Bioluminescent Reporter Research Articles

The in vivo timeline of differentiation of engrafted human neural progenitor cells

Understanding the individual timeline of stem cell differentiation in vivo is critical for evaluating stem cell properties in animal models. However, with conventional ex vivo techniques, such as histology, the individual timeline of differentiation is not accessible. Therefore, we designed lentiviral plasmids with cell-specific promoters to control the expression of bioluminescence and fluorescence imaging reporters. Promoter-dependent reporter expression in transduced human induced pluripotent stem cell-derived neural progenitor cells (hNPCs) was an effective indicator of differentiation in cell culture. A 12-week in vivo imaging observation period revealed the time profile of differentiation of engrafted hNPCs in the mouse brain into astrocytes and mature neurons which was verified by immunostainings, patch-clamp electrophysiology, and light-sheet fluorescence microscopy. The lentiviral vectors validated in this study provide an efficient imaging toolbox for non-invasive and longitudinal characterization of stem cell differentiation, in vitro screenings, and in vivo studies of cell therapy in animal models.

Identification of Factors Complicating Bioluminescence Imaging.

In vivo bioluminescence imaging (BLI) has become a standard, non-invasive imaging modality for following gene expression or the fate of proteins and cells in living animals. Currently, bioluminescent reporters used in laboratories are mostly derivatives of two major luciferase families: ATP-dependent insect luciferases and ATP-independent marine luciferases. Inconsistent results of experiments using different bioluminescent reporters, such as Akaluc and Antareas2, have been reported. Herein, we re-examined the inconsistency in several experimental settings and identified the factors, such as ATP dependency, stability in serum, and molecular sizes of luciferases, that contributed to the observed differences. We expect this study will make the research community aware of these factors and facilitate more accurate interpretation of BLI data by considering the nature of each bioluminescent reporter.

Isolation of photoprotective signal transduction mutants by systematic bioluminescence screening in Chlamydomonas reinhardtii

In photosynthetic organisms, photoprotection to avoid overexcitation of photosystems is a prerequisite for survival. Green algae have evolved light-inducible photoprotective mechanisms mediated by genes such as light-harvesting complex stress-related (LHCSR). Studies on the light-dependent regulation of LHCSR expression in the green alga Chlamydomonas reinhardtii have revealed that photoreceptors for blue light (phototropin) and ultraviolet light perception (UVR8) play key roles in initiating photoprotective signal transduction. Although initial light perception via phototropin or UVR8 is known to result in increased LHCSR3 and LHCSR1 gene expression, respectively, the mechanisms of signal transduction from the input (light perception) to the output (gene expression) remain unclear. In this study, to further elucidate the signal transduction pathway of the photoprotective response of green algae, we established a systematic screening protocol for UV-inducible LHCSR1 gene expression mutants using a bioluminescence reporter assay. Following random mutagenesis screening, we succeeded in isolating mutants deficient in LHCSR1 gene and protein expression after UV illumination. Further characterization revealed that the obtained mutants could be separated into 3 different phenotype groups, the “UV-specific”, “LHCSR1-promoter/transcript-specific” and “general photoprotective” mutant groups, which provided further insight into photoprotective signal transduction in C. reinhardtii.

Development and Applications of Bioluminescent and Chemiluminescent Reporters and Biosensors.

Although fluorescent reporters and biosensors have become indispensable tools in biological and biomedical fields, fluorescence measurements require external excitation light, thereby limiting their use in thick tissues and live animals. Bioluminescent reporters and biosensors may potentially overcome this hurdle because they use enzyme-catalyzed exothermic biochemical reactions to generate excited-state emitters. This review first introduces the development of bioluminescent reporters, and next, their applications in sensing biological changes in vitro and in vivo as biosensors. Lastly, we discuss chemiluminescent sensors that produce photons in the absence of luciferases. This review aims to explore fundamentals and experimental insights and to emphasize the yet-to-be-reached potential of next-generation luminescent reporters and biosensors.

Group A Streptococcus Subcutaneous Infection-Induced Central Nervous System Inflammation Is Attenuated by Blocking Peripheral TNF.

