Nonparametric estimation of correlation functions in longitudinal and spatial data, with application to colon carcinogenesis experiments

In longitudinal and spatial studies, observations often demonstrate strong correlations that are stationary in time or distance lags, and the times or locations of these data being sampled may not be homogeneous. We propose a nonparametric estimator of the correlation function in such data, using kernel methods. We develop a pointwise asymptotic normal distribution for the proposed estimator, when the number of subjects is fixed and the number of vectors or functions within each subject goes to infinity. Based on the asymptotic theory, we propose a weighted block bootstrapping method for making inferences about the correlation function, where the weights account for the inhomogeneity of the distribution of the times or locations. The method is applied to a data set from a colon carcinogenesis study, in which colonic crypts were sampled from a piece of colon segment from each of the 12 rats in the experiment and the expression level of p27, an important cell cycle protein, was then measured for each cell within the sampled crypts. A simulation study is also provided to illustrate the numerical performance of the proposed method.Comment: Published in at http://dx.doi.org/10.1214/009053607000000082 the Annals of Statistics (http://www.imstat.org/aos/) by the Institute of Mathematical Statistics (http://www.imstat.org

Ultrasensitive Analysis of Binding Affinity of HIV Receptor and Neutralizing Antibodies Using Solution-Phase Electrochemiluminescence Assay

Binding of a few ligand molecules with its receptors on cell surface can initiate cellular signaling transduction pathways, and trigger viral infection of host cells. HIV-1 infects host T-cells by binding its viral envelope protein (gp120) with its receptor (a glycoprotein, CD4) on T cells. Primary strategies to prevent and treat HIV infection is to develop therapies (e.g., neutralizing antibodies) that can block specific binding of CD4 with gp120. The infection often leads to the lower counts of CD4 cells, which makes it an effective biomarker to monitor the AIDS progression and treatment. Despite research over decades, quantitative assays for effective measurements of binding affinities of protein-protein (ligand-receptor, antigen-antibody) interactions remains highly sought. Solid-phase electrochemiluminescence (ECL) immunoassay has been commonly used to capture analytes from the solution for analysis, which involves immobilization of antibody on solid surfaces (micron-sized beads), but it cannot quantitatively measure binding affinities of molecular interactions. In this study, we have developed solution phase ECL assay with a wide dynamic range (0–2 nM) and high sensitivity and specificity for quantitative analysis of CD4 at femtomolar level and their binding affinity with gp120 and monoclonal antibodies (MABs). We found that binding affinities of CD4 with gp120 and MAB (Q4120) are 9.5×108 and 1.2×109 M−1, respectively. The results also show that MAB (Q4120) of CD4 can completely block the binding of gp120 with CD4, while MAB (17b) of gp120 can only partially block their interaction. This study demonstrates that the solution-phase ECL assay can be used for ultrasensitive and quantitative analysis of binding affinities of protein-protein interactions in solution for better understating of protein functions and identification of effective therapies to block their interactions

In Vivo Imaging of Transport and Biocompatibility of Single Silver Nanoparticles in Early Development of Zebrafish Embryos

Real-time study of the transport and biocompatibility of nanomaterials in early embryonic development at single-nanoparticle resolution can offer new knowledge about the delivery and effects of nanomaterials in vivo, and provide new insights into molecular transport mechanisms in developing embryos. In this study, we directly characterized the transport of single silver nanoparticles into an in vivo model system (zebrafish embryos) and investigated their effects on early embryonic development at single-nanoparticle resolution in real time. We designed highly purified and stable (not aggregated and no photodecomposition) nanoparticles and developed single-nanoparticle optics and in vivo assays to enable the study. We found that single Ag nanoparticles (5- 46 nm) transport in and out of embryos through chorion pore canals (CPCs), and exhibit Brownian diffusion (not active transport), with ∼26 times lower diffusion coefficient (3×10-9 cm2/s) inside the chorionic space than that in egg water (7.7×10-8 cm2/s). In contrast, nanoparticles were trapped inside CPCs and the inner mass of the embryos, showing restricted diffusion. Individual Ag nanoparticles were observed inside embryos at each developmental stage and in normally developed, deformed, and dead zebrafish, showing that the biocompatibility and toxicity of Ag nanoparticles and types of abnormalities observed in zebrafish are highly dependent on the dose of Ag nanoparticles, with a critical concentration of 0.19 nM. Rates of passive diffusion and accumulation of nanoparticles in embryos are likely responsible for the dose-dependent abnormalities. Unlike other chemicals, single nanoparticles can be directly imaged inside developing embryos at nanometer (nm) spatial resolution, offering new opportunities to unravel the related pathways that lead to the abnormalities

Silver Nanoparticles Induce Developmental Stage-Specific Embryonic Phenotypes in Zebrafish

