Expression Of Firefly Luciferase Research Articles

Construction and identification of a stable CT26 cell line expressing CD19-FLUC-GFP

Solid tumors lack well-defined targets for chimeric antigen receptor T-cell (CAR-T) therapy. Therefore, introducing a known target molecule, CD19, into solid tumor cell lines via lentiviral transduction to investigate the cytotoxicity of CD19 CAR-T cells can potentially support CAR-T cell therapy against solid tumors. In this study, a stable colon cancer CT26 cell line, CT26-CD19-FLUC-GFP, expressing CD19, firefly luciferase (FLUC), and green fluorescent protein (GFP), was constructed using a triple-plasmid lentiviral system. The growth characteristics of this cell line were consistent with those of the CT26 cell line. Subsequent flow cytometry analysis confirmed stable expression of CD19 and GFP in CT26-CD19-FLUC-GFP cells after serial passaging up to the 5th, 10th, and 22nd generations. Further validation revealed significantly higher levels of CD19 mRNA and FLUC expression in CT26-CD19-FLUC-GFP cells continuously passaged up to the 22nd generation compared to the control CT26 cells. In comparison to T cells, CD19 CAR-T cells demonstrated substantial cytotoxicity against CT26-CD19-FLUC-GFP cells and MC38-CD19 cells. One week after intraperitoneal implantation of CT26-CD19-FLUC-GFP cells into mice, FLUC expression in the peritoneal region could be detected. These results indicate the successful establishment of a stable CT26 cell line expressing CD19-FLUC-GFP, which can be specifically targeted by CD19 CAR-T cells.

Isolation of Cells from Glioblastoma Multiforme Grade 4 Tumors for Infection with Zika Virus prME and ME Pseudotyped HIV-1

This study aimed to isolate cells from grade 4 glioblastoma multiforme tumors for infection experiments with Zika virus (ZIKV) prME or ME enveloped HIV-1 pseudotypes. The cells obtained from tumor tissue were successfully cultured in human cerebrospinal fluid (hCSF) or a mixture of hCSF/DMEM in cell culture flasks with polar and hydrophilic surfaces. The isolated tumor cells as well as the U87, U138, and U343 cells tested positive for ZIKV receptors Axl and Integrin αvβ5. Pseudotype entry was detected by the expression of firefly luciferase or green fluorescent protein (gfp). In prME and ME pseudotype infections, luciferase expression in U-cell lines was 2.5 to 3.5 logarithms above the background, but still two logarithms lower than in the VSV-G pseudotype control. Infection of single cells was successfully detected in U-cell lines and isolated tumor cells by gfp detection. Even though prME and ME pseudotypes had low infection rates, pseudotypes with ZIKV envelopes are promising candidates for the treatment of glioblastoma.

A High-Throughput Amenable Dual Luciferase System for Measuring Toxoplasma gondii Bradyzoite Viability after Drug Treatment.

It is estimated that more than 2 billion people are chronically infected with the intracellular protozoan parasite Toxoplasma gondii (T. gondii). Despite this, there is currently no vaccine to prevent infection in humans, and there is no recognized curative treatment to clear tissue cysts. A major hurdle for identifying effective drug candidates against chronic-stage cysts has been the low throughput of existing in vitro assays for testing the survival of bradyzoites. We have developed a luciferase-based platform for specifically determining bradyzoite survival within in vitro cysts in a 96-well plate format. We engineered a cystogenic type II T. gondii PruΔku80Δhxgpr strain for stage-specific expression of firefly luciferase in the cytosol of bradyzoites and nanoluciferase for secretion into the lumen of the cyst (DuaLuc strain). Using this DuaLuc strain, we found that the ratio of firefly luciferase to nanoluciferase decreased upon treatment with atovaquone or LHVS, two compounds that are known to compromise bradyzoite viability. The 96-well format allowed us to test several additional compounds and generate dose-response curves for calculation of EC50 values indicating relative effectiveness of a compound. Accordingly, this DuaLuc system should be suitable for screening libraries of diverse compounds and defining the potency of hits or other compounds with a putative antibradyzoite activity.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Establishment of stable expression of firefly luciferase and EGFP in a canine inflammatory mammary carcinoma cell line and tumor-bearing model in nude mice.

