Top PDF Microfluidic Technologies for High-Throughput Screening Applications

Microfluidic Technologies for High-Throughput Screening Applications

Microfluidic Technologies for High-Throughput Screening Applications

While affinity-based selection strategies have been effectively used to analyze protein-protein binding interactions using large combinatorial protein libraries consisting of billions of variants 32,33 , screening such libraries for other properties such as catalytic activity has remained a challenge. Large mutant enzyme libraries are typically expressed in organisms like yeast or E. coli. The screening of a large cell population of the basis of biocatalytic activity primarily involves the physical separation of the cells that allows the assay of a single colony or cell 34 . Although libraries consisting of random single DNA point mutations (6 x 10 3 variants for a 300-residue enzyme) are feasibly screened through plate assays and spectroscopic techniques, the number of variants increases exponentially for each additional mutation, outpacing techniques which require the physical isolation of individual mutants. The critical step in the screening process is signal development that comes from exposing the expression host to the selected substrate. Elaborate HTS strategies have been devised to look at enzyme libraries, such as fluorescence-activated cell sorting (FACS) of an enzyme library tethered to the cell surface via an outer membrane protein (OmpA) linker 35 . A FRET substrate is cleaved and the product non- covalently attaches to the cell surface of the active mutants, allowing them to be sorted on the basis of fluorescent activity. However, this technique is not generally applicable to most enzyme systems, as most substrates either do not enter the cell or generate soluble products that rapidly diffuse away from the cell. In such systems, compartmentalization is needed to keep the signal associated with the cell(s) that generate it. Compartment-
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Classification of large circulating tumor cells isolated with ultra- high throughput microfluidic Vortex technology

Classification of large circulating tumor cells isolated with ultra- high throughput microfluidic Vortex technology

The objective cell identification criteria presented here addresses common but widely unreported concerns surrounding immunostains. Since many cells may transition to a mesenchymal state [32], traditional epithelial cell staining techniques may overlook a significant number of candidate cells [47], resulting in underreported performances especially in size-based isolation platforms. While most devices are characterized using probes for CK, CD45, and DAPI, the introduced CTC identification criteria makes use of a sequential checklist that includes well-defined morphological criteria associated with malignancy—which take advantage of accumulated cytopathology knowledge [10, 38, 39, 48]— and may help minimize user-errors in manual enumeration. Morphological characterization may also help classify large cells that stain negative for common CTC markers, which may arise from size-based isolation methods, and cytometric analyses may sufficiently distinguish CTCs from other cell types present in blood, like monocytes, granulocytes, and cancer-associated non-CTCs such as disseminated tumor-activated macrophages [49]. We expect the described cell identification protocol will complement future device performance characterizations, clinical applications, and help standardize existing commercial prognostic and sample preparation tools as well as those in development. To help others who wish to adopt these tools, we provide a comprehensive guide and training worksheets (Supplementary Material) to more effectively convey our accumulated knowledge. As with most available techniques, the introduced enumeration protocol is not fully comprehensive and does not factor in the use of other marker types, including those that are cancer origin-specific (e.g., anti-HER2 staining for breast cancer samples, or anti-PSA for prostate cancer). We expect that the presented criteria will help foster future discussions regarding thorough validation of CTCs, and envision that the described criteria can serve as a starting point for further adaptations to the method as promising new markers or automated imaging software become available.
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Design and Analysis of High-Throughput Microfluidic Devices for Scalable Production of Encapsulated Tissue and Biological Materials.

Design and Analysis of High-Throughput Microfluidic Devices for Scalable Production of Encapsulated Tissue and Biological Materials.

