Quantification of HBsAg:Basic virology for clinical practice

Hepatitis B surface antigen (HBsAg) is produced and secreted through a complex mechanism that is still not fully understood. In clinical fields, HBsAg has long served as a qualitative diagnostic marker for hepatitis B virus infection. Notably, advances have been made in the development of quantitative HBsAg assays, which have allowed viral replication monitoring, and there is an opportunity to make maximal use of quantitative HBsAg to elucidate its role in clinical fields. Yet, it needs to be underscored that a further understanding of HBsAg, not only from clinical point of view but also from a virologic point of view, would enable us to deepen our insights, so that we could more widely expand and apply its utility. It is also important to be familiar with HBsAg variants and their clinical consequences in terms of immune escape mutants, issues resulting from overlap with corresponding mutation in the P gene, and detection problems for the HBsAg variants. In this article, we review current concepts and issues on the quantification of HBsAg titers with respect to their biologic nature, method principles, and clinically relevant topics.

Rice Repetitive DNA Sequence RRD3:a Plant Promoter and Its Application to RNA Interference

Previously, a moderately repetitive DNA sequence （RRD3） was cloned from rice （Oryza sativa L.） by DNA renaturation kinetics. Sequence analysis revealed several conserved promoter motifs, including four TATA-boxes and a CAAT-box, and promoter activity was shown in Escherichia coli and mammalian expression systems. Here, we inserted the RRD3 fragment into the plant promoter-capture vector, pCAMBIA1391Z, and examined whether the RRD3 fragment has promoter activity in plants. Transgenic tobacco and rice calli both showed β-glucuronidase （GUS） activity, indicating that RRD3 can act as a promoter in both monocot and dicot plants. Based on the promoter characteristic of RRD3, we designed a plant universal binary vector, pCRiRRD3, which is suitable for performing researches on plant RNA interference. This vector has two multiple cloning sites to facilitate sense and antisense cloning of the target sequence, separated by an intron fragment of 200 bp. The efficiency of the vector for gene silencing was assayed by histochemical and quantitative fluorometric GUS assays in transgenic tobacco. These research results suggested that this plant RNAi vector pCRiRRD3 can effectively perform gene silencing researches on both monocot and dicot plants.

A quantitative RT-PCR assay for rapid detection of Eurasianlineage H10 subtype influenza A virus

Dear Editor,Influenza A viruses(IAVs)are single-stranded,negative sense RNA viruses.IAV subtype is determined on the basis of the viral surface glycoproteins,hemagglutinin(HA),and neuraminidase(NA).To date,18 HA and 11NA subtypes have been reported(Tong et al.,2012).

Effect of electroacupuncture on the mRNA and protein expression of Rho-A and Rho-associated kinase Ⅱ in spinal cord injury rats

Electroacupuncture is beneficial for the recovery of spinal cord injury, but the underlying mechanism is unclear. The Rho/Rho-associated kinase（ROCK） signaling pathway regulates the actin cytoskeleton by controlling the adhesive and migratory behaviors of cells that could inhibit neurite regrowth after neural injury and consequently hinder the recovery from spinal cord injury. Therefore, we hypothesized electroacupuncture could affect the Rho/ROCK signaling pathway to promote the recovery of spinal cord injury. In our experiments, the spinal cord injury in adult Sprague-Dawley rats was caused by an impact device. Those rats were subjected to electroacupuncture at Yaoyangguan（GV3）, Dazhui（GV14）, Zusanli（ST36） and Ciliao（BL32） and/or monosialoganglioside treatment. Behavioral scores revealed that the hindlimb motor functions improved with those treatments. Real-time quantitative polymerase chain reaction, fluorescence in situ hybridization and western blot assay showed that electroacupuncture suppressed the m RNA and protein expression of Rho-A and Rho-associated kinase Ⅱ（ROCKⅡ） of injured spinal cord. Although monosialoganglioside promoted the recovery of hindlimb motor function, monosialoganglioside did not affect the expression of Rho-A and ROCKⅡ. However, electroacupuncture combined with monosialoganglioside did not further improve the motor function or suppress the expression of Rho-A and ROCKⅡ. Our data suggested that the electroacupuncture could specifically inhibit the activation of the Rho/ROCK signaling pathway thus partially contributing to the repair of injured spinal cord. Monosialoganglioside could promote the motor function but did not suppress expression of Rho A and ROCKⅡ. There was no synergistic effect of electroacupuncture combined with monosialoganglioside.

