Application of nanofiber-packed SPE for determination of salivary-free cortisol using fluorescence precolumn derivatization and HPLC detection.

Salivary cortisol has emerged as an easy-to-collect biologic marker of stress in many researches. In this study, we present a method for the determination of salivary-free cortisol using HPLC method with fluorescence precolumn derivatization, which is based on a novel extraction from the strongly acidic medium (fluorescent derivatives of cortisol in sulfuric acid medium) by electrospun polystyrene nanofibers packed SPE. For high-throughput sample extraction, an array pretreatment device based on nanofibers packed SPE micro-column was designed. The LOD of cortisol was 0.01 microg/L (S/N=3). The RSDs (n=6) for all analytes were below 8.0%, and the recoveries were 110, 102.4, and 99.4% (n=3) for saliva spiked with 0.1, 10, and 20 microg/L of cortisol, respectively. The proposed method was then successfully applied in the determination of free cortisol in human saliva. The salivary cortisol concentrations in the real samples ranged from 0.22 to 7.45 microg/L. The nanofiber-packed SPE overcame the low extraction recovery and bad clean-up effect of the conventional methods, and increased the sensitivity and selectivity of the method.

[Genomic analysis of DNA-protein interaction by chromatin immunoprecipitation].

Chromatin immunoprecipitation (ChIP) is an effective technique to analyze the interactions of DNA binding proteins with the genome in vivo. ChIP coupled with high density microarray (ChIP-chip) or high-throughput sequencing (ChIP-Seq) has generated large amount of data and expected to allow the development of a network describing the cellular transcriptional regulation. Here, we reviewed the ChIP, ChIP-chip, and ChIP-Seq techniques as well as their perspectives. Focus is given to data analysis of ChIP-Seq and the applications of ChIP-chip and ChIP-Seq.

[Electrochemical detection of toxin gene in Listeria monocytogenes].

Listeria monocytogenes (LM) is a food-borne pathogen inducing listeriosis, an illness characterized by encephalitis, septicaemia, and meningitis. Listeriolysin O (LLO) is absolutely required for virulence by L. monocytogenes, and is found only in virulent strains of the species. One of the best ways to detect and confirm the pathogen is detection of one of the virulence factors, LLO, produced by the microorganism. This paper focused on the electrical method used to detect the LLO toxin gene in food products and organism without labeling the target DNA. The electrochemical sensor was obtained by immobilizing single-stranded oligonucleotides onto the gold electrode with the mercaptan activated by N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamion)propyl-N'-ethyl carbodiimidehydrochloride (EDC). The hy-bridization reaction that occurred on the electrode surface was evidenced by Cyclic Voltammetry (CV) analysis using [Co(phen)3](ClO4)3 as an indicator. The covalently immobilized single-stranded DNA could selectively hybridize to its complementary DNA in solution to form double-stranded DNA on the gold surface. A significant increase of the peak cur-rent of Cyclic Voltammetry (CV) upon hybridization of immobilized ssDNA with PCR amplification products in the solu-tion was observed. This peak current change was used to monitor the amount of PCR amplification products. Factors deter-mining the sensitivity of the electrochemical assay, such as DNA target concentration and hybridization conditions, were investigated. The coupling of DNA to the electrochemical sensors has the potential of the quantitative evaluation of gene.

[Characterization of reaction kinetics between ligand and receptor on the solid and liquid interface based on optical waveguide lightmode spectroscopy].

The present paper describes the use of optical waveguide lightmode spectroscopy (OWLS) for study of the binding interaction of the vascular endothelial growth factor (VGEF) with VEGF receptor 2 (VEGFR2). VEGF were immobilized in the surface of an 3-amino 3-propyltriethoxy silane (APTES) modified sensor chip. The solutions with different concentration of VEGFR2 were injected to the system to investigate the kinetic character with OWLS on the solid and liquid interface. The receptor binding and dissociation on the interface, quantified by association and dissociation rate coefficients ka and kd, were determined by the OWLS experiments. The k(a) and k(d) is 6.86 x 10(5) L x mol(-1) x s(-1) and 1.15 x 10(-3) s(-1), respectively. The results show that OWLS method could meet the requirement of kinetic determination of ligand--receptor interaction in applications for related fundamental research and pharmaceutical development.