This dissertation describes a scalable co-axial air flow based high throughput microfluidic device containing eight droplet formation zones capable of producing microcapsules at 8 times the current production rates. This research was motivated by the fact that the two most widely used devices for microencapsulation namely the air-syringe droplet generator and the electrostatic bead generator each of which fitted with a single needle through which microcapsules containing cells and other biological materials are incapable of producing sufficient number of microcapsules in a short-period of time to permit mass production of encapsulated and viable cells for transplantation in large animals and humans. As such there is an urgent need for a new approach to producing viable encapsulated cells in sufficient quantities rapidly for routine application in human cell therapy. In this work, an 8 channel co-axial air flow based high throughput microfluidic device was developed which can be scaled up to even 16, 32, 64 droplet formation zones. In this chapter, a summary of design problem, the approach and the applications of the high throughput microfluidic device are presented. This is followed by suggestions for future work.
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Validation of high throughput sequencing and microbial forensics applications

Validation of high throughput sequencing and microbial forensics applications

Because of the vast and incompletely described biological diversity of microbes and the potential of having to deal with a large number of samples in a microbial forensic case, it is not possible to validate every scenario. Moreover, HTS and bioinformatics technologies are changing rapidly and will continue to be improved in the immediate and long-range future. Lastly, exigent circumstances may require immediate response, and microbial forensics should be able to lend support using all available tools. For such unforeseen circumstances preliminary validation may be ‘carried out to acquire limited test data to enable the evaluation of a method for its investigative-lead value, with the intent of identifying key parameters and operating conditions and of establishing a degree of confidence in the methods of collection, extraction, and analysis’ [74]. However, once general validation is accomplished for instrumentation, bioinformatics data analysis, and Standard Operating Protocols (SOPs), only novel aspects of validation for new targets may be needed to generate informative leads and to make public health decisions with associated levels of confidence. Therefore, it is extremely important to establish comprehensive criteria for validation of HTS technologies with all aspects of the validation study documented. The fact that a validation study is preliminary should be stated clearly, with the limitations of the assay and validation study clearly described. However, validation of finalized SOPs is essential for reliable and defensible use of HTS technologies in microbial forensics. Sample collection and storage have been addressed elsewhere [75] and will not be described here. Validation of the HTS process addressed here relies, in part, on reports available in the literature [59-61,76] that have defined validation requirements for HTS applied to human clinical genetic analyses. The validation guidelines for the three major technical components of HTS (sample preparation, sequencing and data interpretation) as related to the field of microbial forensics, are presented in the following sections.
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Therapy response testing of breast cancer in a 3D high-throughput perfused microfluidic platform

Therapy response testing of breast cancer in a 3D high-throughput perfused microfluidic platform

There will be wide application of the type of technology studied here in the future. A major goal for developing this technology is to help direct and speed up the selection of therapies by predicting responses based on drug testing in 3D cultures grown directly from human tumor samples. In current therapy selection methods, most in vitro drug screens are performed in 2D culture but the results have often proved less than optimal. Alternatively, patient de- rived xenograft models have become a popular in vivo model to capture human tumor heterogeneity. However, PDX models are expensive and labor-intensive to develop. It also takes many months for tumors to develop in mice. These disadvantages limit the use of PDX models in a real-time setting to help predict treatment response and test drugs for patients. Here, we propose the use of 3D microfluidic models as a new approach to overcome the issues faced with existing 2D and PDX models (Fig. 5). A major goal for developing this technology is to create 3D physiologically-relevant cultures of human tissue which might allow improved and real-time selection of therapy and prediction of response based on drug testing using 3D culture grown directly from human tumor samples. This approach would significantly shorten the timeline for drug screening. Equally important, results from the 3D culture screenings are likely to be closer to in vivo results, render- ing them more accurate for directing the selection of ap- propriate drugs and predicting response for individual patients, thus, achieving personalized therapy.
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A high-throughput cellulase screening system based on droplet microfluidics

