| 1. |
- Al-Amin, Rasel A., Researcher, 1983-, et al.
(författare)
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Monitoring drug–target interactions through target engagement-mediated amplification on arrays and in situ
- 2022
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 50:22, s. e129-e129
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Tidskriftsartikel (refereegranskat)abstract
- Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by the dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to fixed adherent cells agreed with expectations from expression profiles of the cells. We also introduce a proximity ligation variant of TEMA to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug–target interactions at spatial resolution in protein arrays, cells and in tissues.
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| 2. |
- Al-Amin, Rasel A., 1983-, et al.
(författare)
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Target Engagement-Mediated Amplification for Monitoring Drug-Target Interactions in Situ
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- It is important to determine the localization of drugs or drug candidates at cellular and subcellular resolution in relevant clinical specimens. This is necessary to evaluate drug candidates from early stages of drug development to clinical evaluation of mutations potentially causing resistance to targeted therapy. We describe a technology where oligonucleotide-conjugated drug molecules are used to visualize and measure target engagement in situ via rolling-circle amplification (RCA) of circularized oligonucleotide probes (padlock probes). We established this target engagement-mediated amplification (TEMA) technique using kinase inhibitor precursor compounds, and we applied the assay to investigate target interactions by microscopy in pathology tissue sections and using flow cytometry for blood samples from patients, as well as in commercial arrays including almost half of all human proteins. In the variant proxTEMAtechnique, in situ proximity ligation assays were performed by combining drug-DNA conjugates with antibody-DNA conjugates to specifically reveal drug binding to particular on- or off-targets in pathological tissues sections. In conclusion, the TEMA methods successfully visualize drug-target interaction by experimental and clinically approved kinase inhibitors in situ and with kinases among a large collection of arrayed proteins.
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| 3. |
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| 4. |
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| 5. |
- Dahlin, Joakim S., et al.
(författare)
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KIT signaling is dispensable for human mast cell progenitor development
- 2017
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Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 130:16, s. 1785-1794
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Tidskriftsartikel (refereegranskat)abstract
- Human hematopoietic progenitors are generally assumed to require stem cell factor (SCF) and KIT signaling during differentiation for the formation of mast cells. Imatinib treatment, which inhibits KIT signaling, depletes mast cells in vivo. Furthermore, the absence of SCF or imatinib treatment prevents progenitors from developing into mast cells in vitro. However, these observations do not mean that mast cell progenitors require SCF and KIT signaling throughout differentiation. Here, we demonstrate that circulating mast cell progenitors are present in patients undergoing imatinib treatment. In addition, we show that mast cell progenitors from peripheral blood survive, mature, and proliferate without SCF and KIT signaling in vitro. Contrary to the prevailing consensus, our results show that SCF and KIT signaling are dispensable for early mast cell development.
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| 6. |
- Doulabi, Ehsan Manouchehri, et al.
(författare)
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Increased levels of thymidine kinase 1 in malignant cell-derived extracellular vesicles.
- 2024
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Ingår i: Biochemistry and biophysics reports. - 2405-5808. ; 39, s. 101761-
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs), whose main subtypes are exosomes, microparticles, and apoptotic bodies, are secreted by all cells and harbor biomolecules such as DNA, RNA, and proteins. They function as intercellular messengers and, depending on their cargo, may have multiple roles in cancer development. Thymidine kinase 1 (TK1) is a cell cycle-dependent enzyme used as a biomarker for cell proliferation. TK1 is usually elevated in cancer patients' serum, making the enzyme a valuable tumor proliferation biomarker that strongly correlates with cancer stage and metastatic capabilities. Here, we investigated the presence of TK1 in EVs derived from three prostate cancer cell lines with various p53 mutation statuses (LNCaP, PC3, and DU145), EVs from the normal prostate epithelial cell line RWPE-1 and EVs isolated from human seminal fluid (prostasomes). We measured the TK1 activity by a real-time assay for these EVs. We demonstrated that the TK1 enzyme activity is higher in EVs derived from the malignant cell lines, with the highest activity from cells deriving from the most aggressive cancer, compared to the prostasomes and RWPE-1 EVs. The measurement of TK1 activity in EVs may be essential in future prostate cancer studies.
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| 7. |
- Doulabi, Ehsan Manouchehri, et al.
