The authors thank Dr. Wietske Kievit for her methodological support and comments on the manuscript.

The study is authorized by the ethics review board of the Radboud University Medical Center (#2015-1742). Subsequent approval of the executive boards from each of the participating institutes is obtained (Trial registration: Dutch Trial Registry, NTR 5475).
1. Enrolling patients in the trial/randomization
<ol>
	<li>Approach all referred patients by general practitioners, surgeons or internal medicine specialists for colonoscopy, to achieve a diverse sample.</li>
	<li>Contact...

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+a+mailed+brochure+on+appointment-keeping+for+screening+colonoscopy:+a+randomized+trial.">Effect of a mailed brochure on appointment-keeping for screening colonoscopy: a randomized trial.</a> Annals of Internal Medicine. 145 (12), 895-900 (2006).</li><li>Morcom, J., Dunn, S. V., Luxford, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+an+Australian+nurse+practitioner-led+colorectal+cancer+screening+clinic.">Establishing an Australian nurse practitioner-led colorectal cancer screening clinic.</a> Gastroenterology Nursing. 28 (1), 33-42 (2005).</li><li>Fox, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+of+the+literature+reporting+on+studies+that+examined+the+impact+of+interactive,+computer-based+patient+education+programs.">A systematic review of the literature reporting on studies that examined the impact of interactive, computer-based patient education programs.</a> Patient Education and Counseling. 77 (1), 6-13 (2009).</li><li>Veldhuijzen, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer-assisted+instruction+before+colonoscopy+is+as+effective+as+nurse+counseling,+a+clinical+pilot+trial.">Computer-assisted instruction before colonoscopy is as effective as nurse counseling, a clinical pilot trial.</a> Endoscopy International Open. 5 (8), E792-E797 (2017).</li><li>Mittal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Boston+bowel+preparation+scale:+reliable+not+only+for+colonoscopy-oriented+research+but+clinical+practice+also.">The Boston bowel preparation scale: reliable not only for colonoscopy-oriented research but clinical practice also.</a> Gastrointestinal Endoscopy. 71 (1), 221 (2010).</li><li>Lai, E. J., Calderwood, A. H., Doros, G., Fix, O. K., Jacobson, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Boston+bowel+preparation+scale:+a+valid+and+reliable+instrument+for+colonoscopy-oriented+research.">The Boston bowel preparation scale: a valid and reliable instrument for colonoscopy-oriented research.</a> Gastrointestinal Endoscopy. 69, 620-625 (2009).</li><li>Bouwmans, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+iMTA+Productivity+Cost+Questionnaire:+A+Standardized+Instrument+for+Measuring+and+Valuing+Health-Related+Productivity+Losses.">The iMTA Productivity Cost Questionnaire: A Standardized Instrument for Measuring and Valuing Health-Related Productivity Losses.</a> Value in Health. 18 (6), 753-758 (2015).</li><li>Spielberger, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual for the State&#8208;Trait anxiety inventory (form Y). , (1987).</li><li>Krol, M. W., de Boer, D., Delnoij, D. M., Rademakers, J. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SF-36+health+survey+update.">SF-36 health survey update.</a> Spine (Phila Pa 1976). 25 (24), 3130-3139 (2000).</li><li>Rex, D. K., Imperiale, T. F., Latinovich, D. R., Bratcher, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+bowel+preparation+on+efficiency+and+cost+of+colonoscopy.">Impact of bowel preparation on efficiency and cost of colonoscopy.</a> American Journal of Gastroenterology. 97 (7), 1696-1700 (2002).</li><li>Rosenfeld, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+patient+education+on+the+quality+of+inpatient+bowel+preparation+for+colonoscopy.">The impact of patient education on the quality of inpatient bowel preparation for colonoscopy.</a> Canadian Journal of Gastroenterology. 24 (9), 543-546 (2010).</li><li>Clark, B. T., Rustagi, T., Laine, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+level+of+bowel+prep+quality+requires+early+repeat+colonoscopy:+systematic+review+and+meta-analysis+of+the+impact+of+preparation+quality+on+adenoma+detection+rate.">What level of bowel prep quality requires early repeat colonoscopy: systematic review and meta-analysis of the impact of preparation quality on adenoma detection rate.</a> American Journal of Gastroenterology. 109 (11), 1714-1723 (2014).</li><li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telephone-based+re-education+on+the+day+before+colonoscopy+improves+the+quality+of+bowel+preparation+and+the+polyp+detection+rate:+a+prospective,+colonoscopist-blinded,+randomised,+controlled+study.">Telephone-based re-education on the day before colonoscopy improves the quality of bowel preparation and the polyp detection rate: a prospective, colonoscopist-blinded, randomised, controlled study.</a> Gut. 63 (1), 125-130 (2014).</li><li>Tae, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+patient+education+with+cartoon+visual+aids+on+the+quality+of+bowel+preparation+for+colonoscopy.">Impact of patient education with cartoon visual aids on the quality of bowel preparation for colonoscopy.</a> Gastrointestinal Endoscopy. 76 (4), 804-811 (2012).</li><li>Prakash, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+quality+of+colonoscopy+bowel+preparation+using+an+educational+video.">Improving the quality of colonoscopy bowel preparation using an educational video.</a> Canadian Journal of Gastroenteroly. 27 (12), 696-700 (2013).</li></ol>

