew S; Pohl, Jan; Hansen, Jason M

2013-10-01

Developmental signals that control growth and differentiation are regulated by environmental factors that generate reactive oxygen species (ROS) and alter steady-state redox environments in tissues and fluids. Protein thiols are selectively oxidized and reduced in distinct spatial and temporal patterns in conjunction with changes in glutathione/glutathione disulfide (GSH/GSSG) and cysteine/cystine (Cys/CySS) redox potentials (E(h)) to regulate developmental signaling. The purpose of this study was to measure compartment-specific thiol redox status in cultured organogenesis-stage rat conceptuses and to evaluate the impact of thiol oxidation on the redox proteome. The visceral yolk sac (VYS) has the highest initial (0 h) total intracellular GSH (GSH+2GSSG) concentration (5.5 mM) and the lowest Eh (-223 mV) as determined by HPLC analysis. Total embryo (EMB) GSH concentrations ranged lower (3.2 mM) and were only slightly more oxidized than the VYS. Total GSH concentrations in yolk sac fluid (YSF) and amniotic fluid (AF) are >500-fold lower than in tissues and are highly oxidized (YSF E(h)=-121 mV and AF E(h)=-49 mV). Steady-state total Cys concentrations (Cys+2CySS) were significantly lower than GSH in tissues but were otherwise equal in VYS and EMB near 0.5 mM. On gestational day 11, total GSH and Cys concentrations in EMB and VYS increase significantly over the 6h time course while E(h) remains relatively constant. The Eh (GSH/GSSG) in YSF and AF become more reduced over time while E(h) (Cys/CySS) become more oxidized. Addition of L-buthionine-S,R-sulfoximine (BS0) to selectively inhibit GSH synthesis and mimic the effects of some GSH-depleting environmental chemicals significantly decreased VYS and EMB GSH and Cys concentrations and increased Eh over the 6h exposure period, showing a greater overall oxidation. In the YSF, BSO caused a significant increase in total Cys concentrations to 1.7 mM but did not significantly change the E(h) for Cys/CySS. A significant net oxidation was seen in the BSO-treated AF compartment after 6 h. Biotinylated iodoacetamide (BIAM) labeling of proteins revealed the significant thiol oxidation of many EMB proteins following BSO treatment. Quantitative changes in the thiol proteome, associated with developmentally relevant pathways, were detected using isotope coded affinity tag (ICAT) labeling and mass spectroscopy. Adaptive pathways were selectively enriched with increased concentrations of proteins involved in mRNA processing (splicesome) and mRNA stabilization (glycolysis, GAPDH), as well as protein synthesis (aminoacyl-tRNA) and protein folding (antigen processing, Hsp70, protein disulfide isomerase). These results show the ability of chemical and environmental modulators to selectively alter compartmental intracellular and extracellular GSH and Cys concentrations and change their corresponding E(h) within the intact viable conceptus. The altered E(h) were also of sufficient magnitude to alter the redox proteome and change relative protein concentrations, suggesting that the mechanistic links through which environmental factors inform and regulate developmental signaling pathways may be discovered using systems developmental biology techniques. Copyright © 2013 Elsevier Inc. All rights reserved.

Exploring changes over time in habilitation professionals' perceptions and applications of the International Classification of Functioning, Disability and Health, version for Children and Youth (ICF-CY).

PubMed

Adolfsson, Margareta; Granlund, Mats; Björck-Akesson, Eva; Ibragimova, Nina; Pless, Mia

2010-07-01

This study explored how professionals in inter-disciplinary teams perceived the implementation of the World Health Organization's International Classification of Functioning, Disability and Health, version for Children and Youth (ICF-CY) in Swedish habilitation services. Descriptive longitudinal mixed-methods design. Following participation in a 2-day in-service training on the ICF-CY, 113 professionals from 14 interdisciplinary teams described their perceptions of the implementation of the ICF-CY at 3 consecutive time-points: during in-service training, after 1 year, and after 2.5 years. Implementation of the ICF-CY in daily work focused on assessment and habilitation planning and required adaptations of routines and materials. The ICF-CY was perceived as useful in supporting analyses and in communication about children's needs. Professionals also perceived it as contributing to new perspectives on problems and a sharpened focus on participation. Professionals indicated that the ICF-CY enhanced their awareness of families' views of child participation, which corresponded to organizational goals for habilitation services. An implementation finding was a lack of tools fitting the comprehensive ICF-CY perspective. The study points to the need for ICF-CY-based assessment and intervention methods focusing on child participation.

Imaging Lung Clearance of Radiolabeled Tumor Cells to Study Mice with Normal, Activated or Depleted Natural Killer (NK) Cells

NASA Astrophysics Data System (ADS)

Kulkarni, P. V.; Bennett, M.; Constantinescu, A.; Arora, V.; Viguet, M.; Antich, P.; Parkey, R. W.; Mathews, D.; Mason, R. P.; Oz, O. K.

2003-08-01

Lung clearance of 51CR and 125I iododeoxyuridine (IUDR) labeled cancer cells assess NK cell activity. It is desirable to develop noninvasive imaging technique to assess NK activity in mice. We labeled target YAC-1 tumor cells with 125I, 111In, 99mTc, or 67Ga and injected I.V. into three groups of BALB/c mice. Animals were treated with medium (group I), 300mg/kg cyclophosmamide (CY) to kill NK cell (group II), or anti-LY49C/1) (ab')2 mAb to augment NK function (group III). Lungs were removed 15 min or 2 h later for tissue counting. Control and treated mice were imaged every 5 min with a scintillating camera for 1 h after 15 min of infusion of the 111In labeled cells. Lung clearance increased after 15 min (lodging: 60-80%) and (2 h retention: 3-7%). Similar results were obtained with all the isotopes studied. Images distinguished the control and treated mice for lung activity. Cells labeled with 111In, 99mTc or 67Ga are cleared similar to those labeled with 51Cr or 125I. NK cell destruction of tumor cells may be assessed by noninvasive imaging method either by SPECT (99mTc, 111In, 67Ga) or by PET (68Ga).

Dendrimeric coating of glass slides for sensitive DNA microarrays analysis

PubMed Central

Le Berre, Véronique; Trévisiol, Emmanuelle; Dagkessamanskaia, Adilia; Sokol, Serguei; Caminade, Anne-Marie; Majoral, Jean Pierre; Meunier, Bernard; François, Jean

2003-01-01

Successful use and reliability of microarray technology is highly dependent on several factors, including surface chemistry parameters and accessibility of cDNA targets to the DNA probes fixed onto the surface. Here, we show that functionalisation of glass slides with homemade dendrimers allow production of more sensitive and reliable DNA microarrays. The dendrimers are nanometric structures of size-controlled diameter with aldehyde function at their periphery. Covalent attachment of these spherical reactive chemical structures on amino-silanised glass slides generates a reactive ∼100 Å layer onto which amino-modified DNA probes are covalently bound. This new grafting chemistry leads to the formation of uniform and homogenous spots. More over, probe concentration before spotting could be reduced from 0.2 to 0.02 mg/ml with PCR products and from 20 to 5 µM with 70mer oligonucleotides without affecting signal intensities after hybridisation with Cy3- and Cy5-labelled targets. More interestingly, while the binding capacity of captured probes on dendrimer-activated glass surface (named dendrislides) is roughly similar to other functionalised glass slides from commercial sources, detection sensitivity was 2-fold higher than with other available DNA microarrays. This detection limit was estimated to 0.1 pM of cDNA targets. Altogether, these features make dendrimer-activated slides ideal for manufacturing cost-effective DNA arrays applicable for gene expression and detection of mutations. PMID:12907740

Immunomagnetic cell separation, imaging, and analysis using Captivate ferrofluids

NASA Astrophysics Data System (ADS)

Jones, Laurie; Beechem, Joseph M.

2002-05-01

We have developed applications of CaptivateTM ferrofluids, paramagnetic particles (approximately 200 nm diameter), for isolating and analyzing cell populations in combination with fluorescence-based techniques. Using a microscope-mounted magnetic yoke and sample insertion chamber, fluorescent images of magnetically captured cells were obtained in culture media, buffer, or whole blood, while non-magnetically labeled cells sedimented to the bottom of the chamber. We combined this immunomagnetic cell separation and imaging technique with fluorescent staining, spectroscopy, and analysis to evaluate cell surface receptor-containing subpopulations, live/dead cell ratios, apoptotic/dead cell ratios, etc. The acquired images were analyzed using multi-color parameters, as produced by nucleic acid staining, esterase activity, or antibody labeling. In addition, the immunomagnetically separated cell fractions were assessed through microplate analysis using the CyQUANT Cell Proliferation Assay. These methods should provide an inexpensive alternative to some flow cytometric measurements. The binding capacities of the streptavidin- labled Captivate ferrofluid (SA-FF) particles were determined to be 8.8 nmol biotin/mg SA-FF, using biotin-4- fluorescein, and > 106 cells/mg SA-FF, using several cell types labeled with biotinylated probes. For goat anti- mouse IgG-labeled ferrofluids (GAM-FF), binding capacities were established to be approximately 0.2 - 7.5 nmol protein/mg GAM-FF using fluorescent conjugates of antibodies, protein G, and protein A.

Tracking of Engineered Bacteria In Vivo Using Nonstandard Amino Acid Incorporation.

PubMed

Praveschotinunt, Pichet; Dorval Courchesne, Noémie-Manuelle; den Hartog, Ilona; Lu, Chaochen; Kim, Jessica J; Nguyen, Peter Q; Joshi, Neel S

2018-06-15

The rapidly growing field of microbiome research presents a need for better methods of monitoring gut microbes in vivo with high spatial and temporal resolution. We report a method of tracking microbes in vivo within the gastrointestinal tract by programming them to incorporate nonstandard amino acids (NSAA) and labeling them via click chemistry. Using established machinery constituting an orthogonal translation system (OTS), we engineered Escherichia coli to incorporate p-azido-l-phenylalanine (pAzF) in place of the UAG (amber) stop codon. We also introduced a mutant gene encoding for a cell surface protein (CsgA) that was altered to contain an in-frame UAG codon. After pAzF incorporation and extracellular display, the engineered strains could be covalently labeled via copper-free click reaction with a Cy5 dye conjugated to the dibenzocyclooctyl (DBCO) group. We confirmed the functionality of the labeling strategy in vivo using a murine model. Labeling of the engineered strain could be observed using oral administration of the dye to mice several days after colonization of the gastrointestinal tract. This work sets the foundation for the development of in vivo tracking microbial strategies that may be compatible with noninvasive imaging modalities and are capable of longitudinal spatiotemporal monitoring of specific microbial populations.

Microbial detection in microfluidic devices through dual staining of quantum dots-labeled immunoassay and RNA hybridization.

PubMed

Zhang, Qing; Zhu, Liang; Feng, Hanhua; Ang, Simon; Chau, Fook Siong; Liu, Wen-Tso

2006-01-18

This paper reported the development of a microfludic device for the rapid detection of viable and nonviable microbial cells through dual labeling by fluorescent in situ hybridization (FISH) and quantum dots (QDs)-labeled immunofluorescent assay (IFA). The coin sized device consists of a microchannel and filtering pillars (gap=1-2 microm) and was demonstrated to effectively trap and concentrate microbial cells (i.e. Giardia lamblia). After sample injection, FISH probe solution and QDs-labeled antibody solution were sequentially pumped into the device to accelerate the fluorescent labeling reactions at optimized flow rates (i.e. 1 and 20 microL/min, respectively). After 2 min washing for each assay, the whole process could be finished within 30 min, with minimum consumption of labeling reagents and superior fluorescent signal intensity. The choice of QDs 525 for IFA resulted in bright and stable fluorescent signal, with minimum interference with the Cy3 signal from FISH detection.

Cathepsin S-cleavable, multi-block HPMA copolymers for improved SPECT/CT imaging of pancreatic cancer.

PubMed

Fan, Wei; Shi, Wen; Zhang, Wenting; Jia, Yinnong; Zhou, Zhengyuan; Brusnahan, Susan K; Garrison, Jered C

2016-10-01

This work continues our efforts to improve the diagnostic and radiotherapeutic effectiveness of nanomedicine platforms by developing approaches to reduce the non-target accumulation of these agents. Herein, we developed multi-block HPMA copolymers with backbones that are susceptible to cleavage by cathepsin S, a protease that is abundantly expressed in tissues of the mononuclear phagocyte system (MPS). Specifically, a bis-thiol terminated HPMA telechelic copolymer containing 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. Three maleimide modified linkers with different sequences, including cathepsin S degradable oligopeptide, scramble oligopeptide and oligo ethylene glycol, were subsequently synthesized and used for the extension of the HPMA copolymers by thiol-maleimide click chemistry. All multi-block HPMA copolymers could be labeled by (177)Lu with high labeling efficiency and exhibited high serum stability. In vitro cleavage studies demonstrated highly selective and efficient cathepsin S mediated cleavage of the cathepsin S-susceptible multi-block HPMA copolymer. A modified multi-block HPMA copolymer series capable of Förster Resonance Energy Transfer (FRET) was utilized to investigate the rate of cleavage of the multi-block HPMA copolymers in monocyte-derived macrophages. Confocal imaging and flow cytometry studies revealed substantially higher rates of cleavage for the multi-block HPMA copolymers containing the cathepsin S-susceptible linker. The efficacy of the cathepsin S-cleavable multi-block HPMA copolymer was further examined using an in vivo model of pancreatic ductal adenocarcinoma. Based on the biodistribution and SPECT/CT studies, the copolymer extended with the cathepsin S susceptible linker exhibited significantly faster clearance and lower non-target retention without compromising tumor targeting. Overall, these results indicate that exploitation of the cathepsin S activity in MPS tissues can be utilized to substantially lower non-target accumulation, suggesting this is a promising approach for the development of diagnostic and radiotherapeutic nanomedicine platforms. Copyright © 2016 Elsevier Ltd. All rights reserved.

Well-defined N-heterocyclic carbene silver halides of 1-cyclohexyl-3-arylmethylimidazolylidenes: synthesis, structure and catalysis in A3-reaction of aldehydes, amines and alkynes.

PubMed

Li, Yanbo; Chen, Xiaofeng; Song, Yin; Fang, Ling; Zou, Gang

2011-03-07

Structurally well-defined N-heterocyclic carbene silver chlorides and bromides supported by 1-cyclohexyl-3-benzylimidazolylidene (CyBn-NHC) or 1-cyclohexyl-3-naphthalen-2-ylmethylimidazolylidene (CyNaph-NHC) were synthesized by reaction of the corresponding imidazolium halides with silver(I) oxide while cationic bis(CyBn-NHC) silver nitrate was isolated under similar conditions using imidazolium iodide in the presence of sodium nitrate. Single-crystal X-ray diffraction revealed a dimeric structure through a nonpolar weak-hydrogen-bond supported Ag-Ag bond for 1-cyclohexyl-3-benzylimidazolylidene silver halides [(CyBn-NHC)AgX](2) (X = Cl, 1; Br, 2) but a monomeric structure for N-heterocyclic carbene silver halides with the more sterically demanding 1-cyclohexyl-3-naphthalen-2-ylmethylimidazolylidene ligand (CyNaph-NHC)AgX (X = Cl, 4; Br, 5). Cationic biscarbene silver nitrate [(CyBn-NHC)(2)Ag](+)NO(3)(-)3 assumed a cis orientation with respect to the two carbene ligands. The monomeric complexes (CyNaph-NHC)AgX 4 and 5 showed higher catalytic activity than the dimeric [(CyBn-NHC)AgX](2)1 and 2 as well as the cationic biscarbene silver nitrate 3 in the model three component reaction of 3-phenylpropionaldehyde, phenylacetylene and piperidine with chloride 4 performing best and giving product in almost quantitative yield within 2 h at 100 °C. An explanation for the structure-activity relationship in N-heterocyclic carbene silver halide catalyzed three component reaction is given based on a slightly modified mechanism from the one in literature.

Impact on long-term OS of conditioning regimen in allogeneic BMT for children with AML in first CR: TBI+CY versus BU+CY: a report from the Société Française de Greffe de Moelle et de Thérapie Cellulaire.

PubMed

de Berranger, E; Cousien, A; Petit, A; Peffault de Latour, R; Galambrun, C; Bertrand, Y; Salmon, A; Rialland, F; Rohrlich, P-S; Vannier, J-P; Lutz, P; Yakouben, K; Duhamel, A; Bruno, B; Michel, G; Dalle, J-H

2014-03-01

Allogeneic hematopoietic SCT (HSCT) appears to be an efficient tool to cure high-risk AML in first CR but the choice between BU-based or TBI-based conditioning regimens still remains controversial. In order to analyze the impact of conditioning regimen on long-term survival, we conducted a retrospective analysis from French registry data including all consecutive patients under 18 years old (n=226) from 1980 to 2004 transplanted for AML in CR1 from sibling (n=142) or matched unrelated donors and given either TBI-1200 cGy and CY 120 mg/kg (TBI-Cy, n=84) or BU 16 mg/kg and CY 200 mg/kg (BuCy200, n=142). Patient subgroups were comparable for all criteria except for median age at diagnosis and HSCT and for donor type. Both 5-year OS and disease-free survival (DFS) were significantly better in BuCy200 group (P=0.02 and 0.005, respectively). In multivariate analysis, both HLA matching and BuCy200 appeared as good prognostic factors for treatment-related mortality and DFS. Grade 2-4 acute GvHD and chronic GvHD rates were statistically higher in TBI-Cy group than in Bu-Cy200 one with a RR at 2 (P=0.002). In total, Bu-Cy200 conditioning regimen gives better outcome compared with TBI-Cy irrespective of the stem cell source and the donor type.

Ceramide-1-phosphate has protective properties against cyclophosphamide-induced ovarian damage in a mice model of premature ovarian failure.

PubMed

Pascuali, Natalia; Scotti, Leopoldina; Di Pietro, Mariana; Oubiña, Gonzalo; Bas, Diana; May, María; Gómez Muñoz, Antonio; Cuasnicú, Patricia S; Cohen, Débora J; Tesone, Marta; Abramovich, Dalhia; Parborell, Fernanda

2018-05-01

Is ceramide-1-phosphate (C1P) an ovarian protective agent during alkylating chemotherapy? Local administration of C1P drastically reduces ovarian damage induced by cyclophosphamide (Cy) via protection of follicular reserve, restoration of hormone levels, inhibition of apoptosis and improvement of stromal vasculature, while protecting fertility, oocyte quality and uterine morphology. Cancer-directed therapies cause accelerated loss of ovarian reserve and lead to premature ovarian failure (POF). Previous studies have demonstrated that C1P regulates different cellular processes including cell proliferation, cell migration, angiogenesis and apoptosis. This sphingolipid may be capable of modulating vascular development and apoptosis in ovaries affected by chemotherapy. The 6-8-week-old mice were weighed and administered either a single intraperitoneal injection of Cy (75 mg/kg) or an equal volume of saline solution only for control mice. Control and Cy mice underwent sham surgery and received an intrabursal injection of saline solution, while Cy + C1P animal groups received 5 μl C1P, either 0.5 or 1 mM, under the bursa of both ovaries 1 h prior to Cy administration. Animals were euthanized by cervical dislocation or cardiac puncture 2 weeks after surgery for collection of blood orovary and uterus samples, which were cleaned of adhering tissue in culture medium and used for subsequent assays. Ovaries were used for Western blotting or immunohistochemical and/or histological analyses or steroid extraction, as required (n = 5-8 per group). A set of mice (n = 3/group) was destined for oocyte recovery and IVF. Finally, another set (n = 5-6/group) was separated to study fertility parameters. The number of primordial (P < 0.01), primary (P < 0.05) and preantral follicles (P < 0.05) were decreased in Cy-treated mice compared to control animals, while atretic follicles were increased (P < 0.001). In Cy + C1P mice, the ovaries recovered control numbers of these follicular structures, in both C1P doses studied. Cy affected AMH expression, while it was at least partially recovered when C1P is administered as well. Cy caused an increase in serum FSH concentration (P < 0.01), which was prevented by C1P coadministration (P < 0.01). E2 levels in Cy-treated ovaries decreased significantly compared to control ovaries (P < 0.01), whilst C1P restored E2 levels to those of control ovaries (P < 0.01). Cy increased the expression of BAX (P < 0.01) and decreased the expression of BCLX-L compared to control ovaries (P < 0.01). The ovarian BCLX-L:BAX ratio was also lower in Cy-treated mice (P < 0.05). In the Cy + C1P group, the expression levels of BAX, BCLX-L and BCLX-L:BAX ratio were no different than those in control ovaries. In addition, acid sphingomyelinase (A-SMase) expression was higher in Cy-treated ovaries, whilst remaining similar to the control in the Cy + C1P group. Cy increased the apoptotic index (TUNEL-positive follicles/total follicles) in preantral and early antral stages, compared to control ovaries (P < 0.001 and P < 0.01, respectively). C1P protected follicles from this increase. No primordial or primary follicular cells stained for either cleaved caspase-3 or TUNEL when exposed to Cy, therefore, we have found no evidence for follicular reserve depletion in response to Cy being due to apoptosis. Cy caused evident vascular injury, especially in large cortical stromal vessels, and some neovascularization. In the Cy + C1P group, the disruptions in vascular wall continuity were less evident and the number of healthy stromal blood vessels seemed to be restored. In Cy-treated ovaries α-SMA-positive cells showed a less uniform distribution around blood vessels. C1P coadministration partially prevented this Cy-induced effect, with a higher presence of α-SMA-positive cells surrounding vessels. By H&E staining, Cy-treated mice showed endometrial alterations compared to controls, affecting both epithelial and stromal compartments. However, C1P allowed that the stromal tissue to maintain its loose quality and its glandular branches. Cy-treated animals had significantly lower pregnancy rates and smaller litter sizes compared with control mice (P = 0.013 and P < 0.05, respectively), whereas cotreatment with C1P preserved normal fertility. Furthermore, a higher (P < 0.05) proportion of abnormal oocytes was recovered from Cy-treated mice compared to the control, which was prevented by C1P administration. N/A. The results of this study were generated from an in-vivo animal experimental model, already used by several authors. Further studies on C1P functions in female reproduction in pathological conditions such as chemotherapy-induced ovarian failure and on the safety of use of this sphingolipid are required. The present findings showed that C1P administration prior to Cy might be a promising fertility preservation strategy in female patients who undergo chemotherapy. This work was supported by grants from ANPCyT (PICT 2015-1117), CONICET (PIP 380), Cancer National Institute (INC) and Roemmers Foundation, Argentina. The authors declare no conflicts of interest.

Improvement of In Situ PCR by Optimization of PCR Cycle Number and Proteinase K Concentration: Localization of X Chromosome-Linked Phosphoglycerate Kinase-1 Gene in Mouse Reproductive Organs

PubMed Central

Hishikawa, Yoshitaka; An, Shucai; Yamamoto-Fukuda, Tomomi; Shibata, Yasuaki; Koji, Takehiko

2009-01-01

In situ polymerase chain reaction (in situ PCR), which can detect a few copies of genes within a cell by amplifying the target gene, was developed to better understand the biological functions of tissues. In this study, we optimized the protocol conditions for the detection of X chromosome-linked phosphoglycerate kinase-1 (pgk-1) gene in paraffin-embedded sections of mouse reproductive organs. The effects of various concentrations of proteinase K (PK) and PCR cycle numbers were examined. To label the amplified DNA, we used digoxigenin-dUTP (Dig), Cy-3-dUTP (Cy-3), or FluorX-dCTP (FluorX). The optimal concentration of PK was 50 µg/ml for the ovary and 10 µg/ml for the testis. Ten PCR cycles were optimal for Dig and 25 cycles were optimal for FluorX and Cy-3 in the ovary and testis. The signal-to-noise ratio of FluorX and Cy-3 for ovarian tissue was better than that of Dig. Using the above conditions, we detected 1–4 and 1–2 spots of pgk-1 in the nuclei of granulosa and germ cells, respectively. Our results indicate that in situ PCR is useful for detecting a specific gene in paraffin-embedded sections under optimized conditions of both PCR cycle number and PK concentration. PMID:19492023

Hyaluronic acid coated poly-epsilon-caprolactone nanospheres deliver high concentrations of cyclosporine A into the cornea.

PubMed

Yenice, Irem; Mocan, Mehmet C; Palaska, Erhan; Bochot, Amélie; Bilensoy, Erem; Vural, Imran; Irkeç, Murat; Hincal, A Atilla

2008-09-01

The objective of this study was to determine cyclosporine A (Cy A) levels in ocular tissues and fluids after topical administration of poly-epsilon-caprolactone (PCL)/benzalkonium chloride (BKC) nanospheres and hyaluronic acid (HA) coated PCL/BKC nanospheres onto healthy rabbit corneas. Nanospheres were prepared by nanoprecipitation and purified by gradient-rate centrifugation. Cy A (0.1%) in either castor oil solution (group 1), PCL/BKC nanosphere formulation (group 2) or HA coated PCL/BKC nanosphere formulation (group 3) was instilled onto rabbit corneas. Tear samples were adsorbed onto Schirmer tear strips. Cy A concentrations of fluid (blood, aqueous humor, tear) and specimen extracts (cornea, conjunctiva, iris/ciliary body) were determined by high performance liquid chromatography-mass spectrometry (LC-MS). The mean corneal Cy A concentration obtained at 0.5, 1, 2, 4, 8 and 24h following instillation of the formulations ranged between 0.12 and 1.2 ng/mg tissue for group 1, 5.9-15.5 ng/mg tissue for group 2 and 11.4-23.0 ng/mg for group 3 (one-way analysis of variance (ANOVA) and pairwise tests (SNK (Student-Newman-Keuls) and Tukey); p<0.05). Conjunctival Cy A levels of group 2 and 3 were not significantly different at any of the time points tested. However, there was a significant difference between Cy A concentration of castor oil formulation and that of PCL/BKC nanosphere formulation at 1 and 8h (p<0.05). The mean iris/ciliary body concentrations obtained with the three formulations were not significantly different at any time point with the exception of group 2 levels being higher than those of groups 1 and 3 at 1h (p<0.05). The lowest ocular tear Cy A concentrations (16-114 ng/ml) were found following the instillation of HA coated PCL/BKC nanoparticles (group 3) during the time period tested. Cy A loaded PCL/BKC and HA coated PCL/BKC nanospheres are able to achieve high levels of Cy A in the cornea that is 10-15-fold higher than that is achieved with Cy A solution in castor oil. Nanosphere formulation and HA may play an important role in delivering high levels of cyclosporine A into the cornea.

Design of a novel theranostic nanomedicine: synthesis and physicochemical properties of a biocompatible polyphosphazene-platinum(II) conjugate.

PubMed

Avaji, Prakash G; Park, Jung Hyun; Lee, Hyun Jeong; Jun, Yong Joo; Park, Kyung Su; Lee, Kyung Eun; Choi, Soo-Jin; Lee, Hwa Jeong; Sohn, Youn Soo

2016-01-01

To develop a theranostic nanomedicine involving the antitumor-active moiety (dach)Pt(II) (dach: trans-(±)-1,2-diaminocyclohexane) of oxaliplatin (OX), a new biocompatible polyphosphazene carrier polymer was designed by grafting with a methoxy poly(ethylene glycol) (MPEG) to increase duration of circulation in the blood and with aminoethanol (AE) as a spacer group. The antitumor (dach)Pt moiety was conjugated to the carrier polymer using cis-aconitic acid (AA) as a linker, resulting in a polymer conjugate formulated as [NP(MPEG550)(AE-AA)Pt(dach)]n, named "Polyplatin" (PP). PP was found to self-assemble into very stable polymeric nanoparticles with a mean diameter of 55.1 nm and a critical aggregation concentration of 18.5 mg/L in saline. PP could easily be labeled with a fluorescence dye such as Cy5.5 for imaging studies. The time-dependent ex vivo image studies on organ distributions and clearance of Cy-labeled PP have shown that PP accumulated in the tumor with high selectivity by the enhanced permeability and retention effect but was cleared out from all the major organs including the liver in about 4 weeks postinjection. Another time-dependent bioimaging study on distribution and clearance of PP in mouse kidney using laser ablation inductively coupled plasma mass spectroscopy has shown that PP accumulates much less in kidney and is more rapidly excreted than monomeric OX, which is in accord with the very low acute toxicity of PP as shown by its high LD50 value of more than 2000 mg/kg. The pharmacokinetic study of PP has shown that it has a much longer half-life (t 1/2β) of 13.3 hours compared with the 5.21 hours of OX and about a 20 times higher area under the curve value of 42,850.8 ng h/mL compared with the 2,320.4 ng h/mL of OX. The nude mouse xenograft trials of PP against the gastric MKN-28 tumor cell line exhibited remarkably better tumor efficacy compared with OX at the higher tolerated dose, with lower systemic toxicity.

Long-term salvage therapy with cyclosporin A in refractory idiopathic thrombocytopenic purpura.

PubMed

Emilia, Giovanni; Morselli, Monica; Luppi, Mario; Longo, Giuseppe; Marasca, Roberto; Gandini, Giovanna; Ferrara, Leonardo; D'Apollo, Nicola; Potenza, Leonardo; Bertesi, Marcello; Torelli, Giuseppe

2002-02-15

Treatment of severe, chronic idiopathic thrombocytopenic purpura (ITP) refractory to most usual therapies is a difficult challenge. Little information exists on the clinical use of cyclosporin A (CyA) in the treatment of ITP. This report describes long-term treatment with CyA (median, 40 months) and follow-up (median, 36.8 months) in 12 adult patients with resistant ITP. CyA used in relatively low doses (2.5-3 mg/kg of body weight per day) led to a clinical improvement in 10 patients (83.3%). Five had a complete response (41.1%), 4 a complete response to maintenance therapy (33.3%), and one a partial response (8.3%). Two patients had no response. Most patients with a response (60%) had a long-term remission (mean, 28.6 months) after discontinuation of CyA. One patient had a relapse of ITP 4 years after CyA therapy was stopped. Side effects were moderate and transient, even in patients dependent on continued CyA treatment. CyA seems to represent reasonable salvage treatment in severe, potentially life-threatening, refractory ITP.

Identification of genes modulated in rheumatoid arthritis using complementary DNA microarray analysis of lymphoblastoid B cell lines from disease-discordant monozygotic twins.

PubMed

Haas, Christian S; Creighton, Chad J; Pi, Xiujun; Maine, Ira; Koch, Alisa E; Haines, G Kenneth; Ling, Song; Chinnaiyan, Arul M; Holoshitz, Joseph

2006-07-01

To identify disease-specific gene expression profiles in patients with rheumatoid arthritis (RA), using complementary DNA (cDNA) microarray analyses on lymphoblastoid B cell lines (LCLs) derived from RA-discordant monozygotic (MZ) twins. The cDNA was prepared from LCLs derived from the peripheral blood of 11 pairs of RA-discordant MZ twins. The RA twin cDNA was labeled with cy5 fluorescent dye, and the cDNA of the healthy co-twin was labeled with cy3. To determine relative expression profiles, cDNA from each twin pair was combined and hybridized on 20,000-element microarray chips. Immunohistochemistry and real-time polymerase chain reaction were used to detect the expression of selected gene products in synovial tissue from patients with RA compared with patients with osteoarthritis and normal healthy controls. In RA twin LCLs compared with healthy co-twin LCLs, 1,163 transcripts were significantly differentially expressed. Of these, 747 were overexpressed and 416 were underexpressed. Gene ontology analysis revealed many genes known to play a role in apoptosis, angiogenesis, proteolysis, and signaling. The 3 most significantly overexpressed genes were laeverin (a novel enzyme with sequence homology to CD13), 11beta-hydroxysteroid dehydrogenase type 2 (a steroid pathway enzyme), and cysteine-rich, angiogenic inducer 61 (a known angiogenic factor). The products of these genes, heretofore uncharacterized in RA, were all abundantly expressed in RA synovial tissues. Microarray cDNA analysis of peripheral blood-derived LCLs from well-controlled patient populations is a useful tool to detect RA-relevant genes and could help in identifying novel therapeutic targets.

Matrix metalloproteinase triggered size-shrinkable gelatin-gold fabricated nanoparticles for tumor microenvironment sensitive penetration and diagnosis of glioma

NASA Astrophysics Data System (ADS)

Ruan, Shaobo; He, Qin; Gao, Huile

2015-05-01

To improve glioma targeting delivery efficiency and to monitor drug delivery and treatment outcome, a novel tumor microenvironment sensitive size-shrinkable theranostic system was constructed and evaluated. The G-AuNPs-DC-RRGD system was constructed by fabricating small sized gold nanoparticles (AuNPs) onto matrix metalloproteinase-2 (MMP-2) degradable gelatin nanoparticles (GNPs), doxorubicin (DOX) and Cy5.5 were decorated onto AuNPs through a hydrazone bond to enable the system with pH triggered cargoes release, and RRGD, a tandem peptide of RGD and octarginine was surface-modified onto the system to enable it with glioma active targeting ability. In vitro, the size of G-AuNPs-DC-RRGD could effectively shrink from 188.2 nm to 55.9 nm after incubation with MMP-2, while DOX and Cy5.5 were released in a pH dependent manner. Cellular uptake demonstrated that G-AuNPs-DC-RRGD could be effectively taken up by cells with higher intensity than G-AuNPs-DC-PEG. A study of tumor spheroids further demonstrated that the particles with smaller size showed better penetration ability, while RRGD modification could further improve permeability. In vivo, G-AuNPs-DC-RRGD displayed the best glioma targeting and accumulation efficiency, with good colocalization with neovessels. Cy5.5 also was colocalized well with DOX, indicating that Cy5.5 could be used for imaging of DOX delivery.To improve glioma targeting delivery efficiency and to monitor drug delivery and treatment outcome, a novel tumor microenvironment sensitive size-shrinkable theranostic system was constructed and evaluated. The G-AuNPs-DC-RRGD system was constructed by fabricating small sized gold nanoparticles (AuNPs) onto matrix metalloproteinase-2 (MMP-2) degradable gelatin nanoparticles (GNPs), doxorubicin (DOX) and Cy5.5 were decorated onto AuNPs through a hydrazone bond to enable the system with pH triggered cargoes release, and RRGD, a tandem peptide of RGD and octarginine was surface-modified onto the system to enable it with glioma active targeting ability. In vitro, the size of G-AuNPs-DC-RRGD could effectively shrink from 188.2 nm to 55.9 nm after incubation with MMP-2, while DOX and Cy5.5 were released in a pH dependent manner. Cellular uptake demonstrated that G-AuNPs-DC-RRGD could be effectively taken up by cells with higher intensity than G-AuNPs-DC-PEG. A study of tumor spheroids further demonstrated that the particles with smaller size showed better penetration ability, while RRGD modification could further improve permeability. In vivo, G-AuNPs-DC-RRGD displayed the best glioma targeting and accumulation efficiency, with good colocalization with neovessels. Cy5.5 also was colocalized well with DOX, indicating that Cy5.5 could be used for imaging of DOX delivery. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr01408e

Cyclophilin A as a potential genetic adjuvant to improve HIV-1 Gag DNA vaccine immunogenicity by eliciting broad and long-term Gag-specific cellular immunity in mice.

PubMed

Hou, Jue; Zhang, Qicheng; Liu, Zheng; Wang, Shuhui; Li, Dan; Liu, Chang; Liu, Ying; Shao, Yiming

2016-01-01

Previous research has shown that host Cyclophilin A (CyPA) can promote dendritic cell maturation and the subsequent innate immune response when incorporated into an HIV-1 Gag protein to circumvent the resistance of dendritic cells to HIV-1 infection. This led us to hypothesize that CyPA may improve HIV-1 Gag-specific vaccine immunogenicity via binding with Gag antigen. The adjuvant effect of CyPA was evaluated using a DNA vaccine with single or dual expression cassettes. Mouse studies indicated that CyPA specifically and markedly promoted HIV-1 Gag-specific cellular immunity but not an HIV-1 Env-specific cellular response. The Gag/CyPA dual expression cassettes stimulated a greater Gag-specific cellular immune response, than Gag immunization alone. Furthermore, CyPA induced a broad Gag-specific T cell response and strong cellular immunity that lasted up to 5 months. In addition, CyPA skewed to cellular rather than humoral immunity. To investigate the mechanisms of the adjuvant effect, site-directed mutagenesis in CyPA, including active site residues H54Q and F60A resulted in mutants that were co-expressed with Gag in dual cassettes. The immune response to this vaccine was analyzed in vivo. Interestingly, the wild type CyPA markedly increased Gag cellular immunity, but the H54Q and F60A mutants drastically reduced CyPA adjuvant activation. Therefore, we suggest that the adjuvant effect of CyPA was based on Gag-CyPA-specific interactions. Herein, we report that Cyclophilin A can augment HIV-1 Gag-specific cellular immunity as a genetic adjuvant in multiplex DNA immunization strategies, and that activity of this adjuvant is specific, broad, long-term, and based on Gag-CyPA interaction.

Cycloheximide: No Ordinary Bitter Stimulus

PubMed Central

Hettinger, Thomas P.; Formaker, Bradley K.; Frank, Marion E.

2007-01-01

Cycloheximide (CyX), a toxic antibiotic with a unique chemical structure generated by the actinomycete, Streptomyces griseus, has emerged as a primary focus of studies on mammalian bitter taste. Rats and mice avoid it at concentrations well below the thresholds for most bitter stimuli and T2R G-protein-coupled receptors specific for CyX with appropriate sensitivity are identified for those species. Like mouse and rat, golden hamsters, Mesocricetus auratus, also detected and rejected micromolar levels of CyX, although 1 mM CyX failed to activate the hamster chorda tympani nerve. Hamsters showed an initial tolerance for 500 μM CyX, but after that, avoidance of CyX dramatically increased, plasticity not reported for rat or mouse. As the hamster lineage branches well before division of the mouse-rat lineage in evolutionary time, differences between hamster and mouse-rat reactions to CyX are not surprising. Furthermore, unlike hamster LiCl-induced learned aversions, the induced CyX aversion neither specifically nor robustly generalized to other non-ionic bitter stimuli; and unlike adverse reactions to other chemosensory stimuli, aversions to CyX were not mollified by adding a sweetener. Thus, CyX is unlike other bitter stimuli. The gene for the high-affinity CyX receptor is a member of a cluster of 5 orthologous T2R genes that are likely rodent specific; this “CyX clade” is found in the mouse, rat and probably hamster, but not in the human or rabbit genome. The rodent CyX-T2R interaction may be one of multiple lineage-specific stimulus-receptor interactions reflecting a response to a particular environmental toxin. The combination of T2R multiplicity, species divergence and gene duplication results in diverse ligands for multiple species-specific T2R receptors, which confounds definition of ‘bitter’ stimuli across species. PMID:17400304

[Chiparray-based identification of gene expression in HUVECs treated with low frequency electric fields].

PubMed

Ulrich, D; Ulrich, F; Silny, J; Unglaub, F; Pallua, N

2006-06-01

After high-voltage electric injury, patients often show progressive tissue necrosis and thrombosis of blood vessels even remote from the entry and exit sites of electrical current. Recently, we were able to demonstrate IN VIVO and VITRO the release of several prothrombotic factors. In this study, we report on IN VITRO studies performed to characterize gene expression profiles using a DNA-microarray in HUVECs (human umbilical vein endothelial cells) exposed to low frequency electrical current. HUVECs were plated and grown to confluence in a culture chamber. They were exposed to 25 periods of 50 Hz sinusoidal waves. The periods had field strength of 60 V/cm and duration of 100 ms. Periods were interrupted by 10-second intervals to prevent significant joule heating. Control HUVECs were treated identically except that no electric field was applied. Samples from control and treated cells were taken after six and 24 hours. A PIQOR Immunology Array (Milteny Biotech) containing 1076 cDNAs was used for gene expression analysis. Hybridization of Cy3- and Cy5-labelled samples, image capture, and signal quantification of hybridized arrays were performed. Local background was subtracted from the signal to obtain the net signal intensity and the ratio of Cy5/Cy3. The ratios were normalized to the median of all ratios and the mean of the ratios of four corresponding spots was computed. More than two-fold increases or decreases of the gene expression were regarded as relevant. A total of 413 genes (1s + s) respectively 345 genes (2s + s) could be detected. The results obtained display a distinct expression pattern of up-regulated genes known to be important for hemostasis (e.g. UPA, UPAR, ECE1, PAFAH1B1, PGT, INOS, ENOS, TPA, ICAM1, VCAM1, PAI1, PAI2, VWF, PTGDR, F3, THBD), which was most evident after 24 hours. This expression profile might lead to a hypercoagulated state. Furthermore, the expression of genes involved in angiogenesis was reduced whereas the expression of those involved in platelet formation was increased. Our results indicate that low frequency electrical fields induce a distinct signature of differential gene expression in exposed HUVECs. This might explain the clinical observation of thrombosis and progressive tissue necrosis after electrical injury.

Methyl-DEAE-dextran: a candidate biomaterial.

PubMed

Zambito, Ylenia; Baggiani, Andrea; Carelli, Vera; Serafini, Maria Francesca; Di Colo, Giacomo

2004-01-01

The full quaternisation of DEAE-dextran was successfully attempted and an application of the quaternised product was suggested. Commercial DEAE-dextran was reacted with iodomethane at 60 degrees C in the presence of NaOH. The raw product was purified by dialysis, during which the iodide ion was replaced by chloride. N-methylation and O-methylation resulted from the reaction. A second methylation step produced no further changes in the molecule. Alkalimetry indicated the absence of amino groups in the methylated polymer molecule, thus testifying to a complete quaternisation. N-acetylcysteine (AcCy) was neutralised with the polymer in the hydroxide form, thus obtaining the methyl DEAE-dextran salt of AcCy (Me-DEAE-dextran/AcCy), whereby an ophthalmic formulation for the treatment of the dry eye syndrome was prepared. For comparison, the neutral AcCy salt of commercial DEAE-dextran (DEAE-dextran/AcCy) was prepared. The AcCy content in Me-DEAE-dextran/AcCy was higher than in DEAE-dextran/AcCy (23 vs 13%), while the viscosity of a solution containing the salt concentration corresponding to the therapeutic AcCy concentration (4%w/v) was lower with the former compared to the latter salt (20.5 vs 23.9 mPa s). Both solutions were ipotonic (245 mOsm/kg), whereas the commercial Tirocular is strongly hypertonic (900 mOsm/kg) and irritant.

3-Chlorotyrosine and 3,5-Dichlorotyrosine as biomarkers of respiratory tract exposure to chlorine gas

EPA Science Inventory

Modification of tyrosine by reactive chlorine can produce both 3-chlorotyrosine (CY) and 3,5-dichlorotyrosine (dCY). Both of these amino acids have proven to be promising biomarkers for assessing the extent of myeloperoxidase-catalyzed chlorine stress in a number of adverse physi...

Reactivity of a dearomatised pincer CoIIBr complex with PNCNHC donors: alkylation and Si-H bond activation via metal-ligand cooperation.

PubMed

Simler, Thomas; Choua, Sylvie; Danopoulos, Andreas A; Braunstein, Pierre

2018-05-18

The double aminolysis reaction of [Co{N(SiMe3)2}2] by the salt 1-(6-((dicyclohexylphosphaneyl)methyl)pyridin-2-yl)-3-(2,6-diisopropylphenyl)-1H-imidazol-3-ium bromide, which contains one phosphane, one pyridine and one imidazolium groups, of formula [o-Cy2PCH2(C5H3N)(o-C3H3N2DiPP)]Br and abbreviated as (CyPNpyrCim)Br, was previously shown to afford the Co(ii) complex [Co(CyP*NaCNHC)Br] (1) containing a dearomatised picolyl moiety in the tridentate, anionic donor ligand CyP*NaCNHC (Na = anionic amido, P* = vinylic P donor). We now report that formation of 1 is preceded by an intermediate tentatively assignable to the 5-coordinate Co(ii) complex [Co(CyPNpyrCNHC){N(SiMe3)2}Br] (2). The reaction of 1 with LiCH2SiMe3 afforded the dark purple, paramagnetic [Co(CyP*NaCNHC)CH2SiMe3] (3) with a low spin d7 CoII; the electronic configurations of 1 and 3 were corroborated by EPR spectroscopy. Addition of excess (≥4 equiv.) H2SiPh2 to a solution of 1 gave the diamagnetic [Co(CyP(SiHPh2)NCNHC)Br] (4) following Si-H activation and silylation of the ligand backbone at the α-CHP. Reduction of CoII to CoI by silanes is uncommon.

Tumour volume response, initial cell kill and cellular repopulation in B16 melanoma treated with cyclophosphamide and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea.

PubMed Central

Stephens, T. C.; Peacock, J. H.

1977-01-01

The relationship between tumour volume response and cell kill in B16 melanoma following treatment in vivo with cyclophosphamide (CY) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) was investigated. Tumour volume response, expressed as growth delay, was estimated from measurements of tumour dimensions. Depression of in vitro colony-forming ability of cells from treated tumours was used as the measure of tumour cell kill. The relationship between these parameters was clearly different for the two agents studied. CY produced more growth delay (7.5 days) per decade of tumour cell kill than CCNU (2 to 3.5 days). The possibility that this was due to a technical artefact was rejected in favour of an alternative explanation that different rates of cellular repopulation in tumours treated with CY and CCNU might be responsible. Cellular repopulation was measured directly, by performing cell-survival assays at various times after treatment with doses of CY and CCNU which produced about 3 decades of cell kill. The rate of repopulation by clonogenic cells was much slower after treatment with CY than with CCNU, and this appears to account for the longer duration of the growth delay obtained with CY. PMID:921888

A novel medium for the enhanced production of cyclosporin A by Tolypocladium inflatum MTCC 557 using solid state fermentation.

PubMed

Survase, Shrikant A; Shaligram, Nikhil S; Pansuriya, Ruchir C; Annapure, Uday S; Singhal, Rekha S

2009-05-01

Cyclosporin A (CyA) produced by Tolypocladium inflatum is a promising drug owing to its immunosuppressive and antifungal activities. From an industrial point of view, the necessity to obtain a suitable and economic medium for higher production of CyA was the aim of this work. The present study evaluated the effect of different fermentation parameters in solid state fermentation, such as selection of solid substrate, hydrolysis of substrates, initial moisture content, supplementation of salts, additional carbon, and nitrogen sources, as well as the inoculum age and size, on production of CyA by Tolypocladium inflatum MTCC 557. The fermentation was carried out at 25+/-2 degrees for 9 days. A combination of hydrolyzed wheat bran flour and coconut oil cake (1:1) at 70% initial moisture content supported a maximum production of 3,872+/-156 mg CyA/kg substrate as compared with 792+/-33 mg/kg substrate before optimization. Furthermore, supplementation of salts, glycerol (1%w/w), and ammonium sulfate (1%w/w) increased the production of CyA to 5,454+75 mg/kg substrate. Inoculation of 5 g of solid substrate with 6 ml of 72-h-old seed culture resulted in a maximum production of 6,480+95 mg CyA/kg substrate.

Clinico-hematological and micronuclear changes induced by cypermethrin in broiler chicks: Their attenuation with vitamin E and selenium.

PubMed

Sharaf, Summaira; Khan, Ahrar; Khan, Muhammad Zargham; Aslam, Faiza; Saleemi, Muhammad Kashif; Mahmood, Fazal

2010-07-01

This study was carried out on 90 one-day-old broiler chicks to know clinico-hematological alterations, DNA damage caused by cypermethrin (CY), and attenuation of toxic effects by vitamin E (Vit E) and selenium (Se). Birds were randomly divided into five equal groups. Groups 1-4 received CY (600mlkg(-1)b.wt) daily for 30 days by crop tubing. In addition to CY, groups 2, 3 and 4 received Vit E (150mgkg(-1)b.wt), Se (0.25mgkg(-1)b.wt), and Vit E (150mgkg(-1)b.wt)+Se (0.25mgkg(-1)b.wt), respectively. Group 5 served as control. Birds were monitored twice daily for clinical signs. They were weighed and blood samples were collected at experimental days 10, 20 and 30 for hematological studies. CY-treated birds showed more prominent signs of toxicity compared to CY+Vit E, CY+Se and CY+Vit E+Se birds. Body weight in groups 1-3 was significantly (P<0.05) smaller at days 20 and 30 when compared with the control group. Significantly (P<0.001) higher numbers of micronuclei appeared in chicks treated with CY compared to CY+Vit E- and CY+Se-treated birds. Significantly decreased total erythrocyte counts (TEC), hemoglobin (Hb) concentration and packed cell volume (PCV) in all treated groups were recorded. Treated birds suffered from macrocytic hypochromic anemia. Leukocytosis in early stage and later leucopenia was seen in treated birds. It can be concluded that CY induces toxic effects in broilers chicks; however, these toxic effects can be ameliorated by Vit E or Se. Combination of Vit E and Se was more effective to ameliorate toxic effects of cypermethrin.

Near-infrared optical imaging in glioblastoma xenograft with ligand targeting α3 integrin

PubMed Central

Xiao, Wenwu; Yao, Nianhuan; Peng, Li; Liu, Ruiwu; Lam, Kit S

2010-01-01

Purpose Patients with glioblastoma usually have a very poor prognosis. Even with a combination of radiotherapy plus temozolomide, the median survival of these patients is only 14.6 months. New treatment approaches to this cancer are needed. Our purpose is to develop new cell-surface binding ligands for glioblastoma cells, and use them as targeted imaging and therapeutic agents for this deadly disease. Methods One-bead one-compound combinatorial cyclic peptide libraries were screened with live human glioblastoma U-87MG cells. The binding affinity and targeting specificity of peptides identified were tested with in vitro experiments on cells and in vivo, and ex vivo experiments on U-87MG xegnograft mouse model. Results A cyclic peptide, LXY1, was identified and shown to be binding to the α3 integrin of U-87MG cells with moderately high affinity (Kd = 0.5+/−0.1 μM) and high specificity. Biotinylated LXY1, when complexed with streptavidin-Cy5.5 (SA-Cy5.5) conjugate, targeted both subcutaneous and orthotopic U-87MG xenograft implants in nude mice. The in vivo targeting specificity was further verified by strong inhibition of tumor uptake of LXY1-biotin-SA-Cy5.5 complex when intravenously injecting the animals with anti-α3 integrin antibody or excess unlabeled LXY1 prior to administrating the imaging probe. The smaller univalent LXY1-Cy5.5 conjugate (2279 Da) was found to have a faster accumulation in the U-87MG tumor and shorter retention time compared with the larger tetravalent LXY1-biotin-SA-Cy5.5 complex (~ 64 KDa). Conclusions Collectively, the data reveals that LXY1 has the potential to be developed into an effective imaging and therapeutic targeting agent for human glioblastoma. PMID:18712382

Synthesis of Enantiopure Alicyclic Ethers and Their Activity on the Chemosensory Organ of the Ectoparasite of Honey Bees, Varroa destructor.

PubMed

Pinnelli, Govardhana R; Singh, Nitin K; Soroker, Victoria; Plettner, Erika

2016-11-16

The preparation of enantiopure conformationally restricted alicyclic ethers and their inhibitory activities on the chemosensory organ of the Varroa destructor, a parasite of honey bees, are reported in this article. We tested the effect of enantiopure ethers of cis-5-(2'-hydroxyethyl)cyclopent-2-en-1-ol on the Varroa chemosensory organ by electrophysiology, for their ability to inhibit the responses to two honey bee-produced odors that are important for the mite to locate its host: nurse bee head space odor and (E)-β-ocimene, a honey bee brood pheromone. Previous work with the racemic compounds showed that they suppress the mite's olfactory response to its bee host, which led to incorrect host choice. Based on a structure-activity relationship, we predicted that the two most active compounds-cis-1-butoxy-5-(2'-methoxyethyl)cyclopent-2-ene, cy{4,1}, and (cis-1-ethoxy-5-(2'ethoxyethyl)cyclopent-2-ene, cy{2,2}-could have opposite active enantiomers. Here we studied the enantiomers of both ethers, whose preparation involved enzymatic resolution of racemic diol cis-5-(2'-hydroxyethyl)cyclopent-2-en-1-ol using Lipase AK with vinyl acetate. The racemic diol was prepared from commercially available 2,5-norbornadiene. We observed that the responses of the chemosensory organ to honey bee head space volatiles were significantly decreased by both enantiomers of cy{4,1} and cy{2,2}, but that responses to (E)-β-ocimene were decreased significantly only by (+)-cy{4,1} (1R,5S) and (-)-cy{2,2} (1S,5R) and not by their respective enantiomers. The importance of this result is that the racemates could be used to inhibit olfactory detection of bee odors by mites, without a loss in activity relative to the more expensive enantiopure compounds.

DIGE compatible labelling of surface proteins on vital cells in vitro and in vivo.

PubMed

Mayrhofer, Corina; Krieger, Sigurd; Allmaier, Günter; Kerjaschki, Dontscho

2006-01-01

Efficient methods for profiling of the cell surface proteome are desirable to get a deeper insight in basic biological processes, to localise proteins and to uncover proteins differentially expressed in diseases. Here we present a strategy to target cell surface exposed proteins via fluorescence labelling using CyDye DIGE fluors. This method has been applied to human cell lines in vitro as well as to a complex biological system in vivo. It allows detection of fluorophore-tagged cell surface proteins and visualisation of the accessible proteome within a single 2-D gel, simplifying subsequent UV MALDI-MS analysis.

Spectral study and protein labeling of inclusion complex between dye and calixarene sulfonate.

