Cas9-Mediated Poxvirus Recombinant Recovery (CASPRR) for Fast Recovery of Recombinant Vaccinia Viruses.

Generation of recombinant vaccinia viruses opens many avenues for poxvirus research; however current methods for virus production can be laborious. Traditional methods rely on recombination strategies that produce engineered viruses at a low frequency, which then need to be identified and isolated from a large background of parent virus. For this reason, marker and reporter genes are often included, but in many cases these require removal in subsequent steps and the entire process is relatively inefficient.

3D Printed Devices for the Separation of Blood Plasma from Capillary Samples.

Sample preparation is a critical requirement for many clinical tests and diagnostic procedures, but it is difficult to perform on a lab-on-a-chip platform. The analytical side of microfluidic technologies has been gradually catching up with laboratory methods in terms of sensitivity, selectivity, and reliability. There is a growing need for the development of sample preparation modules that can either be connected or embedded into such devices and extract blood plasma in a fast, safe, and automated way.

The expression and function of HSV ICP47 and its promoter in mice.

Immune evasion and latency are key mechanisms that underlie the success of herpesviruses. In each case, interactions between viral and host proteins are required and due to co-evolution, not all mechanisms are preserved across host species, even if infection is possible. This is highlighted by the herpes simplex virus (HSV) protein immediate early-infected cell protein (ICP)47, which inhibits the detection of infected cells by killer T cells and acts with high efficiency in humans, but poorly, if at all in mouse cells.

Design and Fabrication of a Fully-Integrated, Miniaturised Fluidic System for the Analysis of Enzyme Kinetics.

The lab-on-a-chip concept, enabled by microfluidic technology, promises the integration of multiple discrete laboratory techniques into a miniaturised system. Research into microfluidics has generally focused on the development of individual elements of the total system (often with relatively limited functionality), without full consideration for integration into a complete fully optimised and miniaturised system. Typically, the operation of many of the reported lab-on-a-chip devices is dependent on the support of a laboratory framework.

Selection of Vaccinia Virus Recombinants Using CRISPR/Cas9.

The engineering of poxvirus genomes is fundamental to primary and applied virology research. Indeed, recombinant poxviruses form the basis for many novel vaccines and virotherapies but producing and purifying these viruses can be arduous. In recent years, CRISPR/Cas9 has become the favoured approach for genome manipulation due to its speed and high success rate.

In vivo application of an implantable tri-anchored methylene blue-based electrochemical pH sensor.

The development of robust implantable sensors is important in the successful advancement of personalised medicine as they have the potential to provide in situ real-time data regarding the status of health and disease and the effectiveness of treatment. Tissue pH is a key physiological parameter and herein, we report the design, fabrication, functionalisation, encapsulation and protection of a miniaturised, self-contained, electrochemical pH sensor system and characterisation of sensor performance. Notably for the first time in this environment the pH sensor was based on a methylene blue redox reporter which showed remarkable robustness, accuracy and sensitivity.

Viperin has species-specific roles in response to herpes simplex virus infection.

Viperin is a gene with a broad spectrum of antiviral functions and various mechanisms of action. The role of viperin in herpes simplex virus type 1 (HSV-1) infection is unclear, with conflicting data in the literature that is derived from a single human cell type. We have addressed this gap by investigating viperin during HSV-1 infection in several cell types, spanning species and including immortalized, non-immortalized and primary cells.

Direct Priming of CD8 T Cells Persists in the Face of Cowpox Virus Inhibitors of Antigen Presentation.

Vaccinia virus (VACV) was the vaccine used to eradicate smallpox and is being repurposed as a vaccine vector. CD8 T cells are key anti-viral mediators, but require priming to become effector or memory cells. Priming requires an interaction with dendritic cells that are either infected (direct priming), or that have acquired virus proteins but remain uninfected (cross priming).

Rapid poxvirus engineering using CRISPR/Cas9 as a selection tool.

In standard uses of CRISPR/Cas9 technology, the cutting of genomes and their efficient repair are considered to go hand-in-hand to achieve desired genetic changes. This includes the current approach for engineering genomes of large dsDNA viruses. However, for poxviruses we show that Cas9-guide RNA complexes cut viral genomes soon after their entry into cells, but repair of these breaks is inefficient.

Surprisingly Effective Priming of CD8 T Cells by Heat-Inactivated Vaccinia Virus Virions.

Robust priming of CD8 T cells by viruses is considered to require infection and expression of viral antigens. A corollary of this is that inactivated viruses are thought of as being inevitably poor vaccines for eliciting these responses. In contrast to this dogma, we found that some antigens present in vaccinia virus (VACV) virions prime strong CD8 T cell responses when the virus was rendered noninfectious by heat.

Experimental and Numerical Study of Electroporation Induced by Long Monopolar and Short Bipolar Pulses on Realistic 3D Irregularly Shaped Cells.

In this article, the reversible electroporation induced by rectangular long unipolar and short bipolar voltage pulses on 3D cells is studied. The cell geometry was reconstructed from 3D images of real cells obtained using the confocal microscopy technique. A numerical model based on the Maxwell and the asymptotic Smoluchowski equations has been developed to calculate the induced transmembrane voltage and pore density on the plasma membrane of real cells exposed to the pulsed electric field.