Canine inflammatory mammary carcinoma (CIMC) is a type of canine malignant mammary tumor with a poor prognosis and high mortality. We transduced firefly luciferase and enhanced green fluorescent protein (EGFP) into CHMp, a CIMC cell line, and established CHMp-Luc-EGFP cells. We investigated the characteristics of this cell line in vitro and in vivo. CHMp-Luc-EGFP was passaged continuously 75 times, with stable expression of luciferase and EGFP. Compared with the wild-type, CHMp-Luc-EGFP had similar proliferation, metastasis, histopathology characteristics, and expression of E-cadherin, N-cadherin, and Ki-67. A tumor-bearing model was established by implantation of CHMp-Luc-EGFP cells, and the dynamic changes of tumors were visualized and quantified using the IVIS imaging system. In summary, the cell line we established could reflect the biological characteristics of CHMp cells, visualize the tumor progression in vivo, and provide a powerful tool for the study of CIMC.

Molecular Imaging of Tumor Progression and Angiogenesis by Dual Bioluminescence.

Angiogenesis is a prerequisite for tumor growth and invasion, and anti-angiogenesis has become a highlight in tumor treatment research. However, so far, there is no reliable solution for how to simultaneously visualize the relationship between tumor progression and angiogenesis. Bioluminescence imaging (BLI) has been broadly utilized and is a very promising non-invasive imaging technique with the advantages of low cost, high sensitivity, and robust specificity. In this chapter, we describe a dual bioluminescence imaging BLI protocol for tumor progression and angiogenesis through implanting murine breast cancer cell line 4T1 which stably expressing Renilla luciferase (RLuc) into the transgenic mice with angiogenesis-induced firefly luciferase (FLuc) expression. This modality enables us to synchronously monitor the tumor progression and angiogenesis in the same mouse, which has broad applicability in oncology studies.

Non-invasive In Vivo Tracking of Mammalian Cells Stably Expressing Firefly Luciferase.

Firefly luciferase (FLuc)-based in vivo optical imaging technology exerts the non-invasive monitoring of transplanted cells in experimental animal models. This chapter introduces an established cell line that stably expresses a retrovirus-delivered FLuc protein gene. The stable expression does not affect the cell morphology, proliferation, migration, and invasion abilities of the parental cells. After implantation, the bioluminescence signal of FLuc cells truly reflects cell proliferation and survival in vivo, which can provide a reliable method for dynamic detection of in vivo cell transplantation.

Visualizing CAR-T cell Immunotherapy Using 3 Tesla Fluorine-19 MRI

PurposeChimeric antigen receptor (CAR) T cell cancer immunotherapies have shown remarkable results in patients with hematological malignancies and represent the first approved genetically modified cellular therapies. However, not all blood cancer patients respond favorably, serious side effects have been reported, and the treatment of solid tumors has been a challenge. An imaging tool for visualizing the variety of CAR-T cell products in use and being explored could provide important patient-specific data on CAR-T cell location to inform on potential success or failure of treatment as well as off-target toxicities. Fluorine-19 (19F) magnetic resonance imaging (MRI) allows for the noninvasive detection of 19F perfluorocarbon (PFC) labeled cells. Our objective was to visualize PFC-labeled (PFC +) CAR-T cells in a mouse model of leukemia using clinical field strength (3 Tesla) 19F MRI and compare the cytotoxicity of PFC + versus unlabeled CAR-T cells.ProceduresNSG mice (n = 17) received subcutaneous injections of CD19 + human B cell leukemia cells (NALM6) expressing firefly luciferase in their left hind flank (1 × 106). Twenty-one days later, each mouse received an intratumoral injection of 10 × 106 PFC + CD19-targeted CAR-T cells (n = 6), unlabeled CD19-targeted CAR-T cells (n = 3), PFC + untransduced T cells (n = 5), or an equivalent volume of saline (n = 3). 19F MRI was performed on mice treated with PFC + CAR-T cells days 1, 3, and 7 post-treatment. Bioluminescence imaging (BLI) was performed on all mice days − 1, 5, 10, and 14 post-treatment to monitor tumor response.ResultsPFC + CAR-T cells were successfully detected in tumors using 19F MRI on days 1, 3, and 7 post-injection. In vivo BLI data revealed that mice treated with PFC + or PFC − CAR-T cells had significantly lower tumor burden by day 14 compared to untreated mice and mice treated with PFC + untransduced T cells (p < 0.05). Importantly, mice treated with PFC + CAR-T cells showed equivalent cytotoxicity compared to mice receiving PFC − CAR-T cells.ConclusionsOur studies demonstrate that clinical field strength 19F MRI can be used to visualize PFC + CAR-T cells for up to 7 days post–intratumoral injection. Importantly, PFC labeling did not significantly affect in vivo CAR-T cell cytotoxicity. These imaging tools may have broad applications for tracking emerging CAR-T cell therapies in preclinical models and may eventually be useful for the detection of CAR-T cells in patients where localized injection of CAR-T cells is being pursued.

