Emerald Green Fluorescent Protein Research Articles

Impact of Fab1/Vac14 inhibition on β-1,3-glucanase localization at the tip in Saccharomyces cerevisiae

Deep mycosis is a severe fungal disease that could result in fatal outcomes. However, there is still a demand for highly effective and safe antifungal drugs, given the side effects of the existing treatments and the increase in the resistance to them. In this study, we evaluated the involvement of the lipid kinase Fab1 and its activator Vac14 (Fab1/Vac14) in tip growth in Saccharomyces cerevisiae INVSc1, along with their impact on cell proliferation, using a genetic approach to inhibit them. The results revealed that Fab1/Vac14 inhibition suppressed growth and caused an increase in the rate of β-1,3-glucanase (BGL2) fused with emerald green fluorescent protein (EmGFP) (BGL2-EmGFP) localization at the tip. The inhibition of the endocytic pathway using a lysosome inhibitor also resulted in an increased localization of BGL2-EmGFP at the tip. The overexpression of wild-type BGL2-EmGFP, but not that of the inactive mutant BGL2, led to a complete loss of the cell proliferation ability. These findings suggested that the Fab1/Vac14 complex could be a novel target for the development of antifungal drugs based on tip growth regulation, possibly via excessive cell wall degradation.

B cells secrete functional antigen-specific IgG antibodies on extracellular vesicles

B cells and the antibodies they produce are critical in host defense against pathogens and contribute to various immune-mediated diseases. B cells responding to activating signals in vitro release extracellular vesicles (EV) that carry surface antibodies, yet B cell production of EVs that express antibodies and their function in vivo is incompletely understood. Using transgenic mice expressing the Cre recombinase in B cells switching to IgG1 to induce expression of fusion proteins between emerald green fluorescent protein (emGFP) and the EV tetraspanin CD63 as a model, we identify emGFP expression in B cells responding to foreign antigen in vivo and characterize the emGFP+ EVs they release. Our data suggests that emGFP+ germinal center B cells undergoing immunoglobulin class switching to express IgG and their progeny memory B cells and plasma cells, also emGFP+, are sources of circulating antigen-specific IgG+ EVs. Furthermore, using a mouse model of influenza virus infection, we find that IgG+ EVs specific for the influenza hemagglutinin antigen protect against virus infection. In addition, crossing the B cell Cre driver EV reporter mice onto the Nba2 lupus-prone strain revealed increased circulating emGFP+ EVs that expressed surface IgG against nuclear antigens linked to autoimmunity. These data identify EVs loaded with antibodies as a novel route for antibody secretion in B cells that contribute to adaptive immune responses, with important implications for different functions of IgG+ EVs in infection and autoimmunity.

Identification of an N-terminal tag (580N) that improves the biosynthesis of fluorescent proteins in Francisella tularensis and other Gram-negative bacteria

Utilization of fluorescent proteins is widespread for the study of microbial pathogenesis and host-pathogen interactions. Here, we discovered that linkage of the 36 N-terminal amino acids of FTL_0580 (a hypothetical protein of Francisella tularensis) to fluorescent proteins increases the fluorescence emission of bacteria that express these recombinant fusions. This N-terminal peptide will be referred to as 580N. Western blotting revealed that the linkage of 580N to Emerald Green Fluorescent Protein (EmGFP) in F. tularensis markedly improved detection of this protein. We therefore hypothesized that transcripts containing 580N may be translated more efficiently than those lacking the coding sequence for this leader peptide. In support, expression of emGFPFt that had been codon-optimized for F. tularensis, yielded significantly enhanced fluorescence than its non-optimized counterpart. Furthermore, fusing emGFP with coding sequence for a small N-terminal peptide (Serine-Lysine-Isoleucine-Lysine), which had previously been shown to inhibit ribosomal stalling, produced robust fluorescence when expressed in F. tularensis. These findings support the interpretation that 580N enhances the translation efficiency of fluorescent proteins in F. tularensis. Interestingly, expression of non-optimized 580N-emGFP produced greater fluorescence intensity than any other construct. Structural predictions suggested that RNA secondary structure also may be influencing translation efficiency. When expressed in Escherichia coli and Klebsiella pneumoniae bacteria, 580N-emGFP produced increased green fluorescence compared to untagged emGFP (neither allele was codon optimized for these bacteria). In conclusion, fusing the coding sequence for the 580N leader peptide to recombinant genes might serve as an economical alternative to codon optimization for enhancing protein expression in bacteria.

