Mouse cDNA Research Articles

Interleukin-18 Binding Protein (IL-18BP): A Long Journey From Discovery to Clinical Application.

IL-18 binding protein (IL-18BP) was originally discovered in 1999 while attempting to identify an IL-18 receptor ligand binding chain (also known as IL-18Rα) by subjecting concentrated human urine to an IL-18 ligand affinity column. The IL-18 ligand chromatography purified molecule was analyzed by protein microsequencing. The result revealed a novel 40 amino acid polypeptide. To isolate the complete open reading frame (ORF), various human and mouse cDNA libraries were screened using cDNA probe derived from the novel IL-18 affinity column bound molecule. The identified entire ORF gene was thought to be an IL-18Rα gene. However, IL-18BP has been proven to be a unique soluble antagonist that shares homology with a variety of viral proteins that are distinct from the IL-18Rα and IL-18Rβ chains. The IL-18BP cDNA was used to generate recombinant IL-18BP (rIL-18BP), which was indispensable for characterizing the role of IL-18BP in vitro and in vivo. Mammalian cell lines were used to produce rIL-18BP due to its glycosylation-dependent activity of IL-18BP (approximately 20 kDa). Various forms of rIL-18BP, intact, C-terminal his-tag, and Fc fusion proteins were produced for in vitro and in vivo experiments. Data showed potent neutralization of IL-18 activity, which seems promising for clinical application in immune diseases involving IL-18. However, it was a long journey from discovery to clinical use although there have been various clinical trials since IL-18BP was discovered in 1999. This review primarily covers the discovery of IL-18BP along with how basic research influences the clinical development of IL-18BP.

Determination of sex in cDNA samples from mice.

The need to take sex into account in biomedical research is now recognized and mandated by funding institutions. In laboratory rodents, such as mice, sexing is usually performed anatomically or by genotyping using multiplex or simplex PCR techniques on genomic DNA. Here we present a simple RT-PCR-based method targeting Kdm5c and Kdm5d to determine genetic sex in mouse cDNA samples, allowing for retrospective sex determination.

Novel GPR87/SDC‐1 Complex Modulates Lacritin Rescue of Homeostasis

IntroductionOcular tears refract 80% of light and are essential for epithelial homeostasis. 7% of the world’s population suffer from tear insufficiency associated with compromised visual acuity and chronic ocular surface inflammation. Lacritin, a prosecretory mitogenic tear and plasma glycoprotein, restores homeostasis in a mouse dry eye model, and in human corneal epithelial cells (HCET) subjected to dry eye‐like IFNG and TNF stress. It does so by transiently accelerating epithelial autophagy (FOXO1/ATG7; FOXO3/ATG101) thereby restoring oxidative phosphorylation, as validated in a recent 204 patient phase 2 clinical trial of human Sjögren's Syndrome Dry Eye. Lacritin also promotes basal tearing by increasing the sensitivity of ion channel TRPM8 on corneal sensory nerves to tear drying. Here we report the identification of G protein‐coupled receptor 87 (GPR87) as the lacritin signaling receptor out of a genome‐wide CRISPR/Cas9 death screen using the C‐terminal lacritin synthetic peptide N‐94 as probe. GPR87 is expressed in the cornea and has a critical role in cellular proliferation and survival. Years ago, GPR87 was thought to be an LPA receptor, later disproven by Arfelt et al, ′17. Finally, we also report that GPR87 interacts with syndecan‐1 (SDC1), a cell surface co‐receptor for lacritin. We observed a direct binding of recombinant lacritin to GPR87, corroborating with the hypothesis that GPR87 is a critical receptor for lacritin homeostatic signaling.MethodsValidation studies of GPR87 were performed by generation of GPR87 knockdown cell lines by shRNA depletion by transducing HCE‐T cells with lentiviral GPR87 shRNA and non‐target controls. Depletion of GPR87 protein level was confirmed by western blot. Complementation assays were performed by transducing depleted HCE‐T’s with lentiviral GPR87 mouse cDNA. Lacritin affinity binding to GPR87 was first studied out of recombinant lacritin‐ or lacritin C‐25‐intein pulldowns. Truncation mutant ‘C‐25’, lacks lacritin's ‘N‐94’ bioactive domain. Lacritin pulldowns were challenged using each of eleven synthetic peptides corresponding to or together spanning all eight extracellular or intracellular strands or loops of GPR87 ‐ each at increasing concentrations (20, 100, 400 µM). To explore whether GPR87 can couple with SDC1, we blotted for GPR87 out of SDC‐1 pulldowns from HEK2936E cells co‐transfected with wild type or mutated GPR87 and SDC1 cDNA’s.ResultsLacritin, but not C‐25, binds GPR87 via GPR87's outer three loops with outer loop 3 peptide most inhibitory. GPR87 complexes with SDC1 in the absence of N‐94, but not with SDC1 lacking the PDZ binding domain nor with GPR87 lacking the G‐protein binding site “DRY” motif. SDC‐1 lacking N‐terminal domain heparan sulfate at S15 or membrane proximal chondroitin sulfate at S184 and S185 failed to bind GPR87. SDC‐1 also failed to complex with GPR87 in cells treated with chlorate to suppress sulfation.ConclusionsLacritin appears to bind directly to the outer loop domains of GPR87 as part of a preformed SDC1‐GPR87 complex ‐ the latter linked in part by glycosaminoglycans and apparently by cytoplasmic SDC1 and GPR87 domains to regulate ocular surface health.

