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  • 1.
    Aljadi, Zenib
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Inst, Dept Clin Sci & Educ, Stockholm, Sweden; Soder Sjukhuset, Stockholm, Sweden .
    Kalm, Frida
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Inst, Dept Clin Sci & Educ, Stockholm, Sweden; Soder Sjukhuset, Stockholm, Sweden.
    Ramachandraiah, Harisha
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nopp, Anna
    Karolinska Inst, Dept Clin Sci & Educ, Stockholm, Sweden.;Soder Sjukhuset, Stockholm, Sweden..
    Lundahl, Joachim
    Karolinska Inst, Dept Clin Sci & Educ, Stockholm, Sweden.;Soder Sjukhuset, Stockholm, Sweden..
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Microfluidic Immunoaffinity Basophil Activation Test for Point-of-Care Allergy Diagnosis2019In: Journal of Applied Laboratory Medicine (JALM), ISSN 2475-7241, Vol. 4, no 2, p. 152-163Article in journal (Refereed)
    Abstract [en]

    Background: The flow cytometry-based basophil activation test (BAT) is used for the diagnosis of allergic response. However, flow cytometry is time-consuming, requiring skilled personnel and cumbersome processing, which has limited its use in the clinic. Here, we introduce a novel microfluidic-based immunoaffinity BAT (miBAT) method. Methods: The microfluidic device, coated with anti-CD203c, was designed to capture basophils directly from whole blood. The captured basophils are activated by anti-FceRI antibody followed by optical detection of CD63 expression (degranulation marker). The device was first characterized using a basophil cell line followed by whole blood experiments. Weevaluated the device with ex vivo stimulation of basophils in whole blood from healthy controls and patients with allergies and compared it with flow cytometry. Results: The microfluidic device was capable of capturing basophils directly from whole blood followed by in vitro activation and quantification of CD63 expression. CD63 expression was significantly higher (P = 0.0002) in on-chip activated basophils compared with nonactivated cells. The difference in CD63 expression on anti-FceRI-activated captured basophils in microfluidic chip was significantly higher (P = 0.03) in patients with allergies compared with healthy controls, and the results were comparable with flow cytometry analysis (P = 0.04). Furthermore, there was no significant difference of CD63% expression in anti-FceRI-activated captured basophils in microfluidic chip compared with flow cytometry. Conclusions: We report on the miBAT. This device is capable of isolating basophils directly from whole blood for on-chip activation and detection. The new miBAT method awaits validation in larger patient populations to assess performance in diagnosis and monitoring of patients with allergies at the point of care.

  • 2.
    Banerjee, Indradumna
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Salih, Tagrid
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Ramachandraiah, Harisha
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Erlandsson, J.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science.
    Petterson, T.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Surface and Corrosion Science.
    Silva, AC
    Karlsson, M
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    LDH based neonatal diagnostics on a low-cost slipdisc based sample preparation platform.2016Conference paper (Refereed)
    Abstract [en]

    INTRODUCTION

    Slipdisc is developed as a sample preparation platform based on slipchip technology [1], using a handwinded clockwork mechanism allowing sample processing from one spot to another with defined precision without the need for sophisticated tools or alignment (Fig.1). An ordinary smartphone or camera can be used to image and analyse the results making it an ideal tool for resource limited settings. Here, we demonstrate a bioassay for detecting LDH (Fig.2), a crucial enzyme found in all living cells which leaks out when the cellular membrane is damaged. This makes LDH a biomarker for several medical conditions in newborns, such as Ozkiraz-13, necrotizing enterocolitis (NEC), and Asphyxia.

    EXPERIMENTAL

    For assembling the slipdisc optically transparent, robust and disposable CD like polycarbonate discs were used with superhydrophobic coating on all except the embedded microfluidic channels. For the LDH assay, heparinized plasma samples were spiked with 7 different concentrations of the LDH enzyme (Lee Biosolutions, USA). These concentrations ranged from clinically normal to abnormal concentrations and used to construct a standard curve for LDH enzyme.

    RESULTS AND DISCUSSION

    The ability of the SlipDisc to quantify LDH enzyme levels from plasma samples was evaluated (Fig.3). Using 7 different concentrations, a standard curve with clinically relevant LDH concentrations was obtained (Fig4). Image and data analyses, including linear regression and Pearson’s correlation, were completed using Image processing tool in Matlab.

    CONCLUSION

    We demonstrate a low-cost neonatal diagnostics platform for the detection of LDH from plasma using a novel SlipDisc platform. The SlipDisc can further be modified to separate plasma from whole blood samples in order to fully integrate the assay. Its simple operation and smartphone based detection capabilities make it an ideal device for point-of-care neonatal diagnostics.

