A supramolecular red to near-infrared fluorescent probe for the detection of drugs in urine.

Water-soluble and fluorescent perylene dyes PMI1 and PMI2 with red to near-infrared (red-NIR) emission and a large Stokes shift were designed and synthesized. These dyes were designed to have two binding units (an aromatic perylene-core and a cationic side group) that allow PMI1 and PMI2 to form strong host-guest complexes with cucurbit[8]uril (CB8) through hydrophobic and electrostatic interactions. As a result, the binding constant of the resulting complexes was determined to be in the range of 106 M-1 which increased about 2 orders of magnitude compared to the previously reported perylene dye with only one binding unit. The results also revealed that the fluorescence emission of PMI1 or PMI2 only in aqueous solution is very low due to the aggregation effect. Upon complexation with CB8, the fluorescence intensity increased about 10-fold while the red-NIR emission and the large Stokes shift were preserved. These host-guest complexes can serve as red-NIR fluorescent displacement probes for the detection of CB8 binding guests. The successful detection of addictive drugs in urine was further demonstrated using the host-guest CB8·PMI1 complex and the interference of autofluorescence from the urine sample was successfully eliminated.

A nickel nanoparticle/nafion-graphene oxide modified screen-printed electrode for amperometric determination of chemical oxygen demand.

A nickel nanoparticle/nafion-graphene oxide (NiNP/Nf-GO) modified screen-printed electrode (SPE) was developed for rapid and environmentally friendly electrochemical determination of chemical oxygen demand (COD). The morphology and the electrochemical performance of the SPEs with different surface modifications were investigated by scanning electron microscopy, electrochemical impedance spectroscopy, amperometry, and cyclic voltammetry, respectively. Interestingly, incorporation of graphene oxide as supporting materials to the NiNP/Nf-GO modified SPE enables high catalyst loading and electrode contact, leading to excellent electrocatalytic oxidation ability. A flow detection system was constructed based the newly designed NiNP/Nf-GO modified SPE with USB connection, a 3D-printed thin-layer flow cell (TLFC), and a peristaltic pump. The flow detection system showed an excellent performance for COD analysis with a linear detection range of 0.1~400 mg L-1 and a lower detection limit of 0.05 mg L-1 with an oxidation potential of 0.45 V. The system was further applied to determine the COD in surface water samples. The results were consistent with those obtained by using the standard method (ISO 6060). Graphical abstract A novel nickel nanoparticle/nafion-graphene oxide (NiNP/Nf-GO) modified screen-printed electrode (SPE) with excellent electrocatalytic oxidation ability was designed and fabricated. This electrode with USB connection was applied in a flow detection system equipped with a 3D-printed thin-layer flow cell and a peristaltic pump for environmentally friendly electrochemical determination of chemical oxygen demand.

A New Electrochemical System Based on a Flow-Field Shaped Solid Electrode and 3D-Printed Thin-Layer Flow Cell: Detection of Pb2+ Ions by Continuous Flow Accumulation Square-Wave Anodic Stripping Voltammetry.

Here we describe a new and sensitive flow electrochemical detection system that employs a novel flow-field shaped solid electrode (FFSSE). The system was constructed with a 3D-printed thin-layer flow cell (TLFC) and a flat screen-printed FFSSE with USB connection. This interface facilitates continuous flow accumulation square-wave anodic stripping voltammetry (ASV). The flow distribution in the working space of TLFC was simulated using the finite element method (FEM) and the shape and configuration of electrodes were optimized accordingly. We demonstrated the electrochemical determination of Pb2+ using this newly designed TLFC-FFSSE detection system without removal of oxygen from samples. This TLFC-FFSSE based system showed an attractive stripping voltammetric performance compared to a traditional ASV based method. A linear range for detection of Pb2+ was found to be 0.5-100 µg/L (0.5 to 100 ppb) and a detection limit of 0.2 µg/L (0.2 ppb) was achieved in the presence of bismuth as codeposition metal. The system was further applied to detect Pb2+ in biological broths of methane fermentation. The electrochemical detection results were consistent with that obtained from atomic fluorescence spectroscopy (AFS) analysis and the average recovery was found to be 95.5-106.5% using a standard addition method. This new flow electrochemical detection system showed better sensitivity and reproducibility compared to a traditional ASV based method. Such a system offers great potential for on-site and real-time detection of heavy metals where compact, inexpensive, robust, and low-volume analysis is required.

