Methods and Protocols doi: 10.3390/mps7020025
Authors: Krishna Pillai Javed Akhter Ahmed H. Mekkawy Sarah J. Valle David L. Morris
The proteolytic activity of enzymes may be evaluated by a colorimetric method with azocasein. Hence, we developed a micro-assay to quantify bromelain using azocasein. A total of 250 µL of 1.0% azocasein in dH2O was added to 250 µL of test solution, vortexed and incubated at ambient room temperature/30 min. The reaction was terminated with 1500 µL of 5% trichloroacetic acid, vortexed and centrifuged. A total of 150 µL of 0.5M NaOH was added to 150 µL of supernatant in triplicates, and absorbance was recorded at 410 nm. The linearity of the calibration curve was tested with 200–800 µg/mL serial dilutions. The detection limit, precision, accuracy, and robustness were tested along with the substrate enzyme reaction time and solvent matrix effect. Good linearity was seen with serially diluted 200 µg/mL bromelain. The limit of quantification and limit of detection were 5.412 and 16.4 µg/mL, respectively. Intra-day and inter-day analyses showed a relative standard deviation below 2.0%. The assay was robust when tested over 400–450 nm wavelengths. The assays performed using dH2O or PBS diluents indicated a higher sensitivity in dH2O. The proteolytic activity of bromelain was enhanced with L-cysteine or N-acetylcysteine. Hence, this micro-azocasein assay is reliable for quantifying bromelain.
]]>Methods and Protocols doi: 10.3390/mps7020024
Authors: Felipe Martínez Carla Taramasco Manuel Espinoza Johanna Acevedo Carolina Goic Bruno Nervi
Cancer, a pervasive global health challenge, necessitates chemotherapy or radiotherapy treatments for many prevalent forms. However, traditional follow-up approaches encounter limitations, exacerbated by the recent COVID-19 pandemic. Consequently, telemonitoring has emerged as a promising solution, although its clinical implementation lacks comprehensive evidence. This report depicts the methodology of a randomized trial which aims to investigate whether leveraging a smartphone app called Contigo for disease monitoring enhances self-reported quality of life among patients with various solid cancers compared to standard care. Secondary objectives encompass evaluating the app’s impact on depressive symptoms and assessing adherence to in-person appointments. Randomization will be performed independently using an allocation sequence that will be kept concealed from clinical investigators. Contigo offers two primary functions: monitoring cancer patients’ progress and providing educational content to assist patients in managing common clinical situations related to their disease. The study will assess outcomes such as quality of life changes and depressive symptom development using validated scales, and adherence to in-person appointments. Specific scales include the EuroQol Group’s EQ-5D questionnaire and the Patient Health Questionnaire (PHQ-9). We hypothesize that the use of Contigo will assist and empower patients receiving cancer treatment, which will translate to better quality of life scores and a reduced incidence of depressive symptoms. All analyses will be undertaken with the intention-to-treat principle by a statistician unaware of treatment allocation. This trial is registered in ClinicalTrials under the registration number NCT06086990.
]]>Methods and Protocols doi: 10.3390/mps7020023
Authors: Imad Alex Awada Adina Magda Florea Alexandru Scafa-Udriște
Gaining practical experience is indispensable for medical students. Therefore, when medical students were prevented access to hospitals during the COVID-19 pandemic in Romania, there was an urgent need to find a solution that would allow medical students to develop the skills they would usually develop in hospitals but without the need to be physically present in a hospital. This was the reason behind the idea of developing a Virtual Case Presentation Platform. The platform offers the possibility for medical students to reproduce virtually, in clinically valid scenarios, the diagnostic process and treatment recommendation, as well as the interactions with patients that usually take place in hospitals using natural language through speech and text. On the platform, the students receive valuable feedback from the professors about their performance. In order to reproduce the whole targeted experience for students, without missing anything, before starting the development of the platform, it was mandatory to identify and understand all the aspects that should be covered by the platform. The proposed platform covers the different aspects that have been identified for the diagnostic process and treatment recommendation. It enables medical students to develop essential skills for their future careers as doctors.
]]>Methods and Protocols doi: 10.3390/mps7020022
Authors: Michaela Kearney David E. McReynolds Henry F. Duncan
The dental pulp has critical functions in tooth development as well as an ongoing role in promoting and maintaining the vitality of teeth. In particular, its regenerative ability allows dental tissues to be restored following damage caused by traumatic injury or caries. Regenerative endodontic procedures aim to utilise these processes to stimulate dental pulp repair in a minimally invasive manner and reduce the need for more invasive procedures such as root canal treatment. Dental pulp is a source of dental pulp cells (DPCs), which has a subpopulation of dental pulp stem cells (DPSCs), which are attractive for use in regenerative medicine due to their high proliferation rate, ability to differentiate into multiple cell types, and their preserved vitality following cryopreservation. The development of next-generation clinical therapeutics that maximise the potential of dental pulp relies on strong empirical evidence arising from in vitro experimentation. Here, we describe a modified method for the efficient isolation of primary human DPCs from sound third molar teeth for culture using an explant outgrowth method on basement membrane-coated flasks, as well as using high-resolution macro-photography to illustrate the methods. Critically, steps are taken to minimise potential physical and mechanical trauma to the cells and maximise yield. Human DPCs cultured using this method can be further expanded in cell culture flasks to facilitate their use in various in vitro experimental procedures.
]]>Methods and Protocols doi: 10.3390/mps7020021
Authors: Evonne Shanita Singh Ashika Naicker Shivneta Singh
As an important way to translate cardiovascular disease prevention efforts, worksite intervention programs can be used to effectively facilitate healthy food choices, health education, and social support among employees, in a targeted approach to improve health outcomes and physical activity levels of employees. In this study, the effectiveness of a canteen and a behavioral intervention on cardiometabolic risk among prediabetic and prehypertensive employees at two multinational worksites in South Africa will be measured. This two-arm randomized controlled trial (RCT) will be structured to provide a six-week intervention at two multinational companies spread across eight worksites and will include a canteen and behavioral arm (CB) and a canteen only (CO) arm. Participants who are either prediabetic or prehypertensive will complete the baseline assessments, which will include anthropometry, a demographic and lifestyle survey, the global physical activity questionnaire (GPAQ) and the 24 h food recall. Participants will be randomized into the CO and the canteen and CB intervention groups. The CO group will receive six weeks of canteen intervention [changes to enable a healthy food environment], while the CB group will receive six weeks of canteen intervention along with a behavioral intervention. The behavioral intervention will include an intense six-week lifestyle program aligned to the Diabetes Prevention Program (DPP). This study will assess the added benefit of environmental-level changes aimed at lowering cardiometabolic risk in a low–middle-income country (LMIC) and has the potential for scale-up to other worksites in South Africa and globally.
]]>Methods and Protocols doi: 10.3390/mps7020020
Authors: Runqiu Song Mariam Bafit Kirsteen M. Tullett Peck Szee Tan Mireille H. Lahoud Meredith O’Keeffe Anthony W. Purcell Asolina Braun
The generation of bone-marrow-derived dendritic cells is a widely used approach in immunological research to study antigen processing and presentation, as well as T-cell activation responses. However, the initial step of isolating the bone marrow can be time-consuming, especially when larger numbers of precursor cells are required. Here, we assessed whether an accelerated bone marrow isolation method using centrifugation is suitable for the differentiation of FMS-like tyrosine kinase 3 ligand-driven dendritic cells. Compared to the conventional flushing method, the centrifugation-based isolation method resulted in a similar bone marrow cell yield on Day 0, increased cell numbers by Day 8, similar proportions of dendritic cell subsets, and consequently a higher number of type 1 conventional dendritic cells (cDC1) from the culture. Although the primary purpose of this method of optimization was to improve experimental efficiency and increase the output of cDC1s, the protocol is also compatible with the differentiation of other dendritic cell subsets such as cDC2 and plasmacytoid dendritic cells, with an improved output cell count and a consistent phenotype.