  • 3.
    Banerjee, Indradumna
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Salih, Tagrid
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Ramachandraiah, Harisha
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Erlandsson, Johan
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Fibre Technology.
    Pettersson, Torbjörn
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center. KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Araújo, A. C.
    Karlsson, M.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Slipdisc: A versatile sample preparation platform for point of care diagnostics2017In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 7, no 56, p. 35048-35054Article in journal (Refereed)
    Abstract [en]

    We report a microfluidic sample preparation platform called "Slipdisc" based on slipchip technology. Slipdisc is a rotational slipchip that uses a unique hand-wound clockwork mechanism for precise movement of specially fabricated polycarbonate discs. In operation, the microchannels and microchambers carved on the closely aligned microfluidic discs convert from continuous filled paths to defined compartments using the slip movement. The clockwork mechanism introduced here is characterised by a food dye experiment and a conventional HRP TMB reaction before measuring lactate dehydrogenase (LDH) enzyme levels, which is a crucial biomarker for neonatal diagnostics. The colorimetry based detection of LDH was performed with an unmodified camera and an image analysis procedure based on normalising images and observing changes in red channel intensity. The analysis showed a close to unity coefficient of determination (R2 = 0.96) in detecting the LDH concentration when compared with a standard Chemical Analyser, demonstrating the excellent performance of the slipdisc platform with colorimetric detection. The versatile point of care sample preparation platform should ideally be suited for a multitude of applications at resource-limited settings.

  • 4.
    Etcheverry, S.
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Faridi, A.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Ramachandraiah, H.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Margulis, W.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Laurell, F.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Russom, A.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Optofludics in microstructured fibers combining particle elasto-inertial focusing and fluorescence2016In: 2016 CONFERENCE ON LASERS AND ELECTRO-OPTICS (CLEO), IEEE conference proceedings, 2016Conference paper (Refereed)
    Abstract [en]

    Optofluidics is exploited in an all-fiber component to detect and identify through fluorescence particles flowing at high rate and inertially focused in a capillary. The system represents a first step towards an in-fiber flow cytometer.

  • 5.
    Etcheverry, Sebastián
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Laser Physics. RISE Acreo AB, Sweden.
    Faridi, Muhammad Asim
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ramachandraiah, Harisha
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kumar, T.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Margulis, Walter
    KTH, School of Engineering Sciences (SCI), Applied Physics, Laser Physics. RISE Acreo AB, Sweden.
    Laurell, Fredrik
    KTH, School of Engineering Sciences (SCI), Applied Physics, Laser Physics.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    High performance micro-flow cytometer based on optical fibres2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 5628Article in journal (Refereed)
    Abstract [en]

    Flow cytometry is currently the gold standard for analysis of cells in the medical laboratory and biomedical research. Fuelled by the need of point-of-care diagnosis, a significant effort has been made to miniaturize and reduce cost of flow cytometers. However, despite recent advances, current microsystems remain less versatile and much slower than their large-scale counterparts. In this work, an all-silica fibre microflow cytometer is presented that measures fluorescence and scattering from particles and cells. It integrates cell transport in circular capillaries and light delivery by optical fibres. Single-stream cell focusing is performed by Elasto-inertial microfluidics to guarantee accurate and sensitive detection. The capability of this technique is extended to high flow rates (up to 800 mu l/min), enabling a throughput of 2500 particles/s. The robust, portable and low-cost system described here could be the basis for a point-of-care flow cytometer with a performance comparable to commercial systems.

  • 6.
    Etcheverry, Sebastián
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics. Dept. of Fiber Optics, Acreo Swedish ICT AB, Sweden .
    Faridi, Muhammad Asim
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Ramachandraiah, Harisha
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Margulis, W.
    Laurell, Fredrik
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    All fiber based micro-flow cytometer by combining optical fiber with inertial focusing2016In: 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016, Chemical and Biological Microsystems Society , 2016, p. 1655-1656Conference paper (Refereed)
    Abstract [en]

    Towards a portable point of care flow cytometry platform, we present here an integrated all optical fiber-based optofluidic system capable of counting and discriminating fluorescent particles and cells. The robust and compact device incorporates optical fibers and circular capillaries to build an all-fiber optofluidic device to enable counting particles based on their fluorescent and back-scatter light emission. Here, we combine this with inertial- and elasto-inertial microfluidics for sheathless particle and cell focusing for integrated detection with scattering and fluorescence detections - all necessary components of standard cytometers. We validated the system for cell counting based on scattering and fluorescence.

