A novel thermostable beetle luciferase based cytotoxicity assay.

Cytotoxicity assays are essential for the testing and development of novel immunotherapies for the treatment of cancer. We recently described a novel cytotoxicity assay, termed the Matador assay, which was based on marine luciferases and their engineered derivatives. In this study, we describe the development of a new cytotoxicity assay termed 'Matador-Glo assay' which takes advantage of a thermostable variant of Click Beetle Luciferase (Luc146-1H2). Matador-Glo assay utilizes Luc146-1H2 and D-luciferin as the luciferase-substrate pair for luminescence detection. The assay involves ectopic over-expression of Luc146-1H2 in the cytosol of target cells of interest. Upon damage to the membrane integrity, the Luc146-1H2 is either released from the dead and dying cells or its activity is preferentially measured in dead and dying cells. We demonstrate that this assay is simple, fast, specific, sensitive, cost-efficient, and not labor-intensive. We further demonstrate that the Matador-Glo assay can be combined with the marine luciferase-based Matador assay to develop a dual luciferase assay for cell death detection. Finally, we demonstrate that the Luc146-1H2 expressing target cells can also be used for in vivo bioluminescence imaging applications.

A Fast and Sensitive Luciferase-based Assay for Antibody Engineering and Design of Chimeric Antigen Receptors.

Success of immunotherapeutic approaches using genetically engineered antibodies and T cells modified with chimeric antigen receptors (CARs) depends, among other things, on the selection of antigen binding domains with desirable expression and binding characteristics. We developed a luciferase-based assay, termed Malibu-Glo Assay, which streamlines the process of optimization of an antigen binding domain with desirable properties and allows the sensitive detection of tumor antigens. The assay involves a recombinant immunoconjugate, termed Malibu-Glo reagent, comprising an immunoglobulin or a non-immunoglobulin based antigen binding domain genetically linked to a marine luciferase. Malibu-Glo reagent can be conveniently produced in mammalian cells as a secreted protein that retains the functional activity of both the antigen binding domain and the luciferase. Moreover, crude supernatant containing the secreted Malibu-Glo reagent can directly be used for detection of cell surface antigens obviating the laborious steps of protein purification and labeling. We further demonstrate the utility of Malibu-Glo assay for the selection of optimal single chain fragment variables (scFvs) with desired affinity characteristics for incorporation into CARs. In summary, Malibu-Glo assay is a fast, simple, sensitive, specific and economical assay for antigen detection with multiple applications in the fields of antibody engineering, antibody humanization and CAR-T cell therapy.

A novel luciferase-based assay for the detection of Chimeric Antigen Receptors.

Chimeric Antigen Receptor-T (CAR-T) cell immunotherapy has produced dramatic responses in hematologic malignancies. One of the challenges in the field is the lack of a simple assay for the detection of CARs on the surface of immune effector cells. In this study, we describe a novel luciferase-based assay, termed Topanga Assay, for the detection of CAR expression. The assay utilizes a recombinant fusion protein, called Topanga reagent, generated by joining the extra-cellular domain of a CAR-target in frame with one of the marine luciferases or their engineered derivatives. The assay involves incubation of CAR expressing cells with the Topanga reagent, a few washes and measurement of luminescence. The assay can detect CARs comprising either immunoglobulin- or non-immunoglobulin-based antigen binding domains. We further demonstrate that addition of epitope tags to the Topanga reagent not only allows its convenient one step purification but also extends its use for detection of CAR cells using flow cytometry. However, crude supernatant containing the secreted Topanga reagent can be directly used in both luminescence and flow-cytometry based assays without prior protein purification. Our results demonstrate that the Topanga assay is a highly sensitive, specific, convenient, economical and versatile assay for the detection of CARs.

Development and characterization of a novel luciferase based cytotoxicity assay.

A simple, accurate, sensitive and robust assay that can rapidly and specifically measure the death of target cells would have applications in many areas of biomedicine and particularly for the development of novel cellular- and immune-therapeutics. In this study, we describe a novel cytotoxicity assay, termed the Matador assay, which takes advantage of the extreme brightness, stability and glow-like characteristics of recently discovered novel marine luciferases and their engineered derivatives. The assay involves expression of a luciferase of interest in target cells in a manner so that it is preferentially retained within the healthy cells but is either released from dead and dying cells or whose activity can be preferentially measured in dead and dying cells. We demonstrate that this assay is highly sensitive, specific, rapid, and can be performed in a single-step manner without the need for any expensive equipment. We further validate this assay by demonstrating its ability to detect cytotoxicity induced by several cellular and immune-therapeutic agents including antibodies, natural killer cells, chimeric antigen receptor expressing T cells and a bispecific T cell engager.

Chronic unpredictable stress impairs endogenous antioxidant defense in rat brain.

Many studies have shown that chronic stress can cause neuronal damage and depression, but this exact mechanism still remains unknown. Neurons are vulnerable to lipid peroxidation-induced damage because the major part of neuronal cell membrane is polyunsaturated fatty acids that are substrate for reactive oxygen species. Since endogenous antioxidant defense systems normally eliminate production of reactive oxygen species, deficient antioxidant defense can cause oxidative stress-induced damage. In the present study, to understand the role of endogenous antioxidant defense in chronic stress-induced neuronal damage, we analyzed lipid peroxidation, total antioxidant capacity, and activities of catalase and glutathione peroxidase in frontal cortex, hippocampus and striatum of rats exposed to chronic unpredictable stress. We found that chronic unpredictable stress for four weeks in rats induced depressive-like behaviors such as anhedonia, despair and decreased exploration. Malondialdehyde, a lipid peroxidation product, is increased, but total antioxidant capacity, glutathione peroxidase activity and catalase activity are decreased in brain of rats exposed to chronic unpredictable stress. Our findings suggest that down regulation of endogenous antioxidant defense induces lipid peroxidation contributing a role to chronic stress and depression.