]]>Methods and Protocols doi: 10.3390/mps7020019
Authors: Jhon Jairo Melchor-Moncada Alejandra García-Barco Augusto Zuluaga-Vélez Luz Angela Veloza Juan Carlos Sepúlveda-Arias
Serratiopeptidase, a bacterial metalloprotease known for its pain-relieving and anti-inflammatory properties, can be produced through fermentation with S. marcescens. This study aimed to identify key factors related to nutrient composition and physicochemical conditions for production in Erlenmeyer flasks and to scale up the mixture to a bioreactor to obtain the maximum proteolytic activity. A Plackett–Burman design was used to determine whether the presence of silkworm pupae (at 1.5%) was a significant parameter for serratiopeptidase production. Along with the variables pH, temperature, and time, they were optimized using a Taguchi experimental design, resulting in values of 7, 25 °C, and 36 h, respectively. Scaling up with a kLa of 25.45 ± 3.12 h−1 showed the highest serratiopeptidase production at 24 h. A factorial design was used for ultrafiltration, resulting in an LMH (liters per square meter per hour) of 960 L/m2h, a TMP (transmembrane pressure) of 15 psi, and a concentration factor of five, with a specific activity of 24,325.81 ± 1515.69 U/mg. Afterward, the retentate was purified using strong anion exchange chromatography and ultrafiltration, yielding a 19.94 ± 3.07% recovery and a purification factor of 1.59 ± 0.31. In conclusion, waste from the sericulture industry can be used for serratiopeptidase production.
]]>Methods and Protocols doi: 10.3390/mps7020018
Authors: Boróka Bárdos Vilmos Altbacker Henrietta Kinga Török István Nagy
European ground squirrel (Spermophilus citellus) populations have declined precipitously over the last 70 years. Its protection cannot be ensured solely by protecting its habitat; it is also necessary to protect the animals ex situ. In our study, within a European ground squirrel species protection program, we examined two elements of indoor housing technology. Knowledge of the animals’ needs is essential for captive housing and breeding success, so in our tests, the animals could freely choose both nest-building materials and feed. In the nest material preference test, the animals could choose from three materials with different structures: paper, Lignocel and hay. In the feed preference test, the animals could also choose from three types of feed: commercial rabbit feed, complete rabbit feed and a natural feed mixture. The first two feeds were in granulated format, and the third was a grain feed mix. Among the nesting materials, they preferred hay, which allowed them to build better-quality nests. Among the feeds, they preferred the grain feed mix, the composition closest to their natural feed, and it was the only one that contained animal protein. Our results contribute to the successful maintenance and breeding the European ground squirrel in captivity.
]]>Methods and Protocols doi: 10.3390/mps7010017
Authors: Christopher R. Stone Dwight D. Harris Mark Broadwin Meghamsh Kanuparthy Sharif A. Sabe Cynthia Xu Jun Feng M. Ruhul Abid Frank W. Sellke
The past several decades have borne witness to several breakthroughs and paradigm shifts within the field of cardiovascular medicine, but one component that has remained constant throughout this time is the need for accurate animal models for the refinement and elaboration of the hypotheses and therapies crucial to our capacity to combat human disease. Numerous sophisticated and high-throughput molecular strategies have emerged, including rational drug design and the multi-omics approaches that allow extensive characterization of the host response to disease states and their prospective resolutions, but these technologies all require grounding within a faithful representation of their clinical context. Over this period, our lab has exhaustively tested, progressively refined, and extensively contributed to cardiovascular discovery on the basis of one such faithful representation. It is the purpose of this paper to review our porcine model of chronic myocardial ischemia using ameroid constriction and the subsequent myriad of physiological and molecular–biological insights it has allowed our lab to attain and describe. We hope that, by depicting our methods and the insight they have yielded clearly and completely—drawing for this purpose on comprehensive videographic illustration—other research teams will be empowered to carry our work forward, drawing on our experience to refine their own investigations into the pathogenesis and eradication of cardiovascular disease.
]]>Methods and Protocols doi: 10.3390/mps7010016
Authors: Carolina López-Guzmán Ana María García Paula Marín Ana María Vásquez
Human placental explants (HPEs) culture has generated significant interest as a valuable in vitro model for studying tissue functions in response to adverse conditions, such as fluctuations in oxygen levels, nutrient availability, exposure to pathogenic microorganisms, and toxic compounds. HPEs offers the advantage of replicating the intricate microenvironment and cell-to-cell communication involved in this critical and transient organ. Although HPEs culture conditions have been extensively discussed, a protocol for assessing the viability and function of HPEs during short-term culture has not been previously outlined. In this study, we have developed a short-term HPEs culture protocol, specifically up to 72 h, and have employed quantitative, semi-quantitative, and qualitative analyses to evaluate tissue viability and function over time. Under our standardized conditions, placental villi explants began to regain their structural properties (the integrity of the trophoblast and villous stroma) and the functionality of the HPEs (production of angiogenic, endocrine, and immunological factors) starting from 48 h of culture. This restoration ensures a suitable environment for several applications. The data presented here can be highly valuable for laboratories aiming to implement an HPEs model, whether in the process of standardization or seeking to enhance and optimize working conditions and timing with placental tissue.
]]>Methods and Protocols doi: 10.3390/mps7010015
Authors: Alexandre Tronel Anne-Sophie Silvent Elena Buelow Joris Giai Corentin Leroy Marion Proust Donald Martin Audrey Le Gouellec Thomas Soranzo Nicolas Mathieu
The connection between imbalances in the human gut microbiota, known as dysbiosis, and various diseases has been well established. Current techniques for sampling the small intestine are both invasive for patients and costly for healthcare facilities. Most studies on human gut microbiome are conducted using faecal samples, which do not accurately represent the microbiome in the upper intestinal tract. A pilot clinical investigation, registered as NCT05477069 and sponsored by the Grenoble Alpes University Hospital, is currently underway to evaluate a novel ingestible medical device (MD) designed for collecting small intestinal liquids by Pelican Health. This study is interventional and monocentric, involving 15 healthy volunteers. The primary objective of the study is to establish the safety and the performance of the MD when used on healthy volunteers. Secondary objectives include assessing the device’s performance and demonstrating the difference between the retrieved sample from the MD and the corresponding faecal sample. Multi-omics analysis will be performed, including metagenomics, metabolomics, and culturomics. We anticipate that the MD will prove to be safe without any reported adverse effects, and we collected samples suitable for the proposed omics analyses in order to demonstrate the functionality of the MD and the clinical potential of the intestinal content.
]]>Methods and Protocols doi: 10.3390/mps7010014
Authors: Carlos Henrique Ferrari Lara Steffany de Carvalho Caroline Trefiglio Rocha Amjad Abu Hasna
This study aimed to assess the relationship between apical fenestration—a defect in the alveolar bone involving the root apex—and tooth position in all tooth groups, excluding the third molars, utilizing cone-beam computed tomography (CBCT) images. A total of 800 CBCT scans (400 maxillary and 400 mandibular) from patients undergoing various treatments were examined by a single professional (radiologist and endodontist). Statistical analyses, including the chi-square test or Fisher’s exact test, were conducted using R software 2.7.3 (R Foundation, Vienna, Austria). Results indicated a significant association (p ≤ 0.05) between apical fenestration and tooth position. In the upper teeth, apical fenestrations were notably present in the mesio-buccal (17.17%) and disto-buccal (11.07%) roots of the first molars. Conversely, apical fenestrations in the lower teeth were relatively less frequent. The study revealed a negative correlation between apical fenestration and mesial inclination, rotation, and extrusion in the upper teeth. However, a positive correlation was observed between apical fenestration and lingual inclination in the upper teeth. In conclusion, this study illuminates the distribution of apical fenestration and its correlation with tooth positions, offering insights into factors influencing this defect in dental anatomy. The findings enhance our understanding of nuanced relationships between tooth position and apical fenestration in the upper and lower dental arches.