The E-Patient Counseling (E-PACO) trial aims to study the utility, practicality, and patient-perceived usefulness of computer-based education (CBE), in comparison to conventional nurse counseling. In this manuscript the CBE is demonstrated together with the methodology used to evaluate the hypotheses.
It is established that high quality colonoscopy is the golden standard for prevention of colorectal cancer. Inadequate bowel preparation is related to the missing of neoplasm&#8217;s and increase...

A complete colonoscopy is the procedure for detection of precancerous lesions in the colon1. For adequate examination of the colon mucosa, optimal bowel cleanliness is crucial. A poorly prepared colon leads to insufficient adenoma detection rate and therefore the need for repeated procedures. In previous studies, better patient understanding of how to prepare clearly results in a higher quality of bowel preparation2. To achieve a clean colon, patients have a restricted diet for 1-2 days and use purgatives to induce diarrhea. This elicits abdominal discomfort and interrupts daily routine. In view of these barriers, inadequate bowel preparation is not infrequent3. Optimal patient compliance to the protocol enhances effective bowel preparation and subsequent efficacy of colonoscopy.
There is appreciable variation in the way information for a colonoscopy is administered to patients4. Some patients receive information directly from their health care professional during consultation, or are informed by auxiliary personnel (nurses, technicians, or administrators), while other units provide information through printed leaflets5. The effect of any information transfer is compounded by patient dependent factors such as educational level, comprehensive capacities, and cultural aspects. This results in a mixed understanding of the information that can negatively affect compliance to instructions.
A pivotal element in patient preparation is that every patient is thoroughly informed about risks and benefits of the procedure including the bowel preparation steps for colonoscopy. In addition, the routine use of sedative and analgesics requires a risk assessment of the individual patient. Many centers rely on nurse counseling to obtain informed consent before the procedure. This results in patient improved adherence to the instructions for bowel preparation. However, while effective, it is time-consuming for the nurse, repetitive, and results in patient-to-patient variability of information. More importantly, it demands an extra hospital visit for the patient, implicating absence of the patient at work6. In summary, it is an economically challenging practice in cost-conscious healthcare environments. Previous studies show that focused e-learning paths enable good comprehension and learning and enhance patients satisfaction7. Web-based education is used successfully for increasing knowledge of patients and it has become an accepted mechanism for obtaining informed consent. This has led to the development of tailored instruction programs for bowel preparation that combines the advantages of flexibility in time and environment, yet maintains consistency in delivery of information. Previously, the authors developed a tool that allows computer assisted instruction (CAI) for colonoscopy8. This tool employs a computer animation that captures the viewers&#8217; attention while adequately informing him/her of objectives for colonoscopy. Written in comprehensible language in logical order, the module educates patients on different aspects of colonoscopy. It provides basic anatomical teaching points and step-by-step instructs the patient how to perform bowel preparation. In our pilot study we showed that CAI for colonoscopy enhanced bowel preparation to the level that is comparable to nurse counseling.
The research group sought to enhance the efficacy of the developed CAI. Its limitation was that it was a unidirectional tool that delivered information but did not allow acquiring patient specific information concerning medical history and medication use. This is an important part of the nurse counseling visit, as it allows a pre-sedation risk assessment when judged by the nurse. Therefore, a dedicated questionnaire was developed, designed to collect data points for structured risk assessment. This questionnaire is completed by the patient at the end of the CAI. This eliminates the need for a face-to-face meeting with a nurse or physician at this point in time. The use of two-way communication (combining CAI with a questionnaire) is practical and provides high quality information to the patient whilst at the same time attending to the need of the endoscopist for information on sedation risks. This combined instruction and acquiring of information is known as computer based education (CBE)7.
The goal of this trial is to test the utility, practicality, and patient-perceived usefulness of CBE off-center, in comparison to conventional nurse counseling. The hypothesis is that CBE is non-inferior to nurse counseling in achieving high quality of bowel preparation during colonoscopy. This process is independent of time and space and therefore can be viewed in the comfort of the patients&#8217; home. Accordingly, the chosen secondary outcomes are patient related outcome measures such as a short leave absence, anxiety, satisfaction and comprehension of information, as these might benefit from delivery through this digital channel. Included process measures are patient activation, health and e-health literacy to determine which patients benefit most from this tool.
