scholarly journals Parasite Burden Measurement in the Leishmania major Infected Mice by Using the Direct Fluorescent Microscopy, Limiting Dilu-tion Assay, and Real-Time PCR Analysis

2020 ◽  
Author(s):  
Sepideh HAGHDOUST ◽  
Mahdieh AZIZI ◽  
Mostafa HAJI MOLLA HOSEINI ◽  
Mojgan BANDEHPOUR ◽  
Mandana MOHSENI MASOOLEH ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Major ◽  
Fluorescent Protein ◽  
Fluorescent Microscopy ◽  
Parasite Burden ◽  
Pcr Analysis ◽  
Green Fluorescent ◽  
Limiting Dilution

Background: We aimed to compare parasite burden in BALB/c mice, using three methods including the direct fluorescent microscopic using recombinant Leishmania major expressing an enhanced green fluorescent protein, limiting dilution assay, and real-time PCR technique. Methods: The current study was carried out in 2018, to induce stable enhanced green fluorescent protein (EGFP) production. Initially, the linearized DNA expression cassette (pLEXSY-egfp-sat2) was integrated into the ssu locus of L. major. The expression of EGFP in recombinant parasite was analyzed using direct fluorescent microscopy. Afterward, BALB/c mice were infected with the L. majorEGFP, and the infection was evaluated in the foot-pads and inguinal lymph-nodes using an in vivo imaging system. Subsequently, eight BALB/c mice were infected with L. majorEGFP, and the results of evaluating parasite burden by a SYBR-Green based real-time PCR analysis and the limiting dilution assays were compared to the results obtained from the direct fluorescent microscopy. Results: The results of the direct fluorescent microscopy showed that EGFP gene stably was expressed in parasites. Moreover, the in vivo imaging analysis of foot-pad lesions revealed that the infection caused by L. majorEGFP was progressing over time. Additionally, significant correlations were observed between the results of parasite burden assay using the direct fluorescent microscopy and either limiting dilution assay (r=0.976, P<0.0001) or quantitative real-time PCR assay (r=0.857, P<0.001). Conclusion: Ultimately, the utilization of the direct fluorescent microscopy by employing a stable EGFP-expressing L. major is a suitable substitution for the existing methods to quantify parasite burden.

scholarly journals Characterization of NGF, trkANGFR, and p75NTRin Retina of Mice Lacking Reelin Glycoprotein

2014 ◽  
Vol 2014 ◽  
pp. 1-13 ◽  
Author(s):  
Bijorn Omar Balzamino ◽  
Filippo Biamonte ◽  
Graziana Esposito ◽  
Ramona Marino ◽  
Francesca Fanelli ◽  
...  
Keyword(s):  
Real Time ◽  
Cross Talk ◽  
Real Time Pcr ◽  
Fluorescent Protein ◽  
Information Source ◽  
Bipolar Cells ◽  
Pcr Analysis ◽  
Additional Information ◽  
Ngf Receptor ◽  
Green Fluorescent

Both Reelin and Nerve Growth Factor (NGF) exert crucial roles in retinal development. Retinogenesis is severely impaired inE-reelermice, a model of Reelin deficiency showing specific Green Fluorescent Protein expression in Rod Bipolar Cells (RBCs). Since no data are available on Reelin and NGF cross-talk, NGF andtrkANGFR/p75NTRexpression was investigated in retinas fromE-reelerversus control mice, by confocal microscopy, Western blotting, and real time PCR analysis. A scattered increase of NGF protein was observed in the Ganglion Cell Layer and more pronounced in the Inner Nuclear Layer (INL). A selective increase ofp75NTRwas detected in most of RBCs and in other cell subtypes of INL. On the contrary, a slight trend towards a decrease was detected fortrkANGFR, albeit not significant. Confocal data were validated by Western blot and real time PCR. Finally, the decreasedtrkANGFR/p75NTRratio, representative ofp75NTRincrease, significantly correlated withE-reelerversus E-control. These data indicate that NGF-trkANGFR/p75NTRis affected inE-reelerretina and thatp75NTRmight represent the main NGF receptor involved in the process. This first NGF-trkANGFR/p75NTRcharacterization suggests thatE-reelermight be suitable for exploring Reelin-NGF cross-talk, representing an additional information source in those pathologies characterized by retinal degeneration.