]]>Methods and Protocols doi: 10.3390/mps7010013
Authors: Ziyi Liu Dokyun Kim Seokmin Kang Jae U. Jung
The development of monoclonal antibodies (mAbs) represents a significant milestone in both basic research and clinical applications due to their target specificity and versatility in therapeutic and diagnostic applications. The innovative strategy of mAb screening, utilizing phage display, facilitates the in vitro screening of antibodies with high affinity to target antigens. The single-chain variable fragment (scFv) is a subset of mAb derivatives, known for its high binding affinity and smaller size—just one-third of that of human IgG. This report outlines a detailed and comprehensive procedure for constructing a scFv phagemid library derived from human patients, followed by screening via phage display affinity selection. The protocol utilizes 348 primer combinations spanning the entire human antibody repertoire to minimize sequence bias and maintain library diversity during polymerase chain reaction (PCR) for scFv generation, resulting in a library size greater than 1 × 108. Furthermore, we describe a high-throughput phage display screening protocol using enzyme-linked immunosorbent assay (ELISA) to evaluate more than 1200 scFv candidates. The generation of a highly diverse scFv library, coupled with the implementation of a phage display screening methodology, is expected to provide a valuable resource for researchers in pursuit of scFvs with high affinity for target antigens, thus advancing both research and clinical endeavors.
]]>Methods and Protocols doi: 10.3390/mps7010012
Authors: Vasiliy V. Pavlichenko Marina V. Protopopova
The rapid advancement of genetic technologies has made it possible to modify various plants through both genetic transformation and gene editing techniques. Poplar, with its rapid in vitro growth and regeneration enabling high rates of micropropagation, has emerged as a model system for the genetic transformation of woody plants. In this study, Populus × berolinensis K. Koch. (Berlin poplar) was chosen as the model organism due to its narrow leaves and spindle-shaped crown, which make it highly suitable for in vitro manipulations. Various protocols for the Agrobacterium-mediated transformation of poplar species have been developed to date. However, the genetic transformation procedures are often constrained by the complexity of the nutrient media used for plant regeneration and growth, which could potentially be simplified. Our study presents a cheaper, simplified, and relatively fast protocol for the Agrobacterium-mediated transformation of Berlin poplar. The protocol involved using internode sections without axillary buds as explants, which were co-cultivated in 10 µL droplets of bacterial suspension directly on the surface of a solid agar-based medium without rinsing and sterile paper drying after inoculation. We used only one regeneration Murashige and Skoogbased medium supplemented with BA (0.2 mg·L−1), TDZ (0.02 mg·L−1), and NAA (0.01 mg·L−1). Acetosyringone was not used as an induction agent for vir genes during the genetic transformation. Applying our protocol and using the binary plasmid pBI121 carrying the nptII selective and uidA reporter genes, we obtained the six transgenic lines of poplar. Transgenesis was confirmed through a PCR-based screening of kanamycin-selected regenerants for the presence of both mentioned genes, Sanger sequencing, and tests for detecting the maintained activity of both genes. The transformation efficiency, considering the 100 explants taken originally, was 6%.
]]>Methods and Protocols doi: 10.3390/mps7010011
Authors: Ion Udroiu
Knowledge of the geometric quantities of the erythrocyte is useful in several physiological studies, both for zoologists and veterinarians. While the diameter and volume (MCV) are easily obtained from observations of blood smears and complete blood count, respectively, the thickness and surface area are instead much more difficult to measure. The precise description of the erythrocyte geometry is given by the equation of the oval of Cassini, but the formulas deriving from it are very complex, comprising elliptic integrals. In this article, three solids are proposed as models approximating the erythrocyte: sphere, cylinder and a spheroid with concave caps. The volumes and surface areas obtained with these models are compared to those effectively measured. The spheroid with concave caps gives the best approximation and can be used as a simple model to determine the erythrocyte surface area. With this model, a simple method that allows one to estimate the surface area by knowing only the diameter and MCV is proposed.
]]>Methods and Protocols doi: 10.3390/mps7010010
Authors: Kristián Slíž Juraj Piešťanský Peter Mikuš
Ostarine is frequently misused as a selective androgen receptor modulator (SARM) in sports. Consequently, there is a pressing need for reliable and simple approaches to monitor its presence in biological systems. In this work, we developed a two-dimensional analytical method utilizing online solid-phase extraction (online-SPE) in conjunction with ultra-high-performance liquid chromatography and tandem mass spectrometry (triple quadrupole). This automated 2D separation approach is characterized by minimum manual steps in the sample preparation (only dilute-and-shoot), reflecting high sample throughput and the reliability of analytical data. It provides favorable performance parameters, including a limit of detection of 0.5 pg/mL, high accuracy (relative error = 1.6–7.5%), precision (relative standard deviation = 0.8–4.5%), and sensitivity. Additionally, it demonstrates excellent linearity (r2 = 0.9999) in the calibration range of 0.05 to 25 ng/mL and robustness, with no carryover effects observed. This comparative study revealed a two-decadic-order-lower LOD of the SPE-UHPLC-MS/MS method to the corresponding UHPLC-MS/MS method and the lowest one in the group of currently published LC-MS methods. The World Anti-Doping Agency screening and confirmation criteria were met through the analysis of spiked urine samples from ten healthy volunteers. Accordingly, the proposed method is suitable for routine use in antidoping laboratories.
]]>Methods and Protocols doi: 10.3390/mps7010009
Authors: Haya Al-Hilal Marianna Maretina Anna Egorova Andrey Glotov Anton Kiselev
Spinal muscular atrophy is a neuromuscular disorder caused by mutations in both copies of the survival motor neuron gene 1 (SMN1), which lead to reduction in the production of the SMN protein. Currently, there are several therapies that have been approved for SMA, with many more undergoing active research. While various biomarkers have been proposed for assessing the effectiveness of SMA treatment, a universally accepted one still has not been identified. This study aimed to describe a fast and reliable method using the number of gems in cell nuclei as a potential tool for assessment of splicing correction of oligonucleotide efficacy in SMA cells. To gain insight into whether the number of gems in cell nuclei varies based on their SMN genotype and whether the increase in gem number is associated with therapeutic response, we utilized fibroblast cell cultures obtained from a patient with SMA type II and from a healthy individual. We discovered a remarkable difference in the number of gems found in the nuclei of these cells, specifically when counting gems per 100 nuclei. The SMA fibroblasts treated with antisense oligonucleotide showed beneficial effects in correcting the abnormal splicing of SMN2 exon 7. It was observed that there was a significant increase in the number of gems in the treated cells compared to the intact SMA cells. The results obtained significantly correlate with an increase of full-length SMN transcript sharing. Based on our findings, we propose using the quantity of gems as a reliable biomarker for SMA drug development.
]]>Methods and Protocols doi: 10.3390/mps7010008
Authors: Sara C. Zapico Gabriela Roca
Lateral flow immunochromatographic (LFI) tests are widely used in both biomedical and forensic sciences for different applications. In forensic sciences, their main use is to detect body fluids at crime scenes. However, there are situations in which the amount of potential biological evidence is so low that DNA extraction is favored with respect to the identification of body fluids. Here, an efficient and quick protocol is presented to integrate the detection of body fluids through LFI with DNA extraction from a sample swab and buffer, providing a complete characterization of the biological evidence. This protocol is a modification of a general DNA extraction silica-based kit, whose main application is for blood and tissues. Thus, it could be carried out in different settings (forensic labs, hospitals, other testing labs) without the necessity of buying a specific kit for swabs. The validation of this protocol is supported by the results presented here and previous publications from our group, obtaining DNA in good quantity and with good quality. This proves the potential application of the protocol in both forensic scenarios, to fully characterize biological evidence, and biomedical settings, to molecularly confirm the results of LFI tests.