PubMed

Fei, Xuening; Zhang, Yong; Zhu, Sen; Liu, Lijuan; Yu, Lu

2013-05-01

The host-guest inclusion complex of calix[6]arene sulfonate (SCA6) with thiazole orange (TO) formed in aqueous solution was studied. Absorption and fluorescence techniques were used for the analysis of this inclusion complex. The addition of calixarene sulfonate leads to a decrease in both absorption and fluorescence intensity of the dye, indicating that the inclusion complex was formed. Simultaneously, the inclusion phenomenon of another cyanine dye, Cy3, with calixarene sulfonate was investigated. The stability constant of the two complexes was determined, and the results were compared. The water solubility of TO dye was increased in the presence of calixarene sulfonate, and further protein labeling experiments suggested that this TO-SCA6 complex can act as a fluorescent probe for labeling of biomolecules.

Evaluation of the Leaching Potential of Anthranilamide Insecticides Through the Soil.

PubMed

Vela, Nuria; Pérez-Lucas, Gabriel; Navarro, María J; Garrido, Isabel; Fenoll, José; Navarro, Simón

2017-10-01

The mobility of two relatively new antranilic diamide insecticides, cyanantraniliprole (CY) and cholantraniliprole (CH) in soil was examined, by means of disturbed columns loaded with a typical semiarid Mediterranean soil (Calcaric fluvisol) under laboratory conditions. Both insecticides appeared in leachates, with 52% of CY and 41% of CH of the initial mass added (1 µg g -1 ) present. For CY, 21% and 19% were recovered from the upper and bottom layers of the soil, respectively, while for CH, 33% and 22% were recovered from the upper and bottom layers respectively. Based on the calculated half-lives (29 and 27 days for CY and CH, respectively) and their log K OC (about 2.5 for both), the calculated Groundwater Ubiquity Score (GUS) index was higher than 5 for both, indicating they have the potential to leach. Two transformation products, C 13 H 9 Cl 2 N 2 O (IN-ECD73) and C 19 H 12 BrClN 6 O (IN-J9Z38) corresponding to the degradation of CH and CY, respectively were also identified and detected in leachates and soil.

Assessing the feasibility of hydrate deposition on pipeline walls--adhesion force measurements of clathrate hydrate particles on carbon steel.

PubMed

Nicholas, Joseph W; Dieker, Laura E; Sloan, E Dendy; Koh, Carolyn A

2009-03-15

Adhesive forces between cyclopentane (CyC5) hydrates and carbon steel (CS) were measured. These forces were found to be substantially lower than CyC5 hydrate-CyC5 hydrate particle measurements and were also lower than ice-CS measurements. The measured adhesive forces were used in a force balance to predict particle removal from the pipeline wall, assuming no free water was present. The force balance predicted entrained hydrate particles of 3 microns and larger diameter would be removed at typical operating flow rates in offshore oil and gas pipelines. These predictions also suggest that hydrate deposition will not occur in stabilized (cold) flow practices.

A New Biomarker Proxy for Palaeo-pCO2 Reconstruction in Ancient Sediments

NASA Astrophysics Data System (ADS)

Pancost, R. D.; Magness, S.; Maxwell, J. R.

2001-12-01

The carbon isotopic composition of marine organic matter has commonly been used in chemostratigraphy or as a proxy for ancient pCO2 levels. Both of these goals require that the source of organic matter be well defined, and in the case of palaeo-pCO2 investigations, the organic matter must be derived ultimately from aquatic photoautotrophs. However, additional sources, including terrestrial biomass, heterotrophs, or bacteria, can also contribute to total organic carbon (TOC). In the past decade, numerous workers have attempted to refine organic carbon isotope records using the isotopic composition of individual compounds (biomarkers) rather than the TOC. The appeal of this approach is that by examining specific biomarkers, a signal diagnostic for photoautotrophic organisms can be obtained. For compound-specific isotope analyses to be most effective, the compounds analysed must have a relatively specific source. Among the most commonly used biomarkers in palaeo-pCO2 investigations are alkenones, long-chain ketones derived exclusively from certain species of haptophyte algae. However, alkenones are absent in rocks older than the Jurassic and either absent or present in low abundances in rocks older than the Miocene. Thus, in older rocks, other biomarkers, including steranes (derived from eukaryotic sterols), phytane (presumably derived from chlorophyll), and n-alkanes (derived from algal macromolecules), are used. Unfortunately, these compounds can have alternative sources and become less reliable as isotopic proxies for photoautotrophs with increasing thermal maturity and complexity of the hydrocarbon distribution. Here we propose the use of a maleimides (1H-pyrrole-2,5-diones) as a new biomarker class for evaluating past changes in photoautotroph carbon isotopic compositions. Maleimides have three key advantages over other biomarkers in ancient rocks. First, they are degradation products of chlorophyll and have no known alternative origins in marine sediments. Second, because of their unique structure, they can be readily isolated from other organic components facilitating the determination of accurate carbon isotope ratios. Finally, the pyrrole structure is relatively stable insuring that maleimides survive even in thermally mature rocks. We have applied the analysis of maleimides to investigations of sediments from the Kupferschiefer (Permian), Vena del Gesso (Messinian) and Livello Bonarelli (Cenomanian-Turonian boundary) formations. In all three cases, the carbon isotopic compositions of selected maleimides exhibit shifts predicted by either carbonate or other biomarker carbon isotope profiles.

Onset of carbon-carbon bonding in the Nb(5)C(y) (y = 0-6) clusters: a threshold photo-ionisation and density functional theory study.

PubMed

Dryza, Viktoras; Gascooke, Jason R; Buntine, Mark A; Metha, Gregory F

2009-02-21

We have used photo-ionisation efficiency spectroscopy to determine the ionisation potentials (IPs) of the niobium-carbide clusters, Nb(5)C(y) (y = 0-6). Of these clusters Nb(5)C(2) and Nb(5)C(3) exhibit the lowest IPs. Complementary density functional theory calculations have been performed to locate the lowest energy isomers for each cluster. By comparing the experimental IPs with those calculated for candidate isomers, the structures of the Nb(5)C(y) clusters observed in the experiment are inferred. For all these structures, the underlying Nb(5) cluster has either a "prolate" or "oblate" trigonal bipyramid geometry. Both Nb(5)C(5) and Nb(5)C(6) are shown to contain carbon-carbon bonding in the form of one and two molecular C(2) units, respectively.

Photoaffinity labelling and solubilization on the central 5-HT1A receptor binding site.

PubMed

Gozlan, H; Emerit, M B; el Mestikawy, S; Cossery, J M; Marquet, A; Besselievre, R; Hamon, M

1987-01-01

Two complementary approaches, covalent labelling and solubilization, have been used to study the biochemical properties of the central 5-HT1A receptor binding site. We have first designed a photoaffinity ligand containing the structure of 8-OH-DPAT, a potent and specific agonist of 5-HT1A sites. Thus, 8-methoxy-2[N-n-propyl,N-3-(2-nitro-4-azido-phenyl)- aminopropyl]aminotetralin or 8-methoxy-3'-NAP-amino-PAT, was found to displace, in the dark, [3H]8-OH-DPAT from 5-HT1A sites in rat hippocampal membranes with an IC50 of 6.6 nM. Under two cumulative UV irradiations (366 nm, for 20 min at 4 degrees C), 8-methoxy-3-'-NAP-amino-PAT (30 nM) blocked irreversibly 55-60% of 5-HT1A binding sites. This blockade was specific of 5-HT1A sites since the other serotoninergic sites, 5-HT1B, 5-HT2 and also the presynaptic 5-HT3 sites were not affected by the treatment. In addition, the binding of [3H]Spiperone and [3H]7-OH-DPAT to striatal dopamine sites remained unchanged under similar photolysis conditions. The tritiated derivative of the photoaffinity ligand (92 Ci/mmol) was then synthesized for the identification of the covalently bound protein(s). SDS-PAGE of solubilized membranes irradiated in the presence of 20 nM 3H-8-methoxy-3'-NAP-amino-PAT allowed the detection of a 63 kD protein whose labelling appeared specific. Thus, 3H-incorporation into the 63 kD band could be prevented by microM concentrations of 5-HT, 8-OH-DPAT and other selective 5-HT1A ligands such as isapirone. In contrast, the 5-HT2 antagonist ketanserin, norepinephrine and dopamine-related ligands (including 7-OH-DPAT) were ineffective. Direct solubilization of 5-HT1A receptor binding sites was also attempted from rat hippocampal membranes. The best results were obtained using CHAPS (10 mM) plus NaCl (0.2 M), which led to 50% recovery of 5-HT1A sites in the 100,000 g supernatant. The pharmacological properties and sensitivity to N-ethyl-maleimide and GppNHp of soluble sites appeared near identical to those of membrane-bound 5-HT1A sites.

Construction of Benzene Rings by Copper-Catalyzed Cycloaddition Reactions of Oximes and Maleimides: An Access to Fused Phthalimides.

PubMed

Yang, Jie; Zhao, Bo; Xi, Yue; Sun, Si; Yang, Zhen; Ye, Ying; Jiang, Kun; Wei, Ye

2018-02-16

A useful Cu-catalyzed cycloaddition protocol for the construction of benzene rings has been achieved. The reactions, utilizing readily available oximes and maleimides as starting materials, proceed under mild reaction conditions to generate a series of structurally interesting fused-phthalimides that are difficult to be prepared by conventional methods.

Bromo- and thiomaleimides as a new class of thiol-mediated fluorescence 'turn-on' reagents.

PubMed

Youziel, Judith; Akhbar, Ahmed R; Aziz, Qadeer; Smith, Mark E B; Caddick, Stephen; Tinker, Andrew; Baker, James R

2014-01-28

Bromo- and thiomaleimides are shown to serve as highly effective quenchers of a covalently attached fluorophore. Reactions with thiols that lead to removal of the maleimide conjugation, or detachment of the fluorophore from the maleimide, result in 'turn-on' of the fluorescence. These reagents thus offer opportunities in thiol sensing and intracellular reporting.

Synthesis and Evaluation of 64Cu-DOTA-NT-Cy5.5 as a Dual-Modality PET/Fluorescence Probe to Image Neurotensin Receptor-Positive Tumor.

PubMed

Deng, Huaifu; Wang, Hui; Wang, Mengzhe; Li, Zibo; Wu, Zhanhong

2015-08-03

Overexpression of neurotensin receptors (NTRs) has been suggested to play important roles in the growth and survival of a variety of tumor types. The aim of this study is to develop a dual-modality probe (64Cu -DOTA-NT-Cy5.5) for imaging NTR1 expression in vivo with both positron emission tomography (PET) and fluorescence. In this approach, the thiol group and N terminal amino group of neurotensin analogue (Cys-NT) were chemically modified with Cy5.5 dye and DOTA chelator, respectively. After radiolabeling with 64Cu, the resulting probe (64Cu-DOTA-NT-Cy5.5) was evaluated in NTR1 positive HT-29 tumor model. Small animal PET quantification analysis demonstrated that the tumor uptake was 1.91±0.22 and 1.79±0.16%ID/g at 1 and 4 h postinjection (p.i.), respectively. The tumor-to-muscle ratio was 17.44±3.25 at 4 h p.i. based on biodistribution. Receptor specificity was confirmed by the successful blocking experiment at 4 h p.i. (0.42±0.05%ID/g). In parallel with PET experiment, fluorescence imaging was also performed, which demonstrated prominent tumor uptake in HT-29 model. As a proof of concept, an imaging guided surgery was performed to the fluorescent moiety of this probe and could provide potential surgery guidance for NTR positive patients. In summary, our results clearly indicated that the dual-modality probe, 64Cu-DOTA-NT-Cy5.5, could serve as a promising agent to image NTR positive tumors in vivo.

Novel calibration tools and validation concepts for microarray-based platforms used in molecular diagnostics and food safety control.

PubMed

Brunner, C; Hoffmann, K; Thiele, T; Schedler, U; Jehle, H; Resch-Genger, U

2015-04-01

Commercial platforms consisting of ready-to-use microarrays printed with target-specific DNA probes, a microarray scanner, and software for data analysis are available for different applications in medical diagnostics and food analysis, detecting, e.g., viral and bacteriological DNA sequences. The transfer of these tools from basic research to routine analysis, their broad acceptance in regulated areas, and their use in medical practice requires suitable calibration tools for regular control of instrument performance in addition to internal assay controls. Here, we present the development of a novel assay-adapted calibration slide for a commercialized DNA-based assay platform, consisting of precisely arranged fluorescent areas of various intensities obtained by incorporating different concentrations of a "green" dye and a "red" dye in a polymer matrix. These dyes present "Cy3" and "Cy5" analogues with improved photostability, chosen based upon their spectroscopic properties closely matching those of common labels for the green and red channel of microarray scanners. This simple tool allows to efficiently and regularly assess and control the performance of the microarray scanner provided with the biochip platform and to compare different scanners. It will be eventually used as fluorescence intensity scale for referencing of assays results and to enhance the overall comparability of diagnostic tests.

Efficient Site-Specific Labeling of Proteins via Cysteines

PubMed Central

Kim, Younggyu; Ho, Sam O.; Gassman, Natalie R.; Korlann, You; Landorf, Elizabeth V.; Collart, Frank R.; Weiss, Shimon

2011-01-01

Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70–90%, and specificities are better than ~95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis. PMID:18275130

Efficient site-specific labeling of proteins via cysteines.

PubMed

Kim, Younggyu; Ho, Sam O; Gassman, Natalie R; Korlann, You; Landorf, Elizabeth V; Collart, Frank R; Weiss, Shimon

2008-03-01

Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70-90%, and specificities are better than approximately 95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis.

Engineering strategies for enhancing the production of eicosapentaenoic acid (EPA) from an isolated microalga Nannochloropsis oceanica CY2.

PubMed

Chen, Chun-Yen; Chen, Yu-Chun; Huang, Hsiao-Chen; Huang, Chieh-Chen; Lee, Wen-Lung; Chang, Jo-Shu

2013-11-01

Microalgae have emerged as promising resources for highly unsaturated fatty acids. In this study, an indigenous microalga identified as Nannochloropsis oceanica CY2 was grown photoautotrophically to produce eicosapentaenoic acid (EPA; 20:5, n-3). Specific engineering strategies were employed to stimulate EPA accumulation in the microalgal cells. The results show that BG-11 was the most effective medium to grow N. oceanica CY2, giving an EPA content and biomass concentration of 2.38% (per dry cell weight) and 1.53 g/l. The EPA content nearly doubled when using the optimal nitrogen source (NaNO3) at a concentration of 1.50 g/l. The illumination system also markedly affected the EPA content for the photoautotrophic microalga. When the microalgal culture was illuminated with a red LED, an impressively high EPA content of 5.5% was obtained. Finally, using semi-batch cultures operations with LED-blue illumination, the EPA content of N. oceanica CY2 was stably maintained at 5.0%. Copyright © 2013 Elsevier Ltd. All rights reserved.

Quadrivalent meningococcal (MenACWY-TT) conjugate vaccine or a fourth dose of H. influenzae-N. meningitidis C/Y conjugate vaccine (HibMenCY-TT) is immunogenic in toddlers who previously received three doses of HibMenCY-TT in infancy.

PubMed

Leonardi, Michael; Latiolais, Thomas; Sarpong, Kwabena; Simon, Michael; Twiggs, Jerry; Lei, Paul; Rinderknecht, Stephen; Blatter, Mark; Bianco, Veronique; Baine, Yaela; Friedland, Leonard R; Miller, Jacqueline M

2015-02-11

Immunogenicity and safety of a single dose of MenACWY-TT or a fourth dose of HibMenCY-TT were evaluated in the second year of life in HibMenCY-TT-primed toddlers. Healthy infants were randomized (5:1) and primed at 2, 4 and 6 months of age with HibMenCY-TT and diphtheria-tetanus-acellular pertussis-hepatitis B-inactivated poliovirus (DTaP-HBV-IPV) vaccine; or Hib-TT and DTaP-HBV-IPV (control). Recipients of HibMenCY-TT+DTaP-HBV-IPV were re-randomized (2:2:1) to receive MenACWY-TT at 12-15 months and DTaP at 15-18 months; MenACWY-TT co-administered with DTaP at 15-18 months; or HibMenCY-TT at 12-15 months and DTaP at 15-18 months. Controls received DTaP only at 15-18 months due to Hib conjugate vaccine shortage. Serum bactericidal activity using human complement (hSBA) and safety were assessed one month after meningococcal vaccination. After vaccination with MenACWY-TT at 12-15 months or MenACWY-TT+DTaP at 15-18 months, all subjects previously primed for serogroups C/Y had hSBA ≥1:8 for these serogroups. At least 96.1% also had hSBA ≥1:8 for serogroups A/W. All subjects in the HibMenCY-TT group had hSBA ≥1:8 for serogroups C/Y. All pre-defined statistical criteria for meningococcal immunogenicity were satisfied. All vaccination regimens had acceptable safety profiles. Children primed with three doses of HibMenCY-TT who then received a single dose of MenACWY-TT or a fourth dose of HibMenCY-TT had robust increases in hSBA titers for serogroups C/Y. These data provide support that MenACWY-TT, given with or without the fourth scheduled dose of DTaP could be administered as an alternative to a fourth dose of HibMenCY-TT in the second year of life. This study (110870/110871) is registered at www.clinicaltrials.gov NCT00614614. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

A vitamin B12 conjugate of exendin-4 improves glucose tolerance without associated nausea or hypophagia in rodents.

PubMed

Mietlicki-Baase, Elizabeth G; Liberini, Claudia G; Workinger, Jayme L; Bonaccorso, Ron L; Borner, Tito; Reiner, David J; Koch-Laskowski, Kieran; McGrath, Lauren E; Lhamo, Rinzin; Stein, Lauren M; De Jonghe, Bart C; Holz, George G; Roth, Christian L; Doyle, Robert P; Hayes, Matthew R

2018-05-01

While pharmacological glucagon-like peptide-1 receptor (GLP-1R) agonists are FDA-approved for treating type 2 diabetes mellitus (T2DM) and obesity, a major side effect is nausea/malaise. We recently developed a conjugate of vitamin B12 (B12) bound to the GLP-1R agonist exendin-4 (Ex4), which displays enhanced proteolytic stability and retention of GLP-1R agonism. Here, we evaluate whether the conjugate (B12-Ex4) can improve glucose tolerance without producing anorexia and malaise. We evaluated the effects of systemic B12-Ex4 and unconjugated Ex4 on food intake and body weight change, oral glucose tolerance and nausea/malaise in male rats, and on intraperitoneal glucose tolerance in mice. To evaluate whether differences in the profile of effects of B12-Ex4 vs unconjugated Ex4 are the result of altered CNS penetrance, rats received systemic injections of fluorescein-Ex4 (Flex), Cy5-B12 or Cy5-B12-Ex4 and brain penetrance was evaluated using confocal microscopy. Uptake of systemically administered Cy5-B12-Ex4 in insulin-containing pancreatic beta cells was also examined. B12-Ex4 conjugate improves glucose tolerance, but does not elicit the malaise and anorexia produced by unconjugated Ex4. While Flex robustly penetrates into the brain (dorsal vagal complex, paraventricular hypothalamus), Cy5-B12 and Cy5-B12-Ex4 fluorescence were not observed centrally, supporting an absence of CNS penetrance, in line with observed reduction in CNS-associated Ex4 side effects. Cy5-B12-Ex4 colocalizes with insulin in the pancreas, suggesting direct pancreatic action as a potential mechanism underlying the hypoglycaemic effects of B12-Ex4. These novel findings highlight the potential clinical utility of B12-Ex4 conjugates as possible future T2DM therapeutics with reduced incidence of adverse effects. © 2018 John Wiley & Sons Ltd.

Evaluating the role of acidic, basic, and polar amino acids and dipeptides on a molecular electrocatalyst for H 2 oxidation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boralugodage, Nilusha Priyadarshani; Arachchige, Rajith Jayasingha; Dutta, Arnab

Amino acids and peptides have been shown to have a significant influence on the H2 production and oxidation reactivity of Ni(P R 2N R’ 2) 2, where P R 2N R’ 2 = 1,5-diaza-3,7-diphosphacyclooctane, R is either phenyl (Ph) or cyclohexyl (Cy), and R’ is either an amino acid or peptide. Most recently, the Ni(P Cy 2Naminoacid 2) 2 complexes (CyAA) have shown enhanced H 2 oxidation rates, water solubility, and in the case of arginine (CyArg) and phenylalanine (CyPhe), electrocatalytic reversibility. Both the backbone –COOH and side chain interactions were shown to be critical to catalytic performance. Here wemore » further investigate the roles of the outer coordination sphere by evaluating amino acids with acidic, basic, and hydrophilic side chains, as well as dipeptides which combine multiple successful features from previous complexes. Six new complexes were prepared, three containing single amino acids: aspartic acid (CyAsp), lysine (CyLys), and serine (CySer) and three containing dipeptides: glycine-phenylalanine (Cy(GlyPhe)), phenylalanine-glycine (Cy(PheGly)), and aspartic acid-phenylananine (Cy(AspPhe)). The resulting catalytic performance demonstrates that complexes need both interactions between side chain and –COOH groups for fast, efficient catalysis. The fastest of all of the catalysts, Cy(AspPhe), had both of these features, while the other dipeptide complexes with an amide replacing the -COOH were both slower; however, the amide group was demonstrated to participate in the proton pathway when side chain interactions are present to position it. Both the hydrophilic and basic side chains, notably lacking in side chain interactions, significantly increased the overpotential, with only modest increases in TOF. Of all of the complexes, only CyAsp was reversible at room temperature, and only in water, the first of these complexes to demonstrate room temperature reversibility in water. These results continue to provide and solidify design rules for controlling reactivity and efficiency of Ni(P 2N 2) 2 complexes with the outer coordination sphere.« less

Eosinophils, pruritus and psoriasis: effects of treatment with etretinate or cyclosporin-A.

PubMed

Schopf, R E; Hultsch, T; Lotz, J; Bräutigam, M

1998-11-01

The antipsoriatic drugs cyclosporin A (CyA) and etretinate have been found to influence proinflammatory eosinophilic leukocytes and pruritus. We compared the number of blood eosinophils, concentration of serum eosinophil cationic protein (ECP), and pruritus in patients with psoriasis treated with either CyA or etretinate. Patients with psoriasis vulgaris were randomly assigned to treatment for 10 weeks with either CyA (n = 21) or etretinate (n = 10). The psoriasis area-and-severity index (PASI-score) and pruritus (according to a 0-3 scale) served as clinical parameters, the blood esosinophil counts (Coulter Counter) and the serum ECP (RIA, Pharmacia) as laboratory parameters. After CyA treatment the PASI-score amounted to 24 +/- 4%, after etretinate to 56 +/- 6% of the initial values (mean +/- SEM). One week after CyA treatment, esosinophils dropped from 190 +/- 21 to 137 +/- 16/microliter (P = 0.038, Wilcoxon test), after 10 weeks to 127 +/- 18/microliter (P = 0.006). By contrast, under etretinate blood eosinophil counts only changed marginally. Before treatment, ECP concentrations of 15.71 +/- 1.30 (CyA) and 15.3 +/- 5.53 micrograms/l (etretinate) were measured (normal range 3-16 micrograms/l), ECP remained constant under both CyA and etretinate or tended to increase after 10 weeks; about 50% of the patients exhibited elevated ECP concentrations. Pruritus diminished more with CyA than etretinate therapy. PASI-scores and pruritus were directly proportional. We conclude that treatment of psoriasis with CyA leads to a rapid drop of blood eosinophils and that the activation state of eosinophils does not decrease after antipsoriatic treatment. Pruritus in psoriasis is coupled to disease severity. The underlying antipsoriatic mechanisms of CyA may be linked to lowering the number of blood eosinophils.

The Bank of Westminster and Hyland Park Construction Contracts as Engineering Student Classroom Projects; Construction Phase.

DTIC Science & Technology

1984-12-13

8217-- - - -- - - - - - ----- - - - - - - - .101 Clear at Building Clear&Grub @ Bldg. 2000.00 CY .50 1,0(,() Mass Bldg. Excav. 26000.00 CY 2.50 65.00, Grade Beam Excav. 2100.00 CY 4.00...95,112 Sail Investigation Compaction Tests 20.00 EA 150.00 3.000 Perimeter Drainage 2000.00 LF 16.00 32,00 Under Floor Drain LF Clean Walks /Street 1.00 LS...EA4 Equip. Curbs 3500.00 Sf 5.00 17.5:00 -. Grade Beams 7200.00 SF 11.50 82,800 Sumo Pits 1.00 EA 500.00 500 Cooling Tower Sump 600.00 SF 11.50 6,900

Identification of cellular and viral factors related to anti-hepatitis C virus activity of cyclophilin inhibitor.

PubMed

Goto, Kaku; Watashi, Koichi; Inoue, Daisuke; Hijikata, Makoto; Shimotohno, Kunitada

2009-10-01

We have so far reported that an immunosuppressant cyclosporin A (CsA), a well-known cyclophilin (CyP) inhibitor (CPI), strongly suppressed hepatitis C virus (HCV) replication in cell culture, and that CyPB was a cellular cofactor for viral replication. To further investigate antiviral mechanisms of CPI, we here developed cells carrying CsA-resistant HCV replicons, by culturing the HCV subgenomic replicon cells for 4 weeks in the presence of CsA with G418. Transfection of total RNA from the isolated CsA-resistant cells to naïve Huh7 cells conferred CsA resistance, suggesting that the replicon RNA itself was responsible for the resistant phenotype. Of the identified amino acid mutations, D320E in NS5A conferred the CsA resistance. The replicon carrying the D320E mutation was sensitive to interferon-alpha, but was resistant to CsA and other CPIs including NIM811 and sanglifehrin A. Knockdown of individual CyP subtypes revealed CyP40, in addition to CyPA and CyPB, contributed to viral replication, and CsA-resistant replicons acquired independence from CyPA for efficient replication. These data provide important evidence on the mechanisms underlying the regulation of HCV replication by CyP and for designing novel and specific anti-HCV strategies with CPIs.

Monitoring DNA triplex formation using multicolor fluorescence and application to insulin-like growth factor I promoter downregulation.

PubMed

Hégarat, Nadia; Novopashina, Darya; Fokina, Alesya A; Boutorine, Alexandre S; Venyaminova, Alya G; Praseuth, Danièle; François, Jean-Christophe

2014-03-01

Inhibition of insulin-like growth factor I (IGF-I) signaling is a promising antitumor strategy and nucleic acid-based approaches have been investigated to target genes in the pathway. Here, we sought to modulate IGF-I transcriptional activity using triple helix formation. The IGF-I P1 promoter contains a purine/pyrimidine (R/Y) sequence that is pivotal for transcription as determined by deletion analysis and can be targeted with a triplex-forming oligonucleotide (TFO). We designed modified purine- and pyrimidine-rich TFOs to bind to the R/Y sequence. To monitor TFO binding, we developed a fluorescence-based gel-retardation assay that allowed independent detection of each strand in three-stranded complexes using end-labeling with Alexa 488, cyanine (Cy)3 and Cy5 fluorochromes. We characterized TFOs for their ability to inhibit restriction enzyme activity, compete with DNA-binding proteins and inhibit IGF-I transcription in reporter assays. TFOs containing modified nucleobases, 5-methyl-2'-deoxycytidine and 5-propynyl-2'-deoxyuridine, specifically inhibited restriction enzyme cleavage and formed triplexes on the P1 promoter fragment. In cells, deletion of the R/Y-rich sequence led to 48% transcriptional inhibition of a reporter gene. Transfection with TFOs inhibited reporter gene activity to a similar extent, whereas transcription from a mutant construct with an interrupted R/Y region was unaffected, strongly suggesting the involvement of triplex formation in the inhibitory mechanisms. Our results indicate that nuclease-resistant TFOs will likely inhibit endogenous IGF-I gene function in cells. © 2014 FEBS.

Modeling Individual Cyclic Variation in Human Behavior.

PubMed

Pierson, Emma; Althoff, Tim; Leskovec, Jure

2018-04-01

Cycles are fundamental to human health and behavior. Examples include mood cycles, circadian rhythms, and the menstrual cycle. However, modeling cycles in time series data is challenging because in most cases the cycles are not labeled or directly observed and need to be inferred from multidimensional measurements taken over time. Here, we present Cyclic Hidden Markov Models (CyH-MMs) for detecting and modeling cycles in a collection of multidimensional heterogeneous time series data. In contrast to previous cycle modeling methods, CyHMMs deal with a number of challenges encountered in modeling real-world cycles: they can model multivariate data with both discrete and continuous dimensions; they explicitly model and are robust to missing data; and they can share information across individuals to accommodate variation both within and between individual time series. Experiments on synthetic and real-world health-tracking data demonstrate that CyHMMs infer cycle lengths more accurately than existing methods, with 58% lower error on simulated data and 63% lower error on real-world data compared to the best-performing baseline. CyHMMs can also perform functions which baselines cannot: they can model the progression of individual features/symptoms over the course of the cycle, identify the most variable features, and cluster individual time series into groups with distinct characteristics. Applying CyHMMs to two real-world health-tracking datasets-of human menstrual cycle symptoms and physical activity tracking data-yields important insights including which symptoms to expect at each point during the cycle. We also find that people fall into several groups with distinct cycle patterns, and that these groups differ along dimensions not provided to the model. For example, by modeling missing data in the menstrual cycles dataset, we are able to discover a medically relevant group of birth control users even though information on birth control is not given to the model.

Modeling Individual Cyclic Variation in Human Behavior

PubMed Central

Pierson, Emma; Althoff, Tim; Leskovec, Jure

2018-01-01

Cycles are fundamental to human health and behavior. Examples include mood cycles, circadian rhythms, and the menstrual cycle. However, modeling cycles in time series data is challenging because in most cases the cycles are not labeled or directly observed and need to be inferred from multidimensional measurements taken over time. Here, we present Cyclic Hidden Markov Models (CyH-MMs) for detecting and modeling cycles in a collection of multidimensional heterogeneous time series data. In contrast to previous cycle modeling methods, CyHMMs deal with a number of challenges encountered in modeling real-world cycles: they can model multivariate data with both discrete and continuous dimensions; they explicitly model and are robust to missing data; and they can share information across individuals to accommodate variation both within and between individual time series. Experiments on synthetic and real-world health-tracking data demonstrate that CyHMMs infer cycle lengths more accurately than existing methods, with 58% lower error on simulated data and 63% lower error on real-world data compared to the best-performing baseline. CyHMMs can also perform functions which baselines cannot: they can model the progression of individual features/symptoms over the course of the cycle, identify the most variable features, and cluster individual time series into groups with distinct characteristics. Applying CyHMMs to two real-world health-tracking datasets—of human menstrual cycle symptoms and physical activity tracking data—yields important insights including which symptoms to expect at each point during the cycle. We also find that people fall into several groups with distinct cycle patterns, and that these groups differ along dimensions not provided to the model. For example, by modeling missing data in the menstrual cycles dataset, we are able to discover a medically relevant group of birth control users even though information on birth control is not given to the model. PMID:29780976

Assessment of Collagen-Induced Arthritis Using Cyanine 5.5 Conjugated with Hydrophobically Modified Glycol Chitosan Nanoparticles: Correlation with 18F-Fluorodeoxyglucose Positron Emission Tomography Data

PubMed Central

Cha, Ji Hyeon; Lee, Sheen-Woo; Park, Kyeongsoon; Moon, Dae Hyuk; Kim, Kwangmeyung; Biswal, Sandip

2012-01-01

Objective To evaluate the potential and correlation between near-infrared fluorescence (NIRF) imaging using cyanine 5.5 conjugated with hydrophobically modified glycol chitosan nanoparticles (HGC-Cy5.5) and 18F-fluorodeoxyglucose-positron emission tomography (18F-FDG-PET) imaging of collagen-induced arthritis (CIA). Materials and Methods We used 10 CIA and 3 normal mice. Nine days after the injecting collagen twice, microPET imaging was performed 40 minutes after the intravenous injection of 9.3 MBq 18F-FDG in 200 µL PBS. One day later, NIRF imaging was performed two hours after the intravenous injection of HGC-cy5.5 (5 mg/kg). We assessed the correlation between these two modalities in the knees and ankles of CIA mice. Results The mean standardized uptake values of 18F-FDG for knees and ankles were 1.68 ± 0.76 and 0.79 ± 0.71, respectively, for CIA mice; and 0.57 ± 0.17 and 0.54 ± 0.20 respectively for control mice. From the NIRF images, the total photon counts per 30 mm2 for knees and ankles were 2.32 ± 1.54 × 105 and 2.75 ± 1.51 × 105, respectively, for CIA mice, and 1.22 ± 0.27 × 105 and 0.88 ± 0.24 × 105, respectively, for control mice. These two modalities showed a moderate correlation for knees (r = 0.604, p = 0.005) and ankles (r = 0.464, p = 0.039). Moreover, both HGC-Cy5.5 (p = 0.002) and 18F-FDG-PET (p = 0.005) imaging also showed statistically significant differences between CIA and normal mice. Conclusion NIRF imaging using HGC-Cy5.5 was moderately correlated with 18F-FDG-PET imaging in the CIA model. As such, HGC-Cy5.5 imaging can be used for the early detection of rheumatoid arthritis. PMID:22778567

Glutathione S-transferase P protects against cyclophosphamide-induced cardiotoxicity in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Conklin, Daniel J., E-mail: dj.conklin@louisville.edu; Institute of Molecular Cardiology, University of Louisville, Louisville, KY 40292; Haberzettl, Petra

2015-06-01

High-dose chemotherapy regimens using cyclophosphamide (CY) are frequently associated with cardiotoxicity that could lead to myocyte damage and congestive heart failure. However, the mechanisms regulating the cardiotoxic effects of CY remain unclear. Because CY is converted to an unsaturated aldehyde acrolein, a toxic, reactive CY metabolite that induces extensive protein modification and myocardial injury, we examined the role of glutathione S-transferase P (GSTP), an acrolein-metabolizing enzyme, in CY cardiotoxicity in wild-type (WT) and GSTP-null mice. Treatment with CY (100–300 mg/kg) increased plasma levels of creatine kinase-MB isoform (CK·MB) and heart-to-body weight ratio to a significantly greater extent in GSTP-null thanmore » WT mice. In addition to modest yet significant echocardiographic changes following acute CY-treatment, GSTP insufficiency was associated with greater phosphorylation of c-Jun and p38 as well as greater accumulation of albumin and protein–acrolein adducts in the heart. Mass spectrometric analysis revealed likely prominent modification of albumin, kallikrein-1-related peptidase, myoglobin and transgelin-2 by acrolein in the hearts of CY-treated mice. Treatment with acrolein (low dose, 1–5 mg/kg) also led to increased heart-to-body weight ratio and myocardial contractility changes. Acrolein induced similar hypotension in GSTP-null and WT mice. GSTP-null mice also were more susceptible than WT mice to mortality associated with high-dose acrolein (10–20 mg/kg). Collectively, these results suggest that CY cardiotoxicity is regulated, in part, by GSTP, which prevents CY toxicity by detoxifying acrolein. Thus, humans with low cardiac GSTP levels or polymorphic forms of GSTP with low acrolein-metabolizing capacity may be more sensitive to CY toxicity. - Graphical abstract: Cyclophosphamide (CY) treatment results in P450-mediated metabolic formation of phosphoramide mustard and acrolein (3-propenal). Acrolein is either metabolized and detoxified by glutathione S-transferase P (GSTP) via conjugation with GSH or acrolein can react with circulating and cardiac proteins to form protein–acrolein adducts that may contribute to cardiac injury, increased vascular permeability and edema and acute cardiotoxicity. Under low dose exposure, this event is reversible but at high levels of CY treatment and/or in susceptible individuals (e.g., hGSTP1 polymorphism), CY-induced cardiotoxicity is augmented and sudden death may occur. - Highlights: • Acute cardiotoxicity of cyclophosphamide (CY) is exacerbated in GSTP-null mice. • CY altered cardiac contractility, vascular leak and protein–acrolein adducts. • Cardiotoxicity of CY is recapitulated by acrolein only exposure. • Acrolein-induced cardiotoxicity and mortality is enhanced in male GSTP-null mice.« less

Amplified detection of cocaine based on strand-displacement polymerization and fluorescence resonance energy transfer.

PubMed

Huang, Jin; Chen, Yan; Yang, Liu; Zhu, Zhi; Zhu, Guizhi; Yang, Xiaohai; Wang, Kemin; Tan, Weihong

2011-10-15

Cocaine is one of the most abused drugs in the United States and is potentially dangerous when consumed in excess. Its detection is thus important in many areas in the fight against drug trafficking. We have developed an amplified detection method for cocaine based on a strand-displacement polymerization reaction using aptamer recognition. The system mainly consists of a hairpin probe with Cy5 labeled on its 3' end, a primer with FAM labeled on its 5' end, and polymerase. The aptamer sequence is integrated into the 5'-section of the hairpin probe. The primer is designed complementary to the 3' end of the hairpin probe, which is also part of the hairpin stem region. The cocaine induced reaction cycle generates product for detection and thus for signal amplification. The detection limit of this method is 200 nM in about 16 min and the specificity of this approach is excellent. We believe that this strategy will be useful for the development of analytical schemes for a variety of aptamers for small molecules, metal ions, and proteins. This simple scheme employing the strand-displacement polymerization reaction may find wide application in forensic analysis, environmental monitoring, and clinical diagnostics. Copyright © 2011 Elsevier B.V. All rights reserved.

A Paper-Based Sandwich Format Hybridization Assay for Unlabeled Nucleic Acid Detection Using Upconversion Nanoparticles as Energy Donors in Luminescence Resonance Energy Transfer.

PubMed

Zhou, Feng; Noor, M Omair; Krull, Ulrich J

2015-09-24

Bioassays based on cellulose paper substrates are gaining increasing popularity for the development of field portable and low-cost diagnostic applications. Herein, we report a paper-based nucleic acid hybridization assay using immobilized upconversion nanoparticles (UCNPs) as donors in luminescence resonance energy transfer (LRET). UCNPs with intense green emission served as donors with Cy3 dye as the acceptor. The avidin functionalized UCNPs were immobilized on cellulose paper and subsequently bioconjugated to biotinylated oligonucleotide probes. Introduction of unlabeled oligonucleotide targets resulted in a formation of probe-target duplexes. A subsequent hybridization of Cy3 labeled reporter with the remaining single stranded portion of target brought the Cy3 dye in close proximity to the UCNPs to trigger a LRET-sensitized emission from the acceptor dye. The hybridization assays provided a limit of detection (LOD) of 146.0 fmol and exhibited selectivity for one base pair mismatch discrimination. The assay was functional even in undiluted serum samples. This work embodies important progress in developing DNA hybridization assays on paper. Detection of unlabeled targets is achieved using UCNPs as LRET donors, with minimization of background signal from paper substrates owing to the implementation of low energy near-infrared (NIR) excitation.

[Cloning and expressing of cyclophilin B gene from Schistosoma japonnicum and the analysis of immunoprotective effect].

PubMed

Peng, Jinbiao; Han, Hongxiao; Hong, Yang; Wang, Yan; Guo, Fanji; Shi, Yaojun; Fu, Zhiqiang; Liu, Jinming; Cheng, Guofeng; Lin, Jiaojiao

2010-03-01

The present study was intend to clone and express the cDNA encoding Cyclophilin B (CyPB) of Schistosoma japonicum, its preliminary biological function and further immunoprotective effect against schistosome infection in mice. RT-PCR technique was applied to amplify a full-length cDNA encoding protein Cyclophilin B (Sj CyPB) from schistosomula cDNA. The expression profiles of Sj CyPB were determined by Real-time PCR using the template cDNAs isolated from 7, 13, 18, 23, 32 and 42 days parasites. The cDNA containing the Open Reading Frame of CyPB was then subcloned into a pGEX-6P-1 vector and transformed into competent Escherichia coli BL21 for expressing. The recombinant protein was renaturated, purified and its antigenicity were detected by Western blotting, and the immunoprotective effect induced by recombinant Sj CyPB was evaluated in Balb/C mice. The cDNA containing the ORF of Sj CyPB was cloned with the length of 672 base pairs, encoding 223 amino acids. Real-time PCR analysis revealed that the gene had the highest expression in 18-day schistosomula, suggesting that Sj CyPB was schistosomula differentially expressed gene. The recombinant protein showed a good antigenicity detected by Western blotting. Animal experiment indicated that the vaccination of recombinant CyPB protein in mice led to 31.5% worm and 41.01% liver egg burden reduction, respectively, compared with those of the control. A full-length cDNA differentially expressed in schistosomula was obtained. The recombinant Sj CyPB protein could induce partial protection against schistosome infection.

A Communication Protocol for CyAMS and the Cyber Fighter Associate Interface

DTIC Science & Technology

2015-05-01

by David Harman , Scott Brown, Brian Henz, and Lisa M Marvel Approved for public release; distribution unlimited...Laboratory A Communication Protocol for CyAMS and the Cyber Fighter Associate Interface by David Harman College Qualified Leaders Student...CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) David Harman , Scott Brown, Brian Henz, and Lisa M Marvel 5d. PROJECT

Rhodium-catalyzed asymmetric construction of quaternary carbon stereocenters: ligand-dependent regiocontrol in the 1,4-addition to substituted maleimides.

PubMed

Shintani, Ryo; Duan, Wei-Liang; Hayashi, Tamio

2006-05-03

A rhodium-catalyzed asymmetric 1,4-addition of arylboronic acids to substituted maleimides has been described. The regioselectivity in this reaction is controlled by the choice of ligand (dienes or bisphosphines), and 1,4-adducts with a quaternary stereocenter can be obtained with high regio- and enantioselectivity by the use of (R)-H8-binap.

An Antibody-Immobilized Silica Inverse Opal Nanostructure for Label-Free Optical Biosensors.

PubMed

Lee, Wang Sik; Kang, Taejoon; Kim, Shin-Hyun; Jeong, Jinyoung

2018-01-20

Three-dimensional SiO₂-based inverse opal (SiO₂-IO) nanostructures were prepared for use as biosensors. SiO₂-IO was fabricated by vertical deposition and calcination processes. Antibodies were immobilized on the surface of SiO₂-IO using 3-aminopropyl trimethoxysilane (APTMS), a succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester (NHS-PEG₄-maleimide) cross-linker, and protein G. The highly accessible surface and porous structure of SiO₂-IO were beneficial for capturing influenza viruses on the antibody-immobilized surfaces. Moreover, as the binding leads to the redshift of the reflectance peak, the influenza virus could be detected by simply monitoring the change in the reflectance spectrum without labeling. SiO₂-IO showed high sensitivity in the range of 10³-10⁵ plaque forming unit (PFU) and high specificity to the influenza A (H1N1) virus. Due to its structural and optical properties, SiO₂-IO is a promising material for the detection of the influenza virus. Our study provides a generalized sensing platform for biohazards as various sensing strategies can be employed through the surface functionalization of three-dimensional nanostructures.

Fate of Flumioxazin in Aquatic Plants: Two Algae (Pseudokirchneriella subcapitata, Synechococcus sp.), Duckweed (Lemna sp.), and Water Milfoil (Myriophyllum elatinoides).

PubMed

Ando, Daisuke; Fujisawa, Takuo; Katagi, Toshiyuki

2017-10-11

Flumioxazin separately 14 C-labeled at 1,2-positions of the tetrahydrophthalimide moiety or uniformly labeled at the phenyl ring was exposed to two algae and duckweed via the water layer and water milfoil via the water layer or bottom sediment for 14 days to investigate uptake and metabolic profiles in these aquatic plants. While 14 C-flumioxazin received immediate hydrolysis through maleimide ring opening and amide bond cleavage with its hydrolytic half-life of <1 day in both water and sediment, the 14 C-plant uptake was ≤4.7% of the applied radioactivity (%AR) with water exposure for all plants and 0.9%AR with sediment exposure for water milfoil. No 14 C-translocation between shoot/leaves and roots occurred in water milfoil. The components of 14 C residues in plants were common among the species, which were the above hydrolysates and their transformation products, that is, dicarboxylic acid derivative metabolized via hydroxylation at the double bond of the cyclohexene ring followed by sugar conjugation with its counterpart amine derivative via acid conjugations.

Detection of proteolytic activity by covalent tethering of fluorogenic substrates in zymogram gels.

PubMed

Deshmukh, Ameya A; Weist, Jessica L; Leight, Jennifer L

2018-05-01

Current zymographic techniques detect only a subset of known proteases due to the limited number of native proteins that have been optimized for incorporation into polyacrylamide gels. To address this limitation, we have developed a technique to covalently incorporate fluorescently labeled, protease-sensitive peptides using an azido-PEG3-maleimide crosslinker. Peptides incorporated into gels enabled measurement of MMP-2, -9, -14, and bacterial collagenase. Sensitivity analysis demonstrated that use of peptide functionalized gels could surpass detection limits of current techniques. Finally, electrophoresis of conditioned media from cultured cells resulted in the appearance of several proteolytic bands, some of which were undetectable by gelatin zymography. Taken together, these results demonstrate that covalent incorporation of fluorescent substrates can greatly expand the library of detectable proteases using zymographic techniques.

Claudin-4-targeted optical imaging detects pancreatic cancer and its precursor lesions.

PubMed

Neesse, Albrecht; Hahnenkamp, Anke; Griesmann, Heidi; Buchholz, Malte; Hahn, Stefan A; Maghnouj, Abdelouahid; Fendrich, Volker; Ring, Janine; Sipos, Bence; Tuveson, David A; Bremer, Christoph; Gress, Thomas M; Michl, Patrick

2013-07-01

Novel imaging methods based on specific molecular targets to detect both established neoplasms and their precursor lesions are highly desirable in cancer medicine. Previously, we identified claudin-4, an integral constituent of tight junctions, as highly expressed in various gastrointestinal tumours including pancreatic cancer. Here, we investigate the potential of targeting claudin-4 with a naturally occurring ligand to visualise pancreatic cancer and its precursor lesions in vitro and in vivo by near-infrared imaging approaches. A non-toxic C-terminal fragment of the claudin-4 ligand Clostridium perfringens enterotoxin (C-CPE) was labelled with a cyanine dye (Cy5.5). Binding of the optical tracer was analysed on claudin-4 positive and negative cells in vitro, and tumour xenografts in vivo. In addition, two genetically engineered mouse models for pancreatic intraepithelial neoplasia (PanIN) and pancreatic cancer were used for in vivo validation. Optical imaging studies were conducted using 2D planar fluorescence reflectance imaging (FRI) technology and 3D fluorescence-mediated tomography (FMT). In vitro, the peptide-dye conjugate showed high binding affinity to claudin-4 positive CAPAN1 cells, while claudin-4 negative HT1080 cells revealed little or no fluorescence. In vivo, claudin-4 positive tumour xenografts, endogenous pancreatic tumours, hepatic metastases, as well as preinvasive PanIN lesions, were visualised by FRI and FMT up to 48 h after injection showing a significantly higher average of fluorochrome concentration as compared with claudin-4 negative xenografts and normal pancreatic tissue. C-CPE-Cy5.5 combined with novel optical imaging methods enables non-invasive visualisation of claudin-4 positive murine pancreatic tumours and their precursor lesions, representing a promising modality for early diagnostic imaging.

Eicosapentaenoic acid production from Nannochloropsis oceanica CY2 using deep sea water in outdoor plastic-bag type photobioreactors.

PubMed

Chen, Chun-Yen; Nagarajan, Dillirani; Cheah, Wai Yan

2018-04-01

In this study, Nannochloropsis oceanica CY2 was grown in deep-sea water (DSW)-based medium in 5-L plastic bag-type photobioreactors (PBRs) for the autotrophic production of Eicosapentaenoic acid (EPA, 20:5n-3). EPA production of N. oceanica CY2 was stimulated when it was grown in 100% DSW amended with 1.5 g L -1 NaNO 3 , achieving a EPA content of 3.1% and a biomass concentration of 3.3 g L -1 . An outdoor-simulated microalgae cultivation system was also conducted to validate the feasibility of outdoor cultivation of the CY2 strain in plastic bag-type PBRs. Using an inoculum size of 0.6 g/L, the biomass concentration in the PBR culture was 3.5 g L -1 , while the EPA content and productivity reached a maximal level of 4.12% and 7.49 mg L -1  d -1 , respectively. When the PBRs were operated on semi-batch mode, the EPA productivity could further increase to 9.9 mg L -1  d -1 with a stable EPA content of 4.1%. Copyright © 2017 Elsevier Ltd. All rights reserved.

Dynamic analysis of the blood-brain barrier disruption in experimental stroke using time domain in vivo fluorescence imaging.

PubMed

Abulrob, Abedelnasser; Brunette, Eric; Slinn, Jacqueline; Baumann, Ewa; Stanimirovic, Danica

2008-01-01

The blood-brain barrier (BBB) disruption following cerebral ischemia can be exploited to deliver imaging agents and therapeutics into the brain. The aim of this study was (a) to establish novel in vivo optical imaging methods for longitudinal assessment of the BBB disruption and (b) to assess size selectivity and temporal patterns of the BBB disruption after a transient focal ischemia. The BBB permeability was assessed using in vivo time domain near-infrared optical imaging after contrast enhancement with either free Cy5.5 (1 kDa) or Cy5.5 conjugated with bovine serum albumin (BSA) (67 kDa) in mice subjected to either 60- or 20-minute transient middle cerebral artery occlusion (MCAO) and various times of reperfusion (up to 14 days). In vivo imaging observations were corroborated by ex vivo brain imaging and microscopic analyses of fluorescent tracer extravasation. The in vivo optical contrast enhancement with Cy5.5 was spatially larger than that observed with BSA-Cy5.5. Longitudinal studies after a transient 20-minute MCAO suggested a bilateral BBB disruption, more pronounced in the ipsilateral hemisphere, peaking at day 7 and resolving at day 14 after ischemia. The area differential between the BBB disruption for small and large molecules could potentially be useful as a surrogate imaging marker for assessing perinfarct tissues to which neuroprotective therapies of appropriate sizes could be delivered.

Design and application of fiber optic evanescent wave biosensor

NASA Astrophysics Data System (ADS)

Huang, Huijie; Zhai, Junhui; Zhao, Yongkai; Yang, Ruifu; Ren, Bingqiang; Cheng, Zhaogu; Du, Longlong; Lu, Dunwu

2003-12-01

A fiber-optic biosensor is developed based on the principle of evanescent wave while light propagates in optical fiber. The biosensor uses a red laser diode at 636.85 nm for exciting Cy5 fluorescent dye. Sensitivity limit of 0.01 nnmol/l is obtained from the detection of serial Cy5 solutions with various concentrations. In log-to-log plot, excellent linear response characteristic is seen in the Cy5 concentrations ranging from 0.01 nmlo/l to 100 nmol/l. And a good result of signal-to-noise ratio of 4.61 is obtained when the biosensor is used to measure Legionella pneumophila solution of 0.01 μmol/l. All the results are comparable with those that are obtained by a commercial biochip scanner GeneTAC 1000.

Catalytic hydrogenation using complexes of base metals with tridentate ligands

DOEpatents

Hanson, Susan K.; Zhang, Guoqi; Vasudevan, Kalyan V.

2017-02-14

Complexes of cobalt and nickel with tridentate ligand PNHP.sup.R are effective for hydrogenation of unsaturated compounds. Cobalt complex [(PNHP.sup.Cy)Co(CH.sub.2SiMe.sub.3)]BAr.sup.F.sub.4 (PNHP.sup.Cy=bis[2-(dicyclohexylphosphino)ethyl]amine, BAr.sup.F.sub.4=B(3,5-(CF.sub.3).sub.2C.sub.6H.sub.3).sub.4)) was prepared and used with hydrogen for hydrogenation of alkenes, aldehydes, ketones, and imines under mild conditions (25-60.degree. C., 1-4 atm H.sub.2). Nickel complex [(PNHP.sup.Cy)Ni(H)]BPh.sub.4 was used for hydrogenation of styrene and 1-octene under mild conditions. (PNP.sup.Cy)Ni(H) was used for hydrogenating alkenes.

Catalytic hydrogenation using complexes of base metals with tridentate ligands

DOEpatents

Vasudevan, Kalyan V.; Zhang, Guoqi; Hanson, Susan K.

2016-09-06

Complexes of cobalt and nickel with tridentate ligand PNHP.sup.R are effective for hydrogenation of unsaturated compounds. Cobalt complex [(PNHP.sup.Cy)Co(CH.sub.2SiMe.sub.3)]BAr.sup.F.sub.4 (PNHP.sup.Cy=bis[2-(dicyclohexylphosphino)ethyl]amine, BAr.sup.F.sub.4=B(3,5-(CF.sub.3).sub.2C.sub.6H.sub.3).sub.4)) was prepared and used with hydrogen for hydrogenation of alkenes, aldehydes, ketones, and imines under mild conditions (25-60.degree. C., 1-4 atm H.sub.2). Nickel complex [(PNHP.sup.Cy)Ni(H)]BPh.sub.4 was used for hydrogenation of styrene and 1-octene under mild conditions. (PNP.sup.Cy)Ni(H) was used for hydrogenating alkenes.

