An enzymatic paper-based biosensor for ultrasensitive detection of DNA.

Point-of-care Nucleic acid testing (POCNAT) has become an attractive technique for DNA identification in resource-limited settings, offering a rapid system for urgent clinical applications. In this study, a chemiluminescence-based lateral flow biosensor (CL-LFB) was developed for the quantitative analysis of DNA, without labeling and amplification. The developed biosensor employs a two-step hybridization, a primary hybridization of 5'-biotinylated detector probe to the target DNA and a secondary hybridization of the resulting complex with the immobilized capture probe. Quantitative analysis of DNA was provided via HRP-catalyzed reaction with the chemiluminescense substrate followed by imaging with a complementary metal-oxide-semiconductor (CMOS) digital camera. The assay performance was investigated using a synthetic target, 16S rRNA gene (775 bp) and the whole genome derived from Escherichia coli (E.coli). A detection limit of 1.5 pM for the synthetic target and 0.4 ng/ml for 16S rRNA gene was obtained. In spite of LFBs limitations for the detection of large DNA fragments, the proposed assay provided a low-cost, fast, and sensitive tool for PCR-free diagnosis of small and larger fragments of nucleic acids.

Designing a new biosensor "DNA ELISA" to detect Escherichia coli using genomic DNA and comparison of this method to PCR-ELISA.

In this experiment, DNA-ELISA biosensor was introduced, bearing the ability to detect specific bacteria in about 4 h. This is a more rapid system in comparison to conventional methods, like colony counting method. Moreover, this method does not require any amplification and directly detects genomic DNA of bacteria, giving a lower limit to the sensitivity of 40,000 bacteria. In this study, two specific probes capture (biotin labelled) and detector (dig labelled), were used against special regions of 16s rRNA gene of Escherichia coli ATCC 25922. The capture probe has the ability to trap the target bacterial DNA from a pool of other kinds of bacteria under specific conditions. The detector probe then was used to hybridize to the genome of trapped bacteria. The detection proceeds by adding HRP-anti dig enzyme and its substrate, ABTS to emit light. Light absorbance is measured for verifying the detection.

Voltammetric determination of the Escherichia coli DNA using a screen-printed carbon electrode modified with polyaniline and gold nanoparticles.

The authors describe an electrochemical assay for fast detection of Escherichia coli (E. coli). It is based on a dual signal amplification strategy and the use of a screen-printed carbon electrode (SPCE) whose surface was modified with a polyaniline (PANI) film and gold nanoparticles (AuNPs) via cyclic voltammetry (CV). In the next step, avidin was covalently immobilized on the PANI/AuNP composite on the SPCE surface. Subsequently, the biotinylated DNA capture probe was immobilized onto the PANI/AuNP/avidin-modified SPCE by biotin-avidin interaction. Then, DNA of E.coli, digoxigenin-labeled DNA detector probe and anti-digoxigenin-labeled horseradish peroxidase (HRP) were placed on the electrode. 3,3',5,5'-Tetramethylbenzidine (TMB) and H2O2 solution were added and the CV electrochemical signal was generated at a potential of -0.1 V (vs. Ag/AgCl) and a scan rate 50 mV.s-1. The assay can detect 4 × 106 to 4 CFU of E. coli without DNA amplification. The biosensor is highly specific over other pathogens including Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis, Staphylococcus haemolyticus and Pseudomonas aeruginosa. It can be concluded that this genosensor has an excellent potential for rapid and accurate diagnosis of E.coli inflicted infections. Graphical Abstract Schematic of an electrochemical E. coli genosensor based on sandwich assay on a polyaniline/gold nanoparticle-modified screen printed carbon electrode (SPCE). The biosensor can detect 4 × 106 to 4 CFU of E. coli without DNA amplification.

Detection of Cryptosporidium spp. in free ranging animals of Tehran, Iran.

Cryptosporidium is a world widely distributed parasite which comparatively has a high prevalence in developing countries. The zoonotic potential of some Cryptosporidium species has made the cryptosporidiosis a significant concern to physicians and veterinarians. The occurrence and zoonotic potential of Cryptosporidium species in probable reservoir hosts for man infections was determined by examining faeces of symptomatic and asymptomatic animals. The aim of this study is to screen the presence of Cryptosporidium in fecal sample of free ranging animals in Tehran using Ziehl-Neelsen staining method. The findings indicate that Cryptosporidium are present in 9/50 (18 %) stray cat (Felis catus), 12/50 (24 %) hooded crows (Corvus cornix), 23/180 (12.7 %) rat (Rattus norvegicus and R. rattus) and 1/40 (2.5 %) pigeons (Columba livia). This investigation confirms the potential role of rats, cats, crows and pigeons for zoonotic transmission of human cryptosporidiosis and they must be considered as reservoir hosts which can endanger public health.