A high-throughput cellulase screening system based on droplet microfluidics

Emulsions were formed using a co-flow flow-focusing Polydimethylsiloxane device prepared by soft lithography as previously described 8 and using fluorocarbon oil containing 1% (v/v) Krytox- PEG-Krytox detergent synthesized as reported in an earlier study. 11,14 The solutions, one containing library cells (S. cerevisiae YPH500 cells, Agilent Technologies, Santa Clara, USA) and the other with the substrate, 14 were mixed at the same flow rate, giving a one-to-one mixing ratio. The library cells were a defined mixture of cells transformed with cel5A pESC-Trp (positive cells) or empty pESC-Trp (negative cells). The two solutions therefore mixed just prior to encapsulation, minimizing the chance that fluorescent products would enter neighboring droplets. The substrate solution con- tained carboxymethyl cellulose (CMC), which has a high viscosity. To prevent fluctuations in the flow of substrate during the emulsification process, we optimized the flow rate and the concentration of CMC and found that a CMC concentration of 0.33% (w/v) produced monodisperse emulsions.
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High throughput miniaturized screening of nanoparticle formation via inkjet printing

High throughput miniaturized screening of nanoparticle formation via inkjet printing

Nevertheless, conventional methods need materials in the milligram scale, require days for the full removal of the solvent, face issues with scalability and batch-to-batch variability and frequently result in NPs with wide size distributions. Employing more sophisticated technologies to test new materials in an easy, fast and automated way that will enable the modulation of the NPs properties is still an unresolved challenge in this field. In this context, there is a real need for high throughput technologies to screen rapidly a large number of materials and optimise their formulation conditions. For example, the use of inkjet technology to obtain polymeric microspheres with defined sizes, [7] protein encapsulated polymeric micro- structures [8] and loaded drug-polymer micro-particles [9] has been well established in the literature. On this basis, inkjet printing could potentially be a promising alternative to the conventional methods used for the production of NPs. Inkjet printing is a versatile, scalable and relatively inexpensive method of depositing small volumes of solutions, even down to the picoliter range, with remarkable accuracy and reproducibility. As inkjet printers become more commercially available, their use in the field of drug delivery has increased.
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High-throughput high-volume nuclear imaging for preclinical in vivo compound screening§

High-throughput high-volume nuclear imaging for preclinical in vivo compound screening§

Multi-atlas segmentation has proven useful for auto- mated region identification especially in clinical neuro- applications [24–27]. To overcome the limitation of many available software packages designed for clinical brain imaging, this approach has been implemented and employed for full and subregion segmentation of other organs (for whole body distribution and radiation dosim- etry) in several non-human species. Data variability—due to factors such as animal positioning, the non-rigidity of non-brain regions, general shape/distribution outliers and fluctuating contrast-to-noise—may occasionally cause segmentation failures, thus requiring strict QC and manual corrections. Nevertheless, we found in tim- ing experiments (not shown here) that multi-atlas seg- mentation followed by user editing of the automatically generated ROIs still significantly decreases processing time and observer variability compared to segmenting regions manually de novo.
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Pitfalls in optical on-line monitoring for high-throughput screening of microbial systems

Pitfalls in optical on-line monitoring for high-throughput screening of microbial systems

protein. Compared to the YFP clone, the delay after the induction is with 2.5 h longer, but from this point a steady increase of the fluorescence intensity can be ob- served without any conspicuous behavior. The maximum product concentration is reached simultaneously with the stationary phase. It can be concluded that oxygen inde- pendent fluorescent proteins simplify the generation of re- liable datasets for product formation kinetics. It should be mentioned that undesired oxygen limitation is not only disadvantageous for maturing of GFP and its derivatives but for bioprocess development in general. Besides misleading fluorescence signals, it can cause inhibited growth and unwanted by product formation which de- creases the feasibility of a bioprocess. Consequently, oxygen non-limiting conditions should be ensured even in micro-scale experiments. This can be achieved e.g. by in- creasing the shaking frequency or decreasing the filling vol- ume per well. Performing cultivations in fed-batch mode avoids oxygen limitations, too. Controlled release systems [44], enzyme based fed-batch media [45], or microfluidic systems for MTPs [40] are convenient solutions. Nonethe- less, this study does not aim for kinetic results so that an adjustment of the conditions was not necessary.
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Metaphor-Based Design of High-Throughput Screening Process Interfaces

