Investigation of the interactions between methylene blue and intramolecular G-quadruplexes: an explicit distinction in electrochemical behavior.

G-quadruplex sequences exist in eukaryotic organisms and prokaryotes, and the investigation of the interactions between G-quadruplexes and small molecule ligands is important for gene therapy, biosensor fabrication, fluorescence imaging and so on. Here, we investigated the behaviour of methylene blue (MB), an electroactive molecule, in the presence of different intramolecular G-quadruplexes by an electrochemical method using a miniaturized electrochemical device based on its intrinsic electrochemical properties. Although the effects of MB on different intramolecular G-quadruplex structures are not obvious by circular dichroism spectroscopy, distinct differences in the binding affinities of MB with different intramolecular G-quadruplexes were quickly and easily observed by an electrochemical technique. At the same time, for the human telomerase G-rich sequence (HT), the diffusion current of MB changed sensitively under different ionic conditions due to the formation of different conformations of HT, which indicated that our electrochemical method has the potential to study the influence of metal ions on the conformations of the G-quadruplexes with simplicity, rapid response and low cost. From all these, a new stacking mechanism and rule were obtained, which were also validated by docking studies and isothermal titration calorimetry (ITC).

Portable, Easy-to-Operate, and Antifouling Microcapsule Array Chips Fabricated by 3D Ice Printing for Visual Target Detection.

Herein, we proposed a portable, easy-to-operate, and antifouling microcapsule array chip for target detection. This prepackaged chip was fabricated by innovative and cost-effective 3D ice printing integrating with photopolymerization sealing which could eliminate complicated preparation of wet chemistry and effectively resist outside contaminants. Only a small volume of sample (2 µL for each microcapsule) was consumed to fulfill the assay. All the reagents required for the analysis were stored in ice form within the microcapsule before use, which guaranteed the long-term stability of microcapsule array chips. Nitrite and glucose were chosen as models for proof of concept to achieve an instant quantitative detection by naked eyes without the need of external sophisticated instruments. The simplicity, low cost, and small sample consumption endowed ice-printing microcapsule array chips with potential commercial value in the fields of on-site environmental monitoring, medical diagnostics, and rapid high-throughput point-of-care quantitative assay.

A smart tailor-made G-clip reporter for sensitive detection of G-triplet-containing sequences.

Taking advantage of the intrinsic characteristics of G-triplet-containing sequences, a pioneering tailor-made clip-like reporter containing three-fourths of a G-quadruplex is established. The reporter can clip the G triplet in the target sequence through a recognition process to form a complete G-quadruplex structure.

Methylene blue as a G-quadruplex binding probe for label-free homogeneous electrochemical biosensing.

Herein, G-quadruplex sequence was found to significantly decrease the diffusion current of methylene blue (MB) in homogeneous solution for the first time. Electrochemical methods combined with circular dichroism spectroscopy and UV-vis spectroscopy were utilized to systematically explore the interaction between MB and an artificial G-quadruplex sequence, EAD2. The interaction of MB and EAD2 (the binding constant, K ≈ 1.3 × 10(6) M(-1)) was stronger than that of MB and double-stranded DNA (dsDNA) (K ≈ 2.2 × 10(5) M(-1)), and the binding stoichiometry (n) of EAD2/MB complex was calculated to be 1.0 according to the electrochemical titration curve combined with Scatchard analysis. MB was proved to stabilize the G-quadruplex structure of EAD2 and showed a competitive binding to G-quadruplex in the presence of hemin. EAD2 might mainly interact with MB, a positive ligand of G-quadruplex, through the end-stacking with π-system of the guanine quartet, which was quite different from the binding mechanism of dsDNA with MB by intercalation. A novel signal read-out mode based on the strong affinity between G-quadruplex and MB coupling with aptamer/G-quadruplex hairpin structure was successfully implemented in cocaine detection with high specificity. G-quadruplex/MB complex will function as a promising electrochemical indicator for constructing homogeneous label-free electrochemical biosensors, especially in the field of simple, rapid, and noninvasive biochemical assays.

A G-quadruplex based platform for label-free monitoring of DNA reaction kinetics.

Research on the kinetic characteristics and mechanisms of DNA reactions is crucial for bioengineering and biosensing. A G-quadruplex, which can form a peroxidase-mimicking DNAzyme with hemin, was for the first time used to establish a versatile platform for kinetic investigations on DNA reactions. G-quadruplex sequence EAD2 was incorporated into the corresponding nucleic acid reaction as product. The kinetic curves can be obtained rapidly and simply via the quantification of created DNAzyme. In this paper, the kinetics of isothermal linear strand displacement amplification reactions with different DNA lengths and isothermal exponential amplification reactions were successfully elucidated via the G-quadruplex based monitoring platform. As a safe and accessible alternative to the traditional methods, this robust, label-free, time-saving and high-throughput platform shows great potential for the exploration of more novel DNA reactions or circuits in an ingenious manner.

