Alphaxolone-alphadolone anaesthesia in laboratory animals

The anaesthetic steroid combination alphaxolone-alphadolone is a well-established short-acting injectable agent for cats and primates. It can be recommended for intravenous administration to rats, rabbits, neonatal pigs, mice and hamsters. It has limited value in mice and hamsters by the intraperitoneal route, but provides sedation in ferrets and neonatal pigs when injected intramuscularly. It can be given repeatedly or continuously to maintain anaesthesia for long periods without the development of tolerance or cumulation.     

Dimerization of an aptamer generated from Ligand-guided selection (LIGS) yields a high affinity scaffold against B-cells

Nucleic Acid Aptamers (NAAs) are a class of synthetic DNA or RNA molecules that bind specifically to their target. We recently introduced an aptamer termed R1.2 against membrane Immunoglobulin M (mIgM) expressing B-cell neoplasms using Ligand Guided Selection (LIGS). While LIGS-generated aptamers are highly specific, their lower affinity prevents aptamers from being used for translational applications. Highly specific aptamers with higher affinity can increase targetability, boosting the application of aptamers as diagnostic and therapeutic molecules. Herein, we report that dimerization of R1.2, an aptamer generated from LIGS, leads to high affinity variants without compromising the specificity. Three dimeric aptamer analogues with variable linker lengths were designed to evaluate the effect of linker length in affinity. The optimized dimeric R1.2 against cultured B-cell neoplasms, four donor B-cell samples and mIgM-positive Waldenström's Macroglobulinemia (WM) showed specificity. Furthermore, confocal imaging of dimeric aptamer and anti-IgM antibody in purified B-cells suggests co-localization. Binding assays against IgM knockout Burkitt's Lymphoma cells utilizing CRISPR/Cas9 further validated specificity of dimeric R1.2. Collectively, our findings show that LIGS-generated aptamers can be re-engineered into dimeric aptamers with high specificity and affinity, demonstrating wide-range of applicability of LIGS in developing clinically practical diagnostic and therapeutic aptamers.     

The Effects of Thermal Radiation on an Unsteady MHD Axisymmetric Stagnation-Point Flow over a Shrinking Sheet in Presence of Temperature Dependent Thermal Conductivity with Navier Slip

In this paper, the magnetohydrodynamic (MHD) axisymmetric stagnation-point flow of an unsteady and electrically conducting incompressible viscous fluid in with temperature dependent thermal conductivity, thermal radiation and Navier slip is investigated. The flow is due to a shrinking surface that is shrunk axisymmetrically in its own plane with a linear velocity. The magnetic field is imposed normally to the sheet. The model equations that describe this fluid flow are solved by using the spectral relaxation method. Here, heat transfer processes are discussed for two different types of wall heating; (a) a prescribed surface temperature and (b) a prescribed surface heat flux. We discuss and evaluate how the various parameters affect the fluid flow, heat transfer and the temperature field with the aid of different graphical presentations and tabulated results.     

The Effect of Thermophoresis on Unsteady Oldroyd-B Nanofluid Flow over Stretching Surface

There are currently only a few theoretical studies on convective heat transfer in polymer nanocomposites. In this paper, the unsteady incompressible flow of a polymer nanocomposite represented by an Oldroyd-B nanofluid along a stretching sheet is investigated. Recent studies have assumed that the nanoparticle fraction can be actively controlled on the boundary, similar to the temperature. However, in practice, such control presents significant challenges and in this study the nanoparticle flux at the boundary surface is assumed to be zero. We have used a relatively novel numerical scheme; the spectral relaxation method to solve the momentum, heat and mass transport equations. The accuracy of the solutions has been determined by benchmarking the results against the quasilinearisation method. We have conducted a parametric study to determine the influence of the fluid parameters on the heat and mass transfer coefficients.     