Metaphor-Based Design of High-Throughput Screening Process Interfaces

requirements for task decisions, as identified through the GDTA, were compared with specification of current interface action sequences and display content based on the AH models to identify potential usability issues with the existing software interfaces (for example, SAMI®). That is, we were able to determine whether particular screening method editor displays and action sequences led to the information operators needed for certain process decisions. In our previous work, we made direct comparison of the GDTA results and AH models to formulate interface design and automation functionality recommendations for enhancing the existing software applications used in the HTS process at CELISCA (Kaber et al., 2006).
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Clinical application of high throughput molecular screening techniques for pharmacogenomics

Clinical application of high throughput molecular screening techniques for pharmacogenomics

In the context of high throughput mutation detection in the clinical setting, turnaround time is an important factor. While the above TaqMan- and LightCycler-based assays only require approximately 1–2 hours to produce analyzable data once a sample is placed on the instrument, there are many preceding steps of DNA extraction and reaction preparation. Thus, these tests are typically batched and run relatively infrequently (eg, once a week), leading to relatively long turnaround times from when a sample is received. Recently, technologies have been developed which provide “all-in-one” hands-off sample preparation and real-time PCR analysis with results provided in under an hour from sample receipt. The most widespread technology is the GeneXpert System (Cepheid, Sunnyvale, CA). After minimal processing, a patient sample is added to the proprietary cartridge, where a microfluidic system allows for automated DNA extraction and subsequent real-time PCR detection of a genetic target. Operation of the instrument requires minimal training and could potentially be used either in the clinical laboratory for on-demand testing or even as a point-of-care test in the clinic. The main drawback of this approach is the relatively high cost of consumables with only one patient sample per cartridge, plus a test menu currently focused largely on microbiology applications. 30,31
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Applications of High-Throughput Sequencing Data Analysis in Transcriptional Studies

Applications of High-Throughput Sequencing Data Analysis in Transcriptional Studies

The wide application of HTS technologies in RNA-protein interaction identification has produced a large amount of data, and the reliable RBP binding sites identified by the HTS technologies have become invaluable resources for understanding the RBP binding specificities [76, 77, 79]. However, CLIP-Seq-based methods also have some shortcom- ings. One of the most severe ones is that the binding sites often have high false-negative rates [80]. As CLIP-Seq sequences the RNA molecules that are bound by the RBPs, it is highly sensitive to the gene expression level [81]. If a gene is not expressed in a sample, there is no way for CLIP-Seq based methods to identify the potential binding sites within that gene. Furthermore, the false negative rate it also inflated by the mapabilty difficulty of the sequences spanning the splicing junctions [82]. These together prevent the CLIP-Seq method from becoming an universal tool for identifying all possible RBP binding sites across the transcriptome.
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High Throughput Screening (HTS) - Global Strategic Business Report

High Throughput Screening (HTS) - Global Strategic Business Report

The report profiles 103 companies including many key and niche players such as Agilent Technologies Inc., Aurora Biomed Inc., Axxam SpA, Beckman Coulter, Inc., BioFocus DPI Ltd., Bio-Rad Laboratories, Inc., BMG LABTECH GmbH, Caliper Life Sciences, Inc., Corning Inc., DiscoveRx Corporation, EVOTEC BioSystems, ForteBio, Inc., Genedata AG, IntelliCyt Corporation, Life Technologies Corporation, Luminex Corporation, Molecular Devices Inc., PerkinElmer Inc., Roche Applied Sciences, Sigma-Aldrich Corporation, and Tecan Group Ltd. Market data and analytics are derived from primary and secondary research. Company profiles are primarily based upon search engine sources in the public domain.
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High-throughput system for screening of Monascus purpureus high-yield strain in pigment production

High-throughput system for screening of Monascus purpureus high-yield strain in pigment production

