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Subsequently, the in vivo antipseudomonal activity of the diastereomer was further evaluated after a longer-term treatment (i.e. 24 hours from the bacterial administration). Excitingly, in this case a more noticeable antibacterial effectiveness than that from 4 hours AMP treatment was observed, with approximately 2-log reduction in the lung bacterial burden compared to the control PBS-treated infected mice (Fig. 5a). Interestingly, a second instillation of the diastereomer (0.1 mg/kg) at 12 hours after bacterial inoculation did not further enhance the therapeutic efficacy against P. aeruginosa-induced respiratory infection. Similarly, the second administration of this AMP did not further decrease the infection level of the spleen (Fig. 5a).Figure 5Effect of single or double administration of Esc(1–21)-1c (panel a); LL-37 and colistin (panel b) on the number of viable P. aeruginosa. The effect of single or double dosage of Esc(1–21)-1c on total leukocytes was also investigated (panel c). Bacterial burden (CFU) in the lung and spleen of infected mice was determined at 24 hours after bacterial infection. Animals were intra-tracheally infected with PAO1 cells. The peptides were instilled intra-tracheally, at 2 μg (0.1 mg/kg) at 2 hours (for single administration) and at 2 hours and 12 hours (for double administration) after bacterial infection. The numbers of viable Pseudomonas cells in the lung and spleen as well as the number of inflammatory cells in the BAL were counted at 24 hours after the infection, as described above. Results are mean ± SEM from three independent experiments; n = 4–6 mice for each treatment group. Following one-way analysis of variance (ANOVA), posthoc comparisons were made using the Dunnett’s multiple comparison test when the P-value was significant (p < 0.05). *p < 0.05, **p < 0.01 for peptide-treated animals versus PBS-treated mice.
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| 100.0 |
Effect of single or double administration of Esc(1–21)-1c (panel a); LL-37 and colistin (panel b) on the number of viable P. aeruginosa. The effect of single or double dosage of Esc(1–21)-1c on total leukocytes was also investigated (panel c). Bacterial burden (CFU) in the lung and spleen of infected mice was determined at 24 hours after bacterial infection. Animals were intra-tracheally infected with PAO1 cells. The peptides were instilled intra-tracheally, at 2 μg (0.1 mg/kg) at 2 hours (for single administration) and at 2 hours and 12 hours (for double administration) after bacterial infection. The numbers of viable Pseudomonas cells in the lung and spleen as well as the number of inflammatory cells in the BAL were counted at 24 hours after the infection, as described above. Results are mean ± SEM from three independent experiments; n = 4–6 mice for each treatment group. Following one-way analysis of variance (ANOVA), posthoc comparisons were made using the Dunnett’s multiple comparison test when the P-value was significant (p < 0.05). *p < 0.05, **p < 0.01 for peptide-treated animals versus PBS-treated mice.
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| 100.0 |
Importantly, the efficacy of Esc(1–21)-1c in reducing lung bacterial burden at 24 hours after Pseudomonas challenge revealed to be comparable (about 2-log reduction of lung burden) to that found for the last resort of antibiotics, i.e. colistin, in another set of experiments (Fig. 5b). In comparison, the human LL-37 almost completely lost its antimicrobial potency (Fig. 5b), presumably due to its higher susceptibility to proteolytic degradation29.
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| 100.0 |
In consistence with the described results of significantly lower bacterial burden in the lungs after treatment with Esc(1–21)-1c, a substantial decrease in the total number of inflammatory cells was also recorded in the BAL of infected mice after a longer-term treatment with a single or double dosage of the diastereomer (at 0.1 mg/kg) compared to the control infected animals receiving the vehicle PBS only (Fig. 5c).
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| 100.0 |
The therapeutic efficacy of the diastereomer closely correlated with the diminished number of inflammatory cells in the BAL of the animals, even though the percentage of macrophages was slightly higher than that of controls. This very low percentage of macrophages within the total leukocytes presumably reflected the lower amount of bacterial burden and the minimally recruited neutrophils to the site of infection.
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| 100.0 |
Compared to infected mice treated with PBS, a marked reduction in the expression level of pro-inflammatory chemokines (e.g. CXCL-1, CXCL-2), cytokines (i.e. IL-6 and IL-17), and other inflammatory-related genes (Tnf-α, Nf-κb) was detected in the lungs of mice at 24 hours after treatment with a single or a double dosage of Esc(1–21)-1c at 0.1 mg/kg (Fig. 6a). Indeed, the gene-expression level was similar to that of untreated non-infected mice (control, Fig. 6a). This suggests that therapeutic treatments with the diastereomer led to a substantially attenuated inflammatory response upon P. aeruginosa-induced respiratory infection. Furthermore, bacterial infection resulted in noticeable induction of airway epithelial gene Ccsp but only mild changes in Pseudomonas-induced lung infection, after administration of Esc(1–21)-1c (Fig. 6b). Ccsp gene encodes for Clara/Club cell secretory protein, and serves as a marker for assessing the cellular integrity and permeability of the lung epithelium that could be induced after epithelial injury38. Our results clearly supported that bacterial infection-induced lung injury was significantly less after one or two dosage of Esc(1–21)-1c administration. The gene expression difference in foxj1 among various treatments was not significant.Figure 6Effect of Esc(1–21)-1c on the expression level of different inflammatory-related genes (panel a) and airway epithelial-associated genes (panel b). Gene expression in the lungs of P. aeruginosa infected mice after a single or a double peptide administration (at 0.1 mg/kg) was investigated at 24 hours after bacterial infection. Mice were intra-tracheally infected with 3 million PAO1 cells. The peptides were administered intra-tracheally at 2 μg (0.1 mg/kg) at 2 hours and 12 hours after the infection. After 24 hours from bacterial challenge, the RNA was extracted from the lung tissue and quantitative PCR was performed as described54. Results are mean ± SEM from three independent experiments; n = 4–6 mice for each treatment group. The t-test was used to compare the means of PBS-receiving non-infected mice (control) versus PBS-treated infected animals. One-way analysis of variance (ANOVA) was used peptide-treated infected mice and PBS-treated infected animals, posthoc comparisons were made using the Dunnett’s multiple comparison test when the P-value was significant (p < 0.05). *p < 0.05, **p < 0.01, ***p < 0.001 for peptide-treated infected mice or PBS-receiving non-infected mice (control) versus PBS-treated infected animals.
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| 100.0 |
Effect of Esc(1–21)-1c on the expression level of different inflammatory-related genes (panel a) and airway epithelial-associated genes (panel b). Gene expression in the lungs of P. aeruginosa infected mice after a single or a double peptide administration (at 0.1 mg/kg) was investigated at 24 hours after bacterial infection. Mice were intra-tracheally infected with 3 million PAO1 cells. The peptides were administered intra-tracheally at 2 μg (0.1 mg/kg) at 2 hours and 12 hours after the infection. After 24 hours from bacterial challenge, the RNA was extracted from the lung tissue and quantitative PCR was performed as described54. Results are mean ± SEM from three independent experiments; n = 4–6 mice for each treatment group. The t-test was used to compare the means of PBS-receiving non-infected mice (control) versus PBS-treated infected animals. One-way analysis of variance (ANOVA) was used peptide-treated infected mice and PBS-treated infected animals, posthoc comparisons were made using the Dunnett’s multiple comparison test when the P-value was significant (p < 0.05). *p < 0.05, **p < 0.01, ***p < 0.001 for peptide-treated infected mice or PBS-receiving non-infected mice (control) versus PBS-treated infected animals.
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| 100.0 |
The efficacy of Esc(1–21)-1c in alleviating respiratory Pseudomonal infection was further assessed by mouse lung histopathological evaluation. We observed the increased number of inflammatory cells after Pseudomonas infection in both airways and alveolar regions, but the increase was more prominent in the alveolar region (Fig. 7). There was clearly more neutrophilic infiltration in the alveoli of PBS-treated mice than in AMP-treated mice at 24 hours after infection (Fig. 7).Figure 7Histologic analysis of lung tissues with PAO1 challenge at 24 hours after bacterial infection. Mouse lung tissues were harvested, fixed and stained for histological evaluation without peptide treatment (B) or with a single (C) and double (D) administration of Esc(1–21)-1c at 0.1 mg/kg in comparison with non-infected and untreated samples. (A) Infected but untreated mice (B) exhibited more inflammation, airway lumen leukocyte accumulation (black arrow).
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| 100.0 |
Histologic analysis of lung tissues with PAO1 challenge at 24 hours after bacterial infection. Mouse lung tissues were harvested, fixed and stained for histological evaluation without peptide treatment (B) or with a single (C) and double (D) administration of Esc(1–21)-1c at 0.1 mg/kg in comparison with non-infected and untreated samples. (A) Infected but untreated mice (B) exhibited more inflammation, airway lumen leukocyte accumulation (black arrow).
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| 100.0 |
Respiratory infection is the most common lung disease, especially in CF sufferers39, 40 and P. aeruginosa is the most predominant lung pathogen in the CF population where it drives to progressive loss of respiratory functions and shortened survival of these patients. This is mainly due to its increasing resistance to the available antibiotics and adaptation within the lung environment, favoring persistence and chronic lung colonization by P. aeruginosa 41, 42.
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| 99.94 |
AMPs hold promise to act as lead compounds for the generation of novel therapeutic agents with fast and wide spectrum of activity, low capacity to induce resistance while displaying relevant immunomodulatory functions (e.g. LPS detoxification; wound-healing). However, a lot of AMPs loss their antimicrobial activity in biological environments and only a limited number of studies have been carried out to demonstrate the in vivo effect(s) of AMPs or derivatives in animal models of P. aeruginosa-induced lung infection.