  • 7.
    Faridi, Muhammad Asim
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. mafaridi@kth.se.
    Ramachandraiah, Harisha
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Banerjee, Indradumna
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ardabli, Sahar
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Zelenin, Sergey
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Elasto-inertial microfluidics for bacteria separation from whole blood for sepsis diagnostics2017In: Journal of Nanobiotechnology, ISSN 1477-3155, E-ISSN 1477-3155, Vol. 15, article id 3Article in journal (Refereed)
    Abstract [en]

    Background: Bloodstream infections (BSI) remain a major challenge with high mortality rate, with an incidence that is increasing worldwide. Early treatment with appropriate therapy can reduce BSI-related morbidity and mortality. However, despite recent progress in molecular based assays, complex sample preparation steps have become critical roadblock for a greater expansion of molecular assays. Here, we report a size based, label-free, bacteria separation from whole blood using elasto-inertial microfluidics.

    Results: In elasto-inertial microfluidics, the viscoelastic flow enables size based migration of blood cells into a non- Newtonian solution, while smaller bacteria remain in the streamline of the blood sample entrance and can be sepa- rated. We first optimized the flow conditions using particles, and show continuous separation of 5 μm particles from 2 μm at a yield of 95% for 5 μm particle and 93% for 2 μm particles at respective outlets. Next, bacteria were continu- ously separated at an efficiency of 76% from undiluted whole blood sample.

    Conclusion: We demonstrate separation of bacteria from undiluted while blood using elasto-inertial microfluidics. The label-free, passive bacteria preparation method has a great potential for downstream phenotypic and molecular analysis of bacteria. 

  • 8. Kjellander, Marcus
    et al.
    Billinger, Erika
    Ramachandraiah, Harisha
    KTH, School of Biotechnology (BIO). Uppsala University, Sweden.
    Boman, Mats
    Lind, Sara Bergström
    Johansson, Gunnar
    A flow-through nanoporous alumina trypsin bioreactor for mass spectrometry peptide fingerprinting2018In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 172, p. 165-172Article in journal (Refereed)
    Abstract [en]

    Mass spectrometry-based proteomics benefits from efficient digestion of protein samples. In this study, trypsin was immobilized on nanoporous anodized alumina membranes to create an enzyme reactor suitable for peptide mass fingerprinting. The membranes were derivatized with 3-aminopropyltriethoxysilane and the amino groups were activated with carbonyldiimidazole to allow coupling of porcine trypsin via c-amino groups. The function was assessed using the artificial substrate Na-Benzoyl-L-arginine 4-nitroanilide hydrochloride, bovine ribonuclease A and a human plasma sample. A 10-membrane flow-through reactor was used for fragmentation and MS analysis after a single pass of substrate both by collection of product and subsequent off-line analysis, and by coupling on-line to the instrument. The peptide pattem allowed correct identification of the single target protein in both cases, and of > 70 plasma proteins in single pass mode followed by LC-MS analysis. The reactor retained 76% of the initial activity after 14 days of storage and repeated use at room temperature. Significance: This manuscript describes the design of a stable enzyme reactor that allows efficient and fast digestion with negligible leakage of enzyme and enzyme fragments. The high stability facilitates the use in an online-setup with MS detection since it allows the processing of multiple samples within an extended period of time without replacement.

  • 9.
    Pettersson, Torbjörn
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Ramachandraiah, Harisha
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Nanocellulose mediated layer-by-layer chip modification for cellular in-vitro diagnostics2017In: Abstracts of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 253Article in journal (Other academic)
  • 10.
    Ramachandraiah, Harisha
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Kugiejko, Karol
    KTH, School of Biotechnology (BIO).
    Pettersson, Torbjörn
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Heuchel, Rainer
    Karolinska Institutet.
    Löhr, Matthias
    karolinska Institute.
    Hedhammar, My
    KTH, School of Biotechnology (BIO), Protein Technology.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Microfluidic based circulating tumor cell isolation and release from whole blood of pancreatic cancer patients using bio-functionalized recombinant spider silkManuscript (preprint) (Other academic)
    Abstract [en]

    A bio-functionalized microsystem was developed for the capture and release of cancer cells from whole blood. Effective isolation and purification of circulating tumor cells from whole blood provides important capability for clinical application and biological research. Here, we demonstrate a single step surface modification procedure for a microfluidic device based on self-assembly of recombinant spider silk harbouring an affinity domain for antibody binding. The surfaces of microfluidic devices were conjugated/equipped with anti-EpCAM antibody for selective isolation of pancreatic cancer cells from spiked whole blood and finally circulating tumor cells from pancreatic cancer patients. Moreover, a protease-cleavage site in the recombinant spider silk proteins provides the unique option to release the captured cancer cells on command from the device without compromising the cell’s viability. Our approach offers a simple, easy and robust surface modification process with a 85% cancer cell capture efficiency. Subsequent addition of a site-specific protease results in the release of 95% of captured cells from the bio functionalized microfluidic systems. 

