Evaluation of an in-house indirect immunoperoxidase test for detection of antibodies against African swine fever virus.

African swine fever (ASF) is a high-consequence transboundary animal disease caused by African swine fever virus (ASFV). Given that vaccines are not widely available, ASFV detection, including by molecular and serologic assays, is paramount to efficacious control and mitigation of ASF. ASFV-specific antibodies can be detected as early as 7-10 d postinfection in infected animals and may persist for several months or longer.

Generation of Mammalian Cells Devoid of Mitochondrial DNA (ρ cells).

To cope with DNA damage, mitochondria have developed a pathway whereby severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules can be discarded and degraded, after which new molecules are synthesized using intact templates. In this unit, we describe a method that harnesses this pathway to eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil-N-glycosylase (mUNG1) in mitochondria. We also provide alternate protocols for mtDNA elimination using either combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC) or clustered regulatory interspersed short palindromic repeat (CRISPR)-Cas9-mediated knockout of TFAM or other genes essential for mtDNA replication.

A Method for In Situ Reverse Genetic Analysis of Proteins Involved mtDNA Replication.

The unavailability of tractable reverse genetic analysis approaches represents an obstacle to a better understanding of mitochondrial DNA replication. Here, we used CRISPR-Cas9 mediated gene editing to establish the conditional viability of knockouts in the key proteins involved in mtDNA replication. This observation prompted us to develop a set of tools for reverse genetic analysis in situ, which we called the GeneSwap approach.

FAK Activation Promotes SMC Dedifferentiation via Increased DNA Methylation in Contractile Genes.

Rationale: Vascular smooth muscle cells (SMCs) exhibit remarkable plasticity and can undergo dedifferentiation upon pathological stimuli associated with disease and interventions.

Objective: Although epigenetic changes are critical in SMC phenotype switching, a fundamental regulator that governs the epigenetic machineries regulating the fate of SMC phenotype has not been elucidated.

Methods And Results: Using SMCs, mouse models, and human atherosclerosis specimens, we found that FAK (focal adhesion kinase) activation elicits SMC dedifferentiation by stabilizing DNMT3A (DNA methyltransferase 3A).

Validation of a binary ethylenimine (BEI) inactivation procedure for biosafety treatment of foot-and-mouth disease viruses (FMDV), vesicular stomatitis viruses (VSV), and swine vesicular disease virus (SVDV).

Binary ethylenimine (BEI) has been widely used as a virucide to inactivate viruses. For regulatory exclusion of a select agent, the United States Federal Select Agent Program (FSAP) requires an inactivation procedure that renders a select agent non-viable but allows the select agent to retain antigenic characteristics for future use must be validated, and the inactivated agent must be confirmed by a viability testing. In this curve-based validation study, we examined impacts of BEI concentration, treatment temperature, and time on our in-house inactivation procedures of Foot-and-Mouth Disease Virus (FMDV), Vesicular Stomatitis Virus (VSV), and Swine Vesicular Disease Virus (SVDV).

Targeting focal adhesion kinase in cancer cells and the tumor microenvironment.

Focal adhesion kinase (FAK) is an integrin-associated protein tyrosine kinase that is frequently overexpressed in advanced human cancers. Recent studies have demonstrated that aside from FAK's catalytic activity in cancer cells, its cellular localization is also critical for regulating the transcription of chemokines that promote a favorable tumor microenvironment (TME) by suppressing destructive host immunity. In addition to the protumor roles of FAK in cancer cells, FAK activity within cells of the TME may also support tumor growth and metastasis through various mechanisms, including increased angiogenesis and vascular permeability and effects related to fibrosis in the stroma.

Antigenic regions of African swine fever virus phosphoprotein P30.

African swine fever (ASF) is one of the most complex and lethally haemorrhagic viral diseases of swine, affecting all breeds and ages of pigs. In the absence of ASF vaccines, reliable laboratory diagnosis and restricted biosecurity are critical for disease prevention and control. A detection of ASF-specific antibodies in an unvaccinated pig is a good marker for the diagnosis of ASF.

A Quantitative Method to Measure Low Levels of ROS in Nonphagocytic Cells by Using a Chemiluminescent Imaging System.

Chemiluminescence (CL) is one of the most useful methods for detecting reactive oxygen species (ROS). Although fluorescence dyes or genetically encoded biosensors have been developed, CL is still used due to its high sensitivity, ease of use, and low cost. While initially established and used to measure high levels of ROS in phagocytic cells, CL assays are not ideal for measuring low levels of ROS.

