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Methods Protoc., Volume 4, Issue 3 (September 2021) – 23 articles

Cover Story (view full-size image): The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) opens up the possibility of modeling human diseases and developing new therapeutics. Using human iPSC-derived cells for preclinical and clinical research, however, requires a constant supply of well-characterized iPSC lines. To address this issue, we developed a multistep workflow to assess our newly generated iPSCs through growth rate profiling, testing of genome integrity, evaluation of pluripotency, and testing their ability to form the three germline layers, with a particular focus on cortical neurons of the ectodermal lineage. This workflow helps to streamline work processes and offers the potential to add new tests to it as technologies evolve, thus ensuring that researchers employ high-quality iPSCs for experimental reproducibility and robustness.View this paper
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10 pages, 1816 KiB  
Protocol
Touch Imprint Intraoperative Flow Cytometry as a Complementary Tool for Detailed Assessment of Resection Margins and Tumor Biology in Liver Surgery for Primary and Metastatic Liver Neoplasms
by Georgios S. Markopoulos, Georgios K. Glantzounis, Anna C. Goussia, Georgios D. Lianos, Anastasia Karampa, George A. Alexiou and George Vartholomatos
Methods Protoc. 2021, 4(3), 66; https://doi.org/10.3390/mps4030066 - 15 Sep 2021
Cited by 12 | Viewed by 2203
Abstract
Liver resection is the main treatment for primary and metastatic liver tumors in order to achieve long-term survival with good quality of life. The ultimate goal of surgical oncology is to achieve complete tumor removal with adequate clear surgical margins. Flow cytometry is [...] Read more.
Liver resection is the main treatment for primary and metastatic liver tumors in order to achieve long-term survival with good quality of life. The ultimate goal of surgical oncology is to achieve complete tumor removal with adequate clear surgical margins. Flow cytometry is a powerful analytical technique with applications such as phenotypic analysis and quantification of DNA content. Intraoperative flow cytometry (iFC) is the application of flow cytometry for DNA content/ploidy and cell cycle distribution analysis during surgery for tumor cell analysis and margin evaluation. It has been used for cell analysis of intracranial tumors and recently of head and neck carcinomas and breast carcinomas, as well as for tumor margin evaluation. Herein, we present a novel touch imprint iFC protocol for the detailed assessment of tumor margins during excision of malignant hepatic lesions. The protocol aims to offer information on surgical margins after removal of malignant liver tumors based on DNA content of cancer cells and to corroborate the results of iFC with that of histopathological analysis. Based on the established role of iFC in other types of malignancies, our specialized protocol has the potential, through characterization of cells in liver transection surface post hepatectomy, to offer significant information on the type of resection and tumor biology. This information can be used to effectively guide intra- and postoperative patient management. Full article
(This article belongs to the Section Biomedical Sciences and Physiology)
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14 pages, 3167 KiB  
Protocol
Improvement of the Collection, Maintenance, and Analysis of Neoplastic Cells from Urine Specimens with the Use of CytoMatrix
by Simone Minasi, Daniela Bosco, Bernardo Moretti, Felice Giangaspero, Antonio Santoro and Francesca Romana Buttarelli
Methods Protoc. 2021, 4(3), 65; https://doi.org/10.3390/mps4030065 - 10 Sep 2021
Cited by 1 | Viewed by 2778
Abstract
Urine cytology is a non-invasive test used in combination with cystoscopy for screening and follow-up of urothelial carcinoma (UC). Although cytology can be used to efficiently identify high-grade UC, it has a lower accuracy for the diagnosis of low-grade UC or patients with [...] Read more.
Urine cytology is a non-invasive test used in combination with cystoscopy for screening and follow-up of urothelial carcinoma (UC). Although cytology can be used to efficiently identify high-grade UC, it has a lower accuracy for the diagnosis of low-grade UC or patients with presence of atypical urothelial cells (AUC). For these reasons, ancillary tests have been added to urine cytology in order to improve the accuracy. However, the poor abundance of neoplastic cells in most samples and the absence of a “tissue-like” structure remains a major challenge. We used a novel synthetic support called CytoMatrix which has the property of capturing and storing cells and micro-macro aggregates within its three-dimensional structure. The urine specimens were obtained from 12 patients: 6 with suspected urothelial neoplasia (low- and high-grade) and 6 with AUC or non-neoplastic samples. The first step is the urine samples preparation, through several centrifugation passages; the second step consists in absorbing cells on the CytoMatrix, and in the subsequent formalin fixation, standard processing and paraffin embedding to prepare FFPE-CytoMatrix block. In the final step, sections are consecutively cut, stained with hematoxylin-eosin (H&E), and analyzed via UroVysion FISH and immunohistochemistry (IHC). Using our simple and reliable protocol, we can improve the quality of urine specimens, allowing a better collection, maintenance, and analysis of cells, with the advantage of using ancillary tests to support cytological diagnosis and the advantage of storing cellular material in a FFPE-CytoMatrix block. Full article
(This article belongs to the Section Molecular and Cellular Biology)
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10 pages, 893 KiB  
Study Protocol
Study Protocol to Determine Association between Environmental Triggers and Asthma among Children in King Williams Town
by Rasaq A. Yusuf, Phoka C. Rathebe and Wells Utembe
Methods Protoc. 2021, 4(3), 64; https://doi.org/10.3390/mps4030064 - 10 Sep 2021
Viewed by 2022
Abstract
Asthma affects over 330 million people worldwide, with relatively higher disease burdens in Australia, New Zealand, Africa, the Middle East, and South America. The symptoms associated with asthma were reported to be prevalent in children from the period of 1993 to 2013, in [...] Read more.