Self-assembled biosensor with universal reporter and dual-quenchers for detection of unlabelled nucleic acids.

A novel biosensor with universal reporter and dual quenchers was developed for rapid, sensitive, selective, and inexpensive detection of unlabelled nucleic acids. The biosensor is based on a single-strand DNA stem-loop motif with an extended universal reporter-binding region, a G-base rich stem region, and a universal address-binding region. The self-assembly of these stem-loop probes with fluorescence labeled universal reporter and a universal address region conjugated to gold nanoparticles forms the basis of a biosensor for DNA or microRNA targets in solution. The introduction of dual quenchers (G-base quenching and gold surface plasmon resonance-induced quenching) significantly reduces the fluorescence background to as low as 12% of its original fluorescence intensity and hence enhances the detection limit to 0.01 picomoles without signal ampilication.

Microparticulate ß-glucan vaccine conjugates phagocytized by dendritic cells activate both naïve CD4 and CD8 T cells in vitro.

Microparticulate ß-glucan (MG) conjugated to vaccine antigen has been shown to serve as an effective adjuvant in vivo. To further study antigen presentation by MG:vaccine conjugates, bone marrow-derived dendritic cells (BMDC) were treated with MG conjugated to ovalbumin (OVA), then interacted with splenocytes from DO11.10 transgenic mice expressing an OVA peptide-specific T cell receptor. BMDC treated with MG:OVA induced significantly higher numbers of activated (CD25+CD69+) OVA-specific CD4+ T cells than BMDC treated with OVA alone. BMDC treated with MG:OVA upregulated CD86 and CD40 expression as well as MG alone, indicating that conjugation of OVA does not alter the immunostimulatory capacity of MG. Activation of CD8+ OVA-specific OT-1 cells showed that MG:OVA is also capable of enhancing cross-presentation by BMDC to CD8+ cytotoxic T cells. These results show that MG acts as an adjuvant to enhance antigen presentation by dendritic cells to naïve, antigen-specific CD4 and CD8 T cells.

Fabrication of MnFe2O4-CuInS2/ZnS Magnetofluorescent Nanocomposites and Their Characterization.

Magnetofluorescent nanocomposites (MFNCs) providing a single nanoscale platform with multimodal properties are gaining momentum in biological manipulation, biomedical imaging and therapy. In this work, we report the preparation of MFNCs integrating MnFe2O4 magnetic nanoparticles (MNPs), CuInS2/ZnS quantum dots (QDs) and poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-PLGA) in a tetrahydrofuran (THF)/water solvent system. Through sonication and quick solvent displacement, multiple nanoparticles of each type are co-encapsulated within the hydrophobic core of PEG-PLGA micelles. The developed fabrication process is simple and fast. Moreover, due to the low toxicity of CuInS2/ZnS QDs, the fabrication process is environmentally benign. The fabricated MFNCs were further characterized regarding their fundamental physical, chemical and biological properties. Results reveal that the MFNCs possess high (Mn + Fe) recovery rates, and the optical properties and magnetic relaxivity of the MFNCs are sensitive to the MNP:QD mass ratios in the fabrication. Furthermore, the MFNCs present excellent stability in aqueous solutions, minimal cytotoxicity, and capability for bioconjugation. This study opens an avenue for the MFNCs to be employed in broad biological or biomedical applications.

Heparin sensing based on multisite-binding induced highly ordered perylene nanoaggregates.

Highly ordered perylene nanoaggregates with ultra-low fluorescence were employed for the selective and sensitive fluorescence sensing of heparin. A supramolecular host-guest complex was used as a displacement probe to improve the sensitivity.

A colorimetric and fluorescent dual-modal displacement probe based on host-assisted modulation of intramolecular charge transfer and deaggregation.

A new colorimetric and fluorescent dual-modal displacement probe was developed based on a supramolecular host-guest complex of cucurbit[8]uril (CB8) and a new functionalized perylene derivative involving macrocycle encapsulation that modulated intramolecular charge transfer and deaggregation.