Group A streptococcus (GAS) infection causes a strong inflammatory response associated with cytokine storms, leading to multiorgan failure, which is characterized as streptococcal toxic shock syndrome. However, little is known about GAS subcutaneous infection-mediated brain inflammation. Therefore, we used a bioluminescent GAS strain and reporter mice carrying firefly luciferase under transcriptional control of the nuclear factor-kappa B (NF-κB) promoter to concurrently monitor the host immune response and bacterial burden in a single mouse. Notably, in addition to the subcutaneous inoculation locus at the back of mice, we detected strong luminescence signals from NF-κB activation and increased inflammatory cytokine production in the brain, implying the existence of central nervous system inflammation after GAS subcutaneous infection. The inflamed brain exhibited an increased expression of glial fibrillary acidic protein and nicotinamide adenine dinucleotide phosphate oxidase components and greater microglial activation and blood–brain barrier (BBB) disruption. Furthermore, Fluoro-Jade C positive cells increased in the brain, indicating that neurons underwent degeneration. Peripheral tumor necrosis factor (TNF), which contributes to pathology in brain injury, was elevated in the circulation, and the expression of its receptor was also increased in the inflamed brain. Blockage of peripheral TNF effectively reduced brain inflammation and injury, thereby preventing BBB disruption and improving survival. Our study provides new insights into GAS-induced central nervous system inflammation, such as encephalopathy, which can be attenuated by circulating TNF blockage.

Abstract PD7-02: Novel human cell line xenograft models of ERα-positive metastatic invasive lobular breast carcinoma as pre-clinical platforms for validating candidate genetic drivers

Abstract Invasive lobular breast carcinoma (ILC) is the second most common subtype of breast cancer following invasive ductal breast carcinoma, accounting for 10-15% of all cases. Despite the intriguing histological and clinical differences between these two subtypes, ILC has been chronically understudied. Human ILC cell lines and xenografts are regarded to be of limited utility for ILC research due to slow proliferation rates in culture and low reported intake rates. Data on human ILC cell line xenografts remains limited and knowledge of their metastatic capacity is currently lacking. Using Estrogen Receptor alpha (ER)-positive human ILC cell lines labeled with dual bioluminescent and fluorescent reporters, herein we performed a comprehensive in vivo characterization of their growth as primary tumors orthotopically in the mammary fat pad, as well as at secondary sites following spontaneous or experimental metastasis. Primary tumors exhibited the characteristic dyscohesive, single-file growth of ILC cells, faithfully recapitulating the histology of human ILC tumors. In vivo bioluminescence imaging coupled with ex vivo fluorescence analysis revealed spontaneous metastases to the bone, brain, lungs and ovaries, closely mirroring the clinical patterns of ILC tumor dissemination. This was in stark contrast to experimental metastasis, where none of the cell lines colonized the lungs when injected into the tail vein and bone colonization was observed with only one cell line following intracardiac injections. The metastatic lesions harbored the classical histological features of ILC, including single strand growth, loss of E-cadherin-mediated adherens junctions and mislocalization of p120-catenin to the cytoplasm. Importantly, both the primary tumors and the metastases exhibited high ER expression and significant response to the anti-endocrine agent fulvestrant - a selective ER downregulator. Using these models, we will also present data from functional studies validating candidate genetic drivers of ILC disease progression, which are associated with poor recurrence-free survival in patients with ILC. Ongoing work focused on genomic and transcriptional analyses of metastatic lesions and isolated cell lines from these models will pinpoint additional candidate drivers of ILC dissemination. This is the first report of a hormone responsive ER+ metastatic xenograft model faithfully representing unique ILC features such as ovarian metastases, which will serve as a valuable pre-clinical platform for testing novel therapeutics towards translation into the clinic. Citation Format: Tasdemir N, Scott J, Laotche J, Hou W, Bossart EA, Atkinson J, Sflomos G, Sreekumar S, Castro C, Anderson C, Lee AV, Brisken C, Lucas PC, Davidson NE, Oesterreich S. Novel human cell line xenograft models of ERα-positive metastatic invasive lobular breast carcinoma as pre-clinical platforms for validating candidate genetic drivers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD7-02.

Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications.

Copepod luciferases-a family of small secretory proteins of 18.4-24.3kDa, including a signal peptide-are responsible for bright secreted bioluminescence of some marine copepods. The copepod luciferases use coelenterazine as a substrate to produce blue light in a simple oxidation reaction without any additional cofactors. They do not share sequence or structural similarity with other identified bioluminescent proteins including coelenterazine-dependent Renilla and Oplophorus luciferases. The small size, strong luminescence activity and high stability, including thermostability, make secreted copepod luciferases very attractive candidates as reporter proteins which are particularly useful for nondisruptive reporter assays and for high-throughput format. The most known and extensively investigated representatives of this family are the first cloned GpLuc and MLuc luciferases from copepods Gaussia princeps and Metridia longa, respectively. Immediately after cloning, these homologous luciferases were successfully applied as bioluminescent reporters invivo and invitro, and since then, the scope of their applications continues to grow. This review is an attempt to systemize and critically evaluate the data scattered through numerous articles regarding the main structural features of copepod luciferases, their luminescent and physicochemical properties. We also review the main trends of their application as bioluminescent reporters in cell and molecular biology.

Imaging Bioluminescent Exogenous Stem Cells in the Intact Guinea Pig Cochlea.

ABSTRACTStem‐cell‐based therapy may be used to replace damaged or lost neurons in the cochlear nerve of patients suffering from severe‐to‐profound sensorineural hearing loss. In order to achieve functional recovery in future clinical trials, knowledge about survival of grafted cells and their differentiation into functional neurons is a prerequisite. This calls for non‐invasive in vivo visualization of cells and long‐term monitoring of their survival and fate after cochlear transplantation. We have investigated if molecular optical imaging enables visualization of exogenous cells in the intact cochlea of guinea pig cadaver heads. Transduced (stem) cells, stably co‐expressing fluorescent (copGFP) and bioluminescent (Luc2) reporter molecules, were injected into the internal auditory meatus or directly into the cochlea through the round window. After injection of the cells into the internal auditory meatus, a bright bioluminescent signal was observed in the cavum conchae of the auricle, indicating that light generated by Luc2 is passing through the tympanic membrane and the external auditory meatus. Similar results were obtained after injection of the cells through the round window membrane, either directly into the scala tympani or in Rosenthal's canal within the modiolus of the basal cochlear turn. Imaging of the auditory bulla demonstrated that the bioluminescent signal passes through the tympanic membrane and crevices in the bony wall of the bulla. After opening the auditory bulla, the bioluminescent signal was emanating from the round window. This is the first study demonstrating that bioluminescence imaging enables visualization of luciferase‐expressing cells injected into the intact guinea pig cochlea. Anat Rec, 303:427–440, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Establishment of an in vitro reporter system for screening HBx-targeting molecules.

Chronic hepatitis B virus (HBV) infection remains a global public health problem. HBV-encoded X protein (HBx) is a multifunctional regulator that is required to initiate and maintain productive HBV infection, and is involved in HBV-related hepatocellular carcinoma (HCC). Inhibitors that interfere with the functions of HBx could be useful not only for the inhibition of HBV replication but also for the prevention or treatment of HBV-related HCC. To screen molecules that target HBx on a large scale remains a technical challenge due to a lack of sensitive and high-throughput system. In this work, we established an in vitro bioluminescent reporter system for screening HBx-targeting molecules. The system is based on a secretory fusion protein that combines HBx and NanoLuc (HBx-Nluc). The measured activity of NanoLuc in the culture supernatant of HBx-Nluc-expressing cells directly reflects the level of secreted HBx-Nluc. HBx protein-targeting intracellular anti-HBx single-chain variable fragment and RNA-targeting shRNA significantly reduced the secreted NanoLuc activity in HBx-Nluc-expressing cells. This system is simple and sensitive, and compatible with continuous non-disruptive screening, suggesting its potential usefulness for high-throughput screening and evaluating HBx-targeting molecules.