Epidemics of HIV Infection among Heavy Drug Users of Depressants Only, Stimulants Only, and Both Depressants and Stimulants in Mainland China: A Series, Cross-Sectional Studies.

Background: Heavy drug users was a global consensus high-risk population of HIV infection. However, the specific impact of drug on HIV infection has not yet been established. Depressants and stimulants were most widely used drugs in mainland China, and mix use of the two drugs was also serious. We assessed the HIV infection rate and trends in heavy drug users by analyzing data from the National Dynamic Management and Control Database for Drug Users (NDMCDDU). Methods: All heavy drug users with HIV test results in NDMCDDU from 2008 to 2016 were grouped into depressants only group (DOG), stimulants only group (SOG), and both depressants and stimulants group (DSG). We used joinpoint regression to examine trends of HIV infection rates. Multivariable logistic regression was used to examine factors related to HIV infection. Results: A total of 466,033 heavy drug users with 9522 cases of HIV infection were included in this analysis. HIV infection rate was estimated at 2.97% (95% CI 2.91-3.04%) of 265,774 users in DOG, 0.45% (95% CI 0.42-0.49%) of 140,895 users in SOG, and 1.65% (95% CI 1.55-1.76%) of 59,364 users in DSG. In DOG, a U-shaped curve of HIV infection rate decreased from 3.85% in 2008 to 2.19% in 2010 (annual percent change (APC) -12.9, 95% CI -19.3--6.0, p < 0.05), then increased to 4.64% in 2016 (APC 8.3, 95% CI 6.1-10.4, p < 0.05) was observed. However, SOG and DSG showed consistent increases from 0.15% in 2008 to 0.54% in 2016 (APC 8.2, 95% CI 4.8-11.8, p < 0.05) and from 0.78% in 2008 to 2.72% in 2016 (APC 13.5, 95% CI 10.7-16.4, p < 0.05), respectively. HIV infection rate of DOG in the southwest region presented a U-shaped trend. All groups showed significant increases in HIV infection in east and central regions. Conclusions: The U-shaped curve for HIV infection rate among DOG users and consistent increases among SOG and DSG users implies drug abuse is still a critical focus of HIV infection in China. It is urgently needed to reassess the effectiveness of current strategies on HIV prevention and control among drug users.

Gel-pad microarrays templated by patterned porous silicon for dual-mode detection of proteins.

A proof-of-concept study demonstrated the feasibility of a novel gel-pad microarray on porous silicon chips, by initiation of an atom transfer radical propagation (ATRP) polymerisation of (polyethylene glycol) methacrylate (PEGMA) with surface Si-H species, stepwise chemical conversions of the gel membrane to an NTA-Ni2+/histidine-tagged protein system, and matrix-assisted laser desorption/ionisation mass spectroscopy (MALDI MS) and fluorescence detections.

Ligand-based neutral ruthenium(II) arene complex: selective anticancer action.

Two new ruthenium(II) arene complexes, 2a (C(24)H(34)B(10)FeRuS(2)) and 2b (C(15)H(26)B(10)O(2)RuS(2)), bearing a carborane unit and other different functional groups were synthesized, and their cytostatic effects on cancerous cells were evaluated. Our observations illustrate that a structural change from a ferrocene unit to a carboxyl group could lead to high selectivity toward cancer cells and facilitate the efficient inhibition of the proliferation of target cells, indicating that the tuning of the overall properties of the ruthenium(II) arene complex by appropriate ligand tagging is critical to creating a selective antineoplastic agent.

[Study on the genotyping of single nucleotide polymorphisms for a large number of samples by three-dimensional polyacrylamide gel-based microarray method].