Microenvironment of the murine mammary carcinoma 4T1: endogenous IFN-gamma affects tumor phenotype, growth, and metastasis.

IFN-gamma has a profound influence on growth and metastasis of solid tumors. This is true for the murine mammary carcinoma 4T1 which grows faster and metastasizes much more readily when transplanted into the mammary fatpads of IFN-gamma(-/-) mice. We were interested in determining which infiltrating hematopoietic cells produce IFN-gamma within the 4T1 tumor microenvironment. 4T1 tumors were infiltrated with progressively increasing numbers of F4/80(+)/CD11c(+) myeloid cells, many of which were also Gr-1(+), and Gr-1(+)/CD11b(+) granulocytes. Only small numbers of CD4 T cells, CD8 T cells, NK cells, and gammadelta T cells, the most likely IFN-gamma-producing cells, were seen at any time point. Sensitive intracellular cytokine staining and flow cytometry revealed no tumor-infiltrating hematopoietic cells with detectable levels of intracellular IFN-gamma, although IFN-gamma mRNA transcripts were detected in tumor tissue. However, a progressive increase in the expression of three IFN-gamma-inducible surface membrane proteins (B7-H1, I-A(d), and ICAM-1) on growing 4T1 tumor cells indicated the presence of biologically active IFN-gamma in the tumor microenvironment. Moreover, 4T1 tumor cells from in vitro culture expressed these surface molecules 48 h after intratumoral injection into mature tumors. These data suggest that very low amounts of endogenous IFN-gamma elaborated by infiltrating hematopoietic cells within the microenvironment of a solid tumor can achieve biologically active concentrations and affect tumor phenotype, growth, and metastasis.

Western blot can distinguish natural and acquired antibodies to Mycoplasma agassizii in the desert tortoise (Gopherus agassizii).

Mycoplasma agassizi has been identified as a cause of upper respiratory tract disease (URTD) in the threatened Mojave population of the desert tortoise (Gopherus agassizii), and anti-M. agassizii antibodies have been found by ELISA in as many as 15% of these animals across their geographic range. Here we report that a cohort of 16 egg-reared desert tortoises never exposed to M. agassizii had ELISA antibody titers to this organism that overlapped with titers obtained from some M. agassizii-infected tortoises. These natural antibodies were predominantly of the IgM class. Western blots of plasma from these non-infected tortoises produced a characteristic banding pattern against M. agassizii antigens. A group of 38 wild-caught desert tortoises was tested by ELISA, and although some of these tortoises had antibody titers significantly higher than the non-infected tortoises, there was considerable overlap at the lower titer levels. However, Western blot analysis revealed distinct banding patterns that could readily distinguish between the non-infected tortoises and tortoises with acquired antibodies, regardless of ELISA antibody titers. We conclude that desert tortoises have natural antibodies to M. agassizii that can compromise the determination of infection status by ELISA. However, the Western blot technique can distinguish between natural and acquired antibody patterns and can be used to confirm the diagnosis of M. agassizii infections in the desert tortoise.

Conjugation of protein antigen to microparticulate beta-glucan from Saccharomyces cerevisiae: a new adjuvant for intradermal and oral immunizations.

Immunostimulatory glucose polymers known as beta-glucans have been studied for many years. Our laboratory has prepared and characterized a novel microparticulate beta-glucan (MG) from the budding yeast Saccharomyces cerevisiae. Because MG particles are rapidly phagocytized by murine peritoneal macrophages and induce the expression of B7 costimulatory molecules, we hypothesized that MG could serve as a vaccine adjuvant to enhance specific immune responses. Here, we describe a procedure for conjugating the test vaccine antigen bovine serum albumin (BSA) to MG via water-soluble carbodiimide linkage. Conjugates with up to 0.4 mg of BSA/mg MG were prepared. MG/BSA conjugates were still actively phagocytized by mouse peritoneal macrophages. When used to immunize mice by the intradermal route, these conjugates enhanced the primary IgG antibody response to BSA in a manner comparable to the prototypic complete Freund's adjuvant. Although primary oral immunization with MG/BSA caused no increase in serum anti-BSA antibody titers, booster immunization elicited a significant anti-BSA antibody response. These results suggest that protein antigens can be conjugated to MG via a carbodiimide linkage and that these conjugates provide an adjuvant effect for stimulating the antibody response to the protein antigens.