Hyperphosphorylation of Tau Due to the Interference of Protein Phosphatase Methylesterase-1 Overexpression by MiR-125b-5p in Melatonin Receptor Knockout Mice.

Melatonin has been indicated to ameliorate tau hyperphosphorylation in the pathogenesis of tau diseases, but the role of melatonin-receptor signal transduction has not been clearly discovered. In this study, we found intensive tau hyperphosphorylation in melatonin receptor knockout mice. Bielschowsky silver staining showed ghostlike neurofibrillary tangles in melatonin receptor-2 knockout (MT2KO) as well as melatonin receptors-1 and -2 knockout (DKO) mice, and an argyrophilic substance was deposited in melatonin receptor-1 knockout (MT1KO) mice. Furthermore, we found significantly decreased activity of protein phosphatase 2A (PP2A) by Western blot and enzyme-linked immunosorbent assay (ELISA), which was partly due to the overexpression of protein phosphatase methylesterase-1 (PME-1), but not glycogen synthase kinase-3β (GSK-3β), cyclin-dependent kinase 5 (CDK5) or protein kinase B (Akt). Finally, we observed a significant increase in cyclic adenosine monophosphate (cAMP) and a decrease in miR-125b-5p levels in MT1KO, MT2KO and DKO mice. Using a luciferase reporter assay, we discovered that miR-125b-5p largely decreased the expression of firefly luciferase by interfering with the 3′UTR of PME-1. Furthermore, miR-125b-5p mimics significantly decreased the expression of PME-1, while miR-125b-5p inhibitor induced tau hyperphosphorylation. These results show that melatonin-receptor signal transduction plays an important role in tau hyperphosphorylation and tangle formation.

Casein Kinase II Inhibition By CX-4945 and Epigenetic Modulation By Decitabine Demonstrate Significant Antiproliferative Activity As Single Agents As Well As in Combination in Acute B-Lymphoblastic Leukemia Cells