Novel homogeneous label-free electrochemical aptasensor based on functional DNA hairpin for target detection.

We first developed a label-free and immobilization-free homogeneous electrochemical aptasensor, which combined a smart functional DNA hairpin and a designed miniaturized electrochemical device. Cocaine was chosen as a model target. The anticocaine aptamer and peroxidase-mimicking DNAzyme were integrated into one single-stranded DNA hairpin. Both aptamer and G-quadruplex were elaborately blocked by the stem region. The conformation switching induced by the affinity interaction between aptamer and cocaine released G-quadruplex part and turned on DNAzyme activity. The designed electrochemical device, constructed by a disposable micropipet tip and a reproducible carbon fiber ultramicroelectrode, was applied to the detection of homogeneous DNAzyme catalytic activity at the microliter level. The aptasensor realized the quantification of cocaine ranging from 1 to 500 µM with high specificity. The clever combination of the functional DNA hairpin and the novel device achieved an absolutely label-free electrochemical aptasensor, which showed excellent performance like low cost, easy operation, rapid detection, and high repeatability.

Identification of the VIL2 enhancer in human embryonic kidney cells.

We previously demonstrated that the VIL2 -87/+134 region exhibited promoter activity in some human cells, and a region further upstream of this promoter might contain an enhancer. However, the properties and location of this VIL2 enhancer remain unclear. In this study, we cloned the VIL2 -1541/-706 segment and investigated its transcriptional regulatory properties via luciferase assays in transiently transfected HEK-293 cells (human embryonic kidney cells). The VIL2 -1541/-706 was found to exhibit promoter activity. Furthermore, when this segment was located upstream of the VIL2 or SV40 (simian virus 40) promoters in the forward orientation, the expression levels of luciferase were dramatically enhanced. However, this transcriptional enhancement disappeared when this segment was located upstream of the promoter in the reverse orientation or downstream of the reporter gene in the forward or reverse orientation. In deletion experiments, we found several potential regulatory regions within the VIL2 -1541/-706. When these regions were separately located upstream of the VIL2 or SV40 promoters, only the -1297/-1186 considerably enhanced the activity of these promoters. Although the other regulatory regions exhibited significant transcriptional regulation in deletion experiments, they weakly enhanced VIL2 promoter activity and/or did not regulate SV40 promoter activity. These results suggest that the DNA sequence upstream of the VIL2 promoter functions as an enhancer in a position- and orientation-dependent manner, and the VIL2 -1297/-1186, which acts as a key enhancer, probably regulates VIL2 transcription in combination with other potential regulatory regions located upstream of the VIL2 promoter.

Development of neonatal mouse and fetal human testicular tissue as ectopic grafts in immunodeficient mice.

AIM: To investigate the stepwise development and germ cell gene expression in allografted neonatal mouse testes and the differentiation of immature human testicular cells in xenografted human testes. METHODS: Immunodeficient nude mice were used as hosts for allografting of neonatal mouse testes and xenografting of human fetal testicular tissues. Stepwise development and stage-specific gene expression of germ cells in allografts were systematically evaluated and parallel compared with those in intact mice by periodically monitoring the graft status with measurement of graft weight, histological analysis and determination of five stage-specific genes. Human testicular tissues from 20 and 26 weeks fetuses were used for the xenografting study. Histological analysis of xenografts was performed 116 and 135 d after the grafting procedure. RESULTS: In the allografting study, progressive increase in tissue volume and weight as well as in tubule diameter in grafts was observed; the appearance time of various germ cells in seminiferous tubules, including spermatogonia, spermatocytes, round and elongate spermatids and sperm, was comparable with that in intact donors; the initiation of gene transcription in grafts showed a similar trend as in normal mice. Graft weight ceased to increase after 7-8 weeks and degradation of grafts was observed after 5 weeks with progressive damage to seminiferous epithelium. In the xenografting study using immature human testicular tissues, graft survival and development was indicated by increasing graft weight, Sertoli cells differentiation into advanced stage, germ cells migration and location to the basal lamina and formation of a niche-like structure. CONCLUSION: The developmental course and gene expression pattern of germ cells in allografts were similar to those in intact mice. The best time point for retrieval of mouse sperm from grafts was 5-7 weeks after grafting procedure. An accelerated development of immature human testicular cells could be achieved by ectopic xenografting of human testes.