Restoring FAS Expression via Lipid-Encapsulated FAS DNA Nanoparticle Delivery Is Sufficient to Suppress Colon Tumor Growth In Vivo.

Simple SummaryA key feature of human colorectal tumor is loss of FAS expression. FAS is the death receptor for FASL of activated T cells. Loss of FAS expression therefore may promote tumor cell immune escape. We aimed at determining whether restoring FAS expression is sufficient to suppress colorectal tumor growth. Mouse and human FAS cDNA was synthesized and encapsulated into cationic lipid nanoparticle DOTAP-Cholesterol to formulate DOTAP-Chol-mFAS and DOTAP-Chol-hFAS, respectively. Restoring FAS expression in metastatic mouse colon-tumor cells enabled FASL-induced elimination of FAS+ tumor cells in vitro and suppressed colon-tumor growth and progression in tumor-bearing mice in vivo. Restoring FAS expression induced FAS receptor auto-oligomerization and tumor cell auto-apoptosis in metastatic human colon-tumor cells in vitro. DOTAP-Chol-hFAS therapy is also sufficient to suppress metastatic human colon tumor xenograft growth in athymic mice. Tumor-selective delivery of FAS DNA nanoparticle is potentially an effective therapy for human colorectal cancer.A hallmark of human colorectal cancer is lost expression of FAS, the death receptor for FASL of cytotoxic T lymphocytes (CTLs). However, it is unknown whether restoring FAS expression alone is sufficient to suppress csolorectal-cancer development. The FAS promoter is hypermethylated and inversely correlated with FAS mRNA level in human colorectal carcinomas. Analysis of single-cell RNA-Seq datasets revealed that FAS is highly expressed in epithelial cells and immune cells but down-regulated in colon-tumor cells in human colorectal-cancer patients. Codon usage-optimized mouse and human FAS cDNA was designed, synthesized, and encapsulated into cationic lipid to formulate nanoparticle DOTAP-Chol-mFAS and DOTAP-Chol-hFAS, respectively. Overexpression of codon usage-optimized FAS in metastatic mouse colon-tumor cells enabled FASL-induced elimination of FAS+ tumor cells in vitro, suppressed colon tumor growth, and increased the survival of tumor-bearing mice in vivo. Overexpression of codon-optimized FAS-induced FAS receptor auto-oligomerization and tumor cell auto-apoptosis in metastatic human colon-tumor cells. DOTAP-Chol-hFAS therapy is also sufficient to suppress metastatic human colon tumor xenograft growth in athymic mice. DOTAP-Chol-mFAS therapy exhibited no significant liver toxicity. Our data determined that tumor-selective delivery of FAS DNA nanoparticles is sufficient for suppression of human colon tumor growth in vivo.

Abstract PS13-28: Pre-treating TNBC with docetaxel and il-12 enhances anti-PD1 efficacy

Abstract Introduction: Amongst all breast cancers, Triple Negative Breast Cancer (TNBC) account for 15-20% of all the cases. TNBC affect younger patients, and is more prevalent in African-American women. The poor prognosis for this very aggressive tumor subtype is exacerbated by the lack of specific targeted therapy against the disease. Although TNBC initially respond very well to chemotherapy, paradoxically the disease-free survival is very short. It has been showed that TNBC have higher rates of CD8+ T-cells infiltration, and express high level of PD-L1. Together, these data provide a strong rationale for the combination of chemotherapy and immunotherapy to treat TNBC patients. In this study, we investigated the response to pre-priming the tumor with one round of docetaxel and IL-12, followed by anti-PD1 maintenance in mouse E0771 and 4T1 TNBC syngeneic models. We hypothesized that docetaxel will promote the release of neo-antigens, while IL-12 will activate immunity specific to these antigens and anti-PD1 therapy prevent the exhaustion of those T-cells. Materials/Methods: Mouse TNBC E0771 and 4T1 cell lines were injected in the mammary fat pad of C57BL/6, and Balb/c mice respectively. On day 1, the mice received a single dose (20mg/kg) of docetaxel and one intratumoral injection (1.25x109) of mAdv.IL-12, a replication defective adenoviral vector containing mIL-12 (mouse) cDNA under the transcriptional control of Rous sarcoma virus long terminal repeat (provided by Dr. Chen, HMRI). Anti-PD1 (InVivoMab anti-mouse PD-1 CD279) was administered 3 times a week (2 cycles) starting 5 days post docetaxel and Il-12 treatment. At the end of the study, IFN-gamma levels were measured from blood and tumor samples; tumor sizes were compared between treatment groups (Control/mAdv.IL-12/anti-PD1/and various Combination), as well as survival curves. The metastatic burden to the lungs (H&E), as well as the apoptosis in the tumor (TUNNEL) were assessed by IHC. Results: In both 4T1 and E0771 tumor models, triple combination of docetaxel + IL-12 followed by anti-PD1 significantly reduced tumor size compared to both single agents, and double combination. In the Triple Combo group, 1 mice had lung metastasis vs all of them in the other treatment groups. IHC data indicate a higher level of TILs in the treatment groups, with a statistically significant difference in the combination groups compared to single agents. There was more apoptosis in the triple combo group as indicated by TUNNEL. Conclusion:Our preliminary data strongly supports that treating TNBC models with docetaxel and mAdv.IL-12 followed by anti-PD1 significantly slows down tumor growth, and decrease lung metastasis incidence. We are actively investigating the mechanism through which the response is achieved. Citation Format: Ann Cassany Anselme, Wei Qian, Jianying Zhou, Roberto R. Rosato, Jenny C. Chang. Pre-treating TNBC with docetaxel and il-12 enhances anti-PD1 efficacy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS13-28.