]]>Methods and Protocols doi: 10.3390/mps7010007
Authors: Alice Baek Asif Rayhan Ga-Eun Lee Sarah Golconda Hannah Yu Shihyoung Kim Patrick A. Limbach Balasubrahmanyam Addepalli Sanggu Kim
The biological significance of chemical modifications to the ribonucleic acid (RNA) of human immunodeficiency virus type-1 (HIV-1) has been recognized. However, our understanding of the site-specific and context-dependent roles of these chemical modifications remains limited, primarily due to the absence of nucleotide-resolution mapping of modification sites. In this study, we present a method for achieving nucleotide-resolution mapping of chemical modification sites on HIV-1 RNA using liquid chromatography and tandem mass spectrometry (LC–MS/MS). LC–MS/MS, a powerful tool capable of directly analyzing native RNAs, has proven effective for mapping RNA modifications in small RNA molecules, including ribosomal RNA and transfer RNA. However, longer RNAs have posed challenges, such as the 9 Kb HIV-1 virion RNA, due to the complexity of and ambiguity in mass differences among RNase T1-cleaved RNA fragments in LC-MS/MS data. Here, we introduce a new target RNA enrichment method to isolate small local RNA fragments of HIV-1 RNA that potentially harbor site-specific N6-methyladenosine (m6A) modifications. In our initial trial, we used target-specific DNA probes only and encountered insufficient RNA fragmentation due to inefficient S1 digestion near the target site. Recognizing that inefficient S1 digestion by HIV-1 RNA is likely due to the formation of secondary structures in proximity to the target site, we designed multiple DNA probes annealing to various sites of HIV-1 RNA to better control the structures of RNA substrates for S1 digestion. The use of these non-target DNA probes significantly improved the isolation of more homogeneous target RNA fragments of approximately 50 bases in length. Oligonucleotide LC-MS/MS analysis of these isolated target RNA fragments successfully separated and detected both m6A-methylated and non-methylated oligomers at the two m6A-predicted sites. The principle of this new target enrichment strategy holds promise and should be broadly applicable to the analysis of any lengthy RNA that was previously deemed infeasible for investigation using oligonucleotide LC-MS/MS.
]]>Methods and Protocols doi: 10.3390/mps7010006
Authors: Yao Yao Yibo Zhu Regina Nogueira Frank Klawonn Markus Wallner
Wastewater-based epidemiology (WBE) has great potential to monitor community public health, especially during pandemics. However, it faces substantial hurdles in pathogen surveillance through WBE, encompassing data representativeness, spatiotemporal variability, population estimates, pathogen decay, and environmental factors. This paper aims to enhance the reliability of WBE data, especially for early outbreak detection and improved sampling strategies within sewer networks. The tool implemented in this paper combines a monitoring model and an optimization model to facilitate the optimal selection of sampling points within sewer networks. The monitoring model utilizes parameters such as feces density and average water consumption to define the detectability of the virus that needs to be monitored. This allows for standardization and simplicity in the process of moving from the analysis of wastewater samples to the identification of infection in the source area. The entropy-based model can select optimal sampling points in a sewer network to obtain the most specific information at a minimum cost. The practicality of our tool is validated using data from Hildesheim, Germany, employing SARS-CoV-2 as a pilot pathogen. It is important to note that the tool’s versatility empowers its extension to monitor other pathogens in the future.
]]>Methods and Protocols doi: 10.3390/mps7010005
Authors: Jasmin Leonie Aeberhard Anda-Petronela Radan Ramin Abolfazl Soltani Karin Maya Strahm Sophie Schneider Adriana Carrié Mathieu Lemay Jens Krauss Ricard Delgado-Gonzalo Daniel Surbek
Artificial intelligence (AI) is gaining increasing interest in the field of medicine because of its capacity to process big data and pattern recognition. Cardiotocography (CTG) is widely used for the assessment of foetal well-being and uterine contractions during pregnancy and labour. It is characterised by inter- and intraobserver variability in interpretation, which depends on the observers’ experience. Artificial intelligence (AI)-assisted interpretation could improve its quality and, thus, intrapartal care. Cardiotocography (CTG) raw signals from labouring women were extracted from the database at the University Hospital of Bern between 2006 and 2019. Later, they were matched with the corresponding foetal outcomes, namely arterial umbilical cord pH and 5-min APGAR score. Excluded were deliveries where data were incomplete, as well as multiple births. Clinical data were grouped regarding foetal pH and APGAR score at 5 min after delivery. Physiological foetal pH was defined as 7.15 and above, and a 5-min APGAR score was considered physiologic when reaching ≥7. With these groups, the algorithm was trained to predict foetal hypoxia. Raw data from 19,399 CTG recordings could be exported. This was accomplished by manually searching the patient’s identification numbers (PIDs) and extracting the corresponding raw data from each episode. For some patients, only one episode per pregnancy could be found, whereas for others, up to ten episodes were available. Initially, 3400 corresponding clinical outcomes were found for the 19,399 CTGs (17.52%). Due to the small size, this dataset was rejected, and a new search strategy was elaborated. After further matching and curation, 6141 (31.65%) paired data samples could be extracted (cardiotocography raw data and corresponding maternal and foetal outcomes). Of these, half will be used to train artificial intelligence (AI) algorithms, whereas the other half will be used for analysis of efficacy. Complete data could only be found for one-third of the available population. Yet, to our knowledge, this is the most exhaustive and second-largest cardiotocography database worldwide, which can be used for computer analysis and programming. A further enrichment of the database is planned.
]]>Methods and Protocols doi: 10.3390/mps7010004
Authors: Lucas Banda Olanrewaju Oladimeji
Introduction: Tuberculosis (TB) and Human Immunodeficiency Virus (HIV) remain major public health issues in sub-Saharan Africa. The co-occurrence of these diseases is a growing concern in the region, and social determinants, the circumstances under which people are born, live, work, and age, are known to influence the risk of disease transmission, diagnosis, treatment, and outcomes. Here, we present a protocol for the evidence synthesis on the social determinants of HIV/TB coinfections in sub-Saharan Africa. The high prevalence of Tuberculosis (TB) and Human Immunodeficiency Virus (HIV) in sub-Saharan Africa presents significant public health challenges. TB/HIV comorbidity is influenced by various social determinants, including social, economic, cultural, and environmental factors, impacting disease transmission risk, accurate diagnosis, and treatment outcomes. This study protocol aims to provide an evidence synthesis on the social determinants of HIV/TB coinfection in sub-Saharan Africa. Methods and analysis: The researchers will use the Arksey and O’Malley’s (2005) methodological framework to guide the scoping review. First, databases such as PubMed, MEDLINE, Web of Science, and PsychInfo will be searched. The researchers will then proceed in two steps. Before finalising the study selection, two independent reviewers will examine the article titles and abstracts for eligibility and inclusion. The researchers will then conduct a full-text screening of the articles based on the selected titles and abstracts. The authors’ tool will be used to extract data, ensuring that the articles are properly screened and that the risk of bias is minimized. The chosen studies will be examined using a standardized tool to examine all bibliographic data and study characteristics. Ethics and dissemination: The review will provide an overview of the social determinants influencing the prevalence and outcomes of TB/HIV comorbidity in the region, as well as identify any research gaps. Policymakers, researchers, and healthcare professionals will benefit from the findings in developing targeted interventions to address the social determinants of TB/HIV comorbidity in sub-Saharan Africa.
]]>Methods and Protocols doi: 10.3390/mps7010003
Authors: Hai Dang Ngo Jan Patrick Formanski Vivien Grunwald Birco Schwalbe Michael Schreiber
Flaviviruses are a family of RNA viruses that includes many known pathogens, such as Zika virus (ZIKV), West Nile virus (WNV), dengue virus (DENV), and yellow fever virus (YFV). A pseudotype is an artificial virus particle created in vitro by incorporating the flavivirus envelope proteins into the structure of, for example, a retrovirus such as human immunodeficiency virus type-1 (HIV-1). They can be a useful tool in virology for understanding the biology of flaviviruses, evaluating immune responses, developing antiviral strategies but can also be used as vectors for gene transfer experiments. This protocol describes the generation of a ZIKV/HIV-1 pseudotype developed as a new tool for infecting cells derived from a highly malignant brain tumor: glioblastoma multiforme grade 4.