[Research progress in ectopic grafting of testicular tissues].

Since Nature published the first report in 2002 on using immunodeficient mice as recipients and allogeneous or heterogeneous testes as donor tissues to study the ectopic development of spermatogenic cells, the technique has been widely applied in various species (including human). In comparison with other in vitro maturation methods for male germ cells, testicular allografting or xenografting technique has such advantages as similar environment for the development of germ cells in physiological conditions, and better reproducibility. Up to now, sperm has been successfully produced by this technique from the testicular tisues of the immature mouse, hamster, cat, rabbit, pig, goat, bovine and rhesus monkey, and their offspring have even been generated by ICSI technique using the mouse and rabbit sperm derived from testis grafts. This article comprehensively reviews the development of the technique by discussing the influencing factors on the germ cell development in grafts including the variety and age of donors, the sex, integrity and immunity of recipients, the graft location and grafting time. And the applications of the technique and the existing problems are discussed as well.

[Conversion of human umbilical cord blood-derived cells into hepatocyte-like cells in a culture system mimicking hepatic injury].

OBJECTIVE: To explore the effect of the microenvironment induced by damaged mouse hepatic cells on the conversion of human umbilical cord blood-derived cells into hepatocyte-like cells. METHODS: A hepatic injury-like microenvironment was mimicked using carbon tetrachloride damaged mouse hepatic cells, where mononuclear cells (MNC) from human umbilical cord blood were cultured in a compartment separated by trans-well membrane. Histochemical staining, reversed transcription-polymerase chain reaction (RT-PCR) and gene sequencing were performed for the information on the conversion of human umbilical cord blood MNC. RESULTS: A number of PAS positive stained cells in MNC co-cultured with damaged mouse hepatic cells were observed after 72 h. Cells expressing mature hepatocyte markers, human albumin (hALB) and human GATA-4 (hGATA-4) mRNA were detected by RT-PCR, which was further confirmed with sequencing. Relevant control groups, MNC co-cultured with normal mouse hepatic cells and MNC cultured alone remained negative. CONCLUSION: The culture system using damaged mouse hepatic cells as stimulator could be a potential in vitro system for the conversion of human umbilical cord blood-derived cells into hepatocyte-like cells.

Effect of Modified Roux-en-Y Gastric Bypass Surgery on GLP-1, GIP in Patients with Type 2 Diabetes Mellitus.

The type 2 diabetes mellitus (T2DM) is one of the most serious diseases that threaten public health. Modified gastric bypass surgery has been applied to the treatment of T2DM patients in the 1990s, but the therapeutic mechanism to this function is still unclear. The aim of this study was to further clarify the effect and the mechanism of modified gastric bypass surgery on glucose metabolism in patients with T2DM. In the study, the incretin indexes and blood glucose indexes were analyzed before surgery and 1 week and 1, 3, and 6 months after surgery. The results suggested that modified Roux-en-Y gastric bypass can promote GLP-1 secretion in patients with T2DM, while reducing the secretion of GIP. Thus it could effectively control blood glucose of patients with T2DM.

[Establishment of animal model for in vitro spermatogenesis by heterotopic grafting of neonatal mouse testis].

OBJECTIVE: To establish an in vitro model for the development of mouse spermatogenic cells into sperm by using the immunodefective mouse as the incubator. METHODS: Tissue grafting was performed using testis from 1-2 days old Kun-ming mice as donor tissue and immunodefective mice as recipients; the expression of TESK1 mRNA in grafts was determined by RT-PCR and the spermatogenesis further observed with histological analysis of grafts. RESULTS: Molecular biological and histological analyses showed that grafts post-grafting not only expressed TESK1 mRNA as in normal mouse testis, but also exhibited similarities in the structure of seminiferous tubules and component of spermatogenic cells, including sperms. CONCLUSION: Spermatogenic cells heterotopically grafted in vitro could continuously grow and complete spermatogenesis and finally develop into sperm.

Comparison of different gastric bypass procedures in gastric carcinoma patients with type 2 diabetes mellitus.