1908-P: Glucagon Receptor Expression Evaluated by Antibody-Dependent and -Independent Approaches

Glucagon regulates metabolic functions through the glucagon receptor (GCGR). Agonists as well as antagonists of GCGR are evaluated as treatments of diabetes and obesity. To understand the role of glucagon, knowledge of GCGR localization is important but limited. Twelve commercially available GCGR antibodies were evaluated using HEK293 cells transfected with mouse or human GCGR cDNA transcripts. Eleven of the 12 GCGR antibodies showed staining of GCGR protein from both species, but not in cells transfected with a scrambled vector. Several of the antibodies stained liver sections from both GCGR+/+ and GCGR-/- mice. Staining was absent in fat and muscle tissues but present in the distal tubuli of the kidney. Two antibodies did not react with tissue from GCGR-/- and were further evaluated by western blotting of GCGR transfected cells. Bands corresponding to the predicted size of the GCGR (62kDa) were identified but larger bands were also observed. Liver tissue from healthy subjects and individuals with nonalcoholic steatohepatitis (NASH) was stained using same GCGR antibodies and based on qualitative intensities GCGR staining did not seem to vary between healthy and NASH liver sections. We used autoradiography to confirm the immunohistochemistry findings. In mice injected with 125I-labeled glucagon, grains were found in the liver and in the distal tubuli of the kidney, whereas fat and muscle sections were negative. To investigate the role of renal GCGR’s, we infused glucagon (1µg) in rats with and without blockade of the GLP-1 receptor (GLP-1R) (Exendin 9-39). Glucagon increased diuresis and renal blood flow, but not blood pressure, in a GLP-1R dependent manner. Urea excretion was numerically increased by glucagon (P=0.08). In conclusion, glucagon receptor localization is challenging due to the lack of suitable commercially available GCGR antibodies. The actions of glucagon on renal parameters may be mediated by the GLP-1R. Glucagon’s role in kidney physiology needs further investigation. Disclosure S. Kjeldsen: None. K.D. Galsgaard: None. M. Winther-Soerensen: None. R. Serizawa: None. J. Pedersen: None. C. Orskov: None. R. Albrechtsen: None. C.M. Sorensen: None. L. Gluud: None. J.J. Holst: Advisory Panel; Self; AstraZeneca, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Zealand Pharma A/S. Other Relationship; Spouse/Partner; Antag Therapeutics. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S, Novo Nordisk Foundation. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp. Funding Novo Nordisk Foundation (NNF19OC0055001)

Non-redundant roles in sister chromatid cohesion of the DNA helicase DDX11 and the SMC3 acetyl transferases ESCO1 and ESCO2.

In a process linked to DNA replication, duplicated chromosomes are entrapped in large, circular cohesin complexes and functional sister chromatid cohesion (SCC) is established by acetylation of the SMC3 cohesin subunit. Roberts Syndrome (RBS) and Warsaw Breakage Syndrome (WABS) are rare human developmental syndromes that are characterized by defective SCC. RBS is caused by mutations in the SMC3 acetyltransferase ESCO2, whereas mutations in the DNA helicase DDX11 lead to WABS. We found that WABS-derived cells predominantly rely on ESCO2, not ESCO1, for residual SCC, growth and survival. Reciprocally, RBS-derived cells depend on DDX11 to maintain low levels of SCC. Synthetic lethality between DDX11 and ESCO2 correlated with a prolonged delay in mitosis, and was rescued by knockdown of the cohesin remover WAPL. Rescue experiments using human or mouse cDNAs revealed that DDX11, ESCO1 and ESCO2 act on different but related aspects of SCC establishment. Furthermore, a DNA binding DDX11 mutant failed to correct SCC in WABS cells and DDX11 deficiency reduced replication fork speed. We propose that DDX11, ESCO1 and ESCO2 control different fractions of cohesin that are spatially and mechanistically separated.

