Magnetic protein microbead-aided indirect fluoroimmunoassay for the determination of canine virus specific antibodies

Rabies, canine distemper, and canine parvovirus are common contagious viral diseases of dogs and many other carnivores, and pose a severe threat to the population dynamics of wild carnivores, as well as endangering carnivore conservation. However, clinical diagnosis of these diseases, especially canine distemper and canine parvovirus, is difficult because of the broad spectrum of symptoms that may be confused with other respiratory and enteric diseases of dogs. The most frequently used and proven techniques for diagnosing viral diseases include the conventional enzyme-linked immunosorbent assay (ELISA), rapid fluorescent focus inhibition test (RFFIT), mouse neutralisation test (MNT), and fluorescent antibody virus neutralization (FAVN) test. However, these methods still have some inherent limitations. In this study, a magnetic protein microbead-aided indirect fluoroimmunoassay was developed to detect canine virus specific antibodies, human rabies immunoglobulin, CDV McAbs, and CPV McAbs. In this assay, an avidin-biotin system was employed to combine magnetic microbeads and virus antigens (rabies virus, canine distemper virus, and canine parvovirus). Quantification of the targeted virus antibodies was analyzed through indirect fluoroimmunoassay using the specific antigen-antibody reaction, as well as their corresponding FITC-labeled detection antibodies (mouse anti-human IgG/FITC conjugate or rabbit anti-dog IgG/FITC conjugate). The results indicated that the fluorescence intensity increased when a higher concentration of the targeted analyte was used, but the control had almost no fluorescence, much like the conventional ELISA. For human rabies immunoglobulin, CDV McAbs, and CPV McAbs, the minimum detectable concentrations were 0.2 IU/mL, 0.3 ng/mL, and 0.5 ng/mL, respectively. All of these results indicate that this assay can be employed to determine the presence of canine virus specific antibodies. In addition, the method devised here can be utilized as a general protocol in other bacterial and viral marker analysis.     

Rapid simultaneous screening of seven clinically important enteric pathogens using a magnetic bead based DNA microarray

In this study, we describe a DNA microarray assay by using bead-mediated visible light-assisted signal detection for simultaneous screening of seven clinically important enteric pathogens, including Escherichia coli O157:H7, Vibrio cholerae, Vibrio parahaemolyticus, Salmonella spp., Staphylococcus aureus, Rotavirus, and Norwalk virus (including genogroup I and II). Seven pairs of primers, in which the forward primers were labeled with biotin at the 5′ end, were designed and two sets of multiplex asymmetric PCR system were established to amplify the target genes of the seven pathogens. Twelve type specific oligonucleotides were designed and immobilized onto the aldehyde radical modified glass slide to function as target capture probes. After hybridization and stringency washes, the hybridized biotinylated PCR products were detected by the streptavidin-coated magnetic beads. The final hybridization results were visible to the naked eyes and can be imaged by CCD or digital camera. A total of 86 samples previously identified by conventional microbiological methods and/or PCR method were randomly selected to assess the specificity of this assay by a blind study. A coincidence rate of 100% was obtained. Due to the simplicity and specificity of the magnetic bead based DNA microarray, it is especially appropriate for the diagnosis and monitoring of enteric infectious diseases in the community and seaport.     

Synthesis and optimization of novel 4,4-disubstituted cyclohexylbenzamide derivatives as potent 11β-HSD1 inhibitors

The synthesis and SAR of a series of 4,4-disubstituted cyclohexylbenzamide inhibitors of 11β-HSD1 are described. Optimization rapidly led to potent, highly selective, and orally bioavailable inhibitors demonstrating efficacy in both rat and non-human primate ex vivo pharmacodynamic models.     

MicroRNA-29a regulates pro-inflammatory cytokine secretion and scavenger receptor expression by targeting LPL in oxLDL-stimulated dendritic cells

There is increasing evidence that microRNAs (miRNAs) play important roles in cell proliferation, apoptosis and differentiation that accompany inflammatory responses. However, whether microRNAs are associated with DC immuno-inflammatory responses with oxidized low density lipoprotein (oxLDL) stimulation is not yet known. Our study aims to explore the link of miRNAs with lipid-overload and immuno-inflammatory mechanism for atherosclerosis. In DCs transfected with microRNA-29a mimics or inhibitors, we showed that microRNA-29a plays an important role in proinflammatory cytokine secretion and scavenger receptor expression upon oxLDL-treatment. Furthermore, we suggest an additional explanation for the mechanism of microRNA-29a regulation of its functional target, lipoprotein lipase. We conclude that microRNA-29a could regulate pro-inflammatory cytokine secretion and scavenger receptor expression by targeting lipoprotein lipase in oxLDL-stimulated dendritic cells.     