]]>Methods and Protocols doi: 10.3390/mps7010002
Authors: Inês M. Ferreira Luz Murillo Jean-Marie Le-Corre Marco Correia Rita Anastácio Mário J. Pereira
The scarcity on the Atlantic coast of the African sea turtle population and its dynamics data is well known. This article discusses the nesting ecology methods and analysis of a nascent Angolan project aimed at preserving the nesting female population of the Olive Ridley turtle (Lepidochelys olivacea) on the coast of Lobito. This study examines the nesting ecology of this species from 2020 to 2023. Females had an average CCL of 70.2 cm and CCW of 68.5 cm. These females laid 127 eggs in nests that averaged 47.0 cm deep. The ex situ nest incubation period averaged 60 days, and the hatchling success was 82.1%. Some techniques used in this project require modifications and enhancements. The utilization of photo identification did not yield the anticipated outcomes, prompting the adoption of passive integrated transponders (PITs) in the last season. However, due to limited funding, the success of this method is contingent upon an augmented field effort, allowing for the recapture of a larger number of females. The continuity of this project hinges upon collaboration between higher authorities and the local community. Together, it is possible to deepen the understanding of the nesting ecology of this species and address pivotal issues for its conservation, thereby implementing the most effective preservation measures.
]]>Methods and Protocols doi: 10.3390/mps7010001
Authors: Jong H. Kim Kathleen L. Chan William M. Hart-Cooper DeAngela Ford Kaydren Orcutt Jeffrey D. Palumbo Christina C. Tam William J. Orts
The United States is a principal producer of tree nuts (almonds, pistachios, and walnuts), resulting in the generation of excess of tree-nutshell by-products each year, with few market outlets. A nutshell is an essential, lignocellulosic layer that protects a kernel (seed) from the environment during cultivation. The objective of this study was to develop nutshell by-products as herbicide delivery systems, which would not only enable sustainable weed control in fields but also increases nutshell value and reduce the cost of waste disposal. We recently identified a natural salicylaldehyde (SA) that emits volatiles with both herbicidal and antifungal properties. In this study, walnut shell particles saturated with 0.8 to 1.6 M SA were developed as delivery vehicles for SA to soil, which allowed for the controlled release of an SA fumigant for weed control. The pre- and post-emergent herbicidal efficacy of SA was investigated using model monocot (Lolium arundinaceum (Schreb.) Darbysh; turfgrass) and dicot (Brassica rapa var. pekinensis; Chinese cabbage) plants. We compared (1) the effects of different types of solvents for dissolving SA (dimethyl sulfoxide (DMSO) and ethanol (60%, v/v)), and (2) the effect of covering soil with plastic layers (i.e., soil pasteurization) or not covering soil during SA fumigation using nutshells. Results: In the pre-emergent herbicidal testing with the soil covered, the dicot plants exhibited levels of higher susceptibility to SA in DMSO emitted from nutshells when compared to the monocot plants. The seed germination frequencies in the dicots were 15% and 1% with 0.8 and 1.6 M SA, respectively, while those in the monocots were 32% and 18%, respectively, under the same test conditions. In the post-emergent herbicidal testing with the soil covered, the growth of both the monocot and dicot plants was completely prevented after 5 to 7 days of SA fumigation, resulting in the deaths of entire plants. It was noteworthy that in the post-emergent herbicidal testing, SA dissolved in ethanol (60%, v/v) completely disrupted the growth of the monocot and dicot plants as early as 3 days after SA emission from the nutshells, even without the soil being covered. Tree-nutshell particles could serve as effective SA delivery vehicles with controlled release capabilities for SA. The SA exhibited pre- and post-emergent herbicidal activities against the monocot and dicot plants at most growth stages. SA (0.8 and 1.6 M) dissolved in ethanol (60%, v/v) might exert a synergism for higher herbicidal activity after emission from nutshells. Since tree nuts capture/store a substantial amount of carbon over their life-cycles, the new and sustainable utility of using nutshells not only reduces carbon emissions but also valorizes tree-nut by-products, thus benefitting the tree-nut industry.
]]>Methods and Protocols doi: 10.3390/mps6060119
Authors: Johanna Alm Benoit Fischer Alexandra Emanuela Burger Francesca Moretti
Perturbation of angiogenesis is associated with a variety of diseases and pro- as well as antiangiogenic therapies are being actively explored. Additionally, unintended adverse drug effects on angiogenesis might lead to promotion of tumor progression and cardiovascular complications. Several tri-dimensional microfluidic vessel-on-chip systems have been described that allow a more accurate investigation of vascular physiology and pathology, compared to the two-dimensional static culture of endothelial cells. The OrganoPlate® angiogenesis-on-chip system has been demonstrated to be amenable to high-throughput screening for the antiangiogenic properties of molecules. We set out to adapt this system for high-throughput screening of molecules with proangiogenic properties. Our technical advancement of the OrganoPlate® angiogenesis-on-chip assay expands its applicability in the early screening of both anti- as well as proangiogenic properties of compounds for therapeutic modulation of angiogenesis as well as the identification of angiogenesis-associated drug-induced vascular toxicities.
]]>Methods and Protocols doi: 10.3390/mps6060118
Authors: Junchi Huang Mikael Montelius Jan-Erik Damber Karin Welén
Bone metastases cause morbidity and mortality in several human cancer forms. Experimental models are used to unravel the mechanisms and identify possible treatment targets. The location inside the skeleton complicates accurate assessment. This study evaluates the performance of magnetic resonance imaging (MRI) of prostate cancer tumors growing intratibially in mice. MRI detected intratibial tumor lesions with a sensitivity and specificity of 100% and 89%, respectively, compared to histological evaluation. Location and some phenotypical features could also be readily detected with MRI. Regarding volume estimation, the correlation between MRI and histological assessment was high (p < 0.001, r = 0.936). In conclusion, this study finds MRI to be a reliable tool for in vivo, non-invasive, non-ionizing, real-time monitoring of intratibial tumor growth.
]]>Methods and Protocols doi: 10.3390/mps6060117
Authors: Anouk van Duinkerken Mark Bosmans Christos Baliatsas Nannah Tak Anne Meerdink Noortje Jansen Marjonneke de Vetten-Mc Mahon Elske Marra Michel Dückers
Background: The global COVID-19 pandemic has profoundly affected public health. Directly, the pandemic resulted in over 6.6 million deaths, numerous hospitalizations, and widespread illness. The pandemic has also affected health indirectly through government-imposed protective measures, causing decline in mental well-being and increasing social isolation. Unlike previous disasters or crises, the pandemic’s worldwide and enduring impact necessitates a unique research approach. The Network for Health Research in Disasters in the Netherlands responded by initiating a longitudinal, extensive research project called the Integrated Health Monitor COVID-19. The Integrated Health Monitor COVID-19 explores both the direct and indirect health effects of the pandemic at the population level. Methods: The Integrated Health Monitor COVID-19 employs a dual-pronged monitoring strategy alongside an annual literature review. This strategy comprises short-cycle monitoring (conducted quarterly) and long-cycle monitoring (conducted once every one or two years). This comprehensive approach enables the evaluation of health trends during the pandemic, facilitating comparisons with pre-pandemic levels and identification of risk and protective factors. Both monitoring methods incorporate data from surveys and general practice registries. The integration of annual literature reviews with these measurements enables iterative research, while dialogues on policy and practice improvements enhance the knowledge-to-action process. Discussion: Much of the existing knowledge about the potential impact of the COVID-19 pandemic is derived from research on sudden-onset disasters limited to specific geographical areas. This study is anticipated to provide valuable fresh insights into the evolving dynamics of population health and specific vulnerabilities within the ongoing pandemic context.
]]>Methods and Protocols doi: 10.3390/mps6060116
Authors: Laurie Nemoz-Billet Jacques Brocard Florence Ruggiero Sandrine Bretaud
Quantifying axonal branching is crucial for understanding neural circuit function, developmental and regeneration processes and disease mechanisms. Factors that regulate patterns of axonal arborization and tune neuronal circuits are investigated for their implication in various disorders in brain connectivity. The lack of a reliable and user-friendly method makes the quantitative analysis of axon morphology difficult. Specifically, methods to visualize and quantify the complex axon arborization are challenging to implement and apply practically. Our study was aimed at developing a robust but simple method of quantification that used ImageJ 2D analysis and compared it with Imaris visualization and analysis of 3D images. We used zebrafish fluorescent transgenic lines to perform in vivo imaging of developing motor neuron axons that adequately reflected the complexity of axonal networks. Our new method, developed on ImageJ, is easy and fast, giving access to new information such as collateral distribution along the axonal shaft. This study describes step-by-step procedures that can be easily applied to a variety of organisms and in vitro systems. Our study provides a basis for further exploration of neural circuits to gain new insights into neuronal disorders and potential therapeutic interventions.