Retroviral transduction of fluonanobody and the variable domain of camelid heavy-chain antibodies to chicken embryonic cells.

BACKGROUND: Single domain antibodies from camel heavy chain antibodies (VHH or nanobody), are advantages due to higher solubility, stability, high homology with human antibody, lower immunogenicity and low molecular weight. These criteria make them candidates for production of engineered antibody fragments particularly in transgenic animals. OBJECTIVE: To study the development of transgenic chicken using a recombinant retrovirus containing fluonanobody. METHODS: The retrovirus constructs containing nanobody genes along with secretory signals and GFP gene were established and packed. The virus particle containing the obtained fusion gene was injected into the eggs in stage X. Molecular detection and protein analysis was done in the G0 chickens. RESULTS: The rate of hatched chicken after gene manipulation was estimated to be about 33%. Real-Time PCR assay showed that the nanobody along with GFP gene were integrated in cells of 1.2% of chickens. CONCLUSION: We conclude that although the rate of gene transfer by recombinant viruses in chickens is low, it would be possible to transfect the target camel immunoglobulin gene into chicken genome.

Induction of differentiation by down-regulation of Nanog and Rex-1 in cord blood derived unrestricted somatic stem cells.

Stem cells with high self-renewal and tissue regeneration potentials are the core components of regenerative medicine. Adult stem cells with many available sources, high repairing ability, and also possessing no ethical issues are popular candidates in the clinical field. In this study we looked upon the effects of two transcription factors Nanog and Rex-1 in self-renewal and differentiation abilities of a subpopulation of cord blood stem cells known as unrestricted somatic stem cells (USSCs). USSCs were expanded and transfected in vitro with siRNAs targeting either Nanog, Rex-1, and in combination. Gene suppressions were achieved at both transcript and proteome level. Differentiations were evaluated by specific Real time PCR and differentiating staining. Nanog knock down revealed a significant increase in osteogenic markers, Osteocalcin and Osteopontin expression as well as a positive Alizarin Red staining, which proposes Osteogenesis. This treatment also became positive for Oil Red staining, implying adipogenic differentiation as well. In contrast, Rex-1 knock down showed an increase in MAP II and Nestin expression, which is a hall mark of neural differentiation. Surprisingly, treatment with both siRNAs did not express any changes in any of the assessed markers. Therefore, our results indicated a bilateral mesenchymal differentiation for Nanog and a neural lineage fate for Rex-1 suppression. Considering that both transcription factors are core activators of self-renewal and also are orchestrating with other factors, our results imply a positive feedback in response to changes in the regulatory network of self-renewal.

Production of Pentameric Cholera Toxin B Subunit in Escherichia coli.

Cholera toxin B subunit (CTB) has been extensively studied as an immunogen, adjuvant, and inducer of oral tolerance in many investigations. Production of CTB has been carried out in the bacterial, plant, insect and yeast expression systems. In this study the expression of the CTB containing a 6XHis-tagged was performed by Escherichia coli (E.coli) M15. The yield of purified pentameric recombinant CTB was about 1 mg/l. Western blot analysis demonstrated that the recombinant CTB was antigenically active. In addition, GM1-ganglioside ELISA showed that recombinant CTB binds to GM1-gangelioside receptor, confirming disulfide bond formation and proper folding of the recombinant protein in E.coli. Overall, in regard to the vast applications of CTB in medicine, this bacterial expression system will be a fast, cost-effective and simple system for production of pentameric CTB and CTB conjugated proteins.

Tropisetron upregulates cannabinoid CB1 receptors in cerebellar granule cells: possible involvement of calcineurin.