Targeted Therapies for Myeloma and Metastatic Bone Cancers

DTIC Science & Technology

2010-09-01

to produce NPs for in-vivo studies compatible with the short half life of Tc99m. (Fig 6,7) Developed methods to radiolabel polymer nanoparticles...fully characterize PLA-b-PEG-Maleimide block Polymer (PLA-b-PEG-MAL) Propose: To synthesize Maleimide modified PLGA-b-PEG 2000 for NPs bone-targeted... polymer was synthesized and characterized by H-NMR. (Appendix 2) Improved lyostability of polymer nanoparticles, with and without PEG modification

Improved Dye Stability in Single-Molecule Fluorescence Experiments

NASA Astrophysics Data System (ADS)

EcheverrÍa Aitken, Colin; Marshall, R. Andrew; Pugi, Joseph D.

Complex biological systems challenge existing single-molecule methods. In particular, dye stability limits observation time in singlemolecule fluorescence applications. Current approaches to improving dye performance involve the addition of enzymatic oxygen scavenging systems and small molecule additives. We present an enzymatic oxygen scavenging system that improves dye stability in single-molecule experiments. Compared to the currently-employed glucose-oxidase/catalase system, the protocatechuate-3,4-dioxygenase system achieves lower dissolved oxygen concentration and stabilizes single Cy3, Cy5, and Alexa488 fluorophores. Moreover, this system possesses none of the limitations associated with the glucose oxidase/catalase system. We also tested the effects of small molecule additives in this system. Biological reducing agents significantly destabilize the Cy5 fluorophore as a function of reducing potential. In contrast, anti-oxidants stabilize the Cy3 and Alexa488 fluorophores. We recommend use of the protocatechuate-3,4,-dioxygenase system with antioxidant additives, and in the absence of biological reducing agents. This system should have wide application to single-molecule fluorescence experiments.

Probing the Orientation of Surface-Immobilized Protein G B1 Using ToF-SIMS Sum Frequency Generation and NEXAFS Spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

L Baugh; T Weidner; J Baio

2011-12-31

The ability to orient active proteins on surfaces is a critical aspect of many medical technologies. An important related challenge is characterizing protein orientation in these surface films. This study uses a combination of time-of-flight secondary ion mass spectrometry (ToF-SIMS), sum frequency generation (SFG) vibrational spectroscopy, and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy to characterize the orientation of surface-immobilized Protein G B1, a rigid 6 kDa domain that binds the Fc fragment of IgG. Two Protein G B1 variants with a single cysteine introduced at either end were immobilized via the cysteine thiol onto maleimide-oligo(ethylene glycol)-functionalized gold and baremore » gold substrates. X-ray photoelectron spectroscopy was used to measure the amount of immobilized protein, and ToF-SIMS was used to measure the amino acid composition of the exposed surface of the protein films and to confirm covalent attachment of protein thiol to the substrate maleimide groups. SFG and NEXAFS were used to characterize the ordering and orientation of peptide or side chain bonds. On both substrates and for both cysteine positions, ToF-SIMS data showed enrichment of mass peaks from amino acids located at the end of the protein opposite to the cysteine surface position as compared with nonspecifically immobilized protein, indicating end-on protein orientations. Orientation on the maleimide substrate was enhanced by increasing pH (7.0-9.5) and salt concentration (0-1.5 M NaCl). SFG spectral peaks characteristic of ordered {alpha}-helix and {beta}-sheet elements were observed for both variants but not for cysteine-free wild type protein on the maleimide surface. The phase of the {alpha}-helix and {beta}-sheet peaks indicated a predominantly upright orientation for both variants, consistent with an end-on protein binding configuration. Polarization dependence of the NEXAFS signal from the N 1s to {pi}* transition of {beta}-sheet peptide bonds also indicated protein ordering, with an estimated tilt angle of inner {beta}-strands of 40-50{sup o} for both variants (one variant more tilted than the other), consistent with SFG results. The combined results demonstrate the power of using complementary techniques to probe protein orientation on surfaces.« less

Immunogenicity and safety of an investigational combined haemophilus influenzae type B-Neisseria meningitidis serogroups C and Y-tetanus toxoid conjugate vaccine.

PubMed

Nolan, Terry; Richmond, Peter; Marshall, Helen; McVernon, Jodie; Alexander, Karyn; Mesaros, Narcisa; Aris, Emmanuel; Miller, Jacqueline; Poolman, Jan; Boutriau, Dominique

2011-03-01

Neisseria meningitidis serogroups B, C, and Y cause most meningococcal disease in industrialized countries. A Haemophilus influenzae type b-meningococcal serogroups C and Y-tetanus toxoid conjugate vaccine (HibMenCY-TT) was evaluated. A total of 1104 infants (randomized 3:1:1) were vaccinated at 2, 4, and 6 months with HibMenCY-TT, MenC-CRM197 + Hib-TT, or Hib-TT. At 12 to 15 months, HibMenCY-TT and MenC-CRM-primed children received HibMenCY-TT; Hib-TT-primed received N. meningitidis serogroup B Hib-outer membrane protein complex. Antibody concentrations and rabbit/human complement serum bactericidal antibody titers (rSBA/hSBA) were determined. Safety was monitored after each dose (diary cards for first 31 days) until 6 months postdose 4. Postdose 3, rates of antipolyribosylribitol phosphate ≥ 1 μg/mL and rSBA-MenC ≥1:128 in HibMenCY-TT recipients were noninferior to licensed controls. Percentages reaching 0.15 μg/mL (1.0 μg/mL postdose 3) and antipolyribosylribitol phosphate GMC were significantly higher after HibMenCY-TT than Hib-TT postdose 2 and postdose 3. The GMC remained significantly higher before and after dose 4. Proportions of HibMenCY-TT recipients with rSBA ≥ 1:8 were 95.6% (MenC), 98.6% (MenY) postdose-2, ≥ 99% for MenC/Y postdose 3 and 4; hSBA ≥ 1:4 were 95.5% (MenC), 89.8% (MenY) postdose 2, >97% for MenC/Y postdose 3 and 4. HibMenCY-TT had a similar safety profile to control vaccines. HibMenCY-TT induced noninferior Hib and MenC responses compared with monovalent Hib and MenC conjugates with a comparable safety profile. Bactericidal antibodies against MenC/Y were induced after 2 doses of HibMenCY-TT.

Decrease in Plasma Cyclophilin A Concentration at 1 Month after Myocardial Infarction Predicts Better Left Ventricular Performance and Synchronicity at 6 Months: A Pilot Study in Patients with ST Elevation Myocardial Infarction

PubMed Central

Huang, Ching-Hui; Chang, Chia-Chu; Kuo, Chen-Ling; Huang, Ching-Shan; Lin, Chih-Sheng; Liu, Chin-San

2015-01-01

Background: Cyclophilin A (CyPA) concentration increases in acute coronary syndrome. In an animal model of acute myocardial infarction, administration of angiotensin-converting-enzyme inhibitor was associated with lower left ventricular (LV) CyPA concentration and improved LV performance. This study investigated the relationships between changes in plasma CyPA concentrations and LV remodeling in patients with ST-elevation myocardial infarction (STEMI). Methods and Results: We enrolled 55 patients who underwent percutaneous coronary intervention for acute STEMI. Plasma CyPA, matrix metalloproteinase (MMP), interleukin-6 and high-sensitivity C-reactive protein concentrations were measured at baseline and at one-month follow-up. Echocardiography was performed at baseline and at one-, three-, and six-month follow-up. Patients with a decrease in baseline CyPA concentration at one-month follow-up (n = 28) had a significant increase in LV ejection fraction (LVEF) (from 60.2 ± 11.5% to 64.6 ± 9.9%, p < 0. 001) and preserved LV synchrony at six months. Patients without a decrease in CyPA concentration at one month (n = 27) did not show improvement in LVEF and had a significantly increased systolic dyssynchrony index (SDI) (from 1.170 ± 0.510% to 1.637 ± 1.299%, p = 0.042) at six months. Multiple linear regression analysis showed a significant association between one-month CyPA concentration and six-month LVEF. The one-month MMP-2 concentration was positively correlated with one-month CyPA concentration and LV SDI. Conclusions: Decreased CyPA concentration at one-month follow-up after STEMI was associated with better LVEF and SDI at six months. Changes in CyPA, therefore, may be a prognosticator of patient outcome. PMID:25552928

The effect of synthetic ceramide analogues on gastritis and esophagitis in rats.

PubMed

Kim, Sung Hyo; Um, Seung In; Nam, Yoonjin; Park, Sun Young; Dong, Je Hyun; Ko, Sung Kwon; Sohn, Uy Dong; Lee, Sang Joon

2016-09-01

The effects of ceremide analogues on esophagitis and gastritis in rats were examined. Gastritis induced by indomethacin was significantly reduced after CY3325 and CY3723 treatment, whereas other analogues had no effect. The amount of malondialdehyde in gastritis was significantly reduced by CY3325 or CY 3723. CY3325 or CY 3723 decreased the glutathione levels in gastritis. The myeloperoxidase level in gastritis is increased, and its increment was decreased by CY3325 and CY3723. In reflux esophagitis, the ulceration was decreased by CY3325, CY3723. The gastric volume and acid output are reduced, whereas the pH value is increased by CY3325 or CY3723 after esophagitis. These results suggest that ceramide analogues, CY3325 and CY3723, can prevent the development of gastritis and reflux esophagitis in rats.

Cryopreservation of MHC multimers: Recommendations for quality assurance in detection of antigen specific T cells.

PubMed

Hadrup, Sine Reker; Maurer, Dominik; Laske, Karoline; Frøsig, Thomas Mørch; Andersen, Sofie Ramskov; Britten, Cedrik M; van der Burg, Sjoerd H; Walter, Steffen; Gouttefangeas, Cécile

2015-01-01

Fluorescence-labeled peptide-MHC class I multimers serve as ideal tools for the detection of antigen-specific T cells by flow cytometry, enabling functional and phenotypical characterization of specific T cells at the single cell level. While this technique offers a number of unique advantages, MHC multimer reagents can be difficult to handle in terms of stability and quality assurance. The stability of a given fluorescence-labeled MHC multimer complex depends on both the stability of the peptide-MHC complex itself and the stability of the fluorochrome. Consequently, stability is difficult to predict and long-term storage is generally not recommended. We investigated here the possibility of cryopreserving MHC multimers, both in-house produced and commercially available, using a wide range of peptide-MHC class I multimers comprising virus and cancer-associated epitopes of different affinities presented by various HLA-class I molecules. Cryopreservation of MHC multimers was feasible for at least 6 months, when they were dissolved in buffer containing 5-16% glycerol (v/v) and 0.5% serum albumin (w/v). The addition of cryoprotectants was tolerated across three different T-cell staining protocols for all fluorescence labels tested (PE, APC, PE-Cy7 and Quantum dots). We propose cryopreservation as an easily implementable method for stable storage of MHC multimers and recommend the use of cryopreservation in long-term immunomonitoring projects, thereby eliminating the variability introduced by different batches and inconsistent stability. © 2014 International Society for Advancement of Cytometry.

Cryopreservation of MHC Multimers: Recommendations for Quality Assurance in Detection of Antigen Specific T Cells

PubMed Central

Hadrup, Sine Reker; Maurer, Dominik; Laske, Karoline; Frøsig, Thomas Mørch; Andersen, Sofie Ramskov; Britten, Cedrik M; van der Burg, Sjoerd H; Walter, Steffen; Gouttefangeas, Cécile

2015-01-01

Fluorescence-labeled peptide-MHC class I multimers serve as ideal tools for the detection of antigen-specific T cells by flow cytometry, enabling functional and phenotypical characterization of specific T cells at the single cell level. While this technique offers a number of unique advantages, MHC multimer reagents can be difficult to handle in terms of stability and quality assurance. The stability of a given fluorescence-labeled MHC multimer complex depends on both the stability of the peptide-MHC complex itself and the stability of the fluorochrome. Consequently, stability is difficult to predict and long-term storage is generally not recommended. We investigated here the possibility of cryopreserving MHC multimers, both in-house produced and commercially available, using a wide range of peptide-MHC class I multimers comprising virus and cancer-associated epitopes of different affinities presented by various HLA-class I molecules. Cryopreservation of MHC multimers was feasible for at least 6 months, when they were dissolved in buffer containing 5–16% glycerol (v/v) and 0.5% serum albumin (w/v). The addition of cryoprotectants was tolerated across three different T-cell staining protocols for all fluorescence labels tested (PE, APC, PE-Cy7 and Quantum dots). We propose cryopreservation as an easily implementable method for stable storage of MHC multimers and recommend the use of cryopreservation in long-term immunomonitoring projects, thereby eliminating the variability introduced by different batches and inconsistent stability. © 2014 International Society for Advancement of Cytometry PMID:25297339

Fluorescence turn-on detection of target sequence DNA based on silicon nanodot-mediated quenching.

PubMed

Zhang, Yanan; Ning, Xinping; Mao, Guobin; Ji, Xinghu; He, Zhike

2018-05-01

We have developed a new enzyme-free method for target sequence DNA detection based on the dynamic quenching of fluorescent silicon nanodots (SiNDs) toward Cy5-tagged DNA probe. Fascinatingly, the water-soluble SiNDs can quench the fluorescence of cyanine (Cy5) in Cy5-tagged DNA probe in homogeneous solution, and the fluorescence of Cy5-tagged DNA probe can be restored in the presence of target sequence DNA (the synthetic target miRNA-27a). Based on this phenomenon, a SiND-featured fluorescent sensor has been constructed for "turn-on" detection of the synthetic target miRNA-27a for the first time. This newly developed approach possesses the merits of low cost, simple design, and convenient operation since no enzymatic reaction, toxic reagents, or separation procedures are involved. The established method achieves a detection limit of 0.16 nM, and the relative standard deviation of this method is 9% (1 nM, n = 5). The linear range is 0.5-20 nM, and the recoveries in spiked human fluids are in the range of 90-122%. This protocol provides a new tactic in the development of the nonenzymic miRNA biosensors and opens a promising avenue for early diagnosis of miRNA-associated disease. Graphical abstract The SiND-based fluorescent sensor for detection of S-miR-27a.

Digital detection of multiple minority mutants in stool DNA for noninvasive colorectal cancer diagnosis.

PubMed

Deng, Lili; Qi, Zongtai; Zou, Binjie; Wu, Haiping; Huang, Huan; Kajiyama, Tomoharu; Kambara, Hideki; Zhou, Guohua

2012-07-03

Somatic mutations in stool DNA are quite specific to colorectal cancer (CRC), but a method being able to detect the extraordinarily low amounts of mutants is challengeable in sensitivity. We proposed a hydrogel bead-array to digitally count CRC-specific mutants in stool at a low cost. At first, multiplex amplification of targets containing multiple mutation loci of interest is carried out by a target enriched multiplex PCR (Tem-PCR), yielding the templates qualified for emulsion PCR (emPCR). Then, after immobilizing the beads from emPCR on a glass surface, the incorporation of Cy3-dUTP into the mutant-specific probes, which are specifically hybridized with the amplified beads from emPCR, is used to color the beads coated with mutants. As all amplified beads are hybridized with the Cy5-labeled universal probe, a mutation rate is readily obtained by digitally counting the beads with different colors (yellow and red). A high specificity of the method is achieved by removing the mismatched probes in a bead-array with electrophoresis. The approach has been used to simultaneously detect 8 mutation loci within the APC, TP53, and KRAS genes in stools from eight CRC patients, and 50% of CRC patients were positively diagnosed; therefore, our method can be a potential tool for the noninvasive diagnosis of CRC.

Structure of adducts of isoindolo[2,1-a]benzimidazole derivatives with maleimides

NASA Astrophysics Data System (ADS)

Korolev, Oleksandr; Yegorova, Tatyana; Levkov, Igor; Malytskyy, Volodymyr; Shishkin, Oleg; Zubatyuk, Roman; Palamarchuk, Genadiy; Vedrenne, Marc; Baltas, Michel; Voitenko, Zoia

2015-03-01

The selectivity of formation and some mechanistic insights during the synthesis of substituted isoindolo[2,1-a]benzimidazoles are discussed. Furthermore, the reactions of the obtained products with maleimides were carried out. Two types rearrangement adducts together with intermediate Michael type adducts were isolated. The influence of the reaction conditions and reagents ratio is discussed. Specific spectral criteria for the identification of the Michael type adducts are indicated.

Eosin Y-catalyzed visible-light-mediated aerobic oxidative cyclization of N,N-dimethylanilines with maleimides.

PubMed

Liang, Zhongwei; Xu, Song; Tian, Wenyan; Zhang, Ronghua

2015-01-01

A novel and simple strategy for the efficient synthesis of the corresponding tetrahydroquinolines from N,N-dimethylanilines and maleimides using visible light in an air atmosphere in the presence of Eosin Y as a photocatalyst has been developed. The metal-free protocol involves aerobic oxidative cyclization via sp(3) C-H bond functionalization process to afford good yields in a one-pot procedure under mild conditions.

"Clickable" Polymeric Nanofibers through Hydrophilic-Hydrophobic Balance: Fabrication of Robust Biomolecular Immobilization Platforms.

PubMed

Kalaoglu-Altan, Ozlem I; Sanyal, Rana; Sanyal, Amitav

2015-05-11

Fabrication of hydrophilic polymeric nanofibers that undergo facile and selective functionalization through metal catalyst-free Diels-Alder "click" reaction in aqueous environment is outlined. Electrospinning of copolymers containing an electron-rich furan moiety, hydrophobic methyl methacrylate units and hydrophilic poly(ethylene glycol)s as side chains provide specifically functionalizable yet antibiofouling fibers that remain stable in aqueous media due to appropriate hydrophobic hydrophilic balance. Efficient functionalization of these nanofibers is accomplished through the Diels-Alder reaction by exposing them to maleimide-containing molecules and ligands. Diels-Alder conjugation based functionalization is demonstrated through attachment of fluorescein-maleimide and a maleimide tethered biotin ligand. Biotinylated nanofibers were utilized to mediate immobilization of the protein streptavidin, as well as streptavidin coated quantum dots. Facile fabrication from readily available polymers and their effective functionalization under mild and reagent-free conditions in aqueous media make these "clickable" nanofibers attractive candidates as functionalizable scaffolds for various biomedical applications.

Combining Orthogonal Chain-End Deprotections and Thiol-Maleimide Michael Coupling: Engineering Discrete Oligomers by an Iterative Growth Strategy.

PubMed

Huang, Zhihao; Zhao, Junfei; Wang, Zimu; Meng, Fanying; Ding, Kunshan; Pan, Xiangqiang; Zhou, Nianchen; Li, Xiaopeng; Zhang, Zhengbiao; Zhu, Xiulin

2017-10-23

Orthogonal maleimide and thiol deprotections were combined with thiol-maleimide coupling to synthesize discrete oligomers/macromolecules on a gram scale with molecular weights up to 27.4 kDa (128mer, 7.9 g) using an iterative exponential growth strategy with a degree of polymerization (DP) of 2 n -1. Using the same chemistry, a "readable" sequence-defined oligomer and a discrete cyclic topology were also created. Furthermore, uniform dendrons were fabricated using sequential growth (DP=2 n -1) or double exponential dendrimer growth approaches (DP=22n -1) with significantly accelerated growth rates. A versatile, efficient, and metal-free method for construction of discrete oligomers with tailored structures and a high growth rate would greatly facilitate research into the structure-property relationships of sophisticated polymeric materials. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cytochrome P450 2B6*5 Increases Relapse after Cyclophosphamide-containing Conditioning and Autologous Transplantation for Lymphoma

PubMed Central

Bachanova, Veronika; Shanley, Ryan; Malik, Farhana; Chauhan, Lata; Lamba, Vishal; Weisdorf, Daniel J.; Burns, Linda J.; Lamba, Jatinder Kaur

2016-01-01

Cyclophosphamide (Cy) is a prodrug that depends on bioactivation by hepatic cytochrome P450 (CYP) enzymes for its cytotoxicity. We evaluated the influence of single nucleotide polymorphisms (SNPs) of CYP enzymes on the efficacy of autologous hematopoietic cell transplantation (HCT) for lymphoma. SNPs of 22 genes were analyzed in 93 patients with Hodgkin (n=52) and non-Hodgkin lymphoma (n=41) treated with high-dose Cy followed by autologous HCT between 2004–2012. Preparative regimens contained Cy (120mg/kg) combined with carmustine/etoposide (n=61) or Cy (6000mg/m2) with total body irradiation (n=32). Lack of complete remission as measured by pre-transplant positron emission tomography was the sole clinical factor associated with increased risk of relapse (HR 2.1). In genomic analysis, we identified a single SNP rs3211371 in exon 9 (C >T) of the CYP2B6 gene (allele designation 2B6*5) that significantly impacted patient outcomes. After adjusting for disease status and conditioning regimen, patients with CYP2B6*1/*5 genotype had a higher 2-year relapse rate (HR 3.3; 95%CI 1.6–6.5; p=0.041) and decreased overall survival (HR 13.5; 95%CI 3.5–51.9; p=0.008) than patients with wild-type allele. Patients with two hypo-functional CYP2B6 variant genotypes, *5 and *6, experienced 2-year PFS of only 11% (95%CI 1–39%) compared to 67% (95% CI 55–77%) for patients with the wild-type CYP2B6*1 allele in exon 9. Our results suggest that CYP2B6 SNPs influence the efficacy of high-dose Cy and significantly reduce the success of autologous HCT for lymphoma patients with the CYP2B6*5 variant. PMID:25677220

Receptor-mediated transcytosis of cyclophilin B through the blood-brain barrier.

PubMed

Carpentier, M; Descamps, L; Allain, F; Denys, A; Durieux, S; Fenart, L; Kieda, C; Cecchelli, R; Spik, G

1999-07-01

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein mainly located in intracellular vesicles and secreted in biological fluids. In previous works, we demonstrated that CyPB interacts with T lymphocytes and enhances in vitro cellular incorporation and activity of CsA. In addition to its immunosuppressive activity, CsA is able to promote regeneration of damaged peripheral nerves. However, the crossing of the drug from plasma to neural tissue is restricted by the relative impermeability of the blood-brain barrier. To know whether CyPB might also participate in the delivery of CsA into the brain, we have analyzed the interactions of CyPB with brain capillary endothelial cells. First, we demonstrated that CyPB binds to two types of binding sites present at the surface of capillary endothelial cells from various species of tissues. The first type of binding sites (K(D) = 300 nM; number of sites = 3 x 10(6)) is related to interactions with negatively charged compounds such as proteoglycans. The second type of binding sites, approximately 50,000 per cell, exhibits a higher affinity for CyPB (K(D) = 15 nM) and is involved in an endocytosis process, indicating it might correspond to a functional receptor. Finally, the use of an in vitro model of blood-brain barrier allowed us to demonstrate that CyPB is transcytosed by a receptor-mediated pathway (flux = 16.5 fmol/cm2/h). In these conditions, CyPB did not significantly modify the passage of CsA, indicating that it is unlikely to provide a pathway for CsA brain delivery.

Investigation of injection dose and camera integration time on quantifying pharmacokinetics of a Cy5.5-GX1 probe with dynamic fluorescence imaging in vivo

NASA Astrophysics Data System (ADS)

Dai, Yunpeng; Chen, Xueli; Yin, Jipeng; Kang, Xiaoyu; Wang, Guodong; Zhang, Xianghan; Nie, Yongzhan; Wu, Kaichun; Liang, Jimin

2016-08-01

The aim of this article is to investigate the influence of a tracer injection dose (ID) and camera integration time (IT) on quantifying pharmacokinetics of Cy5.5-GX1 in gastric cancer BGC-823 cell xenografted mice. Based on three factors, including whether or not to inject free GX1, the ID of Cy5.5-GX1, and the camera IT, 32 mice were randomly divided into eight groups and received 60-min dynamic fluorescence imaging. Gurfinkel exponential model (GEXPM) and Lammertsma simplified reference tissue model (SRTM) combined with a singular value decomposition analysis were used to quantitatively analyze the acquired dynamic fluorescent images. The binding potential (Bp) and the sum of the pharmacokinetic rate constants (SKRC) of Cy5.5-GX1 were determined by the SRTM and EXPM, respectively. In the tumor region, the SKRC value exhibited an obvious trend with change in the tracer ID, but the Bp value was not sensitive to it. Both the Bp and SKRC values were independent of the camera IT. In addition, the ratio of the tumor-to-muscle region was correlated with the camera IT but was independent of the tracer ID. Dynamic fluorescence imaging in conjunction with a kinetic analysis may provide more quantitative information than static fluorescence imaging, especially for a priori information on the optimal ID of targeted probes for individual therapy.

Facile and Stabile Linkages through Tyrosine: Bioconjugation Strategies with the Tyrosine-Click Reaction

PubMed Central

Ban, Hitoshi; Nagano, Masanobu; Gavrilyuk, Julia; Hakamata, Wataru; Inokuma, Tsubasa; Barbas, Carlos F.

2013-01-01

The scope, chemoselectivity, and utility of the click-like tyrosine labeling reaction with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs) is reported. To study the utility and chemoselectivity of PTAD derivatives in peptide and protein chemistry, we synthesized PTAD derivatives possessing azide, alkyne, and ketone groups and studied their reactions with amino acid derivatives and peptides of increasing complexity. With proteins we studied the compatibility of the tyrosine click reaction with cysteine and lysine-targeted labeling approaches and demonstrate that chemoselective tri-functionalization of proteins is readily achieved. In particular cases, we noted PTAD decomposition resulted in formation of a putative isocyanate by-product that was promiscuous in labeling. This side reaction product, however, was readily scavenged by the addition of a small amount of 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) to the reaction medium. To study the potential of the tyrosine click reaction to introduce poly(ethylene) glycol chains onto proteins (PEGylation), we demonstrate that this novel reagent provides for the selective PEGylation of chymotrypsinogen whereas traditional succinimide-based PEGylation targeting lysine residues provided a more diverse range of PEGylated products. Finally, we applied the tyrosine click reaction to create a novel antibody drug conjugate. For this purpose, we synthesized a PTAD derivative linked to the HIV entry inhibitor aplaviroc. Labeling of the antibody trastuzumab with this reagent provided a labeled antibody conjugate that demonstrated potent HIV-1 neutralization activity demonstrating the potential of this reaction in creating protein conjugates with small molecules. The tyrosine click linkage demonstrated stability to extremes of pH, temperature and exposure to human blood plasma indicating that this linkage is significantly more robust than maleimide-type linkages that are commonly employed in bioconjugations. These studies support the broad utility of this reaction in the chemoselective modification of small molecules, peptides, and proteins under mild aqueous conditions over a broad pH range using a wide variety of biologically acceptable buffers such as phosphate buffered saline (PBS) and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) buffers as well as others and mixed buffered compositions. PMID:23534985

Relocation of the disulfonic stilbene sites of AE1 (band 3) on the basis of fluorescence energy transfer measurements.

PubMed

Knauf, Philip A; Law, Foon-Yee; Leung, Tze-Wah Vivian; Atherton, Stephen J

2004-09-28

Previous fluorescence resonance energy transfer (FRET) measurements, using BIDS (4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate) as a label for the disulfonic stilbene site and FM (fluorescein-5-maleimide) as a label for the cytoplasmic SH groups on band 3 (AE1), combined with data showing that the cytoplasmic SH groups lie about 40 A from the cytoplasmic surface of the lipid bilayer, would place the BIDS sites very near the membrane's inner surface, a location that seems to be inconsistent with current models of AE1 structure and mechanism. We reinvestigated the BIDS-FM distance, using laser single photon counting techniques as well as steady-state fluorescence of AE1, in its native membrane environment. Both techniques agree that there is very little energy transfer from BIDS to FM. The mean energy transfer (E), based on three-exponential fits to the fluorescence decay data, is 2.5 +/- 0.7% (SEM, N = 12). Steady-state fluorescence measurements also indicate <3% energy transfer from BIDS to FM. These data indicate that the BIDS sites are probably over 63 A from the cytoplasmic SH groups, placing them near the middle or the external half of the lipid bilayer. This relocation of the BIDS sites fits with other evidence that the disulfonic stilbene sites are located farther toward the external membrane surface than Glu-681, a residue near the inner membrane surface whose modification affects the pH dependence and anion selectivity of band 3. The involvement of two relatively distant parts of the AE1 protein in transport function suggests that the transport mechanism requires coordinated large-scale conformational changes in the band 3 protein.

[Change of chart genes expression in small intestines of mouse induced by electromagnetic pulse irradiation].

PubMed

Ren, Dongqing; Jin, Juan; Li, Xiaojuan; Zeng, Guiying

2008-01-01

To explore the bio-effects of electromagnetic pulse(EMP) on mouse small intestines induced by means of gene chip. Twelve BALB/c mice were randomly assigned to the normal control group and the EMP group with 6 in each group. The EMP group was irradiated with 200 kV/m, 200 pulses EMP. 18 hours after the irradiation, the mice were sacrificed and their jejunum of small intestines were eviscerated. The fluorescent cDNA probes labeled with Cy3 and Cy5 were prepared from RNA extracted from the intestines of the two groups. Probes of the two groups were then hybridized against cDNA gene chip, the fluorescent signals were scanned with a scanner and the results were analyzed by computer. Compared with the control, 56 genes in gene expression profile were altered. The expression levels of 37 genes were up-regulated distinctly while 19 genes were down-regulated significantly. Among the 56 genes, 19 were reported with known or inferred functions, 12 up-regulated genes were catenin alpha 1 (alpha-catenin), ly-6 alloantigen(Ly-6E), fructose-6-phosphate transaminase (GF6P), ribosomal protein S17 (rpS17), small proline-rich protein 2A (Sprr2a), glandular kallikrein27 (GK27), lipoxygenase-3, aldo-keto reductase (Akr1c12), GSG1, amylase 2 (Amy2),elastase 2, p6-5 gene and 7 down-regulated genes were junctional adhesion molecule (Jam), protein arginine methyltransferase (Carm1),NNP-1, 2-5 A synthetase L2,Mlark gene, ATP synthase alpha subunit, uncoupling protein-2 (Ucp2) gene; the other 37 were reported with unknown functions. EMP irradiation could induce specific expressions of some genes in mouse small intestines and most of these genes were up-regulated ones.

Probing effects of pressure release on virus capture during virus filtration using confocal microscopy.

PubMed

Dishari, Shudipto K; Venkiteshwaran, Adith; Zydney, Andrew L

2015-10-01

Virus filtration is used to ensure drug safety in the production of biotherapeutics. Several recent studies have shown a dramatic decrease in virus retention as a result of a process disruption, e.g., a transient pressure release. In this work, a novel two-label fluorescence technique was developed to probe virus capture within virus filtration membranes using confocal microscopy. Experiments were performed with Ultipor® DV20, Viresolve® Pro, and Viresolve® NFP membranes using bacteriophage φx174 as a model virus. The filters were challenged with two batches of fluorescently labeled phage: one labeled with red dye (Cy5) and one with green dye (SYBR Gold) to visualize captured phage from before and after the pressure release. The capture patterns seen in the confocal images were a strong function of the underlying membrane morphology and pore structure. The DV20 and Viresolve® NFP showed migration of previously captured phage further into the filter, consistent with the observed loss of virus retention after the pressure release. In contrast, there was no migration of captured virus in the Viresolve® Pro membranes, and these filters were also the only ones to show stable virus retention after a pressure release. The direct visualization of virus capture using the two-label fluorescence technique provides unique insights into the factors controlling the retention characteristics of virus filters with different pore structure. © 2015 Wiley Periodicals, Inc.

In vitro isolation of small-molecule-binding aptamers with intrinsic dye-displacement functionality

PubMed Central

Yu, Haixiang; Yang, Weijuan; Alkhamis, Obtin; Canoura, Juan; Yang, Kyung-Ae; Xiao, Yi

2018-01-01

Abstract Aptamer-based sensors offer a powerful tool for molecular detection, but the practical implementation of these biosensors is hindered by costly and laborious sequence engineering and chemical modification procedures. We report a simple strategy for directly isolating signal-reporting aptamers in vitro through systematic evolution of ligands by exponential enrichment (SELEX) that transduce binding events into a detectable change of absorbance via target-induced displacement of a small-molecule dye. We first demonstrate that diethylthiatricarbocyanine (Cy7) can stack into DNA three-way junctions (TWJs) in a sequence-independent fashion, greatly altering the dye's absorbance spectrum. We then design a TWJ-containing structured library and isolate an aptamer against 3,4-methylenedioxypyrovalerone (MDPV), a synthetic cathinone that is an emerging drug of abuse. This aptamer intrinsically binds Cy7 within its TWJ domain, but MDPV efficiently displaces the dye, resulting in a change in absorbance within seconds. This assay is label-free, and detects nanomolar concentrations of MDPV. It also recognizes other synthetic cathinones, offering the potential to detect newly-emerging designer drugs, but does not detect structurally-similar non-cathinone compounds or common cutting agents. Moreover, we demonstrate that the Cy7-displacement colorimetric assay is more sensitive than a conventional strand-displacement fluorescence assay. We believe our strategy offers an effective generalized approach for the development of sensitive dye-displacement colorimetric assays for other small-molecule targets. PMID:29361056

Labeling Thiols on Proteins, Living Cells, and Tissues with Enhanced Emission Induced by FRET

PubMed Central

Yuan, Yue; Wang, Xijun; Mei, Bin; Zhang, Dongxin; Tang, Anming; An, Linna; He, Xiaoxiao; Jiang, Jun; Liang, Gaolin

2013-01-01

Using N-(2-Aminoethyl)maleimide-cysteine(StBu) (Mal-Cys) as a medium, protein thiols were converted into N-terminal cysteines. After a biocompatible condensation reaction between the N-terminal cysteine and fluorescent probe 2-cyanobenzothiazole-Gly-Gly-Gly-fluorescein isothiocyanate (CBT-GGG-FITC), a new fluorogenic structure Luciferin-GGG-FITC was obtained. The latter exhibits near one order of magnitude (7 folds) enhanced fluorescence emission compared to the precursor moiety due to fluorescence resonance energy transfer (FRET) effect between the newly formed luciferin structure and the FITC motif. Theoretical investigations revealed the underlying mechanism that satisfactorily explained the experimental results. With this method, enhanced fluorescence imaging of thiols on proteins, outer membranes of living cells, translocation of membrane proteins, and endothelial cell layers of small arteries was successfully achieved. PMID:24343586

Labeling Thiols on Proteins, Living Cells, and Tissues with Enhanced Emission Induced by FRET

NASA Astrophysics Data System (ADS)

Yuan, Yue; Wang, Xijun; Mei, Bin; Zhang, Dongxin; Tang, Anming; An, Linna; He, Xiaoxiao; Jiang, Jun; Liang, Gaolin

2013-12-01

Using N-(2-Aminoethyl)maleimide-cysteine(StBu) (Mal-Cys) as a medium, protein thiols were converted into N-terminal cysteines. After a biocompatible condensation reaction between the N-terminal cysteine and fluorescent probe 2-cyanobenzothiazole-Gly-Gly-Gly-fluorescein isothiocyanate (CBT-GGG-FITC), a new fluorogenic structure Luciferin-GGG-FITC was obtained. The latter exhibits near one order of magnitude (7 folds) enhanced fluorescence emission compared to the precursor moiety due to fluorescence resonance energy transfer (FRET) effect between the newly formed luciferin structure and the FITC motif. Theoretical investigations revealed the underlying mechanism that satisfactorily explained the experimental results. With this method, enhanced fluorescence imaging of thiols on proteins, outer membranes of living cells, translocation of membrane proteins, and endothelial cell layers of small arteries was successfully achieved.

The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction.

PubMed

Lu, Ting; Lin, Zongwei; Ren, Jianwei; Yao, Peng; Wang, Xiaowei; Wang, Zhe; Zhang, Qunye

2016-01-01

MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs. Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye) could firmly bind to the surface of adherent cells (Hela) and suspended cells (K562) even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein) to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it. These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.

Lanthanide contraction effect on crystal structures of lanthanide coordination polymers with cyclohexanocucurbit[6]uril ligand

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Li-Mei; Liu, Jing-Xin, E-mail: jxliu411@ahut.edu.cn

A series of compounds based on the macrocyclic ligand cyclohexanocucurbit[6]uril (Cy6Q[6]) with formulas (Ln(H{sub 2}O){sub 6}Cy6Q[6])·2(CdCl{sub 4})·H{sub 3}O·xH{sub 2}O [isomorphous with Ln=La (1), Ce (2), Pr (3) and Nd (4), x=11 (1), 11 (2), 10 (3) and 11 (4)], (Sm(H{sub 2}O){sub 5}Cy6Q[6])·2(CdCl{sub 4})·H{sub 3}O·10H{sub 2}O (5) and (Ln(H{sub 2}O){sub 5}(NO{sub 3})@Cy6Q[6])·2(CdCl{sub 4})·2H{sub 3}O·xH{sub 2}O [isomorphous with Ln=Gd (6), Tb (7) and Dy (8), x=8 (6), 6 (7) and 6 (8)], have been successfully synthesized by the self-assembly of Cy6Q[6] with the corresponding lanthanide nitrate under hydrochloric acid aqueous solution in the presence of CdCl{sub 2}. Single-crystal X-ray diffraction analyses revealedmore » that compounds 1–8 all crystallize in monoclinic space group P2{sub 1}/c, and display 1D coordination polymer structures. The lanthanide contraction effect on the structures of 1–8 has also been investigated and discussed in detail. In contrast, the reaction of Cy6Q[6] with the Ho(NO){sub 3}, Tm(NO){sub 3}, Yb(NO){sub 3} under the same conditions resulted in the compounds 9–11 with formulas Cy6Q[6]·2(CdCl{sub 4})·2H{sub 3}O·xH{sub 2}O [isomorphous with x=10 (9), 10 (10), and 9 (11)], in which no lanthanide cations are observed. The structural difference of these compounds indicates that the Cy6Q[6] may be used in the separation of lanthanide cations. - Graphical abstract: The reaction of cyclohexanocucurbit[6]uril with lanthanide ions (La{sup 3+}, Ce{sup 3+}, Pr{sup 3+}, Nd{sup 3+}, Sm{sup 3+}, Gd{sup 3+}, Tb{sup 3+}, Dy{sup 3+}, Ho{sup 3+}, Tm{sup 3+} and Yb{sup 3+}) under hydrochloric acid in the presence of CdCl{sub 2} resulted in eleven compounds, which demonstrate interesting lanthanide contraction effect and provide a means of separating lanthanide ions. - Highlights: • Eleven compounds of the Ln{sup 3+} with the Cy6Q[6] were synthesized and described. • Compounds 1-8 demonstrate interesting lanthanide contraction effect. • In solid-state structures of compounds 9-11, no lanthanide ions were observed. • This study provides a means of separating lanthanides cations.« less

Purification, characterization, and sequencing of antimicrobial peptides, Cy-AMP1, Cy-AMP2, and Cy-AMP3, from the Cycad (Cycas revoluta) seeds.

PubMed

Yokoyama, Seiya; Kato, Kouji; Koba, Atsuko; Minami, Yuji; Watanabe, Keiichi; Yagi, Fumio

2008-12-01

Novel antimicrobial peptides (AMP), designated Cy-AMP1, Cy-AMP2, and Cy-AMP3, were purified from seeds of the cycad (Cycas revoluta) by a CM cellulofine column, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They had molecular masses of 4583.2 Da, 4568.9 Da and 9275.8 Da, respectively, by MALDI-TOF MS analysis. Half of the amino acid residues of Cy-AMP1 and Cy-AMP2 were cysteine, glycine and proline, and their sequences were similar. The sequence of Cy-AMP3 showed high homology to various lipid transfer proteins. For Cy-AMP1 and Cy-AMP2, the concentrations of peptides required for 50% inhibition (IC(50)) of the growth of plant pathogenic fungi, Gram-positive and Gram-negative bacteria were 7.0-8.9 microg/ml. The Cy-AMP3 had weak antimicrobial activity. The structural and antimicrobial characteristics of Cy-AMP1 and Cy-AMP2 indicated that they are a novel type of antimicrobial peptide belonging to a plant defensin family.

Pharmacokinetics and enhanced oral bioavailability in beagle dogs of cyclosporine A encapsulated in glyceryl monooleate/poloxamer 407 cubic nanoparticles

PubMed Central

Lai, Jie; Lu, Yi; Yin, Zongning; Hu, Fuqiang; Wu, Wei

2010-01-01

Efforts to improve the oral bioavailability of cyclosporine A (CyA) remains a challenge in the field of drug delivery. In this study, glyceryl monooleate (GMO)/poloxamer 407 cubic nanoparticles were evaluated as potential vehicles to improve the oral bioavailability of CyA. Cubic nanoparticles were prepared via the fragmentation of a bulk GMO/poloxamer 407 cubic phase gel by sonication and homogenization. The cubic inner structure formed was verified using Cryo-TEM. The mean diameters of the nanoparticles were about 180 nm, and the entrapment efficiency of these particles for CyA was over 85%. The in vitro release of CyA from these nanoparticles was less than 5% at 12 h. The results of a pharmacokinetic study in beagle dogs showed improved absorption of CyA from cubic nanoparticles as compared to microemulsion-based Neoral®; higher Cmax (1371.18 ± 37.34 vs 969.68 ± 176.3 ng mL−1), higher AUC0–t (7757.21 ± 1093.64 vs 4739.52 ± 806.30 ng h mL−1) and AUC0–∞ (9004.77 ± 1090.38 vs 5462.31 ± 930.76 ng h mL−1). The relative oral bioavailability of CyA cubic nanoparticles calculated on the basis of AUC0–∞ was about 178% as compared to Neoral®. The enhanced bioavailability of CyA is likely due to facilitated absorption by cubic nanoparticles rather than improved release. PMID:20161984

Sulfobutyl ether β-cyclodextrin (Captisol(®) ) and methyl β-cyclodextrin enhance and stabilize fluorescence of aqueous indocyanine green.

PubMed

DeDora, Daniel J; Suhrland, Cassandra; Goenka, Shilpi; Mullick Chowdhury, Sayan; Lalwani, Gaurav; Mujica-Parodi, Lilianne R; Sitharaman, Balaji

2016-10-01

As the only FDA-approved near-infrared fluorophore, indocyanine green (ICG) is commonly used to image vasculature in vivo. ICG degrades rapidly in solution, which limits its usefulness in certain applications, including time-sensitive surgical procedures. We propose formulations that address this shortcoming via complexation with β-cyclodextrin derivatives (β-CyD), which are known to create stabilizing inclusion complexes with hydrophobic molecules. Here, we complexed ICG with highly soluble methyl β-CyD and FDA-approved sulfobutyl ether β-CyD (Captisol(®) ) in aqueous solution. We measured the fluorescence of the complexes over 24 h. We found that both CyD+ICG complexes exhibit sustained fluorescence increases of >2.0× versus ICG in water and >20.0× in PBS. Using transmission electron microscopy, we found evidence of reduced aggregation in complexes versus ICG alone. We thus conclude that this reduction in aggregation helps mitigate fluorescence autoquenching of CyD+ICG complexes compared in ICG alone. We also found that while ICG complexed with methyl β-CyD severely reduced the viability of MRC-5 fibroblasts, ICG complexed with sulfobutyl ether β-CyD had no effect on viability. These results represent an important first step toward enhancing the utility of aqueous ICG by reducing aggregation-dependent fluorescence degradation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1457-1464, 2016. © 2015 Wiley Periodicals, Inc.

Extracellular cyclophilin A activates platelets via EMMPRIN (CD147) and PI3K/Akt signaling, which promotes platelet adhesion and thrombus formation in vitro and in vivo.

PubMed

Seizer, Peter; Ungern-Sternberg, Saskia N I V; Schönberger, Tanja; Borst, Oliver; Münzer, Patrick; Schmidt, Eva-Maria; Mack, Andreas F; Heinzmann, David; Chatterjee, Madhumita; Langer, Harald; Malešević, Miroslav; Lang, Florian; Gawaz, Meinrad; Fischer, Gunter; May, Andreas E

2015-03-01

Cyclophilin A (CyPA) is secreted under inflammatory conditions by various cell types. Whereas the important role of intracellular CyPA for platelet function has been reported, the effect of extracellular CyPA on platelet function has not been investigated yet. Inhibition of extracellular CyPA through a novel specific inhibitor MM284 reduced thrombus after ferric chloride-induced injury in vivo. In vitro extracellular CyPA enhanced thrombus formation even in CyPA(-/-) platelets. Treatment of isolated platelets with recombinant CyPA resulted in platelet degranulation in a time- and dose-dependent manner. Inhibition of the platelet surface receptor extracellular matrix metalloproteinase inducer (cluster of differentiation 147) by an anticluster of differentiation 147 monoclonal antibody significantly reduced CyPA-dependent platelet degranulation. Pretreatment of platelets with CyPA enhanced their recruitment to mouse carotid arteries after arterial injury, which could be inhibited by an anticluster of differentiation 147 monoclonal antibody (intravital microscopy). The role of extracellular CyPA in adhesion could be confirmed by infusing CyPA(-/-) platelets in CyPA(+/+) mice and by infusing CyPA(+/+) platelets in CyPA(-/-) mice. Stimulation of platelets with CyPA induced phosphorylation of Akt, which could in turn be inhibited in the presence of phosphoinositid-3-kinase inhibitors. Akt-1(-/-) platelets revealed a markedly decreased degranulation on CyPA stimulation. Finally, ADP-induced platelet aggregation was attenuated by MM284, as well as by inhibiting paracrine-secreted CyPA without directly affecting Ca(2+)-signaling. Extracellular CyPA activates platelets via cluster of differentiation 147-mediated phosphoinositid-3-kinase/Akt-signaling, leading to enhanced adhesion and thrombus formation independently of intracellular CyPA. Targeting extracellular CyPA via a specific inhibitor may be a promising strategy for platelet inhibition without affecting critical functions of intracellular CyPA. © 2014 American Heart Association, Inc.

[New approaches to the treatment of keratoconjunctivitis sicca].

PubMed

Safonova, T N; Gladkova, O V; Novikov, I A; Boev, V I; Fedorov, A A

A new method has been developed for the treatment of severe forms of keratoconjunctivitis sicca (KCS) that involves the use of an original cyclosporine A (CyA) saturated soft contact lens (SCL) together with preservative-free artificial tears therapy. to evaluate the effectiveness of the newly developed treatment for KCS based on the use of medical SCL saturated with 0.05% CyA. The patients (43 men, 60 eyes) with severe KCS were divided into 2 groups. Group 1 included 21 patients (30 eyes), who received artificial tears and wore 0.05% CyA-saturated silicone-hydrogel SCLs. Group 2 included 22 patients (30 eyes), who wore unsaturated original SCLs and received CyA instillations 2 times daily and, also, artificial tears. Apart from a standard ophthalmic examination, the assessment included Schirmer's test, Norn's test, vital eye stain tests, tear osmometry, laser confocal tomography of the cornea, optical coherence tomography of the anterior segment with meniscometry, impression cytology of the conjunctiva, tear pH measurement, plating of the content of the conjunctival cavity, measurement of the width of the palpebral fissure, and calculation of the ocular surface disease index. Treatment results were followed up at 1, 3, 6, and 12 months. The use of 0.05% CyA-saturated SCLs allows to halve treatment time for patients with severe KSC (down to 1 week - 1 month) as compared to unsaturated original SCLs in combination with 0.05% CyA instillations and to reduce it 5 times as compared to 0.05% CyA instillations only. The new method of KSC treatment that involves the use of medical SCL of original design (ensures even distribution of 0.05% CyA across the ocular surface) and preservative-free artificial tears has demonstrated high therapeutic effectiveness as compared to existing methods.

Interaction with glycosaminoglycans is required for cyclophilin B to trigger integrin-mediated adhesion of peripheral blood T lymphocytes to extracellular matrix

PubMed Central

Allain, Fabrice; Vanpouille, Christophe; Carpentier, Mathieu; Slomianny, Marie-Christine; Durieux, Sandrine; Spik, Geneviève

2002-01-01

Cyclophilins A and B (CyPA and CyPB) are cyclosporin A-binding proteins that are involved in inflammatory events. We have reported that CyPB interacts with two types of cell-surface-binding sites. The first site corresponds to a functional receptor and requires interaction with the central core of CyPB. This region is highly conserved in cyclophilins, suggesting that CyPA and CyPB might share biological activities mediated by interaction with this receptor. The second site is identified with glycosaminoglycans (GAGs), the binding region located in the N terminus of CyPB. The difference in the N-terminal extensions of CyPA and CyPB suggests that a unique interaction with GAGs might account for selective activity of CyPB. To explore this hypothesis, we analyzed the lymphocyte responses triggered by CyPA, CyPB, and CyPBKKK−, a mutant unable to interact with GAGs. The three ligands seemed capable enough to elicit calcium signal and chemotaxis by binding to the same signaling receptor. In contrast, only CyPB enhanced firm adhesion of T cells to the extracellular matrix. This activity depended on the interactions with GAGs and signaling receptor. CyPB-mediated adhesion required CD147 presumably because it was a costimulatory molecule and was related to an activation of α4β1 and α4β7 integrins. Finally, we showed that CyPB was capable mainly to enhance T cell adhesion of the CD4+CD45RO+ subset. The present data indicate that CyPB rather than CyPA is a proinflammatory factor for T lymphocytes and highlight the crucial role of CyPB–GAG interaction in the chemokine-like activity of this protein. PMID:11867726

Interaction with glycosaminoglycans is required for cyclophilin B to trigger integrin-mediated adhesion of peripheral blood T lymphocytes to extracellular matrix.

PubMed

Allain, Fabrice; Vanpouille, Christophe; Carpentier, Mathieu; Slomianny, Marie-Christine; Durieux, Sandrine; Spik, Geneviève

2002-03-05

Cyclophilins A and B (CyPA and CyPB) are cyclosporin A-binding proteins that are involved in inflammatory events. We have reported that CyPB interacts with two types of cell-surface-binding sites. The first site corresponds to a functional receptor and requires interaction with the central core of CyPB. This region is highly conserved in cyclophilins, suggesting that CyPA and CyPB might share biological activities mediated by interaction with this receptor. The second site is identified with glycosaminoglycans (GAGs), the binding region located in the N terminus of CyPB. The difference in the N-terminal extensions of CyPA and CyPB suggests that a unique interaction with GAGs might account for selective activity of CyPB. To explore this hypothesis, we analyzed the lymphocyte responses triggered by CyPA, CyPB, and CyPB(KKK-), a mutant unable to interact with GAGs. The three ligands seemed capable enough to elicit calcium signal and chemotaxis by binding to the same signaling receptor. In contrast, only CyPB enhanced firm adhesion of T cells to the extracellular matrix. This activity depended on the interactions with GAGs and signaling receptor. CyPB-mediated adhesion required CD147 presumably because it was a costimulatory molecule and was related to an activation of alpha4beta1 and alpha4beta7 integrins. Finally, we showed that CyPB was capable mainly to enhance T cell adhesion of the CD4+CD45RO+ subset. The present data indicate that CyPB rather than CyPA is a proinflammatory factor for T lymphocytes and highlight the crucial role of CyPB-GAG interaction in the chemokine-like activity of this protein.

Comparative Genomics of Carp Herpesviruses

PubMed Central

Kurobe, Tomofumi; Gatherer, Derek; Cunningham, Charles; Korf, Ian; Fukuda, Hideo; Hedrick, Ronald P.; Waltzek, Thomas B.

2013-01-01

Three alloherpesviruses are known to cause disease in cyprinid fish: cyprinid herpesviruses 1 and 3 (CyHV1 and CyHV3) in common carp and koi and cyprinid herpesvirus 2 (CyHV2) in goldfish. We have determined the genome sequences of CyHV1 and CyHV2 and compared them with the published CyHV3 sequence. The CyHV1 and CyHV2 genomes are 291,144 and 290,304 bp, respectively, in size, and thus the CyHV3 genome, at 295,146 bp, remains the largest recorded among the herpesviruses. Each of the three genomes consists of a unique region flanked at each terminus by a sizeable direct repeat. The CyHV1, CyHV2, and CyHV3 genomes are predicted to contain 137, 150, and 155 unique, functional protein-coding genes, respectively, of which six, four, and eight, respectively, are duplicated in the terminal repeat. The three viruses share 120 orthologous genes in a largely colinear arrangement, of which up to 55 are also conserved in the other member of the genus Cyprinivirus, anguillid herpesvirus 1. Twelve genes are conserved convincingly in all sequenced alloherpesviruses, and two others are conserved marginally. The reference CyHV3 strain has been reported to contain five fragmented genes that are presumably nonfunctional. The CyHV2 strain has two fragmented genes, and the CyHV1 strain has none. CyHV1, CyHV2, and CyHV3 have five, six, and five families of paralogous genes, respectively. One family unique to CyHV1 is related to cellular JUNB, which encodes a transcription factor involved in oncogenesis. To our knowledge, this is the first time that JUNB-related sequences have been reported in a herpesvirus. PMID:23269803

Whole-genome sequence of a novel Chinese cyprinid herpesvirus 3 isolate reveals the existence of a distinct European genotype in East Asia.