Asthma affects over 330 million people worldwide, with relatively higher disease burdens in Australia, New Zealand, Africa, the Middle East, and South America. The symptoms associated with asthma were reported to be prevalent in children from the period of 1993 to 2013, in many low- and middle-income countries, due to changes in environmental conditions, such as domestic lifestyle, and urban and industrial developments. (1) Background: Several studies have also shown that children are prone to a severe type of asthma, because of their narrow respiratory airways and susceptibility to irritation from environmental agents. This study aimed to assess the association between environmental exposure and asthma among children in King Williams Town, South Africa. (2) Methodology: This study adopted a cross-sectional design method, with an estimated sample size of 262 participants. The eligible study participants were enrolled while attending Grey hospital in King Williams Town, for asthma management. Information will be collected from eligible, stable participants, on asthma treatment, through in-person interviewing in 2021. A semi-structured questionnaire will be administered to the participants. However, as a result of the prevailing COVID-19 pandemic, data may be abstracted from the asthma medical record of the eligible participants. Multivariate regression will be utilized, to describe the correlation between the variables, and the odds ratio will be calculated as well. (3) Discussion and conclusion: The study will objectively identify the local environmental agents that are associated with asthma among children in King Williams Town, in order to reprioritize treatment and preventative strategies. Ethical approval was obtained from the Research Ethics Committee, Faculty of Health Sciences at the University of Johannesburg. Full article
(This article belongs to the Section Public Health Research)
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12 pages, 848 KiB  
Article
Submerged Bioreactor Production of Geobacillus stearothermophilus ATCC 7953 Spores for Use as Bioindicators to Validate Hydrogen Peroxide Inactivation Processes
by Philipp Stier and Ulrich Kulozik
Methods Protoc. 2021, 4(3), 63; https://doi.org/10.3390/mps4030063 - 10 Sep 2021
Cited by 4 | Viewed by 2990
Abstract
In the food and pharmaceutical industries, evaluating the sterilization performance preceding aseptic production processes is of central importance. In the case of hydrogen peroxide sterilization of solid surfaces, bioindicators (BI) consisting of spores of Bacillus atrophaeus or Geobacillus stearothermophilus are used to validate [...] Read more.
In the food and pharmaceutical industries, evaluating the sterilization performance preceding aseptic production processes is of central importance. In the case of hydrogen peroxide sterilization of solid surfaces, bioindicators (BI) consisting of spores of Bacillus atrophaeus or Geobacillus stearothermophilus are used to validate the effectiveness and efficiency of the inactivation procedure. Commercial production of G. stearothermophilus is commonly performed on agar plates, where cultivation and sporulation conditions are not well-defined. Therefore, the produced BI can vary in their resistance, which in turn creates unacceptable uncertainties in the evaluation of aseptic processes. Submerged production in the bioreactor would allow more control over sporulation conditions, while reducing production time, resistance variability, and avoidance of false-positive or false-negative test results. In addition, submerged production of G. stearothermophilus so far was a challenge to achieve sufficiently high spore concentrations for BI production. This study reports on the development of a method for submerged production of G. stearothermophilus spores (pH 7.0, 57 °C, 30% pO2) that can achieve 1.6 × 107 spores/mL with a resistance against 35% H2O2 at 25 °C of D25°C,35% H2O2 = 73 s. This resistance ranks within the range of commercially available BI, making the results directly transferable to industrial applications. Full article
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27 pages, 2061 KiB  
Review
What’s Wrong in a Jump? Prediction and Validation of Splice Site Variants
by Giulia Riolo, Silvia Cantara and Claudia Ricci
Methods Protoc. 2021, 4(3), 62; https://doi.org/10.3390/mps4030062 - 5 Sep 2021
Cited by 12 | Viewed by 4859
Abstract
Alternative splicing (AS) is a crucial process to enhance gene expression driving organism development. Interestingly, more than 95% of human genes undergo AS, producing multiple protein isoforms from the same transcript. Any alteration (e.g., nucleotide substitutions, insertions, and deletions) involving consensus splicing regulatory [...] Read more.
Alternative splicing (AS) is a crucial process to enhance gene expression driving organism development. Interestingly, more than 95% of human genes undergo AS, producing multiple protein isoforms from the same transcript. Any alteration (e.g., nucleotide substitutions, insertions, and deletions) involving consensus splicing regulatory sequences in a specific gene may result in the production of aberrant and not properly working proteins. In this review, we introduce the key steps of splicing mechanism and describe all different types of genomic variants affecting this process (splicing variants in acceptor/donor sites or branch point or polypyrimidine tract, exonic, and deep intronic changes). Then, we provide an updated approach to improve splice variants detection. First, we review the main computational tools, including the recent Machine Learning-based algorithms, for the prediction of splice site variants, in order to characterize how a genomic variant interferes with splicing process. Next, we report the experimental methods to validate the predictive analyses are defined, distinguishing between methods testing RNA (transcriptomics analysis) or proteins (proteomics experiments). For both prediction and validation steps, benefits and weaknesses of each tool/procedure are accurately reported, as well as suggestions on which approaches are more suitable in diagnostic rather than in clinical research. Full article
(This article belongs to the Section Omics and High Throughput)
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12 pages, 935 KiB  
Protocol
Effect of Oral Prophylactic Measures on the Occurrence of Pre-Eclampsia (OP-PE) in High-Risk Pregnant Women: A Cluster Randomized Controlled Trial
by Aida Kanoute, Jocelyne Gare, Nicolas Meda, Stephane Viennot, Paul Tramini, Laurie Fraticelli, Florence Carrouel and Denis Bourgeois
Methods Protoc. 2021, 4(3), 61; https://doi.org/10.3390/mps4030061 - 5 Sep 2021
Cited by 3 | Viewed by 2745
Abstract
Pre-eclampsia (PE), a pregnancy-specific hypertensive disorder, characterized by the development of placental endothelial dysfunction, remains a major source of maternal and perinatal morbidity and mortality, especially in low- and middle-income settings. Periodontal disorders during pregnancy, and particularly periodontal pathogens, may be related to [...] Read more.