(författare)
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Surface protein profiling of prostate-derived extracellular vesicles by mass spectrometry and proximity assays
- 2022
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Ingår i: Communications biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 5:1, s. 1402-
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs) are mediators of intercellular communication and a promising class of biomarkers. Surface proteins of EVs play decisive roles in establishing a connection with recipient cells, and they are putative targets for diagnostic assays. Analysis of the surface proteins can thus both illuminate the biological functions of EVs and help identify potential biomarkers. We developed a strategy combining high-resolution mass spectrometry (HRMS) and proximity ligation assays (PLA) to first identify and then validate surface proteins discovered on EVs. We applied our workflow to investigate surface proteins of small EVs found in seminal fluid (SF-sEV). We identified 1,014 surface proteins and verified the presence of a subset of these on the surface of SF-sEVs. Our work demonstrates a general strategy for deep analysis of EVs’ surface proteins across patients and pathological conditions, proceeding from unbiased screening by HRMS to ultra-sensitive targeted analyses via PLA.
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| 8. |
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| 9. |
- Dubois, Louise, et al.
(författare)
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Human erythrocyte-derived nanovesicles can readily be loaded with doxorubicin and act as anticancer agents
- 2018
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Ingår i: Cancer Research Frontiers. - : Cancer Research Frontiers. - 2328-5249. ; 4:1, s. 13-26
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: In future therapeutics new formulas are needed that assure lower doses, fewer side effects, targeted administration and protection of the drug from degradation. In a first step to fulfil the requirements defined above, we carried out an in vitro study by developing a new procedure to encapsulate drugs using native vesicles first from prostasomes and then from erythrocyte membranes known to be well tolerated. The new method for production of drug delivery vesicles utilized osmotic loading of detergent resistant membranes (DRMs).Materials and methods: DRMs of prostasomes and prepared human erythrocyte membranes were extracted and separated in a sucrose gradient at a density of 1.10 g/mL containing 1% Triton X-100. These DRMs were characterized by electron microscopy (transmission and scanning EM) and loaded with low and high molecular compounds. PC3 prostate cancer cells were treated with doxorubicin loaded DRMs in triplicate. DAPI (nuclear fluorescent stain) was included and fluorescence microscopic pictures were taken before the cells were trypsinized and counted after 48h.Results: The content of the well separated band was observed ultrastructurally as small spherical, double layered membrane vesicles, (DRM vesicles) which harbored hyperosmolar sucrose of the gradient. Encapsulated hyperosmolar sucrose induced a transient osmotic lysis of the DRM vesicles when suspended in isotonic buffer containing loading molecules allowing vesicular inclusion. After this proof of concept, the method was finally employed for doxorubicin loading of DRM vesicles from human erythrocytes. When incubating such vesicles with PC3 cells a complete arrest of growth was observed in sharp contrast to PC3 cells incubated with plain doxorubicin in similar conditions.Conclusion: The present results open up new possibilities for using DRM vesicles as drug delivery vesicles.
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| 10. |
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| 11. |
- Ebai, Tonge, et al.
(författare)
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Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification
- 2017
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 63:9, s. 1497-1505
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Detecting proteins at low concentrations in plasma is crucial for early diagnosis. Current techniques in clinical routine, such as sandwich ELISA, provide sensitive protein detection because of a dependence on target recognition by pairs of antibodies, but detection of still lower protein concentrations is often called for. Proximity ligation assay with rolling circle amplification (PLARCA) is a modified proximity ligation assay (PLA) for analytically specific and sensitive protein detection via binding of target proteins by 3 antibodies, and signal amplification via rolling circle amplification (RCA) in microtiter wells, easily adapted to instrumentation in use in hospitals.METHODS: Proteins captured by immobilized antibodies were detected using a pair of oligonucleotide-conjugated antibodies. Upon target recognition, these PLA probes guided oligonucleotide ligation, followed by amplification via RCA of circular DNA strands that formed in the reaction. The RCA products were detected by horseradish peroxidase-labeled oligonucleotides to generate colorimetric reaction products with readout in an absorbance microplate reader.RESULTS: We compared detection of interleukin (IL)-4, IL-6, IL-8, p53, and growth differentiation factor-15 by PLARCA and conventional sandwich ELISA or immuno RCA. PLARCA detected lower concentrations of proteins and exhibited a broader dynamic range compared ELISA and iRCA using the same antibodies. IL-4 and IL-6 were detected in clinical samples at femtomolar concentrations, considerably lower than for ELISA.CONCLUSIONS: PLARCA offers detection of lower protein levels and increased dynamic ranges compared to ELISA. The PLARCA procedure may be adapted to routine instrumentation available in hospitals and research laboratories.