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review
| 99.75 |
We previously proved that by changing the configuration of only two amino acids in Esc(1–21), with the corresponding d-enantiomers, the resulting diastereomer Esc(1–21)-1c had a (i) significantly higher biostability, (ii) lower cytotoxicity, (iii) better antibiofilm activity, (iv) stronger ability in killing Pseudomonas internalized into bronchial cells expressing a copy of functional or mutated CF-transmembrane conductance regulator (AF508 CFTR) and (v) a more pronounced wound-healing activity in monolayers of epithelial cells29. All these findings contributed to make Esc(1–21)-1c a very attractive candidate for the development of new drugs against Pseudomonas-induced lung infections. Nevertheless, no evidences on its in vivo antimicrobial efficacy were previously reported. Here, for the first time we demonstrated a remarkable in vivo antipseudomonal activity of the diastereomer Esc(1–21)-1c after a single local instillation in the lungs, with a higher potency than the all-L parent peptide and the widely used and characterized human AMP LL-37. Our in vivo studies underlined that 4 hours peptide treatment is not an ideal time for an optimal evaluation of the in vivo antimicrobial effectiveness of esculentin-derived AMPs, which presumably relies on a direct bactericidal activity of the peptide. However, at this stage we cannot exclude that the respiratory epithelial cell-mediated immune modulation could also contribute to the overall host antimicrobial activity. The bacterial killing activity by Esc(1–21) and Esc(1–21)-1c would prevent inhaled/inoculated bacteria from propagate in mouse lung. Nonetheless, the bacterial infection-induced host innate immunity may be able to provide additional killing mechanism to control the severity of bacterial infection and to reduce inflammatory response. Importantly, we have found out that a single intra-tracheal instillation of Esc(1–21)-1c, at a very low peptide dosage i.e. 20 μM (0.1 mg/kg), is sufficient to cause approximately 2-log reduction in the lung Pseudomonas burden, within 24 hours from bacterial challenge. The data suggest that the diastereomer Esc(1–21)-1c likely has a sustained residence time in the lungs, due to its higher biostability and resistance to proteolytic degradation than Esc(1–21) and LL-37, according to our previously published data28, 29. Thus, it would continue to display microbicidal effects at longer-term. In comparison, the in vivo antipseudomonal activity of Esc(1–21)-1c resulted to be similar in bacterial reduction to that of colistin, which is extensively used in clinical practice43. Note however that colistin is a peptide which is active only against Gram-negative bacteria and it easily induces microbial resistance31, 44, 45, most likely because of modifications to the phosphate groups of lipid A and core oligosaccharide moieties of LPS, weakening colistin binding to it. Yet, colistin resistance in humans without prior exposure to this peptide has been recently disclosed; and this is an important concern to public health46.
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| 100.0 |
Differently, the in vivo antibacterial efficacy of LL-37 was mostly lost at longer-term (Fig. 5b), probably due to its higher susceptibility to proteases29. This is in contrast with previous findings by Beagumont and colleagues showing that in a murine model of acute P. aeruginosa lung infection, LL-37 enhanced bacterial clearance in vivo only over 24 hours, by means of neutrophils recruitment, while no effect was obtained 6 hours after infection47. However, in their work, bacteria and peptide were co-administered via intranasal instillation rather than intra-tracheally. This latter administration route provides a more direct introduction of bacteria into the lungs and a more reproducible infection that can be more representative of pneumonia, generally leading to establishment of infection in the lower regions of the lungs48. In addition, intra-tracheal administration of a drug better mimics an aerosol-based therapy. Furthermore, immediate administration of LL-37 following bacterial inoculation as used in the paper47 did not allow sufficient time for the bacteria to adapt to the lung microenvironment before being exposed to LL-37. Note that this previously used experimental procedure could not provide a valuable indication of the antimicrobial effectiveness of AMPs in vivo. Thus, in this study, we purposely delayed the AMP administration at 2 hours after bacterial infection to better reflect the real life infectious conditions.
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| 99.94 |
Finally in our hands, LL-37 negatively disturbed bronchial epithelial cell tight junction (Fig. 1). This could potentially result in elevated cytotoxicity to the lung epithelial cells and limit its practical use in exogenously supplying viable therapy for lung bacterial infection.
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study
| 100.0 |
To the best of our knowledge, no indication on the effect of AMPs on the expression of airway epithelia-associated genes upon their administration to infected or non-infected lungs has been provided so far. This is a fundamental issue that deserves to be investigated in order to explore the clinical safety of a new drug. Indeed, besides experiencing the AMPs effect on the bacterial clearance, it is also meaningful to get insight into their potential beneficial/toxic effect on the lung epithelium, in vivo.
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study
| 99.94 |
Our findings have emphasized that two amino acids substitution in Esc(1–21) with the corresponding d-enantiomers can reduce the peptide’s cytotoxicity and withstand the feasibility of using the diasteremer to alleviate pneumonia severity. Furthermore, we have also shown that in addition to having a therapeutic outcome, the diastereomer does not induce any undesirable side effect at the lung, but it is able to abate the inflammatory response upon bacterial infection, as corroborated by the results of immune cell differential counts, gene expression and lung histology analysis.
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| 100.0 |
In summary, the results of our work suggest a strong therapeutic potential of diastereomer Esc(1–21)-1c in treating bacterial infection and concur to support further advanced preclinical studies aimed at developing it as a viable lead compound for the manufacture of new peptide-based formulation(s) for topical treatment of Pseudomonas lung infection.
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study
| 99.94 |
Synthetic Esc(1–21) and its diastereomer Esc(1–21)-1c (Table 1) were purchased from Chematek Spa (Milan, Italy). Briefly, each peptide was assembled by step-wise solid-phase synthesis using a standard F-moc strategy and purified via RP-HPLC to a purity of 98%, while the molecular mass was verified by mass spectrometry. Colistin sulphate was purchased from Sigma (St. Louis, MO) and the natural AMP human cathelicidin LL-37 (Table 1) was synthesized by Genscript (Piscataway, NJ). BronchialLife Epithelial Airway Medium was purchased from Lifeline Technology (Frederick, MD). B-ALI Bronchial Air Liquid Interface BulletKit was purchased from Lonza (Walkersville, MD). Eagle’s minimum essential medium (EMEM), was purchased from Sigma (St. Louis, MO), and tryptic soy broth (TSB) and trypitc soy agar (TSA) were purchased from MP Biomedicals (Santa Ana, CA).Table 1Primary structure of the peptides under study.PeptidePrimary structurea Esc(1–21)GIFSKLAGKKIKNLLISGLKG-NH2 Esc(1–21)-1cGIFSKLAGKKIKNLLISGLKG-NH2 LL-37LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES a d-amino acids are in italics and bolded.
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study
| 100.0 |
The Pseudomonas aeruginosa strain (PAO1, ATCC BAA-47) was used for all the experiments. For each experiment, an aliquot of bacteria was grown overnight at 37 °C in TSB with shaking at 225 rpm to achieve a stationary phase suspension. Afterwards, an aliquot was diluted 1:5 into fresh TSB and incubated at 37 °C for additional 2 h at 37 °C to reach an exponential growth phase. Bacterial cells were harvested by centrifugation at 1,500 × g for 10 min, washed twice and resuspended in PBS to adjust the concentration at 6 × 107 CFU/ml, for use in the experiments.
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| 100.0 |
Fully differentiated primary human bronchial epithelial (HBE) cell cultures were derived from lungs removed at the time of lung transplantation from the Center for Organ Recovery and Education. Cells were prepared using previously described methods approved by the University of Pittsburgh IRB49, 50. Briefly, bronchi from the 2nd to 6th generations were collected, rinsed, and incubated overnight in EMEM at 4 °C. The bronchi were then digested in MEM containing protease XIV and DNase (0.2%). The epithelial cells were removed and collected by centrifugation and then resuspended in BronchiaLife medium and plated onto collagen-treated tissue culture flasks. When 80–90% confluence was reached, the passage 0 cells were trypsinized and seeded onto collagen-coated transwell permeable supports (Corning #3450, 105 cells/well). B-ALI Bronchial Air Liquid Interface Medium was replaced three times a week on both apical and basolateal sides of the permeable supports up to 8–10 days. Subsequently the apical medium was removed and the cultures were maintained at air liquid interface (ALI) to promote a further polarization and differentiation of the epithelium. The basolateral medium was changed twice weekly.
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study
| 100.0 |
Wild-type C57BL/6J female mice were purchased from Jackson Laboratory (Bar Harbor, ME) and maintained in a specific pathogen-free status in a 12 hours light/dark cycle. All procedures were conducted using mice 6–8 weeks of age maintained in ventilated microisolator cages housed in an American Association for Accreditation of Laboratory Animal Care (AAALAC) approved animal housing facility. Protocols and studies involving animals were conducted in accordance with National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pittsburgh.
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other
| 99.9 |
Lung epithelial integrity in polarized primary bronchial cells was examined by measuring the TEER using an electronic resistance system (Millicell ERS-2, EMD Millipore). Ex vivo-differentiated lung epithelium was grown on ALI in 24-transwell plates (Corning, Tewksbury, MA) using BronchiaLife Epithelial Airway Medium in the basolateral compartment. Peptides dissolved in PBS at different concentrations were added onto the apical surface of the differentiated epithelial cells in a final volume of 100 μl. A sterile electrode was applied onto the apical side of the transwell insert containing the cultured epithelial cells with/without peptide treatments and the electrical resistance was measured three times for comparison at different time intervals.
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study
| 100.0 |
Mice were anesthetized with inhalation of isoflurane and the peptide was intra-tracheally administered in 50 µl of PBS at 20 μM and 64 μM corresponding to a peptide dosage of 0.1 mg/kg and 0.35 mg/kg, respectively. Control mice were instilled with 50 µl of PBS without peptide. After 24 hours of treatment, mice were anesthetized with 2.5% tribromoethanol (Avertin). The trachea was cannulated, the lungs were lavaged twice using 1 ml saline, and the BAL samples collected as previously described51. The number of live immune cells in the BAL was determined by a Vision Cell Analyzer automatic cell counter (Nexcelom, Lawrence, MA). An additional aliquot was placed onto glass microscope slides (Shanon Cytospin; Thermo Fisher, Pittsburgh, PA), stained with Diff-Quick (Thermo Fisher Scientific, Waltham, MA); cell differential was determined microscopically. A total of 400 cells of every slide were counted at least twice independently for inflammatory cell differential counts. In other sets of experiments, cell differential counts were evaluated in the BAL of infected mice treated with or without peptides at 6 hours or 24 hours after peptide administration, as indicated.
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study
| 100.0 |
Mice were anesthetized with inhalation of isoflurane and instilled with PAO1 bacteria (sensitive to all peptides), through intra-tracheal inoculation of ~3 × 106 CFU per mouse in 50 µl PBS. Two hours after the bacterial infection, the peptide was intra-tracheally administered at desired dosage in 50 µl of PBS. Control mice were instilled with 50 µl of PBS without peptide. For repeated dosage experiments, the peptide was intra-tracheally instilled at 2 hours and 12 hours after bacterial infection.
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study
| 100.0 |
After 6 hours or 24 hours of bacterial instillation, the numbers of CFU in lungs, BAL and spleen were determined by serial dilution on TSB agar plates. Mice (5–6 mice/group) were anesthetized with 2.5% tribromoethanol. The trachea was cannulated, the lungs were lavaged twice using 1 ml saline, and the BAL samples collected. The left lung lobe was homogenized in 1 ml saline and placed on ice. Dilution of 100 μl of lung tissue homogenate or BAL was mixed with 900 μl PBS. Four serial 10-fold dilutions in saline were prepared and plated on TSB agar plates and incubated overnight at 37 °C, each dilution plated in triplicate, for counting.
