Bombyx batryticatus Protein-Rich Extract Induces Maturation of Dendritic Cells and Th1 Polarization: A Potential Immunological Adjuvant for Cancer Vaccine

Bombyx batryticatus, a protein-rich edible insect, is widely used as a traditional medicine in China. Several pharmacological studies have reported the anticancer activity of B. batryticatus extracts; however, the capacity of B. batryticatus extracts as immune potentiators for increasing the efficacy of cancer immunotherapy is still unverified. In the present study, we investigated the immunomodulatory role of B. batryticatus protein-rich extract (BBPE) in bone marrow-derived dendritic cells (BMDCs) and DC vaccine-immunized mice. BBPE-treated BMDCs displayed characteristics of mature immune status, including high expression of surface molecules (CD80, CD86, major histocompatibility complex (MHC)-I, and MHC-II), increased production of proinflammatory cytokines (tumor necrosis factor-α and interleukin-12p70), enhanced antigen-presenting ability, and reduced endocytosis. BBPE-treated BMDCs promoted naive CD4⁺ and CD8⁺ T-cell proliferation and activation. Furthermore, BBPE/ovalbumin (OVA)-pulsed DC-immunized mice showed a stronger OVA-specific multifunctional T-cell response in CD4⁺ and CD8⁺ T cells and a stronger Th1 antibody response than mice receiving differently treated DCs, which showed the enhanced protective effect against tumor growth in E.G7 tumor-bearing mice. Our data demonstrate that BBPE can be a novel immune potentiator for a DC-based vaccine in anticancer therapy.     

Anticonvulsant Activity of Bombyx batryticatus and Analysis of Bioactive Extracts Based on UHPLC-Q-TOF MS/MS and Molecular Networking

Bombyx batryticatus (BB) is an anticonvulsant animal medicine in traditional Chinese medicine (TCM) and acts on the central nervous system. This research aimed to study the anticonvulsant effects of different polarity fractions of extracts from BB and to explore the components conferring anticonvulsant activity. Materials and methods: Crude extracts of BB at 20 g/kg were divided into different polarity fractions (petroleum ether, chloroform, ethyl acetate, water) and were administered to groups of mice before injecting pentylenetetrazol (PTZ) to induce convulsions. The animals were placed in chambers, and their behaviors were recorded for 30 min following the injection. Latency time, percent of protection, convulsion, convulsion rate, and convulsion score were determined for these mice. The compounds present in the different fractions were analyzed, and those from the fraction that conferred anticonvulsant activity were identified by high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF MS) and molecular networking (MN). The chloroform extract fractions (B-C) clearly increased the seizure latency time and protection percentage and decreased the convulsion percentage compared to the control group. The anticonvulsant effect of other extract fractions was not significant. Our study shows that the chloroform extract fractions (B-C) of BB have a significant anticonvulsant effect. We also identified 17 compounds including lumichrome, pheophorbide A, and episyringaresinol 4′-O-beta-d-glucopyranose that were found for the first time. The results of this study may lay the groundwork for studying compounds derived from Bombyx batryticatus and their anticonvulsant effect.     

A rapid site‐specific PCR based one‐tube authentication method for saffron in crude drugs and processed herbal medicines

Saffron is one of the world's most precious medicinal herbs and often found to be adulterated with other cheaper materials. The chemical compounds in Crocus sativus L. such as crocin, picrocrocin also exist in other plants, which makes the chemotype‐driven analysis not ideal for the quality control of saffron. Herein, we developed a rapid authentication method for saffron in crude drugs by the site‐specific PCR. In order to realize fast high‐throughput analysis, a one‐tube identification approach was further established by using a universal fluorescent dye to detect the PCR products. In addition, this method was also applied to the authentication of saffron in a processed herbal medicine “Er shi wu wei shan hu wan” which consists of twenty‐five kinds of medicinal materials including plants, minerals and animals. Additionally, this method was also proved to be with high specificity and repeatability. The flexibility of choosing different primers also made this method versatile for other medicinal materials.     