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| 12. |
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| 13. |
- Gholiha, Alex Reza, et al.
(författare)
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Checkpoint CD47 expression in classical Hodgkin lymphoma
- 2022
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Ingår i: British Journal of Haematology. - : John Wiley & Sons. - 0007-1048 .- 1365-2141. ; 197:5, s. 580-589
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Tidskriftsartikel (refereegranskat)abstract
- The glycoprotein CD47 regulates antiphagocytic activity via signal regulatory protein alpha (SIRPa). This study investigated CD47 expression on Hodgkin and Reed–Sternberg (HRS) cells in the classical Hodgkin lymphoma (cHL) tumour microenvironment and its correlation with prognosis, programmed-death (PD) immune markers, and SIRPa+ leukocytes. We conducted immunohistochemistry with CD47 and SIRPa antibodies on diagnostic biopsies (tissue microarrays) from cHL patients from two cohorts (n = 178). In cohort I (n = 136) patients with high expression of CD47 on HRS cells (n = 48) had a significantly inferior event-free survival [hazard ratio (HR) = 5.57; 95% confidence interval (CI), 2.78–11.20; p < 0.001] and overall survival (OS) (HR = 8.54; 95% CI, 3.19–22.90; p < 0.001) compared with patients with low expression (n = 88). The survival results remained statistically significant in multivariable Cox regression adjusted for known prognostic factors. In cohort II (n = 42) high HRS cell CD47 expression also carried shorter event-free survival (EFS) (HR = 5.96; 95% CI, 1.20–29.59; p = 0.029) and OS (HR = 5.61; 95% CI, 0.58–54.15; p = 0.136), although it did not retain statistical significance in the multivariable analysis. Further, high CD47 expression did not correlate with SIRPa+ leukocytes or PD-1, PD-L1 and PD-L2 expression. This study provides a deeper understanding of the role of CD47 in cHL during an era of emerging CD47 therapies.
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| 14. |
- Gholiha, Alex R., et al.
(författare)
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Immune-Proteome Profiling in Classical Hodgkin Lymphoma Tumor Diagnostic Tissue
- 2022
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Ingår i: Cancers. - : MDPI. - 2072-6694. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- In classical Hodgkin Lymphoma (cHL), immunoediting via protein signaling is key to evading tumor surveillance. We aimed to identify immune-related proteins that distinguish diagnostic cHL tissues (=diagnostic tumor lysates, n = 27) from control tissues (reactive lymph node lysates, n = 30). Further, we correlated our findings with the proteome plasma profile between cHL patients (n = 26) and healthy controls (n = 27). We used the proximity extension assay (PEA) with the OlinkTM multiplex Immuno-Oncology panel, consisting of 92 proteins. Univariate, multivariate-adjusted analysis and Benjamini–Hochberg’s false discovery testing (=Padj) were performed to detect significant discrepancies. Proteins distinguishing cHL cases from controls were more numerous in plasma (30 proteins) than tissue (17 proteins), all Padj < 0.05. Eight of the identified proteins in cHL tissue (PD-L1, IL-6, CCL17, CCL3, IL-13, MMP12, TNFRS4, and LAG3) were elevated in both cHL tissues and cHL plasma compared with control samples. Six proteins distinguishing cHL tissues from controls tissues were significantly correlated to PD-L1 expression in cHL tissue (IL-6, MCP-2, CCL3, CCL4, GZMB, and IFN-gamma, all p ≤0.05). In conclusion, this study introduces a distinguishing proteomic profile in cHL tissue and potential immune-related markers of pathophysiological relevance
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| 15. |
- Ikebuchi, Ryoyo, et al.
