Highly sensitive chemiluminescence immunoassay on chitosan membrane modified paper platform using TiO2 nanoparticles/multiwalled carbon nanotubes as label

A highly sensitive chemiluminescence (CL) immunoassay was incorporated into a low‐cost microfluidic paper‐based analytical device (μ‐PAD) to fabricate a facile paper‐based CL immunodevice (denoted as μ‐PCLI). This μ‐PCLI was constructed by covalently immobilizing capture antibody on a chitosan membrane modified μ‐PADs, which was developed by simple wax printing methodology. TiO₂ nanoparticles coated multiwalled carbon nanotubes (TiO₂/MWCNTs) were synthesized as an amplification catalyst tag to label signal antibody (Ab₂). After sandwich‐type immunoreactions, the TiO₂/MWCNTs were captured on the surface of μ‐PADs to catalyze the luminol‐p‐iodophenol‐H₂O₂ CL system, which produced an enhanced CL emission. Using prostate‐specific antigen as a model analyte, the approach provided a good linear response range from 0.001 to 20 ng/mL with a low detection limit of 0.8 pg/mL under optimal conditions. This μ‐PCLI showed good reproducibility, selectivity and stability. The assay results of prostate‐specific antigen in clinical serum samples were in good agreement with that obtained by commercially used electrochemiluminescence methods at the Cancer Research Center of Shandong Tumor Hospital (Jinan, Shandong Province, China). This μ‐PCLI could be very useful to realize highly sensitive, qualitative point‐of‐care testing in developing or developed countries. Copyright © 2013 John Wiley & Sons, Ltd.     

Rational design of microRNA–siRNA chimeras for multifunctional target suppression

MicroRNAs (miRNAs) are involved in a variety of human diseases by simultaneously suppressing many gene targets. Thus, the therapeutic value of miRNAs has been intensely studied. However, there are potential limitations with miRNA-based therapeutics such as a relatively moderate impact on gene target regulation and cellular phenotypic control. To address these issues, we proposed to design new chimeric small RNAs (aiRNAs) by incorporating sequences from both miRNAs and siRNAs. These aiRNAs not only inherited functions from natural miRNAs, but also gained new functions of gene knockdown in an siRNA-like fashion. The improved efficacy of multifunctional aiRNAs was demonstrated in our study by design and testing of an aiRNA that inherited the functions of both miR-200a and an AKT1-targeting siRNA for simultaneous suppression of cancer cell motility and proliferation. The general principles of aiRNA design were further validated by engineering new aiRNAs mimicking another miRNA, miR-9. By regulating multiple cellular functions, aiRNAs could be used as an improved tool over miRNAs to target disease-related genes, thus alleviating our dependency on a limited number of miRNAs for the development of RNAi-based therapeutics.     

Inhibition of hydrogen sulfide synthesis provides protection for severe acute pancreatitis rats via apoptosis pathway

We aimed to investigate the relationship between the synthesis of hydrogen sulfide (H₂S) and the pancreatic acinar cell apoptosis in severe acute pancreatitis (SAP) rats, as well as analyse the potential apoptotic pathway involved in this process. Sixty rats had been equally divided into four groups: sham, SAP, SAP + sodium hydrosulfide (NaHS) and SAP + DL-propargylglycine (PAG). 24 h after SAP induction, all surviving animals of each group were sacrificed to collect blood and tissue samples for the following measurements: the level of serum H₂S as well as the levels of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), H₂S synthesizing activity, CSE mRNA and protein expression, maleic dialdehyde (MDA) and myeloperoxidase (MPO) activity, the expression of Bax, Bcl-2, caspase-3, -8 and -9, the release of cytochrome c and the activation of nuclear factor-kappa B (NF-κB), ERK1/2, JNK1/2 and p38 in pancreas. Furthermore, in situ detection of cell apoptosis was examined and the severity of pancreatic damage was analyzed by pathological grading and scoring. Results Significant differences in every index except IL-10 had been found between the SAP, NaHS and PAG groups (P < 0.05). Treatment with PAG obviously induced the pancreatic acinar cell apoptosis as well as improved all the pathological changes and inflammatory parameters. In contrast, administration of NaHS significantly attenuated apoptosis in the pancreas and aggravated the severity of pancreatic damage. Moreover, the expressions of caspase-3, -8, -9 and the release of cytochrome c were all increased in the apoptotic cells, and the activity of NF-κB as well as the phosphorylation of ERK1/2, JNK1/2 and p38 decreased accompanying with the reduction of the serum H₂S level. H₂S plays a pivotal role in the regulation of pancreatic acinar cell apoptosis in SAP rats. The present results showed that inhibition of H₂S synthesis provided protection for SAP rats via inducing acinar cell apoptosis. This process acted through both extrinsic and intrinsic apoptotic pathways, and may be regulated by reducing the activity of NF-κB.     

A New Coronary Retroinfusion Technique in theRat Infarct Model: Transjugular Cardiac Vein Catheterization

Cell delivery via the retrograde coronary route boasts less vessel embolism, myocardial injury, and arrhythmogenicity when compared with those via antegrade coronary administration or myocardial injection. However, conventional insertion into the coronary sinus and consequent bleeding complication prevent its application in small animals. To overcome the complication of bleeding, we described a modified coronary retroinfusion technique via the jugular vein route in rats with myocardial infarction (MI). A flexible wire with a bent end was inserted into the left internal jugular vein and advanced slowly along the left superior vena cava. Under direct vision, the wire was run into the left cardiac vein by rotating the wire and changing the position of its tip. A fine tube was then advanced along the wire to the left cardiac vein. This modified technique showed less lethal hemorrhage than the conventional technique. Retroinfusion via transjugular catheter enabled efficient fluid or cell dissemination to the majority areas of the free wall of the left ventricle, covering the infarcted anterior wall. In conclusion, transjugular cardiac vein catheterization may make retrocoronary infusion a more safe and practical route for delivering cell, drug, and gene therapy into the infarcted myocardium of rats.     