Introduction: Aberrant PI3K/AKT signaling is frequently observed in B cell precursor acute lymphoblastic leukemia (B-ALL) resulting in increased cell proliferation and survival. Casein kinase II (CK2) is a regulator of both, PI3K signaling and epigenetic mechanisms in hematological neoplasms. CK2 induces downregulation of de novo methylation and DNA damage response control. However, it is unclear if maintenance methylation is also influenced by CK2. We performed extended in vitro and in vivo analyses with CK2 inhibitor CX-4945 (CX) alone and in combination with hypomethylating agent Decitabine (DEC) or PI3K pathway inhibitors to enhance anti-leukemic efficiency. We hypothesized that demethylation and CK2 blockade act synergistically and have anti-leukemic activity.Methods: B-ALL cell lines SEM, RS4;11, REH, NALM-6 and de novo B-ALL cells were exposed to CX alone or combined with DEC, Idelalisib, Ibrutinib, Sorafenib, BKM120, Perifosine or RAD001 for up to 72 h. B-ALL cells had a broad variety of genetic aberrations. PI3K downstream protein, metabolism, apoptosis and proliferation analysis were performed by western blot, WST-1 assay, Annexin V/propidium iodide and trypan blue staining. Synergistic effects were calculated using the Bliss formula. Whole genome methylation and B cell receptor (BCR) signaling expression were analyzed using Illumina Infinium MethylationEPIC BeadChip and AmpliSeq targeted sequencing after 24 h and 48 h, respectively. For in vivo studies NOD scid gamma (NSG) mice were i.v. injected (d0) with dual firefly luciferase (ffluc) and GFP expression plasmid transduced ALL cell lines or de novo patient-derived (PDX) B-ALL cells. Mice were treated with saline, CX (50 mg/kg per dose BID d7-12), DEC (0.4 mg/kg d7-10) or both. Leukemic engraftment and drug response were monitored twice weekly by peripheral blood (PB) flow cytometry and in vivo bioluminescence imaging (BLI) for up to 30 days.Results: CX significantly inhibited proliferation and metabolism in all cell lines (p < 0.05). In addition, apoptosis induction and decreased phosphorylation of PTEN, AKT and ERK were observed. Combination of CX with any PI3K pathway inhibitor resulted in significantly stronger reduced metabolic activity (p < 0.05 vs single CX or inhibitor). Synergistic effects were generally stronger with substances targeting upstream signaling molecules of the pathway than with those affecting downstream targets. DEC alone and in combination with CX significantly reduced proliferation and metabolism while protein phosphorylation remained unchanged.In order to identify potential CX-mediated regulation of gene expression, targeted sequencing of 179 genes related to BCR signaling was performed, identifying expression changes in interleukin, mTOR and cyclin related genes. Combination with DEC barely modulated CX-induced expression patterns. Methylome profiling revealed 54 demethylated and 82 hypermethylated genes after CX incubation. Addition of DEC increased demethylated genes to 1,323, mainly being involved in vesicle transport, NFκB and WNT signaling. Interestingly, DEC alone demethylated 1,974 genes and showed stronger demethylating effects than the combination.In vivo , BLI revealed slower increase of the tumor burden during and shortly after CX therapy in cell line xenograft models (SEM-ffluc d14: 3.6E+08 ± 3.0E+08 (n=6)) compared to saline treated xenograft mice (5.8E+08 ± 6.3E+08 (n=9), p < 0.05). CX also reduced PB blast frequency in SEM-ffluc (d30, 9.5 ± 6.1 % vs 43.7 ± 26.2 %, p < 0.01), RS4;11-ffluc (d30, 8.3 ± 4.3 % vs 13.8 ± 7.0 %, p = 0.21) and PDX xenograft models (d32, 16.5 ± 2.5 % vs 32.3 ± 1.2 %, p = 0.06). Spleen and bone marrow (BM) infiltration remained similar to the controls. Combined therapy of CX and DEC (n=5) significantly decreased leukemic infiltration in spleen (d30, 18.3 ± 13.5 % vs 54.4 ± 14.8 % in CX, p < 0.01) and reduced PB (p < 0.01) and BM (p = 0.09) blast frequency. However, effects were not stronger than achieved by DEC alone.Conclusion: Herein we demonstrate that CK2 inhibitor CX-4945 and hypomethylating agent DEC have significant in vitro and in vivo antiproliferative acitivity in precursor B-ALL cells of different genetic background. While the combination of CX and DEC acted synergistically in vitro , this effect could not be demonstrated in vivo . We conclude that both CK2 and hypermethylation targeting strategies should be explored in B-ALL in a clinical setting. DisclosuresNo relevant conflicts of interest to declare.

Development and validation of a reporter gene assay for bioactivity determination of Anti-CGRP monoclonal antibodies

Calcitonin gene-related peptide (CGRP) is critical for the pathophysiology of migraine, and four therapeutic antibodies targeting CGRP and its corresponding receptors have been approved by the Food and Drug Administration (FDA), while many others are in the different stages of clinical trials. Bioactivity determination is essential for the quality control and clinical application of therapeutic monoclonal antibodies (mAbs). However, no bioassay has been reported to date. In this study, we developed a reporter gene assay (RGA) based on SK-N-MC cells stably expressing firefly luciferase driven by cAMP response element (CRE). The key assay parameters were optimized according to signal-to-noise (SNR), the response value, and the fitted dose-response curve. Validation of the RGA in accordance with ICH-Q2 guidelines showed that the method had good specificity, accuracy, linearity, and precision. The established RGA can be utilized as a reference method for release testing and stability studies of relevant antibodies.

The Inducible lac Operator-Repressor System Is Functional in Zebrafish Cells.

BackgroundZebrafish are a foundational model organism for studying the spatio-temporal activity of genes and their regulatory sequences. A variety of approaches are currently available for editing genes and modifying gene expression in zebrafish, including RNAi, Cre/lox, and CRISPR-Cas9. However, the lac operator-repressor system, an E. coli lac operon component which has been adapted for use in many other species and is a valuable, flexible tool for inducible modulation of gene expression studies, has not been previously tested in zebrafish.ResultsHere we demonstrate that the lac operator-repressor system robustly decreases expression of firefly luciferase in cultured zebrafish fibroblast cells. Our work establishes the lac operator-repressor system as a promising tool for the manipulation of gene expression in whole zebrafish.ConclusionOur results lay the groundwork for the development of lac-based reporter assays in zebrafish, and adds to the tools available for investigating dynamic gene expression in embryogenesis. We believe this work will catalyze the development of new reporter assay systems to investigate uncharacterized regulatory elements and their cell-type specific activities.