Magnetic Janus micromotors for fluorescence biosensing of tacrolimus in oral fluids

Tacrolimus (FK506) is a macrolide lactone immunosuppressive drug that is commonly used in transplanted patients to avoid organ rejection. FK506 exhibits high inter- and intra-patient pharmacokinetic variability, making monitoring necessary for organ graft survival. This work describes the development of a novel bioassay for monitoring FK506. The bioassay is based on using polycaprolactone-based (PCL) magnetic Janus micromotors and a recombinant chimera receptor that incorporates the immunophilin tacrolimus binding protein 1A (FKBP1A) tagged with Emerald Green Fluorescent Protein (EmGFP). The approach relies on a fluorescence competitive bioassay between the drug and the micromotors decorated with a carboxylated FK506 toward the specific site of the fluorescent immunophilin. The proposed homogeneous assay could be performed in a single step without washing steps to separate the unbound receptor. The proposed approach fits the therapeutic requirements, showing a limit of detection of 0.8 ng/mL and a wide dynamic range of up to 90 ng/mL. Assay selectivity was evaluated by measuring the competitive inhibition curves with other immunosuppressive drugs usually co-administered with FK506. The magnetic propulsion mechanism allows for efficient operation in raw samples without damaging the biological binding receptor (FKBP1A-EmGFP). The enhanced target recognition and micromixing strategies hold considerable potential for FK506 monitoring in practical clinical use.

Production and applications of fluorobody from redox-engineered Escherichia coli

Efficient selection and production of antibody fragments in microbial systems remain to be a challenging process. To optimize microbial production of single-chain variable fragments (scFvs), we have chosen five model targets, 1) a hapten, Zearalenone (ZEN) mycotoxin, along with infectious agents 2) rabies virus, 3) Propionibacterium acnes, 4) Pseudomonas aeruginosa, and a cancer cell 5) acute myeloid leukemia cell line (HL-60). The scFv binders were affinity selected from a non-immunized human phage display scFv antibody library and genetically fused to the N-terminus of emerald green fluorescent protein (EmGFP). The scFv-EmGFP fusion constructs were subcloned into an expression vector, under the control of T7 promoter, C-terminally tagged with hexa-histidine and expressed in different Escherichia coli (E. coli) hosts. This enabled the detection of cells that expressed the correct scFv-EmGFP fusion, termed fluorobody, via bright fluorescent signal in the cytoplasm. Among the three E. coli hosts tested, an engineered E. coli B strain called SHuffle B that promotes disulfide bond formation in the cytoplasm appeared to be the most appropriate host. The recombinant fluorobodies were well expressed (2–8 mg/L), possessed the fluorescence property of EmGFP, and retained the ability to bind to their cognate targets. Their specific bindings were demonstrated by ELISA, fluorescence-linked immunosorbent assay (FLISA), flow cytometry, and fluorescent microscope imaging. The fluorobody expression platform in this study could be further adopted as a one-step immunostaining technique based on scFv, isolated from phage display library to numerous desired targets.Key points• E. coli SHuffle express T7 is a suitable expression host for scFv-EmGFP (fluorobody)• Only the clones harboring scFv-EmGFP plasmid will show bright fluorescent signal• This platform can be used to produce fluorobodies for numerous purposesGraphical abstract

Detection of human annexin A1 as the novel N-terminal tag for separation and purification handle