AIM: To determine the effect of different Roux-en-Y gastric bypass procedures in gastric carcinoma patients with type 2 diabetes mellitus. METHODS: A retrospective analysis of the clinical data of 54 patients with gastric cancer and type 2 diabetes mellitus treated in the Department of General Surgery from January 2006 to June 2013 was conducted. The patients underwent gastrectomy using different Roux-en-Y gastric bypass procedures (traditional, n = 26; modified, n = 28). Fasting plasma glucose (FPG), two hour postprandial blood glucose (2 h PBG) and hemoglobin A1c (HbA1c) were analyzed before surgery (0 mo) and 1, 3 and 6 mo after surgery. RESULTS: FPG and 2 h PBG levels were significantly decreased 1 mo after surgery in the traditional Roux-en-Y gastric bypass group (FPG 7.5 ± 1.3 vs 10.7 ± 1.2, P < 0.05) (2 h PBG 10.2 ± 1.8 vs 13.8 ± 3.2, P < 0.05). FPG and 2 h PBG levels were significantly decreased after surgery in the modified Roux-en-Y gastric bypass group (FPG 6.9 ± 1.2 vs 10.5 ± 1.1, 6.5 ± 1.3 vs 10.5 ± 1.1, 6.4 ± 1.2 vs 10.5 ± 1.1, P < 0.05) (2 h PBG 9.9 ± 2.2 vs 14.1 ± 2.9, 9.2 ± 2.4 vs 14.1 ± 2.9, 8.9 ± 2.6 vs 14.1 ± 2.9, P < 0.05). Compared with the levels before surgery, HbA1c levels were significantly decreased 3 and 6 mo after surgery (7.2 ± 1.1 vs 10.5 ± 1.1, 5.5 ± 1.1 vs 10.5 ± 1.1, P < 0.05). Significant differences between the two groups regarding FPG, 2 h PBG and HbA1c concentration were observed 3 and 6 mo after surgery (FPG 10.1 ± 1.5 vs 6.5 ± 1.3, 10.3 ± 1.4 vs 6.4 ± 1.2, P < 0.05) (2 h PBG 13.1 ± 2.8 vs 9.2 ± 2.4, 13.6 ± 3.1 vs 8.9 ± 2.6, P < 0.05) (HbA1c 10.1 ± 1.4 vs 7.2 ± 1.1, 10.5 ± 1.3 vs 5.5 ± 1.1, P < 0.05). CONCLUSION: Modified Roux-en-Y gastric bypass can improve glucose metabolism in type 2 diabetic patients with gastric cancer.

Reporter-triggered isothermal exponential amplification strategy in ultrasensitive homogeneous label-free electrochemical nucleic acid biosensing.

A simple and novel reporter-triggered isothermal exponential amplification reaction (R-EXPAR) integrated with a miniaturized electrochemical device was developed, which achieved excellent improvement (five orders of magnitude) of sensitivity toward reporter, G-quadruplex. This R-EXPAR strategy has been successfully implemented to construct a homogeneous label-free electrochemical sensor for ultrasensitive DNA detection.

A label-free aptasensor for the sensitive and specific detection of cocaine using supramolecular aptamer fragments/target complex by electrochemical impedance spectroscopy.

A simple and label-free aptasensor for sensitive and specific detection of cocaine was developed by measuring the change in electrochemical impedance spectra (EIS), based on the formation of a supramolecular aptamer fragments/substrate complex. An anticocaine aptamer was divided into two fragments, Cx and Cy. Three different sensing interfaces, called Au/Cx5S/MCE, Au/Cy3S/MCE and Au/Cy5S/MCE, were fabricated by immobilizing Cx or Cy on a gold electrode through modifying their 5' or 3' end with a thiolated group followed by the treatment with mercaptoethanol (MCE). The formation of the corresponding supramolecular aptamer fragments/cocaine complex was investigated via monitoring electrochemical impedance spectra in the presence of [Fe(CN)(6)](3-/4-). The interfacial electron transfer resistance (R(et)) was found to depend strongly on the cocaine concentration. Since the supramolecular aptamer fragments/cocaine complex was formed on the electrode surface, the sensing interface strongly affected the sensitivity of the aptasensor. Au/Cx5S/MCE was shown to have good sensitivity within a cocaine detection range of 0.1-20 µM. Moreover, MCE was shown to improve the sensitivity of the aptasensor greatly. Even without the help of amplification or labeling, cocaine concentrations as low as 100 nM could be easily detected by the impedimetric aptasensor developed. The specificity and regeneration of the cocaine aptasensor were also investigated and satisfactory results were obtained. The developed aptasensor was successfully applied to detect the cocaine in biological fluids.

An electrochemical aptasensor based on enzyme linked aptamer assay.