PubMed

Li, Wei; Lee, Xuezhu; Weng, Shaoping; He, Jianguo; Dong, Chuanfu

2015-02-25

Cyprinid herpesvirus 3 (CyHV3), also known as koi herpesvirus (KHV), can be subdivided primarily into European and Asian genotypes, which are represented by CyHV3-U or CyHV3-I and CyHV3-J, respectively. In this study, the whole genome sequence of a novel Chinese CyHV3 isolate (GZ11) was determined and annotated. CyHV3-GZ11 genome was found to contain 295,119 nucleotides with 52.9% G/C content, which is highly similar to those of published CyHV3-U, CyHV3-I, and CyHV3-J strains. With reference to CyHV3-U, CyHV3-I, and CyHV3-J, CyHV3-GZ11 was also classified into 164 open reading frames (ORF), which include eight repeated ORFs. On the basis of the 12 alloherpeviruses core genes, results from phylogenetic analysis showed that CyHV3-GZ11 had closer evolutionary relationships with CyHV3-U and CyHV3-I than with CyHV3/KHV-J, which were also supported by genome wide-based single nucleotide substitution analysis and the use of a series of developed molecular markers. This study was the first to reveal the presence of a distinct European CyHV3 genotype in East and Southeast Asia at a whole genome level, which will evoke new insights on exploring the origin, evolution, and epidemiology of the virus. Copyright © 2014 Elsevier B.V. All rights reserved.

A Supramolecular Approach for Liver Radioembolization

PubMed Central

Spa, Silvia J.; Welling, Mick M.; van Oosterom, Matthias N.; Rietbergen, Daphne D. D.; Burgmans, Mark C.; Verboom, Willem; Huskens, Jurriaan; Buckle, Tessa; van Leeuwen, Fijs W. B.

2018-01-01

Hepatic radioembolization therapies can suffer from discrepancies between diagnostic planning (scout-scan) and the therapeutic delivery itself, resulting in unwanted side-effects such as pulmonary shunting. We reasoned that a nanotechnology-based pre-targeting strategy could help overcome this shortcoming by directly linking pre-interventional diagnostics to the local delivery of therapy. Methods: The host-guest interaction between adamantane and cyclodextrin was employed in an in vivo pre-targeting set-up. Adamantane (guest)-functionalized macro albumin aggregates (MAA-Ad; d = 18 μm) and (radiolabeled) Cy5 and β-cyclodextrin (host)-containing PIBMA polymers (99mTc-Cy50.5CD10PIBMA39; MW ~ 18.8 kDa) functioned as the reactive pair. Following liver or lung embolization with (99mTc)-MAA-Ad or (99mTc)-MAA (control), the utility of the pre-targeting concept was evaluated after intravenous administration of 99mTc-Cy50.5CD10PIBMA39. Results: Interactions between MAA-Ad and Cy50.5CD10PIBMA39 could be monitored in solution using confocal microscopy and were quantified by radioisotope-based binding experiments. In vivo the accumulation of the MAA-Ad particles in the liver or lungs yielded an approximate ten-fold increase in accumulation of 99mTc-Cy50.5CD10PIBMA39 in these organs (16.2 %ID/g and 10.5 %ID/g, respectively) compared to the control. Pre-targeting with MAA alone was shown to be only half as efficient. Uniquely, for the first time, this data demonstrates that the formation of supramolecular interactions between cyclodextrin and adamantane can be used to drive complex formation in the chemically challenging in vivo environment. Conclusion: The in vivo distribution pattern of the cyclodextrin host could be guided by the pre-administration of the adamantane guest, thereby creating a direct link between the scout-scan (MAA-Ad) and delivery of therapy. PMID:29721086

Safety and survival with GVAX pancreas prime and Listeria Monocytogenes-expressing mesothelin (CRS-207) boost vaccines for metastatic pancreatic cancer.

PubMed

Le, Dung T; Wang-Gillam, Andrea; Picozzi, Vincent; Greten, Tim F; Crocenzi, Todd; Springett, Gregory; Morse, Michael; Zeh, Herbert; Cohen, Deirdre; Fine, Robert L; Onners, Beth; Uram, Jennifer N; Laheru, Daniel A; Lutz, Eric R; Solt, Sara; Murphy, Aimee Luck; Skoble, Justin; Lemmens, Ed; Grous, John; Dubensky, Thomas; Brockstedt, Dirk G; Jaffee, Elizabeth M

2015-04-20

GVAX pancreas, granulocyte-macrophage colony-stimulating factor-secreting allogeneic pancreatic tumor cells, induces T-cell immunity to cancer antigens, including mesothelin. GVAX is administered with low-dose cyclophosphamide (Cy) to inhibit regulatory T cells. CRS-207, live-attenuated Listeria monocytogenes-expressing mesothelin, induces innate and adaptive immunity. On the basis of preclinical synergy, we tested prime/boost vaccination with GVAX and CRS-207 in pancreatic adenocarcinoma. Previously treated patients with metastatic pancreatic adenocarcinoma were randomly assigned at a ratio of 2:1 to two doses of Cy/GVAX followed by four doses of CRS-207 (arm A) or six doses of Cy/GVAX (arm B) every 3 weeks. Stable patients were offered additional courses. The primary end point was overall survival (OS) between arms. Secondary end points were safety and clinical response. A total of 90 patients were treated (arm A, n = 61; arm B, n = 29); 97% had received prior chemotherapy; 51% had received ≥ two regimens for metastatic disease. Mean number of doses (± standard deviation) administered in arms A and B were 5.5 ± 4.5 and 3.7 ± 2.2, respectively. The most frequent grade 3 to 4 related toxicities were transient fevers, lymphopenia, elevated liver enzymes, and fatigue. OS was 6.1 months in arm A versus 3.9 months in arm B (hazard ratio [HR], 0.59; P = .02). In a prespecified per-protocol analysis of patients who received at least three doses (two doses of Cy/GVAX plus one of CRS-207 or three of Cy/GVAX), OS was 9.7 versus 4.6 months (arm A v B; HR, 0.53; P = .02). Enhanced mesothelin-specific CD8 T-cell responses were associated with longer OS, regardless of treatment arm. Heterologous prime/boost with Cy/GVAX and CRS-207 extended survival for patients with pancreatic cancer, with minimal toxicity. © 2015 by American Society of Clinical Oncology.

Safety and Survival With GVAX Pancreas Prime and Listeria Monocytogenes–Expressing Mesothelin (CRS-207) Boost Vaccines for Metastatic Pancreatic Cancer

PubMed Central

Le, Dung T.; Wang-Gillam, Andrea; Picozzi, Vincent; Greten, Tim F.; Crocenzi, Todd; Springett, Gregory; Morse, Michael; Zeh, Herbert; Cohen, Deirdre; Fine, Robert L.; Onners, Beth; Uram, Jennifer N.; Laheru, Daniel A.; Lutz, Eric R.; Solt, Sara; Murphy, Aimee Luck; Skoble, Justin; Lemmens, Ed; Grous, John; Dubensky, Thomas; Brockstedt, Dirk G.; Jaffee, Elizabeth M.

2015-01-01

Purpose GVAX pancreas, granulocyte-macrophage colony-stimulating factor–secreting allogeneic pancreatic tumor cells, induces T-cell immunity to cancer antigens, including mesothelin. GVAX is administered with low-dose cyclophosphamide (Cy) to inhibit regulatory T cells. CRS-207, live-attenuated Listeria monocytogenes–expressing mesothelin, induces innate and adaptive immunity. On the basis of preclinical synergy, we tested prime/boost vaccination with GVAX and CRS-207 in pancreatic adenocarcinoma. Patients and Methods Previously treated patients with metastatic pancreatic adenocarcinoma were randomly assigned at a ratio of 2:1 to two doses of Cy/GVAX followed by four doses of CRS-207 (arm A) or six doses of Cy/GVAX (arm B) every 3 weeks. Stable patients were offered additional courses. The primary end point was overall survival (OS) between arms. Secondary end points were safety and clinical response. Results A total of 90 patients were treated (arm A, n = 61; arm B, n = 29); 97% had received prior chemotherapy; 51% had received ≥ two regimens for metastatic disease. Mean number of doses (± standard deviation) administered in arms A and B were 5.5 ± 4.5 and 3.7 ± 2.2, respectively. The most frequent grade 3 to 4 related toxicities were transient fevers, lymphopenia, elevated liver enzymes, and fatigue. OS was 6.1 months in arm A versus 3.9 months in arm B (hazard ratio [HR], 0.59; P = .02). In a prespecified per-protocol analysis of patients who received at least three doses (two doses of Cy/GVAX plus one of CRS-207 or three of Cy/GVAX), OS was 9.7 versus 4.6 months (arm A v B; HR, 0.53; P = .02). Enhanced mesothelin-specific CD8 T-cell responses were associated with longer OS, regardless of treatment arm. Conclusion Heterologous prime/boost with Cy/GVAX and CRS-207 extended survival for patients with pancreatic cancer, with minimal toxicity. PMID:25584002

The Primary Mechanism of Cellular Internalization for a Short Cell- Penetrating Peptide as a Nano-Scale Delivery System.

PubMed

Liu, Betty R; Huang, Yue-Wern; Korivi, Mallikarjuna; Lo, Shih-Yen; Aronstam, Robert S; Lee, Han-Jung

2017-01-01

Development of effective drug delivery systems (DDS) is a critical issue in health care and medicine. Advances in molecular biology and nanotechnology have allowed the introduction of nanomaterial-based drug delivery systems. Cell-penetrating peptides (CPPs) can form the basis of drug delivery systems by virtue of their ability to support the transport of cargoes into the cell. Potential cargoes include proteins, DNA, RNA, liposomes, and nanomaterials. These cargoes generally retain their bioactivities upon entering cells. In the present study, the smallest, fully-active lactoferricin-derived CPP, L5a is used to demonstrate the primary contributor of cellular internalization. The secondary helical structure of L5a encompasses symmetrical positive charges around the periphery. The contributions of cell-specificity, peptide length, concentration, zeta potential, particle size, and spatial structure of the peptides were examined, but only zeta potential and spatial structure affected protein transduction efficiency. FITC-labeled L5a appeared to enter cells via direct membrane translocation insofar as endocytic modulators did not block FITC-L5a entry. This is the same mechanism of protein transduction active in Cy5 labeled DNA delivery mediated by FITC-L5a. A significant reduction of transduction efficiency was observed with structurally incomplete FITC-L5a formed by tryptic destruction, in which case the mechanism of internalization switched to a classical energydependent endocytosis pathway. These results support the continued development of the non-cytotoxic L5a as an efficient tool for drug delivery. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Acetylene terminated aspartimides and resins therefrom

NASA Technical Reports Server (NTRS)

Hergenrother, Paul M. (Inventor); Connell, John W. (Inventor); Havens, Stephen J. (Inventor)

1989-01-01

Acetylene terminated aspartimides are prepared using two methods. In the first, an amino-substituted aromatic acetylene is reacted with an aromatic bismaleimide in a solvent of glacial acetic acid and/or m-cresol. In the second method, an aromatic diamine is reacted with an ethynyl containing maleimide, such an N-(3-ethynyl phenyl) maleimide, in a solvent of glacial acetic acid and/or m-cresol. In addition, acetylene terminated aspartimides are blended with various acetylene terminated oligomers and polymers to yield composite materials exhibiting improved mechanical properties.

ELANE mutant-specific activation of different UPR pathways in congenital neutropenia.

PubMed

Nustede, Rainer; Klimiankou, Maksim; Klimenkova, Olga; Kuznetsova, Inna; Zeidler, Cornelia; Welte, Karl; Skokowa, Julia

2016-01-01

A number of studies have demonstrated induction of the unfolded protein response (UPR) in patients with severe congenital neutropenia (CN) harbouring mutations of ELANE, encoding neutrophil elastase. Why UPR is not activated in patients with cyclic neutropenia (CyN) carrying the same ELANE mutations is unclear. We evaluated the effects of ELANE mutants on UPR induction in myeloid cells from CN and CyN patients, and analysed whether additional CN-specific defects contribute to the differences in UPR induction between CN and CyN patients harbouring identical ELANE mutations. We investigated CN-specific p.C71R and p.V174_C181del (NP_001963.1) and CN/CyN-shared p.S126L (NP_001963.1) ELANE mutants. We found that transduction of haematopoietic cells with p.C71R, but not with p.V174_C181del or p.S126L ELANE mutants induced expression of ATF6, and the ATF6 target genes PPP1R15A, DDIT3 and HSPA5. Recently, we found that levels of secretory leucocyte protease inhibitor (SLPI), a natural ELANE inhibitor, are diminished in myeloid cells from CN patients, but not CyN patients. Combined knockdown of SLPI by shRNA and transduction of ELANE p.S126L in myeloid cells led to elevated levels of ATF6, PPP1R15A and HSPA5 RNA, suggesting that normal levels of SLPI in CyN patients might protect them from the UPR induced by mutant ELANE. In summary, different ELANE mutants have different effects on UPR activation, and SLPI regulates the extent of ELANE-triggered UPR. © 2015 John Wiley & Sons Ltd.

Enhanced extravasation, stability and in vivo cardiac gene silencing via in situ siRNA-albumin conjugation.

PubMed

Lau, Shannen; Graham, Bim; Cao, Nga; Boyd, Ben J; Pouton, Colin W; White, Paul J

2012-01-01

A potential barrier to progression of siRNA therapeutics to the clinic is the ability of these agents to cross the vascular endothelium to reach target cells. This study aimed to bypass the endothelial barrier by harnessing the extravasation capability of the serum protein albumin to allow siRNA to reach cardiomyocytes. A strategy for conjugating siRNA to albumin in vivo was developed that involved activating 3'-amine, 2'-O-methyl, phosphorothioate modified siRNA with succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to yield maleimide-functionalized siRNA ("activated siRNA"); this thiol-reactive species can then irreversibly link to the single surface-exposed cysteine residue of endogenous albumin following intravenous administration. An IGF-I-receptor (IGF-IR) siRNA sequence which was effective in vitro was used to test the ability of the siRNA-albumin conjugate to bypass the endothelial barrier in Balb/C mice and produce silencing. In situ conjugation of maleimide-functionalized siRNA to albumin in mouse serum occurred within minutes of addition, and the resulting conjugate was found to be nuclease stable by SDS-PAGE analysis. In Sprague-Dawley rats, activated siRNA showed a significantly enhanced elimination half-life (75.9 ± 18.2 min) compared to unactivated siRNA (5.1 ± 0.2 min). Intravenous injection of this activated siRNA (1 mg/kg daily for four days) significantly reduced left ventricle IGF-IR mRNA to 64.1 ± 4.1% of that in vehicle-treated animals (mean ± SEM), while the control siRNA (unconjugated) had no effect (n = 4, P > 0.05). Imaging of microvessels from mice treated with fluorescein-labeled activated siRNA showed clear evidence of extravasation and cellular uptake in capillary endothelial cells, cardiomyocytes and vascular smooth muscle cells for mice treated with the activated siRNA but not mice treated with the unactivated siRNA. siRNA-albumin constructs are therefore capable of extravasation to the myocardium resulting in silencing in this otherwise silencing-resistant organ.

An enzymatically-sensitized sequential and concentric energy transfer relay self-assembled around semiconductor quantum dots

NASA Astrophysics Data System (ADS)

Samanta, Anirban; Walper, Scott A.; Susumu, Kimihiro; Dwyer, Chris L.; Medintz, Igor L.

2015-04-01

The ability to control light energy within de novo nanoscale structures and devices will greatly benefit their continuing development and ultimate application. Ideally, this control should extend from generating the light itself to its spatial propagation within the device along with providing defined emission wavelength(s), all in a stand-alone modality. Here we design and characterize macromolecular nanoassemblies consisting of semiconductor quantum dots (QDs), several differentially dye-labeled peptides and the enzyme luciferase which cumulatively demonstrate many of these capabilities by engaging in multiple-sequential energy transfer steps. To create these structures, recombinantly-expressed luciferase and the dye-labeled peptides were appended with a terminal polyhistidine sequence allowing for controlled ratiometric self-assembly around the QDs via metal-affinity coordination. The QDs serve to provide multiple roles in these structures including as central assembly platforms or nanoscaffolds along with acting as a potent energy harvesting and transfer relay. The devices are activated by addition of coelenterazine H substrate which is oxidized by luciferase producing light energy which sensitizes the central 625 nm emitting QD acceptor by bioluminescence resonance energy transfer (BRET). The sensitized QD, in turn, acts as a relay and transfers the energy to a first peptide-labeled Alexa Fluor 647 acceptor dye displayed on its surface. This dye then transfers energy to a second red-shifted peptide-labeled dye acceptor on the QD surface through a second concentric Förster resonance energy transfer (FRET) process. Alexa Fluor 700 and Cy5.5 are both tested in the role of this terminal FRET acceptor. Photophysical analysis of spectral profiles from the resulting sequential BRET-FRET-FRET processes allow us to estimate the efficiency of each of the transfer steps. Importantly, the efficiency of each step within this energy transfer cascade can be controlled to some extent by the number of enzymes/peptides displayed on the QD. Further optimization of the energy transfer process(es) along with potential applications of such devices are finally discussed.The ability to control light energy within de novo nanoscale structures and devices will greatly benefit their continuing development and ultimate application. Ideally, this control should extend from generating the light itself to its spatial propagation within the device along with providing defined emission wavelength(s), all in a stand-alone modality. Here we design and characterize macromolecular nanoassemblies consisting of semiconductor quantum dots (QDs), several differentially dye-labeled peptides and the enzyme luciferase which cumulatively demonstrate many of these capabilities by engaging in multiple-sequential energy transfer steps. To create these structures, recombinantly-expressed luciferase and the dye-labeled peptides were appended with a terminal polyhistidine sequence allowing for controlled ratiometric self-assembly around the QDs via metal-affinity coordination. The QDs serve to provide multiple roles in these structures including as central assembly platforms or nanoscaffolds along with acting as a potent energy harvesting and transfer relay. The devices are activated by addition of coelenterazine H substrate which is oxidized by luciferase producing light energy which sensitizes the central 625 nm emitting QD acceptor by bioluminescence resonance energy transfer (BRET). The sensitized QD, in turn, acts as a relay and transfers the energy to a first peptide-labeled Alexa Fluor 647 acceptor dye displayed on its surface. This dye then transfers energy to a second red-shifted peptide-labeled dye acceptor on the QD surface through a second concentric Förster resonance energy transfer (FRET) process. Alexa Fluor 700 and Cy5.5 are both tested in the role of this terminal FRET acceptor. Photophysical analysis of spectral profiles from the resulting sequential BRET-FRET-FRET processes allow us to estimate the efficiency of each of the transfer steps. Importantly, the efficiency of each step within this energy transfer cascade can be controlled to some extent by the number of enzymes/peptides displayed on the QD. Further optimization of the energy transfer process(es) along with potential applications of such devices are finally discussed. Electronic supplementary information (ESI) available: This material includes control experimental data and select deconvoluted spectra. See DOI: 10.1039/c5nr00828j

An Antibody-Immobilized Silica Inverse Opal Nanostructure for Label-Free Optical Biosensors

PubMed Central

Lee, Wang Sik; Kim, Shin-Hyun

2018-01-01

Three-dimensional SiO2-based inverse opal (SiO2-IO) nanostructures were prepared for use as biosensors. SiO2-IO was fabricated by vertical deposition and calcination processes. Antibodies were immobilized on the surface of SiO2-IO using 3-aminopropyl trimethoxysilane (APTMS), a succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester (NHS-PEG4-maleimide) cross-linker, and protein G. The highly accessible surface and porous structure of SiO2-IO were beneficial for capturing influenza viruses on the antibody-immobilized surfaces. Moreover, as the binding leads to the redshift of the reflectance peak, the influenza virus could be detected by simply monitoring the change in the reflectance spectrum without labeling. SiO2-IO showed high sensitivity in the range of 103–105 plaque forming unit (PFU) and high specificity to the influenza A (H1N1) virus. Due to its structural and optical properties, SiO2-IO is a promising material for the detection of the influenza virus. Our study provides a generalized sensing platform for biohazards as various sensing strategies can be employed through the surface functionalization of three-dimensional nanostructures. PMID:29361683

Diels-Alder reactions of five-membered heterocycles containing one heteroatom

PubMed Central

Ding, Xiaoyuan; Nguyen, Son T.; Williams, John D.; Peet, Norton P.

2015-01-01

Diels-Alder reactions of five-membered heterocycles containing one heteroatom with an N-arylmaleimide were studied. Cycloaddition of 2,5-dimethylfuran (4) with 2-(4-methylphenyl)maleimide (3) in toluene at 60 °C gave bicyclic adduct 5. Cycloadditions of 3 with 2,5-dimethylthiophene (11) and 1,2,5-trimethylpyrrole (14) were also studied. Interestingly, the bicyclic compound 5 cleanly rearranged, with loss of water, when treated with p-toluenesulfonic acid in toluene at 80 °C to give 4,7-dimethyl-2-p-tolylisoindoline-1,3-dione (6). PMID:25838605

Combination of active targeting, enzyme-triggered release and fluorescent dye into gold nanoclusters for endomicroscopy-guided photothermal/photodynamic therapy to pancreatic ductal adenocarcinoma.

PubMed

Li, Hui; Wang, Ping; Deng, Yunxiang; Zeng, Meiying; Tang, Yan; Zhu, Wei-Hong; Cheng, Yingsheng

2017-09-01

Pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating malignancies in patients, and there is an urgent need for an effective treatment method. Herein, we report a novel gold nanocluster-based platform for confocal laser endomicroscopy-guided photothermal therapy (PTT)/photodynamic therapy (PDT) for PDAC, which consists of four components: the PTT-carrier gold nanocluster, an active targeting ligand U11 peptide, a Cathepsin E (CTSE)-sensitive PDT therapy prodrug, and a CTSE-sensitive imaging agent (cyanine dye Cy5.5). Due to the strong coupling among cross-linked gold nanoparticles (AuNPs), the surface plasmon resonance peak of nanoclusters shifts to the near-infrared (NIR) region, thus making the nanoclusters useful in the effective PTT therapy. In the system, the labeling of nanoclusters with U11 peptide can distinctly increase their affinity and accelerate their uptake by pancreatic cancer cells. Cell apoptosis staining demonstrates that, upon incorporation of the uPAR-targeted unit, the antitumor efficacy of CTSE-sensitive nanocluster AuS-U11 is significantly enhanced with respect to that of the non-targeted nanocluster AuS-PEG and the insensitive nanocluster AuC-PEG. In vivo and ex vivo optical imaging confirms the high accumulation of AuS-U11 in the in situ pancreatic tumor model. Therapeutic studies further show that the combination of active targeting for tumor tissue, enzyme-triggered drug release of 5-ALA and fluorescent dye Cy5.5 in nanoclusters AuS-U11 could achieve optimal therapeutic efficacy with endomicroscopy-guided photothermal/photodynamic therapy with minimal side effects. As a consequence, the delicate gold nanocluster concept provides a promising strategy to enhance the therapy efficiency in the most challenging PDAC treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Selective assay for CyPA and CyPB in human blood using highly specific anti-peptide antibodies.

PubMed

Allain, F; Boutillon, C; Mariller, C; Spik, G

1995-01-13

Cyclophilins A and B (CyPA and CyPB) are known to be the main binding proteins for cyclosporin A (CsA), a potent immunosuppressive drug. Due to the high homology between the two proteins, antibodies to CyPB were found to cross-react with CyPA. In order to avoid this phenomenon, we raised specific antibodies against peptides copying the most divergent parts of the two sequences. These antibodies allowed us to develop an ELISA capture assay selective for either isotype. Thus, we showed that leukocyte CyPB concentration was almost ten times lower than that of CyPA, and that in contrast to the results described in the literature, only CyPB was released in plasma. Moreover, CyPB levels in leukocytes and plasma were found to correlate for the same donor, but no relationship was found with CyPA level.

Sortase A-mediated site-specific labeling of camelid single-domain antibody-fragments: a versatile strategy for multiple molecular imaging modalities.

PubMed

Massa, Sam; Vikani, Niravkumar; Betti, Cecilia; Ballet, Steven; Vanderhaegen, Saskia; Steyaert, Jan; Descamps, Benedicte; Vanhove, Christian; Bunschoten, Anton; van Leeuwen, Fijs W B; Hernot, Sophie; Caveliers, Vicky; Lahoutte, Tony; Muyldermans, Serge; Xavier, Catarina; Devoogdt, Nick

2016-09-01

A generic site-specific conjugation method that generates a homogeneous product is of utmost importance in tracer development for molecular imaging and therapy. We explored the protein-ligation capacity of the enzyme Sortase A to label camelid single-domain antibody-fragments, also known as nanobodies. The versatility of the approach was demonstrated by conjugating independently three different imaging probes: the chelating agents CHX-A"-DTPA and NOTA for single-photon emission computed tomography (SPECT) with indium-111 and positron emission tomography (PET) with gallium-68, respectively, and the fluorescent dye Cy5 for fluorescence reflectance imaging (FRI). After a straightforward purification process, homogeneous single-conjugated tracer populations were obtained in high yield (30-50%). The enzymatic conjugation did not affect the affinity of the tracers, nor the radiolabeling efficiency or spectral characteristics. In vivo, the tracers enabled the visualization of human epidermal growth factor receptor 2 (HER2) expressing BT474M1-tumors with high contrast and specificity as soon as 1 h post injection in all three imaging modalities. These data demonstrate Sortase A-mediated conjugation as a valuable strategy for the development of site-specifically labeled camelid single-domain antibody-fragments for use in multiple molecular imaging modalities. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Genomic Mapping of Human DNA provides Evidence of Difference in Stretch between AT and GC rich regions

NASA Astrophysics Data System (ADS)

Reifenberger, Jeffrey; Dorfman, Kevin; Cao, Han

Human DNA is a not a polymer consisting of a uniform distribution of all 4 nucleic acids, but rather contains regions of high AT and high GC content. When confined, these regions could have different stretch due to the extra hydrogen bond present in the GC basepair. To measure this potential difference, human genomic DNA was nicked with NtBspQI, labeled with a cy3 like fluorophore at the nick site, stained with YOYO, loaded into a device containing an array of nanochannels, and imaged. Over 473,000 individual molecules of DNA, corresponding to roughly 30x coverage of a human genome, were collected and aligned to the human reference. Based on the known AT/GC content between aligned pairs of labels, the stretch was measured for regions of similar size but different AT/GC content. We found that regions of high GC content were consistently more stretched than regions of high AT content between pairs of labels varying in size between 2.5 kbp and 500 kbp. We measured that for every 1% increase in GC content there was roughly a 0.06% increase in stretch. While this effect is small, it is important to take into account differences in stretch between AT and GC rich regions to improve the sensitivity of detection of structural variations from genomic variations. NIH Grant: R01-HG006851.

Multicolor fluorescent biosensor for multiplexed detection of DNA.

PubMed

Hu, Rong; Liu, Tao; Zhang, Xiao-Bing; Huan, Shuang-Yan; Wu, Cuichen; Fu, Ting; Tan, Weihong

2014-05-20

Development of efficient methods for highly sensitive and rapid screening of specific oligonucleotide sequences is essential to the early diagnosis of serious diseases. In this work, an aggregated cationic perylene diimide (PDI) derivative was found to efficiently quench the fluorescence emission of a variety of anionic oligonucleotide-labeled fluorophores that emit at wavelengths from the visible to NIR region. This broad-spectrum quencher was then adopted to develop a multicolor biosensor via a label-free approach for multiplexed fluorescent detection of DNA. The aggregated perylene derivative exhibits a very high quenching efficiency on all ssDNA-labeled dyes associated with biosensor detection, having efficiency values of 98.3 ± 0.9%, 97 ± 1.1%, and 98.2 ± 0.6% for FAM, TAMRA, and Cy5, respectively. An exonuclease-assisted autocatalytic target recycling amplification was also integrated into the sensing system. High quenching efficiency combined with autocatalytic target recycling amplification afforded the biosensor with high sensitivity toward target DNA, resulting in a detection limit of 20 pM, which is about 50-fold lower than that of traditional unamplified homogeneous fluorescent assay methods. The quencher did not interfere with the catalytic activity of nuclease, and the biosensor could be manipulated in either preaddition or postaddition manner with similar sensitivity. Moreover, the proposed sensing system allows for simultaneous and multicolor analysis of several oligonucleotides in homogeneous solution, demonstrating its potential application in the rapid screening of multiple biotargets.

Prevalence of bisphosphonate-related osteonecrosis of the jaw-like lesions is increased in a chemotherapeutic dose-dependent manner in mice.

PubMed

Kuroshima, Shinichiro; Sasaki, Muneteru; Nakajima, Kazunori; Tamaki, Saki; Hayano, Hiroki; Sawase, Takashi

2018-07-01

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) worsens oral health-related quality of life. Most BRONJ occurs in multiple myeloma or metastatic breast cancer patients treated with bisphosphonate/chemotherapeutic combination therapies. Cyclophosphamide (CY), an alkylating chemotherapeutic drug, is used to treat multiple myeloma, although its use has been recently reduced. The aim of this study was to clarify the effects of CY dose on tooth extraction socket healing when CY is used with or without bisphosphonate in mice. Low-dose CY (50 mg/kg; CY-L), moderate-dose CY (100 mg/kg; CY-M), high-dose CY (150 mg/kg; CY-H), and bisphosphonate [Zometa (ZA): 0.05 mg/kg] were administered for 7 weeks. Each dose of CY and ZA in combination was also administered for 7 weeks. Both maxillary first molars were extracted at 3 weeks after the initiation of drug administration. Euthanasia was performed at 4 weeks post-extraction. Gross wound healing, microcomputed tomography analysis, histomorphometry, and immunohistochemistry were used to quantitatively evaluate osseous and soft tissue wound healing of tooth extraction sockets. ZA monotherapy induced no BRONJ-like lesions in mice. CY monotherapy rarely induced open wounds, though delayed osseous wound healing occurred in a CY dose-dependent manner. In contrast, CY/ZA combination therapy prevalently induced BRONJ-like lesions with compromised osseous and soft tissue healing in a CY dose-dependent manner. Interestingly, anti-angiogenesis was noted regardless of CY dose and ZA administration, even though only CY-M/ZA and CY-H/ZA combination therapies induced BRONJ-like lesions. Our findings suggest that high-dose CY may be associated with the development of BRONJ following tooth extraction only when CY is used together with ZA. In addition to anti-angiogenesis, other factors may contribute to the pathoetiology of BRONJ. Copyright © 2018 Elsevier Inc. All rights reserved.

Conjugation Approach To Produce a Staphylococcus aureus Synbody with Activity in Serum.

PubMed

Lainson, John C; Fuenmayor, Mariana Ferrer; Johnston, Stephen Albert; Diehnelt, Chris W

2015-10-21

Synbodies show promise as a new class of synthetic antibiotics. Here, we explore improvements in their activity and production through conjugation chemistry. Maleimide conjugation is a widely used conjugation strategy due to its high yield, selectivity, and low cost. We used this strategy to conjugate two antibacterial peptides to produce a bivalent antibacterial peptide, called a synbody that has bactericidal activity against methicillin resistant Staphylococcus aureus (MRSA). The synbody was prepared by conjugation of a partially d-amino acid substituted synthetic antibacterial peptide to a bis-maleimide scaffold. The synbody slowly degrades in serum, but also undergoes exchange reactions with other serum proteins, such as albumin. Therefore, we hydrolyzed the thiosuccinimide ring using a mild hydrolysis protocol to produce a new synbody with similar bactericidal activity. The synbody was now resistant to exchange reactions and maintained bactericidal activity in serum for 2 h. This work demonstrates that low-cost maleimide coupling can be used to produce antibacterial peptide conjugates with activity in serum.

Cysteine-specific, covalent anchoring of transition organometallic complexes to the protein papain from Carica papaya.

PubMed

Haquette, Pierre; Salmain, Michèle; Svedlung, Karolina; Martel, Annie; Rudolf, Bogna; Zakrzewski, Janusz; Cordier, Stéphane; Roisnel, Thierry; Fosse, Céline; Jaouen, Gérard

2007-01-22

Site-directed and covalent introduction of various transition metal-organic entities to the active site of the cysteine endoproteinase, papain, was achieved by treatment of this enzyme with a series of organometallic maleimide derivatives specially designed for the purpose. Kinetic studies made it clear that time-dependent irreversible inactivation of papain occurred in the presence of these organometallic maleimides as a result of Michael addition of the sulfhydryl of Cys25. The rate and mechanism of inactivation were highly dependent on the structure of the organometallic entity attached to the maleimide group. Combined ESI-MS and IR analysis indicated that all the resulting papain adducts contained one organometallic moiety per protein molecule. This confirmed that chemospecific introduction of the metal complexes was indeed achieved. Thus, three novel reagents for heavy-atom derivatization of protein crystals, which include ruthenium, rhenium and tungsten, are now available for the introduction of electron-dense scatterers for phasing of X-ray crystallographic data.

Functionalized graphene nanomaterials: new insight into direct exfoliation of graphite with supramolecular polymers

NASA Astrophysics Data System (ADS)

Cheng, Chih-Chia; Chang, Feng-Chih; Wang, Jui-Hsu; Chen, Jem-Kun; Yen, Ying-Chieh; Lee, Duu-Jong

2015-12-01

A novel urea-cytosine end-capped polypropylene glycol (UrCy-PPG) can self-assemble into a long-range ordered lamellar microstructure on the surface of graphene, due to the strong specific interactions between UrCy-PPG and graphene. In addition, the graphene composite produced exhibits a high conductivity (~1093 S m-1) with a dramatic thermo-responsive ON/OFF resistance-switching behavior (10 consecutive cycles).A novel urea-cytosine end-capped polypropylene glycol (UrCy-PPG) can self-assemble into a long-range ordered lamellar microstructure on the surface of graphene, due to the strong specific interactions between UrCy-PPG and graphene. In addition, the graphene composite produced exhibits a high conductivity (~1093 S m-1) with a dramatic thermo-responsive ON/OFF resistance-switching behavior (10 consecutive cycles). Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07076g

Development of a microarray-based assay for efficient testing of new HSP70/DnaK inhibitors.

PubMed

Mohammadi-Ostad-Kalayeh, Sona; Hrupins, Vjaceslavs; Helmsen, Sabine; Ahlbrecht, Christin; Stahl, Frank; Scheper, Thomas; Preller, Matthias; Surup, Frank; Stadler, Marc; Kirschning, Andreas; Zeilinger, Carsten

2017-12-15

A facile method for testing ATP binding in a highly miniaturized microarray environment using human HSP70 and DnaK from Mycobacterium tuberculosis as biological targets is reported. Supported by molecular modelling studies we demonstrate that the position of the fluorescence label on ATP has a strong influence on the binding to human HSP70. Importantly, the label has to be positioned on the adenine ring and not to the terminal phosphate group. Unlabelled ATP displaced bound Cy5-ATP from HSP70 in the micromolar range. The affinity of a well-known HSP70 inhibitor VER155008 for the ATP binding site in HSP70 was determined, with a EC 50 in the micromolar range, whereas reblastin, a HSP90-inhibitor, did not compete for ATP in the presence of HSP70. The applicability of the method was demonstrated by screening a small compound library of natural products. This unraveled that terphenyls rickenyl A and D, recently isolated from cultures of the fungus Hypoxylon rickii, are inhibitors of HSP70. They compete with ATP for the chaperone in the range of 29 µM (Rickenyl D) and 49 µM (Rickenyl A). Furthermore, the microarray-based test system enabled protein-protein interaction analysis using full-length HSP70 and HSP90 proteins. The labelled full-length human HSP90 binds with a half-maximal affinity of 5.5 µg/ml (∼40 µM) to HSP70. The data also demonstrate that the microarray test has potency for many applications from inhibitor screening to target-oriented interaction studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ethanol Alters Alveolar Fluid Balance via Nadph Oxidase (NOX) Signaling to Epithelial Sodium Channels (ENaC) in the Lung

PubMed Central

Downs, Charles A.; Kreiner, Lisa H.; Eaton, Amity F.; Johnson, Nicholle M.; Brown, Lou Ann

2013-01-01

Chronic alcohol consumption is associated with increased incidence of ICU-related morbidity and mortality, primarily from acute respiratory distress syndrome (ARDS). However, the mechanisms involved are unknown. One explanation is that alcohol regulates epithelial sodium channels (ENaC) via oxidant signaling to promote a pro- injury environment. We used small rodent models to mimic acute and chronic alcohol consumption and tested the hypothesis that ethanol (EtOH) would affect lung fluid clearance by up-regulating ENaC activity in the lung. Fluorescence labeling of rat lung slices and in vivo mouse lung revealed an increase in ROS production in response to acute EtOH exposure. Using western blots and fluorescein-5-maleimide labeling, we conclude that EtOH exposure modifies cysteines of α-ENaC while data from single channel patch clamp analysis confirm that 0.16% EtOH increased ENaC activity in rat alveolar cells. In vivo lung fluid clearance demonstrated a latent increase in fluid clearance in mice receiving EtOH diet. Ethanol mice given a tracheal instillation of LPS demonstrated early lung fluid clearance compared to caloric control mice and C57Bl/6 mice. Standard biochemical techniques reveal that chronic EtOH consumption resulted in greater protein expression of the catalytic gp91phox subunit and the obligate Rac1 protein. Collectively these data suggest that chronic EtOH consumption may lead to altered regulation of ENaC, contributing to a ‘pro-injury’ environment in the alcohol lung. PMID:23382956

A novel bifunctional metabolizable linker for the conjugation of antibodies with radionuclides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arano, Y.; Matsushima, H.; Tagawa, M.

1991-03-01

A novel heterogeneous bifunctional reagent containing an ester bond, N-((4-(2-maleimidoethoxy)-succinyl)oxy)succinimide (MESS), was designed and synthesized for the conjugation of antibodies with the gallium-67 (67Ga) chelate of succinyldeferoxamine (SDF) via the ester bond. MESS was synthesized by the acylation of N-(2-hydroxyethyl)maleimide with succinic anhydride, followed by the activation of the resulting carboxylic acid to a succinimido ester. MESS possesses a maleimide group for protein conjugation and an active ester group for deferoxamine (DFO) coupling, and the two functional groups are linked via ester bonding. Conjugation of 67Ga-SDF with nonspecific human IgG was performed by reacting freshly thiolated IgG with the reactionmore » product of MESS and DFO, followed by 67Ga labeling of the resulting conjugate using GaCl3 (67Ga-DFO-MESS-IgG). For comparison, 67Ga-DFO conjugated nonspecific human IgG with a nonmetabolizable linkage was synthesized under the same conjugation conditions as those for 67Ga-DFO-MESS-IgG, using a nonmetabolizable heterogenous bifunctional reagent (N-((6-maleimidocaproyl)oxy)succinimide, EMCS) instead of MESS (67Ga-DFO-EMCS-IgG). HPLC size-exclusion chromatography of both preparations showed a single radioactivity and UV peak corresponding to the intact IgG. Generation of 67Ga-SDF from the 67Ga-DFO-MESS-IgG was demonstrated by reverse-phase HPLC analysis and cellulose acetate electrophoresis after the incubation of 67Ga-DFO-MESS-IgG in a buffered solution containing carboxyesterase. After injection of 67Ga-DFO-MESS-IgG into mice, faster radioactivity clearance from the blood and less radioactivity accumulation in the liver, kidney, and spleen was noted than when 67Ga-DFO-EMCS-IgG was injected.« less

Design, Synthesis, and Biological Evaluations of Asymmetric Bow-Tie PAMAM Dendrimer-Based Conjugates for Tumor-Targeted Drug Delivery.

PubMed

Wang, Tao; Zhang, Yaozhong; Wei, Longfei; Teng, Yuhan G; Honda, Tadashi; Ojima, Iwao

2018-04-30

A unique asymmetric bow-tie poly(amidoamine) (PAMAM) dendrimer (ABTD) scaffold was designed and developed as a well-defined macromolecular carrier for tumor-targeted drug delivery. The ABTD scaffold in this study consists of a G3-half-dendron (G3-HD) unit and a G1-half-dendron (G1-HD) unit, bearing thiol moiety in each unit and a bis(maleimide) linker unit, which undergo sequential thiol-maleimide coupling to assemble the scaffold. This assembly methodology is applicable to all other combinations of different generations of PAMAM dendrimers. In the prototype ABTD in this study, 16 biotin moieties were tethered to the G3-HD unit and 4 payloads (new-generation taxoid) to the G1-HD via a self-immolative linker to form an ABTD-tumor-targeting conjugate (ABTD-TTC-1). Two other ABTD-TTCs were synthesized, wherein the G1-HD unit was tethered to a fluorescence-labeled taxoid or to a fluorescent probe. These three ABTD-TTCs were constructed by using a common key ABTD 6 bearing a terminal acetylene group in the G1-HD unit, which was fully characterized as a single molecule by high-resolution mass spectrometry and NMR despite its high molecular weight ( M w : 12 876). Then, the click reaction was employed to couple ABTD 6 with a small-molecule payload or fluorescence probe unit bearing a terminal azide moiety. ABTD-TTC-3, as a surrogate of ABTD-TTC-2, showed substantially enhanced internalization into two cancer cell lines via receptor-mediated endocytosis, attributed to multibinding effect. ABTD-TTC-1 exhibited a remarkable selectivity to cancer cells (1400-7500 times) compared to human normal cells, which demonstrates the salient feature and bright prospect of the ABTD-based tumor-targeted drug-delivery system.

First characterization of three cyclophilin family proteins in the oyster, Crassostrea ariakensis Gould.

PubMed

Xu, Ting; Xie, Jiasong; Yang, Shoubao; Ye, Shigen; Luo, Ming; Wu, Xinzhong

2016-08-01

Cyclophilins (CyPs) are a family of proteins that bind the immunosuppressive agent cyclosporin A (CsA) with high-affinity and belong to one of the three superfamilies of peptidyl-prolyl cis-trans isomerases (PPIase). In this report, three cyclophilin genes (Ca-CyPs), including Ca-CyPA, Ca-CyPB and Ca-PPIL3, were identified from oyster, Crassostrea ariakensis Gould in which Ca-CyPA encodes a protein with 165 amino acid sequences, Ca-CyPB encodes a protein with 217 amino acid sequences and Ca-PPIL3 encodes a protein with 162 amino acid sequences. All of the three Ca-CyPs genes contain a typical CyP-PPIase domain with its signature sequences and Ca-CyPB contains an N-signal peptide sequences. Tissue distribution study revealed that Ca-CyPs were ubiquitously expressed in all examined tissues and the highest levels were observed in hemocytes. RLO incubation upregulated the mRNA expression levels of Ca-CyPs, indicating that three Ca-CyPs might be involved in oyster immune response against RLO infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ophthalmic delivery of Cyclosporine A from Brij-97 microemulsion and surfactant-laden p-HEMA hydrogels.

PubMed

Kapoor, Yash; Chauhan, Anuj

2008-09-01

Cyclosporine A (CyA) is an immunosuppressant drug that is used for treating a variety of ocular diseases and disorders. CyA is commonly delivered via eye drops, which is highly inefficient due to a low bioavailability of less than 5%. The bioavailability of ophthalmic drugs can be substantially improved to about 50% by delivering them via contact lenses. This paper focuses on the development of nanostructured poly (2-hydroxyethyl methacrylate) (p-HEMA) hydrogels containing microemulsions or micelles of Brij 97 (C(18)H(35)(OCH(2)CH(2))(10)) for extended delivery of CyA. Release of CyA from these nanostructured hydrogels was performed in vitro to explore the mechanisms of release and the effects of surfactant concentration, processing conditions and storage on the release kinetics. Results show that the surfactant and microemulsion-laden gels can deliver CyA at therapeutic dosages for a period of about 20 days. Release of the drug is diffusion controlled with effective diffusivities decreasing with increasing surfactant loading. The release kinetics are relatively similar for both surfactant and microemulsion-laden gels with comparable surfactant loading. The results also show that these hydrogels retain their effectiveness even after exposure to all the relevant processing conditions including unreacted monomer extraction, autoclaving and packaging, and so these materials seem to be very promising for ophthalmic delivery of CyA and perhaps other drugs.

Kinetics of antigen binding to arrays of antibodies in different sized spots

NASA Technical Reports Server (NTRS)

Sapsford, K. E.; Liron, Z.; Shubin, Y. S.; Ligler, F. S.

2001-01-01

A fluorescence-based array biosensor has been developed which can measure the binding kinetics of an antigen to an immobilized antibody in real time. A patterned array of antibodies immobilized on the surface of a planar waveguide was used to capture a Cy5-labeled antigen present in a solution that was continuously flowed over the surface. The CCD image of the waveguide was monitored continuously for 25 min. The resulting exponential rise in fluorescence signal was determined by image analysis software and fitted to a reaction-limited kinetics model, giving a kf of 3.6 x 10(5) M(-1) s(-1). Different spot sizes were then patterned on the surface of the waveguide using either a PDMS flow cell or laser exposure, producing width sizes ranging from 80 to 1145 microm. It was demonstrated that under flow conditions, the reduction of spot size did not alter the association rate of the antigen with immobilized antibody; however, as the spot width decreased to < 200 nm, the signal intensity also decreased.

Development of polymer-polysaccharide hydrogels for controlling drug delivery

NASA Astrophysics Data System (ADS)

Baldwin, Aaron David

The use of polymers as biomaterials has evolved over the past several decades, encompassing an expanding synthetic toolbox and many bio-mimetic approaches. Both synthetic and natural polymers have been used as components for biomaterials as their unique chemical structures can provide specific functions for desired applications. Of these materials, heparin, a highly sulfated naturally occurring polysaccharide, has been investigated extensively as a core component in drug delivery platforms and tissue engineering. The goal of this work was to further explore the use of heparin via conjugation with synthetic polymers for applications in drug delivery. We begin by investigating low molecular weight heparin (LMWH), a depolymerized heparin that is used medicinally in the prevention of thrombosis by subcutaneous injection or intravenous drip. Certain disease states or disorders require frequent administration with invasive delivery modalities leading to compliance issues for individuals on prolonged therapeutic courses. To address these issues, a long-term delivery method was developed for LMWH via subcutaneous injection of in situ hydrogelators. This therapy was accomplished by chemical modification of LMWH with maleimide functionality so that it may be crosslinked into continuous hydrogel networks with four-arm thiolated polyethylene glycol (PEG-SH). These hydrogels degrade via hydrolysis over a period of weeks and release bioactive LMWH with first-order kinetics as determined by in vitro and in vivo models, thus indicating the possibility of an alternative means of heparin delivery over current accepted methodologies. Evaluation of the maleimide-thiol chemistries applied in the LMWH hydrogels revealed reversibility for some conjugates under reducing conditions. Addition chemistries, such as maleimide-thiol reactions, are widely employed in biological conjugates and are generally accepted as stable. Here we show that the resulting succinimide thioether formed by the Michael type addition of thiol derivatives to N-ethylmaleimide (NEM) undergoes retro and exchange reactions in the presence of other thiol compounds at physiological pH and temperature. Model studies of NEM conjugated to various thiols (4-mercaptophenylacetic acid (MPA), N-acetylcysteine, or 3-mercaptopropionic acid (MP)), incubated with a naturally occurring reducing agent, glutathione, showed half-lives from 20-80 hrs with extents of conversion from 20-90% for MPA and N-acetylcysteine conjugates. The kinetics of the retro reactions and extent of exchange can be modulated by the Michael donor's reactivity; therefore the degradation of maleimide-thiol adducts could be tuned for controlled release of drugs or degradation of materials at timescales different than those currently possible via disulfide-mediated release. The reduction sensitive maleimide-thiol chemistry was then investigated as a crosslinking mechanism for LMWH hydrogels. Crosslinking maleimide functionalized LMWH with PEG functionalized with thiophenyl functionalities imparted glutathione sensitivity. 4-mercaptophenylpropionic acid and 2,2-dimethyl-3-(4-mercaptophenyl)propionic acid, induced sensitivity to glutathione as shown by a decrease in degradation time of 4x and 5x respectively. The pseudo-first order retro reaction constants were approximately an order of magnitude slower than hydrogels crosslinked via disulfide linkages, indicating the potential use of the retro succinimide-thioether covalent bonds for reduction mediated release and/or degradation with increased blood stability and prolonged drug delivery timescales compared to disulfide chemistries. In summary, this work highlights the use of polymer-polysaccharide hydrogels composed of LMWH and PEG as investigated for drug delivery and as a tool for elucidating a novel reduction sensitive controlled release mechanism.

[Stimulation of DNA molecules association with amphiphilic derivatives of 1,3-diazaadamantane containing hydrophobic side chanins].

PubMed

Mamaeva, O K; Gabrielian, A G; Arutiunian, G L; Bocharova, T N; Smirnova, E A; Volodin, A A; Shchelkina, A K; Kaliuzhnyĭ, D N

2014-01-01

Earlier, a new class of compounds--amphiphilic derivatives of 1,3-diazaadamantanes, capable of facilitating the strand exchange in the system of short oligonucleotides was revealed. Longer hydrophobic side chains of 1,3-diazaadamantanes promoted stronger acceleration of the reaction. In this study, interaction with DNA of two 1,3-diazaadamantane derivatives containing different side chains was investigated by use of optical methods. Concentration of the investigated 1,3-diazaadamantans micelles formation were determined by the means of monitoring fluorescence intensity enhancement of 1-anilinonaphtalene-8-sulphonate probe; as well as the ranges of concentrations where the compounds/water mixtures existed as true solutions. 1,3-diazaadamantanes affinity to DNA was determined with Fluorescent Intercalator Displacement (FID) approach. Significant increase in hydrodynamic volume of short DNA hairpins in the complexes with 1,3-diazaadamantanes was revealed by estimation of the fluorescence polarization of ethidium bromide probe bound to the hairpins. Intermolecular association of DNA hairpins upon binding with 1,3-diazaadamantans was confirmed by Förster resonance energy transfer in system of an equimolar mixture of fluorescently labeled with Cy-3 and Cy-5 hairpins. In this study, the number of positive charges at 1,3-diazaadamantane derivatives containing side chains of different lengths was demonstrated to affect their affinity to DNA, whereas longer length of the hydrophobic side chains ensured more efficient interaction between the DNA duplexes that may facilitate, in particular, DNA strand exchange.

Evidence for two koi herpesvirus (KHV) genotypes in South Korea.

PubMed

Kim, Hyoung Jun; Kwon, Se Ryun

2013-06-13

The geographic distribution of koi herpesvirus (KHV) has recently been analyzed by polymerase chain reaction (PCR, based on the alleles of 3 domains) and sequence analysis using 3 regions of KHV genomic DNA (SphI-5, 9/5, and the thymidine kinase gene). In this study, samples from 6 carp showing symptoms of KHV infection in 2008 were examined for the presence of KHV by using PCR and cell culture isolation methods. KHV was detected in 2 (Pyeongtaek and Buan) of the samples. Sequence analysis revealed that the genotype of the KHV PT-08 isolate was Asia genotype variant 1 (A1), and the genotype of the KHV BA-08 isolate was European genotype variant 4 (E4). In addition, PCR patterns and sequence analysis based on the alleles of 3 domains of an alternate KHV classification system confirmed that the genotype of the KHV PT-08 isolate was CyHV3-J, and the genotype of the KHV BA-08 isolate was CyHV3-third genotype. To our knowledge, this is the first study to demonstrate the presence of 2 genotypes of KHV (genotype A1/CyHV3-J; genotype E4/CyHV3-third genotype) in South Korea.

Poly(2-vinyl pyridine)-block-poly(ethylene oxide) featuring a furan group at the block junction-synthesis and functionalization.

PubMed

Rudolph, Tobias; Barthel, Markus J; Kretschmer, Florian; Mansfeld, Ulrich; Hoeppener, Stephanie; Hager, Martin D; Schubert, Ulrich S; Schacher, Felix H

2014-05-01

Furfuryl glycidyl ether (FGE) represents a highly versatile monomer for the preparation of reversibly cross-linkable nanostructured materials via Diels-Alder reactions. Here, the use of FGE for the mid-chain functionalization of a P2VP-b-PEO diblock copolymer is reported. The material features one furan moiety at the block junction, P2VP68 -FGE-b-PEO390 , which can be subsequently addressed in Diels-Alder reactions using maleimide-functionalized counterparts. The presence of the FGE moiety enables the introduction of dyes as model labels or the formation of hetero-grafted brushes as shell on hybrid Au@Polymer nanoparticles. This renders P2VP68 -FGE-b-PEO390 , a powerful tool for selective functionalization reactions, including the modification of surfaces. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detection of hydroxyapatite in calcified cardiovascular tissues.