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study
| 100.0 |
The peptides’ effect on the expression of different inflammatory genes or airway epithelial cells-related genes after peptide administration at 0.1 mg/kg to non-infected or infected mice was determined, as indicated. Briefly, total mRNA was isolated from the right lung tissues using Trizol reagent. Quantitative PCR was performed using ABI 7900HT (Applied Biosystems, Foster City, CA) and primers of all genes tested including IL-6, IL-10, Tnf-α, Nf-κb, Muc5b, Foxj1. Validation tests were performed to confirm equivalent PCR efficiencies for the target genes. Test and calibrator lung RNAs were reverse transcribed using a high-capacity cDNA reverse transcription kit (Life Technologies) and PCR was amplified as follows: 50 °C for 2 min, 95 °C for 10 min, 40 cycles; 95 °C for 15 s; 60 °C for 1 min. Three replicates were used to calculate the average cycle threshold for the transcript of interest and for a transcript for normalization (β-glucuronidase; Assays on Demand; Applied Biosystems). Relative mRNA abundance was calculated using the ΔΔ cycle threshold (Ct) method.
|
study
| 100.0 |
Lung tissues were harvested from mice at 24 hours after infection and compared with those without infection or after infection and peptide treatment (single or double administration at 0.1 mg/kg, as described above). Afterwards, they were fixed in situ with 4% paraformaldehyde for 10 minutes with the chest cavity open. The right lobe was embedded in paraffin and 5 μm sections were prepared. Sections were stained with hematoxylin and eosin, and histological evaluation was performed to examine bacterial infection-induced pathological severity. The stained lung sections were evaluated in a double-blind fashion under a light microscope, as described previously52, 53.
|
study
| 100.0 |
The vast majority of human hematological malignancies are caused by the clonal expansion of a single cell that has acquired genomic aberrations. Tumor-specific chromosomal translocations are frequent and contribute directly to malignant transformation. Such translocations, and other genetic abnormalities, have been described for many hematological malignancies, including acute or chronic lymphoid and myeloid leukemia, other myeloproliferative disorders, myelodysplastic syndromes, multiple myeloma, and malignant lymphomas.
|
review
| 99.9 |
From the clinical perspective, detection of chromosomal abnormalities in most hematological malignancies is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. A description of chromosomal abnormalities is included in the 2008 World Health Organization Classification of Tumors of Hematopoietic and Lymphoid Tissues and guides the practitioner in choosing the most appropriate treatment. Furthermore, chromosomal abnormalities identification harbors a prognosis value; for example patients bearing the chromosomal translocation t(12;21)(p13;q22) generating ETV6-RUNX1 fusion gene have a better prognosis than those displaying chromosomal rearrangements in MLL gene [2, 3].
|
review
| 99.9 |
From a translational research perspective, the step from genetic identification of a chromosomal translocation to confirmation of the presence of the corresponding fusion protein has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy. Historically, identification of the BCR and ABL genes involved in a balanced translocation between chromosomes 9 and 22 has led to the discovery of the BCR-ABL1 fusion protein, a constitutively active tyrosine kinase. From this discovery, other protein tyrosine kinase inhibitors, which are effective not only against the BCR-ABL1 fusion protein but also against other neoplasms producing protein tyrosine kinases, have been developed .
|
review
| 99.9 |
Current cytogenetic analyses are based on DNA and RNA and consist of karyotyping analyses, fluorescence in situ hybridization (FISH), quantitative real-time polymerase chain reaction (RT-PCR), microarray-based comparative genomic hybridization (array CGH) and more recently next-generation sequencing (NGS) . Those techniques enable the detection of chromosomal abnormalities including translocations, recurrent fusion genes, internal chromosomal amplification, and loss or gain of chromosomal region. Those routine techniques used for the clinical diagnosis such as FISH, array CGH or NGS can be laborious, time-consuming and expensive and therefore may be not available or applicable in all research laboratories.
|
review
| 99.44 |
To remedy these limitations, we introduce an optimized method, the Proximity Ligation Assay (PLA), to identify fusion proteins and their cofactors in non-adherent cells that can be easily handled in research field. PLA is accessible to biological laboratories since it does not necessitate specific skills or knowledge and requires common materials found in any molecular and cellular laboratory such as cell culture incubator or epifluorescence microscope.
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study
| 92.94 |
PLA extends the capabilities of traditional immunoassays and was validated for the first time in 2002 for protein detection and in 2008 for endogenous in situ protein-protein interactions in cell lines . PLA enables detection, visualization and quantification of individual endogenous proteins, protein modifications and protein interactions in tissue and cell samples prepared for microscopy. PLA can be performed on many different samples including protein suspensions (e.g. cell lysates), or fixed tissues (e.g. cell culture slides, cytospin preparations or tissue sections). The readout is a fluorescence signal which is easily visualized under a microscope and quantified. This method has many advantages, notably its high sensitivity and specificity, the relatively short duration of the procedure (2 days), the repeatability, and the small number of cells required. Moreover, identifying a fusion protein at the protein level enables evaluation of the expression level of the endogenous fusion protein within the cell, assessment of the protein subcellular localization, and identification of novel protein partners.
|
review
| 99.8 |
We specifically chose the B-precursor acute lymphoblastic leukemia (B-ALL) as a study model since this disease is the most common childhood malignancy and the leading cause of cancer-related death in children and young adults. The most frequent B-ALL (∼22%) is characterized by the chromosomal translocation t(12;21)(p13;q22) that results in the fusion of two transcription factors, ETV6 and RUNX1, producing a functional fusion protein ETV6-RUNX1 previously known as TEL-AML1 [8, 9]. Specifically, we adapted permeabilization buffers and incubation time to investigate the presence of ETV6-RUNX1 fusion protein in pre-B cells, as well as ETV6-RUNX1 interaction with well-known cofactors.
|
study
| 100.0 |
Using this PLA approach, we were able to confirm or deny the existence of the endogenous ETV6-RUNX1 fusion protein in pre-B lymphoblastic cell lines and, more interestingly, in pre-B lymphoblasts from leukemic patients. Additionally, we were able to demonstrate the molecular proximity of CBFB, a well-known cofactor of RUNX1, with ETV6-RUNX1. The optimized PLA procedure can be readily applied to other non-adherent hematological cells, from cell lines or patients’ cells to detect fusion proteins as well as protein interactions.
|
study
| 100.0 |
The REH cell line is a pre-B ALL cell line initiated from the peripheral blood of a patient with pre-B ALL in first relapse . REH cells carry the chromosomal translocation t(12;21) and chromosomal deletion del(12) producing respectively the ETV6-RUNX1 (previously known as TEL-AML1) fusion gene and the deletion of the residual ETV6 gene (previously known as TEL) . The pre-B Nalm6 cell line was also initiated from ALL relapse and presents a near diploid karyotype with a translocation t(5;12)(q33.2; p13.2) . Nalm6 and REH cells were maintained in RPMI 1640 medium (Gibco, Thermo Fisher Scientific) containing 10% heat-inactivated fetal calf serum (Eurobio) supplemented with antibiotics (100 U/mL penicillin-G and 100 U/mL streptomycin, Gibco). The cells were maintained at 37 °C in a humidified incubator under a 5% CO2 atmosphere.
|
study
| 99.9 |
To obtain stable Nalm6+RUNX1 or Nalm6+ETV6-RUNX1 cell lines, Halotag-RUNX1 human ORF from pFN21A (#FHC01784, Kazusa collection, Promega), or Halotag-ETV6-RUNX1 (ETV6-RUNX1 ORF subcloned from plasmid kindly provided by G. Nucifora ) were cloned into a pLenti CMV-Puro-DEST by Gateway technology. Lenti CMV Puro DEST (w118–1) was a gift from Eric Campeau (Addgene plasmid # 17452) . To produce lentivirus, HEK293T cells were co-transfected with pLenti-CMV-Puro-DEST bearing Halotag-RUNX1 or Halotag-ETV6-RUNX1, pSPAX2 and pCMV-VSV-G for packaging using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific). The plasmid psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260) and pCMV-VSV-G was a gift from Bob Weinberg (Addgene plasmid # 8454) . After 48 h, supernatant was harvested, filtered and added to Nalm6 cells with 4 μg/mL polybrene. All the transduced cells were selected in medium containing 0.25 μg/mL puromycin (Invitrogen) as previously established .
|
study
| 100.0 |
Bone marrow leukemia cells were collected at diagnosis, after informed consent had been obtained, in accordance with the declaration of Helsinki. The protocol was approved by the ethics committee of Rennes Hospital (Rennes, France). Vital mononuclear cells were isolated from bone marrow by successive centrifugations through lymphocytes separation medium (Eurobio). The detection of chromosomal abnormalities was performed at Rennes University Hospital by FISH analysis.
|
other
| 91.0 |
PLA was carried out with Duolink® In Situ Detection Reagents Orange (#DUO92007, Sigma Aldrich). Additional reagents Duolink® In Situ PLA® Probe Anti-Rabbit PLUS/MINUS (#DUO92002/DUO92005, Sigma Aldrich) and Duolink® In Situ PLA® Probe Anti-Mouse PLUS/MINUS (#DUO92001/DUO92004, Sigma Aldrich) were used. Optimization of manufacturer’s procedure applied to non-adherent cells is the goal of this article and is described underneath. One representative experiment of at least three independent experiments is shown.
|
other
| 93.44 |
Duolink™ PLA experiments rely on the selection of two primary antibodies (preferably immunohistochemistry, immunocytochemistry or immunofluorescence validated) that must be raised in two different species (for instance mouse and rabbit). Primary antibodies should be from IgG-class and specific for the target to be detected. PLA can use either monoclonal or polyclonal antibodies. We have tested several antibodies (Table 1).Table 1Antibodies used for PLA assayAntigenSpeciesNameConcentrationReferenceRUNX1mouseAnti-RUNX11 mg/mLab110035 (Abcam)RUNX1rabbitAnti-RUNX11 mg/mLab23980 (Abcam)ETV6mouseAnti-ETV60.5 mg/mLab54705 (Abcam)ETV6rabbitAnti-ETV60.2 mg/mLsc11382 (Santa Cruz biotechnology)ETV6rabbitAnti-ETV60.2 mg/mLsc166865 (Santa Cruz biotechnology)CBFBrabbitAnti-CBFB1 mg/mLab133600 (Abcam)
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study
| 99.06 |
RNA was extracted using the NucleoSpin RNA II kit (Macherey Nagel). cDNA was synthesized using High capacity cDNA RT kit (Life Technologies) according to the manufacturer’s protocol. Real-time PCR was carried out in sealed 384-well microtiter plates using the SYBR™ Green PCR Master Mix (Applied Biosystems), according to Applied Biosystems gene amplification specifications (40 cycles of 15 s at 95 °C and 1 min at 60 °C). The following forward (F) and reverse (R) primers were used for RUNX1: F-RUNX1 (5′-ACAAACCCACCGCAAGTC-3′), R-RUNX1 (5′-CATCTAGTTTCTGCCGATGTCTT-3′); and for ETV6-RUNX1: F-ETV6 (5′-AAGCCCATCAACCTCTCTCA-3′), R-RUNX1 (5′-TCGTGGACGTCTCTAGAAGGA-3′). Data analysis was performed using the ΔΔCT-method . The housekeeping genes GAPDH or ABL were used to normalize the data. The log2 fold change of all genes of interest was calculated compared to Nalm6 control cells.