A novel colorimetric PCR-based biosensor for detection and quantification of hepatitis B virus

Hepatitis B virus (HBV) can cause viral infection that attacks the liver and it is a major global health problem that put people at a high risk of death from cirrhosis of the liver and liver cancer. HBV has infected one third of the worldwide population, and 350 million people suffer from chronic HBV infection. For these reasons, development of an accurate, sensitive and expedient detection method for diagnosing, monitoring and assessing therapeutic response of HBV is very necessary and urgent for public health and disease control. Here we report a new strategy for detection of viral load quantitation of HBV based on colorimetric polymerase chain reaction (PCR) with DNAzyme-containing probe. The special DNAzyme adopting a G-quadruplex structure exhibited peroxidase-like activity in the presence of hemin to report colorimetric signal. This method has shown a broad range of linearity and high sensitivity. This study builds important foundation to achieve the specific and accurate detection level of HBV DNA with a low-cost and effective method in helping diagnosing, preventing and protecting human health form HBV generally all over the world and especially in developing countries.     

Application of a new focused microwave technology with species-specific isotope dilution analysis for the quantitative extraction of organometallic contaminants in solid environmental matrices

A new self-tuning single-mode-focused microwave technology has been evaluated in this work to perform the quantitative routine extraction of organometallic species from solid matrices of environmental interest. Species-specific isotope dilution analysis has been employed to better investigate the real influence of the microwave-assisted extractions on the final results. The advantages of such methodology in comparison with other established microwave units for the routine speciation analysis of organomercury and organotin compounds are discussed (such as the capability of using disposable glass vials, a self-tuning mode to provide an accurate control of the temperature and pressure inside of the vials, and the possibility of performing automated sequence of extractions with low sample size). The results obtained in this work demonstrated that such technology provides a fast and reliable quantitative extraction of the organometallic species in a wide range of extraction conditions even when the multi-elemental (Sn and Hg) species-specific determination is carried out.     

A novel real-time cell electronic analysis technology for the authentication and quality control of natural medicines

The study was designed to explore the potential applications of the real-time cell electronic analysis technology in the quality evaluation of natural medicines. The natural medicinal Flos Carthami was discussed as a methodological example and the specific time/dose-dependent cell response profiles （TCRPs） were produced by the real-time cell electronic analysis technology. The similarity and bioactivity were obtained by analyzing all TCRPs. Meanwhile, an HPLC method according to the Chinese Pharmacopeia （edition 2010） was used to evaluate the quality of Flos Carthami. The correlation was obtained by comparing the results produced by the two different approaches. By analyzing the data, five different samples ofFlos Carthami can produce remarkably similar TCRPs. The quality ofFlos Carthami was evaluated by both the HPLC and the TCRPs analysis-based approaches and similar results were obtained. The results suggest that the same natural medicine from different locations could produce similar TCRPs. By analyzing the TCRPs, the bioactivity and quality evaluation of natural medicines can be obtained. This technology is a physiologically relevant approach for the quality evaluation of natural medicines. The ultimate aim of our study is to establish a new standard for quality evaluation.     

A new single‐step quantitative pathogen detection system: Template‐tagging followed by multiplex asymmetric PCR using common primers and CE‐SSCP

Rapid diagnosis of bacterial infection is important for patient management and appropriate therapy during the early phase of bacteria‐induced disease. Among the existing techniques for identifying microbial, CE‐SSCP combined with 16S ribosomal RNA gene‐specific PCR has the benefits of excellent sensitivity, resolution, and reproducibility. However, even though CE‐SSCP can separate PCR products with high‐resolution, multiplex detection and quantification are complicated by primer‐dimer formation and non‐specific amplification. Here, we describe a novel technique for multiplex detection and quantification of pathogens by template‐tagging followed by multiplex asymmetric PCR and subsequent CE‐SSCP. More specifically, we reverse transcribed 16S ribosomal RNAs from seven septicemia‐inducing pathogens, tagged the templates with common end sequences, and amplified them using common primers. The resulting amplicons could be successfully separated by CE‐SSCP and quantified by comparison to an internal standard. This method yielded results that illustrate the potential of this system for diagnosing infectious disease.     