Pre-eclampsia (PE), a pregnancy-specific hypertensive disorder, characterized by the development of placental endothelial dysfunction, remains a major source of maternal and perinatal morbidity and mortality, especially in low- and middle-income settings. Periodontal disorders during pregnancy, and particularly periodontal pathogens, may be related to the risk of PE. Standard oral hygiene methods, based mainly on the joint use of toothbrushes and interdental brushes, reduce periodontal inflammatory risk and modulate the dysbiosis of the oral microbiome. The aim of this trial is to compare the PE outcomes in high-risk pregnant women receiving oral prophylactic measures to a control group. This trial is designed as a two-arm, parallel, cluster randomized controlled trial with the antenatal obstetric clinic as the unit of randomization and an allocation ratio of 1:1. The pregnant women will be included at 3 months of pregnancy and will be followed throughout the pregnancy. The primary outcome measure will be the incidence of PE from a baseline during the pregnancy. Secondary outcomes measures will include changes from the baseline in quantification of the pathogenic bacterial load of the interdental microbiota, the severity scores of periodontal indicators, and the incidence of adverse perinatal outcomes. This trial should demonstrate that the implementation of daily oral hygiene reduces oral dysbiosis, the incidence of periodontal disease, and the risk of PE. Full article
(This article belongs to the Special Issue Feature Papers 2021)
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13 pages, 456 KiB  
Protocol
Cluster Randomized Controlled Trial on the Effects of 12 Months of Combined Exercise Training during Hemodialysis in Patients with Chronic Kidney Disease—Study Protocol of the Dialysis Training Therapy (DiaTT) Trial
by Gero von Gersdorff, Pia von Korn, André Duvinage, Gabriele Ihorst, Anika Josef, Margit Kaufmann, Thomas Baer, Tim Fellerhoff, Iris Fuhrmann, Elisa Koesel, Sven Zeissler, Lars Bobka, Marion Heinrich, Anette Schindler, Rasmus Weber, Cornelia Breuer, Anna Maria Meyer, M. Cristina Polidori, Sophia M.T. Dinges, Julia Schoenfeld, Mathias Siebenbuerger, Stefan Degenhardt, Kirsten Anding-Rost and Martin Halleadd Show full author list remove Hide full author list
Methods Protoc. 2021, 4(3), 60; https://doi.org/10.3390/mps4030060 - 31 Aug 2021
Cited by 6 | Viewed by 3386
Abstract
Patients with chronic kidney disease (CKD) on hemodialysis (HD) experience treatment-related immobility and physical deconditioning, which is responsible for an increased risk of frailty and a high burden of multi-morbidity. Exercise has been shown to counteract this vicious cycle; however, its effectiveness has [...] Read more.
Patients with chronic kidney disease (CKD) on hemodialysis (HD) experience treatment-related immobility and physical deconditioning, which is responsible for an increased risk of frailty and a high burden of multi-morbidity. Exercise has been shown to counteract this vicious cycle; however, its effectiveness has only been investigated in small cohorts. Therefore, the objective of the Dialysis Training Therapy (DiaTT) trial will be to assess the effects of a 12-month intradialytic exercise program on physical functioning, frailty and health economics in a large cohort of HD patients in a real-world setting. DiaTT will be a prospective, cluster-randomized (1:1), controlled, multi-center, interventional clinical trial across 28 dialysis units, aiming at the recruitment of >1100 CKD patients on HD. The intervention group will receive 12 months’ intradialytic exercise (combined aerobic and resistance training), whereas the usual care group will not receive intervention. The primary endpoint will be a change on the sit-to-stand test (STS60) result between baseline and 12 months. Secondary endpoints will include physical functioning, frailty, quality of life, 3-point MACE, hospitalizations, survival, quality of HD, health literacy and health care costs. By including almost as many patients as previously investigated in smaller trials, DiaTT will be the largest randomized, controlled trial assessing frailty, quality of life and mortality in the field of nephrology. Full article
(This article belongs to the Section Biomedical Sciences and Physiology)
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9 pages, 1078 KiB  
Protocol
A Mating Procedure for Genetic Transfer of Integrative and Conjugative Elements (ICEs) of Streptococci and Enterococci
by Francesco Iannelli, Francesco Santoro, Valeria Fox and Gianni Pozzi
Methods Protoc. 2021, 4(3), 59; https://doi.org/10.3390/mps4030059 - 28 Aug 2021
Cited by 8 | Viewed by 2996
Abstract
DNA sequencing of whole bacterial genomes has revealed that the entire set of mobile genes (mobilome) represents as much as 25% of the bacterial genome. Despite the huge availability of sequence data, the functional analysis of the mobile genetic elements (MGEs) is rarely [...] Read more.
DNA sequencing of whole bacterial genomes has revealed that the entire set of mobile genes (mobilome) represents as much as 25% of the bacterial genome. Despite the huge availability of sequence data, the functional analysis of the mobile genetic elements (MGEs) is rarely reported. Therefore, established laboratory protocols are needed to investigate the biology of this important part of the bacterial genome. Conjugation is a mechanism of horizontal gene transfer which allows the exchange of MGEs among strains of the same or different bacterial species. In streptococci and enterococci, integrative and conjugative elements (ICEs) represent a large part of the mobilome. Here, we describe an efficient and easy-to-perform plate mating protocol for in vitro conjugative transfer of ICEs in streptococci (Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus gordonii, Streptococcus pyogenes), Enterococcus faecalis, and Bacillus subtilis. Conjugative transfer is carried out on solid media and selection of transconjugants is performed with a multilayer plating. This protocol allows the transfer of large genetic elements with a size up to 81 kb, and a transfer frequency up to 6.7 × 10−3 transconjugants/donor cells. Full article
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7 pages, 2551 KiB  
Protocol
Time Mating Guinea Pigs by Monitoring Changes to the Vaginal Membrane throughout the Estrus Cycle and with Ultrasound Confirmation
by Rebecca L. Wilson, Kristin Lampe, Brad J. Matushewski, Timothy R. H. Regnault and Helen N. Jones
Methods Protoc. 2021, 4(3), 58; https://doi.org/10.3390/mps4030058 - 27 Aug 2021
Cited by 4 | Viewed by 7415
Abstract
One of the greatest challenges to the development and implementation of pregnancy therapeutics is the ability to rigorously test treatments in clinically relevant animal models. Guinea pigs offer a unique advantage in studying the placenta, fetal development, and reproductive health as they have [...] Read more.
