The Imaging of Insulinomas Using a Radionuclide-Labelled Molecule of the GLP-1 Analogue Liraglutide: A New Application of Liraglutide

Objective

This study explores a new, non-invasive imaging method for the specific diagnosis of insulinoma by providing an initial investigation of the use of ¹²⁵I-labelled molecules of the glucagon-like peptide-1 (GLP-1) analogue liraglutide for in vivo and in vitro small-animal SPECT/CT (single-photon emission computed tomography/computed tomography) imaging of insulinomas.

Methods

Liraglutide was labelled with ¹²⁵I by the Iodogen method. The labelled ¹²⁵I-liraglutide compound and insulinoma cells from the INS-1 cell line were then used for in vitro saturation and competitive binding experiments. In addition, in a nude mouse model, the use of ¹²⁵I-liraglutide for the in vivo small-animal SPECT/CT imaging of insulinomas and the resulting distribution of radioactivity across various organs were examined.

Results

The labelling of liraglutide with ¹²⁵I was successful, yielding a labelling rate of approximately 95% and a radiochemical purity of greater than 95%. For the binding between ¹²⁵I-liraglutide and the GLP-1 receptor on the surface of INS-1 cells, the equilibrium dissociation constant (K_d) was 128.8±30.4 nmol/L(N = 3), and the half-inhibition concentration (IC₅₀) was 542.4±187.5 nmol/L(N = 3). Small-animal SPECT/CT imaging with ¹²⁵I-liraglutide indicated that the tumour imaging was clearest at 90 min after the ¹²⁵I-liraglutide treatment. An examination of the in vivo distribution of radioactivity revealed that at 90 min after the ¹²⁵I-liraglutide treatment, the target/non-target (T/NT) ratio for tumour and muscle tissue was 4.83±1.30(N = 3). Our study suggested that ¹²⁵I-liraglutide was predominantly metabolised and cleared by the liver and kidneys.

Conclusion

The radionuclide ¹²⁵I-liraglutide can be utilised for the specific imaging of insulinomas, representing a new non-invasive approach for the in vivo diagnosis of insulinomas.     

The function of p120 catenin in filopodial growth and synaptic vesicle clustering in neurons

At the developing neuromuscular junction (NMJ), physical contact between motor axons and muscle cells initiates presynaptic and postsynaptic differentiation. Using Xenopus nerve-muscle cocultures, we previously showed that innervating axons induced muscle filopodia (myopodia), which facilitated interactions between the synaptic partners and promoted NMJ formation. The myopodia were generated by nerve-released signals through muscle p120 catenin (p120ctn), a protein of the cadherin complex that modulates the activity of Rho GTPases. Because axons also extend filopodia that mediate early nerve-muscle interactions, here we test p120ctn's function in the assembly of these presynaptic processes. Overexpression of wild-type p120ctn in Xenopus spinal neurons leads to an increase in filopodial growth and synaptic vesicle (SV) clustering along axons, whereas the development of these specializations is inhibited following the expression of a p120ctn mutant lacking sequences important for regulating Rho GTPases. The p120ctn mutant also inhibits the induction of axonal filopodia and SV clusters by basic fibroblast growth factor, a muscle-derived molecule that triggers presynaptic differentiation. Of importance, introduction of the p120ctn mutant into neurons hinders NMJ formation, which is observed as a reduction in the accumulation of acetylcholine receptors at innervation sites in muscle. Our results suggest that p120ctn signaling in motor neurons promotes nerve-muscle interaction and NMJ assembly.     

Salinity-induced changes in protein expression in the halophytic plant Nitraria sphaerocarpa

Salinity is a major abiotic stress that inhibits plant growth and development. Plants have evolved complex adaptive mechanisms that respond to salinity stress. However, an understanding of how plants respond to salinity stress is far from being complete. In particular, how plants survive salinity stress via alterations to their intercellular metabolic networks and defense systems is largely unknown. To delineate the responses of Nitraria sphaerocarpa cell suspensions to salinity, changes in their protein expression patterns were characterized by a comparative proteomic approach. Cells that had been treated with 150 mM NaCl for 1, 3, 5, 7, or 9 days developed several stress-related phenotypes, including those affecting morphology and biochemical activities. Of ~1100 proteins detected in 2-DE gel patterns, 130 proteins showed differences in abundance with more than 1.5-fold when cells were stressed by salinity. All but one of these proteins was identified by MS and database searching. The 129 spots contained 111 different proteins, including those involved in signal transduction, cell rescue/defense, cytoskeleton and cell cycle, protein folding and assembly, which were the most significantly affected. Taken together, our results provide a foundation to understand the mechanism of salinity response.     