(författare)
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Human proteins incorporated into tick-borne encephalitis virus revealed by in situ proximity ligation
- 2020
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : ACADEMIC PRESS INC ELSEVIER SCIENCE. - 0006-291X .- 1090-2104. ; 525:3, s. 714-719
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Tidskriftsartikel (refereegranskat)abstract
- Host proteins incorporated into virus particles have been reported to contribute to infectivity and tissue-tropism. This incorporation of host proteins is expected to be variable among viral particles, however, protein analysis at single-virus levels has been challenging. We have developed a method to detect host proteins incorporated on the surface of virions using the in situ proximity ligation assay (isPLA) with rolling circle amplification (RCA), employing oligonucleotide-conjugated antibody pairs. The technique allows highly selective and sensitive antibody-based detection of viral and host proteins on the surface of individual virions. We detected recombinant noninfectious sub-viral particles (SVPs) of tick-borne encephalitis virus (TBEV) immobilized in microtiter wells as fluorescent particles detected by regular fluorescence microscopy. Counting the particles in the images enabled us to estimate individual TBEVSVP counts in different samples. Using isPLA we detected individual calnexin-, CD9-, CD81-, CD29-and CD59-positive SVPs among the viral particles. Our data suggests that a diversity of host proteins may be incorporated into TEBV, illustrating that isPLA with digital counting enables single-virus analysis of host protein incorporation. (C) 2020 The Authors. Published by Elsevier Inc.
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| 16. |
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| 17. |
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| 18. |
- Koos, Björn, et al.
(författare)
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Proximity-dependent initiation of hybridization chain reaction
- 2015
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point-of-care devices and high-throughput screening, should be reliable, cost effective and robust. To achieve this, here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence. This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry. As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.
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| 19. |
- Larssen, Pia, et al.
(författare)
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Tracing Cellular Origin of Human Exosomes Using Multiplex Proximity Extension Assay
- 2017
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 16:3, s. 502-511
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs) are membrane-coated objects such as exosomes and microvesicles, released by many cell-types. Their presence in body fluids and the variable surface composition and content render them attractive potential biomarkers. The ability to determine their cellular origin could greatly move the field forward. We used multiplex proximity extension assays (PEA) to identify with high specificity and sensitivity the protein profiles of exosomes of different origins, including seven cell lines and two different body fluids. By comparing cells and exosomes, we successfully identified the cells originating the exosomes. Furthermore, by principal component analysis of protein patterns human milk EVs and prostasomes released from prostate acinar cells clustered with cell lines from breast and prostate tissues, respectively. Milk exosomes uniquely expressed CXCL5, MIA and KLK6, while prostasomes carried NKX31, GSTP1 and SRC, highlighting that EVs originating from different origins express distinct proteins. In conclusion, PEA provides a powerful protein screening tool in exosome research, for purposes of identifying the cell source of exosomes, or new biomarkers in diseases such as cancer and inflammation.
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| 20. |
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| 21. |
- Löf, Liza
(författare)
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Applications of in situ proximity ligation assays for cancer research and diagnostics
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the field of cancer research and diagnostics it is crucial to have reliable methods for detecting molecules involved in the disease. New and better methods for diagnostics, prognostics and drug delivery therefore remain a permanent aim. In this thesis applications of the in situ proximity ligation assay (in situ PLA) were developed for diagnostics and research. Two new methods were developed, one more cost effective proximity assay without the use of enzymes and one method for loading pharmaceuticals in lipid rafts made from detergent resistant membranes (DRMs) to be used as a drug delivery platform.In Paper I the aim was to develop a flow cytometric detection method of the fusion protein BCR-ABL that is the hallmark of chronic myeloid leukemia (CML). By using in situ PLA the malignant cells carrying the fusion protein could be detected in patients in a convenient workflow.Paper II describes an application of multiplex in situ PLA, where extracellular vesicles (EVs) are detected and identified using flow cytometry. Up to five different antigens are targeted on the EVs, reflected in three different colors during detection in the flow cytometer. By using antibodies targeting proteins specific for prostasomes a population of prostasomes could be identified in human blood plasma.In Paper III a new method is described for using lipid raft for drug delivery. In this method, lipid rafts, derived from prostasomes or erythrocytes, are loaded with pharmaceuticals. The vehicles were loaded with doxorubicin, added to cells and counted. Cells that received the vehicle with doxorubicin stopped proliferating and died, while controls that received the lipid raft vehicle without doxorubicin were not affected, suggesting that the vehicles are effectively loaded with the drug and that they are safe. This lipid raft vehicle could provide a safe drug delivery system. Paper IV investigates the crosstalk between the two major signal pathways Hippo and Wnt, and how these are affected in gastric cancer. When looking at different colon cancer tumor stages, we found that the cellular localization of TAZ/β-catenin interactions were different. We also found that protein complexes involved in the crosstalk increased in sparsely growing cells compared to more densely growing cells. On the basis of these results the protein E-cadherin, involved in maintenance of the epithelial integrity, was investigated and was found to have a probable role in regulating the crosstalk between Hippo and Wnt. A new method for localized protein detection is described in paper V. Here a proximity assay, based on the hybridization chain reaction (HCR), was developed. This assay, proxHCR, is more cost effective than in situ PLA because no enzymes are required. ProxHCR successfully detects protein interactions and can be used together with both fluorescence microscopy and flow cytometry.