Improve carbon metabolic flux in Saccharomyces cerevisiae at high temperature by overexpressed TSL1 gene

This study describes a novel strategy to improve the glycolysis flux of Saccharomyces cerevisiae at high temperature. The TSL1 gene-encoding regulatory subunit of the trehalose synthase complex was overexpressed in S. cerevisiae Z-06, which increased levels of trehalose synthase activity in extracts, enhanced stress tolerance and glucose consuming rate of the yeast cells. As a consequence, the final ethanol concentration of 185.5 g/L was obtained at 38 °C for 36 h (with productivity up to 5.2 g/L/h) in 7-L fermentor, and the ethanol productivity was 92.7 % higher than that of the parent strain. The results presented here provide a novel way to enhance the carbon metabolic flux at high temperature, which will be available for the purposes of producing other primary metabolites of commercial interest using S. cerevisiae as a host.     

Subtype Identification in Acutely Dissociated Rat Nodose Ganglion Neurons Based on Morphologic Parameters

Nodose ganglia are composed of A-, Ah- and C-type neurons. Despite their important roles in regulating visceral afferent function, including cardiovascular, pulmonary, and gastrointestinal homeostasis, information about subtype-specific expression, molecular identity, and function of individual ion transporting proteins is scarce. Although experiments utilizing the sliced ganglion preparation have provided valuable insights into the electrophysiological properties of nodose ganglion neuron subtypes, detailed characterization of their electrical phenotypes will require measurements in isolated cells. One major unresolved problem, however, is the difficulty to unambiguously identify the subtype of isolated nodose ganglion neurons without current-clamp recording, because the magnitude of conduction velocity in the corresponding afferent fiber, a reliable marker to discriminate subtypes in situ, can no longer be determined. Here, we present data supporting the notion that application of an algorithm regarding to microscopic structural characteristics, such as neuron shape evaluated by the ratio between shortest and longest axis, neuron surface characteristics, like membrane roughness, and axon attachment, enables specific and sensitive subtype identification of acutely dissociated rat nodose ganglion neurons, by which the accuracy of identification is further validated by electrophysiological markers and overall positive predictive rates is 89.26% (90.04%, 76.47%, and 98.21% for A-, Ah, and C-type, respectively). This approach should aid in gaining insight into the molecular correlates underlying phenotypic heterogeneity of nodose ganglia. Additionally, several critical points that help for neuron identification and afferent conduction calibration are also discussed.     

Pre-induced adult human peripheral blood mononuclear cells migrate widely into the degenerative retinas of rd1 mice

Background aimsRecent advances in stem cell research have raised the possibility of stem cells repairing or replacing retinal photoreceptor cells that are either dysfunctional or lost in many retinal diseases. Various types of stem cells have been used to replace retinal photoreceptor cells. Recently, peripheral blood stem cells, a small proportion of pluripotent stem cells, have been reported to mainly exist in the peripheral blood mononuclear cells (PBMCs).MethodsIn this study, the effects of pre-induced adult human PBMCs (hPBMCs) on the degenerative retinas of rd1 mice were investigated. Freshly isolated adult hPBMCs were pre-induced with the use of the conditioned medium of rat retinas for 4 days and were then labeled with chloromethyl-benzamidodialkylcarbocyanine (CM-DiI) and then transplanted into the subretinal space of the right eye of rd1 mice through a trans-scleral approach. The right eyes were collected 30 days after transplantation. The survival and migration of the transplanted cells in host retinas were investigated by whole-mount retinas, retinal frozen sections and immunofluorescent staining.ResultsAfter subretinal transplantation, pre-induced hPBMCs were able to survive and widely migrate into the retinas of rd1 mice. A few CM-DiI–labeled cells migrated into the inner nuclear layer and the retinal ganglion cell layer. Some transplanted cells in the subretinal space of rd1 host mice expressed the human photoreceptor–specific marker rhodopsin.ConclusionsThis study suggests that pre-induced hPBMCs may be a potential cell source of cell replacement therapy for retinal degenerative diseases.     

S-Adenosyl-Homocysteine Is a Weakly Bound Inhibitor for a Flaviviral Methyltransferase

The methyltransferase enzyme (MTase), which catalyzes the transfer of a methyl group from S-adenosyl-methionine (AdoMet) to viral RNA, and generates S-adenosyl-homocysteine (AdoHcy) as a by-product, is essential for the life cycle of many significant human pathogen flaviviruses. Here we investigated inhibition of the flavivirus MTase by several AdoHcy-derivatives. Unexpectedly we found that AdoHcy itself barely inhibits the flavivirus MTase activities, even at high concentrations. AdoHcy was also shown to not inhibit virus growth in cell-culture. Binding studies confirmed that AdoHcy has a much lower binding affinity for the MTase than either the AdoMet co-factor, or the natural AdoMet analog inhibitor sinefungin (SIN). While AdoMet is a positively charged molecule, SIN is similar to AdoHcy in being uncharged, and only has an additional amine group that can make extra electrostatic contacts with the MTase. Molecular Mechanics Poisson-Boltzmann Sovation Area analysis on AdoHcy and SIN binding to the MTase suggests that the stronger binding of SIN may not be directly due to interactions of this amine group, but due to distributed differences in SIN binding resulting from its presence. The results suggest that better MTase inhibitors could be designed by using SIN as a scaffold rather than AdoHcy.