Nest predation risk and growth strategies of passerine species: grow fast or develop traits to escape risk?

Abstract Different body components are thought to trade off in their growth and development rates, but the causes for relative prioritization of any trait remains a critical question. Offspring of species at higher risk of predation might prioritize development of locomotor traits that facilitate escaping risky environments over growth of mass. We tested this possibility in 12 altricial passerine species that differed in their risk of nest predation. We found that rates of growth and development of mass, wings, and endothermy increased with nest predation risk across species. In particular, species with higher nest predation risk exhibited relatively faster growth of wings than of mass, fledged with relatively larger wing sizes and smaller mass, and developed endothermy earlier at relatively smaller mass. This differential development can facilitate both escape from predators and survival outside of the nest environment. Tarsus growth was not differentially prioritized with respect to nest predation risk, and instead all species achieved adult tarsus size by age of fledging. We also tested whether different foraging modes (aerial, arboreal, and ground foragers) might explain the variation of differential growth of locomotor modules, but we found that little residual variation was explained. Our results suggest that differences in nest predation risk among species are associated with relative prioritization of body components to facilitate escape from the risky nest environment.     

Characterization of germin-like protein with polyphenol oxidase activity from Satsuma mandarine

Polyphenol oxidases (PPOs) catalyzing the oxygen dependent oxidation of phenols to quinones are ubiquitously distributed in plants and are assumed to be involved in plant defense against pests and pathogens. A protein with high PPO activity was identified in Satsuma mandarine, extracted with Tris–HCl buffer, purified by salt precipitation and column chromatography, and characterized by mass spectrometry as germin-like protein (GLP), which belongs to pathogenesis related protein (PR) family. In the present study, the structure and enzymatic properties of GLP were characterized using spectroscopy methods. Based on native PAGE analysis, the molecular weight of GLP was estimated to be 108 kDa and GLP was identified as a pentamer containing five subunits of 22 kDa. The optimum pH and temperature for PPO catalyzing activity of GLP was 6.5 and 65 °C, respectively. Kinetic constants were 0.0365 M and 0.0196 M with the substrates catechol and pyrogallol, respectively. The structural characterization of GLP provided better insights into the regions responsible for its PPO activity.     

Pneumococcal Transmission and Disease In Silico: A Microsimulation Model of the Indirect Effects of Vaccination

Background

The degree and time frame of indirect effects of vaccination (serotype replacement and herd immunity) are key determinants in assessing the net effectiveness of vaccination with pneumococcal conjugate vaccines (PCV) in control of pneumococcal disease. Using modelling, we aimed to quantify these effects and their dependence on coverage of vaccination and the vaccine''s efficacy against susceptibility to pneumococcal carriage.

Methods and Findings

We constructed an individual-based simulation model that explores the effects of large-scale PCV programmes and applied it in a developed country setting (Finland). A population structure with transmission of carriage taking place within relevant mixing groups (families, day care groups, schools and neighbourhoods) was considered in order to properly assess the dependency of herd immunity on coverage of vaccination and vaccine efficacy against carriage. Issues regarding potential serotype replacement were addressed by employing a novel competition structure between multiple pneumococcal serotypes. Model parameters were calibrated from pre-vaccination data about the age-specific carriage prevalence and serotype distribution. The model predicts that elimination of vaccine-type carriage and disease among those vaccinated and, due to a substantial herd effect, also among the general population takes place within 5–10 years since the onset of a PCV programme with high (90%) coverage of vaccination and moderate (50%) vaccine efficacy against acquisition of carriage. A near-complete replacement of vaccine-type carriage by non-vaccine-type carriage occurs within the same time frame.

Conclusions

The changed patterns in pneumococcal carriage after PCV vaccination predicted by the model are unequivocal. The overall effect on disease incidence depends crucially on the magnitude of age- and serotype-specific case-to-carrier ratios of the remaining serotypes relative to those of the vaccine types. Thus the availability of reliable data on the incidence of both pneumococcal carriage and disease is essential in assessing the net effectiveness of PCV vaccination in a given epidemiological setting.     