Study design 
The trial is set up as an endoscopist blinded multicenter randomized controlled trial design. Inclusion criteria are adult age and a referral for elective complete colonoscopy. Exclusion criteria are illiteracy in Dutch and significant audiovisual handicaps and mental disabilities that preclude delivery of CBE. Also, patients were excluded if there is no internet access or a relative with internet access (see Table 1). Patients will be recruited by back office staff at the outpatient&#8217;s clinic in 4 large volume endoscopy centers in the Netherlands. All patients receive a split dose laxative regime based on either polyethylene glycol or sodium picosulfate. After evaluation of in- and exclusion criteria by trained staff, patients are randomized in 1:1 distribution per trial site using a randomization tool (described in the protocol below). Reasons for declining to participate are recorded. The trial flowchart is presented in Figure 2.
Outcome measures 
The primary outcome measure is the quality of bowel preparation during endoscopy. Endoscopists are trained to score the bowel preparation with the Boston Bowel Preparation Scale (BBPS). The BBPS is a cumulative score of three bowel segments, ranging from 0-1 &#8220;unsatisfactory&#8221;, 2-3 &#8220;poor&#8221;, 4-5 &#8220;fair&#8221;, 6-7 &#8220;good&#8221;, and 8-9 &#8220;excellent&#8221;. Scores of &#8805;6 are considered adequate9,10. As secondary outcomes, the focus is on sickness absence, anxiety, satisfaction and information re-call. Information is also collected on patient activation and health literacy.
The cost minimization effect of the intervention is calculated in two ways. The comparison between groups with regard to endoscopy unit costs will be done using a cost-per-visit analysis. The macroeconomic effect of sickness absence is also evaluated, as patients in the intervention group will need less hospital visits. To do so, several items are assessed: socio-economic status, work status and duration of sickness absence, using an adapted iProductivity Cost Questionnaire11.
Patients anticipating invasive medical procedures often experience anxiety that may exceed their coping mechanisms. Anxiety is assessed at T0 and T1 with the State-Trait Anxiety Inventory (STAI)12. The STAI is a widely used 20-item self-report instrument with scores ranging from 20 (absence of anxiety) to 80 (high anxiety). Patient satisfaction is scored using two different measures. Patient experience impacts future behavior and therefore &#8220;willingness to return&#8221; is assessed at T3, ranging from 1 (extremely unwilling to return) to 10 (extremely willing to return). Furthermore, the Net Promoter Score (NPS) is utilized on the question &#8220;Would you recommend this endoscopy unit to your peers?&#8221;. Patient&#8217;s scores range from 1 (Not at all likely) to 10 (Extremely likely). The NPS will be assessed at T0 and T3 and is calculated as % Promoters (scores 9-10) - % Detractors (scores 1-6)13. To evaluate patient comprehension of the information in the CBE patients are asked to reproduce elements of the instruction. The patient information re-call is assessed at T1 (before colonoscopy) using a 10-item test, with questions to be answered with &#8220;yes&#8221; or &#8220;no&#8221;. The effect of patient education in colonoscopy is influenced by the patient ability to understand medical information. The 14-item Dutch validated Health Literacy Scale is used to assess this item, divided in 3 subscales, at T014. A new 21-item questionnaire is added as a measure for e-Health Literacy15. This contains questions regarding the skill and experience of patients in handling medical information online. Patients are confronted with options every day that may have major implications for their health. Effectively managing their choices requires knowledge, skill, and confidence. To this end these elements were mapped at T0 13-item Patient Activation Measure Scale (PAM-13)16. The current health status of patients is evaluated with the Medical Outcomes Study 36-item health survey (RAND-36) at T017.
Statistical analysis 
To statistically compare both groups on the primary outcome, the relative risk for an inadequately prepared colon, defined as a BBPS &#60;6, is used. In literature, a 90% success rate (for an adequately prepared colon) is common, with a 10% non-inferiority margin as the maximum clinically acceptable difference. The non-inferiority power calculation resulted in 180 patients per group, 360 patients in total. This is required to exclude a difference in favor of the standard group of more than 10%. With a margin of &#177; 60% loss of patients before completing the protocol, based on earlier research, the target number of patients to approach is set at 1,000. In addition to the non-inferiority analyses, superiority analyses will be conducted to investigate effects on secondary outcome measures.