一种新型快速检测半胱氨酸的荧光探针EI北大核心CSCD

基于三苯胺母体的强供电子能力,设计合成了一种共轭性良好的新型半胱氨酸(Cys)荧光探针。采用荧光光谱法和紫外-可见光谱法研究了目标探针T-Probe对半胱氨酸(Cys)的光谱响应。结果表明:目标探针分子与Cys作用后,荧光发射波长有约20 nm红移,荧光强度发生明显的增强,在365 nm紫外灯下,溶液由青色变为蓝色;探针分子选择性识别Cys的检测限为98.4 nmol/L,且灵敏度较高。     

A rapid and effective method for silver staining of PCR products separated in polyacrylamide gels

With the development of molecular quantitative genetics, particularly, genetic linkage map construction, quantitative trait loci mapping or genes fine mapping and association analysis etc., more and more PCR products separated in polyacrylamide gels need to be silver‐stained. However, conventional silver‐staining procedures are complicated and time‐consuming as they require a lot of preparation and handling of several solutions prior to use. In this study, a simple and rapid protocol for silver staining of PCR products was developed. The number of steps was reduced compared to conventional protocols, thus achieving detection of PCR products in 7 min, saving time and resources. Fixation and staining solution and developing solution in present staining procedure allowed a reutilization for 12 and 8 times, respectively, reducing the cost greatly. Meanwhile, the sensitivity was significantly improved with the improved method and the minimum of 0.097 ng/μL of DNA amount can be detected in denaturing polyacrylamide gel. The protocol developed in this study will facilitate the development of molecular quantitative genetics.     

Expression Analysis and Characteristics of <Emphasis Type="Italic">Profilin</Emphasis> Gene from Silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

A recombinant Bombyx mori profilin protein (rBmPFN) was overexpressed in Escherichia coli BL21. Purified rBmPFN was used to generate anti-BmPFN polyclonal antibody, which were used to determine the subcellular localization of BmPFN. Immunostaining indicated that profilin can be found in both the nucleus and cytoplasm but is primarily located in the cytoplasm. Real-time RT-PCR and Western blot analyses indicated that, during the larvae stage, profilin expression levels are highest in the silk gland, followed by the gonad, and are lowest in the fatty body. Additionally, BmPFN expression begins during the egg stage, increases during the larvae stage, reaches a peak during the pupa stage, and decreases significantly in the moth. Therefore, we propose that BmPFN may play an important role during larva stage development, especially in the silk gland.     

Comparative study of electrospray and photospray ionization sources coupled to quadrupole time-of-flight mass spectrometer for olive oil authentication

The use of fast and reliable analytical procedures for olive oil authentication is a priority demand due to its wide consumption and healthy benefits. Olive oil adulteration with other cheaper vegetable oils is a common practice that has to be detected and controlled. Rapid screening methods based on high resolution tandem mass spectrometry constitute today the option of choice due to sample handling simplicity and the elimination of the chromatographic step. The selection of the ionization source is critical and the comparison of their reliability necessary. The possibilities of the direct infusion electrospray ionization (ESI) and the recently introduced atmospheric pressure photospray ionization source (APPI), coupled to quadrupole time-of-flight (QqTOF), have been critically studied and compared to control olive oil adulteration. These techniques are very rapid (approximately 1 min per sample) and have high discrimination power to elucidate key components in the edible oils studied (olive, hazelnut, sunflower and corn). Nevertheless, both sources are complementary, being APPI more sensitive for monoacyl- and diacylglycerol fragment ions and ESI for triacylglycerols. In addition, methods reproducibility's are very high, especially for APPI source. Mixtures of olive oil with the others vegetable oils can be easily discriminated which has been tested by using principal components analysis (PCA) with both ESI-MS and APPI-MS spectra. Analogously, linear discriminant analysis (LDA) confirms methods reproducibility and detection of other oils used as adulterants, in particular hazelnut oil, which is especially difficult given its chemical similarity with olive oil.     