An analytical method for quantitative estimation of the cytotoxicity of dental alloys using MTT assay,ICP analysis and effective cytotoxicity calculation

The two most important criteria for dental materials are their biofunctional and biocompatible endurance within the anticipated life-span of the dental restoration in the mouth. Biocompatibility relates mainly to the allergenicity and the toxicity of the material. To test the non-specific toxicity of dental materials, in vitro cell culture assays have been developed. For in vitro screening, such tests are recommended to check the cytotoxicity of dental materials (ISO 10993 5). Various studies have already been performed to quantitatively determine the cytotoxicity level of dental alloys. However, as long as only dental alloys and the cell culture technique are applied, it is not possible to determine which of the alloying elements cause the cytotoxicity. Therefore, an analytical method is needed. Wataha et al determined in 1991 the TC50 values of 9 metal cations of various dental casting alloys, using cell culture methods. Kapert et al reported in 1994 a complex in vitro test concept, where the ICP analysis (inductively coupled plasma emission spectroscopy) was introduced to measure the trace elements extracted from various alloys. Experimentelle Zahnheilkunde, Universitts ZMK Klinik Freiburg, Germany (Lü XY and Kappert HF) The aim of the present study was to find a relation between the ICP results, the TC50 value of metal cations, and the cytotoxicity of dental alloys. The cytotoxicity levels of various dental alloys and the TC50 values of 10 metal cations were established using the MTT assay, an effective cell culture of method. Then, the concentrations of the corrosively soluted metal cations in the extracts of the alloys were measured using the ICP method. From all these experimental results it was found that the relation between the effective cytotoxicity Z eff of an alloy, the concentrations C i of i th trace element and the TC50 values T Ci of the i th metal cation can approximately be expressed by Z eff =∑iC i2·T Ci . Two significant applications of this expression are a) The cytotoxicity of an alloy can be estimated by ICP analysis of the extract if the TC50 values of the trace elements are know. b) The cytotoxicity of a new-developed-alloy can be estimated in advance, according to the alloying components.

Immune fluorescence test strips based on quantum dots for rapid and quantitative detection of carcino-embryonic antigen

At present,many researchers focused on the point-of-care testing（POCT）,a method of disease markers detection without large-scale instruments and specialized persons.However,most POCT diagnostic methods were suffered from poor detection sensitivity or inefficiency in quantitative detection.Herein,we developed a newly QD-immune fluorescence test strips（QD-IFTS） based on quantum dots（QDs） as the fluorescence nanocarrier to prepare the immune fluorescence probes in the classical immunochromatography detection system for sensing carcino-embryonic antigen（CEA）,a kind of glycoprotein produced by intestinal tissue and a broad spectrum of tumor marker for cancer diagnosis.And we designed a homemade strips fluorescence reader for detection of fluorescence intensity of QDs on the QD-IFTS.Under the optimized reaction conditions,chromatographic time of the newly QD-IFTS was only25 min,sample volume of the newly QD-IFTS was only 40 m L and the LOD of the newly QD-IFTS was 0.72 ng/m L.In addition,the efficiency and robustness of the newly QD-IFTS were confirmed by successfully application in 300 clinical serum samples,and the results revealed great potential in clinical POCT of other biomarkers.

AuNP aggregation-induced quantitative colorimetric aptasensing of sulfadimethoxine with a smartphone

A gold nanoparticle(AuNP)aggregation-induced colorimetric aptasensing method for quantitative detection of sulfadimethoxine(SDM)with a smartphone was developed.AuNPs were complexed with aptamers which protected Au NPs from aggregating in high-concentration salt solutions.In the presence of SDM,SDM bound with the aptamer on the surface of Au NPs with higher affinity,which competitively desorbed the aptamer from the AuNP surface and resulted in AuNPs aggregation,accompanied with a color change from red to purple-blue.The R,G and B values of images taken by a smartphone camera were analyzed with an app on the smartphone,and were utilized for quantitative analysis of SDM.Under the optimized conditions,the colorimetric aptasensing method using a smartphone showed high sensitivity for SDM,with the limit of detection of 0.023 ppm,lower than the allowed maximum SDM residue limit.This study provides a simple,fast,and easy to read method for on-site quantitative biochemical and cellular analysis.