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| 22. |
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| 23. |
- Löf, Liza, et al.
(författare)
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Detecting individual extracellular vesicles using a multicolor in situ proximity ligation assay with flow cytometric readout
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Flow cytometry is a powerful method for quantitative and qualitative analysis of individual cells. However, flow cytometric analysis of extracellular vesicles (EVs), and the proteins present on their surfaces has been hampered by the small size of the EVs - in particular for the smallest EVs, which can be as little as 40 nm in diameter, the limited number of antigens present, and their low refractive index. We addressed these limitations for detection and characterization of EV by flow cytometry through the use of multiplex and multicolor in situ proximity ligation assays (in situ PLA), allowing each detected EV to be easily recorded over background noise using a conventional flow cytometer. By targeting sets of proteins on the surface that are specific for distinct classes of EVs, the method allows for selective recognition of populations of EVs in samples containing more than one type of EVs. The method presented herein opens up for analyses of EVs using flow cytometry for their characterization and quantification.
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| 24. |
- Löf, Liza, et al.
(författare)
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Detection of Extracellular Vesicles Using Proximity Ligation Assay with Flow Cytometry Readout – ExoPLA : In Print
- 2017
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Ingår i: Current Protocol in Cytometry. - : John Wiley & Sons. - 1934-9297 .- 1934-9300. ; 81:1
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Forskningsöversikt (refereegranskat)abstract
- Extracellular vesicles (EVs) are continuously released by most cells, and they carry surface markers of their cells of origin. Found in all body fluids, EVs function as conveyers of cellular information, and evidence implicates them as markers of disease. These characteristics make EVs attractive diagnostic targets. However, detection and characterization of EVs is challenging due to their small size. We've established a method, called ExoPLA, that allows individual EVs to be detected and characterized at high specificity and sensitivity. Based on the in situ proximity ligation assay (in situ PLA), proximal oligonucleotide-conjugated antibodies bound to their targets on the surfaces of the EVs allow formation of circular products that can be fluorescently labeled by rolling circle amplification. The intense fluorescent signals produced in this assay allow detection and enumeration of individual EVs by flow cytometry. We describe the procedures for ExoPLA, along with expected results and troubleshooting.
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| 25. |
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| 26. |
- Löf, Liza, et al.
(författare)
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Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia
- 2017
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Chronic myeloid leukemia (CML) is characterized in the majority of cases by a t(9;22)(q34;q11) translocation, also called the Philadelphia chromosome, giving rise to the BCR-ABL1 fusion protein. Current treatment with tyrosine kinase inhibitors is directed against the constitutively active ABL1 domain of the fusion protein, and minimal residual disease (MRD) after therapy is monitored by real-time quantitative PCR (RQ-PCR) of the fusion transcript. Here, we describe a novel approach to detect and enumerate cells positive for the BCR-ABL1 fusion protein by combining the in situ proximity ligation assay with flow cytometry as readout (PLA-flow). By targeting of the BCR and ABL1 parts of the fusion protein with one antibody each, and creating strong fluorescent signals through rolling circle amplification, PLA-flow allowed sensitive detection of cells positive for the BCR-ABL1 fusion at frequencies as low as one in 10,000. Importantly, the flow cytometric results correlated strongly to those of RQ-PCR, both in diagnostic testing and for MRD measurements over time. In summary, we believe this flow cytometry-based method can serve as an attractive approach for routine measurement of cells harboring BCR-ABL1 fusions, also allowing simultaneously assessment of other cell surface markers as well as sensitive longitudinal follow-up.
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| 27. |
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| 28. |
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| 29. |
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| 30. |
- Manouchehri Doulabi, Ehsan, et al.