A novel rapid detection approach for the analysis of radionuclides in environmental samples using graphite MALDI mass spectrometry

In nuclear technology, tri-n-butyl phosphate (TBP) diluted with a hydrocarbon diluent such as n-dodecane or NPH is the most frequently used solvent in liquid–liquid extraction for fuel reprocessing. This extraction, known as the plutonium uranium refining by extraction, is still considered as the most dominant process for the extraction of uranium and plutonium from irradiated fuels. The solubility of pure TBP in water is about 0.4 g/L at 25 °C. This is enough to create trouble during evaporation of raffinate and product solutions. Solubility data for undiluted TBP and TBP (diluted in inert hydrocarbon diluent) in various concentrations of nitric acid is not adequate in the literature. The solubility data generated in the present study provide complete information on the solubility of TBP in various nitric acid concentrations (0–15.7 M) at room temperature. The effect of heavy metal ion concentration such as uranium and various fission products on the solubility of TBP in nitric acid is also presented. The results obtained from gas chromatographic technique were compared with spectrophotometric technique by converting the organic phosphate into inorganic phosphate. The generated data is of direct relevance to reprocessing applications.     

A novel procedure for rapid surface functionalisation and mediator loading of screen-printed carbon electrodes

We report a simple and rapid procedure that leads to incorporation of mediator and introduction of amine functionality onto the surface of screen-printed carbon electrodes (SPCE). The electrodes were doped with cobalt phthalocyanine (CoPc) by enhanced adsorption in a process that uses minimal amounts of this redox mediator as compared with CoPc loaded inks. The CoPc-doped SPCE showed a substantially increased sensitivity to hydrogen peroxide and thiocholine as compared to unmodified electrodes. This greatly facilitated their use as transducers for the construction of amperometric biosensors based on enzymes producing oxidizable products such as hydrogen peroxide or thiols. Immobilisation of enzymes including glucose oxidase, acetylcholinesterase and choline oxidase was achieved through their multi-contact electrostatic interaction with polyethyleneimine (PEI) which was electrodeposited on the surface of CoPc-doped electrodes in one step from ethanolic solution. The efficiency of enzyme immobilisation was shown to depend on the molecular weight of the PEI used, reaching a maximum for 25 kDa PEI. The biosensors shown sensitivity to glucose at 130 nA mM⁻¹ (LOD 0.15 mM) and to acetylcholine at 70 nA mM⁻¹ (LOD 0.10 mM) under +0.6 V. Detection of glucose has been demonstrated at +0.4 V with the sensitivity of 60 nA mM⁻¹ and LOD of 0.33 mM. Possibility of the inhibition analysis of pesticides has been shown for acetylcholinesterase-based sensors.     

A sensitive, rapid and specific technique for the detection of collagenase using zymography

A fast and specific method for the detection of collagenase by electrophoresis is described. The method avoids inclusion of the substrate in the resolving gel and can detect nanogram levels of the enzyme.     

A novel sequential injection--Cold vapour atomic absorption spectrometric system for rapid and reliable determination of mercury

A simple and rapid method is described for reliable determination of ultra-trace concentrations of mercury based on a novel combination of a sequential injection (SI) system with cold vapour atomic absorption spectrometry (CVAAS). The SI-CVAAS method achieved a very high sampling frequency of 180h(-1), a detection limit of 0.2-0.3ngmL(-1), a dynamic range of 0-60ngmL(-1) and requires only 0.2-0.6mL of sample. The presence of common metal ions, such as Cu(2+), Pb(2+), Cd(2+), Zn(2+), Fe(3+), Co(2+), Ni(2+) and Mn(2+), did not interfere with the measurement of mercury by this method. Also the interference from organic matter, such as humic substances, was easily reduced or eliminated by appropriate sample dilution. The method was successfully applied to the determination of mercury in lake sediment samples and a river sediment reference material. An average percentage recovery of 101.2% was achieved by the SI-CVAAS method for mercury in the reference material with a R.S.D. of 1.8%.     