scholarly journals Developing, Modifying, and Validating a TaqMan Real-Time PCR Technique for Accurate Identification of Leishmania Parasites Causing Most Leishmaniasis in Iran

2021 ◽  
Vol 11 ◽  
Author(s):  
Reza Fotouhi-Ardakani ◽  
Seyedeh Maryam Ghafari ◽  
Paul Donald Ready ◽  
Parviz Parvizi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Major ◽  
Taqman Probe ◽  
Amino Acid Permease ◽  
Specific Primers ◽  
Accurate Identification ◽  
Parasitic Load ◽  
Species Specific

Many laboratory methods are used to diagnose leishmaniasis because it is characterized by varied symptoms and caused by different Leishmania species. A quantitative real-time PCR method based on a TaqMan probe was developed and modified for accurate identification of human cutaneous leishmaniasis (caused by Leishmania major or Leishmania tropica) from endemic areas of Iran. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were considered. Six new sets of species-specific primers and probes were designed. A total of 123 samples were examined and employed to evaluate and validate real-time PCR. According to parasitic load of the genesig®Leishmania Advanced Standard Kit, a serial dilution of purified plasmid (2–2×107 copies/reaction) was prepared under the same conditions for both genes. Specific primers and probes were able to detect three and six parasite copies in AAP3 and COII genes, respectively, and were able to detect three copies of parasites for L. major and L. tropica. The sensitivities of the reference kit and our method were 98.7 and 98.1%, respectively, and specificity was 100% for detecting parasite genomes in all assays. Designed primers and probes performed well in terms of efficiency and regression coefficient. For AAP3 and COII genes, respectively, the linear log range was 7 and the correlation coefficient (R2) was 0.749 and 0.996 for the reference kit using the standard generated curve and 0.98 and 0.96 with serial dilutions of parasite DNA. This research detected L. major and L. tropica definitely and opens the horizon for the other scientists in the multiplex reactions in designing and optimization of the conditions in silico and in vivo.

Long-Term Increase in Fetal Hemoglobin Expression in Nonhuman Primates Following Transplantation of Autologous Bcl11a Nuclease-Edited HSCs

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2035-2035 ◽  
Author(s):  
Olivier Humbert ◽  
Hans-Peter Kiem
Keyword(s):  
Flow Cytometry ◽  
Real Time ◽  
Real Time Pcr ◽  
Peripheral Blood ◽  
Nonhuman Primates ◽  
Pcr Analysis ◽  
Beta Globin ◽  
Gamma Globin ◽  
Cd34 Cells