OBJECTIVE: To genotype single nucleotide polymorphisms (SNPs) in a large number of samples by applying three-dimensional polyacrylamide gel-based microarray. METHODS: The method relies on copolymerization of acrylamide-modified PCR products with acrylamide monomers and acryl-modified slides to prepare gel-based microarray. Then array is hybridized with a pair of specific probes and the two universal dual-color fluorescent detectors labeled with Cy3 or Cy5 respectively (Tag1 and Tag2). Electrophoresis is used in post-hybridization to remove the nonspecifically bound targets and mismatches. Finally, genotyping is based on the images captured through two-color fluorescent scanning. RESULTS: The 3-D gel-immobilization of nucleic acids has a high immobilization yield and good hybridization efficiency. As universal dual-color fluorescent detectors are used, it is not required that specific probes be labeled for all SNPs, therefore the expense for synthesis can be reduced considerably. Electrophoresis in post-hybridization can enhance the capability for discriminating a single nucleotide mismatch from the perfectly matched sequence and improve the signal-to-noise ratio significantly. CONCLUSION: The gel-based microarray is a rapid, simple and high-throughput method for SNPs genotyping and may be very competitive in the efficiency, fidelity and cost for constructing DNA microarrays, which will hold significant promise for applications in human DNA diagnostics.

Correlation of the SNPs of FGFR1, FGF10, FGF18 with nonsyndromic cleft lip with or without palate in Chinese population.

OBJECTIVE: To explore the relationship between the polymorphisms in gene FGFR1, FGF10, FGF18 and the nonsyndromic cleft lip with or without cleft palate (NS CLP) in Chinese population. METHODS: Genomic DNA was isolated from peripheral lymphocytes of 75 patients with NS CLP and their parents and 75 unimpaired healthy children. The polymorphisms in FGFR1 gene rs13317, p.E467K, p.M369I and p.S393S, FGF10 gene rs1448037 and FGF18 gene rs4043716 were detected by applying three-dimensional (3-D) polyacrylamide gel microarray technology. The data were performed using statistical analysis: the genotype frequency and allele frequency between patients with NSCL/P and control subjects were performed. Haplotype relative risk (HRR), family based association test (FBAT), and transmission disequilibrium test (TDT) in nuclear family were performed. RESULTS: There were no polymorphism in FGFR1 gene p. E467K, p. M369I and p.S393S site, the corresponding base was all G. The polymorphisms of rs13317 and rs1448037 were detected and their genotype frequency and allele frequency showed no significant difference between 75 patients with NSCL/P and 75 normal children. TDT, HRR and FBAT were also no significant differences. The genotype frequency of gene FGF18 rs4043716 showed significant difference, but allele frequency were no significant difference. TDT, HRR and FBAT were also no significant difference. CONCLUSION: Our studies suggest an association between gene FGF18 rs4043716 and the NS CLP in Chinese population, and no association among gene FGFR1 rs13317, p. E467K, p. M369I, p. S393S and gene FGF10 rs1448037.

Alternate succession of aggregate-forming cyanobacterial genera correlated with their attached bacteria by co-pathways.

Freshwater lakes are threatened by harmful blooms characterized by Cyanobacterial Aggregates (CAs) that are normally aggregated with extracellular polysaccharides released by cyanobacteria to form a phycosphere. It is possible that mutualistic relationships exist between bacteria and cyanobacteria in these CAs wherein bacterial products supplement cyanobacterial growth, and cyanobacterial exudates, in turn, serve as substrates for bacteria, thus augmenting the stability of each constituent. However, little is known about the exact interaction between cyanobacteria and their attached bacteria in CAs. Therefore, in this study, we collected 26 CA samples from Lake Taihu, a large freshwater lake in China from March of 2015 to February of 2016. We then sequenced both the V4 regions of 16S rRNA genes and full metagenomes, resulting in 610 Mb of 16S rRNA gene data and 198.98 Gb of high-quality metagenomic data. We observed that two cyanobacteria genera (Microcystis and Dolichospermum) alternately dominated CAs along the sampling time and specific bacterial genera attached to different cyanobacteria genera dominated CAs. More specifically, Dolichospermum dominates CAs when water temperature is low and total nitrogen is high, while Microcystis dominates CAs when water temperature is high and total nitrogen is low. Moreover, we found specific bacterial genera attached to different cyanobacteria genera dominated CAs. The cyanobacteria-bacteria related pairs Dolichospermum-Burkholderia and Microcystis-Hyphomonas were detected by ecological networks construction. Bacterial communities in CAs were found to be more correlated with the cyanobacterial community (Mantel's r = 0.76, P = 0.001) than with environmental factors (Mantel's r = 0.27, P = 0.017). A potential codependent nitrogen-cycling pathway between cyanobacteria and their attached bacteria was constructed, indicating their functional link. Overall, these results demonstrated that mutualistic relationships do, indeed, exist between cyanobacteria and bacteria in CAs at both taxonomic and gene levels, providing biological clues potentially leading to the control of blooms by interventional strategies to disrupt bacteria-cyanobacteria relationships and co-pathways.