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study
| 99.94 |
RUNX1 and ETV6-RUNX1 proteins were detected via immunoblot using an anti-RUNX1 mouse antibody (#110035, clone 5A1, Abcam) diluted at 1:300. ETV6 proteins were detected via immunoblot using an anti-ETV6 mouse antibody (#54705, Abcam) diluted at 1:1000. As a loading control, HSC70 protein levels were assessed using a mouse-derived antibody (#7298, clone B-6, Santa Cruz) diluted at 1:500. The immunoblots were visualized with enhanced chemiluminescence Western blotting detection system (WBKLS0500, Merck Millipore) according to the manufacturer’s instructions.
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study
| 99.94 |
PLA technology allows the detection of interactions between endogenous proteins. This technique is based on the detection of protein proximity. PLA uses one pair of primary antibodies. Those primary antibodies target proteins of interest, for instance two epitopes of a fusion protein, or two distinct proteins for which we want to study the proximity. Primary antibodies are raised in different species and are detected with secondary antibodies conjugated to short DNA oligonucleotides. If the oligonucleotides are in close proximity (theoretically less than 40 nm) the DNA strands hybridize and participate in rolling circle DNA synthesis. These DNA copies can further be detected through hybridization of fluorescent-labeled oligonucleotides. The resulting high concentration of fluorescence is easily visualized under a microscope and quantified . One dot corresponds to one colocalization.
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study
| 99.56 |
Because PLA can detect endogenous proteins, and requires few cells, we decided to broaden PLA technology to include the identification of fusion proteins and protein interactions in hematological malignant cells. For that purpose, we chose to optimize the detection of ETV6-RUNX1 fusion protein and the interaction between RUNX1 and CBFB, its well-known cofactor, in non-adherent pre-B lymphoblasts. A flowchart of the successive steps of the optimized PLA protocol is presented in Fig. 1. Briefly, the cells are cytospun, fixed, permeabilized, and incubated with two primary antibodies raised in different species. Then, we performed hybridization with PLA probes, ligation of the probes, rolling circle amplification, slide mounting, image acquisition by microscopy and image analysis (Fig. 1).Fig. 1Optimized protocol outlines of Proximity Ligation Assay for non-adherent pre-B lymphoblasts. Schematic outline summarizing the procedure of PLA for detection, visualization and quantification of individual endogenous proteins, protein modifications and protein interactions. Asterisk depicts steps that have been adapted from the original manufacturer’s procedure
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| 100.0 |
Optimized protocol outlines of Proximity Ligation Assay for non-adherent pre-B lymphoblasts. Schematic outline summarizing the procedure of PLA for detection, visualization and quantification of individual endogenous proteins, protein modifications and protein interactions. Asterisk depicts steps that have been adapted from the original manufacturer’s procedure
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other
| 99.9 |
Non-adherent pre-B Nalm6 cells, REH cells and mononuclear cells from human bone marrow were collected by centrifugation. The cells were washed in cold 1X Phosphate Buffer Saline (PBS) (#ET330, Euromedex) and diluted to reach the concentration of 320,000 cells/mL. The Superfrost™ Menzel-Glaser microscope slides (#10143560 W90, Thermo Fisher Scientific) and disposable sample chambers (Shandon™ Cytofunnel™ double, 28mm2, #5991039, Thermo Fisher) were placed into appropriate slots in the Shandon Cytospin® 4 Cytocentrifuge (Thermo Scientific). Then, 250 μL of cell suspension were aliquoted in each chamber to drop approximately 80,000 cells per spot. The optimal cellular confluence for PLA experiments is 40–70% confluency after cytospin. The cytocentrifugation was run at 800 rpm during 5 min under low-acceleration. Then, chambers were removed and the areas with cells were encircled using a hydrophobic delimiting pen (Dako pen, #S200230–2, Agilent). The samples were fixed with 20 μL of 4% paraformaldehyde for 20 min at 4 °C and were washed twice in 1X PBS for 5 min in a staining jar (spots must be well covered) under shaking (60–90 rpm/min). We typically used a 70 mL staining jar for 5 slides. After fixation, the samples should not be left to dry at any case before the final step. The cells were blocked and permeabilized by adding 5% Bovine Serum Albumin Fraction V (BSA, #10735094001, Roche) and 0.2% saponin (#47036, Sigma Aldrich) in 1X PBS for 1 h 20 at 4 °C in a humidity chamber. A washing step was finally performed before incubation with primary antibodies in PBS/0.2% saponin for 5 min under shaking at room temperature in a staining jar.
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study
| 99.94 |
According to the manufacturer’s instructions, primary antibodies should be from IgG-class, specific for the target to be detected and preferably affinity purified. The primary antibodies can be either polyclonal or monoclonal. To maximize the specificity, antibodies should be validated for immunohistochemistry, and/or immunofluorescence. When using two primary antibodies targeting the same protein, they must be directed against different, non-competing epitopes. The two primary antibodies must have been raised in different species. Moreover, both primary antibodies must bind to the target under the same conditions (fixation, buffer etc.).
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other
| 99.9 |
Antibodies used in this protocol are presented in Table 1. Primary antibodies were added at 0.1 mg/mL final in 15 μL preblocking buffer (PBS with 5% BSA) and incubated at 4 °C overnight into a humidity chamber to prevent evaporation. The droplet must cover the reaction area. Typically, for 0.28 cm2, it is not recommended to use less than 15 μL of total reaction volume on the spot.
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other
| 99.75 |
The next day, the slides were washed three times 5 min each in a staining jar under gentle shaking (60–90 rpm/min) at room temperature: first with 1X PBS/0.5% tween-20 (#P9416, Sigma Aldrich), second with 1X PBS/0.2% saponin/0.5% tween-20 and the third with 1X PBS/0.5% tween-20. Then, secondary antibodies conjugated with oligonucleotides (PLA probe anti-species 1 MINUS and PLA probe anti-species 2 PLUS) were diluted 1:5 in preblocking buffer and 15 μL/spot was applied for 1 h at 37 °C in a humidity chamber.
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other
| 98.6 |
The wash buffers should be brought to room temperature before use, as low temperature slows down the enzymatic reactions. The slides were washed four times, 5 min each in a staining jar under gentle shaking (60–90 rpm/min) at room temperature: first with 1X PBS/0.5% tween-20, second with 1X PBS/0.2% saponin/0.5% tween-20, third with 1X PBS/0.5% tween-20, and fourth with 1X PBS. The 5X Ligation buffer was diluted in water for a final concentration of 1X and the ligase (1 U/μL) diluted in 1:40 in the ligation 1X solution. Fresh dilutions should be prepared just before use. Samples were incubated with 15 μL of ligase solution for 1 h at 37 °C in the humidity chamber.
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other
| 99.7 |
Then, the slides were washed three times, 5 min each in a staining jar under gentle shaking (60–90 rpm/min) at room temperature: 1X PBS/0.2% saponin/0.5% tween-20, second with 1X PBS/0.5% tween-20 and the third with 1X PBS. The amplification 5X was diluted extemporaneously in water for a final concentration of 1X and the polymerase (10 U/μL) diluted in 1:80 in the amplification 1X solution. Samples were incubated with 15 μL of amplification solution for 1 h 15 at 37 °C in the humidity chamber. The samples should be protected from light in order to avoid bleaching of the fluorophores.
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other
| 99.4 |
The slides were washed three times 5 min each in a staining jar under gentle shaking (60–90 rpm/min) at room temperature: first 1X PBS/0.2% saponin/0.5% tween-20, second with 1X PBS/0.5% tween-20 and the third with 1X PBS. The outline of circles was dried and slides were mounted with a cover slip using a minimal volume of Mounting Medium with DAPI (#H-1200, Clinisciences). The slide was then sealed with nail polish and analyzed on an epifluorescence microscope.
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other
| 99.94 |
Images of the cells were acquired under a DeltaVision Elite High Resolution epifluorescence microscope, using appropriate filters. The fluorophore for the Amplification Orange kit has an excitation wavelength of 554 nm and an emission wavelength of 579 nm and can be detected using the TRITC filter. A DAPI filter, excitation 360 nm and emission 460 nm, was used for the nuclear staining. Images were captured by a photometrics cool snap HQ2 camera utilizing the image capture software softWoRx version 5.5. Slides were analyzed under a 20X/0.85 oil magnification objective.
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other
| 99.94 |
During image capture, 5–7 images were taken on each spot. The exposure time was set so that PLA signals were easily distinguishable but not overexposed. It varied between 200 and 500 ms for TRITC with 10% exposure and 200–400 ms for DAPI with 5% exposure. Each image was captured on one layer in the focus plane (DAPI). For TRITC filter, few signals can be visualized either above or below the current focus but the low thickness of the cells after the cytospin allowed all dots to be visible in one focus. If necessary, to increase the resolution, 8 layers (separated for instance by 0.3 nm each) can be taken and a "Z-projection" on the maximum intensity can be applied on the picture. The captured images can be saved as .dv or .tiff.
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other
| 86.94 |
With the PLA approach, since each dot represents a high concentration of fluorescence (several hundred–fold replication of the DNA circle) as a result of the probe proximity, the dot number can be quantified independently of the intensity. The fluorescent particle analysis was performed and automated with ImageJ software (https://imagej.nih.gov/ij/). The script for .dv images (DeltaVision) is presented in Table 2. The two channels (for instance DAPI and TRITC) were separated to analyze the image from DAPI-nuclear staining separately from the image of the TRITC-channel associated to the PLA dots. First, on the DAPI-channel image, the filter "Smooth" surface was applied to the image. Then, the best threshold was set to identify nucleus and to convert the image to binary (black and white) image. To help separating touching nuclei, morphological functions were processed as followed: "Close" command (i.e. a "Dilation" operation followed by "Erosion", to fill nuclei) and "Open" command (i.e. "Erosion" operation followed by "Dilation", to smooth the image and remove isolated pixels). The overlapping nuclei were separated using the "Watershed" function. The "Analyze particles" command was used to count the number of separated nuclei on the image, and results were added to ROI (region of interest) manager. In this step, the appropriate minimum and maximum pixel area sizes was set and cells on picture edge were excluded.Table 2ImageJ SCRIPT for .dv image (DeltaVision format) analysisSTEPSSCRIPT1. Separate the different channels (C1- for DAPI-nuclear staining and C2- for TRITC-channel associated to the PLA dots)- imageName = getTitle();- run (“Split Channels”);- selectWindow (“C1-” + imageName);- selectWindow (“C2-” + imageName);- selectWindow (“C1-” + imageName);- run (“Smooth”);- run (“Median...”, “radius = 2”);2. Apply threshold on C1- setAutoThreshold (“Default dark”);- //run (“Threshold...”);- setOption (“BlackBackground”, false);- run (“Convert to Mask”);3. Apply morphological filters on C1- selectWindow (“C1-” + imageName);- run (“Close-”);- run (“Open”);4. Segment nuclei on C1- run (“Watershed”);- run (“Sharpen”);- run (“Clear Results”);5. Count individual nuclear staining on C1- run (“Analyze Particles...”, “size = 75–550 show = [Overlay Outlines] exclude clear add”);6. Count individual PLA dots on C2- selectWindow (“C2-” + imageName);- run (“Find Maxima...”, “noise = 60 output = [Single Points] exclude”);- selectWindow (“C2-” + imageName + “Maxima”);7. Divide by 255 on the “find maxima output”- run (“Divide...”, “value = 255”);- run (“Set Measurements...”, “area integrated redirect = None decimal = 3”);8. Measure the pixel values- roiManager (“Measure”);9. Get the results of the number of PLA dots in each nucleus- String.copyResults();- selectWindow (“C2-” + imageName + “Maxima”);- close();
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study
| 100.0 |
Second, on the FITC-channel image, the "Find maxima" function was applied. The noise tolerance has to be determined initially (depending on the picture resolution), and the output type selected was "Single point". The number of dots in each nucleus was calculated with the "Measure" command from the ROI manager, to allow all regions previously identified to be represented on the single point output image. In the results window, the raw integrated density (RawIntDen) represents the sum of pixel values in each nuclear staining. Because the single point output is in black and white and because we had 8-bit images (28 equals to 256 different pixel values, in the range 0–255), we divided the RawIntDen values by 255 to obtain the number of detected maxima/dots in each region defined by the nuclear stain.