An aptamer is an artificial functional oligonucleic acid, which can interact with its target molecule with high affinity and specificity. Enzyme linked aptamer assay (ELAA) is developed to detect cocaine using aptamer fragment/cocaine configuration based on the affinity interaction between aptamer fragments with cocaine. The aptasensor was constructed by cleaving anticocaine aptamer into two fragments: one was assembled on a gold electrode surface, while the other was modified with biotin at 3'-end, which could be further labelled with streptavidin-horseradish peroxidase (SA-HRP). Upon binding with cocaine, the HRP-labelled aptamer fragment/cocaine complex formed on the electrode would increase the reduction current of hydroquinone (HQ) in the presence of H(2)O(2). The sensitivity and the specificity of the proposed electrochemical aptasensor were investigated by differential pulse voltammetry (DPV). The results indicated that the DPV signal change could be used to sensitively detect cocaine with the dynamic range from 0.1 µM to 50 µM and the detection limit down to 20 nM (S/N=3). The proposed aptasensor has the advantages of high sensitivity and low background current. Furthermore, a new configuration for ELAA requiring only a single aptamer sequence is constructed, which can be generalized for detecting different kinds of targets by cleaving the aptamers into two suitable segments.

Global transcriptional profiling in porcine mammary glands from late pregnancy to peak lactation.

Sow milk yield and quality is crucial for the survival and growth of piglets. To understand the molecular mechanisms of lactogenesis and lactation, mammary tissue samples were taken from six sows at -17(±2), 1 and 17(±2) days relative to parturition. Mammary tissues from two sows in the same stage were used to extract RNA, which were subsequently pooled in equal amounts. Nine pooled samples were hybridized to porcine Affymetrix GeneChips. Totally 1,524 genes were detected as significantly differentially expressed over the time course tested (p<0.01, q<0.01, fold change≥2 or ≤-2), including 709 upregulated and 575 downregulated genes identified at peak lactation compared to late pregnancy. Gene ontology analysis revealed that most of the upregulated genes were involved in transport, biosynthetic processes, and homeostasis, whereas most of the downregulated genes were involved in intracellular signaling cascades, cell cycle, and DNA replication. Furthermore, we identified 64 differentially expressed genes of the solute carrier families. Taken together, our microarray analysis provides insights into previously uncharacterized changes in transcriptome between late pregnancy and peak lactation in the porcine mammary gland. The solute carrier genes and other differentially expressed genes identified in this study will guide further characterization of their function to enhance milk yield and piglet growth.

Transplantation of microencapsulated umbilical-cord-blood-derived hepatic-like cells for treatment of hepatic failure.

AIM: To investigate intraperitoneal transplantation of microencapsulated hepatic-like cells from human umbilical cord blood for treatment of hepatic failure in rats. METHODS: CD34+ cells in umbilical cord blood cells were isolated by magnetic cell sorting. In the in vitro experiment, sorted CD34+ cells were amplified and induced into hepatic-like cells by culturing with a combination of fibroblast growth factor 4 and hepatocyte growth factor. Cultures without growth factor addition served as controls. mRNA and protein levels for hepatic-like cells were analyzed by reverse transcription-polymerase chain reaction, immunohistochemistry and immunofluorescence. In the in vivo experiment, the hepatic-like cells were encapsulated and transplanted into the abdominal cavity of acute hepatic failure (AHF) rats at 48 h after D-galactosamine induction of acute hepatic failure. Transplantation with PBS and unencapsulated hepatic-like cells served as controls. The mortality rate, hepatic pathological changes and serum biochemical indexes were determined. The morphology and structure of microcapsules in the greater omentum were observed. RESULTS: Human albumin, alpha-fetoprotein and GATA-4 mRNA and albumin protein positive cells were found among cultured cells after 16 d. Albumin level in culture medium was significantly increased after culturing with growth factors in comparison with culturing without growth factor addition (P < 0.01). Compared with the unencapsulated group, the mortality rate of the encapsulated hepatic-like cell-transplanted group was significantly lower (P < 0.05). Serum biochemical parameters, alanine aminotransferase, aspartate aminotransferase and total bilirubin in the encapsulated group were significantly improvement compared with the PBS control group (P < 0.01). Pathological staining further supported these findings. At 1-2 wk post-transplantation, free microcapsules with a round clear structure and a smooth surface were observed in peritoneal lavage fluid, surviving cells inside microcapsules were found by trypan blue staining, but some fibrous tissue around microcapsules was also detected in the greater omentum of encapsulated group by hematoxylin and eosin staining. CONCLUSION: Transplantation of microencapsulated hepatic-like cells derived from umbilical cord blood cells could preliminarily alleviate the symptoms of AHF rats.