Intracavity folding of a perylene dye affords a high-affinity complex with cucurbit[8]uril.

A fluorescent perylene dye with two aromatic units was designed for binding to cucurbit[8]uril. The binding affinity of the complexes increased about 3 orders of magnitude compared to the dye without a secondary aromatic unit. The high affinity allows the complexes to act as fluorescent probes for detection of strong binding guests with nanomolar sensitivity.

Evaluation of the behavioral characteristics of the mdx mouse model of duchenne muscular dystrophy through operant conditioning procedures.

The mdx mouse is an important nonhuman model for Duchenne muscular dystrophy (DMD) research. Characterizing the behavioral traits of the strain relative to congenic wild-type (WT) mice may enhance our understanding of the cognitive deficits observed in some humans with DMD and contribute to treatment development and evaluation. In this paper we report the results of a number of experiments comparing the behavior of mdx to WT mice in operant conditioning procedures designed to assess learning and memory. We found that mdx outperformed WT in all learning and memory tasks involving food reinforcement, and this appeared to be related to the differential effects of the food deprivation motivating operation on mdx mice. Conversely, WT outperformed mdx in an escape/avoidance learning task. These results suggest motivational differences between the strains and demonstrate the potential utility of operant conditioning procedures in the assessment of the behavioral characteristics of the mdx mouse.

COMPARISON OF CURRENT METHODS FOR THE DETECTION OF CHRONIC MYCOPLASMAL URTD IN WILD POPULATIONS OF THE MOJAVE DESERT TORTOISE (GOPHERUS AGASSIZII).

Pathogens that cause subclinical diseases or exhibit low infection intensities are difficult to quantify in wild populations. Mojave desert tortoises ( Gopherus agassizii ) have been the focus of much research aimed at measuring the presence of upper respiratory disease (URTD) and URTD-associated pathogens, and techniques used to quantify disease in Gopherus species have also been used for disease surveillance in other species of turtles and tortoises of conservation concern. Published surveys of G. agassizii populations have found a relatively low prevalence of URTD, with most URTD-positive animals exhibiting moderate, intermittent signs of morbidity. Therefore, multiple tests have been developed to quantify URTD including genetic detection of the pathogens Mycoplasma agassizii and Mycoplasma testudineum , detection of M. agassizii -specific antibodies, and standardized quantification of clinical signs of URTD and body condition. These diagnostic tests have only been compared in diseased or moribund, semicaptive animals. We compared diagnostic techniques (TaqMan® and SYBR™ Green qPCR, serology, and visible examination) to detect M. agassizii -associated URTD in 126 wild desert tortoises sampled in Nevada and California, US in 2010. All had healthy body condition indices and none exhibited more than mild-to-moderate visual signs of URTD. Pairwise comparisons of diagnostic techniques indicated poor performance in diagnosing disease in individual animals. We found stronger, but inconsistent, statistical associations among diagnostic techniques at the population level. Our findings have implications for quantifying subclinical respiratory disease in tortoises.

IFN-gamma primes macrophages for enhanced TNF-alpha expression in response to stimulatory and non-stimulatory amounts of microparticulate beta-glucan.

beta-(1-->3)-D-Glucan is an integral cell wall component of a variety of fungi, plants, and bacteria. Like the prototypic inflammatory mediator lipopolysaccharide (LPS), some beta-(1--> 3)-D-glucan-containing preparations have been shown to induce the production of proinflammatory cytokines by macrophages. In the present study, we have tested a new microparticulate form of beta-(1--> 3)-D-glucan (MG) from Saccharomyces cerevisiae for its ability to induce proinflammatory cytokine secretion in mouse peritoneal macrophages in vitro, and we have examined the effect of IFN-gamma. MG was rapidly phagocytized by peritoneal macrophages, and these MG-treated macrophages upregulated TNF-alpha, IL-6, and IL-1beta mRNAs and secreted these proinflammatory cytokines. IFN-gamma treatment alone did not induce unstimulated macrophages to produce TNF-alpha. However, a 4 h IFN-gamma pretreatment augmented TNF-alpha secretion by peritoneal macrophages subsequently treated with an optimally stimulatory dose of MG. IFN-gamma pretreatment for 2 h followed by thorough washing and a further 2 h incubation without IFN-gamma still resulted in enhanced TNF-alpha production in response to MG, suggesting that IFN-gamma can prime macrophages for a subsequent proinflammatory response. Most interestingly, we found that IFN-gamma pretreatment of peritoneal macrophages enhanced the TNF-alpha response to amounts of MG that were poorly stimulatory or non-stimulatory in the absence of IFN-gamma priming. These data suggest that a synergy between IFN-gamma and beta-glucan may have evolved to lower the threshold of sensitivity of the innate immune response to fungal pathogens.