One of the greatest challenges to the development and implementation of pregnancy therapeutics is the ability to rigorously test treatments in clinically relevant animal models. Guinea pigs offer a unique advantage in studying the placenta, fetal development, and reproductive health as they have similar developmental milestones to humans, both throughout gestation and following birth. Tracking the guinea pig estrus cycle is imperative to ensuring appropriately timed mating and can be performed by monitoring the guinea pig vaginal membrane. Here, we describe a methodology to efficiently and accurately time mate guinea pigs, and provide a picture representation of changes to the guinea pig vaginal membrane throughout the estrus cycle. Utilization of this monitoring enabled a 100% pregnancy success rate on the first mating attempt in a cohort of five guinea pigs. This approach, along with early pregnancy ultrasounds as a secondary method to confirm pregnancy, offers a reliable approach to timed mating in the guinea pig. Full article
(This article belongs to the Section Biomedical Sciences and Physiology)
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13 pages, 4120 KiB  
Protocol
The VISTA Approach in Canine Disimpaction
by Gabriella Galluccio, Alessandra Impellizzeri, Alessandra Pietrantoni, Adriana De Stefano, Gerardo La Monaca and Roberto Pippi
Methods Protoc. 2021, 4(3), 57; https://doi.org/10.3390/mps4030057 - 20 Aug 2021
Cited by 3 | Viewed by 3284
Abstract
Canine disimpaction is always a challenging orthodontic treatment overall, even when the impacted permanent canine is in a high position, especially when in tight relation with the upper incisors’ roots. Conventional treatment methods are usually not capable of performing the correct force direction, [...] Read more.
Canine disimpaction is always a challenging orthodontic treatment overall, even when the impacted permanent canine is in a high position, especially when in tight relation with the upper incisors’ roots. Conventional treatment methods are usually not capable of performing the correct force direction, consisting of the contemporary movement in the distal and vestibular directions of the canine crown, often provoking, as side effects, the presence of decubitus on the mucous of the lips and cheeks or a poor final appearance of the periodontal support of the disimpacted canine. Among the different approaches, the vertical incision subperiosteal tunnel access (VISTA) technique shows good performance with regard to the direction of the forces and the canine’s periodontal conditions when erupted; it is usually realized through an elastic chain connected to a temporary anchorage device (TAD) in the posterior area. In this paper, a different protocol for the VISTA method is also presented, to be resorted to in cases of difficult miniscrew positioning due to the anatomic conditions or stage of dentitions. The new protocol also considers the use of nickel–titanium coil springs in order to avoid the need of frequent reactivation of the device and consequent patient discomfort, highlighting its advantages and indications with respect to the traditional approach. Full article
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15 pages, 6462 KiB  
Protocol
Contrast-Enhanced Tissue Processing of Fibrillin-Rich Elastic Fibres for 3D Visualization by Volume Scanning Electron Microscopy
by Philip N. Lewis, Robert D. Young, R. B. Souza, Andrew J. Quantock and Keith M. Meek
Methods Protoc. 2021, 4(3), 56; https://doi.org/10.3390/mps4030056 - 15 Aug 2021
Cited by 4 | Viewed by 2136
Abstract
Elastic fibres constitute an important component of the extracellular matrix and currently are the subject of intensive study in order to elucidate their assembly, function and involvement in cell–matrix interactions and disease. However, few studies to date have investigated the 3D architecture of [...] Read more.
Elastic fibres constitute an important component of the extracellular matrix and currently are the subject of intensive study in order to elucidate their assembly, function and involvement in cell–matrix interactions and disease. However, few studies to date have investigated the 3D architecture of the elastic fibre system in bulk tissue. We describe a protocol for preparation of tissue samples, including primary fixation and backscatter electron contrast-enhancement steps, through dehydration into stable resin-embedded blocks for volume electron microscopy. The use of low molecular weight tannic acid and alcoholic lead staining are critical stages in this procedure. Block preparation by ultramicrotomy and evaporative metal coating prior to microscopical examination are also described. We present images acquired from serial block face scanning electron microscopy of cornea and aorta showing target structures clearly differentiated from cells and other matrix components. The processing method imparts high contrast to fibrillin-containing elastic fibres, thus facilitating their segmentation and rendering into 3D reconstructions by image analysis software from large serial image datasets. Full article
(This article belongs to the Section Tissue Engineering and Organoids)
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13 pages, 372 KiB  
Protocol
Effectiveness of Specialized Nutritious Foods and Social and Behavior Change Communication Interventions to Prevent Stunting among Children in Badakhshan, Afghanistan: Protocol for a Quasi-Experimental Study
by Sajid Bashir Soofi, Gul Nawaz Khan, Shabina Ariff, Arjumand Rizvi, Mohammad Asif Hussainyar, Cecilia Garzon, Martin Ahimbisibwe, Rafiullah Sadeed and Ahmad Reshad
Methods Protoc. 2021, 4(3), 55; https://doi.org/10.3390/mps4030055 - 13 Aug 2021
Cited by 3 | Viewed by 3389
Abstract
Stunting predominantly occurs during the first 1000 days of life and continues to the age of five years. We will aim to assess the effectiveness of specialized nutritious foods (SNF)and social and behavior change communication (SBCC) strategies during the first 1000 days of [...] Read more.