An integrated chromosome map of microsatellite markers and inversion breakpoints for an Asian malaria mosquito, Anopheles stephensi

Anopheles stephensi is one of the major vectors of malaria in the Middle East and Indo-Pakistan subcontinent. Understanding the population genetic structure of malaria mosquitoes is important for developing adequate and successful vector control strategies. Commonly used markers for inferring anopheline taxonomic and population status include microsatellites and chromosomal inversions. Knowledge about chromosomal locations of microsatellite markers with respect to polymorphic inversions could be useful for better understanding a genetic structure of natural populations. However, fragments with microsatellites used in population genetic studies are usually too short for successful labeling and hybridization with chromosomes. We designed new primers for amplification of microsatellite loci identified in the A. stephensi genome sequenced with next-generation technologies. Twelve microsatellites were mapped to polytene chromosomes from ovarian nurse cells of A. stephensi using fluorescent in situ hybridization. All microsatellites hybridized to unique locations on autosomes, and 7 of them localized to the largest arm 2R. Ten microsatellites were mapped inside the previously described polymorphic chromosomal inversions, including 4 loci located inside the widespread inversion 2Rb. We analyzed microsatellite-based population genetic data available for A. stephensi in light of our mapping results. This study demonstrates that the chromosomal position of microsatellites may affect estimates of population genetic parameters and highlights the importance of developing physical maps for nonmodel organisms.     

Identification, fine mapping and characterisation of a dwarf mutant (bnaC.dwf) in Brassica napus

In the present study, we have obtained one dwarf mutant (bnaC.dwf) from the Brassica napus inbred line T6 through chemical mutagen ethyl methanesulfonate (EMS). We have determined the phenotypic effects and genetic characteristics of dwarf mutant (bnaC.dwf). The dwarf mutant was insensitive to exogenous GA(3) for plant height, suggesting that it is significantly playing a crucial role in the gibberellins response pathway. Genetic analysis revealed that one recessive gene is responsible for controlling the phenotypic expression of dwarf mutant. Amplified Fragment Length Polymorphism (AFLP) technique was applied for selecting markers linked to the BnaC.DWF gene which assisted in screening of dwarf and normal individuals in the BC(4) population. We have screened 1,024 primer combinations and then identified nine AFLP markers linked to the BnaC.DWF gene. Identification and linkage of the markers were carried out by analysing 2,000 individuals from a larger population of the BC(4). Two markers EA10MC09 and EA12MC02 were located on the flanking region of the BnaC.DWF gene at a distance of 0.2 and 0.05 cM, respectively. Four AFLP markers EA09MG05, EA02MC07, EA01MC01 and EC04MC07 were successfully converted into Sequence Characterised Amplified Region markers namely SCA9G5, SCA2C7, SCA1C1 and SCC4C7. We further integrated BnaC.DWF linked Simple Sequence Repeat markers into two populations (Piquemal et al. Theor Appl Genet 111:1514-1523, 2005; Cheng et al. Theor Appl Genet 118:1121-1131, 2009). BnaC.DWF was mapped to the linkage region N18. The molecular markers developed from these investigations will greatly accelerate the selection process for developing dwarf varieties in B. napus by Marker Assisted Selection and genetic engineering.     

Anthraquinone derivatives induce G2/M cell cycle arrest and apoptosis in NTUB1 cells

Thirteen anthraquinone derivatives 5-17 including two 3-(3-alkylaminopropoxy)-9,10-anthraquinone (NHA) derivatives 5 and 6, and 11 1-hydroxy-3-(3-alkylaminopropoxy)-9,10-anthraquinone (MHA) derivatives 7-17 were synthesized, evaluated for cytotoxicities against two cancer cell lines, and assayed the generation of reactive oxygen species (ROS) in NTUB1 cells (a human bladder carcinoma cell line). Compound 9 bearing a pyrrolidinyl group induced the stronger cytotoxic effect than those of other synthesized NHA and MHA derivatives. Exposure of NTUB1 cells to 9, 13, and 17 for 24h significantly increased the production of ROS, respectively. Flow cytometric analysis exhibited that the exposure of NTUB1 cells to the selective 9 led to the G2/M phase arrest accompanied by an increase of apoptotic cell death after the incubation for 24h. Compound 9 induced up-regulation of cyclinB1 and p21 expressions. Biological results suggested that the induction of G2/M arrest, apoptosis, and cell death by 9 may associate with increased expression of p21 and cyclin B1, elevation of Bax and p53 levels, and generation of ROS in the cell. In conclusion, these series of compounds may be used as anticancer agents.     

Relationship between gene expression and observed intensities in DNA microarrays—a modeling study

A theoretical study of the physical properties which determine the variation in signal strength from probe to probe on a microarray is presented. A model which incorporates probe-target hybridization, as well as the subsequent dissociation which occurs during stringent washing of the microarray, is introduced and shown to reasonably describe publicly available spike-in experiments carried out at Affymetrix. In particular, this model suggests that probe-target dissociation during the stringent wash plays a critical role in determining the observed hybridization intensities. In addition, it is demonstrated that non-specific hybridization introduces uncertainties which significantly limit the ability of any model to accurately quantify absolute gene expression levels while, in contrast, target folding appears to have little effect on these results. Finally, for data from target spike-in experiments, our model is shown to compare favorably with an existing statistical model in determining target concentration levels.