Much is anticipated from the development and deployment of nanomaterials in biological organisms, but concerns remain regarding their biocompatibility and target specificity. Here we report our study of the transport, biocompatibility and toxicity of purified and stable silver nanoparticles (Ag NPs, 13.1 ± 2.5 nm in diameter) upon the specific developmental stages of zebrafish embryos using single NP plasmonic spectroscopy. We find that single Ag NPs passively diffuse into five different developmental stages of embryos (cleavage, early-gastrula, early-segmentation, late-segmentation, and hatching stages), showing stage-independent diffusion modes and diffusion coefficients. Notably, the Ag NPs induce distinctive stage and dose-dependent phenotypes and nanotoxicity, upon their acute exposure to the Ag NPs (0–0.7 nM) for only 2 h. The late-segmentation embryos are most sensitive to the NPs with the lowest critical concentration (CNP,c ≪ 0.02 nM) and highest percentages of cardiac abnormalities, followed by early-segmentation embryos (CNP,c \u3c 0.02 nM), suggesting that disruption of cell differentiation by the NPs causes the most toxic effects on embryonic development. The cleavage-stage embryos treated with the NPs develop to a wide variety of phenotypes (abnormal finfold, tail/spinal cord flexure, cardiac malformation, yolk sac edema, and acephaly). These organ structures are not yet developed in cleavage-stage embryos, suggesting that the earliest determinative events to create these structures are ongoing, and disrupted by NPs, which leads to the downstream effects. In contrast, the hatching embryos are most resistant to the Ag NPs, and majority of embryos (94%) develop normally, and none of them develops abnormality. Interestingly, early-gastrula embryos are less sensitive to the NPs than cleavage and segmentation stage embryos, and do not develop abnormally. These important findings suggest that the Ag NPs are not simple poisons, and they can target specific pathways in development, and potentially enable target specific study and therapy for early embryonic development

Design and Study of the Efflux Function of the EGFP Fused MexAB-OprM Membrane Transporter in Pseudomonas aeruginosa Using Spectroscopy

Multidrug membrane transporters (efflux pumps) can selectively extrude a variety of structurally and functionally diverse substrates (e.g., chemotoxics, antibiotics), leading to multidrug resistance (MDR) and ineffective treatment of a wide variety of diseases. In this study, we have designed and constructed a fusion gene (egfp-mexB) of N-terminal mexB with C-terminal egfp, inserted it into a plasmid vector (pMMB67EH), and successfully expressed it in the Δ MexB (MexB deletion) strain of Pseudomonas aeruginosato create a new strain that expresses MexA-(EGFP-MexB)-OprM. We characterized the fusion gene using gel electrophoresis and DNA sequencing, and determined its expression in live cells by measuring the fluorescence of EGFP in single live cells using fluorescence microscopy. Efflux function of the new strain was studied by measuring its accumulation kinetics of ethidium bromide (EtBr, a pump substrate) using fluorescence spectroscopy, which was compared with cells (WT, ΔMexM, ΔABM, and nalB1) with various expression levels of MexAB-OprM. The new strain shows 6-fold lower accumulation rates of EtBr (15 μM) than ΔABM, 4-fold lower than ΔMexB, but only 1.1-fold higher than WT. As the EtBr concentration increases to 40 mM, the new strain has nearly the same accumulation rate of EtBr as ΔMexB, but 1.4-fold higher than WT. We observed the nearly same level of inhibitory effect of CCCP (carbonyl cyanide-m-chlorophenylhydrazone) on the efflux of EtBr by the new strain and WT. Antibiotic susceptibility study shows that the minimum inhibitory concentrations (MICs) of aztreonam (AZT) and chloramphenicol (CP) for the new strain are 6-fold or 3-fold lower than WT, respectively, and 2-fold higher than those of Δ MexB. Taken together, the results suggest that the fusion protein partially retains the efflux function of MexAB-OprM. The modeled structure of the fusion protein shows that the position and orientation of the N-terminal fused EGFP domain may either partially block the translocation pore or restrict the movement of the individual pump domains, which may lead to partially restricted efflux activity

Vascular smooth muscle TRPC3 channels facilitate the inverse hemodynamic response during status epilepticus