A fully portable microchip real‐time polymerase chain reaction for rapid detection of pathogen

Point‐of‐care detection for pathogen is of critical need for wide epidemic warning and medical diagnosis. In this work, we have designed and developed a fully portable and integrated microchip based real‐time polymerase chain reaction machine for rapid pathogen detection. The instrument consists of three functional components including heating, optical, and electrical modules, which are integrated into a portable compact box. The microchip is consumable material replaceable to meet various detection needs. Consequently, we demonstrated the outstanding performance of this portable machine for rapid detection of Salmonella and Escherichia coli O157:H7 with the advantage of time‐saving (～25 min), less samples consumption, portability, and user‐friendly operation.     

Pyrazoles—A novel class of blocking agents for isocyanates

Pyrazoles were found as a novel class of blocking groups for isocyanates. Adhesive mixtures of pyrazole blocked isocyanates and amine terminated prepolymers like Jeffamines® (Texaco Chem. Co.) combine excellent reactivities (gelation times within minutes at 100–120°C), good latencies (more than 170 days at 40°C), and good adhesion properties on many substrates. The reactivity of pyrazole blocked isocyanates toward nucleophiles increases with the number of electron donor substituents on the pyrazole nucleus and, thus, can be fine tuned by the appropriate substitution pattern. This behavior contrasts sharply with that of phenolic blocked isocyanates, where reactivities with the same nucleophiles decrease with more and stronger electron donor substituents on the phenol nucleus. Therefore, different deblocking reaction mechanisms were proposed for pyrazole vs. phenol blocked isocyanates. The excellent latency of pyrazole blocked isocyanate/Jeffamine® mixtures is due to the insolubility of the two components at ambient temperature and the slow endothermic dissolution process at higher temperature. The good adhesion of formulations with pyrazole blocked isocyanates as reactive components on most plastic and metal substrates is ascribed to the primer action of the released blocking group. © 1994 John Wiley & Sons, Inc.     

A novel ion‐imprinted hydrogel for recognition of potassium ions with rapid response

A novel ion‐imprinted strategy is developed for synthesizing responsive hydrogels with rapid response to potassium ions. With potassium ions as templates, ion‐imprinted poly(N‐isopropylacrylamide‐co‐benzo‐15‐crown‐5‐acrylamide) (P(NIPAM‐co‐B15C5Am)) hydrogels are synthesized with 15‐crown‐5 crown ethers mounted on the polymer networks in pairs; therefore, it is very easy and fast for the crown ethers to capture potassium ions again by their Venus flytrap action and form stable 2:1 “host–guest” complexes with potassium ions in the ion‐recognition process. As a result, the response rate of the ion‐imprinted hydrogels to potassium ions is significantly faster than that of normal P(NIPAM‐co‐B15C5Am) hydrogels in which 15‐crown‐5 crown ethers are randomly pendent on the polymeric networks. Copyright © 2010 John Wiley & Sons, Ltd.     

A multiplex SNP genotyping by allele‐specificspecific PCR based on stem‐loop and universal fluorescent primers of Chr1daxin mice

Single nucleotide polymorphisms (SNPs) are one of the most common markers in mammals. Rapid, accurate, and multiplex typing of SNPs is critical for subsequent biological and genetic research. In this study, we have developed a novel method for multiplex genotyping SNPs in mice. The method involves allele‐specific PCR amplification of genomic DNA with two stem‐loop primers accompanied by two different universal fluorescent primers. Blue and green fluorescent signals were conveniently detected on a DNA sequencer. We verified four SNPs of 65 mice based on the novel method, and it is well suited for multiplex genotyping as it requires only one reaction per sample in a single tube with multiplex PCR. The use of universal fluorescent primers greatly reduces the cost of designing different fluorescent probes for each SNP. Therefore, this method can be applied to many biological and genetic studies, such as multiple candidate gene testing, genome‐wide association study, pharmacogenetics, and medical diagnostics.     

A rapid and general method for the asymmetric synthesis of 2-substituted pyrrolidines using tert-butanesulfinamide

A new method for the asymmetric synthesis of 2-substituted pyrrolidines in three steps from commercially available starting materials is described. Addition of the Grignard reagent prepared from 2-(2-bromoethyl)-1,3-dioxane to N-tert-butanesulfinyl aldimines proceeds in high yields and with good diastereoselectivities. The sulfinamide products are then cleanly converted into pyrrolidines in one step.