Tropisetron, a selective 5-HT(3) receptor antagonist, is widely used to counteract chemotherapy-induced emesis. Some investigations describe disparate effects including immunomodulatory properties for tropisetron which may be mediated through immunophilin-calcineurin pathway. Calcineurin, a phosphatase involved in immune system signaling, modulates expression of several genes, such as Cannabinoid type one (CB(1)) receptors. On the quest for its underlying mechanisms of action, this study aimed to investigate the effect of tropisetron on calcineurin activity and CB(1) receptor expression and function in cerebellar granule neurons (CGNs). The rat pup CGNs were used as highly calcineurin-rich and devoid of 5-HT(3) receptor neuronal cells. Calcineurin activity was assessed in CGNs treated with tropisetron or the congener granisetron at 1nM-10µM concentrations. Moreover, cannabinoid CB(1) receptor expression at mRNA and protein levels were investigated by real time PCR and western blotting, respectively and its functionality studied by measuring the secondary messenger cAMP in CGNs receiving tropisetron or granisetron. Results indicate that tropisetron, but not granisetron, significantly inhibits the phosphatase activity of calcineurin, over-expresses the CB(1) receptors at both transcriptional and protein levels, and reduces cAMP content. Our investigation shows that tropisetron targets calcineurin in a receptor-independent fashion. Tropisetron-induced CB(1) receptor up-regulation might underlie many pharmacological aspects of tropisetron unrelated to anti-emesis.

Functional Concentrations of BMP4 on Differentiation of Mouse Embryonic Stem Cells to Primordial Germ Cells.

BACKGROUND: Bone morphogenetic protein 4 (BMP4) has a significant role in primordial germ cells (PGCs) differentiation from mouse embryonic stem cell (mESC). The aim of this study is to determine the best concentration of BMP4 at a time of two days on differentiation PGCs from mESC. MATERIALS AND METHODS: To differentiate PGCs, embryoid bodies (EBs) from mESCs were cultured in concentrations of 0, 5 and 10 ng/ml BMP4 for two days. Germ cell markers Oct4 (Pou5f1), Stella (Dppa3) and Mvh (Ddx4) were analyzed by flow cytometry, immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Flow cytometry data demonstrated most Mvh-positive cells were observed only in the treated groups. Immunocytochemistry of EBs in the treated groups identified cells positive for Mvh. PCR results showed expression of Oct4 in the control group and treated groups. Stella and Mvh were expressed only in the treated groups. CONCLUSION: Low concentrations of BMP4 during two days had an optimal effect on differentiation of PGCs from mESC.

Analysis of apoptosis and expression of genes related to apoptosis in cultures of follicles derived from vitrified and non-vitrified ovaries.

The aim of this study was to evaluate the incidence of apoptosis after in vitro culture of isolated follicles derived from vitrified and non-vitrified ovaries. Mouse ovaries were vitrified and their pre-antral follicles were mechanically isolated and cultured for 10 days. Growth and survival rates of the follicles were assessed during the culture period and the ultrastructure of the follicles was studied. The expression of p53, Bcl-2, Bax, Fas, FasL and survivin were analyzed by real-time RT-PCR in different follicular developmental stages. The percentages of apoptotic and necrotic cells were determined using a fluorescein-activated cell sorting (FACS) technique. There were no differences between the growth and survival rates of follicles in the vitrified and non-vitrified groups. All of the evaluated genes were expressed in the pre-antral, large pre-antral and antral follicles in both groups, except Fas mRNA, which was not expressed in the pre-antral follicles. The expression of p53, Bcl2, Bax and FasL mRNA was similar in vitrified and non-vitrified groups; however, Fas mRNAs were more strongly expressed in the antral follicles of the vitrified group than of the control group (P < 0.05). The expression of survivin 140 was lower in the antral follicles of the vitrified group than of the control group (P < 0.05). FACS analysis showed that the percentage of intact cells was lower in the vitrified group than in the non-vitrified group (P < 0.05). This study demonstrated no signs of apoptosis ultrastructurally in cultured follicles; however, vitrification was shown to affect the expression of some genes related to apoptosis.

The effects of N-acetylcysteine on the expression of matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2 in hepatic fibrosis in bile duct ligated rats.

AIM: N-acetylcysteine can inhibit the formation of intracellular reactive oxygen intermediates. Cellular redox state plays a role in regulating the secretion of matrix metalloproteinase-2. We investigated the effects of N-acetylcysteine on the expression of matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2. METHODS: Bile duct ligated rats were used as a model of hepatic fibrosis. We compared the level of gene expression (using real-time reverse transcription polymerase chain reaction [RT-PCR]), liver function parameters, hepatic reactive oxygen production, lipid peroxidation and glutathione state in experimental groups. RESULTS: N-acetylcysteine treatment significantly improved liver function parameters including the plasma levels of aspartate aminotransferase, alkaline phosphatase, gamma-glutamyl transpeptidase and bilirubin. In addition, significant improvement of glutathione state and reactive oxygen production were observed. Hepatic lipid peroxidation was reversed by N-acetylcysteine treatment. Although N-acetylcysteine treatment did not completely normalize the increased matrix metalloproteinase-2 expression, it significantly decreased its level by 65%. N-acetylcysteine treatment also significantly decreased matrix metalloproteinase-2 activity and normalized tissue inhibitor of matrix metalloproteinase-2 expression. CONCLUSION: Collectively, N-acetylcysteine showed inhibition of matrix metalloproteinase-2 expression and activity. In addition, administration of N-acetylcysteine was associated with downregulation of the expression of tissue inhibitor of matrix metalloproteinase-2 and amelioration of oxidative stress in the liver of bile duct ligated rats.