Human status epilepticus (SE) is associated with a pathological reduction in cerebral blood flow termed the inverse hemodynamic response (IHR). Canonical transient receptor potential 3 (TRPC3) channels are integral to the propagation of seizures in SE, and vascular smooth muscle cell (VSMC) TRPC3 channels participate in vasoconstriction. Therefore, we hypothesize that cerebrovascular TRPC3 channels may contribute to seizure-induced IHR. To examine this possibility, we developed a smooth muscle-specific TRPC3 knockout (TRPC3smcKO) mouse. To quantify changes in neurovascular coupling, we combined laser speckle contrast imaging with simultaneous electroencephalogram recordings. Control mice exhibited multiple IHRs, and a limited increase in cerebral blood flow during SE with a high degree of moment-to-moment variability in which blood flow was not correlated with neuronal activity. In contrast, TRPC3smcKO mice showed a greater increase in blood flow that was less variable and was positively correlated with neuronal activity. Genetic ablation of smooth muscle TRPC3 channels shortened the duration of SE by eliminating a secondary phase of intense seizures, which was evident in littermate controls. Our results are consistent with the idea that TRPC3 channels expressed by cerebral VSMCs contribute to the IHR during SE, which is a critical factor in the progression of SE.Fil: Cozart, Michael A.. University of Arkansas for Medical Sciences; Estados UnidosFil: Phelan, Kevin D.. University of Arkansas for Medical Sciences; Estados UnidosFil: Wu, Hong. University of Arkansas for Medical Sciences; Estados UnidosFil: Mu, Shengyu. University of Arkansas for Medical Sciences; Estados UnidosFil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Rusch, Nancy J.. University of Arkansas for Medical Sciences; Estados UnidosFil: Zheng, Fang. University of Arkansas for Medical Sciences; Estados Unido

Angiotensin II stimulates superoxide production by nitric oxide synthase in thick ascending limbs

Angiotensin II (Ang II) causes nitric oxide synthase (NOS) to become a source of superoxide (O2 (-)) via a protein kinase C (PKC)-dependent process in endothelial cells. Ang II stimulates both NO and O2 (-) production in thick ascending limbs. We hypothesized that Ang II causes O2 (-) production by NOS in thick ascending limbs via a PKC-dependent mechanism. NO production was measured in isolated rat thick ascending limbs using DAF-FM, whereas O2 (-) was measured in thick ascending limb suspensions using the lucigenin assay. Consistent stimulation of NO was observed with 1 nmol/L Ang II (P \u3c 0.001; n = 9). This concentration of Ang II-stimulated O2 (-) production by 50% (1.77 ± 0.26 vs. 2.62 ± 0.36 relative lights units (RLU)/s/μg protein; P \u3c 0.04; n = 5). In the presence of the NOS inhibitor L-NAME, Ang II-stimulated O2 (-) decreased from 2.02 ± 0.29 to 1.10 ± 0.11 RLU/s/μg protein (P \u3c 0.01; n = 8). L-arginine alone did not change Ang II-stimulated O2 (-) (2.34 ± 0.22 vs. 2.29 ± 0.29 RLU/s/μg protein; n = 5). In the presence of Ang II plus the PKC α/β1 inhibitor Gö 6976, L-NAME had no effect on O2 (-) production (0.78 ± 0.23 vs. 0.62 ± 0.11 RLU/s/μg protein; n = 7). In the presence of Ang II plus apocynin, a NADPH oxidase inhibitor, L-NAME did not change O2 (-) (0.59 ± 0.04 vs. 0.61 ± ×0.08 RLU/s/μg protein; n = 5). We conclude that: (1) Ang II causes NOS to produce O2 (-) in thick ascending limbs via a PKC- and NADPH oxidase-dependent process; and (2) the effect of Ang II is not due to limited substrate

Single Nanoparticle Plasmonic Spectroscopy for Study of the Efflux Function of Multidrug ABC Membrance Transporters of Single Live Cells

ATP-binding cassette (ABC) membrane transporters exist in all living organisms and play key roles in a wide range of cellular and physiological functions. The ABC transporters can selectively extrude a wide variety of structurally and functionally unrelated substrates, leading to multidrug resistance. Despite extensive study, their efflux molecular mechanisms remain elusive. In this study, we synthesized and characterized purified silver nanoparticles (Ag NPs) (97 ± 13 nm in diameter), and used them as photostable optical imaging probes to study efflux kinetics of ABC membrane transporters (BmrA) of single live cells (B. subtilis). The NPs with concentrations up to 3.7 pM were stable (non-aggregated) in a PBS buffer and biocompatible with the cells. We found a high dependence of accumulation of the intracellular NPs in single live cells (WT, Ct-BmrAEGFP, ΔBmrA) upon the cellular expression level of BmrA and NP concentration (0.93, 1.85 and 3.7 pM), showing the highest accumulation of intracellular NPs in ΔBmrA (deletion of BmrA) and the lowest ones in Ct-BmrA-EGFP (over-expression of BmrA). Interestingly, the accumulation of intracellular NPs in ΔBmrA increases nearly proportionally with the NP concentration, while those in WT and Ct-BmrA-EGFP do not. This result suggests that the NPs enter the cells via passive diffusion driven by concentration gradients across the cellular membrane and they are extruded out of cells by BmrA transporters, similar to conventional pump substrates (antibiotics). This study shows that such large substrates (84-100 nm NPs) can enter into the live cells and be extruded out of the cells by BmrA, and the NPs can serve as nm-sized optical imaging probes to study the size-dependent efflux kinetics of membrane transporters in single live cells in real time