CRISPR-based modular assembly of a UAS-cDNA/ORF plasmid library for more than 5500 Drosophila genes conserved in humans.

Construction of a genome-wide transgenic UAS-cDNA/ORF library in Drosophila based on the binary GAL4/UAS system has been severely hampered by technical difficulties, although genome-wide cDNA or ORF resources of Drosophila, human, and mouse have been publicly available for more than a decade. Here, we developed a new method named CRISPR-based modular assembly (CRISPRmass) for the high-throughput construction of a genome-wide UAS-cDNA/ORF library from publicly available cDNA/ORF resources. Through cleavage of shared vector sequences of cDNA/ORF plasmids by CRISPR/Cas9 and subsequent insertion of UAS modules by Gibson assembly, the procedure of construction of such a library by CRISPRmass is standardized as massively parallel two-step test tube reactions before bacterial transformation. Using CRISPRmass, we generated 5551 UAS-cDNA/ORF constructs covering 83% of the Drosophila genes conserved in humans in the Drosophila Genomics Resource Center (DGRC) Gold Collection, and among them, 5518 were generated within 3 mo by three people. Our results show that CRISPRmass allows modulization, simplicity, efficiency, and adaptability in the generation of a genome-wide UAS-cDNA/ORF plasmid library by using publicly available cDNA/ORF resources. CRISPRmass can be applied to editing various genome-wide libraries in general and is an alternative to Gateway technology in high-throughput plasmid library editing. Furthermore, the more than 5500 UAS-cDNA/ORF plasmids of Drosophila genes serve as a powerful resource for gain-of-function (GOF) screening in cultured cells and for generation of a transgenic UAS-cDNA/ORF library in Drosophila.

Abstract B134: Use of p19Arf/interferon-β immunotherapy in association with chemotherapy permits reduced drug dosage and avoids cardiotoxicity associated with doxorubicin

Abstract Background: A variety of methods have been shown to induce immunogenic cell death (ICD) and overcome the immunosuppressive tumor microenvironment, thus generating an effective immunotherapy. In our previous work, we have shown that non-replicating adenovirus mediated gene transfer of p19Arf and interferon-β (IFNβ) forms a strategic alliance between gene function and the vector, resulting in a multi-modal mechanism of cell death consistent with necroptosis and with the liberation of ICD molecules HMGB1, ATP and calreticulin in B16F10 mouse melanoma cells. We have performed vaccine and immunotherapy assays as well as in situ vaccination in order to show that our gene transfer approach contributes to an antitumor immune response that involves neutrophils, NK, CD4+ and CD8+ T-cells and cytokine production that is consistent with a Th1 profile. While promising so far, we strive to boost tumor cell killing while preserving the well-being of the study subject. Objective: To use our gene transfer approach in association with doxorubicin (Dox), a well-known inducer of ICD, in order to promote cell killing while permitting the use of reduced drug dosages, thus ameliorating collateral effects, including cardiopathy, and possibly benefitting the antitumor immune response. Materials and Methods: MCA205 and B16F10 cells were used in this study. The AdRGDPG vectors are non-replicating recombinant Ad5 viruses containing the RGD tripeptide modification of the virus fiber protein and which utilize a p53-responsive promoter (termed PG) to control transgene expression. The AdRGDPG-GFP, AdRGDPG-p19, and AdRGDPG-IFNβ vectors encode enhanced green fluorescent protein (GFP), the mouse cDNAs for p19Arf (a functional partner of p53) and IFNβ, respectively. Using flow cytometry (Attune, Thermo Fisher), accumulation of hypodiploid cells was revealed upon fixation and propidium iodide staining while caspase 3/7 activity was visualized using Cell Event Caspase 3/7 (Thermo Fisher). Alternatively, BLI (IVIS Spectrum, Perkin Elmer) was used to detect a DEVD-dependent luciferase reporter construct in vivo. Cardiac function was monitored using echocardiography (VisualSonics Vevo 2100 Imaging System) and data analyzed using VevoCQ LV analysis software (VisualSonics). All animal assays were performed according to and with the approval of the Committee for the Ethical Use of Animals, FM-USP. Results: Association of p19Arf/IFNβ gene transfer with Dox treatment in vitro improved cell killing and central composite rotational design analysis revealed that indeed, the association permits the application of less drug and/or virus while preserving high levels of death in either cell line. In vitro, caspase 3/7 activity was additive upon exposure of MCA205 cells to the associated treatments, yet Dox was sufficient in B16F10. In vivo, Dox (20 mg/kg) alone or in combination with in situ gene therapy was correlated with inhibition of MCA205 tumor progression, increased caspase 3/7 activity and prolonged survival. However, 20 mg/kg Dox provoked reduced cardiac function (left ventricular ejection and systolic fractions, stroke and systolic volumes). Strikingly, the sub-therapeutic dose of 10 mg/kg Dox was not correlated with cardiopathy, yet only when used in combination with in situ gene transfer did it effectively block tumor progression. Finally, immunotherapy of previously established MCA205 tumors using a therapeutic vaccine consisting of dying cells treated ex vivo with both gene transfer and Dox resulted in effective challenge tumor inhibition. Conclusions: Here we have clearly shown that the association of gene transfer with Dox enables the use of lower drug doses in order to both preserve cardiac function and inhibit tumor progression. Early evidence also suggests that this combination increases the immunogenicity of treated cells, inviting the development of potential immunotherapeutic strategies. Funding: Sao Paulo Research Foundation (FAPESP). Citation Format: Bryan E. Strauss, Ruan F.V. Medrano, Rodrigo Tamura, Samir A. Mendonça, Valker A. Feitosa, Rafael Dariolli, Thiago A. Salles, Aline Hunger, João P.P. Catani, Elaine G. Rodrigues. Use of p19Arf/interferon-β immunotherapy in association with chemotherapy permits reduced drug dosage and avoids cardiotoxicity associated with doxorubicin [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 B134.