Abstract A089: The effect of lactate dehydrogenase-A (LDH-A) knockdown and human prostate-specific membrane antigen (hPSMA) directed CAR T-cell treatment on hPSMA(+) Myc-CaP tumors

Abstract Objective: Determine whether LDH-A knockdown enhances CAR T-cell tumor-targeting and treatment response.Background: It has been shown that T-cells are restricted from entering many human and murine solid tumors, including both murine and human prostate tumors. CD3(+) T-cells are restricted from Myc-CaP murine prostate tumors and localize around the periphery of the tumor nodules (1). Human prostate specific membrane antigen (hPSMA) targeted CAR T-cells are also restricted from hPSMA(+) Myc-CaP tumors and this is only partially reversed by anti-PD1 treatment (1). This report describes the benefit of LDH-A depletion and a greater response of CAR T-cells targeting Myc-CaP tumors with low expression of LDH-A. Methods: LDH-A depletion in hPSMA(+) Myc-CaP cells bearing a bioluminescence reporter (Renilla Luciferase), was achieved by shRNA knockdown (KD) (1). A scrambled IgG shRNA was used as a control (NC). LDH-A expression was quantified by digital droplet PCR (ddPCR), Western blotting and LDH enzyme activity. The glycolytic activity of KD and NC cells was measured using Seahorse XF96 and XFp analyzers. Intratumoral lactate levels were monitored by magnetic resonance spectroscopy (MRS). The preparation and characterization of “second generation” hPSMA-directed CAR T-cells and the hPSMA(+) Myc-CaP tumor models in SCID mice have been described (1). To monitor CAR T-cell trafficking, T-cells were transduced with a Click Beetle Red luciferase reporter to enable efficient visualization by bioluminescence imaging (BLI) of CAR T-cell trafficking, persistence and viability within the hPSMA(+)Myc-CaP tumor mass. Tumor volume was calculated from caliper measurements. CD31 and PD-L1 expression was quantified with immunofluorescent staining and Metamorph Offline image analysis. Results and Discussion: These studies demonstrated that LDH-A KD was the dominant factor in reducing tumor growth. The difference in tumor doubling time (DT) between KD and NC tumors in the presence of CAR T-cell therapy was significant (p<0.0001): DT= 5.0±0.3 vs 2.9±0.5 days, respectively. A significant difference was also found in the absence of CAR T-cell therapy. Although LDH-A KD produced the larger single-treatment effect, the addition of CAR T-cells was additive. To explain these observations and study the effect of LDH-A KD on tumor metabolism and microenvironment, Seahorse and MRS experiments were performed. LDH-A depletion (KD) resulted in: lower rates of basal (NC: 24.0±1.9 vs KD: 6.9±1.9 pmol H+/min/µg protein; p<0.0001) and compensatory glycolysis (NC: 43.0±2.3 vs KD: 18.3±3.9 pmol H+/min/µg protein, p<0.0001), and lactate production. In contrast, basal and maximal respiration increased following LDH-A KD (NC: 4.0±0.8 vs KD: 8.3±0.6, p=0.0011, and NC: 6.7±1.4 vs KD: 13.8±0.6 pmol O2/min/µg protein, p=0.0003, respectively). However, total tumor lactate, measured in vivo by MRS, showed only marginal differences between KD and NC tumors. Nevertheless, there were significant differences in CAR T-cell trafficking-to and persistence-in KD compared to NC tumors, and the increase in tumor doubling time was directly related to the CAR T-cell expansion ratio. Interestingly, tumor vascularity (CD31), but not PD-L1, was significantly lower in KD compared to NC tumors. Necrosis was minimal in both KD and NC tumors, with or without CAR T-cell treatment. Conclusions: LDHA knockdown (KD) in the Myc-CaP murine prostate tumor model has a significant effect on tumor cell metabolism resulting in reduced glycolysis, reduced lactate production and increased oxidative phosphorylation that impacts the tumor microenvironment. The LDH-A KD induced changes in tumor metabolism and the microenvironment diminish Myc-CaP tumor growth rate and impact CAR T-cell trafficking and persistence in the tumor. Future studies will compare the effects of anti-PD1 and anti-CTLA4 therapy in combination with LDH-A knockdown. Reference: 1. Serganova I, et al. Molecular Therapy: Oncolytics 2017;4:41. Citation Format: Mayuresh M. Mane, Khalid Shalaby, Ivan Cohen, Avi Albeg, Jenny Ijoma, Myat Ko, Masatomo Maeda, Kiranmayi Vemuri, Jaya Satagopan, Anna Moroz, Juan Zurita, Larissa Shenker, Ellen Ackerstaff, Masahiro Shindo, Ekaterina Moroz, Maxim A. Moroz, Inna Serganova, Jason Koutcher, Vladimir Ponomarev, Ronald G. Blasberg. The effect of lactate dehydrogenase-A (LDH-A) knockdown and human prostate-specific membrane antigen (hPSMA) directed CAR T-cell treatment on hPSMA(+) Myc-CaP tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A089.