Among the colonies isolated from the high-yield mixture T33, ten available colonies were selected by our practical experience and their submerged cultiva- tions in shake flasks were carried out with the parent strain (M-403) as the control (Figure 4b). Except col- ony T33-3, the productions of other nine colonies were almost 190.0 U/mL averagely, higher than that of the parent strain (137.0 U/mL). There was only a small part of low-yield mutants mixed in the high-yield mix- ture, so there was a high possibility to isolate the de- sired strain from the high-yield mixture compared with traditional shake flask screening. Repeated studies showed that the production of single colony T33-6 remained at the same level after several consecutive generations by submerged fermentation, three paralle- lisms for shake flasks at least. To the best of our know- ledge, this is the first report to buildup an integrated HTS strategy for M. purpureus applications.
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A microfluidic spheroid culture device with a concentration gradient generator for high-throughput screening of drug efficacy

A microfluidic spheroid culture device with a concentration gradient generator for high-throughput screening of drug efficacy

In conclusion, our μFSCD with a CGG offered a new approach for large-scale drug screening using spheroid microarrays. This approach was based on features of concave microwells connected with a CGG. Given that the μFSCD with the CGG provided a large amount of homogeneous 3D spheroids and several drug concentrations, it can constitute a convenient tool for widespread, parallel processing for the prediction of the effectiveness of the drugs and the determination of the proper cancer drug concentration in patients. Therefore, the proposed μFSCD with a CGG could be useful for clinical samples and could become cost-effective in personalised medicine given that a small number of cells can rapidly form spheroids, and given that the device consumes a small volume of nutrients and drugs.
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A High-Throughput Screening Approach To Repurpose FDA-Approved Drugs for Bactericidal Applications against Staphylococcus aureus Small-Colony Variants

A High-Throughput Screening Approach To Repurpose FDA-Approved Drugs for Bactericidal Applications against Staphylococcus aureus Small-Colony Variants

The objective of this study was to perform a high-throughput screen (HTS) of an FDA-approved drug library to specifically identify candidates that are not only bacte- ricidal against NCP and biofilm but also bactericidal to a mutated small-colony variant strain (SCV) harboring a hemB deletion, UAMS-1112 (ΔhemB). With this objective in mind, we set out to adapt a recently developed and validated adenylate kinase (AK) release reporter assay as an HTS platform to identify agents that display bactericidal activity toward slowly and/or nongrowing bacterial populations, including SCV (35). This HTS has recently been used to screen an FDA-approved drug library for agents that displayed bactericidal activity against Pseudomonas aeruginosa biofilms, identifying 34 compounds that displayed antibiofilm activity (36). The basis for the assay is that AK is a ubiquitous intracellular enzyme that catalyzes the conversion of two ADP molecules into ATP and AMP, which is released extracellularly after cell death. Extracellular AK release from lysed cells can subsequently be measured using commercial ToxiLight reporter cocktail based on the ATP-dependent bioluminescent measurement of AK (Lonza, Basel, Switzerland). In this study, we demonstrate that this AK release assay can be scaled for HTS of a Food and Drug Administration (FDA)-approved drug library containing 853 drug candidates and that it can identify candidates that are bactericidal against UAMS-1112 (ΔhemB). Assay hits were then further validated and characterized for their antimicrobial efficacy against wild-type S. aureus strains, antibiofilm activity, resistance frequency, and human cell cytotoxicity.
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Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica

Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica

Most of the time, in droplet-based microfluidic sys- tems, enzymatic activities are detected using fluorogenic substrates especially adapted to the droplet format. In particular, the fluorescent probe product should not be transported from one compartment to another one over the time scale of the assay [6, 7], and the substrate should be accessible to the expressed enzymes within the w/o droplet. Consequently, the expression system must be carefully chosen and, ideally, should involve enzymes that are expressed extracellularly or at the cell surface. To date, most of the protein libraries screened have relied on cytoplasmic or periplasmic expression in Escherichia coli, which implies that both the substrate and the product travel through the cell membrane [8, 9] or that an addi- tional lysis step is needed to perform the enzymatic assay [10–12]. While horseradish peroxidase libraries were screened displayed at the surface of Saccharomyces cer- evisiae cells [5], none extracellular recombinant expres- sion systems were described up to now in combination with droplet-based microfluidics.
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Applications of Support Vector Machines as a Robust tool in High Throughput Virtual Screening