Stunting predominantly occurs during the first 1000 days of life and continues to the age of five years. We will aim to assess the effectiveness of specialized nutritious foods (SNF)and social and behavior change communication (SBCC) strategies during the first 1000 days of life to prevent stunting among children in two rural districts of Badakhshan, Afghanistan. This will be a quasi-experimental pre-post study with the control group utilizing qualitative and quantitative methods. Before launching the program, formative research will be conducted on the acceptability, appropriate use and SBCC strategies needed to support the introduction of intervention package. Repeated cross-sectional baseline and endline surveys will be conducted in both the intervention and the control districts. After the formative research and baseline household survey, an intervention focusing on the provision of SNF, targeting pregnant and lactating women and children 6–23 months, and SBCC strategies will be implemented for at least 12 months. The primary outcome will be a reduction in the prevalence of stunting among children under two years in the intervention group compared to the control group. We will aim to compare the intervention and control group between the pre- and post-intervention assessments to isolate the effect of the intervention by difference-in-differences estimates. The program monitoring and evaluation component will examine the quality of implementation, acceptability of intervention, identification of potential barriers and to learn how to enhance the program’s effectiveness through ongoing operational improvements. The results will be beneficial to design interventions to prevent stunting within Afghanistan and other low–middle-income countries. Full article
(This article belongs to the Section Public Health Research)
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8 pages, 9726 KiB  
Protocol
Modern Applications of 3D Printing: The Case of an Artificial Ear Splint Model
by Athanasios Argyropoulos and Pantelis N. Botsaris
Methods Protoc. 2021, 4(3), 54; https://doi.org/10.3390/mps4030054 - 6 Aug 2021
Cited by 4 | Viewed by 3174
Abstract
Three-dimensional (3D) printing is a leading manufacturing technique in the medical field. The constantly improving quality of 3D printers has revolutionized the approach to new challenges in medicine for a wide range of applications including otoplasty, medical devices, and tissue engineering. The aim [...] Read more.
Three-dimensional (3D) printing is a leading manufacturing technique in the medical field. The constantly improving quality of 3D printers has revolutionized the approach to new challenges in medicine for a wide range of applications including otoplasty, medical devices, and tissue engineering. The aim of this study is to provide a comprehensive overview of an artificial ear splint model applied to the human auricle for the treatment of stick-out protruding ears. The deformity of stick-out protruding ears remains a significant challenge, where the complex and distinctive shape preservation are key factors. To address this challenge, we have developed a protocol that involves photogrammetry techniques, reverse engineering technologies, a smart prototype design, and 3D printing processes. Specifically, we fabricated a 3D printed ear splint model via fused deposition modelling (FDM) technology by testing two materials, a thermoplastic polyester elastomer material (Z-Flex) and polycaprolactone (PCL 100). Our strategy affords a custom-made and patient-specific artificial ear aligner with mechanical properties that ensures sufficient preservation of the auricular shape by applying a force on the helix and antihelix and enables the ears to pin back to the head. Full article
(This article belongs to the Special Issue 3D Printing for Medical Applications)
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18 pages, 8329 KiB  
Protocol
Dissections of Larval, Pupal and Adult Butterfly Brains for Immunostaining and Molecular Analysis
by Yi Peng Toh, Emilie Dion and Antónia Monteiro
Methods Protoc. 2021, 4(3), 53; https://doi.org/10.3390/mps4030053 - 5 Aug 2021
Viewed by 4874
Abstract
Butterflies possess impressive cognitive abilities, and investigations into the neural mechanisms underlying these abilities are increasingly being conducted. Exploring butterfly neurobiology may require the isolation of larval, pupal, and/or adult brains for further molecular and histological experiments. This procedure has been largely described [...] Read more.
Butterflies possess impressive cognitive abilities, and investigations into the neural mechanisms underlying these abilities are increasingly being conducted. Exploring butterfly neurobiology may require the isolation of larval, pupal, and/or adult brains for further molecular and histological experiments. This procedure has been largely described in the fruit fly, but a detailed description of butterfly brain dissections is still lacking. Here, we provide a detailed written and video protocol for the removal of Bicyclus anynana adult, pupal, and larval brains. This species is gradually becoming a popular model because it uses a large set of sensory modalities, displays plastic and hormonally controlled courtship behaviour, and learns visual mate preference and olfactory preferences that can be passed on to its offspring. The extracted brain can be used for downstream analyses, such as immunostaining, DNA or RNA extraction, and the procedure can be easily adapted to other lepidopteran species and life stages. Full article
(This article belongs to the Special Issue Feature Papers 2021)
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14 pages, 695 KiB  
Study Protocol
Archive for Research in Child Health (ARCH) and Baby Gut: Study Protocol for a Remote, Prospective, Longitudinal Pregnancy and Birth Cohort to Address Microbiota Development and Child Health
by Eliot N. Haddad and Sarah S. Comstock
Methods Protoc. 2021, 4(3), 52; https://doi.org/10.3390/mps4030052 - 3 Aug 2021
Cited by 8 | Viewed by 3268
Abstract
The infant gut microbiome is shaped by numerous factors such as diet and the maternal microbiota and is also associated with later atopy and obesity. The Archive for Research in Child Health and Baby Gut (ARCHBG) cohort was established in 2015 to (1) [...] Read more.