Development and comparison of novel bioluminescent mouse models of pancreatic neuroendocrine neoplasm metastasis

Pancreatic neuroendocrine neoplasms (pNENs) are slow growing cancers of increasing incidence that lack effective treatments once they become metastatic. Unfortunately, nearly half of pNEN patients present with metastatic liver tumors at diagnosis and current therapies fail to improve overall survival. Pre-clinical models of pNEN metastasis are needed to advance our understanding of the mechanisms driving the metastatic process and for the development of novel, targeted therapeutic interventions. To model metastatic dissemination of tumor cells, human pNEN cell lines (BON1 and Qgp1) stably expressing firefly luciferase (luc) were generated and introduced into NSG immunodeficient mice by intracardiac (IC) or intravenous (IV) injection. The efficiency, kinetics and distribution of tumor growth was evaluated weekly by non-invasive bioluminescent imaging (BLI). Tumors formed in all animals in both the IC and IV models. Bioluminescent Qgp1.luc cells preferentially metastasized to the liver regardless of delivery route, mimicking the predominant site of pNEN metastasis in patients. By comparison, BON1.luc cells most commonly formed lung tumors following either IV or IC administration and colonized a wider variety of tissues than Qgp1.luc cells. These models provide a unique platform for testing candidate metastasis genes and anti-metastatic therapies for pNENs.

Abstract 16757: Immunomodulatory Hydrogel Promotes Survival of Mesenchymal Stem Cells in a Rat Model of Myocardial Infarction

Introduction: Stem cell (SC) therapy is a potential method of repairing the heart after injury. However, SCs have been shown to have poor survival after transplantation, at least in part, due to a hostile pro-inflammatory ischemic myocardium. Thus, there is significant interest in the identification of strategies that can increase the retention/survival of SCs after transplantation. Here we seek to investigate whether the use of an immunomodulatory hydrogel, containing anti-inflammatory sinapic acid (SA), promotes the survival of SCs after transplantation. Methods: Myocardial infarction was induced in rats followed by the delayed delivery of A) mesenchymal stem cells (MSCs), B) MSCs in a hydrogel without SA or C) MSCs in a hydrogel containing SA (10μM). Delivery of MSCs (7.5 x 10 5 in 30μL) occurred 7 days after MI, representing the subacute stage and to minimize acute inflammation. Survival of MSCs, stably expressing firefly luciferase under the constitutive CMV promoter, was followed longitudinally using bioluminescence imaging. Results: Panels A-C provide representative images of the viability of MSCs in different groups, with the quantification shown in panel D. There were no differences in survival when MSCs were delivered alone (A) or in a hydrogel without SA (B). However, the survival of MSCs was significantly increased when they were transplanted in a hydrogel with the anti-inflammatory SA (C). Conclusions: Here we provide evidence of the potential for an immunomodulatory hydrogel to enhance the viability of MSCs after infarction, which may lead to improvement of cardiac function. Furthermore, use of this hydrogel in combination with stem cells could be a potential therapeutic strategy for ameliorating cardiac repair after significant damage.

Abstract 6086: Integrin beta3 is a target for treating breast cancer skeletal metastasis