BackgroundSeveral fusion tags for separation handle have been developed, but the fused tag for simply and cheaply separating the target protein is still lacking.ResultsSeparation conditions for the human annexin A1 (hanA1) tagged emerald green fluorescent protein (EmGFP) in Escherichia coli were optimized via precipitation with calcium chloride (CaCl2) and resolubilization with ethylenediamine tetraacetic acid disodium salt (EDTA-Na2). The HanA1-EmGFP absorbing with other three affinity matrix was detected, only it was strongly bound to heparin Sepharose. The separation efficiency of the HanA1-EmGFP was comparable with purification efficiency of the His6-tagged HanA1-EmGFP via metal ion affinity chromatography. Three fluorescent proteins for the EmGFP, mCherry red fluorescent protein and flavin-binding cyan-green fluorescent protein LOV from Chlamydomonas reinhardtii were used for naked-eye detection of the separation and purification processes, and two colored proteins including a red protein for a Vitreoscilla hemoglobin (Vhb), and a brown protein for maize sirohydrochlorin ferrochelatase (mSF) were used for visualizing the separation process. The added EDTA-Na2 disrupted the Fe–S cluster in the mSF, but it showed little impact on heme in Vhb.ConclusionsThe selected five colored proteins were efficient for detecting the applicability of the highly selective hanA1 for fusion separation and purification handle. The fused hanA1 tag will be potentially used for simple and cheap affinity separation of the target proteins in industry and diagnosis.

Biosensing Tacrolimus in Human Whole Blood by Using a Drug Receptor Fused to the Emerald Green Fluorescent Protein.

Tacrolimus (FK506) is an immunosuppressant drug (ISD) used to prevent organ rejection after transplantation that exhibits a narrow therapeutic window and is subject to wide inter- and intra-individual pharmacokinetic fluctuations requiring careful monitoring. The immunosuppressive capacity of FK506 arises from the formation of a complex with immunophilin FKBP1A. This paper describes the use of FKBP1A as an alternative to common antibodies for biosensing purposes. Bioassays use recombinant FKBP1A fused to the emerald green fluorescent protein (FKBP1A-EmGFP). Samples containing the immunosuppressant are incubated with the recombinant protein, and free FKBP1A-EmGFP is captured by magnetic beads functionalized with FK506 to generate a fluorescence signal. Recombinant receptor-drug interaction is evaluated by using a quartz crystal microbalance and nuclear magnetic resonance. The limit of detection (3 ng mL-1) and dynamic range thus obtained (5-70 ng mL-1) fulfill therapeutic requirements. The assay is selective for other ISD usually coadministered with FK506 and allows the drug to be determined in human whole blood samples from organ transplant patients with results comparing favorably with those of an external laboratory.

Loss-of-Function Approach in the Embryonic Chick Retina by using Tol2 Transposon-Mediated Transgenic Expression of Artificial microRNAs.

The chick retina has long been an important model system in developmental neurobiology, with advantages including its large size, rapid development, and accessibility for visualization and experimental manipulations. However, its major technical limitation had been the lack of robust loss-of-function approaches for gene function analyses. This protocol describes a methodology of gene silencing in the developing chick retina that involves transgenic expression of artificial microRNAs (miRNAs) by using the Tol2 transposon system. In this approach, a Tol2 transposon plasmid that contains an expression cassette for the EmGFP (emerald green fluorescent protein) marker and artificial pre-miRNA sequences against a target gene is introduced into the embryonic chick retina with a Tol2 transposase expression construct by in ovo electroporation. In the transfected retinal cells, the transposase catalyzes the excision of the expression cassette from the transposon vector and its integration into host chromosomes, leading to the stable expression of miRNAs and the EmGFP protein. In our previous study, we have demonstrated that the expression of Nel, a glycoprotein that exerts multiple functions in neural development, can be significantly suppressed in the developing chick retina by using this technique. Our results indicate that this methodology induces a stable and robust suppression of gene expression and thus provides an efficient loss-of-function approach for studies of retinal development.

The role of three putative transcriptional regulators of Francisella tularensis during replication within host cells

Francisella tularensis is a pathogenic bacterium capable of replication within host monocytes and macrophages and is the causative agent of Tularemia. We utilized the attenuated live vaccine strain to model the infection and replication of F. tularensis in the mouse macrophage RAW 264.7 cell line. Our lab has previously identified three genes predicted to encode transcriptional regulators that are upregulated in the presence of human blood cells: FTL_0671, FTL_1199, and FTL_1665. The objective of this project is to determine if these genes play a role in the pathogenesis of F. tularensis. 
 Wild type F. tularensis LVS and mutant bacteria with FTL_0671, FTL_1199, and FTL_1665 gene deletions were constructed and engineered to express an extremely bright variant of Emerald Green Fluorescent Protein (encoded in the plasmid, pKHEG). These strains were used to infect RAW 264.7 macrophages and fluorescence was monitored over time using a plate reader. Our results suggest that FTL_1199 and FTL_1665 are attenuated for intracellular replication indicating that the products of the mutated genes may be important for pathogenesis.