Abstract Elevated levels of fetal hemoglobin (HbF) ameliorate the clinical symptoms of beta-thalassemia and sickle cell anemia. The transcription factor B-cell lymphoma/leukemia 11A (BCL11A) is required for silencing of gamma-globin expression in adult erythroid cells and functions as a switch from fetal to adult hemoglobin production in humans. BCL11A therefore constitutes a therapeutic target for the treatment of hemoglobinopathies. We inactivated BCL11A function by double-strand DNA break-induced mutagenesis using Transcription Activator-Like Effector Nucleases (TALENs). 20 to 30% gene editing could be achieved in vitro in human and nonhuman primate CD34+ cells by TALEN mRNAs electroporation targeting exon 2 of Bcl11a. Colony-forming efficiency was slightly lower in Bcl11a-edited CD34+ cells but lineage differentiation potential was unchanged. Erythroid differentiation of CD34+ cells in culture showed increased Fetal to Beta hemoglobin ratio in both human and primate Bcl11a-modified cells as compared to control cells, thus validating our editing approach to increase HbF production. To determine if Bcl11a-edited hematopoietic stem cells (HSCs) could be engrafted and give rise to HbF-producing erythrocytes, we transplanted a pigtail macaque with autologous CD34+ electroporated with Bcl11a TALEN mRNA following conditioning by total body irradiation. We detected about 1 % gene disruption in vivo early post-transplant and disruption frequency gradually declined to reach a set point of about 0.3% starting at day 28 post-transplantation. In this analysis, which we have so far taken out to 42 days, single clones could be tracked based on their mutation signature, and we found that several clones persisted over time, confirming engraftment of Bcl11a-modified cells. Since the transplantation procedure and chemo-radiotherapy conditioning can raise HbF production, three control animals that were transplanted using similar conditions as with the Bcl11a-edited HSCs and one untransplanted animal were also included in our analysis. Flow cytometry measurement of HbF in peripheral blood showed a rapid increase in F-cell production in all animals, reaching levels that ranged from 10% to 40% by 30 days, while the untransplanted control showed basal HbF expression of about 0.5% (Fig. 1A). The peak for HbF expression lasted for about 140 days and eventually returned to basal levels that averaged 0.5% for all control animals. In comparison, the animal transplanted with Bcl11a-edited cells showed significantly higher HbF levels starting at day 140 post-treatment (1-1.5%), and HbF production has remained constant for at least 150 days. This result was confirmed by hemoglobin mRNA analysis in peripheral blood using real-time PCR. We found a rapid increase in gamma globin expression following transplantation, before returning to near basal levels. As compared to controls, the animal transplanted with Bcl11a-edited cells showed a 5 to 10-fold increase in gamma to beta globin ratio at day 140 and this ratio has remained constant ever since (Fig. 1B). We are currently working on ways to enhance Bcl11a-editing and to select for Bcl11a-modified HSCs using targeted integration of the chemoselection cassette P140K MGMT to ultimately achieve curative HbF production. Potential TALEN off-target sites will also be examined as well as any side effect associated with the inactivation of BCL11A. Overall, our data demonstrate that transplantation of Bcl11a-edited HSCs results in elevated HbF production in nonhuman primates. Furthermore, we show that nonhuman primates can serve as a useful model for novel gene editing strategies toward the treatment of hemoglobinopathies. Figure 1. In vivo monitoring of HbF expression by flow cytometry and real-time PCR. (A) Intracellular HbF staining of peripheral blood measured by flow cytometry. (B) Real-time PCR analysis of hemoglobin transcripts in RNA isolated from peripheral blood. Expression was normalized to GAPDH and %HbG is calculated as HbG/(HbG+HbB). HbG=gamma globin; HbB=beta globin. Black line=Bcl11a transplant; grey line=control transplant; dashed line=untransplanted control. Figure 1. In vivo monitoring of HbF expression by flow cytometry and real-time PCR. (A) Intracellular HbF staining of peripheral blood measured by flow cytometry. (B) Real-time PCR analysis of hemoglobin transcripts in RNA isolated from peripheral blood. Expression was normalized to GAPDH and %HbG is calculated as HbG/(HbG+HbB). HbG=gamma globin; HbB=beta globin. Black line=Bcl11a transplant; grey line=control transplant; dashed line=untransplanted control. Disclosures No relevant conflicts of interest to declare.

scholarly journals Rapid Screening Method for Compounds That Affect the Growth and Germination of Candida albicans, Using a Real-Time PCR Thermocycler

2011 ◽  
Vol 77 (22) ◽  
pp. 8193-8196 ◽  
Author(s):  
Lucja M. Jarosz ◽  
Bastiaan P. Krom
Keyword(s):  
Candida Albicans ◽  
Real Time ◽  
Real Time Pcr ◽  
Fluorescent Protein ◽  
Screening Method ◽  
Rapid Screening ◽  
Content Type ◽  
Wide Range ◽  
Hyphal Formation ◽  
Green Fluorescent

ABSTRACTWe propose a screening method for compounds affecting growth and germination inCandida albicansusing a real-time PCR thermocycler to quantify green fluorescent protein (GFP) fluorescence. Using PACT1-GFPand PHWP1-GFPreporter strains, the effects of a wide range of compounds on growth and hyphal formation were quantitatively assessed within 3 h after inoculation.

scholarly journals Flavin Mononucleotide-Based Fluorescent Reporter Proteins Outperform Green Fluorescent Protein-Like Proteins as Quantitative In Vivo Real-Time Reporters