PubMed

Lee, Jae Sam; Morrisett, Joel D; Tung, Ching-Hsuan

2012-10-01

The objective of this study is to develop a method for selective detection of the calcific (hydroxyapatite) component in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues ex vivo. This method uses a novel optical molecular imaging contrast dye, Cy-HABP-19, to target calcified cells and tissues. A peptide that mimics the binding affinity of osteocalcin was used to label hydroxyapatite in vitro and ex vivo. Morphological changes in vascular smooth muscle cells were evaluated at an early stage of the mineralization process induced by extrinsic stimuli, osteogenic factors and a magnetic suspension cell culture. Hydroxyapatite components were detected in monolayers of these cells in the presence of osteogenic factors and a magnetic suspension environment. Atherosclerotic plaque contains multiple components including lipidic, fibrotic, thrombotic, and calcific materials. Using optical imaging and the Cy-HABP-19 molecular imaging probe, we demonstrated that hydroxyapatite components could be selectively distinguished from various calcium salts in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues, carotid endarterectomy samples and aortic valves, ex vivo. Hydroxyapatite deposits in cardiovascular tissues were selectively detected in the early stage of the calcification process using our Cy-HABP-19 probe. This new probe makes it possible to study the earliest events associated with vascular hydroxyapatite deposition at the cellular and molecular levels. This target-selective molecular imaging probe approach holds high potential for revealing early pathophysiological changes, leading to progression, regression, or stabilization of cardiovascular diseases. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Detection of Hydroxyapatite in Calcified Cardiovascular Tissues

PubMed Central

Lee, Jae Sam; Morrisett, Joel D.; Tung, Ching-Hsuan

2012-01-01

Objective The objective of this study is to develop a method for selective detection of the calcific (hydroxyapatite) component in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues ex vivo. This method uses a novel optical molecular imaging contrast dye, Cy-HABP-19, to target calcified cells and tissues. Methods A peptide that mimics the binding affinity of osteocalcin was used to label hydroxyapatite in vitro and ex vivo. Morphological changes in vascular smooth muscle cells were evaluated at an early stage of the mineralization process induced by extrinsic stimuli, osteogenic factors and a magnetic suspension cell culture. Hydroxyapatite components were detected in monolayers of these cells in the presence of osteogenic factors and a magnetic suspension environment. Results Atherosclerotic plaque contains multiple components including lipidic, fibrotic, thrombotic, and calcific materials. Using optical imaging and the Cy-HABP-19 molecular imaging probe, we demonstrated that hydroxyapatite components could be selectively distinguished from various calcium salts in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues, carotid endarterectomy samples and aortic valves, ex vivo. Conclusion Hydroxyapatite deposits in cardiovascular tissues were selectively detected in the early stage of the calcification process using our Cy-HABP-19 probe. This new probe makes it possible to study the earliest events associated with vascular hydroxyapatite deposition at the cellular and molecular levels. This target-selective molecular imaging probe approach holds high potential for revealing early pathophysiological changes, leading to progression, regression, or stabilization of cardiovascular diseases. PMID:22877867

Siloxane containing addition polyimides

NASA Technical Reports Server (NTRS)

Maudgal, S.; St. Clair, T. L.

1984-01-01

Addition polyimide oligomers have been synthesized from bis(gamma-aminopropyl) tetramethyldisiloxane and 3, 3', 4, 4'-benzophenonetetracarboxylic dianhydride using a variety of latent crosslinking groups as endcappers. The prepolymers were isolated and characterized for solubility (in amide, chlorinated and ether solvents), melt flow and cure properties. The most promising systems, maleimide and acetylene terminated prepolymers, were selected for detailed study. Graphite cloth reinforced composites were prepared and properties compared with those of graphite/Kerimid 601, a commercially available bismaleimide. Mixtures of the maleimide terminated system with Kerimid 601, in varying proportions, were also studied.

Statistical optimization for improved production of cyclosporin a in solid-state fermentation.

PubMed

Survase, Shrikant A; Annapure, Uday S; Singhal, Rekha S

2009-11-01

This work evaluates the effect of different amino acids on production of CyA production in solid-state fermentation that was previously optimized for different fermentation parameters by one-factor-at-a-time for the maximum production of CyA by Tolypocladium inflatum MTCC 557. Based on the Plackett-Burman design, glycerol, ammonium sulfate, FeCl3, and inoculum size were selected for further optimization by response surface methodology (RSM). After identifying effective nutrients, RSM was used to develop mathematical model equations, study responses, and establish the optimum concentrations of the key nutrients for higher CyA production. It was observed that supplementation of medium containing (% w/w) glycerol, 1.53; ammonium sulfate, 0.95; FeCl3, 0.18; and inoculum size 6.4 ml/5g yielded a maximum of 7,106 mg/kg as compared with 6,480 mg CyA/kg substrate using one factor at a time. In the second step, the effect of amino acids on the production of CyA was studied. Addition of L-valine and L-leucine in combination after 20 h of fermentation resulted in maximum production of 8,166 mg/kg.

The rat red blood cell proteome is altered by priming with 2-butoxyethanol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Palkar, Prajakta S.; Kakhniashvili, David G.; Goodman, Steven R.

2008-08-01

Administration of a low priming dose of 2-butoxyethanol (BE, 500 mg/kg, p.o.) 7 days prior to a larger LD{sub 90} dose (1500 mg BE/kg, p.o.) offers protection against the lethal dose-induced hemolysis and death in female Sprague Dawley rats because of prompt and efficient replacement of red blood cells (RBCs) with new resilient RBCs. The objective of the present work was to analyze the altered proteome of RBCs upon priming with BE in order to identify the potential anti-hemolytic survival proteins induced in the primed rat RBCs (P-RBCs) as opposed to vehicle-treated RBCs (V-RBCs). The RBCs from the two groupsmore » were fractionated into membrane and cytosolic fractions. The cytosolic fractions were further fractionated for proteomic analysis into 3 fractions. The fractions were labeled with Cy3 and Cy5 fluorescent dyes and subjected to 2-dimensional differential gel electrophoresis (DIGE) to analyze the protein profiles. Seven membrane and 8 cytosolic proteins were found to be significantly increased ({>=} 2.5 fold) in P-RBCs as compared to V-RBCs. The identified proteins can be classified into antioxidant, membrane skeleton, protein turnover, lipid raft, and energy metabolism components. Increased levels of the proteins from antioxidant and membrane skeleton groups were confirmed by Western blot analysis. The study provides the first report on protein profiling of rat RBCs as well as on alteration of the proteome upon exposure to a priming dose of hemotoxicant. Further studies are needed to prove the protective role of the identified proteins and will initiate the field of survival/protective/anti-hemolytic proteins in RBCs.« less

Subconjunctival dendrimer-drug therapy for the treatment of dry eye in rabbits with induced autoimmune dacryoadenitis.

PubMed

Lin, Hui; Liu, Ying; Kambhampati, Siva P; Hsu, Chih-Chien; Kannan, Rangaramanujam M; Yiu, Samuel C

2018-05-16

To investigate the efficacy of a single subconjunctival injection of dendrimer-dexamethasone to treat dry eye in a rabbit model of induced autoimmune dacryoadenitis (AID). Dendrimer biodistribution after subconjuntival injection in AID animals was evaluated using Cy5-labelled dendrimer (D-Cy5) and confocal microscopy. Diseased animals were treated with free dexamethasone (Free-Dex), dendrimer-dexamethasone (D-Dex), or saline via a single subconjunctival injection. The efficacy was evaluated using various clinical evaluations, such as Schirmer's test, tear breakup time (TBUT), and fluorescein and rose bengal staining. Histopathology was evaluated by H&E staining and immunostaining. Levels of inflammatory cytokines and aquaporin proteins in the LGs were determined by real-time PCR. Subconjunctivally administered dendrimers selectively localized in the inflamed LGs, and were taken up by the infiltrated cells. At two weeks post-treatment, the D-Dex group showed improved clinical evaluations. No significant changes were observed in other groups. H&E staining demonstrated, less inflammatory cell infiltration and fewer atrophic acini in D-Dex group, compared to those treated with saline or Free-Dex. Immunohistochemistry demonstrated that the intensity of CD-18 (+) and RTLA (+) was weaker in LGs in the D-Dex group than in other treatment rabbits. Pro-inflammatory gene expression levels of MMP9, IL6, IL8, and TNFα were significantly decreased in the D-Dex group compared to the saline group. The dendrimer exhibits pathology-dependent biodistribution in the inflamed LGs. Subconjunctivally administered D-Dex suppressed LG inflammation, leading to partial recovery of LG function with clinical improvement in induced AID. Sjögren's patients may benefit from this targeted nanomedicine approach. Copyright © 2018. Published by Elsevier Inc.

Distribution of cyclophilin B-binding sites in the subsets of human peripheral blood lymphocytes.

PubMed

Denys, A; Allain, F; Foxwell, B; Spik, G

1997-08-01

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway and released in biological fluids. We have recently demonstrated that both free CyPB and CyPB-CsA complex specifically bind to peripheral blood T lymphocytes and are internalized. These results suggest that CyPB might promote the targeting of the drug into sensitive cells. Peripheral blood lymphocytes are subdivided in several populations according to their biological functions and sensitivity to CsA. We have investigated the binding of CyPB to these different subsets using a CyPB derivatized by fluorescein through its single cysteine which retains its binding properties. We have confirmed that only T cells were involved in the interaction with CyPB. The ligand binding was found to be heterogeneously distributed on the different T-cell subsets and surface-bound CyPB was mainly associated with the CD4-positive cells. No significant difference was noted between the CD45RA and CD45RO subsets, demonstrating that CyPB-binding sites were equally distributed between native and memory T cells. CD3 stimulation of T lymphocytes led to a decrease in the CyPB-binding capacity, that may be explained by a down-regulation of the CyPB-receptor expression upon T-cell activation. Finally, we demonstrated that CyPB-receptor-positive cells, isolated on CyPB sulphydryl-coupled affinity matrices, are more sensitive to CyPB-complexed CsA than mixed peripheral blood lymphocytes, suggesting that CyPB potentiates CsA activity through the binding of the complex. Taken together, our results demonstrate that CyPB-binding sites are mainly associated with resting cells of the helper T lymphocyte, and that CyPB might modulate the distribution of CsA through the drug targeting to sensitive cells.

The Communication Supports Inventory-Children & Youth (CSI-CY), a new instrument based on the ICF-CY.

PubMed

Rowland, Charity; Fried-Oken, Melanie; Bowser, Gayl; Granlund, Mats; Lollar, Donald; Phelps, Randall; Simeonsson, Rune J; Steiner, Sandra A M

2016-09-01

Two studies are presented that evaluated the Communication Supports Inventory-Children & Youth (CSI-CY), an instrument designed to facilitate the development of communication-related educational goals for students with complex communication needs (CCN). The CSI-CY incorporates a code set based on the ICF-CY. The studies were designed to determine the effect of using the CSI-CY on IEP goals for students with CCN and to evaluate consumer satisfaction. In Study 1, sixty-one educators and speech-language pathologists were randomly assigned to either (a) provide a student's current IEP (control group) or (b) complete the CSI-CY prior to preparing a student's next IEP and to submit the new IEP (experimental group). Study 2 was a field test to generate consumer satisfaction data. Study 1 showed that IEP goals submitted by participants in the experimental group referenced CSI-CY-related content significantly more frequently than did those submitted by control participants. Study 2 revealed high satisfaction with the instrument. The code set basis of the CSI-CY extends the common language of the ICF-CY to practical educational use for children with CCN across diagnostic groups. The CSI-CY is well regarded as an instrument to inform the content of communication goals related to CCN. Implications for Rehabilitation The CSI-CY will guide rehabilitation professionals to develop goals for children with complex communication impairments. The CSI-CY is a new instrument that is based on the ICF-CY for documentation of communication goals.

Reliable and accurate CD4+ T cell count and percent by the portable flow cytometer CyFlow MiniPOC and "CD4 Easy Count Kit-Dry", as revealed by the comparison with the gold standard dual platform technology.

PubMed

Nasi, Milena; De Biasi, Sara; Bianchini, Elena; Gibellini, Lara; Pinti, Marcello; Scacchetti, Tiziana; Trenti, Tommaso; Borghi, Vanni; Mussini, Cristina; Cossarizza, Andrea

2015-01-01

An accurate and affordable CD4+ T cells count is an essential tool in the fight against HIV/AIDS. Flow cytometry (FCM) is the "gold standard" for counting such cells, but this technique is expensive and requires sophisticated equipment, temperature-sensitive monoclonal antibodies (mAbs) and trained personnel. The lack of access to technical support and quality assurance programs thus limits the use of FCM in resource-constrained countries. We have tested the accuracy, the precision and the carry-over contamination of Partec CyFlow MiniPOC, a portable and economically affordable flow cytometer designed for CD4+ count and percentage, used along with the "CD4% Count Kit-Dry". Venous blood from 59 adult HIV+ patients (age: 25-58 years; 43 males and 16 females) was collected and stained with the "MiniPOC CD4% Count Kit-Dry". CD4+ count and percentage were then determined in triplicate by the CyFlow MiniPOC. In parallel, CD4 count was performed using mAbs and a CyFlow Counter, or by a dual platform system (from Beckman Coulter) based upon Cytomic FC500 ("Cytostat tetrachrome kit" for mAbs) and Coulter HmX Hematology Analyzer (for absolute cell count). The accuracy of CyFlow MiniPOC against Cytomic FC500 showed a correlation coefficient (CC) of 0.98 and 0.97 for CD4+ count and percentage, respectively. The accuracy of CyFlow MiniPOC against CyFlow Counter showed a CC of 0.99 and 0.99 for CD4 T cell count and percentage, respectively. CyFlow MiniPOC showed an excellent repeatability: CD4+ cell count and percentage were analyzed on two instruments, with an intra-assay precision below ± 5% deviation. Finally, there was no carry-over contamination for samples at all CD4 values, regardless of their position in the sequence of analysis. The cost-effective CyFlow MiniPOC produces rapid, reliable and accurate results that are fully comparable with those from highly expensive dual platform systems.

Parents' Assessments of Disability in Their Children Using World Health Organization International Classification of Functioning, Disability and Health, Child and Youth Version Joined Body Functions and Activity Codes Related to Everyday Life.

PubMed

Illum, Niels Ove; Gradel, Kim Oren

2017-01-01

To help parents assess disability in their own children using World Health Organization (WHO) International Classification of Functioning, Disability and Health, Child and Youth Version (ICF-CY) code qualifier scoring and to assess the validity and reliability of the data sets obtained. Parents of 162 children with spina bifida, spinal muscular atrophy, muscular disorders, cerebral palsy, visual impairment, hearing impairment, mental disability, or disability following brain tumours performed scoring for 26 body functions qualifiers (b codes) and activities and participation qualifiers (d codes). Scoring was repeated after 6 months. Psychometric and Rasch data analysis was undertaken. The initial and repeated data had Cronbach α of 0.96 and 0.97, respectively. Inter-code correlation was 0.54 (range: 0.23-0.91) and 0.76 (range: 0.20-0.92). The corrected code-total correlations were 0.72 (range: 0.49-0.83) and 0.75 (range: 0.50-0.87). When repeated, the ICF-CY code qualifier scoring showed a correlation R of 0.90. Rasch analysis of the selected ICF-CY code data demonstrated a mean measure of 0.00 and 0.00, respectively. Code qualifier infit mean square (MNSQ) had a mean of 1.01 and 1.00. The mean corresponding outfit MNSQ was 1.05 and 1.01. The ICF-CY code τ thresholds and category measures were continuous when assessed and reassessed by parents. Participating children had a mean of 56 codes scores (range: 26-130) before and a mean of 55.9 scores (range: 25-125) after repeat. Corresponding measures were -1.10 (range: -5.31 to 5.25) and -1.11 (range: -5.42 to 5.36), respectively. Based on measures obtained at the 2 occasions, the correlation coefficient R was 0.84. The child code map showed coherence of ICF-CY codes at each level. There was continuity in covering the range across disabilities. And, first and foremost, the distribution of codes reflexed a true continuity in disability with codes for motor functions activated first, then codes for cognitive functions, and, finally, codes for more complex functions. Parents can assess their own children in a valid and reliable way, and if the WHO ICF-CY second-level code data set is functioning in a clinically sound way, it can be employed as a tool for identifying the severity of disabilities and for monitoring changes in those disabilities over time. The ICF-CY codes selected in this study might be one cornerstone in forming a national or even international generic set of ICF-CY codes for the benefit of children with disabilities, their parents, and caregivers and for the whole community supporting with children with disabilities on a daily and perpetual basis.

Parents’ Assessments of Disability in Their Children Using World Health Organization International Classification of Functioning, Disability and Health, Child and Youth Version Joined Body Functions and Activity Codes Related to Everyday Life

PubMed Central

Illum, Niels Ove; Gradel, Kim Oren

2017-01-01

AIM To help parents assess disability in their own children using World Health Organization (WHO) International Classification of Functioning, Disability and Health, Child and Youth Version (ICF-CY) code qualifier scoring and to assess the validity and reliability of the data sets obtained. METHOD Parents of 162 children with spina bifida, spinal muscular atrophy, muscular disorders, cerebral palsy, visual impairment, hearing impairment, mental disability, or disability following brain tumours performed scoring for 26 body functions qualifiers (b codes) and activities and participation qualifiers (d codes). Scoring was repeated after 6 months. Psychometric and Rasch data analysis was undertaken. RESULTS The initial and repeated data had Cronbach α of 0.96 and 0.97, respectively. Inter-code correlation was 0.54 (range: 0.23-0.91) and 0.76 (range: 0.20-0.92). The corrected code-total correlations were 0.72 (range: 0.49-0.83) and 0.75 (range: 0.50-0.87). When repeated, the ICF-CY code qualifier scoring showed a correlation R of 0.90. Rasch analysis of the selected ICF-CY code data demonstrated a mean measure of 0.00 and 0.00, respectively. Code qualifier infit mean square (MNSQ) had a mean of 1.01 and 1.00. The mean corresponding outfit MNSQ was 1.05 and 1.01. The ICF-CY code τ thresholds and category measures were continuous when assessed and reassessed by parents. Participating children had a mean of 56 codes scores (range: 26-130) before and a mean of 55.9 scores (range: 25-125) after repeat. Corresponding measures were −1.10 (range: −5.31 to 5.25) and −1.11 (range: −5.42 to 5.36), respectively. Based on measures obtained at the 2 occasions, the correlation coefficient R was 0.84. The child code map showed coherence of ICF-CY codes at each level. There was continuity in covering the range across disabilities. And, first and foremost, the distribution of codes reflexed a true continuity in disability with codes for motor functions activated first, then codes for cognitive functions, and, finally, codes for more complex functions. CONCLUSIONS Parents can assess their own children in a valid and reliable way, and if the WHO ICF-CY second-level code data set is functioning in a clinically sound way, it can be employed as a tool for identifying the severity of disabilities and for monitoring changes in those disabilities over time. The ICF-CY codes selected in this study might be one cornerstone in forming a national or even international generic set of ICF-CY codes for the benefit of children with disabilities, their parents, and caregivers and for the whole community supporting with children with disabilities on a daily and perpetual basis. PMID:28680270

Cross-cultural validation and psychometric evaluation of the Participation and Environment Measure for Children and Youth in Korea.

PubMed

Jeong, Yunwha; Law, Mary; Stratford, Paul; DeMatteo, Carol; Kim, Hwan

2016-11-01

To develop the Korean version of the Participation and Environment Measure for Children and Youth (KPEM-CY) and examine its psychometric properties. The PEM-CY was cross-culturally translated into Korean using a specific guideline: pre-review of participation items, forward/backward translation, expert committee review, pre-test of the KPEM-CY and final review. To establish internal consistency, test-retest reliability and construct validity of the KPEM-CY, 80 parents of children with disabilities aged 5-13 years were recruited in South Korea. Across the home, school and community settings, 76% of participation items and 29% of environment items were revised to improve their fit with Korean culture. Internal consistency was moderate to excellent (0.67-0.92) for different summary scores. Test-retest reliability was excellent (>0.75) in the summary scores of participation frequency and extent of involvement across the three settings and moderate to excellent (0.53-0.95) in all summary scores at home. Child's age, type of school and annual income were the factors that significantly influenced specific dimensions of participation and environment across all settings. Results indicated that the KPEM-CY is equivalent to the original PEM-CY and has initial evidence of reliability and validity for use with Korean children with disabilities. Implications for rehabilitation Because 'participation' is a key outcome of the rehabilitation, measuring comprehensive participation of children with disabilities is necessary. The PEM-CY is a parent-report survey measure to assess comprehensive participation of children and youth and environment, which affect their participation, at home, school and in the community. A cross-cultural adaptation process is mandatory to adapt the measurement tool to a new culture or country. The Korean PEM-CY has both reliability and validity and can therefore generate useful clinical data for Korean children with disabilities.

Intracellularly monitoring/imaging the release of doxorubicin from pH-responsive nanoparticles using Förster resonance energy transfer.

PubMed

Chen, Ko-Jie; Chiu, Ya-Ling; Chen, Yu-Ming; Ho, Yi-Cheng; Sung, Hsing-Wen

2011-04-01

Stimuli-responsive nanoparticles (NPs) have been receiving much attention as a drug-delivery vehicle for therapeutic applications; once internalized into cells, the intracellular fate of NPs and their drug release behavior in response to local stimuli must be understood for efficient delivery of therapeutics. In this study, we prepared pH-responsive doxorubicin (DOX)-loaded NPs, made of N-palmitoyl chitosan bearing a Cy5 moiety (Cy5-NPCS), as an anticancer delivery device. The results of our molecular dynamic simulations showed that the ability of Cy5-NPCS to self-associate offered the close proximity between the donor (DOX) and the acceptor (Cy5) required for Förster resonance energy transfer (FRET), while the pH-driven structure transition prescribed the on-to-off switch of the energy transfer. The caveolae-mediated pathway played a major role in the internalization of NPCS NPs. Using the concept of FRET, we found that the DOX fluorescence in the cytosol was first seen when NPCS NPs were present in the slightly acidic early endosomes. Following NPCS NPs trafficking into a more acidic organelle (late endosomes/lysosomes), a more evident release of DOX into the cytosol was observed; the released DOX was then gradually accumulated in the cell nuclei, leading to a significant cytotoxicity. Understanding the fate of NPs with respect to their intracellular localization and drug release behavior is crucial for the rational design of drug carriers. Copyright © 2010 Elsevier Ltd. All rights reserved.

Cross-Conjugated Nanoarchitectures

DTIC Science & Technology

2013-08-23

compounds were further evaluated by Lippert –Mataga analysis of the fluorescence solvatochromism and measurement of quantum yields and fluorescence...1.9 1.1 A(mP)2A Cy 0.49 5.5 0.90 0.93 D(Th)2D Cy 0.008 1.1 0.07 9 A(Th)2A Tol 0.014 2.1f 0.07 4.7 a Calculated from Lippert –Mataga plots for...Δfʹ region of the Lippert –Mataga plot. d Double exponential fit: τ1 = 21.5 ns (73%) and τ2 = 3.7 ns (27%). e Double exponential fit: τ1 = 0.85 ns

Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acids

PubMed Central

Iqbal, Asif; Arslan, Sinan; Okumus, Burak; Wilson, Timothy J.; Giraud, Gerard; Norman, David G.; Ha, Taekjip; Lilley, David M. J.

2008-01-01

We have found that the efficiency of fluorescence resonance energy transfer between Cy3 and Cy5 terminally attached to the 5′ ends of a DNA duplex is significantly affected by the relative orientation of the two fluorophores. The cyanine fluorophores are predominantly stacked on the ends of the helix in the manner of an additional base pair, and thus their relative orientation depends on the length of the helix. Observed fluorescence resonance energy transfer (FRET) efficiency depends on the length of the helix, as well as its helical periodicity. By changing the helical geometry from B form double-stranded DNA to A form hybrid RNA/DNA, a marked phase shift occurs in the modulation of FRET efficiency with helix length. Both curves are well explained by the standard geometry of B and A form helices. The observed modulation for both polymers is less than that calculated for a fully rigid attachment of the fluorophores. However, a model involving lateral mobility of the fluorophores on the ends of the helix explains the observed experimental data. This has been further modified to take account of a minor fraction of unstacked fluorophore observed by fluorescent lifetime measurements. Our data unequivocally establish that Förster transfer obeys the orientation dependence as expected for a dipole–dipole interaction. PMID:18676615

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsia, Chu Chieh; Chizhikov, Vladimir E.; Yang, Amy X.

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminatedmore » the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.« less

Ultrasensitive near-infrared fluorescence-enhanced probe for in vivo nitroreductase imaging.

PubMed

Li, Yuhao; Sun, Yun; Li, Jiachang; Su, Qianqian; Yuan, Wei; Dai, Yu; Han, Chunmiao; Wang, Qiuhong; Feng, Wei; Li, Fuyou

2015-05-20

Nitroreductase (NTR) can be overexpressed in hypoxic tumors, thus the selective and efficient detection of NTR is of great importance. To date, although a few optical methods have been reported for the detection of NTR in solution, an effective optical probe for NTR monitoring in vivo is still lacking. Therefore, it is necessary to develop a near-infrared (NIR) fluorescent detection probe for NTR. In this study, five NIR cyanine dyes with fluorescence reporting structure decorated with different nitro aromatic groups, Cy7-1-5, have been designed and explored for possible rapid detection of NTR. Our experimental results presented that only a para-nitro benzoate group modified cyanine probe (Cy7-1) could serve as a rapid NIR fluorescence-enhanced probe for monitoring and bioimaging of NTR. The structure-function relationship has been revealed by theoretical study. The linker connecting the detecting and fluorescence reporting groups and the nitro group position is a key factor for the formation of hydrogen bonds and spatial structure match, inducing the NTR catalytic ability enhancement. The in vitro response and mechanism of the enzyme-catalyzed reduction of Cy7-1 have been investigated through kinetic optical studies and other methods. The results have indicated that an electro-withdrawing group induced electron-transfer process becomes blocked when Cy7-1 is catalytically reduced to Cy7-NH2 by NTR, which is manifested in enhanced fluorescence intensity during the detection process. Confocal fluorescence imaging of hypoxic A549 cells has confirmed the NTR detection ability of Cy7-1 at the cellular level. Importantly, Cy7-1 can detect tumor hypoxia in a murine hypoxic tumor model, showing a rapid and significant enhancement of its NIR fluorescence characteristics suitable for fluorescence bioimaging. This method may potentially be used for tumor hypoxia diagnosis.

Genomic Sequencing and Characterization of Cynomolgus Macaque Cytomegalovirus▿

PubMed Central

Marsh, Angie K.; Willer, David O.; Ambagala, Aruna P. N.; Dzamba, Misko; Chan, Jacqueline K.; Pilon, Richard; Fournier, Jocelyn; Sandstrom, Paul; Brudno, Michael; MacDonald, Kelly S.

2011-01-01

Cytomegalovirus (CMV) infection is the most common opportunistic infection in immunosuppressed individuals, such as transplant recipients or people living with HIV/AIDS, and congenital CMV is the leading viral cause of developmental disabilities in infants. Due to the highly species-specific nature of CMV, animal models that closely recapitulate human CMV (HCMV) are of growing importance for vaccine development. Here we present the genomic sequence of a novel nonhuman primate CMV from cynomolgus macaques (Macaca fascicularis; CyCMV). CyCMV (Ottawa strain) was isolated from the urine of a healthy, captive-bred, 4-year-old cynomolgus macaque of Philippine origin, and the viral genome was sequenced using next-generation Illumina sequencing to an average of 516-fold coverage. The CyCMV genome is 218,041 bp in length, with 49.5% G+C content and 84% protein-coding density. We have identified 262 putative open reading frames (ORFs) with an average coding length of 789 bp. The genomic organization of CyCMV is largely colinear with that of rhesus macaque CMV (RhCMV). Of the 262 CyCMV ORFs, 137 are homologous to HCMV genes, 243 are homologous to RhCMV 68.1, and 200 are homologous to RhCMV 180.92. CyCMV encodes four ORFs that are not present in RhCMV strain 68.1 or 180.92 but have homologies with HCMV (UL30, UL74A, UL126, and UL146). Similar to HCMV, CyCMV does not produce the RhCMV-specific viral homologue of cyclooxygenase-2. This newly characterized CMV may provide a novel model in which to study CMV biology and HCMV vaccine development. PMID:21994460

Achieving Reversible H2/H+ Interconversion at Room Temperature with Enzyme-Inspired Molecular Complexes: A Mechanistic Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Priyadarshani, Nilusha; Dutta, Arnab; Ginovska-Pangovska, Bojana

Inspired by the contribution of the protein scaffold to the efficiency with which enzymes function, we report the first molecular complex that is reversible for electrocatalytic H2 production/oxidation at room temperature in methanol. [Ni(PCy2NPhe2)2]2+ (CyPhe; PR2NR’2 = 1,5-diaza-3,7-diphosphacyclooctane, Cy=cyclohexyl, Phe=phenylalanine), shows reversible behavior in acidic methanol with peripheral phenylalanine groups providing key contributions to the catalytic behavior. The importance of the aromatic rings is implicated in achieving reversibility, based on the lack of reversibility of similar complexes, [Ni(PCy2NAmino Acid2)2]2+, containing arginine (CyArg) or glycine (CyGly). A complex with an added OH group on the ring, (CyTyr; Tyr=Tyrosine), also shows similarmore » behavior. NMR studies reveal a significantly slower rate of chair-boat isomerization for the CyPhe relative to other derivatives, suggesting that the aromatic groups provide structural control by interacting with each other, an observation supported by molecular dynamics studies. NMR studies also show extremely fast proton movement, with a proton pathway from the Ni-H through the pendant amine to the –COOH group. Further, studies of acomplex without the –COOH group, [Ni(PCy2NTym2)2]2+ (CyTym; Tym=Tyramine), are not reversible and have slow proton movement from the pendant amine, demonstrating the essential nature of the –COOH group in achieving reversibility. Finally, methanol is demonstrated to play a critical contributing role. The influence of multiple factors on reversibility for this synthetic catalyst is a demonstration of the intricate interplay between the first, second, and outer coordination spheres and resembles the complexity observed in metalloenzymes.« less

Comparing contents of outcome measures in cerebral palsy using the International Classification of Functioning (ICF-CY): a systematic review.

PubMed

Schiariti, Veronica; Klassen, Anne F; Cieza, Alarcos; Sauve, Karen; O'Donnell, Maureen; Armstrong, Robert; Mâsse, Louise C

2014-01-01

The International Classification of Functioning children and youth version (ICF-CY) provides a universal framework for defining and classifying functioning and disability in children worldwide. To facilitate the application of the ICF in practice, ICF based-tools like the "ICF Core Sets" are being developed. In the context of the development of the ICF-CY Core Sets for children with Cerebral Palsy (CP), the aims of this study were as follows: to identify and compare the content of outcome measures used in studies of children with CP using the ICF-CY coding system; and to describe the most frequently addressed areas of functioning in those studies. We searched multiple databases likely to capture studies involving children with CP from January 1998 to March 2012. We included all English language articles that studied children aged 2-18 years and described an interventional or observational study. Constructs of the outcome measures identified in studies were linked to the ICF-CY by two trained professionals. We found 231 articles that described 238 outcome measures. The outcome measures contained 2193 concepts that were linked to the ICF-CY and covered 161 independent ICF-CY categories. Out of the 161 categories, 53 (33.5%) were related to body functions, 75 (46%) were related to activities/participation, 26 (16.1%) were related to environmental factors, and 7 (4.3%) were related to body structures. This systematic review provides information about content of measures that may guide researchers and clinicians in their selection of an outcome measure for use in a study and/or clinical practice with children with CP. Copyright © 2013 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

Rotational dynamics of spin-labeled F-actin during activation of myosin S1 ATPase using caged ATP.

PubMed Central

Ostap, E. M.; Thomas, D. D.

1991-01-01

The most probable source of force generation in muscle fibers in the rotation of the myosin head when bound to actin. This laboratory has demonstrated that ATP induces microsecond rotational motions of spin-labeled myosin heads bound to actin (Berger, C. L. E. C. Svensson, and D. D. Thomas. 1989. Proc. Natl. Acad. Sci. USA. 86:8753-8757). Our goal is to determine whether the observed ATP-induced rotational motions of actin-bound heads are accompanied by changes in actin rotational motions. We have used saturation transfer electron paramagnetic resonance (ST-EPR) and laser-induced photolysis of caged ATP to monitor changes in the microsecond rotational dynamics of spin-labeled F-actin in the presence of myosin subfragment-1 (S1). A maleimide spin label was attached selectively to cys-374 on actin. In the absence of ATP (with or without caged ATP), the ST-EPR spectrum (corresponding to an effective rotational time of approximately 150 microseconds) was essentially the same as observed for the same spin label bound to cys-707 (SH1) on S1, indicating that S1 is rigidly bound to actin in rigor. At normal ionic strength (micro = 186 mM), a decrease in ST-EPR intensity (increase in microsecond F-actin mobility) was clearly indicated upon photolysis of 1 mM caged ATP with a 50-ms, 351-nm laser pulse. This increase in mobility is due to the complete dissociation of Si from the actin filament. At low ionic strength (micro, = 36 mM), when about half the Si heads remain bound during ATP hydrolysis, no change in the actin mobility was detected, despite much faster motions of labeled S1 bound to actin. Therefore, we conclude that the active interaction of Si, actin,and ATP induces rotation of myosin heads relative to actin, but does not affect the microsecond rotational motion of actin itself, as detected at cys-374 of actin. PMID:1651780

Targeted vertical cross-sectional imaging with handheld near-infrared dual axes confocal fluorescence endomicroscope.

PubMed

Qiu, Zhen; Liu, Zhongyao; Duan, Xiyu; Khondee, Supang; Joshi, Bishnu; Mandella, Michael J; Oldham, Kenn; Kurabayashi, Katsuo; Wang, Thomas D

2013-02-01

We demonstrate vertical cross-sectional (XZ-plane) images of near-infrared (NIR) fluorescence with a handheld dual axes confocal endomicroscope that reveals specific binding of a Cy5.5-labeled peptide to pre-malignant colonic mucosa. This view is perpendicular to the tissue surface, and is similar to that used by pathologists. The scan head is 10 mm in outer diameter (OD), and integrates a one dimensional (1-D) microelectromechanical systems (MEMS) X-axis scanner and a bulky lead zirconate titanate (PZT) based Z-axis actuator. The microscope images in a raster-scanning pattern with a ±6 degrees (mechanical) scan angle at ~3 kHz in the X-axis (fast) and up to 10 Hz (0-400 μm) in the Z-axis (slow). Vertical cross-sectional fluorescence images are collected with a transverse and axial resolution of 4 and 5 μm, respectively, over a field-of-view of 800 μm (width) × 400 μm (depth). NIR vertical cross-sectional fluorescence images of fresh mouse colonic mucosa demonstrate histology-like imaging performance with this miniature instrument.

Dual-triggered oxygen self-supply black phosphorus nanosystem for enhanced photodynamic therapy.

PubMed

Liu, Jintong; Du, Ping; Mao, Hui; Zhang, Lei; Ju, Huangxian; Lei, Jianping

2018-07-01

Nonspecific distribution of photosensitizer and the intrinsic hypoxic condition in the tumor microenvironment are two key factors limiting the efficacy of O 2 -dependent photodynamic therapy (PDT). Herein, a dual-triggered oxygen self-supported nanosystem using black phosphorus nanosheet (BPNS) as both photosensitizer and nanocarrier was developed to enhance PDT for tumors within hypoxic microenvironment. The BPNS platform was functionalized with folate and a blocker DNA duplex of 5'-Cy5-aptamer-heme/3'-heme labeled oligonucleotides. The resulting heme dimer could passivate its peroxidase activity. After specific recognition of aptamer-target, the quenched fluorescence is "turned" on by cellular adenosine triphosphate. The passivated nanosystem then activates the catalytic function towards excessive intracellular H 2 O 2 to generate O 2 essential to sustain BPNS-mediated PDT, leading to 8.7-fold and 7.5-fold increase of PDT efficacy in treating the hypoxic cell and tumor, respectively. Therefore, the dual-triggered oxygen self-supply nanosystem not only exerts tumor microenvironment-associated stimulus for enhanced PDT but also surmounts hypoxia-associated therapy resistance. Copyright © 2018 Elsevier Ltd. All rights reserved.

Targeted vertical cross-sectional imaging with handheld near-infrared dual axes confocal fluorescence endomicroscope

PubMed Central

Qiu, Zhen; Liu, Zhongyao; Duan, Xiyu; Khondee, Supang; Joshi, Bishnu; Mandella, Michael J.; Oldham, Kenn; Kurabayashi, Katsuo; Wang, Thomas D.

2013-01-01

We demonstrate vertical cross-sectional (XZ-plane) images of near-infrared (NIR) fluorescence with a handheld dual axes confocal endomicroscope that reveals specific binding of a Cy5.5-labeled peptide to pre-malignant colonic mucosa. This view is perpendicular to the tissue surface, and is similar to that used by pathologists. The scan head is 10 mm in outer diameter (OD), and integrates a one dimensional (1-D) microelectromechanical systems (MEMS) X-axis scanner and a bulky lead zirconate titanate (PZT) based Z-axis actuator. The microscope images in a raster-scanning pattern with a ±6 degrees (mechanical) scan angle at ~3 kHz in the X-axis (fast) and up to 10 Hz (0–400 μm) in the Z-axis (slow). Vertical cross-sectional fluorescence images are collected with a transverse and axial resolution of 4 and 5 μm, respectively, over a field-of-view of 800 μm (width) × 400 μm (depth). NIR vertical cross-sectional fluorescence images of fresh mouse colonic mucosa demonstrate histology-like imaging performance with this miniature instrument. PMID:23412564

Ibuprofen-in-cyclodextrin-in-W/O/W emulsion - Improving the initial and long-term encapsulation efficiency of a model active ingredient.

PubMed

Hattrem, Magnus N; Kristiansen, Kåre A; Aachmann, Finn L; Dille, Morten J; Draget, Kurt I

2015-06-20

A challenge in formulating water-in-oil-in-water (W/O/W) emulsions is the uncontrolled release of the encapsulated compound prior to application. Pharmaceuticals and nutraceuticals usually have amphipathic nature, which may contribute to leakage of the active ingredient. In the present study, cyclodextrins (CyDs) were used to impart a change in the relative polarity and size of a model compound (ibuprofen) by the formation of inclusion complexes. Various inclusion complexes (2-hydroxypropyl (HP)-β-CyD-, α-CyD- and γ-CyD-ibuprofen) were prepared and presented within W/O/W emulsions, and the initial and long-term encapsulation efficiency was investigated. HP-β-CyD-ibuprofen provided the highest encapsulation of ibuprofen in comparison to a W/O/W emulsion with unassociated ibuprofen confined within the inner water phase, with a four-fold increase in the encapsulation efficiency. An improved, although lower, encapsulation efficiency was obtained for the inclusion complex γ-CyD-ibuprofen in comparison to HP-β-CyD-ibuprofen, whereas α-CyD-ibuprofen had a similar encapsulation efficiency to that of unassociated ibuprofen. The lower encapsulation efficiency of ibuprofen in combination with α-CyD and γ-CyD was attributed to a lower association constant for the γ-CyD-ibuprofen inclusion complex and the ability of α-CyD to form inclusion complexes with fatty acids. For the W/O/W emulsion prepared with HP-β-CyD-ibuprofen, the highest encapsulation of ibuprofen was obtained at hyper- and iso-osmotic conditions and by using an excess molar ratio of CyD to ibuprofen. In the last part of the study, it was suggested that the chemical modification of the HP-β-CyD molecule did not influence the encapsulation of ibuprofen, as a similar encapsulation efficiency was obtained for an inclusion complex prepared with mono-1-glucose-β-CyD. Copyright © 2015 Elsevier B.V. All rights reserved.

Thiol surface functionalization via continuous phase plasma polymerization of allyl mercaptan, with subsequent maleimide-linked conjugation of collagen.

PubMed

Stynes, Gil D; Gengenbach, Thomas R; Kiroff, George K; Morrison, Wayne A; Kirkland, Mark A

2017-07-01

Thiol groups can undergo a large variety of chemical reactions and are used in solution phase to conjugate many bioactive molecules. Previous research on solid substrates with continuous phase glow discharge polymerization of thiol-containing monomers may have been compromised by oxidation. Thiol surface functionalization via glow discharge polymerization has been reported as requiring pulsing. Herein, continuous phase glow discharge polymerization of allyl mercaptan (2-propene-1-thiol) was used to generate significant densities of thiol groups on a mixed macrodiol polyurethane and tantalum. Three general classes of chemistry are used to conjugate proteins to thiol groups, with maleimide linkers being used most commonly. Here the pH specificity of maleimide reactions was used effectively to conjugate surface-bound thiol groups to amine groups in collagen. XPS demonstrated surface-bound thiol groups without evidence of oxidation, along with the subsequent presence of maleimide and collagen. Glow discharge reactor parameters were optimized by testing the resistance of bound collagen to degradation by 8 M urea. The nature of the chemical bonding of collagen to surface thiol groups was effectively assessed by colorimetric assay (ELISA) of residual collagen after incubation in 8 M urea over 8 days and after incubation with keratinocytes over 15 days. The facile creation of useable solid-supported thiol groups via continuous phase glow discharge polymerization of allyl mercaptan opens a route for attaching a vast array of bioactive molecules. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1940-1948, 2017. © 2017 Wiley Periodicals, Inc.

Evidence that human milk isolated cyclophilin B corresponds to a truncated form.

PubMed

Mariller, C; Allain, F; Kouach, M; Spik, G

1996-03-07

Cyclophilin B (CyPB) is a member of the cyclophilin family (cyclosporin A-binding proteins) with specific N- and C-terminal extensions. In contrast to cyclophilin A, CyPB owns a signal sequence leading to its translocation in the endoplasmic reticulum. CyPB was reported to be present in human blood and milk, suggesting it is secreted. For this purpose, CyPB was purified to homogeneity from human milk and compared to recombinant CyPB expressed in E. coli. Ion spray mass spectrometry revealed that CyPB secreted in human milk exhibits a lower molecular mass than the one expected. Identification of phenylalanine as the C-terminus amino-acid residue of human milk CyPB indicates that the difference in molecular mass may be explained by the absence of the five C-terminal amino-acid residues AIAKE. These results suggest that in the sequence VEKPFAIAKE known to be responsible for retention of CyPB in the endoplasmic reticulum, the sequence AIAKE is more particularly necessary. Our findings raise the possibility that the CyPB may be processed to promote its release. As recombinant CyPB was shown to bind specifically to Jurkat cells, a lymphoblastic T-cell line, we then wanted to investigate the binding of human milk CyPB to these cells. Despite lacking the five C-terminal amino-acid residues, human milk CyPB is able to inhibit the binding of recombinant CyPB to Jurkat T cells.

B-branes and supersymmetric quivers in 2d

NASA Astrophysics Data System (ADS)

Closset, Cyril; Guo, Jirui; Sharpe, Eric

2018-02-01

We study 2d N = (0, 2) supersymmetric quiver gauge theories that describe the low-energy dynamics of D1-branes at Calabi-Yau fourfold (CY4) singularities. On general grounds, the holomorphic sector of these theories — matter content and (classical) superpotential interactions — should be fully captured by the topological B-model on the CY4. By studying a number of examples, we confirm this expectation and flesh out the dictionary between B-brane category and supersymmetric quiver: the matter content of the supersymmetric quiver is encoded in morphisms between B-branes (that is, Ext groups of coherent sheaves), while the superpotential interactions are encoded in the A ∞ algebra satisfied by the morphisms. This provides us with a derivation of the supersymmetric quiver directly from the CY4 geometry. We also suggest a relation between triality of N = (0 ,2) gauge theories and certain mutations of exceptional collections of sheaves. 0d N = 1 supersymmetric quivers, corresponding to D-instantons probing CY5 singularities, can be discussed similarly.

Toughening of BIS maleimide resins: Synthesis and characterization of maleimide terminated poly(arylene ether) oligomers and polymers

NASA Technical Reports Server (NTRS)

Mcgrath, J. E.; Lyle, G. D.; Jurek, M. J.; Mohanty, D.; Hedrick, J. C.

1986-01-01

Amine functional poly(arylene ether) sulfones were previously reported. Herein, the chemistry was extended to amorphous poly(arylene ether) ketones because of their higher fracture toughness values, relative to the polysulfones. It was demonstrated that the amino functional oligomers undergo a self-crosslinking reaction at temperatures above about 220 C. This produces an insoluble, but ductile network that has excellent resistance. A ketamine structure hypothesis was proposed and verified using solid state magic angle NMR. In most cases, the water generated upon ketamine formation is too low to produce porosity and solid networks are obtained. The stability of the ketamine networks towards hydrolysis is excellent. The chemistry was further demonstrated to be able to crosslink preformed nonfunctional poly(arylene ether) ketones if a difunctional amine was utilized. This concept has the possibility of greatly improving the creep resistance of thermoplastics. Also, a new technique was developed for converting the amine functional oligomers cleanly into maleimide structures. This method involves reacting maleic anhydride with monomeric aminophenols in the presence of solvent mixtures.

Antifouling Thermoplastic Composites with Maleimide Encapsulated in Clay Nanotubes.

PubMed

Fu, Ye; Gong, Congcong; Wang, Wencai; Zhang, Liqun; Ivanov, Evgenii; Lvov, Yuri

2017-09-06

An antifouling ethylene-vinyl acetate copolymer (EVA) coating with halloysite clay nanotubes loaded with maleimide (TCPM) is prepared. Such antifoulant encapsulation allowed for extended release of TCPM and a long-lasting, efficient protection of the coated surface against marine microorganisms proliferation. Halloysite also induces the composite's anisotropy due to parallel alignment of the nanotubes. The maleimide loaded halloysite incorporated into the polymer matrix allowed for 12-month release of the bacterial inhibitor preventing fouling; it is much longer than the 2-3 month protection when TCPM is directly admixed into EVA. The antifouling properties of the EVA-halloysite nanocomposites were tested by monitoring surface adhesion and proliferation of marine V. natriegens bacteria with SEM. As compared to the composite directly doped with TCPM-antifoulant, there were much less bacteria accumulated on the EVA-halloysite-TCPM coating after a 2-month exposure to seawater. Field tests at South China Sea marine station further confirmed the formulation efficiency. The doping of 28 wt % TCPM loaded halloysite drastically enhanced material antifouling property, which promises wide applications for protective marine coating.

Genetic Manipulation of Schistosoma haematobium, the Neglected Schistosome

PubMed Central

Rinaldi, Gabriel; Okatcha, Tunika I.; Popratiloff, Anastas; Ayuk, Mary A.; Suttiprapa, Sutas; Mann, Victoria H.; Liang, Yung-san; Lewis, Fred A.; Loukas, Alex; Brindley, Paul J.

2011-01-01

Background Minimal information on the genome and proteome of Schistosoma haematobium is available, in marked contrast to the situation with the other major species of human schistosomes for which draft genome sequences have been reported. Accordingly, little is known about functional genomics in S. haematobium, including the utility or not of RNA interference techniques that, if available, promise to guide development of new interventions for schistosomiasis haematobia. Methods/Findings Here we isolated and cultured developmental stages of S. haematobium, derived from experimentally infected hamsters. Targeting different developmental stages, we investigated the utility of soaking and/or square wave electroporation in order to transfect S. haematobium with nucleic acid reporters including Cy3-labeled small RNAs, messenger RNA encoding firefly luciferase, and short interfering RNAs (siRNAs). Three hours after incubation of S. haematobium eggs in 50 ng/µl Cy3-labeled siRNA, fluorescent foci were evident indicating that labeled siRNA had penetrated into miracidia developing within the egg shell. Firefly luciferase activity was detected three hours after square wave electroporation of the schistosome eggs and adult worms in 150 ng/µl of mRNA. RNA interference knockdown (silencing) of reporter luciferase activity was seen following the introduction of dsRNA specific for luciferase mRNA in eggs, schistosomules and mixed sex adults. Moreover, introduction of an endogenous gene-specific siRNA into adult schistosomes silenced transcription of tetraspanin 2 (Sh-tsp-2), the apparent orthologue of the Schistosoma mansoni gene Sm-tsp-2 which encodes the surface localized structural and signaling protein Sm-TSP-2. Together, knockdown of reporter luciferase and Sh-tsp-2 indicated the presence of an intact RNAi pathway in S. haematobium. Also, we employed laser scanning confocal microscopy to view the adult stages of S. haematobium. Conclusions These findings and approaches should facilitate analysis of gene function in S. haematobium, which in turn could facilitate the characterization of prospective intervention targets for this neglected tropical disease pathogen. PMID:22022628

Gonadal function and fertility after stem cell transplantation in childhood: comparison of a reduced intensity conditioning regimen containing melphalan with a myeloablative regimen containing busulfan.

PubMed

Panasiuk, Anna; Nussey, Stephen; Veys, Paul; Amrolia, Persis; Rao, Kanchan; Krawczuk-Rybak, Maryna; Leiper, Alison

2015-09-01

The occurrence of late sequelae after myeloablative conditioning regimens for stem-cell transplantation (SCT) has prompted the introduction of reduced-intensity chemotherapy (RIC) regimens in an attempt to reduce toxicity and spare fertility. We retrospectively evaluated gonadal function in survivors of SCT in childhood by comparing patients conditioned with a myeloablative regimen containing busulfan and cyclophosphamide (BuCy, N = 51, 28 boys) and a RIC regimen containing fludarabine and melphalan (FluMel, N = 40, 19 boys). Spontaneous puberty occurred in 56% of girls and 89% of boys after BuCy, whereas 90% of females and all males in the FluMel group entered puberty spontaneously (P = 0·012). Significantly more females (61%) conditioned with BuCy required hormone replacement compared with the FluMel group (10·5%, P = 0·012). Females in the FluMel group took significantly longer to develop elevation of serum follicle-stimulating hormone (FSH) concentrations (>10 iu/l) from the onset of puberty than females in the BuCy group (median 5·2 years vs. 2·7 years respectively, P = 0·0135). In males no difference was noted between the two conditioning groups in time to FSH elevation (median 4 years in FluMel versus 6 years in BuCy). Whilst the two regimens have similar effects on the testis, ovarian function seems to be better preserved in females undergoing SCT with RIC. © 2015 John Wiley & Sons Ltd.

Chemically engineered papain as artificial formate dehydrogenase for NAD(P)H regeneration.

PubMed

Haquette, Pierre; Talbi, Barisa; Barilleau, Laure; Madern, Nathalie; Fosse, Céline; Salmain, Michèle

2011-08-21

Organometallic complexes of the general formula [(η(6)-arene)Ru(N⁁N)Cl](+) and [(η(5)-Cp*)Rh(N⁁N)Cl](+) where N⁁N is a 2,2'-dipyridylamine (DPA) derivative carrying a thiol-targeted maleimide group, 2,2'-bispyridyl (bpy), 1,10-phenanthroline (phen) or ethylenediamine (en) and arene is benzene, 2-chloro-N-[2-(phenyl)ethyl]acetamide or p-cymene were identified as catalysts for the stereoselective reduction of the enzyme cofactors NAD(P)(+) into NAD(P)H with formate as a hydride donor. A thorough comparison of their effectiveness towards NAD(+) (expressed as TOF) revealed that the Rh(III) complexes were much more potent catalysts than the Ru(II) complexes. Within the Ru(II) complex series, both the N⁁N and arene ligands forming the coordination sphere had a noticeable influence on the activity of the complexes. Covalent anchoring of the maleimide-functionalized Ru(II) and Rh(III) complexes to the cysteine endoproteinase papain yielded hybrid metalloproteins, some of them displaying formate dehydrogenase activity with potentially interesting kinetic parameters.

Passive potassium transport in LK sheep red cells. Modification by N- ethyl maleimide

PubMed Central

1983-01-01

Passive K transport, as modified by N-ethyl maleimide (NEM), was studied in erythrocytes of the low-K (LK) phenotype of sheep. Brief (5- min) treatment with NEM at less than 0.5 mM caused inhibition of passive K influx; NEM at concentrations greater than 0.5 mM caused stimulation of K influx. NEM had similar effects on K efflux. The treatments with NEM did not affect cell volumes (passive K transport in LK cells is sensitive to changes in cell volume). The stimulation of K transport by high [NEM] was also not a consequence of an effect on the metabolic state of the cells. Passive K transport in LK cells is dependent on Cl (it is inhibited in Cl-free media; it may be K/Cl cotransport). NEM had no effect on K influx in Cl-free (NO3- substituted) media. Pretreatment of the cells with anti-L antiserum (L antigen is found on LK cells and not on HK cells) prevented stimulation of K influx by NEM, but did not prevent inhibition. Therefore, NEM modifies the Cl-dependent K transport pathway at two separate sites, a low-affinity site, at which it stimulates, and a high-affinity site, at which it inhibits. Anti-L antibody prevents NEM's action, but only at the low-affinity site. PMID:6875508

Protective effect of ALDH2 against cyclophosphamide-induced acute hepatotoxicity via attenuating oxidative stress and reactive aldehydes.

PubMed

Zhai, Xiaoxuan; Zhang, Zhenxiao; Liu, Wenwen; Liu, Baoshan; Zhang, Rui; Wang, Wenjun; Zheng, Wen; Xu, Feng; Wang, Jiali; Chen, Yuguo

2018-04-30

Cyclophosphamide (CY) is a widely used chemotherapeutic agent that is associated with severe side effects, such as hepatotoxicity and nephrotoxicity. However, the extent, mechanisms and potential prevention and treatment strategies of CY-induced acute hepatotoxicity and nephrotoxicity are largely unknown. In this study, we determined the existence and extent of CY-induced acute hepatotoxicity and nephrotoxicity, and demonstrated the effect of ALDH2 on CY-induced acute tissue toxicity and related mechanisms. Adult male C57BL/6J (wide-type, WT) and ALDH2 -/- (KO) mice were divided into four groups: WT, WT + CY, KO + CY and WT + CY + Alda-1. Biochemical analysis showed that plasma ALT was increased by 35.8% in KO + CY group and decreased by 21.1% in WT + CY + Alda-1 group compared to WT + CY group (P < 0.05, respectively). However, there was no significant difference among WT, WT + CY and KO + CY groups regarding plasma renal marker enzymes, including blood urea nitrogen (BUN), creatinine and cystatin C (CysC). Levels of reactive oxygen species (ROS) and toxic aldehydes (acrolein, 4-hydroxynonenol and malondialdehyde) were increased significantly in KO + CY group and decreased significantly in WT + CY + Alda-1 group compared to WT + CY group (P < 0.05, respectively). These findings demonstrate that CY could induce acute hepatotoxicity without nephrotoxicity, and ALDH2 plays a protective role in CY-induced acute hepatotoxicity. The underlying mechanisms are associated with attenuating oxidative stress and detoxifying reactive aldehydes. Copyright © 2018 Elsevier Inc. All rights reserved.