Altered expression of long non-coding RNA GAS5 in digestive tumors.

Cancer has become one of the most important diseases that affect human health and life. The effects of cancer in the digestive system are particularly prominent. Recently, long non-coding RNA (lncRNA) has attracted the attention of more and more researchers and has become an emerging field of gene research. The lncRNA growth arrest-specific 5 (GAS5) is a novel lncRNA that has attracted the attention of researchers in recent years and plays an important role in the development of tumors, especially in digestive system tumors. GAS5 was first identified in a mouse cDNA library. It was generally considered that it has the role of tumor suppressor genes, but there are still studies that have a certain ability to promote cancer. Furthermore, the 5-bp indel polymorphism (rs145204276) in the GAS5 promoter region also has a carcinogenic effect. The discovery of GAS5 and in-depth study of single nucleotide polymorphism (SNP) mechanism can provide a new way for the prevention and treatment of digestive system tumors.

Identification of Novel Binding Partners for Transcription Factor Emx2.

The mammalian homolog of Drosophila empty spiracles 2 (Emx2) is a homeobox transcription factor that plays central roles in early development of the inner ear, pelvic and shoulder girdles, cerebral cortex, and urogenital organs. The role for Emx2 is best understood within the context of the development of the neocortical region of the cortex, where Emx2 is expressed in a high posterior-medial to low anterior-lateral gradient that regulates the partitioning of the neocortex into different functional fields that perform discrete computational tasks. Despite several lines of evidence demonstrating an Emx2 concentration-dependent mechanism for establishing functional areas within the developing neocortex, little is known about how Emx2 physically carries out this role. Although several binding partners for Emx2 have been identified (including Sp8, eIF4E, and Pbx1), no screens have been used to identify potential protein binding partners for this protein. We utilized a yeast two-hybrid screen using a library constructed from embryonic mouse cDNA in an attempt to identify novel binding partners for Emx2. This initial screen isolated two potential Emx2-binding partner proteins, Cnot6l and QkI-7. These novel Emx2-binding proteins are involved in multiple levels of mRNA metabolism that including splicing, mRNA export, translation, and destruction, thus making them interesting targets for further study.

Oligomerization and cell surface expression of recombinant GABAA receptors tagged in the δ subunit.

The γ-Aminobutyric acid type A receptors (GABAARs) are heteropentameric chloride channels responsible for primary inhibition in the mammalian brain. Studies have shown the expression of recombinant GABAAR subunits tagged with the green fluorescent protein (GFP), a 26.9 kDa protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. This allows the formation of recombinant proteins essential for the development of relevant in-vitro and in-vivo methodologies. Among the GABAAR subunits, the δ subunit was never tagged in its cytoplasmic domain, an evolutionary conserved domain found in between the third and the fourth transmembrane domains. In this study, first, we have cloned the mouse cDNAs encoding for the δ, α1, β2 subunits of GABAARs, and then developed two fusion proteins of δ subunit each tagged with the GFP variant, EGFP (enhanced GFP) at unique sites in the cytoplasmic domain. The recombinant proteins were expressed alone or in combination with α1 and/or β2 subunits in neuroblastoma 2a cells. Live cell confocal microscopy indicated that the cytoplasmically tagged δ subunits were targeted to the cell membrane when expressed in the presence of α1 and β2 subunits in neuroblastoma 2a cells. However, this was not observed when they were expressed alone or only with α1 or β2 subunits in the same cell line. These results confirm the general oligomerization and targeting pattern of GABAAR subtypes described in the other in-vitro studies in the literature. Thus, our results suggest that the EGFP tagging in the ctoplasmic domain did not interfere with the oligomerization and cell surface expression of recombinant δ subunits. To our knowledge, this is the first study showing the generation, expression and preliminary analysis of the δ-GABAARs tagged in the cytoplasmic domain of the δ subunit which can be further elaborated to probe intracellular protein interactions of GABAARs via the δ subunit.

Screening and Identification of the Host Proteins Interacting with Toxoplasma gondii Rhoptry Protein ROP16.