Deficits in water maze performance and oxidative stress in the hippocampus and striatum induced by extremely low frequency magnetic field exposure.

The exposures to extremely low frequency magnetic field (ELF-MF) in our environment have dramatically increased. Epidemiological studies suggest that there is a possible association between ELF-MF exposure and increased risks of cardiovascular disease, cancers and neurodegenerative disorders. Animal studies show that ELF-MF exposure may interfere with the activity of brain cells, generate behavioral and cognitive disturbances, and produce deficits in attention, perception and spatial learning. Although, many research efforts have been focused on the interaction between ELF-MF exposure and the central nervous system, the mechanism of interaction is still unknown. In this study, we examined the effects of ELF-MF exposure on learning in mice using two water maze tasks and on some parameters indicative of oxidative stress in the hippocampus and striatum. We found that ELF-MF exposure (1 mT, 50 Hz) induced serious oxidative stress in the hippocampus and striatum and impaired hippocampal-dependent spatial learning and striatum-dependent habit learning. This study provides evidence for the association between the impairment of learning and the oxidative stress in hippocampus and striatum induced by ELF-MF exposure.

P38-Nrf-2 signaling pathway of oxidative stress in mice caused by nanoparticulate TiO2.

Some recent studies have been previously suggested that nanoparticulate titanium dioxide (TiO(2)) damaged liver function and decreased immunity of mice, but the spleen injury and its oxidative stress mechanism are still unclear. To understand the spleen injury induced by intragastric administration of nanoparticulate anatase TiO(2) for consecutive 30 days, the spleen pathological changes, the oxidative stress, and p38 and c-Jun N-terminal kinase signaling pathways, along with nuclear factor-κB and nuclear factor-E2-related factor-2 (Nrf-2), were investigated as the upstream events of oxidative stress in the mouse spleen from exposure to nanoparticulate TiO(2). The results suggested that nanoparticulate TiO(2) caused congestion and lymph nodule proliferation of spleen tissue, which might exert its toxicity through oxidative stress, as it caused significant increases in the mouse spleen reactive oxygen species accumulations, subsequently leading to the strong lipid peroxidation and the significant expression of heme oxygenase-1 via the p38-Nrf-2 signaling pathway. The studies on the mechanism by which nanoparticulate TiO(2) induced the p38-Nrf-2 signaling pathway are helpful to a better understanding of the nanoparticulate TiO(2)-induced oxidative stress and reduction of immune capacity.

Cerium relieves the inhibition of photosynthesis of maize caused by manganese deficiency.

It had been proved that manganese (Mn) deficiency could damage the photosynthesis of plants, and lanthanides could improve photosynthesis and greatly promote plant growth. However, the mechanisms on how Mn deficiency and cerium (Ce) addition affects the photosynthetic carbon reaction of plants under manganese deficiency are still poorly understood. In this study, the main aim was to determine Mn deficiency and cerium addition effects in key enzymes of CO(2) assimilation of maize. Maize plants were cultivated in Hoagland's solution. They were subjected to Mn deficiency and to Ce administered in the Mn-present Hoagland's media and Mn-deficient Hoagland's media. The growth condition, chlorophyll synthesis, and oxygen evolution were significantly destroyed by manganese deficiency, the activities of ribulose-1, 5-bisphosphate caroxylase/oxygenase (Rubisco), and Rubisco activase, and their genes expressions were inhibited by Mn deficiency. However, Ce treatment promoted the chlorophyll synthesis, oxygen evolution, and the activities of two key enzymes in CO(2) assimilation. Reverse transcription polymerase chain reaction was carried out, and the results showed that the mRNA expressions of Rubisco small subunit (rbcS), Rubisco large subunit (rbcL), and Rubisco activase subunit (rca) in the cerium-treated maize were obviously increased. One of the possible mechanisms of carbon reaction promoted by Ce is that the Ce treatment resulted in the enhancements of Rubisco and Rubisco activase mRNA amounts, the protein levels, and activities of Rubisco and Rubisco activase, thereby leading to the high rate of photosynthetic carbon reaction and enhancement of maize growth under Mn-deficient conditions. Together, the experimental study implied that Ce could partly substitute for magnesium and increase the oxidative stress-resistance of spinach chloroplast grown in Mn-deficiency conditions, but the mechanisms need further study.

Spleen injury and apoptotic pathway in mice caused by titanium dioxide nanoparticules.

Nanoparticulate titanium dioxide (TiO(2)) has been demonstrated to decrease immunity of mice, but very little is known about the injury of spleen involved immunomodulation and its molecular mechanism. In order to understand the spleen injury induced by intraperitoneal injection of TiO(2) nanoparticules (NPs) for consecutive 45 days, the spleen pathological changes, apoptosis, the expression levels of the apoptotic genes and their proteins, and oxidative stress in the mouse spleen were investigated. The results demonstrated that TiO(2) NPs had obvious accumulation in the mouse spleen, leading to congestion and lymph nodule proliferation of spleen tissue, and splenocyte apoptosis. TiO(2) NPs effectively activated caspase-3 and -9, decreased the Bcl-2 the levels of gene and protein, and increase the levels of Bax, and cytochrome c genes and their protein expression, promoted ROS accumulation. Taken together, this study indicated that TiO(2) NPs-induced apoptosis in the mouse splenocyte via mitochondrial-mediated pathway. These findings provide strong evidence that the TiO(2) NPs can induce the spleen pathological changes, apoptosis, leading to the reduction of immunity of mice.