2010 ◽  
Vol 76 (17) ◽  
pp. 5990-5994 ◽  
Author(s):  
Thomas Drepper ◽  
Robert Huber ◽  
Achim Heck ◽  
Franco Circolone ◽  
Anne-Kathrin Hillmer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Real Time ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Flavin Mononucleotide ◽  
Fluorescence Signal ◽  
Reporter Protein ◽  
Reporter Proteins ◽  
Green Fluorescent

ABSTRACT Fluorescent proteins of the green fluorescent protein (GFP) family are commonly used as reporter proteins for quantitative analysis of complex biological processes in living microorganisms. Here we demonstrate that the fluorescence signal intensity of GFP-like proteins is affected under oxygen limitation and therefore does not reflect the amount of reporter protein in Escherichia coli batch cultures. Instead, flavin mononucleotide (FMN)-binding fluorescent proteins (FbFPs) are suitable for quantitative real-time in vivo assays under these conditions.

scholarly journals Real-Time PCR and Immunocytochemical Study of Chondroitin Sulfate Proteoglycans after Scratch Wounding in Cultured Astrocytes / PCR I IMUNOCITOHEMIJSKA STUDIJA EKSPRESIJE HONDROITIN-SULFATNIH PROTEOGLIKANA NAKON POVREDE ASTROCITA U KULTURI

2013 ◽  
Vol 32 (4) ◽  
pp. 398-405
Author(s):  
Ana Parabucki ◽  
Anja Santrač ◽  
Danijela Savić ◽  
Sanja Dacić ◽  
Ivana Bjelobaba ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Complex Disease ◽  
In Vitro Models ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Cultured Astrocytes ◽  
Scratch Wound

Summary Background: Various in vivo and in vitro models have been described in order to elucidate the pathobiology underlying the traumatic brain injury (TBI) and test potentially suitable treatments. Since TBI is a complex disease, models differ in regard to the aspect of TBI that is being investigated. One of the used in vitro models is the scratch wound assay, first established as a reproducible, low-cost assay for the analysis of cell migration in vitro. The aim of the present study was to further investigate the relevancy of this model as a counter- part of in vivo TBI models. Methods: We have examined the astrocytic response to a mechanical injury in terms of expression of chondroitin sul- fate proteoglycans (CSPGs) - phosphacan, neurocan and brevican, using real-time PCR and immunocytochemistry. Results: Our results indicate that in vitro scratch wounding alters the expression profile of examined CSPGs. Four hours after the scratch injury of the astrocytic monolayer, real-time PCR analysis revealed upregulation of mRNA levels for phos- phacan (3-fold) and neurocan (2-fold), whereas brevican mRNA was downregulated (2-fold). Immunofluorescent sig- nal for phosphacan and neurocan was more intense in astro- cytes close to the injury site, while brevican was scarcely present in cultured astrocytes. Conclusions: Obtained results indicate that CSPGs are differ- entially expressed by astrocytes after scratch wounding, demonstrating that the scratch wound model might be suit- able for investigation of astrocyte-derived response to injury.

scholarly journals Genetically-encoded fluorescent biosensor for rapid detection of protein expression

2020 ◽  
Author(s):  
Matthew G Eason ◽  
Antonia T Pandelieva ◽  
Marc M Mayer ◽  
Safwat T Khan ◽  
Hernan G Garcia ◽  
...  
Keyword(s):  
Protein Expression ◽  
Real Time ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Live Cells ◽  
Spatiotemporal Resolution ◽  
E Coli ◽  
Fluorescent Biosensor ◽  
Green Fluorescent

Fluorescent proteins are widely used as fusion tags to detect protein expression in vivo. To become fluorescent, these proteins must undergo chromophore maturation, a slow process with a half-time of 5 to >30 min, which causes delays in real-time detection of protein expression. Here, we engineer a genetically-encoded fluorescent biosensor to enable detection of protein expression within seconds in live cells. This sensor for transiently-expressed proteins (STEP) is based on a fully matured but dim green fluorescent protein in which pre-existing fluorescence increases 11-fold in vivo following the specific and rapid binding of a protein tag (Kd 120 nM, kon 1.7 x 10^5 M-1s-1). In live E. coli cells, our STEP biosensor enables detection of protein expression twice as fast as the use of standard fluorescent protein fusions. Our biosensor opens the door to the real-time study of short-timescale processes in research model animals with high spatiotemporal resolution.