Disilane-based cyclic deposition/etch of Si, Si:P and Si1-yCy:P layers: I. The elementary process steps

NASA Astrophysics Data System (ADS)

Hartmann, J. M.; Benevent, V.; Barnes, J. P.; Veillerot, M.; Deguet, C.

2013-02-01

We have benchmarked the 550 °C, 20 Torr growth of Si:P and Si1-yCy:P using SiH4 and Si2H6. P segregation has prevented us from reaching P+ ion concentrations in Si higher than a few 1019 cm-3 using SiH4; the resulting surface ‘poisoning’ led to a severe growth rate reduction. Meanwhile, [P+] increased linearly with the phosphine flow when using Si2H6 as the Si precursor; values as high as 1.7 × 1020 cm-3 were obtained. The Si:P growth rate using Si2H6 was initially stable then increased as the PH3 flow increased. Mono-methylsilane flows 6.5-10 times higher were needed with Si2H6 than with SiH4 to reach the same substitutional C concentrations in intrinsic Si1-yCy layers ([C]subst. up to 1.9%). Growth rates were approximately six times higher with Si2H6 than with SiH4, however. 30 nm thick Si1-yCy layers became rough as [C]subst. exceeded 1.6% (formation of increasing numbers of islands). We have also studied the structural and electrical properties of ‘low’ and ‘high’ C content Si1-yCy:P layers (˜ 1.5 and 1.8%, respectively) grown with Si2H6. Adding significant amounts of PH3 led to a reduction of the tensile strain in the films. This was due to the incorporation of P atoms (at the expense of C atoms) in the substitutional sites of the Si matrix. Si1-yCy:P layers otherwise became rough as the PH3 flow increased. Resistivities lower than 1 mΩ cm were nevertheless associated with those Si1-yCy:P layers, with P atomic concentrations at most 3.9 × 1020 cm-3. Finally, we have quantified the beneficial impact of adding GeH4 to HCl for the low-temperature etching of Si. Etch rates 12-36 times higher with HCl + GeH4 than with pure HCl were achieved at 20 Torr. Workable etch rates close to 1 nm min-1 were obtained at 600 °C (versus 750 °C for pure HCl), enabling low-temperature cyclic deposition/etch strategies for the selective epitaxial growth of Si, Si:P and Si1-yCy:P layers on patterned wafers.

Soluble Variants of Rhodobacter capsulatus Membrane-anchored Cytochrome cy Are Efficient Photosynthetic Electron Carriers*

PubMed Central

Öztürk, Yavuz; Lee, Dong-Woo; Mandaci, Sevnur; Osyczka, Artur; Prince, Roger C.; Daldal, Fevzi

2008-01-01

Photosynthetic (Ps) electron transport pathways often contain multiple electron carriers with overlapping functions. Here we focus on two c-type cytochromes (cyt) in facultative phototrophic bacteria of the Rhodobacter genus: the diffusible cyt c2 and the membrane-anchored cyt cy. In species like R. capsulatus, cyt cy functions in both Ps and respiratory electron transport chains, whereas in other species like R. sphaeroides, it does so only in respiration. The molecular bases of this difference was investigated by producing a soluble variant of cyt cy (S-cy), by fusing genetically the cyt c2 signal sequence to the cyt c domain of cyt cy. This novel electron carrier was unable to support the Ps growth of R. capsulatus. However, strains harboring cyt S-cy regained Ps growth ability by acquiring mutations in its cyt c domain. They produced cyt S-cy variants at amounts comparable with that of cyt c2, and conferred Ps growth. Chemical titration indicated that the redox midpoint potential of cyt S-cy was about 340 mV, similar to that of cyts c2 or cy. Remarkably, electron transfer kinetics from the cyt bc1 complex to the photochemical reaction center (RC) mediated by cyt S-cy was distinct from those seen with the cyt c2 or cyt cy. The kinetics exhibited a pronounced slow phase, suggesting that cyt S-cy interacted with the RC less tightly than cyt c2. Comparison of structural models of cyts c2 and S-cy revealed that several of the amino acid residues implicated in long-range electrostatic interactions promoting binding of cyt c2 to the RC are not conserved in cyt cy, whereas those supporting short-range hydrophobic interactions are conserved. These findings indicated that attaching electron carrier cytochromes to the membrane allowed them to weaken their interactions with their partners so that they could accommodate more rapid multiple turnovers. PMID:18343817

Electrochemical detection of Francisella tularensis genomic DNA using solid-phase recombinase polymerase amplification.

PubMed

del Río, Jonathan Sabaté; Yehia Adly, Nouran; Acero-Sánchez, Josep Lluis; Henry, Olivier Y F; O'Sullivan, Ciara K

2014-04-15

Solid-phase isothermal DNA amplification was performed exploiting the homology protein recombinase A (recA). The system was primarily tested on maleimide activated microtitre plates as a proof-of-concept and later translated to an electrochemical platform. In both cases, forward primer for Francisella tularensis holarctica genomic DNA was surface immobilised via a thiol or an amino moiety and then elongated during the recA mediated amplification, carried out in the presence of specific target sequence and reverse primers. The formation of the subsequent surface tethered amplicons was either colorimetrically or electrochemically monitored using a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the elongated strand. The amplification time was optimised to amplify even low amounts of DNA copies in less than an hour at a constant temperature of 37°C, achieving a limit of detection of 1.3×10(-13) M (4×10(6) copies in 50 μL) for the colorimetric assay and 3.3×10(-14) M (2×10(5) copies in 10 μL) for the chronoamperometric assay. The system was demonstrated to be highly specific with negligible cross-reactivity with non-complementary targets or primers. © 2013 Elsevier B.V. All rights reserved.

PET/CT with 18F-FDG- and 18F-FBEM-labeled leukocytes for metabolic activity and leukocyte recruitment monitoring in a mouse model of pulmonary fibrosis.

PubMed

Bondue, Benjamin; Sherer, Félicie; Van Simaeys, Gaetan; Doumont, Gilles; Egrise, Dominique; Yakoub, Yousof; Huaux, François; Parmentier, Marc; Rorive, Sandrine; Sauvage, Sébastien; Lacroix, Simon; Vosters, Olivier; De Vuyst, Paul; Goldman, Serge

2015-01-01

Idiopathic pulmonary fibrosis is characterized by a progressive and irreversible respiratory failure. Validated noninvasive methods able to assess disease activity are essential for prognostic purposes as well as for the evaluation of emerging antifibrotic treatments. C57BL/6 mice were used in a murine model of pulmonary fibrosis induced by an intratracheal instillation of bleomycin (control mice were instilled with a saline solution). At different times after instillation, PET/CT with (18)F-FDG- or (18)F-4-fluorobenzamido-N-ethylamino-maleimide ((18)F-FBEM)-labeled leukocytes was performed to assess metabolic activity and leukocyte recruitment, respectively. In bleomycin-treated mice, a higher metabolic activity was measured on (18)F-FDG PET/CT scans from day 7 to day 24 after instillation, with a peak of activity measured at day 14. Of note, lung mean standardized uptake values correlated with bleomycin doses, histologic score of fibrosis, lung hydroxyproline content, and weight loss. Moreover, during the inflammatory phase of the model (day 7), but not the fibrotic phase (day 23), bleomycin-treated mice presented with an enhanced leukocyte recruitment as assessed by (18)F-FBEM-labeled leukocyte PET/CT. Autoradiographic analysis of lung sections and CD45 immunostaining confirm the higher and early recruitment of leukocytes in bleomycin-treated mice, compared with control mice. (18)F-FDG- and (18)F-FBEM-labeled leukocyte PET/CT enable monitoring of metabolic activity and leukocyte recruitment in a mouse model of pulmonary fibrosis. Implications for preclinical evaluation of antifibrotic therapy are expected. © 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Chromenoquinoline-based thiol probes: a study on the quencher position for controlling fluorescent Off-On characteristics.

PubMed

Kand, Dnyaneshwar; Kalle, Arunasree Marasanapalli; Talukdar, Pinaki

2013-02-13

The design, synthesis and thiol sensing ability of chromenoquinoline-based fluorescent probes 4, 5 and 6 and are reported here. The relative position of the maleimide moiety was varied along the chromenoquinoline fluorophore to decrease the background fluorescence. Lower background fluorescence in probes 4 and 6 was rationalized by the smaller k(r)/k(nr) values compared to that of probe 5. An intramolecular charge transfer (ICT) mechanism was proposed for quenching and the extent was dependent on the position of the maleimide quencher. Fluorescent Off-On characteristics were evaluated by theoretical calculations. All probes were selective only towards thiol containing amino acids. Thiol sensing by probes 4 and 6 were much better compared to 5. Probe 4 displayed a better fluorescence response for less hindered thiol (185-, 223- and 156-fold for Hcy, Cys and GSH, respectively), while for probe 6, a higher enhancement in fluorescence was observed with more hindered thiols (180-, 205- and 245-fold for Hcy, Cys and GSH, respectively). The better response to bulkier thiol, GSH by probe 6 was attributed to the steric crowding at the C-4 position and bulkiness of the GSH group which force the succinimide unit to be in a nearly orthogonal conformation. This spatial arrangement was important in reducing the fluorescence quenching ability of the succinimide moiety. The application of probes 4, 5 and 6 was demonstrated by naked eye detection thiols using a 96-well plate system as well as by live-cell imaging.

Immunogenicity and reactogenicity of Infanrix™ when co-administered with meningococcal MenACWY-TT conjugate vaccine in toddlers primed with MenHibrix™ and Pediarix™.

PubMed

Leonardi, Michael; Latiolais, Thomas; Sarpong, Kwabena; Simon, Michael; Twiggs, Jerry; Lei, Paul; Rinderknecht, Stephen; Blatter, Mark; Bianco, Veronique; Baine, Yaela; Friedland, Leonard R; Baccarini, Carmen; Miller, Jacqueline M

2015-02-11

Co-administration of an investigational quadrivalent meningococcal serogroups A, C, W and Y tetanus toxoid conjugate vaccine (MenACWY-TT) with the fourth dose of diphtheria-tetanus-acellular pertussis vaccine (DTaP) at age 15-18 months was investigated in 3-dose Haemophilus influenzae type b-meningococcal serogroups C/Y conjugate vaccine (HibMenCY-TT)-primed toddlers. Infants were randomized (5:1) and primed at 2, 4 and 6 months of age with HibMenCY-TT and DTaP-hepatitis B-inactivated poliovirus (DTaP-HBV-IPV) vaccine, or Hib-TT and DTaP-HBV-IPV (Control). HibMenCY-TT+ DTaP-HBV-IPV vaccinees were re-randomized (2:2:1) to receive MenACWY-TT at 12-15 months and DTaP at 15-18 months (MenACWY-TT group); MenACWY-TT co-administered with DTaP at 15-18 months (Coad group); or HibMenCY-TT at 12-15 months and DTaP at 15-18 months (HibMenCY-TT group). Controls received DTaP at 15-18 months. Only children in the HibMenCY-TT group received a fourth dose of Hib conjugate vaccine due to Hib conjugate vaccine shortage at the time of the study. DTaP immunogenicity and reactogenicity were assessed one month post-vaccination. Pre-defined statistical non-inferiority criteria between Coad and Control groups were met for diphtheria, tetanus and filamentous haemagglutinin but not pertussis toxoid and pertactin. Following vaccination ≥99% of children had anti-diphtheria/anti-tetanus concentrations ≥1.0 IU/ml. Pertussis GMCs were lower in all investigational groups versus Control. In post hoc analyses, pertussis antibody concentrations were above those in infants following 3-dose DTaP primary vaccination in whom efficacy against pertussis was demonstrated (Schmitt, von König, et al., 1996; Schmitt, Schuind, et al., 1996). The reactogenicity profile of the Coad group was similar to DTaP administered alone. Routine booster DTaP was immunogenic with an acceptable safety profile when co-administered with MenACWY-TT vaccine in HibMenCY-TT-primed toddlers. These data support the administration of a fourth DTaP dose following a 4-dose HibMenCY-TT vaccination series, or co-administered with MenACWY-TT in HibMenCY-TT-primed children. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Facile and High-Throughput Synthesis of Functional Microparticles with Quick Response Codes.

PubMed

Ramirez, Lisa Marie S; He, Muhan; Mailloux, Shay; George, Justin; Wang, Jun

2016-06-01

Encoded microparticles are high demand in multiplexed assays and labeling. However, the current methods for the synthesis and coding of microparticles either lack robustness and reliability, or possess limited coding capacity. Here, a massive coding of dissociated elements (MiCODE) technology based on innovation of a chemically reactive off-stoichimetry thiol-allyl photocurable polymer and standard lithography to produce a large number of quick response (QR) code microparticles is introduced. The coding process is performed by photobleaching the QR code patterns on microparticles when fluorophores are incorporated into the prepolymer formulation. The fabricated encoded microparticles can be released from a substrate without changing their features. Excess thiol functionality on the microparticle surface allows for grafting of amine groups and further DNA probes. A multiplexed assay is demonstrated using the DNA-grafted QR code microparticles. The MiCODE technology is further characterized by showing the incorporation of BODIPY-maleimide (BDP-M) and Nile Red fluorophores for coding and the use of microcontact printing for immobilizing DNA probes on microparticle surfaces. This versatile technology leverages mature lithography facilities for fabrication and thus is amenable to scale-up in the future, with potential applications in bioassays and in labeling consumer products. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chain Collapse of an Amyloidogenic Intrinsically Disordered Protein

PubMed Central

Jain, Neha; Bhattacharya, Mily; Mukhopadhyay, Samrat

2011-01-01

Natively unfolded or intrinsically disordered proteins (IDPs) are under intense scrutiny due to their involvement in both normal biological functions and abnormal protein misfolding disorders. Polypeptide chain collapse of amyloidogenic IDPs is believed to play a key role in protein misfolding, oligomerization, and aggregation leading to amyloid fibril formation, which is implicated in a number of human diseases. In this work, we used bovine κ-casein, which serves as an archetypal model protein for amyloidogenic IDPs. Using a variety of biophysical tools involving both prediction and spectroscopic techniques, we first established that monomeric κ-casein adopts a collapsed premolten-globule-like conformational ensemble under physiological conditions. Our time-resolved fluorescence and light-scattering data indicate a change in the mean hydrodynamic radius from ∼4.6 nm to ∼1.9 nm upon chain collapse. We then took the advantage of two cysteines separated by 77 amino-acid residues and covalently labeled them using thiol-reactive pyrene maleimide. This dual-labeled protein demonstrated a strong excimer formation upon renaturation from urea- and acid-denatured states under both equilibrium and kinetic conditions, providing compelling evidence of polypeptide chain collapse under physiological conditions. The implication of the IDP chain collapse in protein aggregation and amyloid formation is also discussed. PMID:21961598

Monochrome Multiplexing in Polymerase Chain Reaction by Photobleaching of Fluorogenic Hydrolysis Probes.

PubMed

Schuler, Friedrich; Trotter, Martin; Zengerle, Roland; von Stetten, Felix

2016-03-01

Multiplexing in polymerase chain reaction (PCR) is a technique widely used to save cost and sample material and to increase sensitivity compared to distributing a sample to several singleplex reactions. One of the most common methods to detect the different amplification products is the use of fluorogenic probes that emit at different wavelengths (colors). To reduce the number of detection channels, several methods for monochrome multiplexing have been suggested. However, they pose restrictions to the amplifiable target length, the sequence, or the melting temperature. To circumvent these limitations, we suggest a novel approach that uses different fluorophores with the same emission maximum. Discrimination is achieved by their different fluorescence stability during photobleaching. Atto488 (emitting at the same wavelength as 6-carboxyfluorescein, FAM) and Atto467N (emitting at the same wavelength as cyanine 5, Cy5) were found to bleach significantly less than FAM and Cy5; i.e., the final fluorescence of Atto dyes was more than tripled compared to FAM and Cy5. We successfully applied this method by performing a 4-plex PCR targeting antibiotic resistance genes in S. aureus using only 2 color channels. Confidence of discrimination between the targets was >99.9% at high copy initial copy numbers of 100 000 copies. Cases where both targets were present could be discriminated with equal confidence for Cy5 channel and reduced levels of confidence (>68%) for FAM channel. Moreover, a 2-plex digital PCR reaction in 1 color channel was shown. In the future, the degree of multiplexing may be increased by adding fluorogenic probe pairs with other emission wavelengths. The method may also be applied to other probe and assay formats, such as Förster resonance energy transfer (FRET) probes and immunoassays.

Reliable and Accurate CD4+ T Cell Count and Percent by the Portable Flow Cytometer CyFlow MiniPOC and “CD4 Easy Count Kit-Dry”, as Revealed by the Comparison with the Gold Standard Dual Platform Technology

PubMed Central

Nasi, Milena; De Biasi, Sara; Bianchini, Elena; Gibellini, Lara; Pinti, Marcello; Scacchetti, Tiziana; Trenti, Tommaso; Borghi, Vanni; Mussini, Cristina; Cossarizza, Andrea

2015-01-01

Background An accurate and affordable CD4+ T cells count is an essential tool in the fight against HIV/AIDS. Flow cytometry (FCM) is the “gold standard” for counting such cells, but this technique is expensive and requires sophisticated equipment, temperature-sensitive monoclonal antibodies (mAbs) and trained personnel. The lack of access to technical support and quality assurance programs thus limits the use of FCM in resource-constrained countries. We have tested the accuracy, the precision and the carry-over contamination of Partec CyFlow MiniPOC, a portable and economically affordable flow cytometer designed for CD4+ count and percentage, used along with the “CD4% Count Kit-Dry”. Materials and Methods Venous blood from 59 adult HIV+ patients (age: 25–58 years; 43 males and 16 females) was collected and stained with the “MiniPOC CD4% Count Kit-Dry”. CD4+ count and percentage were then determined in triplicate by the CyFlow MiniPOC. In parallel, CD4 count was performed using mAbs and a CyFlow Counter, or by a dual platform system (from Beckman Coulter) based upon Cytomic FC500 (“Cytostat tetrachrome kit” for mAbs) and Coulter HmX Hematology Analyzer (for absolute cell count). Results The accuracy of CyFlow MiniPOC against Cytomic FC500 showed a correlation coefficient (CC) of 0.98 and 0.97 for CD4+ count and percentage, respectively. The accuracy of CyFlow MiniPOC against CyFlow Counter showed a CC of 0.99 and 0.99 for CD4 T cell count and percentage, respectively. CyFlow MiniPOC showed an excellent repeatability: CD4+ cell count and percentage were analyzed on two instruments, with an intra-assay precision below ±5% deviation. Finally, there was no carry-over contamination for samples at all CD4 values, regardless of their position in the sequence of analysis. Conclusion The cost-effective CyFlow MiniPOC produces rapid, reliable and accurate results that are fully comparable with those from highly expensive dual platform systems. PMID:25622041

Effects of Growth Temperature and Postharvest Cooling on Anthocyanin Profiles in Juvenile and Mature Brassica oleracea.

PubMed

Socquet-Juglard, Didier; Bennett, Alexandra A; Manns, David C; Mansfield, Anna Katharine; Robbins, Rebecca J; Collins, Thomas M; Griffiths, Phillip D

2016-02-24

The effects of growth temperatures on anthocyanin content and profile were tested on juvenile cabbage and kale plants. The effects of cold storage time were evaluated on both juvenile and mature plants. The anthocyanin content in juvenile plants ranged from 3.82 mg of cyanidin-3,5-diglucoside equivalent (Cy equiv)/g of dry matter (dm) at 25 °C to 10.00 mg of Cy equiv/g of dm at 16 °C, with up to 76% diacylated anthocyanins. Cold storage of juvenile plants decreased the total amount of anthocyanins but increased the diacylated anthocyanin content by 3-5%. In mature plants, cold storage reduced the total anthocyanin content from 22 to 12.23 mg/g after 5 weeks of storage in red cabbage, while the total anthocyanin content increased after 2 weeks of storage from 2.34 to 3.66 mg of Cy equiv/g of dm in kale without having any effect on acylation in either morphotype. The results obtained in this study will be useful for optimizing anthocyanin production.

Optofluidic FRET Lasers Using Aqueous Quantum Dots as Donors

PubMed Central

Chen, Qiushu; Kiraz, Alper; Fan, Xudong

2015-01-01

An optofluidic FRET (fluorescence resonance energy transfer) laser is formed by putting FRET pairs inside a microcavity acting as gain medium. This integration of optofluidic laser and FRET mechanism provides novel research frontiers, including sensitive biochemical analysis and novel photonic devices, such as on-chip coherent light sources and bio-tunable lasers. Here we investigated an optofluidic FRET laser using quantum dots (QDs) as FRET donors. We achieved lasing from Cy5 as the acceptor in the QD-Cy5 pair when excited at 450 nm where Cy5 has negligible absorption by itself. The threshold was approximately 14 µJ/mm2. The demonstrated capability of QDs as the donor in a FRET laser greatly improves the versatility of optofluidic laser operation due to the broad and large absorption cross section of QDs in the blue and UV spectral region. The excitation efficiency of the acceptor molecules through FRET channel was also analyzed, showing that the energy transfer rate and the non-radiative Auger recombination rate of QDs plays a significant role in FRET laser performance. PMID:26659274

Optofluidic FRET lasers using aqueous quantum dots as donors.

PubMed

Chen, Qiushu; Kiraz, Alper; Fan, Xudong

2016-01-21

An optofluidic FRET (fluorescence resonance energy transfer) laser is formed by putting FRET pairs inside a microcavity acting as a gain medium. This integration of an optofluidic laser and the FRET mechanism provides novel research frontiers, including sensitive biochemical analysis and novel photonic devices, such as on-chip coherent light sources and bio-tunable lasers. Here, we investigated an optofluidic FRET laser using quantum dots (QDs) as FRET donors. We achieved lasing from Cy5 as the acceptor in a QD-Cy5 pair upon excitation at 450 nm, where Cy5 has negligible absorption by itself. The threshold was approximately 14 μJ mm(-2). The demonstrated capability of QDs as donors in the FRET laser greatly improves the versatility of optofluidic laser operation due to the broad and large absorption cross section of the QDs in the blue and UV spectral regions. The excitation efficiency of the acceptor molecules through a FRET channel was also analyzed, showing that the energy transfer rate and the non-radiative Auger recombination rate of QDs play a significant role in FRET laser performance.

Novel compound heterozygous SPTA1 mutations in a patient with hereditary elliptocytosis

PubMed Central

Ma, Shiyue; Qin, Jinqiu; Wei, Aiqiu; Li, Xiaohong; Qin, Yuanyuan; Liao, Lin; Lin, Faquan

2018-01-01

Hereditaryelliptocytosis (HE) is a hereditary hemolytic disease, characterized by the presence of many elliptical erythrocytes in the peripheral blood that is caused by abnormal cytoskeletal proteins in the erythrocyte membrane. In the present study, a novel, causal HE mutation was reported. Routine blood examinations were performed on the proband and their family, and the fluorescence intensity of eosin-5-maleimide (EMA)-labeled erythrocytes was determined via flow cytometry. Subsequently, DNA was extracted from the peripheral blood of the proband and their family members, and amplified by quantitative polymerase chain reaction. The Sanger sequencing approach was used to determine and identify gene mutations, which were verified by matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry. To exclude genetic polymorphisms, newly identified mutations were subjected to large-scale gene screening using high-resolution melt analysis. Protein expression levels in the erythrocyte membrane of the proband were determined via SDS-PAGE, which demonstrated that, compared with healthy controls, the proband exhibited a reduction in EMA-labeled erythrocytes. In addition, DNA analysis demonstrated that the proband carried three mutations in the spectrin α chain erythrocytic 1 (SPTA1) gene: c.161A>C, c.5572C>G and 6531-12C>T. The corresponding mutant polypeptides were also analyzed by MALDI-TOF mass spectroscopy. SDS-PAGE analysis indicated that the proband exhibited normal levels of erythrocyte membrane proteins. In the present study, a novel HE case with a His54Pro mutation in the SPTA1 gene was reported. The results suggested that the His54Pro mutation influenced the role of erythrocyte membrane proteins without reducing its level of expression. PMID:29484404

Fluoresceination of FepA during colicin B killing: effects of temperature, toxin and TonB.

PubMed

Smallwood, Chuck R; Marco, Amparo Gala; Xiao, Qiaobin; Trinh, Vy; Newton, Salete M C; Klebba, Phillip E

2009-06-01

We studied the reactivity of 35 genetically engineered Cys sulphydryl groups at different locations in Escherichia coli FepA. Modification of surface loop residues by fluorescein maleimide (FM) was strongly temperature-dependent in vivo, whereas reactivity at other sites was much less affected. Control reactions with bovine serum albumin showed that the temperature dependence of loop residue reactivity was unusually high, indicating that conformational changes in multiple loops (L2, L3, L4, L5, L7, L8, L10) transform the receptor to a more accessible form at 37 degrees C. At 0 degrees C colicin B binding impaired or blocked labelling at 8 of 10 surface loop sites, presumably by steric hindrance. Overall, colicin B adsorption decreased the reactivity of more than half of the 35 sites, in both the N- and C- domains of FepA. However, colicin B penetration into the cell at 37 degrees C did not augment the chemical modification of any residues in FepA. The FM modification patterns were similarly unaffected by the tonB locus. FepA was expressed at lower levels in a tonB host strain, but when we accounted for this decrease its FM labelling was comparable whether TonB was present or absent. Thus we did not detect TonB-dependent structural changes in FepA, either alone or when it interacted with colicin B at 37 degrees C. The only changes in chemical modification were reductions from steric hindrance when the bacteriocin bound to the receptor protein. The absence of increases in the reactivity of N-domain residues argues against the idea that the colicin B polypeptide traverses the FepA channel.

Fluoresceination of FepA during Colicin B Killing: Effects of Temperature, Toxin and TonB

PubMed Central

Smallwood, Chuck R.; Marco, Amparo Gala; Xiao, Qiaobin; Trinh, Vy; Newton, Salete M. C.; Klebba, Phillip E.

2009-01-01

We studied the reactivity of 35 genetically engineered Cys sulfhydryl groups at different locations in Escherichia coli FepA. Modification of surface loop residues by fluorescein maleimide (FM) was strongly temperature-dependent in vivo, whereas reactivity at other sites was much less affected. Control reactions with bovine serum albumin showed that the temperature dependence of loop residue reactivity was unusually high, indicating that conformational changes in multiple loops (L2, L3, L4, L5, L7, L8, L10) transform the receptor to a more accessible form at 37 °C. At 0 °C colicin B binding impaired or blocked labeling at 8 of 10 surface loop sites, presumably by steric hindrance. Overall, colicin B adsorption decreased the reactivity of more than half of the 35 sites, in both the N - and C- domains of FepA. However, colicin B penetration into the cell at 37 °C did not augment the chemical modification of any residues in FepA. The FM modification patterns were similarly unaffected by the tonB locus. FepA was expressed at lower levels in a tonB host strain, but when we accounted for this decrease its FM-labeling was comparable whether TonB was present or absent. Thus we did not detect TonB-dependent structural changes in FepA, either alone or when it interacted with colicin B at 37 °C. The only changes in chemical modification were reductions from steric hindrance when the bacteriocin bound to the receptor protein. The absence of increases in the reactivity of N-domain residues argues against the idea (Devanathan and Postle, Mol. Microbiol. 65: 441–453, 2007) that the colicin B polypeptide traverses the FepA channel. PMID:19432807

Fortification of Yogurts with Vitamin D and Calcium Enhances the Inhibition of Serum Parathyroid Hormone and Bone Resorption Markers: A Double Blind Randomized Controlled Trial in Women over 60 Living in a Community Dwelling Home.

PubMed

Bonjour, J-P; Benoit, V; Atkin, S; Walrand, S

2015-05-01

To evaluate whether fortification of yogurts with vitamin D and calcium exerts an additional lowering effect on serum parathyroid hormone (PTH) and bone resorption markers (BRM) as compared to iso-caloric and iso-protein dairy products in aged white women at risk of fragility fractures. A randomized double-blind controlled trial. A community dwelling home. Forty-eight women over 60 years (mean age 73.4). Consumption during 84 days of two 125 g servings of either vitamin D and calcium-fortified yogurts (FY) at supplemental levels of 10 µg vitamin D3/d and 520 mg/d of calcium (total=800 mg/d), or non fortified control yogurts (CY) providing 280 mg/d of calcium. Serum changes from baseline (D0) to D28, D56 and D84 in 25OHD, PTH and in two BRM: Tartrate-resistant-acid-phosphatase-isoform-5b (TRAP5b) and carboxy-terminal-cross-linked-telopeptide of type-I-collagen (CTX). The 10 years risk of major and hip fractures were 13.1 and 5.0%, and 12.9 and 4.2 %, in FY and CY groups, respectively. From D0 to D84, serum 25OHD increased (mean±SE) from 34.3±2.4 to 56.3±2.4 nmol/L in FY (n=24) and from 35.0±2.5 to 41.3±3.0 nmol/L in CY (n=24), (P=0.00001). The corresponding changes in PTH were from 64.1±5.1 to 47.4±3.8 ng/L in FY and from 63.5±4.6 to 60.7±4.2 ng/L in CY (P=0.0011). After D84, TRAP5b was reduced significantly (P=0.0228) and CTX fell though not significantly (P=0.0773) in FY compared to CY. This trial in aged white women living in a community dwelling home at risk for osteoporotic fractures confirms that fortification of dairy products with vitamin D3 and calcium should provide a greater prevention of secondary hyperparathyroidism and accelerated bone resorption as compared to non-fortified equivalent foods.

Self-Healing Organic Fluorophore of Cyanine-Conjugated Amphiphilic Polypeptide for Near-Infrared Photostable Bioimaging.

PubMed

Li, Tuanwei; Liu, Le; Jing, Titao; Ruan, Zheng; Yuan, Pan; Yan, Lifeng

2018-05-02

Photobleaching and biotoxicity are the main bottlenecks for organic fluorescent dyes applied in real-time dynamic monitoring of living cells. Here, an unnatural amino acid, 4-nitro-3-phenyl-l-alanine (NPA), was used as a scaffold to covalently link a near-infrared fluorophore Cy5.5 and an amphiphilic polypeptide, poly[oligo(ethylene glycol) methyl ether methacrylate]- block-poly[2-amino-N 4 -(2-diisopropylamino-ethyl)-l-aspartic acid] (P(OEGMA) 21 -P(Asp) 16 -iPr), was then conjugated for increasing the photostability and improving the biocompatibility simultaneously. The protective agent of NPA can service as an effective triplet state quenching by intramolecular electron transfer between Cy5.5 and NPA. The less sensitivity of the electron-transfer process for molecular oxygen makes it an ideal photostabilized strategy for fluorophores applied in live-cell imaging. Bonding to copolymer is a common way for hydrophobic dyes to expand their application in biomedical imaging and increase their functionality, depending on the delivery system. The results indicate that Cy5.5-NPA-linked polypeptide copolymer exhibited an enhanced photostability and an excellent biocompatibility, which means this scaffolding strategy has a potential application in fluorescence-guided surgery, lived-cell imaging, and super-resolution microscopy.

Analysis of cyclophosphamide and carboxyethylphosphoramide mustard enantiomers in human plasma and application to clinical pharmacokinetics.

PubMed

Attié de Castro, Francine; Scatena, Gabriel dos Santos; Cass, Quézia Bezerra; Simões, Belinda Pinto; Lanchote, Vera Lucia

2014-11-15

This study describes for the first time a method for the sequential analysis of the enantiomers of cyclophosphamide (CY) and its metabolite carboxyethylphosphoramide mustard (CEPM) in human plasma. The CY and CEPM enantiomers were extracted from plasma using only ethyl acetate and separated on a Chiralpak(®) AD-RH column using a mixture of water:acetonitrile:ethanol (45:30:25, v/v/v) plus 0.1% trifluoroacetic acid as the mobile phase at a flow rate of 0.5mL/min. No matrix effect was observed in the analysis of the enantiomers of both analytes and the analytical method was linear in the range of 0.05-25.0μg and 250-1000ng of each enantiomer/mL plasma. The coefficients of variation and relative errors obtained for the assessment of intra- and interassay precision and accuracy were less than 15%. CY and CEPM were found to be stable in human plasma after three successive freeze/thaw cycles, during storage for 4h at room temperature, and after 24h inside the autosampler at 4°C, with deviations less than 15%. The method was applied to the study of the pharmacokinetics of CY and its metabolite CEPM in patients with multiple sclerosis (n=10) who received a CY pretransplant conditioning regimen for hematopoietic stem cell transplantation. The pharmacokinetic parameters showed plasma accumulation of the (S)-(-)-CY enantiomer (S/R ratio=1.3) and lack of enantioselective exposure to the CEPM metabolite (S/R ratio=1.0). Copyright © 2014 Elsevier B.V. All rights reserved.

Sensitive immunoassay detection of multiple environmental chemicals on protein microarrays using DNA/dye conjugate as a fluorescent label.

PubMed

Fan, Ziyan; Keum, Young Soo; Li, Qing X; Shelver, Weilin L; Guo, Liang-Hong

2012-05-01

Indirect competitive immunoassays were developed on protein microarrays for the sensitive and simultaneous detection of multiple environmental chemicals in one sample. In this assay, a DNA/SYTOX Orange conjugate was employed as an antibody label to increase the fluorescence signal and sensitivity of the immunoassays. Epoxy-modified glass slides were selected as the substrate for the production of 4 × 4 coating antigen microarrays. With this signal-enhancing system, competition curves for 17β-estradiol (E2), benzo[a]pyrene (BaP) and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) were obtained individually on the protein microarray. The IC(50) and calculated limit of detection (LOD) are 0.32 μg L(-1) and 0.022 μg L(-1) for E2, 37.2 μg L(-1) and 24.5 μg L(-1) for BaP, and 31.6 μg L(-1) and 2.8 μg L(-1) for BDE-47, respectively. LOD of E2 is 14-fold lower than the value reported in a previous study using Cy3 labeled antibody (Du et al., Clin. Chem, 2005, 51, 368-375). The results of the microarray immunoassay were within 15% of chromatographic analysis for all three pollutants in spiked river water samples, thus verifying the immunoassay. Simultaneous detection of E2, BaP and BDE-47 in one sample was demonstrated. There was no cross-reaction in the immunoassay between these three environmental chemicals. These results suggest that microarray-based immunoassays with DNA/dye conjugate labels are useful tools for the rapid, sensitive, and high throughput screening of multiple environmental contaminants.

Measurement of nucleotide exchange rate constants in single rabbit soleus myofibrils during shortening and lengthening using a fluorescent ATP analog.

PubMed Central

Shirakawa, I; Chaen, S; Bagshaw, C R; Sugi, H

2000-01-01

The kinetics of displacement of a fluorescent nucleotide, 2'(3')-O-[N[2-[[Cy3]amido]ethyl]carbamoyl]-adenosine 5'-triphosphate (Cy3-EDA-ATP), bound to rabbit soleus muscle myofibrils were studied using flash photolysis of caged ATP. Use of myofibrils from this slow twitch muscle allowed better resolution of the kinetics of nucleotide exchange than previous studies with psoas muscle myofibrils (, Biophys. J. 73:2033-2042). Soleus myofibrils in the presence of Cy3-EDA-nucleotides (Cy3-EDA-ATP or Cy3-EDA-ADP) showed selective fluorescence staining of the A-band. The K(m) for Cy3-EDA-ATP and the K(d) for Cy3-EDA-ADP binding to the myofibril A-band were 1.9 microM and 3.8 microM, respectively, indicating stronger binding of nucleotide to soleus cross-bridges compared to psoas cross-bridges (2.6 microM and 50 microM, respectively). After flash photolysis of caged ATP, the A-band fluorescence of the myofibril in the Cy3-EDA-ATP solution under isometric conditions decayed exponentially with a rate constant of 0.045 +/- 0.007 s(-1) (n = 32) at 10 degrees C, which was about seven times slower than that for psoas myofibrils. When a myofibril was allowed to shorten with a constant velocity, the nucleotide displacement rate constant increased from 0.066 s(-1) (isometric) to 0.14 s(-1) at 20 degrees C with increasing shortening velocity up to 0.1 myofibril length/s (V(max), the shortening velocity under no load was approximately 0. 2 myofibril lengths/s). The rate constant was not significantly affected by an isovelocity stretch of up to 0.1 myofibril lengths/s. These results suggest that the cross-bridge kinetics are not significantly affected at higher strain during lengthening but depend on the lower strain during shortening. These data also indicate that the interaction distance between a cross-bridge and the actin filament is at least 16 nm for a single cycle of the ATPase. PMID:10653804

Screening and identification of gastric adenocarcinoma metastasis-related genes by using cDNA microarray coupled to FDD-PCR.

PubMed

Wang, Jian-Hua; Chen, Shi-Shu

2002-07-01

To clone gastric adenocarcinoma metastasis related genes, RF-1 cell line (primary tumor of a gastric adenocarcinoma patient ) and RF-48 cell line (its metastatic counterpart) were used as a model for studying the molecular mechanism of tumor metastasis. Two fluorescent cDNA probes, labeled with Cy3 and Cy5 dyes, were prepared from RF-1 and RF-48 mRNA samples by reverse transcription method. The two color probes were then mixed and hybridized to the cDNA chip constructed by double-dots of 4 096 human genes, and scanned at two wavelengths. The experiment was repeated for 2 times. Differential expression genes from the above two cells were analyzed using the computer. 138 in all genes (3.4%) revealed differential expression in RF-48 cells compared with RF-1 cells: 81(2.1%) genes revealed apparent up-regulation, and 56(1.3%) genes revealed down-regulation. 45 genes involved in gastric adenocarcinoma metastasis were cloned using fluorescent differential display-PCR (FDD-PCR), including 3 novel genes. There were 7 differential expression genes that agreed with each other in two detection methods. The possible roles of some differential expressed genes, which maybe involved in the mechanism of tumor metastasis, were discussed. cDNA chip was used to analyze gene expression in a high-throughput and large scale manner, in combination with FDD-PCR for cloning unknown novel genes. In conclusion, some genes related to metastasis were preliminarily scanned, which would contribute to disclose the molecular mechanism of gastric adenocarcinoma metastasis.

Detection of Subclinical Arthritis in Mice by a Thrombin Receptor-Derived Imaging Agent.

PubMed

Friedman, Beth; Whitney, Michael A; Savariar, Elamprakash N; Caneda, Christa; Steinbach, Paul; Xiong, Qing; Hingorani, Dina V; Crisp, Jessica; Adams, Stephen R; Kenner, Michael; Lippert, Csilla N; Nguyen, Quyen T; Guma, Monica; Tsien, Roger Y; Corr, Maripat

2018-01-01

Functional imaging of synovitis could improve both early detection of rheumatoid arthritis (RA) and long-term outcomes. Given the intersection of inflammation with coagulation protease activation, this study was undertaken to examine coagulation protease activities in arthritic mice with a dual-fluorescence ratiometric activatable cell-penetrating peptide (RACPP) that has a linker, norleucine (Nle)-TPRSFL, with a cleavage site for thrombin. K/BxN-transgenic mice with chronic arthritis and mice with day 1 passive serum-transfer arthritis were imaged in vivo for Cy5:Cy7 emission ratiometric fluorescence from proteolytic cleavage and activation of RACPP NleTPRSFL . Joint thickness in mice with serum-transfer arthritis was measured from days 0 to 10. The cleavage-evoked release of Cy5-tagged tissue-adhesive fragments enabled microscopic correlation with immunohistochemistry for inflammatory markers. Thrombin dependence of ratiometric fluorescence was tested by ex vivo application of RACPP NleTPRSFL and argatroban to cryosections obtained from mouse hind paws on day 1 of serum-transfer arthritis. In chronic arthritis, RACPP NleTPRSFL fluorescence ratios of Cy5:Cy7 emission were significantly higher in diseased swollen ankles of K/BxN-transgenic mice than in normal mouse ankles. A high ratio of RACPP NleTPRSFL fluorescence in mouse ankles and toes on day 1 of serum-transfer arthritis correlated with subsequent joint swelling. Foci of high ratiometric fluorescence localized to inflammation, as demarcated by immune reactivity for citrullinated histones, macrophages, mast cells, and neutrophils, in soft tissue on day 1 of serum-transfer arthritis. Ex vivo application of RACPP NleTPRSFL to cryosections obtained from mice on day 1 of serum-transfer arthritis produced ratiometric fluorescence that was inhibited by argatroban. RACPP NleTPRSFL activation detects established experimental arthritis, and the detection of inflammation by RACPP NleTPRSFL on day 1 of serum-transfer arthritis correlates with disease progression. © 2017, American College of Rheumatology.

Visualizing epithelial expression of EGFR in vivo with distal scanning side-viewing confocal endomicroscope

NASA Astrophysics Data System (ADS)

Duan, Xiyu; Li, Haijun; Zhou, Juan; Zhou, Quan; Oldham, Kenn R.; Wang, Thomas D.

2016-11-01

Confocal endomicroscopy is an emerging imaging technology that has recently been introduced into the clinic to instantaneously collect “optical biopsies” in vivo with histology-like quality. Here, we demonstrate a fast scanner located in the distal end of a side-viewing instrument using a compact lens assembly with numerical aperture of 0.5 to achieve a working distance of 100 μm and field-of-view of 300 × 400 μm2. The microelectromechanical systems (MEMS) mirror was designed based on the principle of parametric resonance and images at 5 frames per second. The instrument has a 4.2 mm outer diameter and 3 cm rigid length, and can pass through the biopsy channel of a medical endoscope. We achieved real time optical sections of NIR fluorescence with 0.87 μm lateral resolution, and were able to visualize in vivo binding of a Cy5.5-labeled peptide specific for EGFR to the cell surface of pre-cancerous colonocytes within the epithelium of dysplastic crypts in mouse colon. By performing targeted imaging with endomicroscopy, we can visualize molecular expression patterns in vivo that provide a biological basis for disease detection.

A Practical Theory of Micro-Solar Power Sensor Networks

DTIC Science & Technology

2009-04-20

Simulation Platform TOSSIM [LLWC03] ns-2 Matlab C++ AVRORA [TLP05] Reference Hardware Mica2 WINS, Medusa Mica Mica2, Medusa Mica2 Simulated Power Power...scale. From this raw data, we can 162 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 2 4 Correlation coefficient F re qu en cy Histogram of correlation...0.5 0.6 0.7 0.8 0.9 1 0 1 2 Correlation coefficient F re qu en cy Histogram of correlation coefficient with solar radiation measurement (Turbidity at

Thiotepa-based conditioning versus total body irradiation as myeloablative conditioning prior to allogeneic stem cell transplantation for acute lymphoblastic leukemia: A matched-pair analysis from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation.

PubMed

Eder, Sandra; Canaani, Jonathan; Beohou, Eric; Labopin, Myriam; Sanz, Jaime; Arcese, William; Or, Reuven; Finke, Juergen; Cortelezzi, Agostino; Beelen, Dietrich; Passweg, Jakob; Socié, Gerard; Gurman, Gunhan; Aljurf, Mahmoud; Stelljes, Matthias; Giebel, Sebastian; Mohty, Mohamad; Nagler, Arnon

2017-10-01

The optimal conditioning regimen to employ before hematopoietic stem cell transplantation in acute lymphoblastic leukemia (ALL) is still undecided, and while cyclophosphamide/total body irradiation (Cy/TBI) is the most commonly used myeloablative regimen, there are concerns regarding long-term toxicity for patients conditioned with this regimen. Thiotepa-based conditioning is an emerging radiation-free regimen with recent publications indicative of comparable clinical outcomes to TBI-based conditioning. In this analysis of the acute leukemia working party of the EBMT, we performed a retrospective matched-pair analysis, evaluating the outcome of adult patients with ALL who received thiotepa-based conditioning (n = 180) with those receiving Cy/TBI conditioning (n = 540). The 2-year leukemia-free survival and overall survival (OS) rates of both conditioning regimens were comparable, 33% for thiotepa [95% confidence interval (CI): 26.4-42.8] versus 39% for Cy/TBI (95% CI: 34.8-44.5] (P = .33) and 46.5% [95% CI: 38.6-56.1] versus 48.8% [95% CI: 44.2-54] (P = .9), respectively. There was no significant difference between the two regimens in the incidence of either acute graft versus host disease (GVHD) or chronic GVHD. Multivariate analysis demonstrated increased relapse incidence for thiotepa conditioning compared to Cy/TBI (HR = 1.78, 95% CI, 1.07-2.95; P = .03) which did not affect OS. Our results indicate that thiotepa-based conditioning may not be inferior to Cy/TBI for adult patients with ALL. © 2017 Wiley Periodicals, Inc.

CD147 is a signaling receptor for cyclophilin B.

PubMed

Yurchenko, V; O'Connor, M; Dai, W W; Guo, H; Toole, B; Sherry, B; Bukrinsky, M

2001-11-09

Cyclophilins A and B (CyPA and CyPB) are cyclosporin A binding proteins that can be secreted in response to inflammatory stimuli. We recently identified CD147 as a cell-surface receptor for CyPA and demonstrated that CD147 is an essential component in the CyPA-initiated signaling cascade that culminates in ERK activation and chemotaxis. Here we demonstrate that CD147 also serves as a receptor for CyPB. CyPB induced Ca(2+) flux and chemotaxis of CD147-transfected, but not control, CHO cells, and the chemotactic response of primary human neutrophils to CyPB was blocked by antibodies to CD147. These results suggest that CD147 serves as a receptor for extracellular cyclophilins. Copyright 2001 Academic Press.

42 CFR 433.10 - Rates of FFP for program services.

Code of Federal Regulations, 2013 CFR

2013-10-01

...; (B) 95 percent, for calendar quarters in CY 2017; (C) 94 percent, for calendar quarters in CY 2018... quarters in CY 2018; and (F) 100 percent, for calendar quarters in CY 2019 and all subsequent calendar...

Factors affecting variation of different measures of cheese yield and milk nutrient recovery from an individual model cheese-manufacturing process.

PubMed

Cipolat-Gotet, C; Cecchinato, A; De Marchi, M; Bittante, G

2013-01-01

Cheese yield (CY) is the most important technological trait of milk, because cheese-making uses a very high proportion of the milk produced worldwide. Few studies have been carried out at the level of individual milk-producing animals due to a scarcity of appropriate procedures for model-cheese production, the complexity of cheese-making, and the frequent use of the fat and protein (or casein) contents of milk as a proxy for cheese yield. Here, we report a high-throughput cheese manufacturing process that mimics all phases of cheese-making, uses 1.5-L samples of milk from individual animals, and allows the simultaneous processing of 15 samples per run. Milk samples were heated (35°C for 40 min), inoculated with starter culture (90 min), mixed with rennet (51.2 international milk-clotting units/L of milk), and recorded for gelation time. Curds were cut twice (10 and 15 min after gelation), separated from the whey, drained (for 30 min), pressed (3 times, 20 min each, with the wheel turned each time), salted in brine (for 60 min), weighed, and sampled. Whey was collected, weighed, and sampled. Milk, curd, and whey samples were analyzed for pH, total solids, fat content, and protein content, and energy content was estimated. Three measures of percentage cheese yield (%CY) were calculated: %CY(CURD), %CY(SOLIDS), and %CY(WATER), representing the ratios between the weight of fresh curd, the total solids of the curd, and the water content of the curd, respectively, and the weight of the milk processed. In addition, 3 measures of daily cheese yield (dCY, kg/d) were defined, considering the daily milk yield. Three measures of nutrient recovery (REC) were computed: REC(FAT), REC(PROTEIN), and REC(SOLIDS), which represented the ratio between the weights of the fat, protein, and total solids in the curd, respectively, and the corresponding components in the milk. Energy recovery, REC(ENERGY), represented the energy content of the cheese compared with that in the milk. This procedure was used to process individual milk samples obtained from 1,167 Brown Swiss cows reared in 85 herds of the province of Trento (Italy). The assessed traits exhibited almost normal distributions, with the exception of REC(FAT). The average values (± SD) were as follows: %CY(CURD)=14.97±1.86, %CY(SOLIDS)=7.18±0.92, %CY(WATER)=7.77±1.27, dCY(CURD)=3.63±1.17, dCY(SOLIDS)=1.74±0.57, dCY(WATER)=1.88±0.63, REC(FAT)=89.79±3.55, REC(PROTEIN)=78.08±2.43, REC(SOLIDS)=51.88±3.52, and REC(ENERGY)=67.19±3.29. All traits were highly influenced by herd-test-date and days in milk of the cow, moderately influenced by parity, and weakly influenced by the utilized vat. Both %CY(CURD) and dCY(CURD) depended not only on the fat and protein (casein) contents of the milk, but also on their proportions retained in the curd; the water trapped in curd presented an higher variability than that of %CY(SOLIDS). All REC traits were variable and affected by days in milk and parity of the cows. The described model cheese-making procedure and the results obtained provided new insight into the phenotypic variation of cheese yield and recovery traits at the individual level. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Development of Cy5.5-Labeled Hydrophobically Modified Glycol Chitosan Nanoparticles for Protein Delivery

NASA Astrophysics Data System (ADS)

Chin, Amanda

Therapeutic proteins are often highly susceptible to enzymatic degradation, thus restricting their in vivo stability. To overcome this limitation, delivery systems designed to promote uptake and reduce degradation kinetics have undergone a rapid shift from macro-scale systems to nanomaterial based carriers. Many of these nanomaterials, however, elicit immune responses and may have cytotoxic effects both in vitro and in vivo. The naturally derived polysaccharide chitosan has emerged as a promising biodegradable material and has been utilized for many biomedical applications; nevertheless, its function is often constrained by poor solubility. Glycol chitosan, a derivative of chitosan, can be hydrophobically modified to impart amphiphilic properties that enable the self-assembly into nanoparticles in aqueous media at neutral pH. This nanoparticle system has shown initial success as a therapeutic agent in several model cell culture systems, but little is known about its stability against enzymatic degradation. Therefore, the goal of this research was to investigate the resistance of hydrophobically modified glycol chitosan against enzyme-catalyzed degradation using an in vivo simulated system containing lysozyme. To synthesize the nanoparticles, hydrophobic cholanic acid was first covalently conjugated to glycol chitosan using of N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Conjugates were purified by dialysis, lyophilized, and ultra-sonicated to form nanoparticles. Fourier transform infrared (FT-IR) spectroscopy confirmed the binding of 5beta-cholanic acid to the glycol chitosan. Particle size and stability over time were determined with dynamic light scattering (DLS), and particle morphology was evaluated by transmission electron microscopy (TEM). The average diameter of the nanoparticles was approximately 200 nm, which remained stable at 4°C for up to 10 days. Additionally, a near infrared fluorescent (NIRF) dye, Cy5.5, was used to label the glycol chitosan nanoparticles to enable the noninvasive imaging of living cells. A model protein (bovine serum albumin, BSA) was encapsulated within the glycol chitosan nanoparticles, and its loading efficiency was calculated to be 88%. Release profile of the BSA showed that only 4% (cumulative mass) was achieved by day 7. Minimal cytotoxicity was observed after delivery of the chitosan vehicle alone. To test degradation kinetics, the BSA-loaded nanoparticles were incubated with lysozyme for up to 3 hours and were applied in SDS-PAGE to determine if enzyme-catalyzed degradation triggered premature release of the encapsulated protein. Confocal laser scanning microscopy was used to visualize the spatiotemporal distribution of FITC-BSA-loaded glycol chitosan nanoparticles after delivery to the rat osteosarcoma (ROS17/2.8) and mouse calvaria-derived (MC3T3-E1) cells.

Site-specific chemical conjugation of human Fas ligand extracellular domain using trans-cyclooctene - methyltetrazine reactions.

PubMed

Muraki, Michiro; Hirota, Kiyonori

2017-07-03

Fas ligand plays a key role in the human immune system as a major cell death inducing protein. The extracellular domain of human Fas ligand (hFasLECD) triggers apoptosis of malignant cells, and therefore is expected to have substantial potentials in medical biotechnology. However, the current application of this protein to clinical medicine is hampered by a shortage of the benefits relative to the drawbacks including the side-effects in systemic administration. Effective procedures for the engineering of the protein by attaching useful additional functions are required to overcome the problem. A procedure for the site-specific chemical conjugation of hFasLECD with a fluorochrome and functional proteins was devised using an inverse-electron-demand Diels-Alder reaction between trans-cyclooctene group and methyltetrazine group. The conjugations in the present study were attained by using much less molar excess amounts of the compounds to be attached as compared with the conventional chemical modification reactions using maleimide derivatives in the previous study. The isolated conjugates of hFasLECD with sulfo-Cy3, avidin and rabbit IgG Fab' domain presented the functional and the structural integrities of the attached molecules without impairing the specific binding activity toward human Fas receptor extracellular domain. The present study provided a new fundamental strategy for the production of the engineered hFasLECDs with additional beneficial functions, which will lead to the developments of the improved diagnostic systems and the effective treatment methods of serious diseases by using this protein as a component of novel molecular tools.

A comprehensive scoping review of ability and disability in ADHD using the International Classification of Functioning, Disability and Health-Children and Youth Version (ICF-CY).