(författare)
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Targeted proteome analysis of extracellular vesicles from gastric cancer cell lines
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Gastric cancer (GC) is the third leading cause of cancer death. Early detection and efficient monitoring of tumor dynamics are prerequisites for reducing disease burden and mortality. Blood-based biomarker assays for the detection of early-stage GC could be of great relevance for population-wide or risk-group-based screening programs. Extracellular vesicles (EVs) are nano-sized membrane-enclosed particles released from all cells, which play an essential role as mediators of intercellular communication and are a promising class of biomarkers. We aimed to determine the putative panel of protein targets by collecting EVs from GC cell lines for future diagnostic assays. We measured levels of over 430 proteins using highly sensitive and high-throughput proximity extension assays (PEA). The protein network and function of each protein were analyzed. In conclusion, despite a similarity between GC EVs and EVs from the prostate as a control, we identified a set of 39 proteins in GC EVs that are involved in different cellular pathways and are not expressed or have low expression in exosomes from the prostate.
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| 31. |
- Schiffer, Tomas A., et al.
(författare)
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Mitochondrial Respiration-Dependent ANT2-UCP2 Interaction
- 2022
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Ingår i: Frontiers in Physiology. - : Frontiers Media S.A.. - 1664-042X. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Adenine nucleotide translocases (ANTs) and uncoupling proteins (UCPs) are known to facilitate proton leak across the inner mitochondrial membrane. However, it remains to be unravelled whether UCP2/3 contribute to significant amount of proton leak in vivo. Reports are indicative of UCP2 dependent proton-coupled efflux of C4 metabolites from the mitochondrial matrix. Previous studies have suggested that UCP2/3 knockdown (KD) contributes to increased ANT-dependent proton leak. Here we investigated the hypothesis that interaction exists between the UCP2 and ANT2 proteins, and that such interaction is regulated by the cellular metabolic demand. Protein-protein interaction was evaluated using reciprocal co-immunoprecipitation and in situ proximity ligation assay. KD of ANT2 and UCP2 was performed by siRNA in human embryonic kidney cells 293A (HEK293A) cells. Mitochondrial and cellular respiration was measured by high-resolution respirometry. ANT2-UCP2 interaction was demonstrated, and this was dependent on cellular metabolism. Inhibition of ATP synthase promoted ANT2-UCP2 interaction whereas high cellular respiration, induced by adding the mitochondrial uncoupler FCCP, prevented interaction. UCP2 KD contributed to increased carboxyatractyloside (CATR) sensitive proton leak, whereas ANT2 and UCP2 double KD reduced CATR sensitive proton leak, compared to UCP2 KD. Furthermore, proton leak was reduced in double KD compared to UCP2 KD. In conclusion, our results show that there is an interaction between ANT2-UCP2, which appears to be dynamically regulated by mitochondrial respiratory activity. This may have implications in the regulation of mitochondrial efficiency or cellular substrate utilization as increased activity of UCP2 may promote a switch from glucose to fatty acid metabolism.
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| 32. |
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| 33. |
- Zhao, Hongxing, et al.
(författare)
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Detection of SARS-CoV-2 antibodies in serum and dried blood spot samples of vaccinated individuals using a sensitive homogeneous proximity extension assay.
- 2022
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Ingår i: New biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 72, s. 139-148
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Tidskriftsartikel (refereegranskat)abstract
- A homogeneous PCR-based assay for sensitive and specific detection of antibodies in serum or dried blood spots (DBS) is presented and the method is used to monitor individuals infected with or vaccinated against SARS-CoV-2. Detection probes were prepared by conjugating the recombinant spike protein subunit 1 (S1), containing the receptor binding domain (RBD) of SARS-CoV-2, to each of a pair of specific oligonucleotides. The same was done for the nucleocapsid protein (NP). Upon incubation with serum or DBS samples, the bi- or multivalency of the antibodies (IgG, IgA or IgM) brings pairs of viral proteins with their conjugated oligonucleotides in proximity, allowing the antibodies to be detected by a modified proximity extension assay (PEA). Anti-S1 and anti-NP antibodies could be detected simultaneously from one incubation reaction. This Antibody PEA (AbPEA) test uses only 1µl of neat or up to 100,000-fold diluted serum or one ø1.2mm disc cut from a DBS. All 100 investigated sera and 21 DBS collected prior to the COVID-19 outbreak were negative, demonstrating a 100% specificity. The area under the curve, as evaluated by Receiver Operating Characteristic (ROC) analysis reached 0.998 (95%CI: 0.993-1) for samples taken from 11 days after symptoms onset. The kinetics of antibody responses were monitored after a first and second vaccination using serially collected DBS from 14 individuals. AbPEA offers highly specific and sensitive solution-phase antibody detection without requirement for secondary antibodies, no elution step when using DBS sample in a simple procedure that lends itself to multiplex survey of antibody responses.
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