]]>Methods and Protocols doi: 10.3390/mps6060115
Authors: Christian Wong Christina Ystrøm Bjerge Ales Jurca Michael Mørk Petersen Soren Boedtker Andreas Balslev-Clausen Steen Harsted
Background: The health of children’s lower extremities and feet is a focus area for caregivers and healthcare professionals such as doctors, school nurses, and podiatrists. Our study aims to investigate the general health status of Danish children’s lower extremities and feet to identify anthropometric parameters that might be preconditions for pain and evaluate for foot diseases and whether they are associated with pain intensity and location, three-dimensional foot dimensions and foot pressure mapping, shoe dimensions, types and intensity of sports activity, quality of life, and foot health. The aim is that we will be able to identify parameters pre-dispositioning for pain, thus providing recommendations for sports activities in relation to the anthropometric conditions of a child as a potential preventive measure for pain. This analysis will be stratified by socioeconomic status on a group level, and this perspective will be able to provide preventative recommendations to prevent pain. Methods: This study is a cross-sectional examination of a thousand children in the first, fifth, and ninth grades in randomized selected Danish primary schools. We will perform a clinical examination of the lower extremities and feet for misalignments, deformities, and diseases as well as rotational status and range of motion. Moreover, we will evaluate their pain levels, sports activities, three-dimensional foot dimensions, plantar pressure, footwear, and patient-related outcome measures (PROMs) for foot health and quality of life. Results: We aim to provide an anthropometrical overview of the lower extremities and feet in children. The obtained basic understanding of healthy normal material in children will be analyzed for its relationships with pain level, sports activities, and socioeconomic status on a group level. This could potentially provide us with an understanding of the factors that impact lower extremity and foot diseases in children. In conclusion, examining children’s lower extremities and feet in Danish primary schools is a step toward identifying areas of improvement in self-care and shoe fitting, mapping podiatry-related needs of care in children’s feet, and providing parental recommendations for preventive actions on shoe fitting and the choice and intensity of sports activity concerning pain. Conclusions: The tenet of this study is a long-term follow-up to evaluate the long-term socioeconomic course on a group level, foot status, and sports activity, using patient-related outcome measures evaluating quality of life and other lifestyle factors such as emotional functioning, social functioning and interaction, and school functioning. Potentially, this will improve children’s quality of life and prevent future diseases.
]]>Methods and Protocols doi: 10.3390/mps6060114
Authors: Hongmin Chu Weonjin Kim Seongsu Joo Eunsik Park Yeong Won Kim Cheol-Hyun Kim Sangkwan Lee
Background. The aim of this study was to compare shoulder movement measurements between a Kinect-based markerless ROM assessment device (POM-Checker) and a 3D motion capture analysis system (BTS SMART DX-400). Methods. This was a single-visit clinical trial designed to evaluate the validity and reliability of the POM-Checker. The primary outcome was to assess the equivalence between two measurement devices within the same set of participants, aiming to evaluate the validity of the POM-Checker compared to the gold standard device (3D Motion Analysis System). As this was a pilot study, six participants were included. Results. The intraclass correlation coefficient (ICC) and the corresponding 95% confidence intervals (CIs) were used to assess the reproducibility of the measurements. Among the 18 movements analyzed, 16 exhibited ICC values of >0.75, indicating excellent reproducibility. Conclusion. The results showed that the POM-checker is reliable and validated to measure the range of motion of the shoulder joint.
]]>Methods and Protocols doi: 10.3390/mps6060113
Authors: Pragya Yadav V. V. Santosh Kumar Jyoti Priya Shashank Kumar Yadav Shivani Nagar Meenu Singh Viswanathan Chinnusamy
Rice is one of the apex food crops in terms of meeting the daily calorific and dietary requirement of the majority of the world population. However, rice productivity is severely limited by various biotic and abiotic attributes, causing a severe threat to global food security. In the use of functional genomics and genome editing for the generation of trait-enhanced genotypes, it is necessary to have an efficient genetic transformation and regeneration protocol. The recalcitrant nature and paucity of efficient and versatile genetic transformation and regeneration protocols for indica cultivars remains a constraint. In the present study, we have optimized a tissue culture method for MTU1010, a mega indica rice variety. We conducted a combinatorial analysis of different plant growth regulators on embryogenic callus induction efficiency, and it was observed that MSB5 medium supplemented with 2.5 mg/L 2-4D and 0.25 mg/L 6-BAP results in maximum embryogenic callus induction, i.e., 92%. The regeneration efficiency of a transformed callus can be enhanced by up to 50% with the supplementation of 1 mg/L kinetin alongside 2.5 mg/L BAP and 0.5 mg/L NAA in the shooting medium. Furthermore, our results unveiled that the pre-activation of Agrobacterium culture for 30 min with 150 µM acetosyringone significantly increased the transformation efficiency of calli. Additionally, descaling the salt concentration to half strength in resuspension and co-cultivation increased the efficiency of transformation up to 33%. Thus, the protocol developed in this study will be instrumental for the genome editing and genetic engineering of indica rice cultivars for functional genomics studies and crop improvement.
]]>Methods and Protocols doi: 10.3390/mps6060112
Authors: Rownock Afruza Nicole Minerva Justin B. Lack Moumita Chakraborty James A. Haddad Rabab O. Ali Christopher Koh Elliot B. Levy Ohad Etzion Theo Heller
Cell-free RNAs (cfRNAs) are promising analytes as non-invasive biomarkers and have even greater potential if tied in with metabolomics. Plasma is an optimal source for cfRNAs but is often derived from a variety of anticoagulants. Plasma obtained in heparin is suitable for metabolomics but is difficult to utilize for qPCR-based downstream analysis. In the present study, we aimed to develop a simple, time-efficient, and cost-effective heparinase protocol, followed by library preparation and sequencing of human plasma cfRNAs drawn and stored in heparin at −80 °C for several years. Blood was collected in CPT™ sodium heparin tubes from patients with chronic HCV infection (NCT02400216) at the National Institutes of Health (NIH) Clinical Center. Plasma cfRNAs were treated with heparinase I and used for library preparation and next-generation sequencing (NGS). Heparinase treatment maintained RNA integrity and allowed for successful library preparation for all the study subjects even with 7 ng of cfRNAs as starting material. The classification report derived from Pavian R package v1.2.0 showed no artificial reads. The abundance of chordate over microbial reads suggests no addition of experimental error through heparinase I treatment. We report a novel and practical approach to heparinase treatment for human plasma collected and frozen in sodium heparin for several years. This is an effective demonstration of utilizing heparin plasma for NGS and downstream transcriptomic research, which could then be integrated with metabolomics from the same samples, maximizing efficiency and minimizing blood draws.
]]>Methods and Protocols doi: 10.3390/mps6060111
Authors: Fernando Albericio Philip Hublitz
As our readers know, Methods and Protocols is a multidisciplinary peer-reviewed scientific journal that provides a forum to the publication of novel approaches in the fields of Life Sciences, Chemistry, and Biomedical Sciences and their intersection with other related scientific fields such as Physics, Earth Sciences, and Environmental Research [...]
]]>Methods and Protocols doi: 10.3390/mps6060110
Authors: Tanya Román Gerardo Acosta Constanza Cárdenas Beatriz G. de la Torre Fanny Guzmán Fernando Albericio
One approach to enhance the bioavailability and half-life of peptides in vivo is through N-methylation of one or more of the amino acids within the peptide sequence. However, commercially available Fmoc-N-Me-AA-OHs are limited and often expensive. In this study, a solid-phase synthesis method for Fmoc-N-Me-AA-OH was developed using a 2-chlorotrityl chloride (2-CTC) resin as a temporary protective group for the carboxylic acid strategy. Two strategies for the alkylation step were compared, employing either dimethyl sulfate or methyl iodide in the Biron−Kessler method. In this work we tested the protocol with two amino acids: Fmoc-Thr(tBu)-OH and Fmoc-βAla-OH. The first one is an alpha amino acid, very hindered and with the amine group directly influenced by the electronic effects of the carboxy group, whereas in Fmoc-βAla-OH, the presence of a methylene group weakens this influence due to the intervening carbon atoms. The desired amino acids, Fmoc-N-Me-Thr(tBu)-OH and Fmoc-N-Me-βAla-OH, were synthesized by both strategies with high yield and purity.