The infant gut microbiome is shaped by numerous factors such as diet and the maternal microbiota and is also associated with later atopy and obesity. The Archive for Research in Child Health and Baby Gut (ARCHBG) cohort was established in 2015 to (1) understand how the development of the infant gut microbiota is associated with atopy, obesity, and gastrointestinal disease and (2) characterize the associations of maternal pre-pregnancy BMI and infant diet with the development of the gut microbiota. Study participants for ARCHBG are convenience samples recruited through two pipelines in Lansing and Traverse City, Michigan: (1) Archive for Research in Child Health (ARCHGUT) and (2) BABYGUT. A total of (n = 51) mother–infant dyads have been enrolled to date. This prospective cohort study collects maternal pre-pregnancy fecal samples, maternal data, child fecal samples at four timepoints (one week, six months, 12 months, and 24 months), and child data up to five years of age. All samples and data are collected remotely by mail, phone, or drop-off at select locations. Of all participants enrolled, 76.5% (n = 39) of infants have a complete record of stool samples. At least 88.2% (n = 45) of fecal samples were submitted at each timepoint. ARCHBG will allow for a nuanced understanding of the temporal development of the infant gut microbiome and numerous child health outcomes. Full article
(This article belongs to the Section Public Health Research)
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13 pages, 1950 KiB  
Article
Pichia pastoris and the Recombinant Human Heterodimeric Amino Acid Transporter 4F2hc-LAT1: From Clone Selection to Pure Protein
by Satish Kantipudi, Daniel Harder, Sara Bonetti, Dimitrios Fotiadis and Jean-Marc Jeckelmann
Methods Protoc. 2021, 4(3), 51; https://doi.org/10.3390/mps4030051 - 24 Jul 2021
Cited by 3 | Viewed by 3237
Abstract
Heterodimeric amino acid transporters (HATs) are protein complexes composed of two subunits, a heavy and a light subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. HATs transport amino acids and derivatives thereof across the plasma membrane. The human HAT 4F2hc-LAT1 [...] Read more.
Heterodimeric amino acid transporters (HATs) are protein complexes composed of two subunits, a heavy and a light subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. HATs transport amino acids and derivatives thereof across the plasma membrane. The human HAT 4F2hc-LAT1 is composed of the type-II membrane N-glycoprotein 4F2hc (SLC3A2) and the L-type amino acid transporter LAT1 (SLC7A5). 4F2hc-LAT1 is medically relevant, and its dysfunction and overexpression are associated with autism and tumor progression. Here, we provide a general applicable protocol on how to screen for the best membrane transport protein-expressing clone in terms of protein amount and function using Pichia pastoris as expression host. Furthermore, we describe an overexpression and purification procedure for the production of the HAT 4F2hc-LAT1. The isolated heterodimeric complex is pure, correctly assembled, stable, binds the substrate L-leucine, and is thus properly folded. Therefore, this Pichia pastoris-derived recombinant human 4F2hc-LAT1 sample can be used for downstream biochemical and biophysical characterizations. Full article
(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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21 pages, 6161 KiB  
Article
A Multistep Workflow to Evaluate Newly Generated iPSCs and Their Ability to Generate Different Cell Types
by Carol X.-Q. Chen, Narges Abdian, Gilles Maussion, Rhalena A. Thomas, Iveta Demirova, Eddie Cai, Mahdieh Tabatabaei, Lenore K. Beitel, Jason Karamchandani, Edward A. Fon and Thomas M. Durcan
Methods Protoc. 2021, 4(3), 50; https://doi.org/10.3390/mps4030050 - 19 Jul 2021
Cited by 25 | Viewed by 5950
Abstract
Induced pluripotent stem cells (iPSCs) derived from human somatic cells have created new opportunities to generate disease-relevant cells. Thus, as the use of patient-derived stem cells has become more widespread, having a workflow to monitor each line is critical. This ensures iPSCs pass [...] Read more.
Induced pluripotent stem cells (iPSCs) derived from human somatic cells have created new opportunities to generate disease-relevant cells. Thus, as the use of patient-derived stem cells has become more widespread, having a workflow to monitor each line is critical. This ensures iPSCs pass a suite of quality-control measures, promoting reproducibility across experiments and between labs. With this in mind, we established a multistep workflow to assess our newly generated iPSCs. Our workflow tests four benchmarks: cell growth, genomic stability, pluripotency, and the ability to form the three germline layers. We also outline a simple test for assessing cell growth and highlight the need to compare different growth media. Genomic integrity in the human iPSCs is analyzed by G-band karyotyping and a qPCR-based test for the detection of common karyotypic abnormalities. Finally, we confirm that the iPSC lines can differentiate into a given cell type, using a trilineage assay, and later confirm that each iPSC can be differentiated into one cell type of interest, with a focus on the generation of cortical neurons. Taken together, we present a multistep quality-control workflow to evaluate newly generated iPSCs and detail the findings on these lines as they are tested within the workflow. Full article
(This article belongs to the Section Molecular and Cellular Biology)
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19 pages, 10554 KiB  
Protocol
HiChIP and Hi-C Protocol Optimized for Primary Murine T Cells
by Tomas Zelenka and Charalampos Spilianakis
Methods Protoc. 2021, 4(3), 49; https://doi.org/10.3390/mps4030049 - 16 Jul 2021
Cited by 1 | Viewed by 6503
Abstract
The functional implications of the three-dimensional genome organization are becoming increasingly recognized. The Hi-C and HiChIP research approaches belong among the most popular choices for probing long-range chromatin interactions. A few methodical protocols have been published so far, yet their reproducibility and efficiency [...] Read more.