Ten-Eleven Translocation Ablation Impairs Cardiac Differentiation of Mouse Embryonic Stem Cells.

Ten-eleven Translocation (TET) dioxygenases mediated DNA methylation oxidation plays an important role in regulating the embryonic stem cells (ESCs) differentiation. Herein, we utilized a CRISPR/Cas9 based genome editing method to generate single, double, and triple Tet-deficient mouse ESCs (mESCs) and differentiated these cells toward cardiac progenitors. By using emerald green fluorescent protein (GFP; emGFP) expression under the control of Nkx2.5 promoter as marker for cardiac progenitor cells, we discovered that Tet1 and Tet2 depletion significantly impaired mESC-to-cardiac progenitor differentiation. Single-cell RNA-seq analysis further revealed that Tet deletion resulted in the accumulation of mesoderm progenitors to hamper cardiac differentiation. Re-expression of the Tet1 catalytic domain (Tet1CD) rescued the differentiation defect in Tet-triple knockout mESCs. Dead Cas9 (dCas9)-Tet1CD mediated loci-specific epigenome editing at the Hand1 loci validated the direct involvement of Tet-mediated epigenetic modifications in transcriptional regulation during cardiac differentiation. Our study establishes that Tet-mediated epigenetic remodeling is essential for maintaining proper transcriptional outputs to safeguard mESC-to-cardiac progenitor differentiation.

Highly Efficient SARS-CoV-2 Infection of Human Cardiomyocytes: Spike Protein-Mediated Cell Fusion and Its Inhibition.

ABSTRACTSevere cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cardiomyocytes express the angiotensin-converting enzyme 2 (ACE2) receptor but not the transmembrane protease serine 2 (TMPRSS2) that mediates spike protein cleavage in the lungs. Nevertheless, SARS-CoV-2 infection of hiPSC-CMs was prolific; viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CMs, smooth-walled exocytic vesicles contained numerous 65- to 90-nm particles with canonical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand how SARS-CoV-2 spreads in hiPSC-CMs, we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm cell-to-cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin and furin-like proteases abolished cell fusion. A spike mutant with the single amino acid change R682S that disrupts the multibasic furin cleavage motif was fusion inactive. Thus, SARS-CoV-2 replicates efficiently in hiPSC-CMs and furin, and/or furin-like-protease activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform enables target-based drug discovery in cardiac COVID-19.IMPORTANCE Cardiac complications frequently observed in COVID-19 patients are tentatively attributed to systemic inflammation and thrombosis, but viral replication has occasionally been confirmed in cardiac tissue autopsy materials. We developed an in vitro model of SARS-CoV-2 spread in myocardium using induced pluripotent stem cell-derived cardiomyocytes. In these highly differentiated cells, viral transcription levels exceeded those previously documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread, we expressed a fluorescent version of its spike protein that allowed us to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin/furin-like protease cleavage site lost cytopathic function. Of note, the fusion activities of the spike protein of other coronaviruses correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 may cause cardiac damage by fusing cardiomyocytes.