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study
| 99.94 |
Statistical analyses were performed with GraphPad Prism 6.0 software. Mean values ± standard deviation (S.D.) are presented. The assay cut-off value is set to two standard deviations over the background signal according to Nordengrahn et al. . The background signal is estimated with a pair of antibodies that is known to not interact. Samples with values below this cut-off are considered to be negative for the interaction of interest while samples with values higher than the threshold are positive.
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study
| 99.94 |
We first performed Proximity Ligation Assay (PLA) on Nalm6 lymphoblasts. Those cells express both RUNX1 and ETV6 transcripts and proteins (Fig. 2a). Specificity of the assay was shown by lack of non-specific staining in the negative control, displayed by the use of mouse-RUNX1 antibody alone (Fig. 2b). On the contrary, incubation with two different-species-antibodies directed against RUNX1 showed on average 21.0 (±12.2, n = 425) fluorescent dots per cell validating the presence of RUNX1 proteins in Nalm6 cells. As expected, RUNX1 proteins were localized in the nuclei. Detection of RUNX1 and CBFB proximity showed on average 13.1 (±6.6, n = 299) dots per cell. This colocalization between RUNX1 and CBFB is in concordance with the literature [20, 21]. We next wanted to assess the proximity of the proteins RUNX1 and ETV6. PLA with anti-mouse RUNX1 antibodies and anti-rabbit ETV6 antibodies did not show any relevant interaction (about 1.2 ± 1.5 dot per nucleus, n = 366) in Nalm6 cells. This result is consistent with the literature and expression results; we did not expect to detect interactions between endogenous ETV6 and RUNX1 proteins in Nalm6 cells. According to this result, the cut-off assigned in this study was a mean of 4.2 PLA dots per nucleus (two standard deviations over the background signal ), representing the fluorescent background or the probability of the 2 proteins to be in a close proximity by chance.Fig. 2Validation of PLA on pre-B lymphoblasts. a Quantitative real-time RT-PCR and western blot analysis showed respectively mRNA and protein expression of RUNX1, ETV6 or ETV6-RUNX1 in Nalm6 and REH cells. All RT-PCR (left panel) were performed in triplicate and gene expression was normalized to ABL1 expression (error bars are S.D.) while western blot analyses (right panel) are representative images from the whole-cell lysates. b Technical controls demonstrate the specificity of PLA signals in Nalm6 cells and the proximity between two proteins (RUNX1 and CBFB). Nuclei were stained with DAPI. RUNX1 ab110035 antibodies were incubated alone (−) or with RUNX1 ab23980, CBFB ab133600 or ETV6 sc11382 antibodies. Each picture (upper panel) is representative of a typical cell staining observed in 5 fields randomly chosen. The quantification of the number of PLA dot per nucleus is presented with the mean values ± S.D
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study
| 100.0 |
Validation of PLA on pre-B lymphoblasts. a Quantitative real-time RT-PCR and western blot analysis showed respectively mRNA and protein expression of RUNX1, ETV6 or ETV6-RUNX1 in Nalm6 and REH cells. All RT-PCR (left panel) were performed in triplicate and gene expression was normalized to ABL1 expression (error bars are S.D.) while western blot analyses (right panel) are representative images from the whole-cell lysates. b Technical controls demonstrate the specificity of PLA signals in Nalm6 cells and the proximity between two proteins (RUNX1 and CBFB). Nuclei were stained with DAPI. RUNX1 ab110035 antibodies were incubated alone (−) or with RUNX1 ab23980, CBFB ab133600 or ETV6 sc11382 antibodies. Each picture (upper panel) is representative of a typical cell staining observed in 5 fields randomly chosen. The quantification of the number of PLA dot per nucleus is presented with the mean values ± S.D
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study
| 100.0 |
Our results show that our optimized PLA protocol is selective and suitable for detection of molecular proximity between two distinct proteins in non-adherent cells. Here, we validated the efficiency of the optimized procedure to detect the well-known protein interaction between the protein RUNX1 and its canonical molecular co-factor CBFB in Nalm6 pre-B lymphoblasts.
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study
| 100.0 |
To evaluate the sensitivity of the assay, we performed PLA on Nalm6 cells displaying a gradient of expression of RUNX1. We generated Nalm6 cell lines depleted for RUNX1 transcript and protein (Nalm6shRUNX1) or overexpressing RUNX1 (Nalm6+RUNX1) (Fig. 3a and 3b). Quantification of RUNX1 protein level by PLA positively correlated with quantification of RUNX1 protein level demonstrated by western blot (Fig. 3b and 3c). This result demonstrates that PLA may be sensitive to protein level.Fig. 3PLA is sensitive to total protein level. a Nalm6 wild type, depleted for RUNX1 protein (Nalm6shRUNX1) or overexpressing RUNX1 (Nalm6+RUNX1) cell lines were validated using RT-qPCR. Results are presented in terms of a fold change after normalizing RUNX1 mRNA levels with GAPDH mRNA. Each value represents the mean of ± S.D. of three independent transduced cells. b Representative images of western blot (left panel) and densitometry analysis (right panel) showing the quantification of RUNX1 protein level normalized to HSC70 c Quantification of the PLA signal (dots plots) on Nalm6 cells displaying a gradient of expression of RUNX1 is represented. Nalm6, Nalm6shRUNX1 or Nalm6+RUNX1 cells were incubated with a pair of RUNX1 antibodies (the mean values ± S.D. are presented)
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study
| 100.0 |
PLA is sensitive to total protein level. a Nalm6 wild type, depleted for RUNX1 protein (Nalm6shRUNX1) or overexpressing RUNX1 (Nalm6+RUNX1) cell lines were validated using RT-qPCR. Results are presented in terms of a fold change after normalizing RUNX1 mRNA levels with GAPDH mRNA. Each value represents the mean of ± S.D. of three independent transduced cells. b Representative images of western blot (left panel) and densitometry analysis (right panel) showing the quantification of RUNX1 protein level normalized to HSC70 c Quantification of the PLA signal (dots plots) on Nalm6 cells displaying a gradient of expression of RUNX1 is represented. Nalm6, Nalm6shRUNX1 or Nalm6+RUNX1 cells were incubated with a pair of RUNX1 antibodies (the mean values ± S.D. are presented)
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study
| 100.0 |
Having proven the selectivity and the sensitivity of the optimized PLA protocol, we next addressed the question of the detectability of fusion proteins (i.e. ETV6-RUNX1) using our optimized PLA protocol. To that aim, we overexpressed ETV6-RUNX1 transcript and protein (Fig. 4a and 4b) in Nalm6 cells (Nalm6+ETV6-RUNX1 cells) and carried out PLA (Fig. 4c). The negative control (RUNX1 antibodies alone) displayed 0.7 (± 1.8, n = 296) dots demonstrating a very low background. The positive control (detection of RUNX1 protein using two anti-RUNX1 antibodies) showed 23.6 (± 27.6, n = 302) dots per nucleus. In those Nalm6+ETV6-RUNX1 cells, protein proximity between RUNX1 and CBFB was maintained (10.2 dots per nucleus) as observed in Nalm6 cells. Detection of ETV6-RUNX1 fusion protein showed 23.3 (± 25.0, n = 419) dots per nucleus in Nalm6+ETV6-RUNX1 cells whilst the same mix of antibodies detected less than 1.2 dots per cells in Nalm6 cells (Fig. 2b and 4c). This result convincingly demonstrates that PLA can be a powerful tool to detect overexpressed fusion proteins. We wanted further to detect the endogenous ETV6-RUNX1 fusion protein. For that purpose, we used REH cells which harbor the chromosomal translocation t(12;21)(p13;q22) that generates the fusion gene and protein ETV6-RUNX1 (Fig. 2a). PLA using single-species RUNX1 antibodies showed no background (0.4 dots per nucleus; negative control) whereas incubation with two species-different antibodies directed against RUNX1 displayed on average 29.8 dots (± 17.6, n = 426) (positive control) (Fig. 4d) confirming the efficiency of the protocol on REH cells. Importantly, PLA using a mix of anti-RUNX1 and anti-ETV6 antibodies showed 21.6 ± 12.0 (n = 415) dots per cells. We observed that the majority of ETV6 and RUNX1 proximities were localized within the nucleus. Because the non-translocated allele of ETV6 is absent in REH cells, this result of ETV6 and RUNX1 proximity means the presence of ETV6-RUNX1 fusion protein in those cells. We were next interested in assaying the molecular proximity between the ETV6-RUNX1 fusion protein and CBFB. To the best of our knowledge, even if this interaction was suspected , it has never been formally demonstrated so far. PLA using a mix of anti-CBFB and anti-RUNX1 antibodies, as well as PLA using a mix of anti-CBFB and anti-ETV6 antibodies revealed a molecular proximity between ETV6-RUNX1 and CBFB (Fig. 4d). Those data demonstrate that PLA can be a powerful tool to detect overexpressed as well as endogenous fusion proteins in cell lines.Fig. 4Our optimized PLA protocol effectively detects overexpressed or endogenous fusion proteins in pre-B lymphoblasts. The Nalm6 cell line overexpressing ETV6-RUNX1 (Nalm6+ETV6-RUNX1) was validated using RT-qPCR (a) and western blot (b). a Results are presented in terms of a fold change after normalizing ETV6-RUNX1 mRNA levels with GAPDH mRNA. Each value represents the mean of ± S.D. of three independent transduced cells. b Representative images of western blot showing expression of RUNX1 or HSC70 proteins in both cell lines are represented. c Pictures and quantification of PLA signals on Nalm6 cells overexpressing ETV6-RUNX1 protein (Nalm6+ETV6-RUNX1 cells) (a) or on REH cells that expressed endogenous ETV6-RUNX1 protein (b). Nuclei were stained with DAPI. RUNX1 ab110035 antibodies were incubated alone (−) or with RUNX1 ab23980, CBFB ab133600 or ETV6 sc11382 antibodies. Each picture (upper panel) is representative of a typical cell staining observed in 5 fields chosen at random. The quantification of the number of PLA dots per nucleus is presented with the mean values ± S.D
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study
| 100.0 |
Our optimized PLA protocol effectively detects overexpressed or endogenous fusion proteins in pre-B lymphoblasts. The Nalm6 cell line overexpressing ETV6-RUNX1 (Nalm6+ETV6-RUNX1) was validated using RT-qPCR (a) and western blot (b). a Results are presented in terms of a fold change after normalizing ETV6-RUNX1 mRNA levels with GAPDH mRNA. Each value represents the mean of ± S.D. of three independent transduced cells. b Representative images of western blot showing expression of RUNX1 or HSC70 proteins in both cell lines are represented. c Pictures and quantification of PLA signals on Nalm6 cells overexpressing ETV6-RUNX1 protein (Nalm6+ETV6-RUNX1 cells) (a) or on REH cells that expressed endogenous ETV6-RUNX1 protein (b). Nuclei were stained with DAPI. RUNX1 ab110035 antibodies were incubated alone (−) or with RUNX1 ab23980, CBFB ab133600 or ETV6 sc11382 antibodies. Each picture (upper panel) is representative of a typical cell staining observed in 5 fields chosen at random. The quantification of the number of PLA dots per nucleus is presented with the mean values ± S.D
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study
| 100.0 |
Various antibodies against ETV6 and RUNX1 are available. Three different pairs of antibodies against ETV6 and RUNX1 were tested in REH cells expressing the ETV6-RUNX1 fusion protein to define the most suitable couple for the assay in patients (Additional file 1: Figure S1). The couple anti-mouse RUNX1 with anti-rabbit ETV6 is the most effective and will be used for ETV6-RUNX1 detection in patients.