The functional implications of the three-dimensional genome organization are becoming increasingly recognized. The Hi-C and HiChIP research approaches belong among the most popular choices for probing long-range chromatin interactions. A few methodical protocols have been published so far, yet their reproducibility and efficiency may vary. Most importantly, the high frequency of the dangling ends may dramatically affect the number of usable reads mapped to valid interaction pairs. Additionally, more obstacles arise from the chromatin compactness of certain investigated cell types, such as primary T cells, which due to their small and compact nuclei, impede limitations for their use in various genomic approaches. Here we systematically optimized all the major steps of the HiChIP protocol in T cells. As a result, we reduced the number of dangling ends to nearly zero and increased the proportion of long-range interaction pairs. Moreover, using three different mouse genotypes and multiple biological replicates, we demonstrated the high reproducibility of the optimized protocol. Although our primary goal was to optimize HiChIP, we also successfully applied the optimized steps to Hi-C, given their significant protocol overlap. Overall, we describe the rationale behind every optimization step, followed by a detailed protocol for both HiChIP and Hi-C experiments. Full article
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16 pages, 3263 KiB  
Study Protocol
Experimental Protocol to Assess Neuromuscular Plasticity Induced by an Exoskeleton Training Session
by Roberto Di Marco, Maria Rubega, Olive Lennon, Emanuela Formaggio, Ngadhnjim Sutaj, Giacomo Dazzi, Chiara Venturin, Ilenia Bonini, Rupert Ortner, Humberto Antonio Cerrel Bazo, Luca Tonin, Stefano Tortora, Stefano Masiero, Alessandra Del Felice and on behalf of the PRO GAIT Consortium
Methods Protoc. 2021, 4(3), 48; https://doi.org/10.3390/mps4030048 - 13 Jul 2021
Cited by 9 | Viewed by 4591
Abstract
Exoskeleton gait rehabilitation is an emerging area of research, with potential applications in the elderly and in people with central nervous system lesions, e.g., stroke, traumatic brain/spinal cord injury. However, adaptability of such technologies to the user is still an unmet goal. Despite [...] Read more.
Exoskeleton gait rehabilitation is an emerging area of research, with potential applications in the elderly and in people with central nervous system lesions, e.g., stroke, traumatic brain/spinal cord injury. However, adaptability of such technologies to the user is still an unmet goal. Despite important technological advances, these robotic systems still lack the fine tuning necessary to adapt to the physiological modification of the user and are not yet capable of a proper human-machine interaction. Interfaces based on physiological signals, e.g., recorded by electroencephalography (EEG) and/or electromyography (EMG), could contribute to solving this technological challenge. This protocol aims to: (1) quantify neuro-muscular plasticity induced by a single training session with a robotic exoskeleton on post-stroke people and on a group of age and sex-matched controls; (2) test the feasibility of predicting lower limb motor trajectory from physiological signals for future use as control signal for the robot. An active exoskeleton that can be set in full mode (i.e., the robot fully replaces and drives the user motion), adaptive mode (i.e., assistance to the user can be tuned according to his/her needs), and free mode (i.e., the robot completely follows the user movements) will be used. Participants will undergo a preparation session, i.e., EMG sensors and EEG cap placement and inertial sensors attachment to measure, respectively, muscular and cortical activity, and motion. They will then be asked to walk in a 15 m corridor: (i) self-paced without the exoskeleton (pre-training session); (ii) wearing the exoskeleton and walking with the three modes of use; (iii) self-paced without the exoskeleton (post-training session). From this dataset, we will: (1) quantitatively estimate short-term neuroplasticity of brain connectivity in chronic stroke survivors after a single session of gait training; (2) compare muscle activation patterns during exoskeleton-gait between stroke survivors and age and sex-matched controls; and (3) perform a feasibility analysis on the use of physiological signals to decode gait intentions. Full article
(This article belongs to the Section Public Health Research)
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8 pages, 1181 KiB  
Protocol
Sensitive and Specific Immunohistochemistry Protocol for Nucleocapsid Protein from All Common SARS-CoV-2 Virus Strains in Formalin-Fixed, Paraffin Embedded Tissues
by Yunguang Sun, Linna Ge, Sameer S. Udhane, John F. Langenheim, Mary J. Rau, Mollie D. Patton, Alexander J. Gallan, Juan C. Felix and Hallgeir Rui
Methods Protoc. 2021, 4(3), 47; https://doi.org/10.3390/mps4030047 - 10 Jul 2021
Cited by 7 | Viewed by 3594
Abstract
Human coronavirus disease 2019 (COVID-19) is a life-threatening and highly contagious disease caused by coronavirus SARS-CoV-2. Sensitive and specific detection of SARS-CoV-2 viral proteins in tissues and cells of COVID-19 patients will support investigations of the biologic behavior and tissue and cell tropism [...] Read more.
Human coronavirus disease 2019 (COVID-19) is a life-threatening and highly contagious disease caused by coronavirus SARS-CoV-2. Sensitive and specific detection of SARS-CoV-2 viral proteins in tissues and cells of COVID-19 patients will support investigations of the biologic behavior and tissue and cell tropism of this virus. We identified commercially available affinity-purified polyclonal antibodies raised against nucleocapsid and spike proteins of SARS-CoV-2 that provide sensitive and specific detection of the virus by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. Two immunohistochemistry protocols are presented that are mutually validated by the matched detection patterns of the two distinct viral antigens in virus-infected cells within autopsy lung tissue of COVID-19 deceased patients. Levels of nucleocapsid protein in the lungs of COVID-19 decedents, as measured by quantitative histo-cytometry of immunohistochemistry images, showed an excellent log–linear relationship with levels of viral nucleocapsid RNA levels, as measured by qRT-PCR. Importantly, since the nucleocapsid protein sequence is conserved across all known viral strains, the nucleocapsid immunohistochemistry protocol is expected to recognize all common variants of SARS-CoV-2. Negative controls include autopsy lung tissues from patients who died from non-COVID-19 respiratory disease and control rabbit immunoglobulin. Sensitive detection of SARS-CoV-2 in human tissues will provide insights into viral tissue and cell distribution and load in patients with active infection, as well as provide insight into the clearance rate of virus in later COVID-19 disease stages. The protocols are also expected to be readily transferable to detect SARS-CoV-2 proteins in tissues of experimental animal models or animals suspected to serve as viral reservoirs. Full article
(This article belongs to the Section Public Health Research)
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14 pages, 453 KiB  
Protocol
Perilla Oil and Bifidobacterium for Alleviating Fear of Cancer Recurrence in Breast Cancer Survivors: Study Protocol for a Three-Arm Phase II Randomized Controlled Study (POB Study)
by Yohei Sasaki, Mina Honyashiki, Takayuki Kinoshita, Akira Matsui, Ayako Nakashoji, Takuma Inagawa, Satoru Ikezawa, Naoki Yoshimura, Ryodai Yamamura, Mizuki Amano, Yui Tomo, Hisateru Tachimori, Yutaka J. Matsuoka and Ryo Okubo
Methods Protoc. 2021, 4(3), 46; https://doi.org/10.3390/mps4030046 - 6 Jul 2021
Cited by 1 | Viewed by 3284
Abstract
The fear of cancer recurrence (FCR) is the most common and most severe unmet need among cancer survivors. Safe treatments for the FCR that are easily disseminated are greatly needed. Our primary aim is a preliminary evaluation of the efficacy and effect size [...] Read more.