Abstract 1543: Development of a novel ECAD-EmGFP reporter line for pancreatic cancer MET study and drug discovery

Abstract World-wide, metastasis continues to be the leading cause of death in cancer patients1. Although epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) have been implicated in the progression of cancer metastasis and drug resistance, their mechanistic pathway and specific role in disease progression is not fully understood2. As such, the development of a reporter line that enables the real-time monitoring of the changing status of cells will not only aid in dissecting the EMT/MET pathway in the research field, but could also become a robust platform for new cancer drug development. E-cadherin, an adhesion protein expressed in epithelial cells, is upregulated in pancreatic cancer cells during MET and is associated with an increase in tight junctions and apico-basal polarity, as well as a change in morphology3,4. Here, we developed a novel PANC-1 ECAD-EmGFP reporter line using CRISPR/Cas9 genome-editing technology. In this cell line, the emerald green fluorescent protein (EmGFP) reporter was incorporated into the last exon of the endogenous E-cadherin gene, enabling real-time monitoring of MET status in live cells. The ECAD-EmGFP knock-in allele was confirmed at genomic, transcriptional, and translational levels. Functional data revealed that miRNA-200 treatment induced the increased expression of ECAD-EmGFP, and decreased expression of Snail, a transcription factor associated with mesenchymal traits. These changes in marker gene expression, as well as the decrease in invasive capacity upon induction, suggest that cells have undergone MET. This cell line is a valuable tool for dissecting the molecular mechanisms underlying EMT and MET and for evaluating or screening compounds targeting EMT and MET in pancreatic cancer. 1. Dizon DS, et al. Clinical Cancer Advances 2016: Annual Report on Progress Against Cancer from the American Society of Clinical Oncology. J Clin Oncol 34: 987-1011, 2016. 2. Nieto MA, et al. EMT: 2016. Cell 166: 21-45, 2016. 3. Polireddy K, et al, Targeting Epithelial-Mesenchymal Transition for Identification of Inhibitors for Pancreatic Cancer Cell Invasion and Tumor Spheres Formation. PLOS ONE 11(10): e0164811, 2016 4. Wong S, et al. E-cadherin: Its dysregulation in carcinogenesis and clinical implications. Critical Reviews in Oncology / Hematology 121: 11- 22, 2018 Citation Format: Diana Douglas, Sangeeta Kumari, Jarkko Huuskonen, Weiguo Shu. Development of a novel ECAD-EmGFP reporter line for pancreatic cancer MET study and drug discovery [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 1543.

Abstract 1885: CRISPR/Cas9 engineered immortalized breast epithelial MCF10A reporter line for EMT studies and anti-cancer drug discovery

Abstract Metastasis is responsible for most cancer-related deaths. One model for the mechanism of metastasis involves epithelial-to-mesenchymal transition (EMT), a process characterized by the decrease in cell adhesion and increase in cell motility. Cells undergoing EMT often display downregulation of epithelial markers (such as, E-cadherin) and upregulation of mesenchymal markers (such as, Vimentin). Besides metastasis, EMT has also been reported to be associated with other pathological conditions, such as acquired therapeutic drug resistance. Given the roles that EMT may play in these various pathological processes, it is of increasing interest as a target for anti-cancer treatment and drug discovery. In vitro reporter models have proven to be a valuable tool for dissecting the signaling pathways that regulate the EMT process and for screening compounds targeting EMT. In previously developed EMT reporter cell lines, the reporter gene was driven by a truncated EMT marker gene promoter. Therefore, the establishment of a more physiologically relevant reporter cell models is critical for advancing our knowledge of EMT. E-cadherin, a hallmark of epithelial cells, has been implicated in the onset of metastatic dissemination. Utilizing CRISPR/Cas9 knock-in technology, we have generated an E-cadherin (ECAD)-emerald green fluorescent protein (EmGFP) reporter model using immortalized breast epithelial MCF10A cells. In the reporter cells, EmGFP gene was tagged at the C-terminus of E-cadherin, allowing for real-time monitoring of EMT progression in live cells. The targeted knock-in of ECAD-EmGFP allele was verified at the genomic DNA, transcript (mRNA), and protein levels. Bio-functional evaluation of the reporter cell line revealed that treatment of ECAD-EmGFP reporter cells with TGFβ led to EMT induction, as demonstrated by a reduction in E-cadherin-GFP expression and increase in vimentin and fibronectin expressions. Additional functional characterization revealed that the reporter cells possessed an enhanced migration capacity upon EMT induction with TGF-β. In summary, this MCF10A-Ecad-EmGFP reporter cell line can serve as a physiologically relevant in vitro cell model for studying EMT cancer biology and anti-EMT drug discovery. Citation Format: Weiguo Shu, Sangeeta Kumari, Diana Douglas, Luis G. Rodriguez, Robert Newman. CRISPR/Cas9 engineered immortalized breast epithelial MCF10A reporter line for EMT studies and anti-cancer drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1885.