Comparison of gene expression profiles in erythroid-like cells derived from mouse embryonic stem cells differentiated in simple and co-culture systems.

The feeder layer and the presence of specific growth factors are thought to induce the differentiation of embryonic stem cells (ESCs) in culture. The aim of this study was to evaluate the effect of erythropoietin (EPO) on the differentiation of ESCs into erythroid colonies in simple and co-culture systems. Embryoid bodies were dissociated and replated in semisolid medium in simple culture or in a co-culture system with bone-marrow stromal cells (BMSCs), both in the presence or absence of EPO. Colony assays, benzidine staining, and ultrastructural studies were carried out until day 10 of culture. Expression of the epsilon globin, betaH1 globin, runt-related transcription factor 1 (RUNX1), betamajor globin, and erythropoietin receptor (EPOR) genes was evaluated using semi-quantitative RT-PCR. A comparison with the corresponding controls showed that colony size increased in both systems (P

Detection of four beta-thalassemia point mutations in Iranians using a PCR-ELISA genotyping system.

Development of molecular techniques with analytical capability of mutation detection can realize the medical diagnosis of diseases and improve people's health. beta-Thalassemia is one of the most prevalent genetic disorders in Iran and using a simple and rapid test in laboratories for the mass screening and prenatal diagnosis is essential. Here, we described a simple method for rapid detection of four common beta-thalassemia point mutations in Iranians (IVS-II-1 (G-->A), IVS-I-5 (G-->C), FSC 8/9 (+G), IVS-I-110 (G-->A)) using a PCR-ELISA genotyping system. After DNA isolation from whole blood, a segment of beta-globin gene was amplified by DIG-labeling PCR. The DIG-labeled PCR amplicons were denatured and added to biotinylated normal probe (for normal gene allele) and mutant probe (for mutant gene allele). The hybrids were detected by colorimetric ELISA method. The optical densities obtained using normal and mutant probes with heterozygous PCR products were very similar. The optical densities obtained using mutant probes were higher than normal probes with homozygous PCR products. In vice versa, the optical densities obtained using normal probes were higher than mutant probes with normal PCR products. All the results demonstrated that the PCR-ELISA has similar specificity in comparison to the amplification refractory mutation system.

Production of anti-digoxigenin antibody HRP conjugate for PCR-ELISA DIG detection system.

There are several methods used to visualize the end product of polymerase chain reactions. One of these methods is an ELISA-based detection system (PCR-ELISA) which is very sensitive and can be used to measure the PCR products quantitatively by a colorimetric method. According to this technique, copies of DNA segments from genomic DNA are amplified by PCR with incorporation of digoxigenin-11-dUTP. Samples are analyzed in a microtiter plate format by alkaline denaturation and are hybridized to biotinylated allele-specific capture probes bound to streptavidin coated plates. Use of the produced anti-digoxigenin antibody horseradish peroxidase conjugate and the substrate 2,2'-azino-di-3-ethylbenzthiazolinsulfonate (ABTS) detected the hybridized DNA. One of the key components in this procedure is the anti-digoxigenin antibody HRP conjugate. Described here is the preparation, purification, and characterization of anti-digoxigenin antibody HRP conjugate for use in the PCR-ELISA DIG detection system. Several biochemical protocols and modifications were applied to increase the sensitivity and specificity of this conjugate for an efficient and cost-effective product.

Changes in hepatic cytosolic glutathione S-transferase activity and expression of its class-P during prenatal and postnatal period in rats treated with aflatoxin B1.