A New Functional Site W115 in CdtA Is Critical for Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin

Aggregatibacter actinomycetemcomitans, a specific pathogen of localized aggressive periodontitis, produces a cytolethal distending toxin (CDT) that arrests eukaryotic cells irreversibly in G0/G1 or G2/M phase of the cell cycle. Although structural studies show that the aromatic patch region of CdtA plays an important role in its biological activity, the functional sites of CdtA have not been firmly established. In this study, site-specific mutagenesis strategy was employed for cdtA point mutations construction so as to examine the contributions of individual amino acids to receptor binding and the biological activity of holotoxin. The binding ability was reduced in CdtA^Y181ABC holotoxin and the biological function of CDT was not weaken in CdtA^Y105ABC, CdtA^Y125ABC, CdtA^F109ABC and CdtA^S106NBC holotoxin suggesting that these sites were not critical to CDT. But the binding activity and cell cycle arrest ability of holotoxin complexes were inhibited in CdtA^W115GBC. And this site did not affect the holotoxin assembly by size exclusion chromatography. Therefore, W115 might be a critical site of CdtA binding ability. These findings suggest that the functional sites of CdtA are not only in the aromatic patch region. W115, the new functional site is critical for receptor binding and cell cycle arrest, which provides potential targets for pharmacological disruption of CDT activity.     

Synthesis,docking, and biological studies of phenanthrene β-diketo acids as novel HIV-1 integrase inhibitors

In the present study we report the synthesis of halogen-substituted phenanthrene β-diketo acids as new HIV-1 integrase inhibitors. The target phenanthrenes were obtained using both standard thermal- and microwave-assisted synthesis. 4-(6-Chlorophenanthren-2-yl)-2,4-dioxobutanoic acid (18) was the most active compound of the series, inhibiting both 3′-end processing (3′-P) and strand transfer (ST) with IC₅₀ values of 5 and 1.3 μM, respectively. Docking studies revealed two predominant binding modes that were distinct from the binding modes of raltegravir and elvitegravir, and suggest a novel binding region in the IN active site. Moreover, these compounds are predicted not to interact significantly with some of the key amino acids (Q148 and N155) implicated in viral resistance. Therefore, this series of compounds can further be investigated for a possible chemotype to circumvent resistance to clinical HIV-1 IN inhibitors.     

Structural basis of a flavivirus recognized by its neutralizing antibody: solution structure of the domain III of the Japanese encephalitis virus envelope protein

The flavivirus envelope protein is the dominant antigen in eliciting neutralizing antibodies and plays an important role in inducing immunologic responses in the infected host. We have determined the solution structure of the major antigenic domain (domain III) of the Japanese encephalitis virus (JEV) envelope protein. The JEV domain III forms a beta-barrel type structure composed of six antiparallel beta-strands resembling the immunoglobulin constant domain. We have also identified epitopes of the JEV domain III to its neutralizing antibody by chemical shift perturbation measurements. Site-directed mutagenesis experiments are performed to confirm the NMR results. Our study provides a structural basis for understanding the mechanism of immunologic protection and for rational design of vaccines effective against flaviviruses.     

K物质对心肌细胞收缩幅度的影响及其机制探讨

目的 :探讨K物质 (SK)对心肌细胞收缩的影响及机制。方法 :原代培养幼鼠心肌细胞 ,利用计算机图像分析系统测定SK处理前后心肌细胞收缩频率和收缩幅度的变化 ,同时观察预先加入速激肽受体拮抗剂DSP、β受体阻断剂心得安、α受体阻断剂酚妥拉明对SK作用的影响。结果 :当加入SK (1 .78× 1 0 - 5mol/L)到培养细胞中时 ,心肌细胞收缩幅度增强 ,但收缩频率变化不显著 ;且在 1 0 - 8～ 1 0 - 5mol/L浓度范围内心肌细胞收缩幅度变化呈剂量 效应关系 ;预先加入DSP可抑制SK对心肌细胞的影响 ,而心得安、酚妥拉明对SK的作用无影响。结论 :SK使心肌细胞的收缩幅度增强 ,其作用是由速激肽受体介导的