<table>
	<tbody>
		<tr>
			<td>Computer Based Education</td>
			<td>Medify BV</td>
			<td>n/a</td>
			<td>Computer Based Education tool for patient instruction prior to colonoscopy</td>
		</tr>
		<tr>
			<td>Computer / tablet</td>
			<td>Any</td>
			<td></td>
			<td>A computer or tablet should be used to complete the e-learning and fill out the questionnaires</td>
		</tr>
		<tr>
			<td>Medify Content Management System</td>
			<td>Medify BV</td>
			<td>n/a</td>
			<td>A content management system to process the e-learning content</td>
		</tr>
	</tbody>
</table>

e-patient counseling trial (e-paco): computer based education versus nurse counseling for patients to prepare for colonoscopy

Improving patient education focusing on bowel preparation before a colonoscopy leads to cleaner colons. Endoscopy units must obtain informed consent and perform a risk assessment for sedative use prior to a colonoscopy. The current practice in the Netherlands to achieve these goals is nurse counseling in an outpatient setting. This is costly and has disadvantages in terms of uniformity and time consumption for both the patient and the hospital. The hypothesis is that computer-based education with use of video and 3D animations may replace nurse counseling in most cases, without losing quality of bowel cleanliness during colonoscopy.
This multicenter, randomized, endoscopist blinded clinical trial evaluates a primary outcome measure (bowel preparation) during colonoscopy. Secondary outcome measures are sickness absence, patient anxiety after instruction and prior to colonoscopy, patient satisfaction and information re-call. The study will be performed in four endoscopy units of different levels (rural, urban, and tertiary). Inclusion criteria are adult age and referral for complete colonoscopy. Exclusion criteria are Dutch illiteracy, audiovisual handicaps or mental disabilities and no (peers with) internet access.
This trial aims to establish online computer-based education as tool for patient education prior to a colonoscopy. By choosing a direct comparison with the standard of care (nurse counseling), both endoscopic quality measures and patient related outcome measures can be evaluated.

The earlier mentioned pilot study compared nurse instruction to CAI using the same interactive tool as used in this protocol8. As the goals of this study were comparable to the outcomes used in this protocol, a short explanation of the results of the pilot are provided here in more detail. See also Table 28.
In this pilot study 385 patients were enrolled. The CAI g...

Watch this Scientific Journal Video about E-Patient Counseling Trial E-PACO: Computer Based Education versus Nurse Counseling for Patients to Prepare for Colonoscopy at JoVE.com

E-Patient Counseling Trial E-PACO: Computer Based Education versus Nurse Counseling for Patients to Prepare for Colonoscopy

The aim of this trial is to establish the position of online computer-based education as a tool for patient preparation prior to a colonoscopy. Computer based education is compared with the standard of care, nurse counseling, evaluating endoscopic quality measures and patient related outcome measures.

E-Patient Counseling Trial (E-PACO): Computer Based Education versus Nurse Counseling for Patients to Prepare for Colonoscopy

Improving patient education focusing on bowel preparation before a colonoscopy leads to cleaner colons. Endoscopy units must obtain informed consent and ...

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视频: E-Patient Counseling Trial E-PACO: Computer Based Education versus Nurse Counseling for Patients to Prepare for Colonoscopy

The aim of this trial is to establish the position of online computer-based education as a tool for patient preparation prior to a colonoscopy. Computer based education is compared with the standard of care, nurse counseling, evaluating endoscopic quality measures and patient related outcome...

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synthesis of infectious bacteriophages in an e. coli-based cell-free expression system

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL...

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

in vitro method to observe e-selectin-mediated interactions between prostate circulating tumor cells derived from patients and human endothelial cells

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow...

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

mutagenesis and functional selection protocols for directed evolution of proteins in e. coli

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

rnai plating for c. elegans feeding: a technique to induce target dsrna expression in e. coli

This video describes the principles behind RNAi treatment in C. elegans and demonstrates a protocol to knockdown lin-35 in a transgenic worm...

RNAi Plating for C. elegans Feeding: A Technique to Induce Target dsRNA Expression in E. coli

computer vision-based biomass estimation for invasive plants

Author Spotlight: UAV Remote Sensing for Efficient Invasive Plant Biomass Estimation

We report detailed procedures for an invasive plant biomass estimation method that utilizes data obtained from unmanned aerial vehicle (UAV) remote sensing to assess biomass and capture the spatial distribution of invasive species. This approach proves highly beneficial for conducting hazard assessment and early warning of invasive...