The effect of aflatoxin B1 (AFB1) on the expression of glutathione S-transferase-P (GST-P) which is the major isoform of GST in developmental stages has been investigated in rat liver during prenatal and postnatal stages. Following administration of AFB1 (0, 0.5, 1.0, 2.0, 3.0 or 4.0 mg/kg bw) injected I.P on day 8.5 of gestation the number of dead or reabsorbed fetuses and malformed embryos were recorded. Then the fetal livers were processed for measurement of total GST and GST-P activities, using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETA) as substrates respectively. RT-PCR using rat GST-P specific primers was performed on mRNA extracted from livers. Besides, the effects of AFB1 on hepatic GST and GST-P were assessed in groups of suckling rats directly injected with the toxin. The results show that a single dose of AFB1 (1.0 or 2.0 mg/kg bw) caused approximately 50-60% depletion in fetal liver GST towards CDNB. Postnatal experiments revealed that liver GST (using CDNB as substrate) was significantly induced (approximately 40%) in suckling rats injected with a single dose of AFB1 (3.0 mg AFB1/kg) 24 h before killing. Liver GST-P expression was unaffected due to AFB1 exposures of rats before and after the birth. This finding was substantiated by western blotting and RT-PCR techniques. These data suggest that AFB1-related induction in rat liver total GST after birth may be implicated in protective mechanisms against AFB1. In contrast, inhibition of this enzyme in fetal liver following placental transfer of the carcinogen may explain high susceptibility of fetal cells to trans-plancental aflatoxins. Furthermore, lack of influence of AFB1 on GST-P expression in developmental stages can role out the involvement of this class of GST in AFB1 biotransformation.

Over expression of anti-MUC1 single-domain antibody fragments in the yeast Pichia pastoris.

The methylotrophic yeast Pichia pastoris has become a highly popular expression host system for the recombinant production of a wide variety of proteins, such as antibody fragments. Camelids produce functional antibodies devoid of light chains and constant heavy-chain domain (CH1). The antigen binding fragments of such heavy chain antibodies are therefore comprised in one single domain, the so-called VH of the camelid heavy chain antibody (VHH). To test the feasibility of expressing VHHs in the yeast, which on account of their small size and antigen recognition properties would have a major impact on antibody engineering strategies, we constructed two VHH genes encoding the single-domain antibody fragments with specificity for a cancer associated mucin, MUC1. The recombinant strains of the yeast P. pastoris were developed which secrete single-domain antibody fragment to the culture supernatant as a biologically active protein. Supplementation of medium with sorbitol (in pre-induction phase) and casamino acid or EDTA (in induction phase) provided ideal condition of increasing the yield of VHH production compared to culture condition devoid of above recipe. The secreted protein was purified following a 80% ammonium sulfate precipitation step, followed by a affinity chromatography column. The specific activity in enzyme-linked immunosorbant assay (ELISA) of the purified yeast VHH was higher than that of a bacterial periplasmic counterpart. These results reaffirm that the yeast P. pastoris is a suitable host for high level and correctly folded production of VHH antibody fragments with potential in vivo diagnostic and therapeutic applications. This is the first report of expression of VHH in P. pastoris.

The production and characterization of novel heavy-chain antibodies against the tandem repeat region of MUC1 mucin.

Camelidae are known to produce immunoglobulins (Igs) devoid of light chains and constant heavy-chain domains (CH1). Antigen-specific fragments of these heavy-chain IgGs (VHH) are of great interest in biotechnology applications. This paper describes the first example of successfully raised heavy-chain antibodies in Camelus dromedarius (single-humped camel) and Camelus bactrianus (two-humped camel) against a MUC1 related peptide that is found to be an important epitope expressed in cancerous tissue. Camels were immunized against a synthetic peptide corresponding to the tandem repeat region of MUC1 mucin and cancerous tissue preparation obtained from patients suffering from breast carcinoma. Three IgG subclasses with different binding properties to protein A and G were purified by affinity chromatography. Both conventional and heavy-chain IgG antibodies were produced in response to MUC1-related peptide. The elicited antibodies could react specifically with the tandem repeat region of MUC1 mucin in an enzyme linked immunosorbant assay (ELISA). Anti-peptide antibodies were purified after passing antiserum over two affinity chromatography columns. Using ELISA, immunocytochemistry and Western blotting, the interaction of purified antibodies with different antigens was evaluated. The antibodies were observed to be selectively bound to antigens namely: MUC1 peptide (tandem repeat region), human milk fat globule membrane (HMFG), deglycosylated human milk fat globule membrane (D-HMFG), homogenized cancerous breast tissue and a native MUC1 purified from ascitic fluid. Ka values of specific polyclonal antipeptide antibodies were estimated in C. dromedarius and C. bactrianus, as 7 x 10(10) M(-1) and 1.4 x 10(10) M(-1) respectively.