PubMed

de Schipper, Elles; Lundequist, Aiko; Wilteus, Anna Löfgren; Coghill, David; de Vries, Petrus J; Granlund, Mats; Holtmann, Martin; Jonsson, Ulf; Karande, Sunil; Levy, Florence; Al-Modayfer, Omar; Rohde, Luis; Tannock, Rosemary; Tonge, Bruce; Bölte, Sven

2015-08-01

This is the first in a series of four empirical investigations to develop International Classification of Functioning, Disability and Health (ICF) Core Sets for Attention Deficit Hyperactivity Disorder (ADHD). The objective here was to use a comprehensive scoping review approach to identify the concepts of functional ability and disability used in the scientific ADHD literature and link these to the nomenclature of the ICF-CY. Systematic searches were conducted using Medline/PubMed, PsycINFO, ERIC and Cinahl, to extract the relevant concepts of functional ability and disability from the identified outcome studies of ADHD. These concepts were then linked to ICF-CY by two independent researchers using a standardized linking procedure. Data from identified studies were analysed until saturation of ICF-CY categories was reached. Eighty studies were included in the final analysis. Concepts contained in these studies were linked to 128 ICF-CY categories. Of these categories, 68 were considered to be particularly relevant to ADHD (i.e., identified in at least 5 % of the studies). Of these, 32 were related to Activities and participation, 31 were related to Body functions, and five were related to environmental factors. The five most frequently identified categories were school education (53 %), energy and drive functions (50 %), psychomotor functions (50 %), attention functions (49 %), and emotional functions (45 %). The broad variety of ICF-CY categories identified in this study underlines the necessity to consider ability and disability in ADHD across all dimensions of life, for which the ICF-CY provides a valuable and universally applicable framework. These results, in combination with three additional preparatory studies (expert survey, focus groups, clinical study), will provide a scientific basis to define the ICF Core Sets for ADHD for multi-purpose use in basic and applied research, and every day clinical practice.

Anti-inflammatory activity of Cymbopogon citratus leaves infusion via proteasome and nuclear factor-κB pathway inhibition: contribution of chlorogenic acid.

PubMed

Francisco, Vera; Costa, Gustavo; Figueirinha, Artur; Marques, Carla; Pereira, Paulo; Miguel Neves, Bruno; Celeste Lopes, Maria; García-Rodríguez, Carmen; Teresa Cruz, Maria; Teresa Batista, Maria

2013-06-21

Cymbopogon citratus (DC.) Stapf leaves infusion is used in traditional medicine for the treatment of inflammatory conditions, however little is known about their bioactive compounds. Investigate the compounds responsible for anti-inflammatory potential of Cymbopogon citratus (Cy) on cytokines production induced by lipopolysaccharide (LPS) in human and mouse macrophages, and the action mechanisms involved. An essential oil-free infusion of Cy was prepared and polyphenol-rich fractions (PFs) were obtained from it by column chromatography. Chlorogenic acid (CGA) was identified, by HPLC/PDA/ESI-MS(n). The expression of cytokines, namely TNF-α and CCL5, was analyzed by real-time RT-PCR, on LPS-stimulated human macrophages. Activation of nuclear factor (NF)-κB, a master regulator of inflammation, was investigated by western blot and gene reporter assay. Proteasome activity was assessed using a fluorogenic peptide. Cymbopogon citratus extract and its polyphenols inhibited the cytokine production on human macrophages. This supports the anti-inflammatory activity of Cy polyphenols in physiologically relevant cells. Concerning the effect on the activation of NF-κB pathway, the results pointed to an inhibition of LPS-induced NF-κB activation by Cy and PFs. CGA was identified, by HPLC/PDA/ESI-MS(n), as the main phenolic acid of the Cy infusion, and it demonstrated to be, at least in part, responsible by that effect. Additionally, it was verified for the first time that Cy and PFs inhibited the proteasome activity, a complex that controls NF-κB activation, having CGA a strong contribution. The results evidenced, for the first time, the anti-inflammatory properties of Cymbopogon citratus through proteasome inhibition and, consequently NF-κB pathway and cytokine expression. Additionally, Cy polyphenols, in particular chlorogenic acid, were highlighted as bioactive compounds. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Bispecific Antibody Conjugated Manganese-Based Magnetic Engineered Iron Oxide for Imaging of HER2/neu- and EGFR-Expressing Tumors

PubMed Central

Wu, Shou-Cheng; Chen, Yu-Jen; Wang, Hsiang-Ching; Chou, Min-Yuan; Chang, Teng-Yuan; Yuan, Shyng-Shiou; Chen, Chiao-Yun; Hou, Ming-Feng; Hsu, John Tsu-An; Wang, Yun-Ming

2016-01-01

The overexpression of HER2/neu and EGFR receptors plays important roles in tumorigenesis and tumor progression. Targeting these two receptors simultaneously can have a more widespread application in early diagnosis of cancers. In this study, a new multifunctional nanoparticles (MnMEIO-CyTE777-(Bis)-mPEG NPs) comprising a manganese-doped iron oxide nanoparticle core (MnMEIO), a silane-amino functionalized poly(ethylene glycol) copolymer shell, a near infrared fluorescence dye (CyTE777), and a covalently conjugated anti-HER2/neu and anti-EGFR receptors bispecific antibody (Bis) were successfully developed. In vitro T2-weighted MR imaging studies in SKBR-3 and A431 tumor cells incubated with MnMEIO-CyTE777-(Bis)-mPEG NPs showed - 94.8 ± 3.8 and - 84.1 ± 2.8% negative contrast enhancement, respectively. Pharmacokinetics study showed that MnMEIO-CyTE777-(Bis)-mPEG NPs were eliminated from serum with the half-life of 21.3 mins. In vivo MR imaging showed that MnMEIO-CyTE777-(Bis)-mPEG NPs could specifically and effectively target to HER2/neu- and EGFR-expressing tumors in mice; the relative contrast enhancements were 11.8 (at 2 hrs post-injection) and 61.5 (at 24 hrs post-injection) fold higher in SKBR-3 tumors as compared to Colo-205 tumors. T2-weighted MR and optical imaging studies revealed that the new contrast agent (MnMEIO-CyTE777-(Bis)-mPEG NPs) could specifically and effectively target to HER2/neu- and/or EGFR-expressing tumors. Our results demonstrate that MnMEIO-CyTE777-(Bis)-mPEG NPs are able to recognize the tumors expressing both HER2/neu and/or EGFR, and may provide a novel molecular imaging tool for early diagnosis of cancers expressing HER2/neu and/or EGFR. PMID:26722378

1-(2-Hy-droxy-eth-yl)-3-[(2-hy-droxy-eth-yl)amino]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione.

PubMed

Xie, Zhi-Xiong; Zhao, Sheng-Yin

2011-04-01

There are four molecules in the asymmetric unit of the title compound, C(16)H(17)N(3)O(4), in which the dihedral angles between the indole ring system and maleimide ring are 4.5 (3), 8.3 (3), 8.4 (2) and 10.4 (2)°. In the crystal, mol-ecules are linked by numerous N-H⋯O and O-H⋯O hydrogen bonds, generating a three-dimensional network.

Single-Molecule Three-Color FRET with Both Negligible Spectral Overlap and Long Observation Time

PubMed Central

Hohng, Sungchul

2010-01-01

Full understanding of complex biological interactions frequently requires multi-color detection capability in doing single-molecule fluorescence resonance energy transfer (FRET) experiments. Existing single-molecule three-color FRET techniques, however, suffer from severe photobleaching of Alexa 488, or its alternative dyes, and have been limitedly used for kinetics studies. In this work, we developed a single-molecule three-color FRET technique based on the Cy3-Cy5-Cy7 dye trio, thus providing enhanced observation time and improved data quality. Because the absorption spectra of three fluorophores are well separated, real-time monitoring of three FRET efficiencies was possible by incorporating the alternating laser excitation (ALEX) technique both in confocal microscopy and in total-internal-reflection fluorescence (TIRF) microscopy. PMID:20808851

Chemical control of rate and onset temperature of nadimide polymerization

NASA Technical Reports Server (NTRS)

Lauver, R. W.

1985-01-01

The chemistry of norbornenyl capped imide compounds (nadimides) is briefly reviewed with emphasis on the contribution of Diels-Alder reversion in controlling the rate and onset of the thermal polymerization reaction. Control of onset temperature of the cure exotherm by adjusting the concentration of maleimide is demonstrated using selected model compounds. The effects of nitrophenyl compounds as free radical retarders on nadimide reactivity are discussed. A simple copolymerization model is proposed for the overall nadimide cure reaction. An approximate numerical analysis is carried out to demonstrate the ability of the model to simulate the trends observed for both maleimide and nitrophenyl additions.

Cyclosporine induced biochemical remission in childhood autoimmune hepatitis.

PubMed

Franulović, Orjena Zaja; Rajacić, Nada; Lesar, Tatjana; Kuna, Andrea Tesija; Morić, Bernardica Valent

2012-09-01

The conventional treatment of autoimmune hepatitis (AIH) in children, which includes prednisone alone or in combination with azathioprine, induces remission in most cases but is often associated with poorly tolerated side effects. To avoid the adverse effects, Alvarez et al. introduced an alternative treatment regimen, using cyclosporine A (CyA) as primary immunosuppression. We carried out a retrospective study to evaluate the efficacy and tolerance of CyA treatment in children and adolescents with AIH treated in our center. During 2000-2010 period, nine children (6 female) aged 5-17.5 years, were diagnosed with AIH according to established international criteria. Following the suggested protocol, CyA was administered orally and when the transaminases tended to normalise, dose was adjusted to lover serum levels. Conversion to low dose of prednisone and azathioprine was started after 6 months, with gradual tapering and discontinuation of CyA. All nine patient had elevated transaminases and gammaglobulin levels, with proven histological changes typical for AIH in 8 patients that underwent liver biopsy (in one patient biopsy was contraindicated due to the prolonged prothrombin time). Serum ANA/SMA autoantibodies were positive in all but one patient, who had positive anti-LKM1. Complete or near complete and persistent normalisation of transaminase activity was observed in 8/9 patients within first 6 to 12 months. In one patient with partial response, an overlap syndrome was established. After ursodeoxycholic acid was added complete remission was observed. All patients had excellent clinical course and histological improvement. During the long-term follow-up (1.5-9 yrs; median 4.5 yrs), biochemical relapse occured in one patient after discontinuation of maintenance corticosteroid dose. Despite registered improvement, none of the patients fulfilled the criteria for therapy discontinuation, so all of them are still receiving maintenance doses of prednisone or azathioprine. The applied protocol allowed for the control of the liver inflammatory disease in all of our patients and protected them from the side effects related to steroid treatment. Side effects of CyA were minimal and were well tolerated.

Cyclophilin B attenuates the expression of TNF-α in lipopolysaccharide-stimulated macrophages through the induction of B cell lymphoma-3.

PubMed

Marcant, Adeline; Denys, Agnès; Melchior, Aurélie; Martinez, Pierre; Deligny, Audrey; Carpentier, Mathieu; Allain, Fabrice

2012-08-15

Extracellular cyclophilin A (CyPA) and CyPB have been well described as chemotactic factors for various leukocyte subsets, suggesting their contribution to inflammatory responses. Unlike CyPA, CyPB accumulates in extracellular matrixes, from which it is released by inflammatory proteases. Hence, we hypothesized that it could participate in tissue inflammation by regulating the activity of macrophages. In the current study, we confirmed that CyPB initiated in vitro migration of macrophages, but it did not induce production of proinflammatory cytokines. In contrast, pretreatment of macrophages with CyPB attenuated the expression of inflammatory mediators induced by LPS stimulation. The expression of TNF-α mRNA was strongly reduced after exposure to CyPB, but it was not accompanied by significant modification in LPS-induced activation of MAPK and NF-κB pathways. LPS activation of a reporter gene under the control of TNF-α gene promoter was also markedly decreased in cells treated with CyPB, suggesting a transcriptional mechanism of inhibition. Consistent with this hypothesis, we found that CyPB induced the expression of B cell lymphoma-3 (Bcl-3), which was accompanied by a decrease in the binding of NF-κB p65 to the TNF-α promoter. As expected, interfering with the expression of Bcl-3 restored cell responsiveness to LPS, thus confirming that CyPB acted by inhibiting initiation of TNF-α gene transcription. Finally, we found that CyPA was not efficient in attenuating the production of TNF-α from LPS-stimulated macrophages, which seemed to be due to a modest induction of Bcl-3 expression. Collectively, these findings suggest an unexpected role for CyPB in attenuation of the responses of proinflammatory macrophages.

Excited state free energy calculations of Cy3 in different environments

NASA Astrophysics Data System (ADS)

Sawangsang, Pilailuk; Buranachai, Chittanon; Punwong, Chutintorn

2015-05-01

Cy3, a cyanine dye, is one of the most widely used dyes in investigating the structure and dynamics of biomolecules by means of fluorescence methods. However, Cy3 fluorescence emission is strongly competed by trans-cis isomerization, whose efficiency is dictated by the isomerization energy barrier and the environment of Cy3. The fluorescence quantum yield of Cy3 is very low when the dye is free in homogeneous solution but it is considerably enhanced in an environment that rigidifies the structure, e.g. when it is attached to a DNA strand. In this work, the barriers for isomerization on the excited state of free Cy3, and Cy3 attached to single- and double-stranded DNA in methanol, are presented. The free energy and subsequently the isomerization barrier calculations are performed using the umbrella sampling technique with the weighted histogram analysis method. The hybrid quantum mechanics/molecular mechanics (QM/MM) approach is employed to provide the potential energy surfaces for the excited state dynamics simulations in umbrella sampling. The semiempirical floating occupation molecular orbital configuration interaction method is used for electronic excited state calculations of the QM region (Cy3). From the free energy calculations, the barrier of Cy3 attached to the single-stranded DNA is highest, in agreement with previously reported experimental results. This is likely due to the stacking interaction between Cy3 and DNA. Such a stacking interaction is likely associated with steric hindrance that prevents the rotation around the conjugated bonds of Cy3. If Cy3 experiences high steric hindrance, it has a higher isomerization barrier and thus the efficiency of fluorescence emission increases.

Impact of cyclophosphamide dose of conditioning on the outcome of allogeneic hematopoietic stem cell transplantation for aplastic anemia from human leukocyte antigen-identical sibling.

PubMed

Mori, Takehiko; Koh, Hideo; Onishi, Yasushi; Kako, Shinichi; Onizuka, Makoto; Kanamori, Heiwa; Ozawa, Yukiyasu; Kato, Chiaki; Iida, Hiroatsu; Suzuki, Ritsuro; Ichinohe, Tatsuo; Kanda, Yoshinobu; Maeda, Tetsuo; Nakao, Shinji; Yamazaki, Hirohito

2016-04-01

The standard conditioning regimen in allogeneic hematopoietic stem cell transplantation (HSCT) for aplastic anemia from a human leukocyte antigen (HLA)-identical sibling has been high-dose cyclophosphamide (CY 200 mg/kg). In the present study, results for 203 patients with aplastic anemia aged 16 years or older who underwent allogeneic HSCT from HLA-identical siblings were retrospectively analyzed using the registry database of Japan Society for Hematopoietic Cell Transplantation. Conditioning regimens were defined as a (1) high-dose CY (200 mg/kg or greater)-based (n = 117); (2) reduced-dose CY (100 mg/kg or greater, but less than 200 mg/kg)-based (n = 38); and (3) low-dose CY (less than 100 mg/kg)-based (n = 48) regimen. Patient age and the proportion of patients receiving fludarabine were significantly higher in the reduced- and low-dose CY groups than the high-dose CY group. Engraftment was comparable among the groups. Five-year overall survival (OS) tended to be higher in the low-dose CY group [93.0 % (95 % CI 85.1-100.0 %)] than the high-dose CY [84.2 % (95 % CI 77.1-91.3 %)] or reduced-dose CY groups [83.8 % (95 % CI 71.8-95.8 %); P = 0.214]. Age-adjusted OS was higher in the low-dose CY group than the high- and reduced-dose CY groups with borderline significance (P = 0.067). These results suggest that CY dose can safely be reduced without increasing graft rejection by adding fludarabine in allogeneic HSCT for aplastic anemia from an HLA-identical sibling.

Polymeric nanotheranostics for real-time non-invasive optical imaging of breast cancer progression and drug release.

PubMed

Ferber, Shiran; Baabur-Cohen, Hemda; Blau, Rachel; Epshtein, Yana; Kisin-Finfer, Einat; Redy, Orit; Shabat, Doron; Satchi-Fainaro, Ronit

2014-09-28

Polymeric nanocarriers conjugated with low molecular weight drugs are designed in order to improve their efficacy and toxicity profile. This approach is particularly beneficial for anticancer drugs, where the polymer-drug conjugates selectively accumulate at the tumor site, due to the enhanced permeability and retention (EPR) effect. The conjugated drug is typically inactive, and upon its pH- or enzymatically-triggered release from the carrier, it regains its therapeutic activity. These settings lack information regarding drug-release time, kinetics and location. Thereby, real-time non-invasive intravital monitoring of drug release is required for theranostics (therapy and diagnostics). We present here the design, synthesis and characterization of a theranostic nanomedicine, based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer, owing its fluorescence-based monitoring of site-specific drug release to a self-quenched near-infrared fluorescence (NIRF) probe. We designed two HPMA copolymer-based systems that complement to a theranostic nanomedicine. The diagnostic system consists of self-quenched Cy5 (SQ-Cy5) as a reporter probe and the therapeutic system is based on the anticancer agent paclitaxel (PTX). HPMA copolymer-PTX/SQ-Cy5 systems enable site-specific release upon enzymatic degradation in cathepsin B-overexpressing breast cancer cells. The release of the drug occurs concomitantly with the activation of the fluorophore to its Turn-ON state. HPMA copolymer-SQ-Cy5 exhibits preferable body distribution and drug release compared with the free drug and probe when administered to cathepsin B-overexpressing 4T1 murine mammary adenocarcinoma-bearing mice. This approach of co-delivery of two complementary systems serves as a proof-of-concept for real-time deep tissue intravital orthotopic monitoring and may have the potential use in clinical utility as a theranostic nanomedicine. Copyright © 2014. Published by Elsevier Ireland Ltd.

Target cell cyclophilins facilitate human papillomavirus type 16 infection.

PubMed

Bienkowska-Haba, Malgorzata; Patel, Hetalkumar D; Sapp, Martin

2009-07-01

Following attachment to primary receptor heparan sulfate proteoglycans (HSPG), human papillomavirus type 16 (HPV16) particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB) facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV-induced diseases.

[Characteristics of cationic polymers PEI-CyD, PEI-PHPA, PEE-PHPA and PEI25kD in vitro and in vivo].

PubMed

Yao, Qi; Jin, Xue; Hu, Tian-nan; Wang, Qi-wen; Wang, Xun-shi; Hu, Qi-da; Xu, Sang; Zhou, Jun; Tang, Gu-ping

2012-11-01

To study the characteristics of cationic polymers polyethylenimine-β-cyclodextrin (PEI-CyD), polyethylenimine-poly-(3-hydroxypropyl)-aspartamide (PEI-PHPA), N,N-Dimethyldipropylenetriamine-Bis(3-aminopropyl)amine-aspartamide (PEE-PHPA) in vitro and in vivo. PEI-PHPA, PEI-CyD and PEE-PHPA were synthesized and the chemistry structure of PEI-PHPA, PEI-CyD and PEE-PHPA was confirmed by (1)H-NMR. The particle size and zeta potential of these polymers were measured, and capacity of plasmid DNA condensation was tested. The inhibition of COS-7, A549, HEK293 and C6 cells was measured by MTT assay. The transfection efficiency was determined in HEK293 cell lines. The toxicity, tissue distribution and transfection efficiency of cationic polymers were tested in vivo. When the N/P of polymers/DNA at 30, the particle sizes were close 250 nm and the zeta-potential were near 35 mv. They were able to condense DNA at N/P ratio < 5. The MTT assay showed that the IC(50) of PEE-PHPA was 21.5, 20.2, 7.30 and 37.1 μg/ml, and that of PEI25kD was 15.8, 18.3, 11.4 and 36.7 μg/ml in C6, COS-7, A549 and HEK293cell lines, respectively. The cell viability of PEI-CyD and PEI-PHPA in above cell lines was over 60%. They had high transfection efficiency in HEK293 cell lines. The LD(50) of PEI25Kd, PEI-CyD, PEI-PHPA and PEE-PHPA in vivo was 19.50, 100.4, 521.2 and 630.0, respectively by intraperitoneal (ip) injection. The contractions of these polymers were higher in kidney than in other organs and tissues.PEE-PHPA had slight effect on kidney and liver function. PEE and PEI25kD have higher transfection efficiency and higher toxicity; while PC and PHPA-PEI have lower toxicity and higher transfection efficiency to be used as non-viral gene vector.

On-chip transduction of nucleic acid hybridization using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer.

PubMed

Tavares, Anthony J; Noor, M Omair; Vannoy, Charles H; Algar, W Russ; Krull, Ulrich J

2012-01-03

The glass surface of a glass-polydimethylsiloxane (PDMS) microfluidic channel was modified to develop a solid-phase assay for quantitative determination of nucleic acids. Electroosmotic flow (EOF) within channels was used to deliver and immobilize semiconductor quantum dots (QDs), and electrophoresis was used to decorate the QDs with oligonucleotide probe sequences. These processes took only minutes to complete. The QDs served as energy donors in fluorescence resonance energy transfer (FRET) for transduction of nucleic acid hybridization. Electrokinetic injection of fluorescent dye (Cy3) labeled oligonucleotide target into a microfluidic channel and subsequent hybridization (within minutes) provided the proximity for FRET, with emission from Cy3 being the analytical signal. The quantification of target concentration was achieved by measurement of the spatial length of coverage by target along a channel. Detection of femtomole quantities of target was possible with a dynamic range spanning an order of magnitude. The assay provided excellent resistance to nonspecific interactions of DNA. Further selectivity of the assay was achieved using 20% formamide, which allowed discrimination between a fully complementary target and a 3 base pair mismatch target at a contrast ratio of 4:1. © 2011 American Chemical Society

Cyclophilin B mediates cyclosporin A incorporation in human blood T-lymphocytes through the specific binding of complexed drug to the cell surface.

PubMed

Allain, F; Denys, A; Spik, G

1996-07-15

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein located within intracellular vesicles and released in biological fluids. We recently reported the specific binding of this protein to T-cell surface receptor which is internalized even in the presence of CsA. These results suggest that CyPB might target the drug to lymphocytes and consequently modify its activity. To verify this hypothesis, we have first investigated the binding capacity and internalization of the CsA-CyPB complex in human peripheral blood T-lymphocytes and secondly compared the inhibitory effect of both free and CyPB-complexed CsA on the CD3-induced activation and proliferation of T-cells. Here, we present evidence that both the CsA-CyPB complex and free CyPB bind to the T-lymphocyte surface, with similar values of Kd and number of sites. At 37 degrees C, the complex is internalized but, in contrast to the protein, the drug is accumulated within the cell. Moreover, CyPB receptors are internalized together with the ligand and rapidly recycled to the cell surface. Finally, we demonstrate that CyPB-complexed CsA remains as efficient as uncomplexed CsA and that CyPB enhances the immunosuppressive activity of the drug. Taken together, our results support the hypothesis that surface CyPB receptors may be related to the selective and variable action of CsA, through specific binding and targeting of the CyPB-CsA complex to peripheral blood T-lymphocytes.

Subclass-specific labeling of protein-reactive natural products with customized nucleophilic probes.

PubMed

Rudolf, Georg C; Koch, Maximilian F; Mandl, Franziska A M; Sieber, Stephan A

2015-02-23

Natural products represent a rich source of bioactive compounds that constitute a large fraction of approved drugs. Among those are molecules with electrophilic scaffolds, such as Michael acceptors, β-lactams, and epoxides that irreversibly inhibit essential enzymes based on their catalytic mechanism. In the search for novel bioactive molecules, current methods are challenged by the frequent rediscovery of known chemical entities. Herein small nucleophilic probes that attack electrophilic natural products and enhance their detection by HPLC-UV and HPLC-MS are introduced. A screen of diverse probe designs revealed one compound with a desired selectivity for epoxide- and maleimide-based antibiotics. Correspondingly, the natural products showdomycin and phosphomycin could be selectively targeted in extracts of their natural producing organism, in which the probe-modified molecules exhibited superior retention and MS detection relative to their unmodified counterparts. This method may thus help to discover small, electrophilic molecules that might otherwise easily elude detection in complex samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Toughening and healing of continuous fibre reinforced composites with bis-maleimide based pre-pregs

NASA Astrophysics Data System (ADS)

Kostopoulos, V.; Kotrotsos, A.; Tsantzalis, S.; Tsokanas, P.; Christopoulos, A. C.; Loutas, T.

2016-08-01

Unidirectional (UD) pre-pregs containing self-healing materials based on Diels-Alder reaction bis-maleimide (BMI) polymers were successfully incorporated on the mid-plane of UD carbon fibre reinforced polymers. The fracture toughness of these composites and the introduced healing capability were measured under mode I loading. The interlaminar fracture toughness was enhanced considerably, since the maximum load (P max) of the modified composite increased approximately 1.5 times and the mode I fracture energy (G IC) displayed a significant increase of almost 3.5 times when compared to the reference composites. Furthermore the modified composites displayed a healing efficiency (HE) value of about 30% for P max and 20% for G IC after the first healing, appearing to be an almost stable behaviour after the third healing cycle. The HE displayed a decrease of 20% and 15% for P max and G IC values, respectively, after the fifth healing cycle. During the tests, the monitored acoustic emission (AE) activity of the samples showed that there is no significant difference due to the presence of BMI polymer in terms of AE hits. Moreover, optical microscopy not only showed that the epoxy matrix at the interface is partly infiltrated by the BMI polymer, but it also revealed the presence of pulled out fibres at the fractured surface, indicating ductile behaviour.

A DNA Crystal Designed to Contain Two Molecules per Asymmetric Unit

DOE Office of Scientific and Technical Information (OSTI.GOV)

T Wang; R Sha; J Birktoft

2011-12-31

We describe the self-assembly of a DNA crystal that contains two tensegrity triangle molecules per asymmetric unit. We have used X-ray crystallography to determine its crystal structure. In addition, we have demonstrated control over the colors of the crystals by attaching either Cy3 dye (pink) or Cy5 dye (blue-green) to the components of the crystal, yielding crystals of corresponding colors. Attaching the pair of dyes to the pair of molecules yields a purple crystal.

EDB Fibronectin Specific Peptide for Prostate Cancer Targeting.

PubMed

Han, Zheng; Zhou, Zhuxian; Shi, Xiaoyue; Wang, Junpeng; Wu, Xiaohui; Sun, Da; Chen, Yinghua; Zhu, Hui; Magi-Galluzzi, Cristina; Lu, Zheng-Rong

2015-05-20

Extradomain-B fibronectin (EDB-FN), one of the oncofetal fibronectin (onfFN) isoforms, is a high-molecular-weight glycoprotein that mediates cell adhesion and migration. The expression of EDB-FN is associated with a number of cancer-related biological processes such as tumorigenesis, angiogenesis, and epithelial-to-mesenchymal transition (EMT). Here, we report the development of a small peptide specific to EDB-FN for targeting prostate cancer. A cyclic nonapeptide, CTVRTSADC (ZD2), was identified using peptide phage display. A ZD2-Cy5 conjugate was synthesized to accomplish molecular imaging of prostate cancer in vitro and in vivo. ZD2-Cy5 demonstrated effective binding to up-regulated EDB-FN secreted by TGF-β-induced PC3 cancer cells following EMT. Following intravenous injections, the targeted fluorescent probe specifically bound to and delineated PC3-GFP prostate tumors in nude mice bearing the tumor xenografts. ZD2-Cy5 also showed stronger binding to human prostate tumor specimens with a higher Gleason score (GS9) compared to those with a lower score (GS 7), with no binding in benign prostatic hyperplasia (BPH). Thus, the ZD2 peptide is a promising strategy for molecular imaging and targeted therapy of prostate cancer.

Potential use of glucuronylglucosyl-β-cyclodextrin/dendrimer conjugate (G2) as a DNA carrier in vitro and in vivo.

PubMed

Anno, Takayuki; Higashi, Taishi; Motoyama, Keiichi; Hirayama, Fumitoshi; Uekama, Kaneto; Arima, Hidetoshi

2012-04-01

In this study, we evaluated the polyamidoamine starburst dendrimer (dendrimer, generation 2: G2) conjugate with 6-O-α-(4-O-α-D-glucuronyl)-D-glucosyl-β-cyclodextrin (GUG-β-CDE (G2)) as a gene transfer carrier. The in vitro gene transfer activity of GUG-β-CDE (G2, degree of substitution (DS) of cyclodextrin (CyD) 1.8) was remarkably higher than that of dendrimer (G2) conjugate with α-CyD (α-CDE (G2, DS 1.2)) and that with β-CyD(β-CDE (G2, DS 1.3)) in A549 and RAW264.7 cells. The particle size, ζ-potential, DNase I-catalyzed degradation, and cellular association of plasmid DNA (pDNA) complex with GUG-β-CDE (G2, DS 1.8) were almost the same as those of the other CDEs. Fluorescent-labeled GUG-β-CDE (G2, DS 1.8) localized in the nucleus 6 h after transfection of its pDNA complex in A549 cells, suggesting that nuclear localization of pDNA complex with GUG-β-CDE (G2, DS 1.8), at least in part, contributes to its high gene transfer activity. GUG-β-CDE (G2, DS 1.8) provided higher gene transfer activity than α-CDE (G2, DS 1.2) and β-CDE (G2, DS 1.3) in kidney with negligible changes in blood chemistry values 12 h after intravenous injection of pDNA complexes with GUG-β-CDE (G2, DS 1.8) in mice. In conclusion, the present findings suggest that GUG-β-CDE (G2, DS 1.8) has the potential for a novel polymeric pDNA carrier in vitro and in vivo.

Aptamer based SERS detection of Salmonella typhimurium using DNA-assembled gold nanodimers.

PubMed

Xu, Xumin; Ma, Xiaoyuan; Wang, Haitao; Wang, Zhouping

2018-06-12

The authors describe a surface-enhanced Raman scattering (SERS) based aptasensor for Salmonella typhimurium (S. typhimurium). Gold nanoparticles (AuNPs; 35 nm i.d.) were functionalized with the aptamer (ssDNA 1) and used as the capture probe, while smaller (15 nm) AuNPs were modified with a Cy3-labeled complementary sequence (ssDNA 2) and used as the signalling probe. The asymmetric gold nanodimers (AuNDs) were assemblied with the Raman signal probe and the capture probe via hybridization of the complementary ssDNAs. The gap between two nanoparticles is a "hot spot" in which the Raman reporter Cy3 is localized. It experiences a strong enhancement of the electromagnetic field around the particle. After addition of S. typhimurium, it will be bound by the aptamer which therefore is partially dehybridized from its complementary sequence. Hence, Raman intensity drops. Under the optimal experimental conditions, the SERS signal at 1203 cm -1 increases linearly with the logarithm of the number of colonies in the 10 2 to 10 7  cfu·mL -1 concentration range, and the limit of detection is 35 cfu·mL -1 . The method can be performed within 1 h and was successfully applied to the analysis of spiked milk samples and performed very well and with high specificity. Graphical abstract DNA-assembled asymmetric gold nanodimers (AuNDs) were synthesized and appllied in a SERS-based aptasensor for S. typhimurium. Capture probe was preferentially combined with S. typhimurium and the structure of the AuNDs was destroyed. The "hot spot" vanished partly, this resulting in the decreased Raman intensity of Cy3.

76 FR 13292 - Medicare Program: Changes to the Hospital Outpatient Prospective Payment System and CY 2011...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-03-11

... Prospective Payment System and CY 2011 Payment Rates; Changes to the Ambulatory Surgical Center Payment System and CY 2011 Payment Rates; Changes to Payments to Hospitals for Graduate Medical Education Costs..., 2010, entitled ``Medicare Program: Hospital Outpatient Prospective Payment System and CY 2011 Payment...

Effects of low-dose cyclophosphamide with piroxicam on tumour neovascularization in a canine oral malignant melanoma-xenografted mouse model.

PubMed

Choisunirachon, N; Jaroensong, T; Yoshida, K; Saeki, K; Mochizuki, M; Nishimura, R; Sasaki, N; Nakagawa, T

2015-12-01

Low-dose cyclophosphamide (CyLD) has shown promise in the treatment of several cancers; however, the effect of CyLD on canine oral malignant melanoma has never been explored. In this study, we investigated the effects of CyLD with or without piroxicam (Px) on tumour neovascularization and vascular normalization in a canine oral malignant melanoma-xenografted mice model. After treatment with CyLD, Px or a combination of both (CyPx), the growth of the tumour in the treatment groups was significantly suppressed compared to the control group at 30 days of treatment. Proliferation index was also significantly reduced by all treatments, only CyPx significantly lowered microvessel density and vascular endothelial growth factor (VEGF) levels. Additionally, CyLD significantly reduced the proportion of normal vessels and caused an imbalance between VEGF and thrombospondin-1. These results suggested that CyPx has potent anti-angiogenic effects in terms of both the number and quality of blood vessels in xenografted canine oral malignant melanoma. © 2013 John Wiley & Sons Ltd.

Cytochrome P450 2J2, a new key enzyme in cyclophosphamide bioactivation and a potential biomarker for hematological malignancies.

PubMed

El-Serafi, I; Fares, M; Abedi-Valugerdi, M; Afsharian, P; Moshfegh, A; Terelius, Y; Potácová, Z; Hassan, M

2015-10-01

The role of cytochrome P450 2J2 (CYP2J2) in cyclophosphamide (Cy) bioactivation was investigated in patients, cells and microsomes. Gene expression analysis showed that CYP2J2 mRNA expression was significantly (P<0.01) higher in 20 patients with hematological malignancies compared with healthy controls. CYP2J2 expression showed significant upregulation (P<0.05) during Cy treatment before stem cell transplantation. Cy bioactivation was significantly correlated to CYP2J2 expression. Studies in HL-60 cells expressing CYP2J2 showed reduced cell viability when incubated with Cy (half maximal inhibitory concentration=3.6 mM). Inhibition of CYP2J2 using telmisartan reduced Cy bioactivation by 50% and improved cell survival. Cy incubated with recombinant CYP2J2 microsomes has resulted in apparent Km and Vmax values of 3.7-6.6 mM and 2.9-10.3 pmol/(min·pmol) CYP, respectively. This is the first study demonstrating that CYP2J2 is equally important to CYP2B6 in Cy metabolism. The heart, intestine and urinary bladder express high levels of CYP2J2; local Cy bioactivation may explain Cy-treatment-related toxicities in these organs.

Delineation of the calcineurin-interacting region of cyclophilin B.

PubMed

Carpentier, M; Allain, F; Haendler, B; Slomianny, M C; Spik, G

2000-12-01

The immunosuppressant drug cyclosporin A (CsA) inhibits T-cell function by blocking the phosphatase activity of calcineurin. This effect is mediated by formation of a complex between the drug and cyclophilin (CyP), which creates a composite surface able to make high-affinity contacts with calcineurin. In vitro, the CyPB/CsA complex is more effective in inhibiting calcineurin than the CyPA/CsA and CyPC/CsA complexes, pointing to fine structural differences in the calcineurin-binding region. To delineate the calcineurin-binding region of CyPB, we mutated several amino acids, located in two loops corresponding to CyPA regions known to be involved, as follows: R76A, G77H, D155R, and D158R. Compared to wild-type CyPB, the G77H, D155R, and D158R mutants had intact isomerase and CsA-binding activities, indicating that no major conformational changes had taken place. When complexed to CsA, they all displayed only reduced affinity for calcineurin and much decreased inhibition of calcineurin phosphatase activity. These results strongly suggest that the three amino acids G77, D155, and D158 are directly involved in the interaction of CyPB/CsA with calcineurin, in agreement with their exposed position. The G77, D155, and D158 residues are not maintained in CyPA and might therefore account for the higher affinity of the CyPB/CsA complex for calcineurin.

Comparison of Cyclophosphamide Combined with Total Body Irradiation, Oral Busulfan, or Intravenous Busulfan for Allogeneic Hematopoietic Cell Transplantation in Adults with Acute Lymphoblastic Leukemia.

PubMed

Mitsuhashi, Kenjiro; Kako, Shinichi; Shigematsu, Akio; Atsuta, Yoshiko; Doki, Noriko; Fukuda, Takahiro; Kanamori, Heiwa; Onizuka, Makoto; Takahashi, Satoshi; Ozawa, Yukiyasu; Kurokawa, Mineo; Inoue, Yoshiko; Nagamura-Inoue, Tokiko; Morishima, Yasuo; Mizuta, Shuichi; Tanaka, Junji

2016-12-01

We conducted a retrospective analysis to compare outcomes in adult patients with acute lymphoblastic leukemia (ALL) who underwent allogeneic hematopoietic cell transplantation (allo-HCT) with conditioning regimens containing cyclophosphamide (CY) in combination with total body irradiation (TBI), oral busulfan (p.o. BU), or intravenous busulfan (i.v. BU). We used data for January 2000 to December 2012 from the Transplant Registry Unified Management Program of the Japan Society of Hematopoietic Cell Transplantation. We identified 2130 patients treated with TBI/CY (n = 2028), p.o. BU/CY (n = 60), or i.v. BU/CY (n = 42). Two-year overall survival (OS) and 2-year relapse-free survival rates were 69.0% and 62.1%, respectively, in the TBI/CY group, 55.9% and 54.2% in the p.o. BU/CY group, and 71.0% and 46.8% in the i.v. BU/CY group. In multivariate analysis, compared with TBI/CY, p.o. BU/CY, but not i.v. BU/CY, was associated with lower OS (hazard ratio [HR], 1.46; P = .047) and a higher incidence of sinusoidal obstruction syndrome (HR, 3.36; P = .030). No between-group differences were seen in the incidence of nonrelapse mortality, relapse, acute graft-versus-host disease (GVHD), or chronic GVHD. We suggest that i.v. BU/CY might be a possible alternative allo-HCT conditioning regimen for adults with ALL who are not suitable for TBI. Copyright © 2016 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Enhancement of curcumin wound healing ability by complexation with 2-hydroxypropyl-γ-cyclodextrin in sacran hydrogel film.

PubMed

Wathoni, Nasrul; Motoyama, Keiichi; Higashi, Taishi; Okajima, Maiko; Kaneko, Tatsuo; Arima, Hidetoshi

2017-05-01

Curcumin is one of promising agents to accelerate the wound-healing process. However, the efficacy of curcumin is limited due to its poor water solubility and stability. To enhance the properties of curcumin, 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CyD) can be used through complexation. Recently, we revealed that sacran has the potential to form a hydrogel film (HGF) as a wound dressing material. Therefore, in the present study, we investigated the wound healing ability of curcumin/HP-γ-CyD (Cur/HP-γ-CyD) complex in sacran-based HGF (Sac-HGF). We prepared the Cur/HP-γ-CyD complex in Sac-HGF without surface roughness. Additionally, the amorphous form in the Cur/HP-γ-CyD complex in Sac-HGF were observed. In contrast, the curcumin in Sac-HGF and curcumin/HP-γ-CyD physical mixture in Sac-HGF formed inhomogeneous films due to crystallization of curcumin. Furthermore, HP-γ-CyD played an important role to increase the elastic modulus of the Sac-HGF with high re-swelling ability. The Cur/HP-γ-CyD complex in Sac-HGF maintained antioxidant properties of curcumin. Curcumin was gradually released from the HP-γ-CyD complex in Sac-HGF. Notably, the Cur/HP-γ-CyD complex in Sac-HGF provided the highest wound healing ability in hairless mice. These results suggest that the Cur/HP-γ-CyD complex in Sac-HGF has the potential for use as a new transdermal therapeutic system to promote the wound-healing process. Copyright © 2017 Elsevier B.V. All rights reserved.

Cytochrome bc1-cy Fusion Complexes Reveal the Distance Constraints for Functional Electron Transfer Between Photosynthesis Components*

PubMed Central

Lee, Dong-Woo; Öztürk, Yavuz; Osyczka, Artur; Cooley, Jason W.; Daldal, Fevzi

2008-01-01

Photosynthetic (Ps) growth of purple non-sulfur bacteria such as Rhodobacter capsulatus depends on the cyclic electron transfer (ET) between the ubihydroquinone (QH2): cytochrome (cyt) c oxidoreductases (cyt bc1 complex), and the photochemical reaction centers (RC), mediated by either a membrane-bound (cyt cy) or a freely diffusible (cyt c2) electron carrier. Previously, we constructed a functional cyt bc1-cy fusion complex that supported Ps growth solely relying on membrane-confined ET (Lee, D.-W., Ozturk, Y., Mamedova, A., Osyczka, A., Cooley, J. W., and Daldal, F. (2006) Biochim. Biophys. Acta1757 ,346 -35216781662). In this work, we further characterized this cyt bc1-cy fusion complex, and used its derivatives with shorter cyt cy linkers as “molecular rulers” to probe the distances separating the Ps components. Comparison of the physicochemical properties of both membrane-embedded and purified cyt bc1-cy fusion complexes established that these enzymes were matured and assembled properly. Light-activated, time-resolved kinetic spectroscopy analyses revealed that their variants with shorter cyt cy linkers exhibited fast, native-like ET rates to the RC via the cyt bc1. However, shortening the length of the cyt cy linker decreased drastically this electronic coupling between the cyt bc1-cy fusion complexes and the RC, thereby limiting Ps growth. The shortest and still functional cyt cy linker was about 45 amino acids long, showing that the minimal distance allowed between the cyt bc1-cy fusion complexes and the RC and their surrounding light harvesting proteins was very short. These findings support the notion that membrane-bound Ps components form large, active structural complexes that are “hardwired” for cyclic ET. PMID:18343816

[Expressions of EMMPRIN and its ligand CyPA in gingival crevicular fluid of chronic periodontitis patients].

PubMed

He, Yan-ping; Xie, Ming; Jiao, Ting

2016-02-01

To detect the expressions of EMMPRIN and its ligand CyPA in gingival crevicular fluid (GCF) of chronic periodontitis (CP) patients and explore their possible relation to the status of periodontal inflammation. GCF of CP patients (group CP) and periodontitis-free patients with intact dentition (the control group) were collected and assayed for EMMPRIN and CyPA expressions by ELISA. The clinical periodontal status of these patients were examined. Statistical analysis was performed by use of SPSS 17.0 software package. Spearman's correlation analysis was utilized to determine the relationships between the expressions of EMMPRIN and CyPA in GCF and the clinical parameters. In addition, analysis of variance (ANOVA) was used for comparing the difference between group CP and the control group. In group CP, GCF volume was positively correlated with EMMPRIN total amount, CyPA total amount and some clinical periodontal indexes (GI,SBI,AL). EMMPRIN total amount was positively correlated with GCF volume, CyPA total amount and some of clinical periodontal indexes (GI,SBI,AL), but it was negatively correlated with smoking status (P<0.05). Moreover, CyPA total amount was positively correlated with GCF volume, EMMPRIN total amount and some of clinical periodontal indexes (GI,SBI,AL). In the control group,there were significant positive correlations among GCF volume, EMMPRIN total amount and CyPA total amount. The difference of GCF, EMMPRIN and CyPA between the 2 groups were statistically significant (P<0.05). EMMPRIN and its ligand CyPA in GCF of periodontitis-free patients with intact dentition and CP patients were all detected. As the progress of periodontal inflammation, GCF secretion increases, as well as the expressions of EMMPRIN and CyPA in GCF.

Identification and Characterization of Cyprinid Herpesvirus-3 (CyHV-3) Encoded MicroRNAs

PubMed Central

Donohoe, Owen H.; Henshilwood, Kathy; Way, Keith; Hakimjavadi, Roya; Stone, David M.; Walls, Dermot

2015-01-01

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in post-transcriptional gene regulation. Some viruses encode their own miRNAs and these are increasingly being recognized as important modulators of viral and host gene expression. Cyprinid herpesvirus 3 (CyHV-3) is a highly pathogenic agent that causes acute mass mortalities in carp (Cyprinus carpio carpio) and koi (Cyprinus carpio koi) worldwide. Here, bioinformatic analyses of the CyHV-3 genome suggested the presence of non-conserved precursor miRNA (pre-miRNA) genes. Deep sequencing of small RNA fractions prepared from in vitro CyHV-3 infections led to the identification of potential miRNAs and miRNA–offset RNAs (moRNAs) derived from some bioinformatically predicted pre-miRNAs. DNA microarray hybridization analysis, Northern blotting and stem-loop RT-qPCR were then used to definitively confirm that CyHV-3 expresses two pre-miRNAs during infection in vitro. The evidence also suggested the presence of an additional four high-probability and two putative viral pre-miRNAs. MiRNAs from the two confirmed pre-miRNAs were also detected in gill tissue from CyHV-3-infected carp. We also present evidence that one confirmed miRNA can regulate the expression of a putative CyHV-3-encoded dUTPase. Candidate homologues of some CyHV-3 pre-miRNAs were identified in CyHV-1 and CyHV-2. This is the first report of miRNA and moRNA genes encoded by members of the Alloherpesviridae family, a group distantly related to the Herpesviridae family. The discovery of these novel CyHV-3 genes may help further our understanding of the biology of this economically important virus and their encoded miRNAs may have potential as biomarkers for the diagnosis of latent CyHV-3. PMID:25928140

Various applications of immobilized glucose oxidase and polyphenol oxidase in a conducting polymer matrix.

PubMed

Cil, M; Böyükbayram, A E; Kiralp, S; Toppare, L; Yağci, Y

2007-06-01

In this study, glucose oxidase and polyphenol oxidase were immobilized in conducting polymer matrices; polypyrrole and poly(N-(4-(3-thienyl methylene)-oxycarbonyl phenyl) maleimide-co-pyrrole) via electrochemical method. Fourier transform infrared and scanning electron microscope were employed to characterize the copolymer of (N-(4-(3-thienyl methylene)-oxycarbonyl phenyl) maleimide) with pyrrole. Kinetic parameters, maximum reaction rate and Michealis-Menten constant, were determined. Effects of temperature and pH were examined for immobilized enzymes. Also, storage and operational stabilities of enzyme electrodes were investigated. Glucose and polyphenol oxidase enzyme electrodes were used for determination of the glucose amount in orange juices and human serum and phenolic amount in red wines, respectively.

Structure-guided design of fluorescent S-adenosylmethionine analogs for a high-throughput screen to target SAM-I riboswitch RNAs.

PubMed

Hickey, Scott F; Hammond, Ming C

2014-03-20

Many classes of S-adenosylmethionine (SAM)-binding RNAs and proteins are of interest as potential drug targets in diverse therapeutic areas, from infectious diseases to cancer. In the former case, the SAM-I riboswitch is an attractive target because this structured RNA element is found only in bacterial mRNAs and regulates multiple genes in several human pathogens. Here, we describe the synthesis of stable and fluorescent analogs of SAM in which the fluorophore is introduced through a functionalizable linker to the ribose. A Cy5-labeled SAM analog was shown to bind several SAM-I riboswitches via in-line probing and fluorescence polarization assays, including one from Staphylococcus aureus that controls the expression of SAM synthetase in this organism. A fluorescent ligand displacement assay was developed and validated for high-throughput screening of compounds to target the SAM-I riboswitch class. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification of matrix metalloproteinase inhibitors by chemical arrays.

PubMed

Kawatani, Makoto; Fukushima, Yukako; Kondoh, Yasumitsu; Honda, Kaori; Sekine, Tomomi; Yamaguchi, Yoshiki; Taniguchi, Naoyuki; Osada, Hiroyuki

2015-01-01

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade many extracellular matrix components and that have been implicated in the pathogenesis of various human diseases including cancer metastasis. Here, we screened MMP-9 inhibitors using photo-cross-linked chemical arrays, which can detect small-molecule ligand-protein interactions on a chip in a high-throughput manner. The array slides were probed sequentially with His-MMP-9, anti-His antibody, and a Cy5-labeled secondary antibody and then scanned with a microarray scanner. We obtained 27 hits among 24,275 compounds from the NPDepo library; 2 of the identified compounds (isoxazole compound 1 and naphthofluorescein) inhibited MMP-9 enzyme activity in vitro. We further explored 17 analogs of 1 and found that compound 18 had the strongest inhibitory activity. Compound 18 also inhibited other MMPs, including MMP-2, MMP-12, and MMP-13 and significantly inhibited cell migration in human fibrosarcoma HT1080 cells. These results suggest that 18 is a broad-spectrum MMP inhibitor.

In vivo imaging of advanced glycation end products (AGEs) of albumin: first observations of significantly reduced clearance and liver deposition properties in mice.

PubMed

Tsutsui, Ayumi; Ogura, Akihiro; Tahara, Tsuyoshi; Nozaki, Satoshi; Urano, Sayaka; Hara, Mitsuko; Kojima, Soichi; Kurbangalieva, Almira; Onoe, Hirotaka; Watanabe, Yasuyoshi; Taniguchi, Naoyuki; Tanaka, Katsunori

2016-06-15

Advanced glycation end products (AGEs) are associated with various diseases, especially during aging and the development of diabetes and uremia. To better understand these biological processes, investigation of the in vivo kinetics of AGEs, i.e., analysis of trafficking and clearance properties, was carried out by molecular imaging. Following the preparation of Cy7.5-labeled AGE-albumin and intravenous injection in BALB/cA-nu/nu mice, noninvasive fluorescence kinetics analysis was performed. In vivo imaging and fluorescence microscopy analysis revealed that non-enzymatic AGEs were smoothly captured by scavenger cells in the liver, i.e., Kupffer and other sinusoidal cells, but were unable to be properly cleared from the body. Overall, these results highlight an important link between AGEs and various disorders associated with them, which may serve as a platform for future research to better understand the processes and mechanisms of these disorders.

Content comparison of haemophilia specific patient-rated outcome measures with the international classification of functioning, disability and health (ICF, ICF-CY)

PubMed Central

2010-01-01

Background Patient-Reported Outcomes (PROs) are considered important outcomes because they reflect the patient's experience in clinical trials. PROs have been included in the field of haemophilia only recently. Purpose Comparing the contents of PROs measures used in haemophilia, based on the ICF/ICF-CY as frame of reference. Methods Haemophilia-specific PROs for adults and children were selected on the grounds of international accessibility. The content of the selected instruments were examined by linking the concepts within the items of these instruments to the ICF/ICF-CY. Results Within the 5 selected instruments 365 concepts were identified, of which 283 concepts were linked to the ICF/ICF CY and mapped into 70 different categories. The most frequently used categories were "b152: Emotional functions" and "e1101: Drugs". Conclusions The present paper provides an overview on current PROs in haemophilia and facilitates the selection of appropriate instruments for specific purposes in clinical and research settings. This work was made possible by the grant of the European Murinet Project (Multidisciplinary Research Network on Health and Disability in Europe). PMID:21108796

Determination of the orientation of fluorescent labels relative to myosin S1 in solution from time-resolved fluorescence anisotropy experiments

NASA Astrophysics Data System (ADS)

van der Heide, Uulke A.; Gerritsen, Hans C.; Trayer, Ian P.; Levine, Yehudi K.

1992-04-01

The time-resolved fluorescence anisotropy of myosin S1 covalently labeled with Eosin-5- maleimide and 1,5-I-AEDANS was measured in solution. Each probe was specifically attached at one SH-group on the S1. The two most reactive SH sites on the heavy chain of the myosin S1 were used. The fluorescence anisotropy was measured at different excitation wavelengths. In this way, several absorption moments were utilized, each having a distinct orientation in the frame of the dye. The orientations of the transition moments in the dyes were determined in a separate experiment using an angle resolved fluorescence depolarization experiment on dyes embedded in stretched matrices of PVA polymers. The anisotropy decay curves exhibit fast (<3 ns) and slow (> 100 ns) components. The slow decay components reflect the motion of the large protein molecules. The fast anisotropy decay are attributed to a fast, but restricted, motion of the bound dye relative to the protein as experiments on free dyes in solution reveal subnanosecond anisotropy decays. The anisotropy decays have been analyzed in terms of a model which describes the restricted motion of the dye molecule relative to the protein and the overall rotation of the dye-protein complex in solution. An important element in the model is the incorporation of the orientational distribution of the dye relative to the protein. The observed anisotropy decays were analyzed using a global target approach in which the experimental data obtained at different excitation wavelengths are fitted simultaneously to the theoretical model. It is important to note that the orientational distribution of the dye relative to the protein, as well as the rotational correlation times of the motions for a dye attached to a given binding site, are independent of the excitation wavelength used. This leads to a reduction in the number of independent parameters optimized by the nonlinear least squares procedure. The orientational distribution of the dye relative to the protein obtained in this way is particularly useful for the interpretation of fluorescence depolarization data obtained from labeled muscle fibers. Indeed, knowledge of the distribution function of a dye attached to a binding site of the S1 protein is a prerequisite for a probe-independent determination of the orientational distribution of the S1 proteins themselves in the muscle fiber.

Nanoparticle-based brachytherapy spacers for delivery of localized combined chemoradiation therapy.