Facilitated preparation of bioconjugatable zwitterionic quantum dots using dual-lipid encapsulation.

Zwitterionic quantum dots prepared through incorporated zwitterionic ligands on quantum dot surfaces, are being paid significant attention in biomedical applications because of their excellent colloidal stability across a wide pH and ionic strength range, antifouling surface, good biocompatibility, etc. In this work, we report a dual-lipid encapsulation approach to prepare bioconjugatable zwitterionic quantum dots using amidosulfobetaine-16 lipids, dipalmitoyl-sn-glycero-3-phosphoethanolamine lipids with functional head groups, and CuInS2/ZnS quantum dots in a tetrahydrofuran/methanol/water solvent system with sonication. Amidosulfobetaine-16 is a zwitterionic lipid and dipalmitoyl-sn-glycero-3-phosphoethanolamine, with its functional head, provides bioconjugation capability. Under sonication, tetrahydrofuran/methanol containing amidosulfobetaine-16, dipalmitoyl-sn-glycero-3-phosphoethanolamine, and hydrophobic quantum dots are dispersed in water to form droplets. Highly water-soluble tetrahydrofuran/methanol in droplets is further displaced by water, which induces the lipid self-assembling on hydrophobic surface of quantum dots and thus forms water soluble zwitterionic quantum dots. The prepared zwitterionic quantum dots maintain colloidal stability in aqueous solutions with high salinity and over a wide pH range. They are also able to be conjugated with biomolecules for bioassay with minimal nonspecific binding.

Thermal Decomposition Based Synthesis of Ag-In-S/ZnS Quantum Dots and Their Chlorotoxin-Modified Micelles for Brain Tumor Cell Targeting.

Cadmium-free silver-indium-sulfide (Ag-In-S or AIS) chalcopyrite quantum dots (QDs) as well as their core-shell structures (AIS/ZnS QDs) are being paid significant attention in biomedical applications because of their low toxicity and excellent optical properties. Here we report a simple and safe synthetic system to prepare high quality AIS and AIS/ZnS QDs using thermal decomposition. The synthetic system simply involves heating a mixture of silver acetate, indium acetate, and oleic acid in dodecanethiol at 170 °C to produce AIS QDs with a 13% quantum yield (QY). After ZnS shell growth, the produced AIS/ZnS QDs achieve a 41% QY. To facilitate phase transfer and bioconjugation of AIS/ZnS QDs for cellular imaging, these QDs were loaded into the core of PLGA-PEG (5k:5k) based micelles to form AIS/ZnS QD-micelles. Cellular imaging studies showed that chlorotoxin-conjugated QD-micelles can be specifically internalized into U-87 brain tumor cells. This work discloses that the scalable synthesis of AIS/ZnS QDs and the facile surface/interface chemistry for phase transfer and bioconjugation of these QDs may open an avenue for the produced QD-micelles to be applied to the detection of endogenous targets expressed on brain tumor cells, or more broadly to cell- or tissue-based diagnosis and therapy.

Microparticulate beta-glucan upregulates the expression of B7.1, B7.2, B7-H1, but not B7-DC on cultured murine peritoneal macrophages.