Toxoplasma gondii, as a zoonotic protozoan parasite, develops sophisticated strategies to manipulate hosts for efficient intracellular survival. After successful invasion, T. gondii injects many effector proteins into host cells for various purposes. TgROP16 (T. gondii rhoptry protein 16), which is secreted from rhoptries into host cells, can activate the host STAT (signal transducer and activator of transcription) signaling pathway through phosphorylation of STAT3 and STAT6. However, whether there are other host proteins modulated by TgROP16 is currently unknown. In this study, yeast two-hybrid (Y2H) screen was used to look for additional host proteins interacting with TgROP16. Yeast cells expressing a mouse cDNA library cloned into the prey vector were used to mate with yeasts expressing ROP16 without signal peptide. Two mouse proteins, Dnaja1 (DnaJ heat shock protein family member A1) and Gabra4 (gamma-aminobutyric acid A receptor, subunit alpha 4) were identified to interact with ROP16 from this screen. Further analysis suggested that the Predomain of ROP16 played key roles in mediating interactions with these host proteins, whereas the contribution from the Kinase domain was minor. The interactions between Dnaja1 and different parts of ROP16 were also estimated in vivo by co-immunoprecipitation. The results showed that the Predomain of ROP16 was the major region to interact with Dnaja1, which is consistent with the Y2H results. Based on the gene ontology analysis, Dnaja1 is predicted to participate in stress response while Gabra4 is involved in the system development process. The discovery of new host proteins that interact with ROP16 of T. gondii will help us to further investigate the functions of this effector proteins during T. gondii infection.

Amplification of nonspecific products in quantitative polymerase chain reactions (qPCR)

Quantitative PCR allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, a survey with 93 validated assays for genes in the Wnt-pathway showed that the amplification of nonspecific products occurs frequently and is unrelated to Cq or PCR efficiency values. Titration experiments showed that the occurrence of low and high melting temperature artifacts was shown to be determined by annealing temperature, primer concentration and cDNA input. To explore the range of input variations that occur in the normal use of the Cre assay these conditions were mimicked in a complete two-way design of template −plasmid DNA- and non-template −mouse cDNA- concentrations. These experiments showed that the frequency of the amplification of the correct product and the artifact, as well as the valid quantification of the correct product, depended on the concentration of the non-template cDNA. This finding questions the interpretation of dilution series in which template as well as non-template concentrations are simultaneously decreasing. Repetition of this cDNA concentration experiment with other templates revealed that exact reproduction qPCR experiments was affected by the time it takes to complete the pipetting of a qPCR plate. Long bench times were observed to lead to significantly more artifacts. However, the measurement of artifact-associated fluorescence can be avoided by inclusion of a small heating step after the elongation phase in the amplification protocol. Taken together, this trouble-shooting journey showed that reliability and reproducibility of qPCR experiments not only depends on standardization and reporting of the biochemistry and technical aspects but also on hitherto neglected factors as sample dilution and waiting times in the laboratory work flow.

Extracellular and Non-Chaperone Function of Heat Shock Protein−90α Is Required for Skin Wound Healing

Despite years of effort and investment, there are few topical or systemic medications for skin wounds. Identifying natural drivers of wound healing could facilitate the development of new and effective treatments. When skin is injured, there is a massive increase of heat shock protein (Hsp) 90α inside the wound bed. The precise role for these Hsp90α proteins, however, was unclear. The availability of a unique mouse model that lacked the intracellular ATPase-driven chaperoning, but spared the extracellular fragment-5-supported pro-motility function of Hsp90α allowed us to test specifically the role of the non-chaperone function of Hsp90α in normal wound closure. We found that the chaperone-defective Hsp90α-Δ mutant mice showed similar wound closure rate as the wild-type Hsp90α mice. We generated recombinant proteins from the mouse cDNAs encoding the Hsp90α-Δ and wild-type Hsp90α. Topical application of Hsp90α-Δ mutant protein promoted wound closure as effectively as the full-length wild-type Hsp90α protein. More importantly, selective inhibition of the extracellular Hsp90α-Δ protein function by a monoclonal antibody targeting the fragment-5 region disrupted normal wound closure in both wild-type Hsp90α and Hsp90α-Δ mice. Thus, this study provides direct support for non-chaperone, extracellular Hsp90α as a potential driver for normal wound closure.