PubMed

Kumar, Rajiv; Belz, Jodi; Markovic, Stacey; Jadhav, Tej; Fowle, William; Niedre, Mark; Cormack, Robert; Makrigiorgos, Mike G; Sridhar, Srinivas

2015-02-01

In radiation therapy (RT), brachytherapy-inert source spacers are commonly used in clinical practice to achieve high spatial accuracy. These implanted devices are critical technical components of precise radiation delivery but provide no direct therapeutic benefits. Here we have fabricated implantable nanoplatforms or chemoradiation therapy (INCeRT) spacers loaded with silica nanoparticles (SNPs) conjugated containing a drug, to act as a slow-release drug depot for simultaneous localized chemoradiation therapy. The spacers are made of poly(lactic-co-glycolic) acid (PLGA) as matrix and are physically identical in size to the commercially available brachytherapy spacers (5 mm × 0.8 mm). The silica nanoparticles, 250 nm in diameter, were conjugated with near infrared fluorophore Cy7.5 as a model drug, and the INCeRT spacers were characterized in terms of size, morphology, and composition using different instrumentation techniques. The spacers were further doped with an anticancer drug, docetaxel. We evaluated the in vivo stability, biocompatibility, and biodegradation of these spacers in live mouse tissues. The electron microscopy studies showed that nanoparticles were distributed throughout the spacers. These INCeRT spacers remained stable and can be tracked by the use of optical fluorescence. In vivo optical imaging studies showed a slow diffusion of nanoparticles from the spacer to the adjacent tissue in contrast to the control Cy7.5-PLGA spacer, which showed rapid disintegration in a few days with a burst release of Cy7.5. The docetaxel spacers showed suppression of tumor growth in contrast to control mice over 16 days. The imaging with the Cy7.5 spacer and therapeutic efficacy with docetaxel spacers supports the hypothesis that INCeRT spacers can be used for delivering the drugs in a slow, sustained manner in conjunction with brachytherapy, in contrast to the rapid clearance of the drugs when administered systemically. The results demonstrate that these spacers with tailored release profiles have potential in improving the combined therapeutic efficacy of chemoradiation therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

A novel "dual-potential" electrochemiluminescence aptasensor array using CdS quantum dots and luminol-gold nanoparticles as labels for simultaneous detection of malachite green and chloramphenicol.

PubMed

Feng, Xiaobin; Gan, Ning; Zhang, Huairong; Yan, Qing; Li, Tianhua; Cao, Yuting; Hu, Futao; Yu, Hongwei; Jiang, Qianli

2015-12-15

A novel type of "dual-potential" electrochemiluminescence (ECL) aptasensor array was fabricated on a homemade screen-printed carbon electrode (SPCE) for simultaneous detection of malachite green (MG) and chloramphenicol (CAP) in one single assay. The SPCE substrate consisted of a common Ag/AgCl reference electrode, carbon counter electrode and two carbon working electrodes (WE1 and WE2). In the system, CdS quantum dots (QDs) were modified on WE1 as cathode ECL emitters and luminol-gold nanoparticles (L-Au NPs) were modified on WE2 as anode ECL emitters. Then the MG aptamer complementary strand (MG cDNA) and CAP aptamer complementary strand (CAP cDNA) were attached on CdS QDs and L-Au NPs, respectively. The cDNA would hybridize with corresponding aptamer that was respectively tagged with cyanine dye (Cy5) (as quenchers of CdS QDs) and chlorogenic acid (CA) (as quenchers of l-Au NPs) using poly(ethylenimine) (PEI) as a bridging agent. PEI could lead to a large number of quenchers on the aptamer, which increased the quenching efficiency. Upon MG and CAP adding, the targets could induce strand release due to the highly affinity of analytes toward aptamers. Meanwhile, it could release the Cy5 and CA, which recovered cathode ECL of CdS QDs and anode ECL of L-Au NPs simultaneously. This "dual-potential" ECL strategy could be used to detect MG and CAP with the linear ranges of 0.1-100 nM and 0.2-150 nM, with detection limits of 0.03 nM and 0.07 nM (at 3sB), respectively. More importantly, this designed method was successfully applied to determine MG and CAP in real fish samples and held great potential in the food analysis. Copyright © 2015 Elsevier B.V. All rights reserved.

DEVELOPMENT OF SULFHYDRYL-REACTIVE SILICA FOR PROTEIN IMMOBILIZATION IN HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

PubMed Central

Mallik, Rangan; Wa, Chunling; Hage, David S.

2008-01-01

Two techniques were developed for the immobilization of proteins and other ligands to silica through sulfhydryl groups. These methods made use of maleimide-activated silica (the SMCC method) or iodoacetyl-activated silica (the SIA method). The resulting supports were tested for use in high-performance affinity chromatography by employing human serum albumin (HSA) as a model protein. Studies with normal and iodoacetamide-modified HSA indicated that these methods had a high selectivity for sulfhydryl groups on this protein, which accounted for the coupling of 77–81% of this protein to maleimide- or iodacetyl-activated silica. These supports were also evaluated in terms of their total protein content, binding capacity, specific activity, non-specific binding, stability and chiral selectivity for several test solutes. HSA columns prepared using maleimide-activated silica gave the best overall results for these properties when compared to HSA that had been immobilized to silica through the Schiff base method (i.e., an amine-based coupling technique). A key advantage of the supports developed in this work is that they offer the potential of giving greater site-selective immobilization and ligand activity than amine-based coupling methods. These features make these supports attractive in the development of protein columns for such applications as the study of biological interactions and chiral separations. PMID:17297940

Lignin-Based Materials Through Thiol-Maleimide "Click" Polymerization.

PubMed

Buono, Pietro; Duval, Antoine; Averous, Luc; Habibi, Youssef

2017-03-09

In the present report an environmentally friendly approach to transforming renewable feedstocks into value-added materials is proposed. This transformation pathway was conducted under green conditions, without the use of solvents or catalyst. First, controlled modification of lignin, a major biopolymer present in wood and plants, was achieved by esterification with 11-maleimidoundecylenic acid (11-MUA), a derivative from castor oil that contains maleimide groups, following its transformation into 11-maleimidoundecanoyl chloride (11-MUC). Different degrees of substitution were achieved by using various amounts of the 11-MUC, leading to an efficient conversion of lignin hydroxy groups, as demonstrated by 1 H and 31 P NMR analyses. These fully biobased maleimide-lignin derivatives were subjected to an extremely fast (ca. 1 min) thiol-ene "click" polymerization with thiol-containing linkers. Aliphatic and aromatic thiol linkers bearing two to four thiol groups were used to tune the reactivity and crosslink density. The properties of the resulting materials were evaluated by swelling tests and thermal and mechanical analyses, which showed that varying the degree of functionality of the linker and the linker structure allowed accurate tailoring of the thermal and mechanical properties of the final materials, thus providing interesting perspectives for lignin in functional aromatic polymers. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV-3) and possible latency of CyHV-3 by temperature shift in the cells.

PubMed

Imajoh, M; Fujioka, H; Furusawa, K; Tamura, K; Yamasaki, K; Kurihara, S; Yamane, J; Kawai, K; Oshima, S

2015-06-01

A new cell line named CCF-K104 predominantly consisting of fibroblastic cells showed optimal growth at temperatures from 25 °C to 30 °C. Serial morphological changes in the cells induced by Cyprinid herpesvirus 3 (CyHV-3) included cytoplasmic vacuolar formation, cell rounding and detachment. Mature virions were purified from CyHV-3-infected CCF-K104 cells by sucrose gradient ultracentrifugation and had a typical herpesvirus structure on electron microscopy. Infectious CyHV-3 was produced stably in CCF-K104 cells over 30 viral passages. Our findings showed that CCF-K104 is a useful cell line for isolation and productive replication of CyHV-3. A temperature shift from 25 °C to 15 °C or 35 °C did not allow serial morphological changes as observed at 25 °C for 14 days. Under the same conditions, real-time PCR showed that CyHV-3 was present with low viral DNA loads, suggesting that CyHV-3 may establish latent infection in CCF-K104 cells. Amplification of the left and right terminal repeat sequences of the CyHV-3 genome arranged in a head-to-tail manner was detected by nested PCR following an upshift in temperature from 25 °C to 35 °C. The PCR results suggested that the circular genome may represent a latent form of CyHV-3. © 2014 John Wiley & Sons Ltd.

The Novel Extracellular Cyclophilin A (CyPA) - Inhibitor MM284 Reduces Myocardial Inflammation and Remodeling in a Mouse Model of Troponin I -Induced Myocarditis.

PubMed

Heinzmann, David; Bangert, Anna; Müller, Anna-Maria; von Ungern-Sternberg, Saskia N I; Emschermann, Frederic; Schönberger, Tanja; Chatterjee, Madhumita; Mack, Andreas F; Klingel, Karin; Kandolf, Reinhard; Malesevic, Miroslav; Borst, Oliver; Gawaz, Meinrad; Langer, Harald F; Katus, Hugo; Fischer, Gunter; May, Andreas E; Kaya, Ziya; Seizer, Peter

2015-01-01

Cyclophilins are a group of highly conserved cytosolic enzymes that have a peptidylprolyl cis/trans isomerase activity. Cyclophilin A (CyPA) can be secreted in the extracellular space by inflammatory cells and upon cell death. The presence of CyPA in patients with non-ischemic cardiomyopathy is associated with poor clinical prognosis. Here, we investigated the inhibition of extracellular CyPA in a mouse model of troponin I-induced autoimmune myocarditis using the strictly extracellular CyPA-inhibitor MM284. Since A/J mice develop severe inflammation and fibrosis after immunization with murine cardiac troponin I (mcTn I), we used this model to analyze the effects of an extracellular CyPA inhibition. As extracellular CyPA-inhibitor we used the recently described CsA-derivate MM284. In vitro studies confirmed that MM284 inhibits CyPA-induced monocytic migration and adhesion. A/J mice immunized with mcTnI were treated with MM284 or vehicle every second day. After 28 days, we found a considerable reduction of myocardial injury and fibrosis. Further analysis revealed a reduced myocardial presence of T-cells and macrophages compared to control treated animals. Whereas MMP-9 expression was reduced significantly by MM284, we observed no significant reduction of inflammatory cytokines such as IL-6 or TNFα. Extracellular CyPA plays an important role in autoimmune myocarditis for myocardial damage and fibrosis. Our data suggest a new pharmacological approach for the treatment of myocardial inflammation and reduction of cardiac fibrosis by inhibition of extracellular CyPA.

Structural and functional characterization of a new recombinant histidine-tagged acyl coenzyme A binding protein (ACBP) from mouse

PubMed Central

Petrescu, Anca D.; Huang, Huan; Hostetler, Heather A.; Schroeder, Friedhelm; Kier, Ann B.

2008-01-01

Acyl-coenzyme A binding protein (ACBP) has been proposed to transport fatty acyl-CoAs intracellularly, facilitating their metabolism. In this study, a new mouse recombinant ACBP was produced by insertion of a histidine (his) tag at the C-terminus to allow efficient purification by Ni-affinity chromatography. The his-tag was inserted at the C-terminus since ACBP is a small molecular size (10 kDa) protein whose structure and activity are sensitive to amino acid substitutions in the N-terminus. The his tag had no or little effect on ACBP structure or ligand binding affinity and specificity. His-ACBP bound the naturally-occurring fluorescent cis-parinaroyl-CoA with very high affinity (Kd=2.15 nM), but exhibited no affinity for non-esterified cis-parinaric acid. To determine if the presence of the C-terminal his tag altered ACBP interactions with other proteins, direct binding to hepatocyte nuclear factor 4α (HNF-4α), a nuclear receptor regulating transcription of genes involved in lipid metabolism, was examined. His-ACBP and HNF-4α were labeled with Cy5 and Cy3, respectively, and direct interaction was determined by a novel fluorescence resonance energy transfer (FRET) binding assay. FRET analysis showed that his-ACBP directly interacted with HNF-4α (intermolecular distance of 73 Å) at high affinity (Kd=64-111 nM) similar to native ACBP. The his-tag also had no effect on ACBPs ability to interact with and stimulate microsomal enzymes utilizing or forming fatty acyl CoA. Thus, C-terminal his-tagged-ACBP maintained very similar structural and functional features of the untagged native protein and can be used in further in vitro experiments that require pure recombinant ACBP. PMID:18178100

Paper-based solid-phase multiplexed nucleic acid hybridization assay with tunable dynamic range using immobilized quantum dots as donors in fluorescence resonance energy transfer.

PubMed

Noor, M Omair; Krull, Ulrich J

2013-08-06

A multiplexed solid-phase nucleic acid hybridization assay on a paper-based platform is presented using multicolor immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize two types of QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) and red-emitting QDs (rQDs) served as donors with Cy3 and Alexa Fluor 647 (A647) acceptors. The gQD/Cy3 FRET pair served as an internal standard, while the rQD/A647 FRET pair served as a detection channel, combining the control and analytical test zones in one physical location. Hybridization of dye-labeled oligonucleotide targets provided the proximity for FRET sensitized emission from the acceptor dyes, which served as an analytical signal. Hybridization assays in the multicolor format provided a limit of detection of 90 fmol and an upper limit of dynamic range of 3.5 pmol. The use of an array of detection zones was designed to provide improved analytical figures of merit compared to that which could be achieved on one type of array design in terms of relative concentration of multicolor QDs. The hybridization assays showed excellent resistance to nonspecific adsorption of oligonucleotides. Selectivity of the two-plex hybridization assay was demonstrated by single nucleotide polymorphism (SNP) detection at a contrast ratio of 50:1. Additionally, it is shown that the use of preformed QD-probe oligonucleotide conjugates and consideration of the relative number density of the two types of QD-probe conjugates in the two-color assay format is advantageous to maximize assay sensitivity and the upper limit of dynamic range.

Screening and identification of gastric adenocarcinoma metastasis-related genes using cDNA microarray coupled to FDD-PCR.

PubMed

Wang, Jianhua; Chen, Shishu

2002-10-01

To identify certain gastric adenocarcinoma metastasis-related genes, an RF-1 cell line (primary tumor from a gastric adenocarcinoma patient) and an RF-48 cell line (its metastatic counterpart) were used as a model for studying the molecular mechanism of tumor metastasis. Two fluorescent cDNA probes, labeled with Cy3 and Cy5 dyes, were prepared from RF-1 and RF-48 mRNA samples by the reverse transcription method. The two color probes were then mixed and hybridized to a cDNA chip constructed with double-dots from 4,096 human genes, and scanned at two wavelengths. The experiment was repeated twice. Differentially expressedn genes from the above two cells were analyzed by use of computer. Of the total genes, 138 (3.4%) revealed differential expression in RF-48 cells compared with RF-1 cells: 81 (2.1%) genes revealed apparent up-regulation, and 56 (1.3%) genes revealed down-regulation. Forty-five genes involved in gastric adenocarcinoma metastasis were cloned using fluorescent differential display-PCR (FDD-PCR), including three novel genes. There were seven differentially expressed genes that presented the same behaviour under both detection methods. The possible roles of some differentially expressed genes, which may be involved in the mechanism of tumor metastasis, were discussed. cDNA chip was used to analyze gene expression in a high-throughput and large-scale manner in combination with FDD-PCR for cloning unknown novel genes. Some genes related to metastasis were preliminarily scanned, which would contribute to disclose the molecular mechanism of gastric adenocarcinoma metastasis and provide new targets for therapeutic intervention.

Assessment of DNA replication in central nervous system by Laser Scanning Cytometry

NASA Astrophysics Data System (ADS)

Lenz, Dominik; Mosch, Birgit; Bocsi, Jozsef; Arendt, Thomas; Tárnok, Attila

2004-07-01

μIn neurons of patients with Alzheimers's disease (AD) signs of cell cycle re-entry as well as polyploidy have been reported1, 2, indicating that the entire or a part of the genome of the neurons is duplicated before its death but mitosis is not initiated so that the cellular DNA content remains tetraploid. It was concluded, that this imbalance is the direct cause of the neuronal loss in AD3. Manual counting of polyploidal cells is possible but time consuming and possibly statistically insufficient. The aim of this study was to develop an automated method that detects the neuronal DNA content abnormalities with Laser Scanning Cytometry (LSC).Frozen sections of formalin-fixed brain tissue of AD patients and control subjects were labelled with anti-cyclin B and anti-NeuN antibodies. Immunolabelling was performed using Cy5- and Cy2-conjugated secondary antibodies and biotin streptavidin or tyramid signal amplification. In the end sections of 20m thickness were incubated with propidium iodide (PI) (50μg/ml) and covered on slides. For analysis by the LSC PI was used as trigger. Cells identified as neurons by NeuN expression were analyzed for cyclin B expression. Per specimen data of at least 10,000 neurons were acquired. In the frozen brain sections an automated quantification of the amount of nuclear DNA is possible with LSC. The DNA ploidy as well as the cell cycle distribution can be analyzed. A high number of neurons can be scanned and the duration of measuring is shorter than a manual examination. The amount of DNA is sufficiently represented by the PI fluorescence to be able to distinguish between eu- and polyploid neurons.

Protein expression profile changes in human fibroblasts induced by low dose energetic protons

NASA Astrophysics Data System (ADS)

Zhang, Ye; Clement, Jade Q.; Gridley, Daila S.; Rodhe, Larry H.; Wu, Honglu

2009-12-01

Extrapolation of known radiation risks to the risks from low dose and low dose-rate exposures of human population, especially prolonged exposures of astronauts in the space radiation environment, relies in part on the mechanistic understanding of radiation induced biological consequences at the molecular level. While some genomic data at the mRNA level are available for cells or animals exposed to radiation, the data at the protein level are still lacking. Here, we studied protein expression profile changes using Panorama antibody microarray chips that contain antibodies to 224 proteins (or their phosphorylated forms) involved in cell signaling that included mostly apoptosis, cytoskeleton, cell cycle and signal transduction. Normal human fibroblasts were cultured until fully confluent and then exposed to 2 cGy of 150 MeV protons at high-dose rate. The proteins were isolated at 2 or 6 h after exposure and labeled with Cy3 for the irradiated cells and with Cy5 for the control samples before loading onto the protein microarray chips. The intensities of the protein spots were analyzed using ScanAlyze software and normalized by the summed fluorescence intensities and the housekeeping proteins. The results showed that low dose protons altered the expression of more than 10% of the proteins listed in the microarray analysis in various protein functional groups. Cell cycle (24%) related proteins were induced by protons and most of them were regulators of G1/S-transition phase. Comparison of the overall protein expression profiles, cell cycle related proteins, cytoskeleton and signal transduction protein groups showed significantly more changes induced by protons compared with other protein functional groups.

Lethal and sublethal effects of cypermethrin to Hypsiboas pulchellus tadpoles.

PubMed

Agostini, M Gabriela; Natale, Guillermo S; Ronco, Alicia E

2010-11-01

The study of the effects of the insecticide cypermethrin (CY) technical grade and its Sherpa® commercial formulation on Hypsiboas pulchellus tadpoles assessing lethality, behavior, growth, and abnormalities under standardized laboratory conditions is reported. Observed behaviors were identified and categorized by means of a ranking system according to the loss of mobility. Results of acute lethal effects indicate higher potency for Sherpa® at this level of assessment. All effects on behavior showed an increasing degree of injury as insecticide concentration increased. Organisms exposed to technical grade CY showed lower body length with respect to controls from 3.44 µg CY/L to higher concentrations, whereas those exposed to Sherpa® exhibited lower growth from 0.83 µg CY/L. Both forms of the tested insecticide caused abnormalities between 0.34 and 4.18 µg CY/L, but 100% of malformed individuals was detected from 34.4 µg CY/L for those exposed to the technical grade CY, and from 8.36 µg CY/L for those exposed to Sherpa®. This study proposes the use of easily identifiable and distinguishable sublethal end-points. The high input loads of CY in natural environments, the detected concentrations in the field, in addition to the low levels of this insecticide required to induce sublethal effects (which could eventually lead to death), allow for the conclusion that the insecticide is a risk factor for amphibians inhabiting agroecosystems.

Melatonin attenuates angiotensin II-induced cardiomyocyte hypertrophy through the CyPA/CD147 signaling pathway.

PubMed

Su, Hongyan; Li, Jingyuan; Chen, Tongshuai; Li, Na; Xiao, Jie; Wang, Shujian; Guo, Xiaobin; Yang, Yi; Bu, Peili

2016-11-01

Melatonin is well known for its cardioprotective effects; however, whether melatonin exerts therapeutic effects on cardiomyocyte hypertrophy remains to be investigated, as do the mechanisms underlying these effects, if they exist. Cyclophilin A (CyPA) and its corresponding receptor, CD147, which exists in a variety of cells, play crucial roles in modulating reactive oxygen species (ROS) production. In this study, we explored the role of the CyPA/CD147 signaling pathway in angiotensin II (Ang II)-induced cardiomyocyte hypertrophy and the protective effects exerted by melatonin against Ang II-induced injury in cultured H9C2 cells. Cyclosporine A, a specific CyPA/CD147 signaling pathway inhibitor, was used to manipulate CyPA/CD147 activity. H9C2 cells were then subjected to Ang II or CyPA treatment in either the absence or presence of melatonin. Our results indicate that Ang II induces cardiomyocyte hypertrophy through the CyPA/CD147 signaling pathway and promotes ROS production, which can be blocked by melatonin pretreatment in a concentration-dependent manner, in cultured H9C2 cells and that CyPA/CD147 signaling pathway inhibition protects against Ang II-induced cardiomyocyte hypertrophy. The protective effects of melatonin against Ang II-induced cardiomyocyte hypertrophy depend at least partially on CyPA/CD147 inhibition.

1-(2-Hy­droxy­eth­yl)-3-[(2-hy­droxy­eth­yl)amino]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione

PubMed Central

Xie, Zhi-Xiong; Zhao, Sheng-Yin

2011-01-01

There are four molecules in the asymmetric unit of the title compound, C16H17N3O4, in which the dihedral angles between the indole ring system and maleimide ring are 4.5 (3), 8.3 (3), 8.4 (2) and 10.4 (2)°. In the crystal, mol­ecules are linked by numerous N—H⋯O and O—H⋯O hydrogen bonds, generating a three-dimensional network. PMID:21754135

Changes in the Conformational State of Hemoglobin in Hemodialysed Patients with Chronic Renal Failure

PubMed Central

Pieniazek, Anna; Gwozdzinski, Krzysztof

2015-01-01

The aim of this study was to evaluate the properties of internal components of erythrocytes in chronic renal failure (CRF) patients undergoing hemodialysis (HD) in comparison to control subjects. For investigation of conformational state of hemoglobin and nonheme proteins (NHP) the maleimide spin label (MSL) in electron paramagnetic resonance (EPR) was applied. The studies were performed using MSL in whole cells and hemolysate as well as proteins separated by ion exchange chromatography and checked by electrophoresis. Additionally the level of –SH groups in hemolysate and isolated internal proteins of CRF erythrocytes was determined using 4,4′-dithiodipyridine. All measurements were performed before and after hemodialysis. Oxidative stress accompanying CRF/hemodialysed patients caused a significant decrease in the mobility of internal components inside erythrocytes indicated by MSL (P < 0.02). The significant decrease in mobility of spin labeled HbA1c and HbA both before and after HD (P < 0.0002) as well as in nonheme proteins before hemodialysis (P < 0.05) versus control was indicated. Decrease in mobility of internal components of erythrocytes was accompanied by loss of thiols before and after hemodialysis versus control in NHP (P < 0.05), HbA1c (P < 0.0002), and HbA (P < 0.0005). These findings showed oxidative influence of hemodialysis on hemoglobins and internal nonheme proteins in erythrocytes of CRF patients. PMID:25866600

Global atmospheric carbon budget: results from an ensemble of atmospheric CO2 inversions

NASA Astrophysics Data System (ADS)

Peylin, P.; Law, R. M.; Gurney, K. R.; Chevallier, F.; Jacobson, A. R.; Maki, T.; Niwa, Y.; Patra, P. K.; Peters, W.; Rayner, P. J.; Rödenbeck, C.; Zhang, X.

2013-03-01

Atmospheric CO2 inversions estimate surface carbon fluxes from an optimal fit to atmospheric CO2 measurements, usually including prior constraints on the flux estimates. Eleven sets of carbon flux estimates are compared, generated by different inversions systems that vary in their inversions methods, choice of atmospheric data, transport model and prior information. The inversions were run for at least 5 yr in the period between 1990 and 2009. Mean fluxes for 2001-2004, seasonal cycles, interannual variability and trends are compared for the tropics and northern and southern extra-tropics, and separately for land and ocean. Some continental/basin-scale subdivisions are also considered where the atmospheric network is denser. Four-year mean fluxes are reasonably consistent across inversions at global/latitudinal scale, with a large total (land plus ocean) carbon uptake in the north (-3.3 Pg Cy-1 (±0.6 standard deviation)) nearly equally spread between land and ocean, a significant although more variable source over the tropics (1.6 ± 1.0 Pg Cy-1) and a compensatory sink of similar magnitude in the south (-1.4 ± 0.6 Pg Cy-1) corresponding mainly to an ocean sink. Largest differences across inversions occur in the balance between tropical land sources and southern land sinks. Interannual variability (IAV) in carbon fluxes is larger for land than ocean regions (standard deviation around 1.05 versus 0.34 Pg Cy-1 for the 1996-2007 period), with much higher consistency amoung the inversions for the land. While the tropical land explains most of the IAV (stdev ∼ 0.69 Pg Cy-1), the northern and southern land also contribute (stdev ∼ 0.39 Pg Cy-1). Most inversions tend to indicate an increase of the northern land carbon uptake through the 2000s (around 0.11 Pg Cy-1), shared by North America and North Asia. The mean seasonal cycle appears to be well constrained by the atmospheric data over the northern land (at the continental scale), but still highly dependent on the prior flux seasonality over the ocean. Finally we provide recommendations to interpret the regional fluxes, along with the uncertainty estimates.

CyDiv, a Conserved and Novel Filamentous Cyanobacterial Cell Division Protein Involved in Septum Localization.

PubMed

Mandakovic, Dinka; Trigo, Carla; Andrade, Derly; Riquelme, Brenda; Gómez-Lillo, Gabriela; Soto-Liebe, Katia; Díez, Beatriz; Vásquez, Mónica

2016-01-01

Cell division in bacteria has been studied mostly in Escherichia coli and Bacillus subtilis, model organisms for Gram-negative and Gram-positive bacteria, respectively. However, cell division in filamentous cyanobacteria is poorly understood. Here, we identified a novel protein, named CyDiv (Cyanobacterial Division), encoded by the all2320 gene in Anabaena sp. PCC 7120. We show that CyDiv plays a key role during cell division. CyDiv has been previously described only as an exclusive and conserved hypothetical protein in filamentous cyanobacteria. Using polyclonal antibodies against CyDiv, we showed that it localizes at different positions depending on cell division timing: poles, septum, in both daughter cells, but also in only one of the daughter cells. The partial deletion of CyDiv gene generates partial defects in cell division, including severe membrane instability and anomalous septum localization during late division. The inability to complete knock out CyDiv strains suggests that it is an essential gene. In silico structural protein analyses and our experimental results suggest that CyDiv is an FtsB/DivIC-like protein, and could therefore, be part of an essential late divisome complex in Anabaena sp. PCC 7120.

Rational design of a pH-insensitive cyan fluorescent protein CyPet2 based on the CyPet crystal structure.

PubMed

Liu, Rui; Hu, Xiao-Jian; Ding, Yu

2017-06-01

The emission spectrum of widely used CyPet is pH-sensitive. In order to synthesize a pH-insensitive cyan fluorescent protein by rational design, we solved the crystal structures of CyPet under different pH conditions. The indole group of the CyPet chromophore adopts a cis-coplanar conformation in acidic and neutral conditions, while it converts to trans-coplanar under basic conditions. His148 and Glu222 play a vital role in this isomerization. The pH-sensitive chromophore isomerization and change in the emission spectrum can be explained by the coexistence of several different fluorescent states. We trap the chromophore in the trans conformation by A167I mutation (CyPet2), which also prevents the multiconformation of the seventh β-strand. CyPet2 exhibits an unchanged emission spectral shape as a function of pH. © 2017 Federation of European Biochemical Societies.

Protective immunity of a modified-live cyprinid herpesvirus 3 vaccine in koi (Cyprinus carpio koi) 13 months after vaccination.

PubMed

O'Connor, Matthew R; Farver, Thomas B; Malm, Kirsten V; Yun, Susan C; Marty, Gary D; Salonius, Kira; Dishon, Arnon; Weber, E P Scott

2014-10-01

To evaluate the long-term protective immunity of a cyprinid herpesvirus 3 (CyHV3) vaccine in naïve koi (Cyprinus carpio koi). 72 koi. Procedures-Vaccinated koi (n = 36) and unvaccinated control koi (36) were challenge exposed to a wild-type CyHV3 strain (KHVp8 F98-50) 13 months after vaccination. The CyHV3 vaccine provided substantial protective immunity against challenge exposure. The proportional mortality rate was less in vaccinated koi (13/36 [36%]) than in unvaccinated koi (36/36 [100%]). For koi that died during the experiment, mean survival time was significantly greater in vaccinated than in unvaccinated fish (17 vs 10 days). The CyHV3 vaccine provided substantial protective immunity against challenge exposure with CyHV3 13 months after vaccination. This provided evidence that koi can be vaccinated annually with the CyHV3 vaccine to significantly reduce mortality and morbidity rates associated with CyHV3 infection.

Syntheses of mono- and dinuclear diiodoboryl complexes of platinum.

PubMed

Braunschweig, Holger; Radacki, Krzysztof; Uttinger, Katharina

2007-10-15

Treatment of [Pt(PCy(3))(2)] (Cy = cyclohexyl) with BI(3) afforded trans-[(Cy(3)P)(2)Pt(I)(BI(2))] by the oxidative addition of a B-I bond. The title compound represents the first diiodoboryl complex and was fully characterized by NMR spectroscopy and X-ray diffraction analysis. The latter revealed a very short Pt-B distance, thus indicating a pronounced pi contribution to this bond. By the addition of another 1 equiv of BI(3) to trans-[(Cy(3)P)(2)Pt(I)(BI(2))], a new Pt species [(Cy(3)P)(I(2)B)Pt(mu-I)](2) was formed with concomitant buildup of the phosphine borane adduct [Cy(3)P-BI(3)]. The former is obviously obtained by abstraction of PCy(3) from trans-[(Cy(3)P)(2)Pt(I)(BI(2))] and the subsequent dimerization of two remaining fragments. Interestingly, the dimerization is reversible, and the dinuclear compound can be converted to trans-[(Cy(3)P)(2)Pt(I)(BI(2))] upon the addition of PCy(3).

The use of native chemical functional groups presented by wound beds for the covalent attachment of polymeric microcarriers of bioactive factors

PubMed Central

Jain, Rishabh; Agarwal, Ankit; Kierski, Patricia R.; Schurr, Michael J.; Murphy, Christopher J.; McAnulty, Jonathan F.; Abbott, Nicholas L.

2012-01-01

The development of versatile methods that provide spatial and temporal control over the presentation of physical and biochemical cues on wound beds can lead to new therapeutic approaches that expedite wound healing by favorably influencing cellular behaviors. Towards that goal, we report that native chemical functional groups presented by wound beds can be utilized for direct covalent attachment of polymeric microbeads. Specifically, we demonstrated the covalent attachment of maleimide-functionalized and catechol-functionalized microbeads, made of either polystyrene (non-degradable) or poly(lactic-co-glycolic acid) ((PLGA), degradable), to sulfhydryl and amine groups present on porcine dermis used here as an ex vivo model wound bed. A pronounced increase (10–70 fold) in the density and persistence of the covalently reactive microbeads was observed relative to microbeads that adsorb via non-covalent interactions. Complementary characterization of the surface chemistry of the ex vivo wound beds using Raman microspectroscopy provides support for our conclusion that the increased adherence of the maleimide-functionalized beads results from their covalent bond formation with sulfhydryl groups on the wound bed. The attachment of maleimide-functionalized microbeads to wounds created in live wild-type and diabetic mice led to observations of differential immobilization of microbeads on them and were consistent with anticipated differences in the presentation of sulfhydryl groups on the two different wound types. Finally, the incorporation of maleimide-functionalized microbeads in wounds created in wild-type mice did not impair the rate of wound closure relative to an untreated wound. Overall, the results presented in this paper enable a general and facile approach to the engineering of wound beds in which microbeads are covalently immobilized to wound beds. Such immobilized microbeads could be used in future studies to release bioactive factors (e.g., antimicrobial agents or growth factors) and/or introduce topographical cues that promote cell behaviors underlying healing and wound closure. PMID:23088838

Epidermal growth factor receptor-targeted lipid nanoparticles retain self-assembled nanostructures and provide high specificity

NASA Astrophysics Data System (ADS)

Zhai, Jiali; Scoble, Judith A.; Li, Nan; Lovrecz, George; Waddington, Lynne J.; Tran, Nhiem; Muir, Benjamin W.; Coia, Gregory; Kirby, Nigel; Drummond, Calum J.; Mulet, Xavier

2015-02-01

Next generation drug delivery utilising nanoparticles incorporates active targeting to specific sites. In this work, we combined targeting with the inherent advantages of self-assembled lipid nanoparticles containing internal nano-structures. Epidermal growth factor receptor (EGFR)-targeting, PEGylated lipid nanoparticles using phytantriol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG-maleimide amphiphiles were created. The self-assembled lipid nanoparticles presented here have internal lyotropic liquid crystalline nano-structures, verified by synchrotron small angle X-ray scattering and cryo-transmission electron microscopy, that offer the potential of high drug loading and enhanced cell penetration. Anti-EGFR Fab' fragments were conjugated to the surface of nanoparticles via a maleimide-thiol reaction at a high conjugation efficiency and retained specificity following conjugation to the nanoparticles. The conjugated nanoparticles were demonstrated to have high affinity for an EGFR target in a ligand binding assay.Next generation drug delivery utilising nanoparticles incorporates active targeting to specific sites. In this work, we combined targeting with the inherent advantages of self-assembled lipid nanoparticles containing internal nano-structures. Epidermal growth factor receptor (EGFR)-targeting, PEGylated lipid nanoparticles using phytantriol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG-maleimide amphiphiles were created. The self-assembled lipid nanoparticles presented here have internal lyotropic liquid crystalline nano-structures, verified by synchrotron small angle X-ray scattering and cryo-transmission electron microscopy, that offer the potential of high drug loading and enhanced cell penetration. Anti-EGFR Fab' fragments were conjugated to the surface of nanoparticles via a maleimide-thiol reaction at a high conjugation efficiency and retained specificity following conjugation to the nanoparticles. The conjugated nanoparticles were demonstrated to have high affinity for an EGFR target in a ligand binding assay. Electronic supplementary information (ESI) available: Fig. S1-S4. See DOI: 10.1039/c4nr05200e

Total Body Irradiation Compared With BEAM: Long-Term Outcomes of Peripheral Blood Autologous Stem Cell Transplantation for Non-Hodgkin's Lymphoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Hong-Wei; University of Manitoba, Winnipeg, MB; Seftel, Matthew D.

Purpose: The optimal preparative regimen for non-Hodgkin's lymphoma patients undergoing autologous peripheral blood stem cell transplantation (PBSCT) is unknown. We compared a total body irradiation (TBI)-based regimen with a chemotherapy-alone regimen. Methods and Materials: A retrospective cohort study was performed at a Canadian cancer center. The TBI regimen consisted of cyclophosphamide, etoposide, and TBI 12 Gy in six fractions (CY/E/TBI). The chemotherapy-alone regimen consisted of carmustine, etoposide, cytarabine, and melphalan (BEAM). We compared the acute and long-term toxicities, disease relapse-free survival, and overall survival (OS). Results: Of 73 patients, 26 received CY/E/TBI and 47 received BEAM. The median follow-up formore » the CY/E/TBI group was 12.0 years and for the BEAM group was 7.3 years. After PBSCT, no differences in acute toxicity were seen between the two groups. The 5-year disease relapse-free survival rate was 50.0% and 50.7% in the CY/E/TBI and BEAM groups, respectively (p = .808). The 5-year OS rate was 53.9% and 63.8% for the CY/E/TBI and BEAM groups, respectivey (p = .492). The univariate analysis results indicated that patients with Stage IV, with chemotherapy-resistant disease, and who had received PBSCT before 2000 had inferior OS. A three-way categorical analysis revealed that transplantation before 2000, rather than the conditioning regimen, was a more important predictive factor of long-term outcome (p = .034). Conclusion: A 12-Gy TBI-based conditioning regimen for PBSCT for non-Hodgkin's lymphoma resulted in disease relapse-free survival and OS similar to that after BEAM. PBSCT before 2000, and not the conditioning regimen, was an important predictor of long-term outcomes. TBI was not associated with more acute toxicity or pneumonitis. We found no indication that the TBI regimen was inferior or superior to BEAM.« less

Improved immune responses to a bivalent vaccine of Newcastle disease and avian influenza in chickens by ginseng stem-leaf saponins.

PubMed

Yu, J; Shi, F S; Hu, S

2015-10-15

Our previous investigation demonstrated that ginseng stem-leaf saponins (GSLS) derived from the stems and leaves of Panax ginseng C.A. Meyer promoted humoral and gut mucosal immunity in chickens vaccinated with live infectious bursa disease vaccine. The present study was designed to evaluate the effect of GSLS on the immune response to a bivalent inactive vaccine of Newcastle disease (ND) and avian influenza (AI) in chickens immunosuppressed by cyclophosphamide (Cy). One hundred and sixty-eight specific-pathogen-free (SPF) chickens were randomly divided into 7 groups, each containing 24 birds. Chickens in groups 3-7 received intramuscular injection of Cy at 100mg/kg BW for 3 days to induce immunosuppression. Groups 1 and 2 were injected with saline solution in the same way as groups 3-7. Following injection of Cy, groups 4-7 were orally administrated GSLS (2.5, 5 and 10mg/kg BW) or astragalus polysaccharide (APS) (200mg/L) in drinking water for 7 days; groups 1-3 were not medicated and served as control birds. After administration of GSLS or APS, groups 2-7 were subcutaneously injected with a bivalent inactive vaccine of ND and AI. After that, serum was sampled for detecting antibody titers by HI, spleen was collected for lymphocyte proliferation assay, and duodenum tissues were collected for measurement of IgA-secreting (IgA+) cells and intestinal intraepithelial lymphocytes (iIELs). The results showed that injection of Cy significantly suppressed immunity in chickens; oral administration of GSLS before immunization recovered splenocyte proliferation induced by ConA and LPS, and the numbers of IgA+ cells and iIELs as well as the specific antibody response to a bivalent inactive vaccine of ND and AIin immunosuppressed chickens treated with Cy. Therefore, GSLS may be the potential agent to improve vaccination in immunosuppressed chickens. Copyright © 2015 Elsevier B.V. All rights reserved.

Upregulated Expression of Integrin α1 in Mesangial Cells and Integrin α3 and Vimentin in Podocytes of Col4a3-Null (Alport) Mice

PubMed Central

Steenhard, Brooke M.; Vanacore, Roberto; Friedman, David; Zelenchuk, Adrian; Stroganova, Larysa; Isom, Kathryn; St. John, Patricia L.; Hudson, Billy G.; Abrahamson, Dale R.

2012-01-01

Alport disease in humans, which usually results in proteinuria and kidney failure, is caused by mutations to the COL4A3, COL4A4, or COL4A5 genes, and absence of collagen α3α4α5(IV) networks found in mature kidney glomerular basement membrane (GBM). The Alport mouse harbors a deletion of the Col4a3 gene, which also results in the lack of GBM collagen α3α4α5(IV). This animal model shares many features with human Alport patients, including the retention of collagen α1α2α1(IV) in GBMs, effacement of podocyte foot processes, gradual loss of glomerular barrier properties, and progression to renal failure. To learn more about the pathogenesis of Alport disease, we undertook a discovery proteomics approach to identify proteins that were differentially expressed in glomeruli purified from Alport and wild-type mouse kidneys. Pairs of cy3- and cy5-labeled extracts from 5-week old Alport and wild-type glomeruli, respectively, underwent 2-dimensional difference gel electrophoresis. Differentially expressed proteins were digested with trypsin and prepared for mass spectrometry, peptide ion mapping/fingerprinting, and protein identification through database searching. The intermediate filament protein, vimentin, was upregulated ∼2.5 fold in Alport glomeruli compared to wild-type. Upregulation was confirmed by quantitative real time RT-PCR of isolated Alport glomeruli (5.4 fold over wild-type), and quantitative confocal immunofluorescence microscopy localized over-expressed vimentin specifically to Alport podocytes. We next hypothesized that increases in vimentin abundance might affect the basement membrane protein receptors, integrins, and screened Alport and wild-type glomeruli for expression of integrins likely to be the main receptors for GBM type IV collagen and laminin. Quantitative immunofluorescence showed an increase in integrin α1 expression in Alport mesangial cells and an increase in integrin α3 in Alport podocytes. We conclude that overexpression of mesangial integrin α1 and podocyte vimentin and integrin α3 may be important features of glomerular Alport disease, possibly affecting cell-signaling, cell shape and cellular adhesion to the GBM. PMID:23236390

Resting State and Elementary Steps of the Coupling of Aryl Halides with Thiols Catalyzed by Alkylbisphosphine Complexes of Palladium

PubMed Central

Alvaro, Elsa

2010-01-01

Detailed mechanistic studies on the coupling of aryl halides with thiols catalyzed by palladium complexes of the alkylbisphosphine ligand CyPF-tBu (1-dicyclohexylphosphino-2-di-tert-butylphosphinoethylferrocene) are reported. The elementary steps that constitute the catalytic cycle, i.e. oxidative addition, transmetalation and reductive elimination, have been studied, and their relative rates are reported. Each of the steps of the catalytic process occurs at temperatures that are much lower than those required for the reactions catalyzed by a combination of palladium precursors and CyPF-tBu. To explain these differences in rates between the catalytic and stoichiometric reactions, studies were conducted to identify the resting state of the catalyst of the reactions catalyzed by a combination of Pd(OAc)2 and CyPF-tBu, a combination of Pd(dba)2 and CyPF-tBu, or the likely intermediate Pd(CyPF-tBu)(Ar)(Br). These show that the major palladium complex in each case lies off of the catalytic cycle. The resting state of the reactions catalyzed by Pd(OAc)2 and CyPF-tBu was the palladium bis-thiolate complex [Pd(CyPF-tBu)(SR)2] (R = alkyl or aryl). The resting state in reactions catalyzed by Pd2(dba)3 and CyPF-tBu was the binuclear complex [Pd(CyPF-tBu)]2(μ2, η2-dba) (9). The resting state of reactions of both aromatic and aliphatic thiols catalyzed by [Pd(CyPF-tBu)(p-tolyl)(Br)] (3a) was the hydridopalladium thiolate complex [Pd(CyPF-tBu)(H)(SR)] (R= alkyl and aryl). All these palladium species have been prepared independently, and the mechanisms by which they enter the catalytic cycle have been examined in detail. These features of the reaction catalyzed by palladium and CyPF-tBu have been compared with those of reactions catalyzed by the alkylbisphosphine DiPPF and Pd(OAc)2 or Pd(dba)2. Our data indicate that the resting states of these reactions are similar to each other and that our mechanistic conclusions about reactions catalyzed by palladium and CyPF-tBu can be extrapolated to reactions catalyzed by complexes of other electron-rich bisphosphines. PMID:19453106

The state-led large scale public private partnership 'Chiranjeevi Program' to increase access to institutional delivery among poor women in Gujarat, India: How has it done? What can we learn?

PubMed

De Costa, Ayesha; Vora, Kranti S; Ryan, Kayleigh; Sankara Raman, Parvathy; Santacatterina, Michele; Mavalankar, Dileep

2014-01-01

Many low-middle income countries have focused on improving access to and quality of obstetric care, as part of promoting a facility based intra-partum care strategy to reduce maternal mortality. The state of Gujarat in India, implements a facility based intra-partum care program through its large for-profit private obstetric sector, under a state-led public-private-partnership, the Chiranjeevi Yojana (CY), under which the state pays accredited private obstetricians to perform deliveries for poor/tribal women. We examine CY performance, its contribution to overall trends in institutional deliveries in Gujarat over the last decade and its effect on private and public sector deliveries there. District level institutional delivery data (public, private, CY), national surveys, poverty estimates, census data were used. Institutional delivery trends in Gujarat 2000-2010 are presented; including contributions of different sectors and CY. Piece-wise regression was used to study the influence of the CY program on public and private sector institutional delivery. Institutional delivery rose from 40.7% (2001) to 89.3% (2010), driven by sharp increases in private sector deliveries. Public sector and CY contributed 25-29% and 13-16% respectively of all deliveries each year. In 2007, 860 of 2000 private obstetricians participated in CY. Since 2007, >600,000 CY deliveries occurred i.e. one-third of births in the target population. Caesareans under CY were 6%, higher than the 2% reported among poor women by the DLHS survey just before CY. CY did not influence the already rising proportion of private sector deliveries in Gujarat. This paper reports a state-led, fully state-funded, large-scale public-private partnership to improve poor women's access to institutional delivery - there have been >600,000 beneficiaries. While caesarean proportions are higher under CY than before, it is uncertain if all beneficiaries who require sections receive these. Other issues to explore include quality of care, provider attrition and the relatively low coverage.

Enhanced absorption of cyclosporin A by complexation with dimethyl-beta-cyclodextrin in bile duct-cannulated and -noncannulated rats.

PubMed

Miyake, K; Arima, H; Irie, T; Hirayama, F; Uekama, K

1999-01-01

The enhancing effects of dimethyl-beta-cyclodextrin (DM-beta-CyD) on the absorption of cyclosporin A (CsA) after oral administration to rats under bile duct-cannulated and -noncannulated conditions were investigated. The dissolution rate of CsA was markedly augmented by complexation with DM-beta-CyD. In a closed loop in situ study, DM-beta-CyD considerably increased the cumulative amounts of CsA in the mesenteric venous blood after injection of the aqueous CsA suspension into the small intestinal sac of rats. In addition, the cumulative amount ratio of M1, the dominant metabolite of CsA in rats, to CsA in the mesenteric venous blood for up to 40 min after the injection of the CsA-DM-beta-CyD suspension into the sac was lower than that of the CsA suspension alone. DM-beta-CyD inhibited the bioconversion of CsA in the small intestinal microsomes of rats. These results indicate that the bioconversion of CsA was abated by complexation with DM-beta-CyD. An in vivo study revealed that DM-beta-CyD increased the transfer of CsA to blood, not lymph, with low variability in the absorption after oral administration of the CsA suspension to rats. The variability of bioavailability of DM-beta-CyD complex was lower than that of Sandimmune, although the extent of bioavailability of DM-beta-CyD was only a little higher than that of Sandimmune. The bioavailability of CsA or its DM-beta-CyD complex was appreciably decreased by the cannulation of the bile duct of rats, and the extent of the lowering in the bioavailability in the presence of DM-beta-CyD was much less serious than that of CsA alone. The present results suggest that DM-beta-CyD is particularly useful in designing oral preparations of CsA with an enhanced bioavailability and a reduced variability in absorption.

Fresh vein allograft survival in dogs after cyclosporine treatment.

PubMed

Mingoli, A; Edwards, J D; Feldhaus, R J; Hunter, W J; Naspetti, R; Cavallari, N; Sapienza, P; Kretchmar, D H; Cavallaro, A

1996-04-01

Synthetic grafts are widely used for peripheral arterial reconstructions when autologous veins are not available, but their results have not been satisfactory. Venous allograft may be used as an alternative to synthetic prostheses. The aim of the study was to explore the immunosuppressive efficacy of Cyclosporine A (CyA) as a means of preventing venous allograft failures and rejection. We utilized 56 mongrel dogs. Immunological incompatibility was checked with the skin graft method. Donor inferior vena cava was transplanted into the infrarenal abdominal aorta of recipient animals. One group (group 1, 10 dogs) served as a control and three groups received CyA treatment regimens. Group 2 (10 dogs) received postoperative oral CyA treatment for 30 days. Group 3 (12 dogs) received a vein graft pretreated with a CyA solution without postoperative immunosuppressive therapy. Group 4 (9 dogs) received a vein graft pretreated with a CyA solution and postoperative CyA treatment for 30 days. Allografts were examined at 30 days for patency, aneurysmal dilatation, gross structural changes, inflammatory response, and lymphocytic infiltration. Sex chromatine assessment determined the origin (donor or recipient) of the endothelial cells. The allografts from groups 1 and 3 showed significant aneurysmal dilatation and perivenous inflammation when compared to dogs treated with oral CyA therapy (P < 0.0002). Moreover allografts treated with CyA therapy had a better-developed venous neointima (P < 0.009) with less fibrin (P < 0.02) and thinner medial (P < 0.0009) with less fibrin (P < 0.02), and thinner medial (P < 0.0009) and adventitial layers (P < 0.02). No significant differences were observed in neointimal thickness among the four groups. Lymphocytic infiltration was greater in the group of animals who did not receive oral CyA therapy (P < 0.0004). Barr bodies status showed significant differences between oral CyA treated groups and nontreated groups (P < 0.0003). Oral CyA therapy reduced aneurysmal dilatation and immunological response, promoted the development of a neoendothelium, and preserved the structure of the venous layers. Graft pretreatment with CyA flushing did not have a significant immunosuppressive effect.

The State-Led Large Scale Public Private Partnership ‘Chiranjeevi Program’ to Increase Access to Institutional Delivery among Poor Women in Gujarat, India: How Has It Done? What Can We Learn?

PubMed Central

De Costa, Ayesha; Vora, Kranti S.; Ryan, Kayleigh; Sankara Raman, Parvathy; Santacatterina, Michele; Mavalankar, Dileep

2014-01-01

Background Many low-middle income countries have focused on improving access to and quality of obstetric care, as part of promoting a facility based intra-partum care strategy to reduce maternal mortality. The state of Gujarat in India, implements a facility based intra-partum care program through its large for-profit private obstetric sector, under a state-led public-private-partnership, the Chiranjeevi Yojana (CY), under which the state pays accredited private obstetricians to perform deliveries for poor/tribal women. We examine CY performance, its contribution to overall trends in institutional deliveries in Gujarat over the last decade and its effect on private and public sector deliveries there. Methods District level institutional delivery data (public, private, CY), national surveys, poverty estimates, census data were used. Institutional delivery trends in Gujarat 2000–2010 are presented; including contributions of different sectors and CY. Piece-wise regression was used to study the influence of the CY program on public and private sector institutional delivery. Results Institutional delivery rose from 40.7% (2001) to 89.3% (2010), driven by sharp increases in private sector deliveries. Public sector and CY contributed 25–29% and 13–16% respectively of all deliveries each year. In 2007, 860 of 2000 private obstetricians participated in CY. Since 2007, >600,000 CY deliveries occurred i.e. one-third of births in the target population. Caesareans under CY were 6%, higher than the 2% reported among poor women by the DLHS survey just before CY. CY did not influence the already rising proportion of private sector deliveries in Gujarat. Conclusion This paper reports a state-led, fully state-funded, large-scale public-private partnership to improve poor women’s access to institutional delivery - there have been >600,000 beneficiaries. While caesarean proportions are higher under CY than before, it is uncertain if all beneficiaries who require sections receive these. Other issues to explore include quality of care, provider attrition and the relatively low coverage. PMID:24787692

Functionalization of Lipid-Based Nutrient Supplement with β-Cyclodextrin Inclusions of Oregano Essential Oil.

PubMed

Gaur, Shashank; Lopez, Emely C; Ojha, Ankur; Andrade, Juan E

2018-06-01

Intestinal parasitic infection is one of the main causes of acute undernutrition in children. Oral consumption of oregano essential oil (OEO) can reduce intestinal parasitic infections, however, its addition to therapeutic and supplementary foods is hampered by its undesirable flavor. The objective of this study was to develop a functional lipid-based nutrient supplement (LNS) containing OEO, which is stable, acceptable and provides targeted intestinal delivery of bioactive. β-cyclodextrin (β-CyD) inclusion complexes of OEO (β-CyD-OEO), and carvacrol (β-CyD-CV) (1:1 molar) were prepared using slurry complexation (-20 °C) method and characterized based on encapsulation efficiency, moisture content, morphology, and 2-phase in vitro digestion stability. Carvacrol (CV) content was measured using reverse phase HPLC-UV. LNS containing β-CyD-OEO (27.2 mg encapsulate/20 g LNS) was formulated using Indian staples and ingredients. Discriminatory sensory tests (triangle) were performed with college students (n = 58) and low-income women (n = 25), with young children at home (1 to 6 years), living in Mehsana, India to evaluate differences between LNS with and without bioactive ingredient (β-CyD-OEO only). Moisture of dried complexes ranged 9.1% to 9.7% d.b., whereas water activity 0.35 to 0.412. The complex size and encapsulation efficiency of β-CyD-OEO and β-CyD-CV were 1.5 to 7 μm and 4 to 20 μm, and 86.04 ± 4.48% and 81.39 ± 3.34%, respectively. The bioactive complexes were stable through the gastric and intestinal phases. Bioaccessibility of encapsulated CV ranged 6.0% to 7.7%. Sensory tests revealed no differences (P > 0.05) in color, aroma, and taste between LNS with and without β-CyD-OEO complexes. Functionalization of LNS with β-CyD-OEO is feasible based on in vitro stability and sensory studies. Despite its antiparasitic activities, the addition of oregano essential oil into foods is limited due to its strong flavor and volatility. In this study, we evaluated the encapsulation of oregano essential oil with β-cyclodextrin and its addition into lipid-based nutrition supplements. The results revealed that complex encapsulation efficiency was above 80%. Also, the bioactive complexes were stable under in vitro gastrointestinal conditions. Sensory evaluation of LNS with and without encapsulated essential oil showed no difference in terms of color, aroma, and taste. The functional LNS can both address nutrient insufficiency as well as parasitic infection among malnourished populations in low-resource settings. © 2018 Institute of Food Technologists®.