Computer Vision-Based Biomass Estimation for Invasive Plants

hydra, a computer-based platform for aiding clinicians in cardiovascular analysis and diagnosis

Hydra, a Computer-Based Platform for Aiding Clinicians in Cardiovascular Analysis and Diagnosis

This article presents a protocol based on Hydra &#8212; a web-based system for clinical decision support that integrates a full and detailed set of functionalities and services required by physicians for complete cardiovascular analysis, risk assessment, early diagnosis, treatment, and monitoring over...

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models.
This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone.
The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its applications.

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Transradial Access Chemoembolization for Hepatocellular Carcinoma Patients

Transarterial chemoembolization (TACE) is the most common modality for treatment of hepatocellular carcinoma (HCC) at the intermediate stage. TACE is typically performed via transfemoral access (TFA). However, transradial access (TRA) is preferred in coronary artery interventions due to decreased complications and mortality. Whether the advantages of TRA can be applied to TACE required investigation.
Patients receiving TRA TACE at a single center were retrospectively enrolled for study. Procedural details, technical success, radial artery occlusion (RAO) rate, and access site-related bleeding complications were evaluated. From October 2017 to October 2018, 112 patients underwent 160 TRA TACE procedures. The overall technical success rate was 95.0% (152/160). The rate of crossover from TRA to TFA was 1.9%. No access site-related bleeding complications were found in any cases. Asymptomatic RA occlusion occurred in three patients (2.7%). Compared with TFA, TRA can increase safety and patient satisfaction while decreasing access site-related bleeding complications. Moreover, TRA interventions can benefit patients with advanced age, obesity, or a high risk of bleeding complications.

Transarterial chemoembolization (TACE) is the standard therapy for patients in the intermediate stage of hepatocellular carcinoma and is typically performed through femoral artery access. Compared with transfemoral access, transradial access (TRA) can decrease the rate of bleeding complications and improve patient tolerance. A method is presented here to perform transarterial chemoembolization via radial artery access.

Transarterial chemoembolization (TACE) is the most common modality for treatment of hepatocellular carcinoma (HCC) at the intermediate stage. TACE is ...

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Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides (MF) and S&#233;zary syndrome (SS) represent the most common subtypes. Despite a number of studies on characterizing gene expression, genetic alterations, and epigenetic abnormalities of CTCL, the molecular pathogenesis of MF/SS remains unclear. MF refers to the more common CTCL with a skin-predominance, and is usually limited to skin, whereas&#160;SS is an aggressive leukemic variant of CTCL with widespread skin involvement and is characterized by neoplastic distribution mainly involving blood, skin, and lymph node. Focusing on clinical practice, the identification of gene expression biomarkers has&#160;enormous potential to improve diagnosis and treatment of MF/SS. Indeed, recent transcriptomic studies have identified potential diagnostic biomarkers from differences in gene expression between normal and malignant T cells, which may improve our understanding of SS biology, and reveal potential therapeutic targets. This manuscript describes a detailed reproducible protocol for the isolation of peripheral blood mononuclear cells from fresh whole blood from patients diagnosed with SS, selection of CD4+ memory T cells (CD4+CD45RO+ T cells), chemical stimulation, and preparation of RNA suitable for transcriptomic profiling to discover novel prognostic molecular markers to gain additional insight in disease etiology. The stimulation using chemical agonist to activate nuclear regulation provides more specific assessment for pathways important in the dynamic transcription regulation and gene expression and eliminates confounding defects that may arise from upstream signaling defects arising from TCR antigen loss at the cell membrane. The data obtained from comparison of transcriptome of unstimulated to stimulated SS T cells unmasks functional regulatory gene expression defects not evident from analysis of quiescent unstimulated cells. Furthermore, the method outlined from this approach can be adapted for studying T cell gene expression defects in other T cell immune diseases.

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&amp;#233;zary Syndrome Patients for Transcriptomic Profiling

We present a simple protocol for the isolation of peripheral blood mononuclear cells from whole blood obtained from patients diagnosed with S&#233;zary Syndrome, followed by selection of CD4+ T cells, their stimulation with phorbol12-myristate13-acetate and A23187 ionophore, and preparation of RNA for transcriptomic profiling.

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

Patient-Derived Tumor Explants As a &quot;Live&quot; Preclinical Platform for Predicting Drug Resistance in Patients

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...