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study
| 100.0 |
Given the high selectivity and sensitivity of our optimized PLA protocol for detecting close proximity between two proteins and validating the presence of a fusion protein, we wondered whether it was beneficial to use this protocol on patient cells to confirm the presence of the fusion protein suspected by the identification of a t(12;21)(p13;q22) chromosomal translocation.
|
study
| 99.9 |
To that aim, PLA was performed on samples of mononuclear cells from 4 patients with pre-B-ALL suspicion. Karyotype features and fusion transcript of the patients were presented in Table 3. The cohort was composed of 3 males and 1 female with a median age of 4-year-old. Lymphoblasts from two of them (patients #3 and #4) were positive for t(12;21) chromosomal translocation and ETV6-RUNX1 fusion transcript while the two others patients’ lymphoblasts (patients #1 and #2) did not carry this translocation. PLA was carried out on an aliquot of the bone marrow diagnosis sample. As previously, we used a single antibody anti-RUNX1 as negative control, and two antibodies against RUNX1 as positive control.Table 3Biological and cytogenetic characteristics of the B-acute lymphoblastic patientsPatientSexAge at diagnosis (year)CytogeneticFusion transcript#1Male2Hyperploidy3 copy RUNX12 copy ETV6none#2Female8t(1;19)E2A-PBX1#3Male4t(12;21)del ETV6ETV6-RUNX1#4Male3t(12;21)del ETV6ETV6-RUNX1
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study
| 100.0 |
For patient #1 and patient #2 (Fig. 5a), the negative controls showed no dots, confirming the high specificity of the procedure. The positive controls showed about 16.9 dots per nucleus (±4.1, n = 50) for patient #1, and 15.8 (±11.5, n = 50) for patient #2. PLA with a mix of anti-RUNX1 and anti-ETV6 showed about 2.6 (±2.4, n = 50) for patient #1 and 2.6 (±3.5, n = 48) dots per nucleus for patient #2. Therefore, the mean number of dots per nucleus for ETV6 and RUNX1 proximity is below the cut-off level for both patients. We concluded that both patient #1 and patient #2 lymphoblasts did not express the fusion protein ETV6-RUNX1, which were concordant with FISH analysesFig. 5Proximity Ligation Assay is a reliable tool for the detection of ETV6-RUNX1 fusion protein in patients’ lymphoblasts. Quantification of PLA signals per nucleus in lymphoblasts from two patients negative (a) or positive (b) for the fusion protein ETV6-RUNX1. The cells were incubated with RUNX1 ab110035 antibodies alone (−) or with RUNX1 ab23980, CBFB ab133600 or ETV6 sc11382 antibodies. The quantification of the number of PLA dot per nucleus is presented with the mean values ± S.D. The line represents the cut-off used to determine positive PLA signals
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study
| 99.94 |
Proximity Ligation Assay is a reliable tool for the detection of ETV6-RUNX1 fusion protein in patients’ lymphoblasts. Quantification of PLA signals per nucleus in lymphoblasts from two patients negative (a) or positive (b) for the fusion protein ETV6-RUNX1. The cells were incubated with RUNX1 ab110035 antibodies alone (−) or with RUNX1 ab23980, CBFB ab133600 or ETV6 sc11382 antibodies. The quantification of the number of PLA dot per nucleus is presented with the mean values ± S.D. The line represents the cut-off used to determine positive PLA signals
|
study
| 99.94 |
For patient #3 (Fig. 5b), the controls produced expected results: no dots with the negative control (anti-RUNX1 alone) and 21.2 (±7.3, n = 24) dots per nucleus with the positive control (2 different anti-RUNX1 antibodies). Interestingly, the mix of anti-RUNX1 and anti-ETV6 revealed 34.6 PLA dots (34.6 ± 10.8, n = 49) per nucleus, demonstrating a close proximity between ETV6 and RUNX1 that we allocated to the presence of the ETV6-RUNX1 fusion protein. The presence of the fusion protein was validated by the FISH result later on. Similarly, in patient #4 (Fig. 5b), the non-specific background was negative and the pair of anti-RUNX1 gave a positive result with 49.6 (±14.6, n = 20) dots per cell. PLA with the mix of anti-RUNX1 and anti-ETV6 showed an equivalent level of detected interactions, 33.1 (±13.5, n = 32) dots per nucleus that we attributed to the presence of the fusion protein ETV6-RUNX1. The FISH result was concordant with our PLA result. We conclude that the optimized PLA can also be used on non-adherent cells from patients, facilitating the study of fusion protein and protein-protein interactions and their subcellular localization.
|
study
| 99.1 |
The purpose of the current study was to develop an optimized PLA protocol for non-adherent cells using commercially available materials and kits, and common molecular and cellular biology laboratory materials. By taking the example of ETV6-RUNX1 fusion protein, we present an improved method for the detection of fusion proteins and their partners that can be an important tool in scientific and translational research. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy, specifically the requirement of few cells and in situ visualization, with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm. We provide compelling negative and positive controls, and demonstrate that the optimized PLA procedure is sensitive to total protein level.
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study
| 100.0 |
We overcome the issue of maintaining non-adherent hematological cells by using cytocentrifugation, and optimized buffers, incubation times, and washing steps. Because hematological cells are more resistant to permeabilization and to preserve protein integrity, we increased permeabilization time to 1 h 20 at 4°C, instead of 30 min at 37°C as recommended by the manufacturer. We adapted the primary antibody buffer to our cells. We increased all washing times and the stringency of buffers to reduce non-specific fluorescence. We increased the ligation incubation time from 30 min to 1 h. Finally, we optimized the amplification step (80 min at 37 °C instead of 100 min) to reduce the potential coalescent signals.
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study
| 100.0 |
As expected, the specificity of the assay depends on using an appropriate pair of antobodies. The pair of antibodies works in conjunction, meaning that specificity and efficiency is limited by the least specific and efficient antibody. Therefore, excellent antibody quality is an important parameter for this method. We strongly recommend validating antibody specificity and efficiency. Validation of antibodies by immunofluorescence is also highly recommended. We have also validated pairs of antibodies by PLA. PLA can be performed in a single recognition experiment using different primary antibodies against the same protein. The couple showing the smaller number of dots is supposed to contain at least one limiting antibody that may be avoided for PLA assay.
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study
| 99.94 |
As the PLA technique is very sensitive, special care is needed to keep the incubation times and conditions equal for different samples. In addition, we draw attention on the fact that the number of dots may vary from one experiment to another. Obviously, the duration of each step is crucial and should be strictly respected. We have also observed a slight decrease of efficiency of the kit over time. To prevent enzyme activity degradation, enzymes must be kept at −20 °C and added just before applying the reaction mixture to the sample. To overcome the kit limitation, we also recommend the systematic inclusion of a positive control by a single protein recognition (e.g. detection of total RUNX1 using 2 RUNX1 antibodies) for each experiment. This positive control can be used to normalize PLA data, allowing comparison between different series of experiments.
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other
| 99.3 |
The total workflow lasts 2 days. This duration accepts some variation. Cells can be stored for a few days in PBS after fixation at 4 °C. We recommend using fresh cells whenever possible but cryopreserved cells in fetal calf serum/10% DMSO could also be used. Moreover, before image acquisition, the stained cells can be kept for a few days in the dark at 4 °C before the fluorescent signal decreases.
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other
| 99.9 |
The optimized PLA protocol has been achieved on pre-B lymphoblast cells, and validated also on other non-adherent cells such as hematopoietic stem cells, multipotent progenitors, progenitors of granulocytes and macrophages and multi-lymphoid progenitors (data not shown); demonstrating that the optimized protocol is a robust assay for non-adherent cells. Therefore, our optimized PLA protocol resolves both selectivity and sensitivity issues in hematological non-adherent cells .
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study
| 100.0 |
Our experiments demonstrate that the optimized PLA protocol enables molecular and cellular biologists to detect fusion proteins, subcellular expression, and protein interactions in non-adherent cells, and therefore provides a new tool for leukemia pathogenesis research. In conclusion, the optimized proximity ligation assay for non-adherent cells described here, is simple, fast, reliable, and can be adopted for a wide range of applications in the biological field.
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study
| 99.94 |
Autophagy is a conserved process for engulfing cytosolic components (proteins, organelles, etc.) into a double-membraned structure, the autophagosome, which subsequently fuses with the lysosome (the vacuole in Saccharomyces cerevisiae) (Nair and Klionsky 2005; Glick et al. 2010). Autophagy provides building blocks during starvation and removes damaged or unnecessary organelles. Thus, autophagy is indispensable for intracellular homeostasis (Yang and Klionsky 2010). Autophagy can be divided into selective and nonselective types, depending on the substrates. The nonselective form of autophagy, called macroautophagy, is responsible for the turnover of cytosolic components. In this paper, “autophagy” will be used to refer to macroautophagy.