ID: 1021 Experimental reprogramming of murine embryonic fibroblasts towards induced pluripotent stem cells using a single polycistronic vector

Background: Embryonic stem cells are pluripotent, thus capable of differentiating into all types of cells derived from the three germ layers. However, the application of embryonic stem cells (ESCs) for preclinical and clinical studies is difficult due to ethical concerns. Induced pluripotent stem cells (iPSCs) are derived from differentiation and have many ESC characteristics. The study herein examines the production of iPSCs from reprogramming of mouse embryonic fibroblasts (MEFs) via transduction with defined factors.
 Methods: MEFs were collected from mouse embryos via a previously published protocol. The cells were transduced with a single polycistronic viral vector encoding mouse cDNAs of Oct3/4, Sox2, Klf4 and c-Myc. Transduced cells were treated and sub- cultured with ESC medium. The cells were evaluated as iPSCs with specific morphology, and expression SSEA-1, Oct3/4, Sox2 and Nanog. In addition, they were also evaluated for pluripotency by assessing alkaline phosphatase (AP) activity and in vivo teratoma formation.
 Results: Under the reprogrammed conditions, the transduced cells displayed a change in morphology, forming ESC-like clusters. These cell clusters strongly expressed pluripotent markers as well as ESC-specific genes. Furthermore, the colonies exhibited higher AP activity and formed teratomas when injected into the murine testis.
 Conclusion: The study herein suggests that MEFs can be reprogrammed into iPSCs using a polycistronic viral vector encoding mouse cDNAs for Oct3/4, Sox2, Klf4 and c- Myc

PP.07.24] PLATELET-DERIVED GROWTH FACTOR B

Objective: Renin, a key component in the regulation of blood pressure, is produced by the highly specialized renal juxtaglomerular (JG) cells. These cells may be derived from vascular smooth muscle cells (VSMC) and they can reversibly differentiate in response to certain stimuli. Because these cells rapidly differentiate when removed from the kidney, the biochemical mechanism responsible for this phenotypic plasticity is currently unknown. To overcome this limitation, we quantified gene expression in human renin-producing tumors (reninomas) and subsequently studied the effect of the most promising ligands on renin synthesis in (pro)renin-producing As4.1 cells, which are derived from a mouse JG cell-targeted tumor. Design and method: Transcriptome analysis was performed on four reninomas. The most highly expressed genes common in all reninomas were subsequently used for in situ hybridization in the mouse kidney. This approach yielded 43 genes, from which 12 ligands were selected. As4.1 cells were incubated for 48 hours with conditioned medium derived from human embryonic kidney (HEK) 293 cells transfected for 48 hours with the mouse cDNA encoding these ligands. Subsequently, As4.1 medium, and cell lysate or RNA were collected, and (pro)renin was measured in these samples by enzyme-kinetic assay. Results: Under control conditions, As4.1 cell medium contained predominantly (>95%) prorenin. In contrast, cell lysates contained renin only, at levels corresponding to <1% of the total amount of (pro)renin in the medium (i.e., 161 ± 61 μg angiotensin I/ml.hr, mean ± SEM). Among the tested ligands, only platelet-derived growth factor B (PDGF-B) affected the medium and cellular (pro)renin levels, as well as As4.1 renin gene expression, inducing parallel decreases of 64 ± 5%, 53 ± 10% and 84 ± 5%, respectively. Additionally, PDGF-B-exposed As4.1 cells displayed a more elongated and aligned shape with no apparent alteration in their viability. This was accompanied by a downregulated expression of α-smooth muscle actin (P < 0.01), and an upregulated expression of interleukin-6 (P < 0.0001), suggesting a phenotypic shift from myo-endocrine to inflammatory. No significant changes in the JG cell marker aldo-keto reductase 1B7 (Akr1b7) were observed. Conclusions: PDGF-B might be one of the factors involved in JG cell phenotypic plasticity.

Effect of mmu-miR-30b on mouse FoxO3 mRNA expression

Objective To analyze the direct interaction between mmu-miR-30b and mouse FoxO3 (mFoxO3) mRNA. Methods Three target gene fragments, which were respectively 402, 123 and 299 bp in length, were amplified from mouse cDNA by PCR using specific primers and site-direct mutant primers. A complete mutant fragment was obtained by joining together the 123 and 299 bp fragments. Recombinant plasmids, pmirGLO-mFoxO3 and pmirGLO-mFoxO3 mt, were constructed through inserting wild and mutant fragments of mFoxO3 into pmirGLO vector, respectively. HEK 293T cells were respectively co-transfected with the constructed recombinant plasmids and mmu-miR-30b/mmu-miR-30b inhibitor. Dual-luciferase reporter assay system was used to determine the Firefly-Renilla luciferase activity in different groups. Western blot assay was performed to evaluate the regulatory effect of mmu-miR-30b on mFoxO3 expression. Results Restriction enzyme analysis and gene sequencing showed that the two recombinant plasmids, pmirGLO-mFoxO3 and pmirGLO-mFoxO3 mt, were constructed successfully. The activity of Firefly-Renilla luciferase in HEK 293T cells transfected with pmirGLO, pmirGLO+ mmu-miR-30b, pmirGLO+ mmu-miR-30b inhibitor, pmirGLO-mFoxO3+ mmu-miR-30b, pmirGLO-mFoxO3+ mmu-miR-30b+ mmu-miR-30b inhibitor, or pmirGLO-mFoxO3 mt+ mmu-miR-30b was 13.18±0.97, 14.35±0.99, 12.46±1.20, 9.55±1.11, 13.71±0.89 and 10.99±0.92, respectively. Compared with pmirGLO+ mmu-miR-30b group, the luciferase activity was significantly decreased in pmirGLO-mFoxO3+ mmu-miR-30b group (P 0.05). In addition, the suppressed Firefly-Renilla luciferase activity in pmirGLO-mFoxO3+ mmu-miR-30b group was restored by mmu-miR-30b inhibitor treatment (P<0.05). Enhancing the expression of mmu-miR-30b could markedly inhibit the expression of mFoxO3 at protien level (P<0.05), and that could be significantly attenuated by mmu-miR-30b inhibitor treatmeat (P<0.05). Conclusion mFoxO3 mRNA is a novel target gene of mmu-miR-30b. There is a direct interaction between mmu-miR-30b and mFoxO3 mRNA. Key words: mmu-miR-30b; Mouse FoxO3; Target gene