Beta-1,3-(D)-glucan from a variety of biological sources has been shown to enhance both humoral and cellular immune responses to a variety of antigens, infectious agents, and tumors. Nevertheless, its mode of action has not been fully defined. We sought to determine whether a 1-2 microm diameter microparticulate form of beta-glucan (MG) from the yeast Saccharomyces cerevisiae could regulate expression of B7 family glycoproteins on resident peritoneal macrophages from BALB/c mice. We discovered that MG uregulated B7.2 mRNA expression and enhanced the surface membrane expression of B7.2 glycoprotein. Although B7.1 mRNA was not upregulated above constitutive levels, MG treatment enhanced B7.1 glycoprotein expression on the macrophages, albeit to a lesser extent than B7.2. At the same time, the gene and cell surface expression of B7-H1, a putative negative regulator of T cell activity, was also upregulated by MG. The expression of B7-DC, another B7 family molecule with negative regulatory activity, was not affected by incubation with MG. This study has demonstrated that a microparticulate form of beta-glucan can enhance B7 co-stimulatory molecule expression on macrophages, thereby enabling these antigen-presenting cells to deliver the second signal to T-lymphocytes that express CD28. In addition, because MG also induces the expression of B7-H1, it may enable macrophages to provide a concomitant downregulatory signal to T-lymphocytes expressing PD-1 or related receptors.

Inhibiting TNF-α signaling does not attenuate induction of endotoxin tolerance.

Tumor necrosis factor-alpha (TNF-α) is a central mediator of inflammatory responses elicited by Toll-like receptor agonists, such as the Gram-negative bacterial outer membrane antigen lipopolysaccharide (LPS). TNF-α is responsible for altering vascular permeability and activating infiltrating inflammatory cells, such as monocytes and neutrophils. Interestingly, TNF-α has also demonstrated the ability to induce tolerance to subsequent challenges with TNF-α or LPS in monocyte and macrophage cell populations. Tolerance is characterized by the inability to mount a typical inflammatory response during subsequent challenges following the initial exposure to an inflammatory mediator such as LPS. The ability of TNF-α to induce a tolerant-like state with regard to LPS is most likely a regulatory mechanism to prevent excessive inflammation. We hypothesized that the induction of tolerance or the degree of tolerance is dependent upon the production of TNF-α during the primary response to LPS. To investigate TNF-α-dependent tolerance, human monocytic THP-1 cells were treated with TNF-α-neutralizing antibodies or antagonistic TNF-α receptor antibodies before primary LPS stimulation and then monitored for the production of TNF-α during the primary and challenge stimulation. During the primary stimulation, anti-TNF-α treatment effectively attenuated the production of TNF-α and interleukin-1ß; however, this reduced production did not impact the induction of endotoxin tolerance. These results demonstrate that interfering with TNF-α signaling attenuates production of inflammatory cytokines without affecting the induction of tolerance.

Properties of human blood monocytes. I. CD91 expression and log orthogonal light scatter provide a robust method to identify monocytes that is more accurate than CD14 expression.

BACKGROUND: This study was designed to improve identification of human blood monocytes by using antibodies to molecules that occur consistently on all stages of monocyte development and differentiation. METHODS: We examined blood samples from 200 healthy adults without clinically diagnosed immunological abnormalities by flow cytometry (FCM) with multiple combinations of antibodies and with a hematology analyzer (Beckman LH750). RESULTS: CD91 (α2 -macroglobulin receptor) was expressed only by monocytes and to a consistent level among subjects [mean median fluorescence intensity (MFI) = 16.2 ± 3.2]. Notably, only 85.7 ± 5.82% of the CD91(+) monocytes expressed high levels of the classical monocyte marker CD14, with some CD91(+) CD16(+) cells having negligible CD14, indicating that substantial FCM under-counts will occur when monocytes are identified by high CD14. CD33 (receptor for sialyl conjugates) was co-expressed with CD91 on monocytes but CD33 expression varied by nearly ten-fold among subjects (mean MFI = 17.4 ± 7.7). In comparison to FCM analyses, the hematology analyzer systematically over-counted monocytes and eosinophils while lymphocyte and neutrophil differential values generally agreed with FCM methods. CONCLUSIONS: CD91 is a better marker to identify monocytes than CD14 or CD33. Furthermore, FCM (with anti-CD91) identifies monocytes better than a currently used clinical CBC instrument. Use of anti-CD91 together with anti-CD14 and anti-CD16 supports the identification of the diagnostically significant monocyte populations with variable expression of CD14 and CD16.