RISP: a web-based server for prediction of RNA-binding sites in proteins.

Protein-RNA interactions play significant roles in a number of biological activities, such as protein synthesis, regulation of gene expression. Here we propose a hybrid RISP (RNA-interaction site prediction) method, using support vector machine (SVM) in conjunction with evolutionary information of amino acid sequences in terms of their position-specific scoring matrices (PSSMs) for prediction of RNA-binding sites. The results show that our RISP method has 72.2% net prediction (NP) (61.0% sensitivity and 83.3% specificity). When compared with previous studies, this novel method appears more accurate and better generalization abilities. RISP is freely available at http://grc.seu.edu.cn/RISP. Given a protein sequence, RISP decides whether residue in the protein is RNA-binding or not (optimal prediction), and gives the confidence value, 'high specificity' prediction and 'high sensitivity' prediction.

Surface functionalized exosomes as targeted drug delivery vehicles for cerebral ischemia therapy.

The safe and effective delivery of drugs is a major obstacle in the treatment of ischemic stroke. Exosomes hold great promise as an endogenous drug delivery nanosystem for the treatment of cerebral ischemia given their unique properties, including low immunogenicity, innate stability, high delivery efficiency, and ability to cross the blood-brain barrier (BBB). However, exosome insufficient targeting capability limits their clinical applications. In this study, the c(RGDyK) peptide has been conjugated to the exosome surface by an easy, rapid, and bio-orthogonal chemistry. In the transient middle cerebral artery occlusion (MCAO) mice model, The engineered c(RGDyK)-conjugated exosomes (cRGD-Exo) target the lesion region of the ischemic brain after intravenous administration. Furthermore, curcumin has been loaded onto the cRGD-Exo, and administration of these exosomes has resulted in a strong suppression of the inflammatory response and cellular apoptosis in the lesion region. The results suggest a targeting delivery vehicle for ischemic brain based on exosomes and provide a strategy for the rapid and large-scale production of functionalized exosomes.

An optimized assay for transcription factor NF-kappaB with dsDNA-coupled microplate.

To develop an EMSA-free assay approach for analyzing the sequence-specific DNA-binding proteins (DBPs), an easy cost-effective dsDNA-coupled plate (dcPlate) was developed in our lab for this purpose. In this paper, the assay conditions of such dcPlate were fully optimized for detecting an important transcription factor, NF-kappaB. The optimized parameters of dcPlate for assay of NF-kappaB were as follows: immobilized DNA probe at the concentration of 25 pmol/100 microL-well, incubation time of 90 min for NF-kappaB binding to dcPlate, primary and secondary antibody concentration of 0.1 microL/100 microL dilution, incubation time of 90 min for primary antibody binding to NF-kappaB, temperature of 25 degrees C for the above process, colorimetric developing time for 30 min. After optimization, the signal was improved three times higher than that from not optimized conditions. The linear colorimetric detection ranges of the purified recombinant NF-kappaB p50 and the cell nuclear extract were from 0.59 to 75 ng/well and 0.313 to 10 microg/well, respectively.

[Quantitative microarray-based DNA methylation analysis of E-cadherin gene promoter in acute leukemia].