Detecting Secretory Proteins by Acoustic Droplet Ejection in Multiplexed High-Throughput Applications.

Nearly one-third of the encoded proteome is comprised of secretory proteins that enable communication between cells and organ systems, playing a ubiquitous role in human health and disease. High-throughput detection of secreted proteins would enhance efforts to identify therapies for secretion-related diseases. Using the Z mutant of alpha-1 antitrypsin as a human secretory model, we have developed 1536-well high-throughput screening assays that utilize acoustic droplet ejection to transfer nanoliter volumes of sample for protein quantification. Among them, the acoustic reverse phase protein array (acoustic RPPA) is a multiplexable, low-cost immunodetection technology for native, endogenously secreted proteins from physiologically relevant model systems like stem cells that is compatible with plate-based instrumentation. Parallel assay profiling with the LOPAC1280 chemical library validated performance and orthogonality between a secreted bioluminescent reporter and acoustic RPPA method by consistently identifying secretory modulators with comparable concentration response relationships. Here, we introduce a robust, multiplexed drug discovery platform coupling extracellular protein quantification by acoustic RPPA with intracellular and cytotoxicity analyses from single wells, demonstrating proof-of-principle applications for human induced pluripotent stem cell-derived hepatocytes.

A Dual-Color Bioluminescence Reporter Mouse for Simultaneous in vivo Imaging of T Cell Localization and Function.

Non-invasive imaging technologies to visualize the location and functionality of T cells are of great value in immunology. Here, we describe the design and generation of a transgenic mouse in which all T cells constitutively express green-emitting click-beetle luciferase (CBG99) while expression of the red-emitting firefly luciferase (PpyRE9) is induced by Nuclear Factor of Activated T cells (NFAT) such as during T cell activation, which allows multicolor bioluminescence imaging of T cell location and function. This dual-luciferase mouse, which we named TbiLuc, showed high constitutive luciferase expression in lymphoid organs such as lymph nodes and the spleen. Ex vivo purified CD8+ and CD4+ T cells both constitutively expressed luciferase, whereas B cells showed no detectable signal. We cross-bred TbiLuc mice to T cell receptor-transgenic OT-I mice to obtain luciferase-expressing naïve CD8+ T cells with defined antigen-specificity. TbiLuc*OT-I T cells showed a fully antigen-specific induction of the T cell activation-dependent luciferase. In vaccinated mice, we visualized T cell localization and activation in vaccine-draining lymph nodes with high sensitivity using two distinct luciferase substrates, D-luciferin and CycLuc1, of which the latter specifically reacts with the PpyRE9 enzyme. This dual-luciferase T cell reporter mouse can be applied in many experimental models studying the location and functional state of T cells.

Infection Assay for Xanthomonas campestris pv. campestris in Arabidopsis thaliana Mimicking Natural Entry via Hydathodes.