Abstract Integrin β3 (ITGB3) is expressed by breast cancer cells and its heterodimer αvβ3 is related to skeletal metastasis. Here we aimed to investigate this mechanism for defining its role as target in anti-metastatic therapy. Therefore, we generated two MDA-MB-231 breast cancer cell clones (I3, I5) with conditional doxycycline-dependent miRNA-mediated ITGB3 knockdown. In the absence of doxycycline the bi-directional Ptet promoter drives the simultaneous expression of firefly luciferase, red fluorescent protein mCherry and a specific miRNA targeting ITGB3 mRNA. In the presence of doxycycline, their expression is switched off. This model allowed monitoring the effects of ITGB3 inhibition at defined time points on cellular and molecular properties by flow cytometry analysis, proliferation and migration assays, as well as expression profiling analysis. Furthermore, the I5 clone was examined in a nude rat model for breast cancer skeletal metastasis for up to 5 weeks of miRNA treatment. Finally, ITGB3 was analyzed in exosomes secreted from breast cancer (MDA-MB-231, MCF7) and from breast epithelial cells (MCF10), as well as from plasma of healthy and MDA-MB-231 implanted rats with skeletal metastasis. MDA-MB-231 cells were incubated with these ex vivo exosomes for 72h and ITGB3 production was analyzed. Also, the levels of ITGB3 were investigated in exosomes isolated from cell clone I5. ITGB3 was well regulated in cell clones I3 and I5, as shown by knockdown at mRNA (78% and 73%) and protein (22% and 40%) levels after 6 days in medium without doxycycline. After this period the proliferation was decreased only in I5 cells (22%), whereas the migration was inhibited in both cell clones, again more pronounced in I5 (87%) than in I3 cells (20%). Furthermore, decreased tumor sizes and even complete remissions were detected in rats bearing the I5 clone after 4 to 5 weeks of miRNA treatment by bioluminescence and magnetic resonance imaging, as well as volume computed tomography. MDA-MB-231 cells secreted higher levels of ITGB3 in exosomes than MCF7 or MCF10 cells. Also, exosomes from rats bearing MDA-MB-231 induced skeletal metastases contained higher levels of ITGB3 than exosomes from healthy controls. MDA-MB-231 cells incubated with these ex vivo exosomes showed increased ITGB3 levels, too. In line, decreased ITGB3 levels were detected in exosomes from cells with conditional knockdown of this protein. The microarray data of cells with ITGB3 knockdown for 3 or 6 days showed a downregulation of genes, which have specific roles in angiogenesis (NPTN, RRM2), tumor growth (NPTN), energy metabolism (ISCA1), cytokinesis (SEPT11), migration (RRM2, STX6), cell proliferation, invasiveness, senescence, tumorigenesis (RRM2) and vesicle trafficking (SEPT11, STX6). In conclusion, ITGB3 has a function related to skeletal metastasis of breast cancer cells and mediates its distant effects via exosomes, which points to this protein as target for treating the disease. Citation Format: Marineta Kovacheva, Michael Zepp, Martin R. Berger. Integrin beta3 is a target for treating breast cancer skeletal metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6086.

Genome-wide DNA methylation meta-analysis in the brains of suicide completers

Suicide is the second leading cause of death globally among young people representing a significant global health burden. Although the molecular correlates of suicide remains poorly understood, it has been hypothesised that epigenomic processes may play a role. The objective of this study was to identify suicide-associated DNA methylation changes in the human brain by utilising previously published and unpublished methylomic datasets. We analysed prefrontal cortex (PFC, n = 211) and cerebellum (CER, n = 114) DNA methylation profiles from suicide completers and non-psychiatric, sudden-death controls, meta-analysing data from independent cohorts for each brain region separately. We report evidence for altered DNA methylation at several genetic loci in suicide cases compared to controls in both brain regions with suicide-associated differentially methylated positions enriched among functional pathways relevant to psychiatric phenotypes and suicidality, including nervous system development (PFC) and regulation of long-term synaptic depression (CER). In addition, we examined the functional consequences of variable DNA methylation within a PFC suicide-associated differentially methylated region (PSORS1C3 DMR) using a dual luciferase assay and examined expression of nearby genes. DNA methylation within this region was associated with decreased expression of firefly luciferase but was not associated with expression of nearby genes, PSORS1C3 and POU5F1. Our data suggest that suicide is associated with DNA methylation, offering novel insights into the molecular pathology associated with suicidality.

A Bifunctional Polyphosphate Kinase Driving the Regeneration of Nucleoside Triphosphate and Reconstituted Cell-Free Protein Synthesis.

Reconstituted cell-free protein synthesis systems (e.g., the PURE system) allow the expression of toxic proteins, hetero-oligomeric protein subunits, and proteins with noncanonical amino acids with high levels of homogeneity. In these systems, an artificial ATP/GTP regeneration system is required to drive protein synthesis, which is accomplished using three kinases and phosphocreatine. Here, we demonstrate the replacement of these three kinases with one bifunctional Cytophaga hutchinsonii polyphosphate kinase that phosphorylates nucleosides in an exchange reaction from polyphosphate. The optimized single-kinase system produced a final sfGFP concentration (∼530 μg/mL) beyond that of the three-kinase system (∼400 μg/mL), with a 5-fold faster mRNA translation rate in the first 90 min. The single-kinase system is also compatible with the expression of heat-sensitive firefly luciferase at 37 °C. Potentially, the single-kinase nucleoside triphosphate regeneration approach developed herein could expand future applications of cell-free protein synthesis systems and could be used to drive other biochemical processes in synthetic biology which require both ATP and GTP.