OBJECTIVE: To quantitatively detect the methylation of E-cadherin gene 5'-CpG islands in acute leukemia by microarray-based DNA analysis and to briefly discuss the role of microarry for detection of methylation in tumors. METHODS: Bisulfite-modified DNA was used as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. Five sets of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of E-cadherin gene CpG islands in acute leukemia. By drawing a standard curve to assess the levels of changes in methylation detected in the examined samples. RESULTS: Microarray assay was successfully used to quantitatively detect methylation changes of E-cadherin gene in 5 acute leukemia samples. Varying degree of methylation was detected in five regions and the hypermethylation region was the same. The result was validated by gene sequencing. CONCLUSION: Microarray assay may be applied as an useful tool for mapping methylation changes in multiple CpG loci and for leukemia research. It is more time-saving and labor-saving than gene sequencing and can be used to quantitatively detect changes in methylation with high throughput.

Resetting blood pressure by a closed-loop implanted chip system in normotensive rats.

A closed-loop implanted chip system was designed to control blood pressure without using drugs. The chip system instantaneously reset blood pressure by stimulating the left aortic depressor nerve according to the feedback signals of arterial blood pressure. The relationship between pressure signals and frequency of stimulation was identified in vitro and in vivo, and the efficiency of the chip system was evaluated in normal anesthetized Wistar rats. To determine whether the depressor effect of the chip was primarily independent on the bradycardia induced by the resetting, the effects of methyl atropine (1.5 g/kg, iv.) and bilateral vagotomy on depressor effect induced by the chip system were determined, respectively. The results indicated that the chip system worked well. The frequency of stimulus linearly increased following the elevation of pressure from 70 to 160 mm Hg. The frequency of the stimulus reached its maximum (100 Hz) when pressure exceeded 160 mm Hg, and the stimulation stopped when MAP was below 70 mm Hg. There were significant decreases in mean arterial pressure (MAP, -20.0+/-4.4 mm Hg) and heart rate (HR, -43.0+/-10.5 bpm) during the resetting in rats. After resetting, both MAP and HR recovered in a minute without any significant rebound. Pretreatment with either methyl atropine or bilateral vagotomy abolished the bradycardia effect but produced no significant effect on hypotension. The results demonstrated that the chip system successfully reset blood pressure in rats, and that the hypotension induced by the chip system was primarily independent on the bradycardia effect.

Notification Rate of Tuberculosis among Migrants in China 2005-2014: A Systematic Review and Meta-analysis.

BACKGROUND: Migrations have been reported to be associated with the high risk of tuberculosis (TB), but there is no systematic analysis of the available data for TB among migrant in China. The aim of this study was to examine the notification rate of active and sputum smear-positive TB by a systematic review and meta-analysis. METHODS: A systematic review and meta-analysis were performed to examine the notification rate of active and sputum smear-positive TB among migrants in China. Two reviewers searched the cross-sectional studies published in PubMed, EMBASE, SciFinder, and Web of Science in English and in CNKI and Wanfang databases in Chinese. Pooled estimates of notification rate of TB among migrants were calculated using a random effects model. Meta-regression analysis and subgroup analysis stratified by year, region were also performed. RESULTS: Seventy eligible studies met the inclusion criteria for the final analysis. The overall notification rate of active TB and sputum smear-positive cases among migrants were 53.12 (95% confidence interval [CI]: 47.32-59.63) and 24.53 (95% CI: 22.01-27.34) per 100,000 populations, respectively. The notification rate of active TB significantly increased from 50.95 (95% CI: 41.11-63.14) per 100,000 populations in 2005 to 84.62 (95% CI: 78.00-91.80) per 100,000 populations in 2014 while that of smear-positive TB was constant during the study time (P = 0.79). The geographic difference was identified both for active and sputum smear-positive TB, with the higher notification rates mainly distributing along the eastern coastal areas. CONCLUSIONS: The pooled estimate of active TB and sputum smear-positive TB among migrants was lower than the national notification rate among general population, but the gap between our data and national notification rate among general population is narrowed down during 2005-2014.

Complete genome sequence and genomic characterization of Microcystis panniformis FACHB 1757 by third-generation sequencing.