An Oligomeric Signaling Platform Formed by the Toll-like Receptor Signal Transducers MyD88 and IRAK-4

Toll-like receptors (TLRs) mediate responses to pathogen-associated molecules as part of the vertebrate innate immune response to infection. Receptor dimerization is coupled to downstream signal transduction by the recruitment of a post-receptor complex containing the adaptor protein MyD88 and the IRAK protein kinases. In this work, we show that the death domains of human MyD88 and IRAK-4 assemble into closed complexes having unusual stoichiometries of 7:4 and 8:4, the Myddosome. Formation of the Myddosome is likely to be a key event for TLR4 signaling in vivo as we show here that pathway activation requires that the receptors cluster into lipid rafts. Taken together, these findings indicate that TLR activation causes the formation of a highly oligomeric signaling platform analogous to the death-inducing signaling complex of the Fas receptor pathway.In vertebrates, the initial responses of innate immunity are mediated by a family of pattern recognition receptors, which are able to sense the presence of a variety of microbial products such as lipids and non-self nucleic acid (1). One important family of pattern recognition receptors is the Toll-like receptors (TLRs)⁴ that are expressed by many immune system cell types such as macrophages and dendritic cells. TLRs are class one transmembrane receptors that are activated by a process of stimulus-induced dimerization of their extracellular domains. This in turn causes the cytoplasmic Toll/interleukin-1 (IL-1) domains (TIRs) to dimerize, forming a scaffold for the recruitment of downstream signaling components (2). TLRs use five signaling adaptor proteins to couple receptor activation to downstream signal transduction (3). All of these adaptors have TIRs and engage with the activated TLRs by TIR-TIR interactions.One of the adaptor proteins, MyD88, is of particular importance because it is used by all but one of the TLRs as well as by the IL-1 and interferon-γ receptors. MyD88-deficient mice have profoundly impaired innate immune responses and are susceptible to a wide range of infectious diseases. The MyD88 sequence is tripartite and is comprised of a death domain (DD) at the N terminus, a short (40-amino-acid) intermediate domain (ID) of unknown structure, and a C-terminal TIR. Evidence from yeast two-hybrid experiments suggests that MyD88 can self-associate with contacts in both the DD and the TIR (4). The current view of post-receptor signal transduction is that two MyD88 TIR domains bind to the activated TLR, and this enables the recruitment of the protein kinases IRAK-4 and IRAK-1 (5). These kinases have DDs at their N termini, and both are recruited into a complex with MyD88 after signal initiation. It appears that IRAK-4 is recruited first, and this binding requires the ID of MyD88 (6, 7). Thus MyD88s, a splice variant that lacks the ID, down-regulates TLR signaling and cannot recruit IRAK-4 into the post-receptor complex. In contrast, IRAK-1 interacts with MyD88s presumably by DD-DD rather than DD-ID interactions. The next step in the signaling process is for IRAK-4 to phosphorylate IRAK-1, causing activation of the latter and hyper-autophosphorylation. IRAK-1 then dissociates from the complex and interacts with the ubiquitin-protein isopeptide ligase (E3) TRAF6 (8, 9).DDs together with the structurally related caspase recruitment domains (CARDs) and death effector domains (DEDs) form the death domain superfamily (10). There are 215 proteins encoded by the human genome that are predicted to have this fold, and they are widely used in cellular signaling including the TLR and apoptotic pathways. Structurally, DDs contain six antiparallel α-helices, and they are predominantly involved in protein-protein interactions with other DDs. Three modes of DD-DD interaction, types 1, 2, and 3 (10), have been characterized and are illustrated by the structures of the Drosophila Tube-Pelle heterodimer (11), the Procaspase-9 homodimer (12), and most remarkably, by the PIDDosome (13). In the latter case, PIDD, RAIDD, and Caspase-2 form a complex, which results in the proximity-induced activation of Caspase-2 protease activity, which in turn leads to cytochrome c release and apoptotic cell death. The DDs of PIDD and RAIDD interact to produce a complex having a stoichiometry of 5:7, and the subunits are arranged in three layers with five PIDDs, five RAIDDs, and then two RAIDDs. The structure is stabilized by 25 DD-DD contacts of which six are type 2, nine are type 1, and 10 are type 3.In this study, we report that like PIDD and RAIDD, the DDs of human MyD88 and IRAK-4 assemble into defined structures having stoichiometries of 7:4 and 8:4. We propose that the structure has two layers with a ring of seven or eight MyD88 subunits and a second layer of four IRAK-4 subunits. The formation of these higher order assemblies provides insight into the complex regulation and cross-talk observed in the TLR signaling pathways.     