]]>Methods and Protocols doi: 10.3390/mps6060109
Authors: Juan David Ospina-Villa Valentina Restrepo-Cano Miryan Margot Sánchez-Jiménez
Bio-SELEX is a revolutionary method for the discovery of novel biomarkers within biological samples, offering profound insights into diagnosing both infectious and non-infectious diseases. This innovative strategy involves three crucial steps: Traditional SELEX, Pull Down, and mass spectrometry. Firstly, Traditional SELEX involves the systematic selection of specific nucleic acid sequences (aptamers) that bind to the target molecules of interest. These aptamers are generated through iterative rounds of selection, amplification, and enrichment, ultimately yielding highly selective ligands. Secondly, the Pull-Down phase employs these aptamers to capture and isolate the target biomarkers from complex biological samples. This step ensures the specificity of the selected aptamers in binding to their intended targets. Lastly, mass spectrometry is utilized to identify and quantify the captured biomarkers, providing precise information about their presence and concentration in the sample. These quantitative data are invaluable in disease diagnosis and monitoring. Bio-SELEX’s significance lies in its ability to discover biomarkers for a wide range of diseases, spanning infectious and non-infectious conditions. This approach holds great promise for early disease detection, personalized medicine, and the development of targeted therapies. By harnessing the power of aptamers and mass spectrometry, Bio-SELEX advances our understanding of disease biology and opens new avenues for improved healthcare.
]]>Methods and Protocols doi: 10.3390/mps6060108
Authors: Youngjae Ryu Yoonju Kim Sang-Joon Park Sung Rae Kim Hyung-Jun Kim Chang Man Ha
Whole-brain imaging is important for understanding brain functions through deciphering tissue structures, neuronal circuits, and single-neuron tracing. Thus, many clearing methods have been developed to acquire whole-brain images or images of three-dimensional thick tissues. However, there are several limitations to imaging whole-brain volumes, including long image acquisition times, large volumes of data, and a long post-image process. Based on these limitations, many researchers are unsure about which light microscopy is most suitable for imaging thick tissues. Here, we compared fast-confocal microscopy with light-sheet fluorescence microscopy for whole-brain three-dimensional imaging, which can acquire images the fastest. To compare the two types of microscopies for large-volume imaging, we performed tissue clearing of a whole mouse brain, and changed the sample chamber and low- magnification objective lens and modified the sample holder of a light-sheet fluorescence microscope. We found out that light-sheet fluorescence microscopy using a 2.5× objective lens possesses several advantages, including saving time, large-volume image acquisitions, and high Z-resolution, over fast-confocal microscopy, which uses a 4× objective lens. Therefore, we suggest that light-sheet fluorescence microscopy is suitable for whole mouse brain imaging and for obtaining high-resolution three-dimensional images.
]]>Methods and Protocols doi: 10.3390/mps6060107
Authors: Noemí Cabré Yongqiang Yang Yanhan Wang Bernd Schnabl
Alcohol-associated liver disease (ALD) is a major global health issue, contributing significantly to morbidity and mortality worldwide. Among the ALD subtypes, alcohol-associated hepatitis poses a severe and urgent medical challenge with high short-term mortality rates. Despite extensive research, the current therapeutic approaches for alcohol-associated hepatitis have limited efficacy, necessitating novel interventions. Recent studies have highlighted the crucial role of the gut microbiota in ALD pathogenesis, particularly Enterococcus faecalis (E. faecalis) and its cytolysin exotoxin. This study presents the development of a standardized real-time quantitative polymerase chain reaction (RT-qPCR) assay to detect and quantify cytolysin in fecal samples from patients with alcohol-associated hepatitis. The diagnostic assay allows for an association analysis between cytolysin-positive E. faecalis and disease severity as well as mortality. This assay was developed to standardize the identification of cytolysin-positive patients who can be selected for clinical trials.
]]>Methods and Protocols doi: 10.3390/mps6060106
Authors: Dong Woo Lim Tae-Sung Yoon Kyung Ho Han Saba Sajjad Heung-Seon Shin Sunghyun Kang
The horizontal flatbed electrophoresis method is employed to separate protein samples, providing greater flexibility for various electrophoretic applications and easier sample loading compared to its vertical counterpart. In the currently available equipment setup, cathode and anode electrodes are positioned on top of a gel at each end. Since an electric field enters the gel from the top, its strength gradually weakens from the top to the bottom of the gel. When examining the interior of gels following electrophoretic separation, the uneven electric field causes the protein bands to lie down forward in the direction of migration, leading to an increase in bandwidth. This issue has remained unaddressed for several decades. To address this problem, new clamp-shaped and double-deck electrodes were developed to apply an electric field simultaneously from both the top and bottom of the gel. Both of these new electrodes facilitated the formation of perpendicular protein band shapes and enhanced resolution at a comparable level. Due to their ease of use, double-deck electrodes are recommended. By combining these new electrodes with the field inversion gel electrophoresis (FIGE) technique, the protein bands could be focused and aligned nearly vertically, resulting in the highest level of electrophoretic resolution. Our electrodes are compatible with polyacrylamide gels of varying sizes, buffer systems, and sample well formats. They can be easily manufactured and seamlessly integrated into existing laboratory instruments for practical use.
]]>Methods and Protocols doi: 10.3390/mps6060105
Authors: Yeison Alberto Garcés-Gómez Sebastián Isaac Pacheco-Gonzalez
This research evaluates extracts from the bark of Heliocarpus popayanensis and Triumfetta bogotensis as coagulating agents for removing turbidity in domestic wastewater, considering the coagulant dosage and pH of the wastewater. ANOVA was conducted to assess differences between the coagulants, dosages, and pH, with three pH levels (5, 8, and 9) and six dosages (7, 9, 11, 13, 15, and 17 mL per 1000 mL of wastewater) at a significance level of α = 0.05, and both the p-value and effect size were evaluated. This study found that the mucilaginous compound from the bark of Triumfetta bogotensis performed better in reducing turbidity levels, with an average reduction of 30.2 NTU (Nephelometric Turbidity Unit) (CI [25.9 NTU; 34.5 NTU], α = 0.05) at a pH of 5, and an average initial NTU of 102.2. This represents an average reduction of 70.45%. The dosage factor did not show significant effects on turbidity reduction, which opens the possibility for further study to determine the optimal dosage of the best coagulant.
]]>Methods and Protocols doi: 10.3390/mps6060104
Authors: Daniélle van Staden Richard K. Haynes Frank Van der Kooy Joe M. Viljoen
We describe the development and validation of a new high performance liquid chromatography (HPLC) method for analysis of a combination of the first-line anti-tubercular drugs isoniazid, pyrazinamide, and rifampicin together with clofazimine. This is a unique challenge since clofazimine and rifampicin are relatively highly lipophilic drugs, whereas isoniazid and pyrazinamide are considerably more hydrophilic. Thus, clear separation of peaks and quantification of four individual drugs can present difficulties during the development of an analytical method. Detection was established at two wavelengths—254 nm for isoniazid and pyrazinamide and 320 nm for clofazimine and rifampicin. Gradient elution was employed using 0.1% aqueous formic acid (A) and acetonitrile (B); clear separation of the four drugs was achieved within 10 min. A linear relationship was indicated by a correlation coefficient (r2) of 0.9999 for each anti-tubercular drug, respectively. The limit of detection (LOD) for the individual drugs was 0.70 µg/mL (isoniazid), 0.30 µg/mL (pyrazinamide), 0.20 µg/mL (rifampicin) and 0.20 µg/mL (clofazimine). Precision experiments rendered a mean recovery percentage of 101.25% (isoniazid), 98.70% (pyrazinamide), 99.68% (rifampicin) and 97.14% (clofazimine). This HPLC method was validated and is reliable, repeatable, and accurate for the purpose of conducting simultaneous HPLC analyses of the four anti-tubercular drugs.