A Cell Fusion-Based Screening Method Identifies Glycosylphosphatidylinositol-Anchored Protein Ly6e as the Receptor for Mouse Endogenous Retroviral Envelope Syncytin-A

Syncytin genes are envelope genes of retroviral origin that have been exapted for a role in placentation. They are involved in the formation of a syncytial structure (the syncytiotrophoblast) at the fetomaternal interface via their fusogenic activity. The mouse placenta is unique among placental mammals since the fetomaternal interface comprises two syncytiotrophoblast layers (ST-I and ST-II) instead of one, as observed in humans and all other hemochorial placentae. Each layer specifically expresses a distinct mouse syncytin, namely, syncytin-A (SynA) for ST-I and syncytin-B (SynB) for ST-II, which have been shown to be essential to placentogenesis and embryo survival. Their cognate cellular receptors, which are necessary to mediate cell-cell fusion and syncytiotrophoblast formation, are still unknown. By devising a sensitive method that combines a cell-cell fusion assay with the screening of a mouse cDNA library, we succeeded in identifying the glycosylphosphatidylinositol (GPI)-anchored membrane protein lymphocyte antigen 6E (Ly6e) as a candidate receptor for SynA. Transfection of cells with the cloned receptor led to their fusion to cells expressing SynA, with no cross-reactive fusion activity with SynB. Knocking down Ly6e greatly reduced SynA-induced cell fusion, thus suggesting that Ly6e is the sole receptor for SynA in vivo Interaction of SynA with Ly6e was further demonstrated by a competition assay using the soluble ectodomain of Ly6e. Finally, reverse transcription-quantitative PCR (RT-qPCR) analysis of Ly6e expression on a representative panel of mouse tissues shows that it is significantly expressed in the mouse placenta together with SynA.IMPORTANCE Syncytin genes are envelope genes of endogenous retroviruses, co-opted for a physiological function in placentation. Syncytins are fusogenic proteins that mediate cell-cell fusion by interacting with receptors present on the partner cells. Here, by devising a sensitive in vitro fusion assay that enables the high-throughput screening of normalized cDNA libraries, we identified the long-sought receptor for syncytin-A (SynA), a mouse syncytin responsible for syncytiotrophoblast formation at the maternofetal interface of the mouse placenta. This protein, Ly6e (lymphocyte antigen 6E), is a GPI-anchored membrane protein, and small interfering RNA (siRNA) experiments targeting its deletion as well as a decoy assay using a recombinant soluble receptor show that Ly6e is the necessary and sufficient partner of SynA. Its profile of expression is consistent with a role in both ancestral endogenization of a SynA founder retrovirus and present-day placenta formation. This study provides a powerful general method to identify genes involved in cell-cell fusion processes.

Experimental reprogramming of murine embryonic fibroblasts towards induced pluripotent stem cells using a single polycistronic vector

Introduction: Embryonic stem cells are pluripotent, thus capable of differentiating into all types of cells derived from the three germ layers. However, the application of embryonic stem cells (ESCs) for preclinical and clinical studies is difficult due to ethical concerns. Induced pluripotent stem cells (iPSCs) are derived from differentiation and have many ESC characteristics. The study herein examines the production of iPSCs from reprogramming of mouse embryonic fibroblasts (MEFs) via transduction with defined factors.&#x0D; Methods: MEFs were collected from mouse embryos via a previously published protocol. The cells were transduced with a single polycistronic viral vector encoding mouse cDNAs of Oct3/4, Sox2, Klf4 and c-Myc. Transduced cells were treated and sub-cultured with ESC medium. The cells were evaluated as iPSCs with specific morphology, and expression SSEA-1, Oct3/4, Sox2 and Nanog. In addition, they also were evaluated for pluripotency by assessing alkaline phosphatase (AP) activity and in vivo teratoma formation.&#x0D; Results: Under the reprogrammed conditions, the transduced cells displayed a change in morphology, forming ESC-like clusters. These cell clusters strongly expressed pluripotent markers as well as ESC-specific genes. Furthermore, the colonies exhibited higher AP activity and formed teratomas when injected into the murine testis.&#x0D; Conclusions: The study herein suggests that MEFs can be reprogrammed into iPSCs using a polycistronic viral vector encoding mouse cDNAs for Oct3/4, Sox2, Klf4 and c-Myc.&#x0D;