Xanthomonas campestris pv. campestris (Xcc) causes the devastating disease Black rot in Brassicaceae. Typically Xcc enters the plant through specialized organs on the leaf margin, called hydathodes, and spreads from there through the vasculature. In order to mimic natural entry as closely as possible, we here describe a "hydathode guttation"-based entry assay for Xcc in Arabidopsis. This disease assay combines spray inoculation with the induction of guttation and allows reabsorption of guttation droplets by the plant. Moreover, our assay relies on a bioluminescent reporter strain of Xcc to allow direct visualization of both entry and subsequent spreading of Xcc in its host. The assay allows the routine infection from one to two hydathodes per Arabidopsis leaf. Infections are scored 14days postinoculation, just before the infection goes systemic.

Real-Time Monitoring of Yersinia pestis Promoter Activity by Bioluminescence Imaging.

Bioluminescence imaging (BLI) has become a major strategy for real-time analysis of dynamic biological processes. In particular, bioluminescent reporter microorganisms have been designed to advance our understanding of infectious diseases. Non-invasive monitoring of light-emitting pathogenic bacteria has revealed novel features of pathogenesis and enabled quantitative and qualitative analysis of antibacterial therapies. Transcriptional gene fusions using the bacterial luciferase operon luxCDABE as a reporter have been successfully used to monitor gene expression in vitro and in vivo, leading to valuable applications and major findings. In this chapter, we describe the construction of Yersinia pestis strains bearing a chromosomal copy of the luxCDABE operon under the control of promoters regulated by temperature and their application to quantify gene expression in real-time in bacteria growing in vitro and in a murine bubonic plague model.

Sensitive ADAR editing reporter in cancer cells enables high-throughput screening of small molecule libraries.

Adenosine to inosine editing is common in the human transcriptome and changes of this essential activity is associated with disease. Children with ADAR1 mutations develop fatal Aicardi-Goutières syndrome characterized by aberrant interferon expression. In contrast, ADAR1 overexpression is associated with increased malignancy of breast, lung and liver cancer. ADAR1 silencing in breast cancer cells leads to increased apoptosis, suggesting an anti-apoptotic function that promotes cancer progression. Yet, suitable high-throughput editing assays are needed to efficiently screen chemical libraries for modifiers of ADAR1 activity. We describe the development of a bioluminescent reporter system that facilitates rapid and accurate determination of endogenous editing activity. The system is based on the highly sensitive and quantitative Nanoluciferase that is conditionally expressed upon reporter-transcript editing. Stably introduced into cancer cell lines, the system reports on elevated endogenous ADAR1 editing activity induced by interferon as well as knockdown of ADAR1 and ADAR2. In a single-well setup we used the reporter in HeLa cells to screen a small molecule library of 33 000 compounds. This yielded a primary hit rate of 0.9% at 70% inhibition of editing. Thus, we provide a key tool for high-throughput identification of modifiers of A-to-I editing activity in cancer cells.

Generation of A Mucor circinelloides Reporter Strain-A Promising New Tool to Study Antifungal Drug Efficacy and Mucormycosis.

Invasive fungal infections caused by Mucorales (mucormycosis) have increased worldwide. These life-threatening infections affect mainly, but not exclusively, immunocompromised patients, and are characterized by rapid progression, severe tissue damage and an unacceptably high rate of mortality. Still, little is known about this disease and its successful therapy. New tools to understand mucormycosis and a screening method for novel antimycotics are required. Bioluminescent imaging is a powerful tool for in vitro and in vivo approaches. Hence, the objective of this work was to generate and functionally analyze bioluminescent reporter strains of Mucor circinelloides, one mucormycosis-causing pathogen. Reporter strains were constructed by targeted integration of the firefly luciferase gene under control of the M. circinelloides promoter Pzrt1. The luciferase gene was sufficiently expressed, and light emission was detected under several conditions. Phenotypic characteristics, virulence potential and antifungal susceptibility were indifferent to the wild-type strains. Light intensity was dependent on growth conditions and biomass, being suitable to determine antifungal efficacy in vitro. This work describes for the first time the generation of reporter strains in a basal fungus that will allow real-time, non-invasive infection monitoring in insect and murine models, and the testing of antifungal efficacy by means other than survival.