The fear of cancer recurrence (FCR) is the most common and most severe unmet need among cancer survivors. Safe treatments for the FCR that are easily disseminated are greatly needed. Our primary aim is a preliminary evaluation of the efficacy and effect size of perilla oil, which is rich in omega-3 fatty acids, and Bifidobacterium, a probiotic, on FCR in breast cancer survivors after the completion of chemotherapy. This study has been planned as an exploratory clinical study (phase II) and will be conducted as a three-arm, 12-week parallel group, masked-rater randomized controlled trial. Fifteen participants will be randomized with 1:1:1 allocation to receive Bifidobacterium plus perilla oil, Bifidobacterium alone, or no intervention (control). Interventions will end within 12 weeks after the random allocation of each participant. The participants will be outpatients with invasive breast cancer aged 20 years or older whose chemotherapy was completed at least 6 months before registration; hormone therapy may be ongoing. The primary outcome will be severity of FCR at 12 weeks assessed by masked raters using the 4-item Concerns about Recurrence Scale concerning overall fear of recurrence. The study protocol for the current study is registered in the Japan Registry of Clinical Trials (jRCTs031200029). Full article
(This article belongs to the Section Biomedical Sciences and Physiology)
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43 pages, 1117 KiB  
Protocol
In Vitro Reconstitution of Yeast Translation System Capable of Synthesizing Long Polypeptide and Recapitulating Programmed Ribosome Stalling
by Riku Nagai, Yichen Xu, Chang Liu, Ayaka Shimabukuro and Nono Takeuchi-Tomita
Methods Protoc. 2021, 4(3), 45; https://doi.org/10.3390/mps4030045 - 4 Jul 2021
Cited by 2 | Viewed by 3895
Abstract
The rates of translation elongation or termination in eukaryotes are modulated through cooperative molecular interactions involving mRNA, the ribosome, aminoacyl- and nascent polypeptidyl-tRNAs, and translation factors. To investigate the molecular mechanisms underlying these processes, we developed an in vitro translation system from yeast, [...] Read more.
The rates of translation elongation or termination in eukaryotes are modulated through cooperative molecular interactions involving mRNA, the ribosome, aminoacyl- and nascent polypeptidyl-tRNAs, and translation factors. To investigate the molecular mechanisms underlying these processes, we developed an in vitro translation system from yeast, reconstituted with purified translation elongation and termination factors, utilizing CrPV IGR IRES-containing mRNA, which functions in the absence of initiation factors. The system is capable of synthesizing not only short oligopeptides but also long reporter proteins such as nanoluciferase. By setting appropriate translation reaction conditions, such as the Mg2+/polyamine concentration, the arrest of translation elongation by known ribosome-stalling sequences (e.g., polyproline and CGA codon repeats) is properly recapitulated in this system. We describe protocols for the preparation of the system components, manipulation of the system, and detection of the translation products. We also mention critical parameters for setting up the translation reaction conditions. This reconstituted translation system not only facilitates biochemical analyses of translation but is also useful for various applications, such as structural and functional studies with the aim of designing drugs that act on eukaryotic ribosomes, and the development of systems for producing novel functional proteins by incorporating unnatural amino acids by eukaryotic ribosomes. Full article
(This article belongs to the Section Molecular and Cellular Biology)
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13 pages, 1599 KiB  
Article
Effect of Using Different Chemical Dispersing Agents in Grain Size Analyses of Fluvial Sediments via Laser Diffraction Spectrometry
by Mubarak Abdulkarim, Haruna M. Grema, Ibrahim H. Adamu, Daniela Mueller, Melanie Schulz, Marius Ulbrich, Johannes M. Miocic and Frank Preusser
Methods Protoc. 2021, 4(3), 44; https://doi.org/10.3390/mps4030044 - 29 Jun 2021
Cited by 15 | Viewed by 3081
Abstract
Laser diffraction spectrometry allows for efficiently obtaining high-resolution grain size data. However, pretreatment and dispersion of aggregates in sediment samples are essential pre-requisites for acquiring accurate results using this method. This study evaluates the effectiveness of five dispersing agents in deflocculating the investigated [...] Read more.
Laser diffraction spectrometry allows for efficiently obtaining high-resolution grain size data. However, pretreatment and dispersion of aggregates in sediment samples are essential pre-requisites for acquiring accurate results using this method. This study evaluates the effectiveness of five dispersing agents in deflocculating the investigated fluvial sediments and the resulting grain size distribution obtained by laser diffraction spectrometry. We also examine the ability of the different dispersing agents to deflocculate sediment samples treated by thermal combustion. Distilled water presented a low efficiency in deflocculating the samples and yielded a near-zero clay content for samples with an expected clay content. The other chemical dispersants were effective in dispersing aggregates and yielding clay, albeit with different efficiencies. Calgon had the highest dispersing ability, followed closely by sodium tripolyphosphate. The performance of chemical treatment with sodium oxalate approaches that of sodium tripolyphosphate. However, it leads to the formation of precipitates in the samples, obscuring the actual grain size data. Sodium pyrophosphate derived the least amount of deflocculation among the four chemical dispersants. Furthermore, all the chemical dispersants were found to be ineffective in dispersing aggregates in samples treated by thermal combustion. Full article
(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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