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review
| 99.8 |
Autophagy is complex and highly regulated. The master regulator is the Target of Rapamycin complex 1 (TORC1), a serine/threonine kinase that functions to sense the cell’s energy status. When cells have sufficient nutrients, TORC1 phosphorylates several substrates and blocks autophagy. In contrast, when cells are starved or treated with rapamycin, the TORC1 kinase is inhibited, triggering autophagy. The elongation of a structure called the phagophore to form the autophagosome requires Atg8, with a covalently-linked molecule of phosphatidylethanolamine (PE) on its C-terminus, which serves to recruit membranes to the phagophore (Klionsky et al. 2007).
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study
| 99.94 |
Autophagy requires several specific proteins, the Atg proteins, but also requires transport of membrane vesicles to the phagophore assembly site (PAS). In yeast, many small GTP-binding proteins, membrane traffic regulators, are essential for different stages of autophagy, from the formation of the autophagosome to the fusion of the autophagosome and the vacuole (Yang and Rosenwald 2014). We recently discovered that two small GTP-binding proteins, Arl1, a member of the Arf/Arl/Sar family, and Ypt6, a member of the Rab family, have novel roles in autophagy in S. cerevisiae (Yang and Rosenwald 2016). Both regulate membrane traffic between the trans-Golgi network (TGN) and endosomes. ARL1 and YPT6 also show synthetic lethality with one another, suggesting the encoded proteins have functional similarities (Costanzo et al. 2010). Mutants lacking one or the other of these genes have similar phenotypes; both the arl1Δ and ypt6Δ strains are unable to grow in the presence of rapamycin and perform autophagy, but only at high temperature (37°). We determined that the high temperature defect is caused by a failure to recruit the Golgi-associated retrograde protein (GARP) complex to the PAS in the absence of arl1Δ or ypt6Δ at 37°. The GARP complex is responsible for recruiting the SNARE Tlg2 to the PAS to deliver membranes derived from the Golgi apparatus to the growing phagophore (Reggiori et al. 2003).
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study
| 100.0 |
Here, we utilized a high copy genomic library (Nasmyth and Reed 1980) to perform a suppressor screen on arl1Δ and ypt6Δ strains. Since we found that neither deletion mutant strain can grow in the presence of rapamycin at 37°, we first used this assay to identify suppressors of the growth defects. Because we are interested in membrane traffic or autophagy regulators that have relationships with Arl1 or Ypt6 during autophagy, a total of seven genes (COG4, SNX4, TAX4, IVY1, PEP3, SLT2, and ATG5) were selected from the sequenced genomic fragments and were transformed into both the arl1Δ and ypt6Δ strains. Autophagy-specific assays were used to determine which genes suppressed the autophagy defects. As a result, we have identified novel partners of Arl1 or Ypt6 during autophagy.
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study
| 100.0 |
Yeast strains arl1Δ (MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met15Δ0/MET15ura3Δ0/ura3Δ0, arl1Δ::KanMX/arl1Δ::KanMX) and ypt6Δ (MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met15Δ0/MET15ura3Δ0/ura3Δ0, ypt6Δ::KanMX/ypt6Δ::KanMX) were used in the high copy suppressor screen for rapamycin sensitivity at 37°. Both strains were obtained from the homozygous diploid deletion collection developed by the Saccharomyces Genome Deletion Project (Winzeler et al. 1999); the parental strain is BY4743. Strains YSA003 (pho8::pho8Δ60 arl1Δ::HIS3) and YSA004 (pho8::pho8Δ60 ypt6Δ::HIS3) in the BY4742 background (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) were constructed previously (Yang and Rosenwald 2016). High copy plasmids containing a single specific yeast ORF were either isolated from the screened library (COG4 and SNX4) or were obtained from the yeast ORF collection (SLT2, ATG5, PEP3, IVY1, and TAX4) (Gelperin et al. 2005). The pRS316-GFP-Atg8 plasmid was a gift from Daniel Klionsky (University of Michigan) (Suzuki et al. 2001). The plasmids used in this study are listed in Supplemental Material, Table S1.
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study
| 100.0 |
Antibodies used included a mouse anti-GFP (green fluorescent protein) primary antibody (Roche Diagnostics, 11814460001); a mouse anti-phosphoglycerate kinase-1 (Pgk-1) antibody (Molecular Probes, A6457); and a sheep anti-mouse IgG horseradish peroxidase-linked secondary antibody (GE Healthcare, NA931). The enhanced chemiluminescence (ECL) prime kit was from GE Healthcare (RPN2236).
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other
| 99.94 |
The YEp13-based yeast high copy number genomic library (ATCC 37323) (Nasmyth and Reed 1980) was used in the screen. Yeast strains arl1Δ and ypt6Δ were transformed with the library by using lithium acetate transformation, as described previously (Gietz and Woods 2002), and were plated onto either SC with glucose without leucine medium to determine number of total transformants (20,000 colonies for each strains) or SC with glucose without leucine containing 5 ng/ml rapamycin (Sigma-Aldrich, R0395). We previously determined using this concentration of rapamycin that the negative control, the atg1Δ strain, cannot grow at either 30 or 37°, while the wild-type (WT) parent can grow in the presence of rapamycin at both temperatures (Yang and Rosenwald 2016). Here, the rapamycin plates were incubated at 37°. Plasmids from the colonies showing resistance to rapamycin at 37° were isolated (Hoffman and Winston 1987) and transformed into Escherichia coli DH5α strain by electroporation, followed by purification from E. coli cells (QIAGEN QIAprep Spin Miniprep Kit). Plasmids were then retransformed into arl1Δ or ypt6Δ strains to confirm that the suppressing function was contained on the plasmid and not as a result of a chromosomal mutation in the original yeast transformant. The plasmids were sequenced with primers (MP10: CTTGGAGCCACTATCGAC, MP11: CCGCACCTGTGGCGCCG) adjacent to the unique BamHI site of YEp13, into which the genomic fragments were cloned. Sequencing was performed by Genewiz (Plainfield, NJ). The sequencing results were analyzed with the Basic Local Alignment Search Tool (BLAST, NCBI) (Altschul et al. 1990). The genomic regions contained in the plasmids were identified with the genome browser tool from the Saccharomyces genome database (SGD; yeastgenome.org). ORF functions as membrane traffic or autophagy regulators were selected and the high copy number plasmids containing a single ORF of interest were either obtained directly from this screen (some of the plasmids that passed the screen contained only a single gene) or from the yeast ORF collection (GE Dharmacon) (Gelperin et al. 2005). These plasmids were transformed into arl1Δ and ypt6Δ strains to test in more detail whether they suppressed the autophagy defect of arl1Δ and ypt6Δ strains at 37°. When the ORF collection plasmids were used, cells were grown in media containing galactose rather than glucose because the genes of interest are under the control of the GAL1 promoter.
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study
| 100.0 |
Yeast strains were cultured in synthetic dropout media (2% glucose and 0.67% yeast nitrogen base without amino acids or uracil, supplemented with appropriate nutrients) (Green and Moehle 2001). For yeast strains containing plasmids from the yeast ORF collection, galactose was used as the carbon source in place of glucose because the genes are under the control of the GAL1 promoter. To induce autophagy, nitrogen starvation medium (SD-N or SGal-N; 2% glucose or 2% galactose, 0.17% yeast nitrogen base without amino acids, ammonium sulfate, or vitamins) was used. Yeast cells were first cultured in synthetic dropout media lacking appropriate amino acids or uracil depending on the plasmids they contained, until OD600 = 0.6. They were then incubated at either 30 or 37° in nonstarvation conditions for 30 min before being washing twice in SD-N (or SGal-N) medium and further incubated in SD-N (or SGal-N) at the same temperature for 3 hr. All chemicals for media were from Fisher Scientific except yeast nitrogen base (Sunrise, 1500-500).
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study
| 100.0 |
Yeast strains containing the pRS316-GFP-Atg8 plasmid were cultured in starvation medium at 30 or 37° to induce autophagy. Cells were collected and subjected to trichloroacetic acid precipitation, used to extract proteins from cells as described previously (Yang and Rosenwald 2016). Total proteins from 0.8 OD600 were separated on precast polyacrylamide gels (Any kD, Bio-Rad, 456–9036). The proteins were transferred onto nitrocellulose membranes and the membrane was incubated with a mouse anti-GFP antibody, followed by an HRP-conjugated anti-mouse secondary antibody. HRP signals were visualized using an ECL prime kit (GE Healthcare) and detected with an ImageQuant LAS 4010 imager (GE Healthcare). Each set of western blot experiments was repeated three times. Representative examples are shown in each figure.
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study
| 100.0 |
Yeast strains containing the pRS316-GFP-Atg8 plasmid were collected from rich medium (SD medium lacking the appropriate nutrients or SGal medium for yeast strains containing the yeast ORF collection plasmids) or starvation medium at 30 or 37°, and washed once with water before imaging. Cells were visualized with a Zeiss AxioImager M2 florescence microscopy system using a 63 × oil lens. Images were captured and deconvolved using Volocity 6.3 (PerkinElmer) software. The fluorescence microscopy experiments were repeated three times.
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study
| 99.94 |
The Pho8Δ60 assay to quantify the magnitude of autophagy was performed as previously described (Noda and Klionsky 2008). Briefly, cell lysates from 0.5 OD600 of cells were incubated with 5.5 mM α-naphthyl phosphate (disodium salt, Sigma-Aldrich, N7255) for 20 min at 30° in reaction buffer (250 mM Tris-HCl, pH 9.0, 10 mM MgSO4, and 10 µM ZnSO4). The reaction was stopped with an equal volume of 2 M glycine-NaOH, pH 11.0. Fluorescence emissions of the product 1-napthol were measured (λex = 330 and λem = 472) using a GloMax plate reader (Promega) with a UV filter. Protein concentrations were determined by the Bradford assay (Bradford 1976). All experiments were repeated three times.
|
study
| 100.0 |
Previously, we found that the deletion of either the ARL1 or YPT6 gene causes sensitivity to the drug rapamycin only at 37°, and not at 30° (the atg1Δ strain, the negative control for autophagy, showed growth defects on rapamycin at both 30 and 37°, while the WT strain was able to grow at both temperatures). We further confirmed that this high temperature growth defect on rapamycin was due to a failure to process autophagy normally (Yang and Rosenwald 2016). To determine what genes might suppress the autophagy defect in these strains, we utilized the rapamycin sensitivity phenotype at 37° as a preliminary way to isolate genomic fragments that contain potential high copy number suppressors. Each deletion strain was transformed with the library and 20,000 colonies each were screened for their ability to grow on rapamycin medium at 37°. As a result, 27 distinct genomic fragments were identified from the screen on the arl1Δ strain (Table S2), and 10 were identified for the ypt6Δ strain (Table S3).
|
study
| 100.0 |
From the screen results for the arl1Δ strain, we selected COG4, TAX4, SNX4, and SLT2 as candidates for further testing based on their cellular functions as important regulators of membrane traffic or autophagy (see Table S2). We obtained ARL1 from the screen, a good internal control. Also, YPT6 was identified from the screen, which was previously confirmed as a suppressor of the arl1Δ strain (Yang and Rosenwald 2016). For genes of interest contained on library plasmids with several genes, we purchased the relevant single genes from the ORF collection (Gelperin et al. 2005). For the COG4 and SNX4 plasmids obtained from the screen, each of these plasmids contained only the gene of interest, thus we used these two plasmids directly in additional assays. For the screen results of the ypt6Δ strain, we selected IVY1, ATG5, and PEP3 (see Table S3). We also obtained a fragment with YPT6, again a good internal control. In summary, we selected a total of seven genes to be tested specifically for suppression of the autophagy defects. All seven plasmids containing single gene candidates were transformed into the arl1Δ and ypt6Δ strains.