Abstract 1045: CRISPR/cas9 mediated generation of an EMT reporter cell line for metastatic breast cancer drug discovery and development

Abstract Among women, breast cancer continues to be the most common cancer, with metastasis being the leading cause of mortality in cancer patients around the world (DIZON et al. 2016). Epithelial to mesenchymal transition (EMT) - the process by which epithelial cells shift to the mesenchymal state, has been implicated in many aspects of breast cancer tumorigenesis, metastasis and drug resistance ). The accumulation of a large body of data on the association of EMT with cancer over the years has not resulted in EMT being an active target for therapeutic development. This is due in part to the lack of appropriate in vitro models. Here we have exploited some of the basic biology of EMT, to create an advanced in vitro metastatic breast cancer reporter cell line model for use in both basic research and discovery of new EMT inhibitors. During EMT, E-cadherin protein expression is down regulated in cancer cells in association with the loss of cell-to-cell adhesion, apico-basal polarity and a change to a spindle-shaped morphology. By installing an emerald green fluorescent protein (EmGFP) tag on the C-terminus of the e-cadherin (ECAD) gene in the epithelial BT-474 breast cancer cell line via CRISPR/Cas9 genome editing, end-point or real-time EMT status of cells can be tracked under defined conditions. The EMT reporter cell line was verified at the nucleic acid (genomic and mRNA) and protein levels as well as in cell-based assays. Bio-functional evaluation of the BT-474 ECAD EmGFP cell line, shows that it responds to EMT induction. The subsequent EMT status of cells can be monitored in real time by observing and measuring ECAD EmGFP expression, in addition, the resulting mesenchymal cells have increased invasive capacities. Furthermore, this EMT reporter cell line shows sensitivity to the MEK1/2 inhibitor U0126 - thereby providing the basis for the use of this cell line in high-throughput screening (HTS) applications including the identification of new anti-EMT drugs for metastatic breast cancer. The BT-474 ECAD EmGFP reporter cell line is also a convenient and sensitive model for basic science research on the mechanisms of metastasis. References DIZON, D. S., L. KRILOV, E. COHEN, T. GANGADHAR, P. A. GANZ et al., 2016 Clinical Cancer Advances 2016: Annual Report on Progress Against Cancer From the American Society of Clinical Oncology. J Clin Oncol 34: 987-1011. HAY, E. D., 1995 An overview of epithelio-mesenchymal transformation. Acta Anat (Basel) 154: 8-20. Citation Format: Metewo S. Enuameh, Weiguo Shu, Robert Newman. CRISPR/cas9 mediated generation of an EMT reporter cell line for metastatic breast cancer drug discovery and development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1045.

A dual signal immunochromatographic strip for the detection of imidaclothiz using a recombinant fluorescent-peptide tracer and gold nanoparticles

Immunochromatographic strips (ICS) attract much attention as practical analytical tools, particularly for point of care testing. However, ICS suffer from insufficient sensitivity, especially for small molecules. Competitive assays developed with analyte peptidomimetics obtained from phage display peptide libraries, usually enable to attain lower detection limits than the widely used chemical haptens, but are rarely used in ICS assay because of the shortcomings of the phage particle. In this work, we used a phage-free fluorescent peptide tracer (C2-15-EmGFP) for ICS, which consists of the emerald green fluorescent protein (EmGFP) fused to a peptidomimetic of imidaclothiz (C2-15). C2-15-EmGFP works as fluorescent donor and colloidal gold nanoparticles (AuNPs) coated with an anti-imidaclohtiz antibody as quencher, which allowed to develop a fluorescence quenching-based ICS (FQICS) for the detection of imidaclothiz in agricultural and environmental samples. The colorimetric and fluorescent signals were determined by both naked eye and imageJ software. Quantitative image analysis showed that the sensitivities obtained using the colorimetric (50% inhibitory concentration (IC50): 9.62 ng mL−1) and fluorescent (50% saturation concentration (SC50): 10.1 ng mL−1) signals were similar. The visual detection by the naked eye showed a sensitivity of 8.00 ng mL−1 with fluorescent readout which was 8-fold lower than that of the colorimetric readout (64.0 ng mL−1). The FQICS performed with excellent recoveries and good correlation with high-performance liquid chromatography in different matrices.