Generation of Tumor Cells Expressing Firefly Luciferase (fLuc) to Evaluate the Effectiveness of CAR in a Murine Model.

Immunotherapy has been showed as a promisor treatment, in special for hematological diseases. Chimeric antigen receptor T cells (CARs) which are showing satisfactory results in early-phase cancer clinical trials can be highlighted. However, preclinical models are critical steps prior to clinical trial. In this way, a well-established preclinical model is an important key in order to confirm the proof of principle. For this purpose, in this chapter will be pointed the methods to generate tumor cells expressing firefly Luciferase. In turn, these modified cells will be used to create a subcutaneous and a systemic murine model of Burkitt's lymphoma in order to evaluate the effectiveness of CAR-T.

Development of a Reporter System Monitoring Regulated Intramembrane Proteolysis of the Transmembrane bZIP Transcription Factor ATF6α.

When endoplasmic reticulum (ER) functions are perturbed, the ER induces several signaling pathways called unfolded protein response to reestablish ER homeostasis through three ER transmembrane proteins: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Although it is important to measure the activity of ATF6 that can indicate the status of the ER, no specific cell-based reporter assay is currently available. Here, we report a new cell-based method for monitoring ER stress based on the cleavage of ATF6α by sequential actions of proteases at the Golgi apparatus during ER stress. A new expressing vector was constructed by using fusion gene of GAL4 DNA binding domain (GAL4DBD) and activation domain derived from herpes simplex virus VP16 protein (VP16AD) followed by a human ATF6α N-terminal deletion variant. During ER stress, the GAL4DBD-VP16AD(GV)-hATF6α deletion variant was cleaved to liberate active transcription activator encompassing GV-hATF6α fragment which could translocate into the nucleus. The translocated GV-hATF6α fragment strongly induced the expression of firefly luciferase in HeLa Luciferase Reporter cell line containing a stably integrated 5X GAL4 site-luciferase gene. The established double stable reporter cell line HLR-GV-hATF6α(333) represents an innovative tool to investigate regulated intramembrane proteolysis of ATF6α. It can substitute active pATF6(N) binding motif-based reporter cell lines.

Development and Validation of Approach for the Detection of Neutralizing Antibodies Against Insulin (Glargine) in Human Blood Plasma

Introduction. IImmunogenicity identification of therapeutic proteins, such as human insulin analogues, is one of the most relevant and significant area in medicine and pharmaceuticals. Determination the possibility of producing neutralizing antibodies to insulin reducing the therapeutic effect of the drug, is an important step to understand the pharmacological profile of the drug. Applying of cell-based methods one allows to determinate neutralizing antibodies to insulin.Aim. Development and validation methods for detection of neutralizing antibodies against insulin in human plasma.Materials and methods. The method is based on the use of the iLiteTM Insulin Assay Ready Cells [1], in the genome of which the firefly luciferase reporter gene is introduced under the control of an insulin-dependent promoter. As the insulin concentration increases, the firefly luciferase expression (Firefly) increases, allowing one to use this cell line to estimate the number of neutralizing antibodies against insulin. For normalization by the number of cells and considering the matrix effect of studied samples, the second reporter gene luciferase Renilla is used, which is expressed under the control of a constitutive promoter. The activity of both luciferases was measured using the DualGlo Luciferase Assay System (Promega) assay [2].Results and discussion. Optimal insulin concentration and plasma/serum dilution were determined to identify neutralizing antibodies to insulin. The long-term stability of neutralizing antibodies to insulin were shown in human plasma for more than 3 months. The developed method was applied in a comparative research of the safety and immunogenicity of insulin analogues (Glargine). Method for the determination of antibodies to insulin was.Conclusion. A method for determination of neutralizing antibodies to insulin in human K2EDTA plasma was developed and validated using iLiteTM Insulin Assay Ready Cells system; based on the binding of the insulin alpha chain to the high-affinity heterodimeric CD220 receptor.