Applications of Support Vector Machines as a Robust tool in High Throughput Virtual Screening

Waaserman and Bajorath [32] have discussed at length various SVM-based target selective searching strategies for virtual screening. They have elucidated a superior approach in terms of enrichment factor as the SVM is trained on the data comprising more than two different classes viz., selective, promiscuously active and non-active compared to the commonly employed binary classification approach. Further, they present a modified preference ranking strategy leading to higher recall of selective compounds. Combinatorial support vector machines have been used as virtual screening tools [33] for searching dual-inhibitors of 11 combinations of 9 anticancer kinase targets (EGFR, VEGFR, PDGFR, Src, FGFR, Lck, CDK1, CDK2, GSK3). In this study, the C-SVM was found either comparable or slightly better than the other conventional method such as Surflex, Dock, Blaster, KNN and PNN. Plewczynski et al. [34] performed an exclusive target-specific supervised SVM analysis for compounds retrieved from MDDR database related to five targets including cyclooxygenase-2, dihydrofolate reductase, thrombin, HIV-reverse transcriptase and antagonists of the estrogen receptor. The SVM model was based on only two dimensional topological descriptors related to atom pairs. The sensitivity and classification for all the protein targets were 80% and 100%, respectively. The literature is replete with more examples of successful application of SVM with both ligand and target based approaches. Table 1 shows a few representative ligands and their corresponding targets; needless to say that SVM modeling has comprehensively mapped chemical diversity and target space in virtual screening.
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THERAPEUTIC APPLICATIONS OF MUSHROOMS AND ITS COMPOSITIONAL ANALYSIS BY HIGH THROUGHPUT SCREENING TECHNIQUES

THERAPEUTIC APPLICATIONS OF MUSHROOMS AND ITS COMPOSITIONAL ANALYSIS BY HIGH THROUGHPUT SCREENING TECHNIQUES

Extracts prepared from mushroom Inonotus hispidus, have isolated phenolic compounds named ergosterol peroxide 4 also available in various mushrooms showed antiviral activity against infl[r]

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Clinical application of high throughput genomic technologies for treatment selection in breast cancer

Clinical application of high throughput genomic technologies for treatment selection in breast cancer

Recognizing that cancer genome sequencing is likely to be integrated into routine clinical decision-making in the near future, many leading cancer research institutions and national cancer agencies have recently launched or are soon to launch broad-scale molecular screening programs for solid tumors, including breast cancer [48]. The majority of institutional programs use a genotyping platform such as Sequenom or SNaPshot and focus their profiling efforts on select tumor types (for example, melanoma, glioblastoma multiforme, breast, lung, colorectal, and ovarian cancer). The size of the gene panel and mutations included vary between each institution; however, in general, the panels include oncogenes linked to targeted drugs that are either approved or in development (Table 3). The Institut Gustav Roussy performs molecular screening as part of their per- sonalized medicine program in several patient subgroups, including metastatic breast cancer (SAFIR01) and potential phase I clinical trial patients (MOlecular Screening for CAncer Treatment and Optimisation, or MOSCATO). Both programs use the Agilent array comparative genome hybridization (Santa Clara, CA, USA) for DNA analysis and the Ion Torrent for mutation analysis. To date, 111 patients underwent a biopsy on MOSCATO; 40% had an actionable mutation and 25 patients received matched therapy [49]. SAFIR01 has screened 423 patients with metastatic breast cancer; of those, 295 samples underwent successful sequen- cing and 140 patients had a targetable genomic alteration [50]. The majority of these mutations were in the PI3K/ AKT pathway.
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