Development of a Cell-Free Optical Biosensor for Detection of a Broad Range of Mercury Contaminants in Water: A Plasmid DNA-Based Approach.

Mercury (Hg) is one of the main water contaminants worldwide. In this study, we have developed both whole-cell and cell-free biosensors to detect Hg. Genetically modified plasmids containing the merR gene were used to design biosensors. Firefly luciferase (LucFF) and emerald green fluorescent protein (EmGFP) genes were separately introduced as a reporter. Both constructs showed a detection limit of 1 ppb (Hg) in Escherichia coli and the cell-free system. We found that higher concentrations of Hg become detrimental to bacteria. This cytotoxic effect shows an anomalous result in high Hg concentrations. This was also observed in the cell-free system. We found that EmGFP fluorescence was decreased in the cell-free system because of a change in pH and quenching effect by Hg excess. Once the pH was adjusted to 7 and a chelating agent was used, the EmGFP fluorescence was partially restored. These adjustments can only be done in the cell-free system after the GFP expression and not in whole cells where their number has been decreased because of toxicity. Therefore, we suggest the use of the cell-free-system, which not only reduces the total experimental time but also allows us to perform these postexperimental adjustments to achieve higher sensitivity. We would also recommend to perform more measurements at a time with different dilution factors to bring down the Hg concentration within the measurable limits or to use some other chelating agents which can further reduce the excess Hg concentration.

Localization of the key cell division determinant FtsZ in Francisella tularensis.

Francisella tularensis, the causative agent of tularemia, is a highly infectious gram-negative bacterium capable of replicating within macrophages, leukocytes, dendritic cells and epithelial cells. In addition, F. tularensis also invades erythrocytes. FtsZ, a homologue of eukaryotic tubulin, is an essential cell division protein in almost all bacteria. Polymerization of FtsZ into a contractile ring is a critical first step in division and the FtsZ ring acts in recruiting the cell wall synthesis machinery to the nascent septum. We have generated a recombinant fusion of ftsZftto emgfp (Emerald Green Fluorescent Protein) which can be expressed, in trans, under its presumed native promotor or a strong Francisella promoter (pGRP). As expected, FtsZft-Emgfp can be seen localizing as rings in F. tularensis. Our aim is to utilize fluorescently tagged FtsZ to investigate replication of F. tularensis in a range of in vitro host cell invasion assays and as a marker for viability to study the morphological and physiological differentiation of F. tularensis as it transitions into a viable but non-culturable (VBNC) state. (This research was made possible by NASA West Virginia Space Grant Consortium Training Grant #NNX15A101H and by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence).

Induction of T7 Promoter at Higher Temperatures May Be Counterproductive

Bacterial expression of recombinant proteins is the most popular and convenient method for obtaining large quantities of pure protein. The induction of T7 promoter with isopropyl-β-d-thiogalactopyranoside (IPTG) is widely used for expression of large quantities of proteins in Escherichia coli. It has been reported that basic T7 promoter is leaky and expresses cloned genes without induction. The effect of T7 promoter induction on expression of proteins at different temperature using flow cytometry has not yet been investigated. Green fluorescent protein (GFP) as a non-peptide tag can be used for protein solubility screening and for high-throughput optimization of expression conditions using flow cytometry. Therefore, flow cytometry was used to study the effect of induction on the expression of T7 promoter driven emerald GFP (emGFP) atvarious temperatures. We noticed that percentage of emGFP positive cells decreased instead of increasing upon induction at higher temperatures. Western blot analysis confirmed that the amount of total and soluble emGFP decreased in induced cells compared uninduced cells at higher temperatures. Our results indicate that induction of basic T7 promoter at higher temperature may not necessarily increase protein expression. While using a basic T7 promoter it is highly recommended to analyze the effect of induction on protein expression at various temperatures.