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study
| 100.0 |
We first transformed high copy plasmids containing COG4, SNX4, SLT2, TAX4, IVY1, ATG5, and PEP3, as well as ARL1 and empty vector (EV, YEp352) into the arl1Δ strain. In plasmids containing SLT2, TAX4, IVY1, ATG5, and PEP3, expression is controlled by the GAL1 promoter, thus, yeast with these plasmids were cultured in medium with galactose as the carbon source. The COG4- and SNX4-containing plasmids were isolated from the YEp13 library as fragments with a single gene and expression of these genes is under the control of their native promoters; thus, yeast containing these two plasmids were cultured using normal growth conditions with glucose as the carbon source. In order to test which of the seven candidates suppressed the autophagy defect at 37°, an assay that followed cleavage of a modified version of the protein Atg8, GFP-Atg8, was used to monitor the transport of Atg8 to the vacuole (Figure 1A). Once autophagy is triggered, Atg4 modifies Atg8 on its C-terminus. This modification enables Atg8 to be conjugated to PE on its C-terminus, which helps expand the membranes of the phagophore to become the autophagosome (Xie et al. 2008). When the autophagosome fuses with the vacuole, Atg8 is degraded. However, when the fusion protein GFP-Atg8 is transferred to the vacuole, Atg8 is degraded, but the GFP moiety is resistant to degradation and can therefore be detected as free GFP on western blots if autophagy proceeds normally. As the results show, at time 0, no free GFP was detected in any of the strains. When the cells were treated in starvation medium for 3 hr at 30°, free GFP could be detected in all strains, demonstrating normal autophagy. As the negative control, no free GFP could be detected in the cell with empty vector at 37°, meaning that Arl1 is required for autophagy at high temperature, as previously found (Yang and Rosenwald 2016). Cells overexpressing COG4, TAX4, SNX4, and ARL1 were able to process GFP-Atg8 normally at 37°, while overexpression of IVY1, ATG5, SLT2, or PEP3 did not suppress the autophagy defect of the arl1Δ strain at 37°.
|
study
| 100.0 |
Autophagy-specific assays show COG4, TAX4, and SNX4 suppress the temperature-sensitive autophagy defect of the arl1Δ strain. (A) GFP-Atg8 degradation was increased at 37° in the arl1Δ strain when transformed with plasmids containing COG4, TAX4, and SNX4, as well as WT ARL1. All strains were cultured at 30° in the appropriate nonstarvation medium (containing either glucose or galactose) until log-phase, then all the strains were incubated at 37 or 30° for 30 min. The cells were then washed twice with SD-N (or SGal-N) medium, and cultured in SD-N (or SGal-N) for 3 hr either at 37 or at 30°. (B) COG4, TAX4, and SNX4 increased the Pho8Δ60 activities in arl1Δ (YSA003) at 37°. Error bars represent SD from three biological replicates. GFP, green fluorescent protein; SD-N, glucose nitrogen starvation medium; SGal-N, galactose nitrogen starvation medium; WT, wild-type.
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study
| 100.0 |
In S. cerevisiae, the activity of a N-terminally truncated form of the vacuolar alkaline phosphatase Pho8 (Pho8Δ60) is widely used to measure the magnitude of autophagy (Noda and Klionsky 2008). Normally, Pho8 is transported to the vacuole via the secretory pathway, where it is processed and activated to form the mature form by proteolysis. Upon removal of the N-terminal 60 amino acids, Pho8 can only be transported to the vacuole by autophagy. Thus, the amount of Pho8 enzymatic activity under these conditions is a measure of autophagy. We performed the Pho8Δ60 assay in the arl1Δ (YSA003) strain, containing the PHO8Δ60 allele after transformation with plasmids bearing the eight genes of interest (Figure 1B). ARL1 and empty vector (YEp352) were used as controls. The results show that, consistent with the GFP-Atg8 assay, overexpression of COG4, TAX4, SNX4, and ARL1 was able to suppress the autophagy defect of the arl1Δ strain at 37°, whereas overexpression of IVY1, ATG5, SLT2, and PEP3 was not.
|
study
| 100.0 |
We also visualized the transport of GFP-Atg8 by fluorescence microscopy (Figure 2). Under nonstarvation conditions, GFP-Atg8 aggregated as single dots in the cells, denoting the PAS (Suzuki et al. 2001) (Figure 2A). When cells were treated with starvation medium at 30°, all of the strains had a vacuolar diffuse green phenotype (Yang and Rosenwald 2016), indicating the normal processing of GFP-Atg8. Under starvation conditions at 37° (Figure 2C), cells overexpressing COG4, SNX4, and TAX4 as well as ARL1 had the green diffuse phenotype, consistent with the conclusion from Figure 1 that these four genes, when overexpressed, are able to suppress the autophagy defect in the arl1Δ strain. On the other hand, cells overexpressing IVY1, PEP3, SLT2, and ATG5 exhibited multiple green dots, suggesting defective autophagy (Figure 2C). After counting the cells with diffuse green phenotype (normal autophagy) at both 30 and 37°, we found that the arl1Δ strains that overexpressed COG4, SNX4, and TAX4 had a significantly higher percentage of cells with the diffuse green phenotype at 37°, while cells overexpressing IVY1, PEP3, SLT2, and ATG5 had a dramatically decreased percentage of cells with this phenotype at high temperature compared with 30°. In conclusion, we identified COG4, SNX4, and TAX4 as high copy suppressors for the high temperature autophagy defect of the arl1Δ strain.
|
study
| 100.0 |
COG4, TAX4, and SNX4 suppress the GFP-Atg8 processing defect at 37° in the arl1Δ strain. Cells were grown then starved for nitrogen as described. (A) Fluorescence images of all strains in nonstarvation conditions. (B) Fluorescence images of all strains in starvation conditions at 30°. (C) Fluorescence images of all strains in starvation conditions at 37°. (D) The percentage of the cells with the green diffusion phenotype in starvation conditions at 30 and 37°. At least 100 cells were counted for each strain. Error bars represent SD from three biological replicates. Scale bar: 3 μm. EV, empty vector.
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study
| 100.0 |
We performed the same set of autophagy assays as for the arl1Δ strain to test which of the seven genes from the screen were able to suppress the high temperature autophagy defect. From the GFP-Atg8 assay, we found that only IVY1 and ATG5, as well as the positive control YPT6, were able to suppress the autophagy defect of the ypt6Δ strain at 37°, as demonstrated by the appearance of free GFP (Figure 3A). This conclusion was further confirmed through Pho8Δ60 assay (Figure 3B); upon overexpression of IVY1, ATG5, or YPT6, the Pho8Δ60 activity in the ypt6Δ strain (YSA004) was increased compared with empty vector. Similarly, these results were confirmed by the GFP-Atg8 fluorescence phenotype (Figure 4, A–C), as we found that the ypt6Δ strain overexpressing IVY1 or ATG5 had an increased percentage of cells with the diffuse green phenotype at 37° (Figure 4D). In conclusion, we identified IVY1 and ATG5 as high copy suppressors for the high temperature autophagy defect of the ypt6Δ strain.
|
study
| 100.0 |
Autophagy-specific assays show IVY1 and ATG5 suppress the temperature-sensitive autophagy defect of the ypt6Δ strain. (A) GFP-Atg8 degradation was increased at 37° in ypt6Δ when transformed with plasmids containing IVY1, ATG5, and WT YPT6. (B) IVY1 and ATG5 increased the Pho8Δ60 activities in ypt6Δ (YSA004) at 37°. Error bars represent SD from three biological replicates. Methods are the same as in Figure 1. GFP, green fluorescent protein; WT, wild-type.
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study
| 100.0 |
IVY1 and ATG5 suppress the GFP-Atg8 processing defect at 37° in the ypt6Δ strain. Cells were grown then starved for nitrogen as described. (A) Fluorescence images of all strains in nonstarvation conditions. (B) Fluorescence images of all strains in starvation conditions at 30°. (C) Fluorescence images of all strains in starvation conditions at 37°. (D) The percentage of the cells with the green diffusion phenotypes in starvation condition at 30 and 37°. At least 100 cells were counted for each strain. Error bars represent SD from three biological replicates. Scale bar: 3 μm. EV, empty vector.
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study
| 100.0 |
From the seven candidates, we found that COG4, TAX4, and SNX4 suppressed the autophagy defect of the arl1Δ strain, while IVY1 and ATG5 suppressed the defect in the ypt6Δ strain. Two genes identified in the primary screen, SLT2 and PEP3, selected from rapamycin sensitivity screens for the arl1Δ and ypt6Δ strains, respectively, did not suppress the defect in either strain as measured by the more specific assays. In order to confirm that these two genes were actually the relevant ones in the genomic fragments, we streaked the arl1Δ strain with a plasmid containing only the SLT2 gene and the ypt6Δ strain with a plasmid containing only the PEP3 gene onto medium containing 5 ng/ml rapamycin, and cultured them at 37°. These genes, SLT2 and PEP3, suppressed the high temperature rapamycin sensitivity of arl1Δ and ypt6Δ, respectively (Figure 5). These results indicate that genes that suppressed the rapamycin phenotype in these strains are not necessarily regulators of autophagy.
|
study
| 100.0 |
Autophagy is an essential cellular process that helps cells overcome stressful environmental conditions. It is also tightly regulated and requires a large number of proteins to perform correctly. The cooperation and interactions between different regulators are particularly important for the correct execution of autophagy. Autophagy relies intensively on membrane resources for the formation of the autophagosome to sequester the targeted cytosolic components. Therefore, many membrane traffic regulators, including many monomeric GTP-binding proteins, have been shown to be essential for this process. Examples include Ypt1 and Sec4. While Ypt1 normally controls the traffic between the ER and the cis-Golgi (Jedd et al. 1995) and Sec4 is required for the exocytic secretion pathway (Kabcenell et al. 1990), both are required for membrane transport to the PAS to form the autophagosome (Geng et al. 2010) (Lynch-Day et al. 2010).
|
review
| 99.2 |
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