Retrovirus Insertion Site Analysis Following In Vivo Drug Selection in a Phase I Clinical Trial Utilizing G156A MGMT for Enhanced Chemotherapy of Advanced Malignancies.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3048-3048
Author(s):  
Colin L. Sweeney ◽  
Karen Lingas ◽  
Jane S. Reese ◽  
Susan Flick ◽  
Stanton L. Gerson
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Insertion Site ◽  
Pcr Analysis ◽  
Site Analysis ◽  
Cd34 Cells ◽  
Vector Copy Number ◽  
Post Infusion

Abstract The G156A mutant of the DNA repair gene O6-methylguanine DNA-methyltransferase (MGMT) confers hematopoietic resistance to O6-benzylguanine (BG) combined with DNA-alkylating agents BCNU or temozolomide, and allows for selective in vivo expansion with drug administration of murine hematopoietic progenitors transduced with G156A MGMT retrovirus. Here we report our latest findings on retroviral vector copy number and insertion site analysis following drug treatment from a Phase I clinical trial utilizing MGMT-mediated chemoprotection for enhanced treatment of advanced solid tumors. Seven patients have entered the trial and 6 have completed the cell infusion process. For all patients, autologous CD34+ cells were transduced ex vivo with an MFG retroviral vector containing the G156A MGMT gene (packaged with PG13 by the National Gene Vector Laboratory, Ken Cornetta, Director) in the presence of the fibronectin fragment CH-296 and the cytokines SCF, Tpo, and Flt-3 ligand for 72 hours with three additions of retroviral supernatant. At 72 hours following patient treatment with BG and BCNU, cells were re-infused. Prior to infusion, the average vector copy number by quantitative real-time PCR analysis for six patients was 0.34 copies per genome, with an average of 24% of CFUs transduced by standard PCR for G156A MGMT, and an average of 9% of CD34+ cells expressing the MGMT transgene by flow cytometry. In one patient with metastatic melanoma we have further analysis of insertions. For this patient, the pre-infusion vector copy number of the bulk CD34+ population was 0.54 copies per genome by real-time PCR, with 27% of CFUs transduced and 8% of CD34+ cells expressing the MGMT transgene prior to infusion. Linear amplification-mediated (LAM)-PCR analysis of retroviral insertion sites in pre-infusion CFUs from this patient confirmed a polyclonal population, with an average of 1.6 retroviral insertions per positive CFU. In this patient, BG (120 mg/m2) and BCNU (33 mg/m2) were administered at 6 weeks post-infusion, and temozolomide (300 mg/day for 5 days) was administered at 13 weeks. Peripheral blood (PB) and bone marrow (BM) granulocyte and mononuclear cells (MNCs) were collected at weeks 5, 11, 15, and 16 for DNA and CFU analysis. Vector copy number at all post-infusion time points was below the limit of detection of SYBR Green probe-based real-time PCR (&lt;100 copies of G156A MGMT per 5000 genomes). LAM-PCR detected the vector in post-treatment samples based on an internal vector control band present in BM MNCs at week 11 and in BM granulocytes at week 16, although specific insertion sites were not detected. Standard PCR revealed 1 out of 100 CFUs from week 11 BM MNCs contained the vector, with 2 out of 30 CFUs from week 15 PB MNCs. LAM-PCR in a subset of week 11 CFUs confirmed a single insertion site present in the same PCR-positive CFU. Sequence analysis of clonal vector insertions pre- and post-infusion is ongoing, and thus far a number of sites have been characterized, adding to the emerging database of clinical retroviral insertions. These are the first data to show emergence of transduced mutant MGMT cells after nonmyeloablative conditioning in humans and suggest that despite a low frequency of vector-marked hematopoietic cells, clinical in vivo drug selection can be observed.

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