The cyanobacterial genus Microcystis is well known as the main group that forms harmful blooms in water. A strain of Microcystis, M. panniformis FACHB1757, was isolated from Meiliang Bay of Lake Taihu in August 2011. The whole genome was sequenced using PacBio RS II sequencer with 48-fold coverage. The complete genome sequence with no gaps contained a 5,686,839 bp chromosome and a 38,683 bp plasmid, which coded for 6,519 and 49 proteins, respectively. Comparison with strains of M. aeruginosa and some other water bloom-forming cyanobacterial species revealed large-scale structure rearrangement and length variation at the genome level along with 36 genomic islands annotated genome-wide, which demonstrates high plasticity of the M. panniformis FACHB1757 genome and reveals that Microcystis has a flexible genome evolution.

[Detection of multidrug-resistance proteins with protein array chips].

OBJECTIVE: To evaluate the use of protein array chips in detection of multidrug-resistance proteins. METHODS: Human erythroleukemic cell line K562 and its doxorubicin-resistant counterpart K562/A02 were used in the study. Monoclonal antibodies against P-glycoprotein (P-gP), multidrug resistance-associated protein (MRP1) and breast cancer resistance protein (BCRP) were immobilized onto agarose film-coated glass. The antibody-cell binding was assessed by capturing K562 and K562/A02 cells. The protein array was observed under a microscope and the image was captured with a CCD camera. The expression levels of the three proteins were also measured by flow cytometry (FCM). RESULTS: The expression of P-gP and BCRP in K562 was very low. However, MRP1 expression was high. P-gP and MRP1 were highly expressed in K562/A02, while the expression of BCRP was low. FCM results showed that the expression rate of P-gP, MRP1 and BCRP in K562 cells was 5.98% +/- 2.19%, 95.80% +/- 3.98%, 1.03% +/- 0.45%, respectively, while that in K562/A02 cells was 92.67% +/- 1.80%, 97.18% +/- 1.02%, 3.98% +/- 0.37%, respectively. The results of protein array method are consistent with those of FCM (P > 0.05). CONCLUSION: It is feasible to develop a new protein array technique and to provide a novel method for multi-drug resistant cell detection, with a high throughput, high specificity, simple procedure and low cost.

B-cell lymphoma 2 rs17757541 C>G polymorphism was associated with an increased risk of coronary artery disease in a Chinese population.

The goal of our study was to evaluate the genetic effects of sixteen single nucleotide polymorphisms (SNPs) in apoptosis-related genes on the development of coronary artery disease (CAD) through a case-control study. A total of 1979 individuals, including 826 CAD cases (aged 67.27 ± 10.26 years) and 1153 non-CAD controls (aged 59.13 ± 10.51 years), were enrolled into the study. The genotypes were determined using a custom-by-design 48-Plex SNPscanTM Kit. The results showed that the BCL2 rs17757541 C>G polymorphism was associated with increased risk of CAD in homozygote comparison and recessive genetic model. However, no association between the other fifteen SNPs and CAD risk was observed. Stratified analyses indicated a significantly increased risk of CAD associated with the BCL2 rs17757541 C>G polymorphism among males and younger patients. Therefore, the results indicated that there is a close correlation between the BCL2 rs17757541 C>G polymorphism and CAD, which suggests that this SNP site should be further studied as a potential biomarker for CAD.

[Strategy of selecting 16S rRNA hypervariable regions for metagenome-phylogenetic marker genes based analysis].

The advent of next generation sequencing technology enables parallel analysis of the whole microbial community from multiple samples. Particularly, sequencing 16S rRNA hypervariable tags has become the most efficient and cost-effective method for assessing microbial diversity. Due to its short read length of the 2nd-generation sequencing methods that cannot cover the full 16S rRNA genomic region, specific hypervariable regions or V-regions must be selected to act as the proxy. Over the past decade, selection of V-regions has not been consistent in assessing microbial diversity. Here we evaluated the current strategies of selecting 16S rRNA hypervariable regions for surveying microbial diversity. The environmental condition was considered as one of the important factors for selection of 16S rRNA hypervariable regions. We suggested that a pilot study to test different V-regions is required in bacterial diversity studies based on 16S rRNA genes.