Development of a competitive binding homogeneous mobility shift assay for the quantification of adalimumab levels in patient serum.

Adalimumab is a TNF specific monoclonal widely used therapeutically. Monitoring adalimumab levels is important for guiding treatment strategies and is predominantly performed using an ELISA. The homogeneous mobility shift assay (HMSA) has many advantages over an ELISA for adalimumab monitoring but current HMSA methodologies do not discriminate between adalimumab and other TNF specific monoclonals such as infliximab.

Modified Vaccinia Virus Ankara Can Induce Optimal CD8 T Cell Responses to Directly Primed Antigens Depending on Vaccine Design.

A variety of strains of vaccinia virus (VACV) have been used as recombinant vaccine vectors with the aim of inducing robust CD8 T cell immunity. While much of the pioneering work was done with virulent strains, such as Western Reserve (WR), attenuated strains such as modified vaccinia virus Ankara (MVA) are more realistic vectors for clinical use. To unify this literature, side-by-side comparisons of virus strains are required.

Most viral peptides displayed by class I MHC on infected cells are immunogenic.

CD8 T cells are essential effectors in antiviral immunity, recognizing short virus-derived peptides presented by MHC class I (pMHCI) on the surface of infected cells. However, the fraction of viral pMHCI on infected cells that are immunogenic has not been shown for any virus. To approach this fundamental question, we used peptide sequencing by high-resolution mass spectrometry to identify more than 170 vaccinia virus pMHCI presented on infected mouse cells.

Discrimination of Anti-drug Antibodies With Neutralizing Capacity in Infliximab- and Adalimumab-Treated Patients: Comparison of the Homogeneous Mobility Shift Assay and the Affinity Capture and Elution Assay.

Background: The measurement of anti-drug antibody (ADA) levels in adalimumab (ADAL)-treated and infliximab (IFX)-treated patients is critical for guiding therapeutic strategies. The homogeneous mobility shift assay (HMSA) and affinity capture elution (ACE) assay provide effective, drug-tolerant formats for measuring total ADA levels. However, their ability to discriminate between ADA from samples with or without neutralizing capacity is unclear and therefore was analyzed in this study.

Novel High-Capacitance-Ratio MEMS Switch: Design, Analysis and Performance Verification.

This paper proposes a novel high-capacitance-ratio radio frequency micro-electromechanical systems (RF MEMS) switch. The proposed RF MEMS mainly consists of serpentine flexure MEMS metallic beam, comprised of coplanar waveguide (CPW) transmission line, dielectric and metal-insulator-metal (MIM) floating metallic membrane. Comparing the proposed high-capacitance-ratio MEMS switch with the ones in available literature, an acceptable insertion loss insulation, acceptable response time and high capacitance ratio (383.

Appraising the "entourage effect": Antitumor action of a pure cannabinoid versus a botanical drug preparation in preclinical models of breast cancer.

Breast cancer is the second leading cause of death among women. Although early diagnosis and development of new treatments have improved their prognosis, many patients present innate or acquired resistance to current therapies. New therapeutic approaches are therefore warranted for the management of this disease.

Intracellular potassium under osmotic stress determines the dielectrophoresis cross-over frequency of murine myeloma cells in the MHz range.

Dielectrophoresis (DEP) has been widely studied for its potential as a biomarker-free method of sorting and characterizing cells based upon their dielectric properties. Most studies have employed voltage signals from ∼1 kHz to no higher than ∼30 MHz. Within this range a transition from negative to positive DEP can be observed at the cross-over frequency f .

Effective Priming of Herpes Simplex Virus-Specific CD8 T Cells Does Not Require Infected Dendritic Cells.

Resolution of virus infections depends on the priming of virus-specific CD8 T cells by dendritic cells (DC). While this process requires major histocompatibility complex (MHC) class I-restricted antigen presentation by DC, the relative contribution to CD8 T cell priming by infected DC is less clear. We have addressed this question in the context of a peripheral infection with herpes simplex virus 1 (HSV).

Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver.

Dysfunction of cell-cell tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. Liver TJs preserve cellular polarity by delimiting functional bile-canalicular structures, forming the blood-biliary barrier. In acetaminophen-hepatotoxicity, the mechanism by which tissue cohesion and polarity are affected remains unclear.

Inter-organisational response to disasters.

Inter-organisational communication failures during times of real-world disasters impede the collaborative response of agencies responsible for ensuring the public's health and safety. In the best of circumstances, communications across jurisdictional boundaries are ineffective. In times of crisis, when communities are grappling with the impact of a disaster, communications become critically important and more complex.

Manufacturing of microcirculation phantoms using rapid prototyping technologies.

In this paper, we describe a method for the manufacturing of a microcirculation phantom that may be used to investigate hemodynamics using optics based methods. We made an Acrylonitrile Butadiene Styrene (ABS) negative mold, manufactured in a Fused Deposition Modelling (FDM) printer, embedded it in Polydimethysilioxane (PDMS) and dissolved it from within using acetone. We successfully made an enlarged three-dimensional (3D) network of microcirculation, and tested it using red blood cell (RBC) analogues.