FAK and Pyk2 activity promote TNF-α and IL-1β-mediated pro-inflammatory gene expression and vascular inflammation.

Protein tyrosine kinase (PTK) activity has been implicated in pro-inflammatory gene expression following tumor necrosis factor-α (TNF-α) or interkeukin-1β (IL-1β) stimulation. However, the identity of responsible PTK(s) in cytokine signaling have not been elucidated. To evaluate which PTK is critical to promote the cytokine-induced inflammatory cell adhesion molecule (CAM) expression including VCAM-1, ICAM-1, and E-selectin in human aortic endothelial cells (HAoECs), we have tested pharmacological inhibitors of major PTKs: Src and the focal adhesion kinase (FAK) family kinases - FAK and proline-rich tyrosine kinase (Pyk2).

Nuclear Focal Adhesion Kinase Controls Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia Through GATA4-Mediated Cyclin D1 Transcription.

Rationale: Neointimal hyperplasia is characterized by excessive accumulation of vascular smooth muscle cells (SMCs) leading to occlusive disorders, such as atherosclerosis and stenosis. Blood vessel injury increases growth factor secretion and matrix synthesis, which promotes SMC proliferation and neointimal hyperplasia via FAK (focal adhesion kinase).

Objective: To understand the mechanism of FAK action in SMC proliferation and neointimal hyperplasia.

The VP1 G-H loop hypervariable epitope contributes to protective immunity against Foot and Mouth Disease Virus in swine.

Foot and Mouth Disease is a highly contagious and economically important disease of livestock. While vaccination is often effective at controlling viral spread, failures can occur due to strain mismatch or viral mutation. Foot and Mouth Disease Virus (FMDV) possesses a hypervariable region within the G-H Loop of VP1, a capsid protein commonly associated with virus neutralization.

FAK inhibition reduces metastasis of α4 integrin-expressing melanoma to lymph nodes by targeting lymphatic VCAM-1 expression.

Malignant melanoma typically metastasizes to lymph nodes (LNs) as a primary or in-transit lesion before secondary metastasis occurs, and LN biopsy is a common procedure to diagnose melanoma progression. Since cancer metastasis is a complex process where various interactions between tumor cells and the stroma play key roles in establishing metastatic lesions, the exact mechanisms underlying melanoma metastasis to LNs remains unknown. It has been known that focal adhesion kinase (FAK) activity promotes the expression of proinflammatory vascular cell adhesion molecule-1 (VCAM-1).

Elimination of Mitochondrial DNA from Mammalian Cells.

To cope with DNA damage, mitochondria developed a pathway by which severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules are abandoned and degraded, and new molecules are resynthesized using intact templates, if available. In this unit, we describe a method that harnesses this pathway to completely eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil-N-glycosylase (mUNG1). We also provide an alternate protocol for mtDNA depletion using combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC).

Alkaline hydrolysis to remove potentially infectious viral RNA contaminants from DNA.

Background: Diagnostics and research of high-consequence animal disease agents is often limited to laboratories with a high level of biosecurity that restrict the transport of biological material. Often, sharing of DNA with external partners is needed to support diagnostics, forensics, or research. Even in the absence of virus, RNA from positive-sense single stranded RNA (+ssRNA) viruses that may contaminate otherwise purified DNA preparations continues to pose a threat due to its potential to be infectious via direct translation to yield viral proteins.

The human IL-13 locus in neonatal CD4+ T cells is refractory to the acquisition of a repressive chromatin architecture.

The Th2 cytokine IL-13 is a major effector molecule in human allergic inflammation. Notably, IL-13 expression at birth correlates with subsequent susceptibility to atopic disease. In order to characterize the chromatin-based mechanisms that regulate IL-13 expression in human neonatal CD4(+) T cells, we analyzed patterns of DNase I hypersensitivity and epigenetic modifications within the IL-13 locus in cord blood CD4(+) T cells, naive or differentiated in vitro under Th1- or Th2-polarizing conditions.

Developmentally restricted genetic determinants of human arsenic metabolism: association between urinary methylated arsenic and CYT19 polymorphisms in children.

We report the results of a screen for genetic association with urinary arsenic metabolite levels in three arsenic metabolism candidate genes, PNP, GSTO, and CYT19, in 135 arsenic-exposed subjects from the Yaqui Valley in Sonora, Mexico, who were exposed to drinking water concentrations ranging from 5.5 to 43.3 ppb.