  • 11.
    Ramachandraiah, Harisha
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Pettersson, Torbjörn
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Layer-by-layer system based cellulose nanofibrils for capture and release of cells in microfluidic deviceManuscript (preprint) (Other (popular science, discussion, etc.))
    Abstract [en]

    Selective isolation of cells, without inducing any phenotypic changes and maintaining cell viability will preserve the information necessary for down stream analysis. Here we present an ultra thin coating on the surface of disposable microfluidic device based on cellulose nanofibrils, that is modified to capture cells and for later release. Layer-by-layer technique facilitates the production of the thin coating of cellulose onto polymeric surfaces and modified to form affinity based cell capture surface. We demonstrate an  efficiently capture and release of cells, the release is done by selectively degrading 

  • 12.
    Ramachandraiah, Harisha
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Svahn Andersson, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Inertial microfluidics combined with selective cell lysis for high throughput separation of nucleated cells from whole blood2017In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, no 47, p. 29505-29514Article in journal (Refereed)
    Abstract [en]

    The ability to rapidly analyze and extract information from peripheral blood cells has the potential of providing a wealth of new information about immune function and general health of the patient. In spite of the tremendous progress achieved in the field of leukocyte analysis, one of the major impediments for routine analysis is the enrichment of cell populations from heterogeneous sources such as blood, as the currently used techniques tend to be laborious. Moreover, the isolation of small and transient cell populations in blood, like circulating tumor cells during cancer metastasis, is even more challenging. Here, we report an integrated device for label-free continuous flow separation of nucleated cells from unprocessed whole blood at high throughput. The method utilizes exposure to hypotonic buffer to completely remove red blood cells and at the same time a size increase of nucleated cells for inertial focusing and separation in spiral microchannel. Using an integrated device with two outlets, we isolated total leukocytes at a high yield of 99%. Furthermore cancer cells spiked into whole blood could be separated at a yield of 88% while 80% of leukocyte could be depleted into separate outlet by simply changing the resistance between the two outlets. Finally, using a three-outlet integrated device, we demonstrate fractionation of leukocyte into subpopulation. The device continuously separates granulocytes at a purity of 86%, monocyte at a purity of 43% and lymphocytes at a purity of 91% simultaneously. Finally, a cell activation study of the immune system using blood from healthy subjects, stimulated ex vivo with lipopolysaccharides (LPS), confirmed that the high operational flow rate of the device does not alter the activation levels of leukocytes or introduce artifacts. Hence, the simple, high-throughput and low-cost integrated device requiring neither external force fields nor mechanical parts to operate should readily be applicable to sort nucleated cells as stand-alone and/or as integrated lab-on-a-chip devices with high-throughput requirements.

  • 13.
    Zelenin, Sergey
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Ramachandraiah, Harisha
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Faridi, Muhammad Asim
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Russom, Aman
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Microfluidic-based bacteria isolation from whole blood for diagnostics of blood stream infection2017In: Methods in Molecular Biology: Microchip Diagnostics, Springer, 2017, p. 175-186Conference paper (Refereed)
    Abstract [en]

    Bacterial blood stream infection (BSI) potentially leads to life-threatening clinical conditions and medical emergencies such as severe sepsis, septic shock, and multi organ failure syndrome. Blood culturing is currently the gold standard for the identification of microorganisms and, although it has been automated over the decade, the process still requires 24–72 h to complete. This long turnaround time, especially for the identification of antimicrobial resistance, is driving the development of rapid molecular diagnostic methods. Rapid detection of microbial pathogens in blood related to bloodstream infections will allow the clinician to decide on or adjust the antimicrobial therapy potentially reducing the morbidity, mortality, and economic burden associated with BSI. For molecular-based methods, there is a lot to gain from an improved and straightforward method for isolation of bacteria from whole blood for downstream processing. We describe a microfluidic-based sample-preparation approach that rapidly and selectively lyses all blood cells while it extracts intact bacteria for downstream analysis. Whole blood is exposed to a mild detergent, which lyses most blood cells, and then to osmotic shock using deionized water, which eliminates the remaining white blood cells. The recovered bacteria are 100% viable, which opens up possibilities for performing drug susceptibility tests and for nucleic-acid-based molecular identification. © Springer Science+Business Media LLC 2017.

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