Bioluminescent reporter assay for monitoring ER stress in human beta cells

During type 1 diabetes development, cells in the islets of Langerhans engage adaptive mechanisms in response to inflammatory signals to cope with stress, to restore cellular homeostasis, and to preserve cell function. Disruption of these mechanisms may induce the formation of a repertoire of stress-induced neoantigens, which are critical in the loss of tolerance to beta cells and the development of autoimmunity. While multiple lines of evidence argue for a critical role of the endoplasmic reticulum in these processes, the lack of tools to specifically monitor beta cell stress hampers the development of therapeutic interventions focusing on maintaining endoplasmic reticulum homeostasis. Here we designed and evaluated a stress-induced reporter in which induction of stress correlates with increased light emission. This Gaussia luciferase-based reporter system employs the unconventional cytoplasmic splicing of XBP1 to report ER stress in cells exposed to known ER-stress inducers. Linking this reporter to a human beta cell-specific promotor allows tracing ER-stress in isolated human beta cells as well as in the EndoC-βH1 cell line. This reporter system represents a valuable tool to assess ER stress in human beta cells and may aid the identification of novel therapeutics that can prevent beta cell stress in human pancreatic islets.

Elevated Circadian Period 2: A Missing Beneficial Factor in Sickle Cell Disease By Lowering Pulmonary Inflammation, Iron Overload and Mortality

Sickle-cell disase (SCD) is a life-threatening hemolytic genetic disorder. Chronic hemolysis and elevated inflammation that underlie SCD pathophysiology is difficult to treat in the clinic due to an unclear mechanism. The role of the circadian clock is required for maintaining inflammatory states which is important for proper cellular and organ function. Circadian clocks are regulated by a series of circadian clock genes which have known functions in inflammation, heme and iron metabolism. However, the function of circadian clocks in SCD remains unknown. Here, using an unbiased and robust microarray screen, we found that genes involved in circadian rhythms, inflammatory response, heme and iron metabolism were significantly altered in the lungs of SCD Berkeley transgenic mice compared to C57BL/6 (WT) mice used as controls (N=3, P<0.05, normalized to WT). Period 2 (Per2) gene plays a role in regulating the circadian clock. Since the role of Per2 is not known in SCD, we transplanted bone marrow (BM) of SCD and WT mice to Per2Luciferase (Per2Luc) bioluminescence reporter mice to generate SCD → Per2 Luc and WT→ Per2Luc mice. Per2 circadian rhythms were determined by amplitude and periodicity in ex-vivo derived lung tissue explants. Although the period of the Per2 rhythms remained persistent throughout three circadian cycles, we demonstrated that the amplitude of circadian rhythm of Per2 was significantly upregulated in SCD mouse lung compared to WT mouse lung (N=3 per group, P<0.05). However, Per2 circadian rhythms in SCD mouse livers and kidneys did not show differences in amplitude or period compared to controls (N= 3 per group). To assess the importance of elevated Per1 and Per2 homologue genes in SCD, we transplanted BM of SCD mice to Per1/Per2 double deficient (Per1/Per2 dKO) and WT mice, respectively. To our surprise, Per1/Per2 dKO transplanted with SCD mouse BM (SCD→ Per1/Per2 dKO) displayed severe irradiation sensitivity and were more susceptible to an early death compared to WT BM transplanted mice. Mechanistically, we revealed that peripheral deletion of Per1/Per2 led to increased systemic inflammation featured with an increase of peripheral white blood cells (WBCs) in SCD→ Per1/Per2 dKO mice compared to SCD→ WT mice (50.47 ± 6.74 and 31.68 ± 1.04, respectfully, N=5 mice per group, P<0.01) without an effect on sickling in both groups of SCD chimeric mice. Moreover, we found that peripheral deletion of Per1/Per2 resulted in severe lung damage characterized by elevated congestion, neutrophil infiltration and secretion of IL-6 in lavage fluid in SCD→ Per1/Per2 dKO mouse lung compared to SCD→ WT mouse lung. Additionally, we identified that peripheral deletion of Per1/Per2 induced iron overload, heme deposits and macrophage infiltration in the lung of SCD→Per1/Per2 dKO mice compared to SCD→ WT mice. In conclusion, we discovered that Per2 is induced in SCD mouse lung but not in other organs such as kidney and liver and that its elevation is beneficial to counteract systemic inflammation, reduce pulmonary inflammatory responses, iron overload, tissue damage and thus increase survival in SCD mice. Overall, our studies reveal new molecular insight applicable to two burgeoning fields, circadian biology and SCD which pave a way for innovative therapeutic avenue for SCD. DisclosuresD'Alessandro:Omix Technologies inc: Equity Ownership; Hemanext inc: Membership on an entity's Board of Directors or advisory committees.