Patterning via Optical Saturable Transitions - Fabrication and Characterization

This protocol describes the fabrication and characterization of nanostructures using a novel nanolithographic technique called Patterning via Optical Saturable Transitions (POST). In this technique the chemical properties of organic photochromic molecules that undergo single-photon reactions are exploited, enabling rapid top-down nanopatterning over large areas at low light intensities, thereby, allowing for the circumvention of the far-field diffraction barrier.⁴ Simple, cost-effective, high throughput and resolution alternatives to nanopatterning are being explored, such as, two-photon polymerization^5,6, beam pen lithography (BPL)⁷, scanning electron beam lithography (SEBL), and focused ion beam (FIB) patterning. However, multi-photon approaches require high light intensities, which limit their potential for high throughput and offer low image contrast. Although, electron and ion beam lithographic processes offer increased resolution, the serial nature of the process is limited to slow writing speeds, which also prevents patterning of features over large areas. Beam-pen lithography is an approach towards parallel near-field optical lithography. However, the gap between the source of the beam and the surface of the photoresist needs to be controlled extremely precisely for good pattern uniformity and this is very challenging to accomplish for large arrays of beams. Patterning via Optical Saturable Transitions (POST) is an alternative optical nanopatterning technique for patterning sub-wavelength features^1-3. Since this technique uses single photons instead of electrons, it is extremely fast and does not require high light intensities^1-3, opening the door to massive parallelization.     

Correlation between NS5A Dimerization and Hepatitis C Virus Replication

Hepatitis C virus (HCV) is the main agent of acute and chronic liver diseases leading to cirrhosis and hepatocellular carcinoma. The current standard therapy has limited efficacy and serious side effects. Thus, the development of alternate therapies is of tremendous importance. HCV NS5A (nonstructural 5A protein) is a pleiotropic protein with key roles in HCV replication and cellular signaling pathways. Here we demonstrate that NS5A dimerization occurs through Domain I (amino acids 1-240). This interaction is not mediated by nucleic acids because benzonase, RNase, and DNase treatments do not prevent NS5A-NS5A interactions. Importantly, DTT abrogates NS5A-NS5A interactions but does not affect NS5A-cyclophilin A interactions. Other reducing agents such as tris(2-carboxyethyl)phosphine and 2-mercaptoethanol also abrogate NS5A-NS5A interactions, implying that disulfide bridges may play a role in this interaction. Cyclophilin inhibitors, cyclosporine A, and alisporivir and NS5A inhibitor BMS-790052 do not block NS5A dimerization, suggesting that their antiviral effects do not involve the disruption of NS5A-NS5A interactions. Four cysteines, Cys-39, Cys-57, Cys-59, and Cys-80, are critical for dimerization. Interestingly, the four cysteines have been proposed to form a zinc-binding motif. Supporting this notion, NS5A dimerization is greatly facilitated by Zn(2+) but not by Mg(2+) or Mn(2+). Importantly, the four cysteines are vital not only for viral replication but also critical for NS5A binding to RNA, revealing a correlation between NS5A dimerization, RNA binding, and HCV replication. Altogether our data suggest that NS5A-NS5A dimerization and/or multimerization could represent a novel target for the development of HCV therapies.     

Neospora caninum: quantification of DNA in the blood of naturally infected aborted and pregnant cows using real-time PCR

This study quantified Neospora caninum DNA in the blood and brain of pregnant and aborted heifers by monitoring PCR product formation in real-time using SYBR Green I, a double-stranded DNA-binding dye. Primers were designed to amplify a 188 bp product specific to N. caninum from the Nc-5 gene fragment of N. caninum. Similarly, a 71 bp product was amplified from the 28S rRNA gene of bovine genomic DNA that served as a control. Agarose gel electrophoresis and analysis of the melting curve for PCR products showed that both primer pairs were specific to their targets. Standard curves were generated for both bovine and N. caninum genomic DNA, and were used to compute the relative concentration of parasite to bovine DNA in the test samples. The concentration of N. caninum DNA in 1 ng of bovine genomic DNA obtained from blood ranged between 0.097 ng at the 1st week of the observation and 0 ng at the 15th week in aborted cows. In pregnant cows, the values ranged between 0.080 ng at the 1st week and 0.155 ng at the 15th week of observation. There was a sustained decrease of DNA concentration in the aborted group after abortion and an increase in DNA concentration in the pregnant group. Comparison of parasite DNA in blood and brain of infected heifers showed a higher DNA concentration in brain than in blood. This study shows that N. caninum DNA can be quantified to obtain the relative concentration of parasite DNA to host genomic DNA in blood. This technique allows testing of a large number of samples at one time and can be done without the need for slaughter of tested animals.