Recombinant, Fluorescent, Peptidomimetic Tracer for Immunodetection of Imidaclothiz.

Peptidomimetic and anti-immunocomplex peptides, which can be readily isolated from a phage-display library, have shown great potential for small-molecule immunoassay development because they typically improve the sensitivity and avoid the use of chemical haptens as coatings or tracer antigens. However, phage-borne peptides are unconventional immunoassay reagents, which greatly limits their use in commercial applications, and require secondary reagents for detection. In order to overcome these limitations, we used C2-15, a peptidomimetic of imidaclothiz, as a model peptide fused to emerald-green fluorescent protein (EmGFP) at the N-terminus (C2-15-EmGFP) and C-terminus (EmGFP-C2-15) to generate novel fluorescent-peptide tracers. Both recombinant fluorophores reacted with similar affinity to the anti-imidaclothiz monoclonal antibody 1E7, but because of its higher expression, C2-15-EmGFP was chosen to develop a competitive magnetic-separation fluorescence immunoassay (MSFIA). After a competitive step with the analyte, the C2-15-EmGFP-antibody complex bound to the magnetic beads was separated with a magnet, and because of the fast dissociation of the peptide-antibody interaction, the fluorescence signal was detected following the spontaneous dissociation of the complex in fresh buffer. The concentration of imidaclothiz causing the 50% inhibitory concentration (IC50) was 11.00 ng mL-1, and the MSFIA performed with excellent recovery and had a good correlation with high-performance liquid chromatography in different matrices.

P.2.005 - Knock-down of G protein-coupled receptor 88 in the striatum reduces psychiatric symptoms in a rat non-motor model of Parkinson’s disease

Although Parkinson's disease (PD) is commonly defined as a progressive motor disorder, it entails many psychiatric non-motor symptoms (NMS) such as depression and cognitive alterations that are irresponsive to L-DOPA treatment and are thus in need of new and more specific targets for their treatment. This underpins interest in Gpr88, a brain-specific orphan G-protein coupled receptor whose expression in the striatal medium spiny neurons is modulated by L-DOPA as well as 6-hydroxydopamine (6OHDA) lesions [1]. Knock-down (KD) of Gpr88 in the ventral striatum (STR) normalizes motor and cognitive alterations in a rat model of psychosis [2], so in this study, we evaluated the impact of Gpr88-KD on depressed mood, sensorimotor gating and social recognition in a model of psychiatric NMS of PD. Partial lesions reproducing several psychiatric symptoms of PD were induced by bilateral stereotaxic injections of 6OHDA (12µg/hemisphere), or control saline, in the dorsolateral STR of rats. Then, Gpr88 was knocked-down using a lentiviral vector co-expressing the emerald Green Fluorescent Protein (emGFP) marker and either a specific (miR-Gpr88) or control (miR-neg) microRNA. These vectors were bilaterally injected into either the ventral (v), dorsomedial (dm), or dorsolateral (dl) STR (2 or 6µl/hemisphere, in the ventral or dorsal regions respectively). The rats were then tested for: depression with the forced swim test (FST); sensorimotor filtering with the Prepulse Inhibition test (PPI, prepulses ranging from 70-80dB and pulse = 110dB); and social recognition with the social novelty discrimination (SND) test, which measures discrimination of a novel from a familiar juvenile rat (score ≥ 3 in controls). The efficacy of the 6-OHDA lesion was quantified by the loss of Tyrosine Hydroxylase (TH) immunofluorescence, and the lentiviral transduction extent and location was validated by emGFP detection. Statistical analyses were performed using one-way ANOVA followed by Bonferroni corrections. Results are expressed as means ± SEM. The 6-OHDA lesion was limited to the dlSTR where it induced a significant loss of TH immunoreactivity (-57.10% ± 4.23, p Taken together, these results indicate that striatal Gpr88 KD moderates striatal dopamine depletion related alterations in mood, sensory motor gating and social discrimination in a model of NMS of PD. This study thus highlights the relevance of GPR88 as a potential target for the control of the psychiatric symptoms of PD, and possibly of other neuropsychiatric diseases.