The Role of the Glyoxylate Shunt in the Acclimation to Iron Limitation in Marine Heterotrophic Bacteria

Iron (Fe) is an essential element for marine microbial growth but is present in trace amounts (<0.1 nM) in surface waters of the ocean. In heterotrophic bacteria, Fe-limitation particularly impacts ATP production as Fe is an essential co-factor of enzymes involved in the electron-transport chain as well as the tricarboxylic acid (TCA) cycle. Fe-limitation can therefore drastically reduce both bacterial growth and respiration, consequently affecting the efficiency of organic carbon remineralization. Heterotrophic bacteria possess various strategies to cope with Fe-limitation. In the present study we tested the hypothesis that the induction of the glyoxylate shunt can represent one such strategy. Genetic approaches were used to gain insight into the potential role the glyoxylate shunt may have in alleviating Fe-stress using the gammaproteobacterium Photobacterium angustum S14. A recombinant bioluminescent reporter of P. angustum S14 (icl-luc) revealed a strong and significant increase in icl expression when cells were subjected to strong Fe-limitation. Although both the growth and respiration rates decreased for the wildtype and an isocitrate lyase knockout mutant (∆icl) under strong Fe-limitation, they were ±30% lower for ∆icl as compared to the wildtype. Complementation of ∆icl restored the growth and respiration rates to those observed in the wild type, further confirming the importance of the glyoxylate shunt under strong Fe-limitation. Due to the ubiquitous nature of the glyoxylate shunt within marine bacteria, our results lead us to propose this pathway as an important acclimation strategy for marine heterotrophic bacteria that are subjected to Fe-limitation.

Chronotype and cellular circadian rhythms predict the clinical response to lithium maintenance treatment in patients with bipolar disorder.

Bipolar disorder (BD) is a serious mood disorder associated with circadian rhythm abnormalities. Risk for BD is genetically encoded and overlaps with systems that maintain circadian rhythms. Lithium is an effective mood stabilizer treatment for BD, but only a minority of patients fully respond to monotherapy. Presently, we hypothesized that lithium-responsive BD patients (Li-R) would show characteristic differences in chronotype and cellular circadian rhythms compared to lithium non-responders (Li-NR). Selecting patients from a prospective, multi-center, clinical trial of lithium monotherapy, we examined morning vs. evening preference (chronotype) as a dimension of circadian rhythm function in 193 Li-R and Li-NR BD patients. From a subset of 59 patientdonors, we measured circadian rhythms in skinfibroblasts longitudinally over 5 days using a bioluminescent reporter (Per2-luc). We then estimated circadian rhythm parameters (amplitude, period, phase) and the pharmacological effects of lithium on rhythms in cells from Li-R and Li-NR donors. Compared to Li-NRs, Li-Rs showed a difference in chronotype, with higher levels of morningness. Evening chronotype was associated with increased mood symptoms at baseline, including depression, mania, and insomnia. Cells from Li-Rs were more likely to exhibit a short circadian period, a linear relationship between period and phase, and period shortening effects of lithium. Common genetic variation in the IP3 signaling pathway may account for some of the individual differences in the effects of lithium on cellular rhythms. We conclude that circadian rhythms may influence response to lithium in maintenance treatment of BD.