]]>Methods and Protocols doi: 10.3390/mps6050103
Authors: Yvonne C. Hornby-Turner Sarah G. Russell Rachel Quigley Veronica Matthews Sarah Larkins Noel Hayman Prabha Lakhan Leon Flicker Kate Smith Dallas McKeown Diane Cadet-James Alan Cass Gail Garvey Dina LoGiudice Gavin Miller Edward Strivens
This protocol describes the methodology and methods for a collaborative project with eight Aboriginal and Torres Strait Islander primary health care (PHC) organisations, across three Australian states and one territory, to increase clinical service performance and access to preventive health and health promotion services for preventing, identifying, treating, and managing dementia risk in Aboriginal and Torres Strait Islander communities. Aboriginal participatory action research (APAR) methodology will be the framework for this project, incorporating continuous quality improvement (CQI), informed by research yarning with stakeholder groups, comprising community members and PHC staff and service providers and data collected from the auditing of client health records and the mapping of existing clinical processes and health services at each partnering PHC organisation. The qualitative and quantitative data will be summarised and discussed with stakeholder groups. Priorities will be identified and broken down into tangible PHC organisation deliverable strategies and programs, which will be co-developed with stakeholder groups and implemented cyclically over 24 months using the Plan, Do, Study, Act model of change. Key project outcome measures include increased clinical service performance and availability of preventive health and health promotion services for safeguarding against dementia. Project implementation will be evaluated for quality and transparency from an Indigenous perspective using an appropriate appraisal tool. The project processes, impact, and sustainability will be evaluated using the RE-AIM framework. A dementia safeguarding framework and accompanying tool kit will be developed from this work to support Aboriginal and Torres Strait Islander PHC organisations to identify, implement, and evaluate dementia safeguarding practice and service improvements on a broader scale.
]]>Methods and Protocols doi: 10.3390/mps6050102
Authors: Miguel A. Rivera-Morán José G. Sampedro
The sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) is a membrane protein that is destabilized during purification in the absence of calcium ions. The disaccharide trehalose is a protein stabilizer that accumulates in the yeast cytoplasm when under stress. In the present work, SERCA was purified by including trehalose in the purification protocol. The purified SERCA showed high protein purity (~95%) and ATPase activity. ATP hydrolysis was dependent on the presence of Ca2+ and the enzyme kinetics showed a hyperbolic dependence on ATP (Km = 12.16 ± 2.25 μM ATP). FITC labeling showed the integrity of the ATP-binding site and the identity of the isolated enzyme as a P-type ATPase. Circular dichroism (CD) spectral changes at a wavelength of 225 nm were observed upon titration with ATP, indicating α-helical rearrangements in the nucleotide-binding domain (N-domain), which correlated with ATP affinity (Km). The presence of Ca2+ did not affect FITC labeling or the ATP-mediated structural changes at the N-domain. The use of trehalose in the SERCA purification protocol stabilized the enzyme. The isolated SERCA appears to be suitable for structural and ligand binding studies, e.g., for testing newly designed or natural inhibitors. The use of trehalose is recommended for the isolation of unstable enzymes.
]]>Methods and Protocols doi: 10.3390/mps6050101
Authors: Sandesh Pantha Martin Jones Pragya Gartoulla Richard Gray
Concept mapping is a phased, mixed-method approach that is increasingly used in health research to develop an understanding of complex phenomena. The six phases of concept mapping are preparation, idea generation, structuring (clustering and prioritization), data analysis, interpretation, and utilization of the map. The reporting of concept mapping research requires the development of a specific reporting guideline. We conducted a systematic review to identify candidate reporting items for inclusion in a reporting guideline. Three databases (MEDLINE, CINAHL, and PsycInfo) were searched to identify studies that used concept mapping methodology. We included 75 concept mapping studies published since 2019 from which we extracted information about the quality of reporting. A third of the studies focused on public health. We identified 71 candidate items that relate to the quality of reporting concept mapping research. The rationale for the study, the focus prompt, procedures for brainstorming, and structuring statements were consistently reported across the included studies. The process for developing the focus prompt, the rationale for the size of the stakeholder groups, and the process for determining the final concept map were generally not reported. The findings from the review will be used to inform the development of our reporting guideline for concept mapping research.
]]>Methods and Protocols doi: 10.3390/mps6050100
Authors: Kirah Jones Ioannis Eleftherianos
Drosophila melanogaster is an excellent model for dissecting innate immune signaling and functions. Humoral and cellular immune mechanisms in the fly take place in the hemolymph, where host defense components are secreted and act in response to microbial invaders. Studying hemolymph factors is critical for understanding the regulation of the host’s antimicrobial immune system. Therefore, methods for extracting the fly hemolymph efficiently and in sufficient quantities are essential for isolating and characterizing immune proteins and peptides. Here, we describe a novel and simple hemolymph isolation protocol for single D. melanogaster male and female adults. This procedure substantially improves the already used technique and allows fly immunologists to explore innate immune hemolymph activity in D. melanogaster individuals.
]]>Methods and Protocols doi: 10.3390/mps6050099
Authors: Céline Clément Denis Bourgeois Flavia Vitiello Herve Perrier Ariane Tabary Florence Carrouel
Hormonal changes and physiological alterations in pregnancy increase the susceptibility of the woman to oral diseases such as plaque-induced gingivitis. In individual oral prophylaxis, effective tooth brushing can reduce gingival inflammation. Therefore, it is necessary to update the scientific evidence to identify which type of toothbrush, manual or sonic-powered, is most effective in reducing the incidence of gingivitis in pregnant women. The aim of this clinical trial is to compare the biofilm control effectiveness of two manual and two sonic toothbrushes in pregnant women. This study is designed as a four-arm, parallel, randomized controlled trial with an allocation ratio of 1:1:1:1. The pregnant woman will be included at 15–18 weeks of amenorrhea and followed for 3 months. The primary outcome will be the change in the incidence of gingival bleeding from a baseline and various follow-up periods of the study. Secondary outcomes measures will be to compare clinical effects of the toothbrushes tested on (i) gingival inflammation, (ii) dental plaque, (iii) gingival attachment and (iv) periodontal pocket; and to assess toothbrush acceptability. Thus, identifying the best device for effective tooth brushing in pregnancy could be helpful in reducing and improving the incidence of gingival inflammation.
]]>Methods and Protocols doi: 10.3390/mps6050098
Authors: Mahsa Pouranayatihosseinabad Maggie Taylor Jason Hawrelak Gregory M. Peterson Felicity Veal Tristan Ling Mackenzie Williams Megan Whatley Kyan Ahdieh Corinne Mirkazemi
Limited epidemiological evidence suggests a link between antibiotic use and developing depression. This study seeks to investigate this association in depth, using a cohort of pregnant individuals. The primary aim is to explore any association between the use of antibiotics during pregnancy and the development of antenatal depressive symptoms up to the third trimester, as well as the use of antibiotics during pregnancy and within 12 months postpartum and the development of postpartum depressive symptoms. A national prospective, observational, longitudinal cohort study has been designed to examine these relationships. A sample size of 1500 pregnant individuals has been sought for this study, assuming 10 potential predictor variables (including antibiotic use) in the final multiple logistic regression model and allowing for a 30% drop-out rate. The development of depressive symptoms is considered either a diagnosis by a medical doctor and/or a scoring 13 or higher on the Edinburgh Postnatal Depression Scale. Data will be collected during the third trimester and at 6 weeks, 6 months, and 12 months postpartum. These surveys include variables previously identified as associated with antenatal and postpartum depression (e.g., level of social support, experience of intimate partner abuse, and obstetric complications), as well as antibiotic and probiotic use. This study will provide an update on the prevalence of the symptoms of depression during pregnancy and postpartum and its associated risk factors. It will also, for the first time, comprehensively explore the potential association between antibiotic use during pregnancy and up to 12 months postpartum and the development of depressive symptoms.
]]>Methods and Protocols doi: 10.3390/mps6050097
Authors: Andreea Creteanu Gabriela Lisa Cornelia Vasile Maria-Cristina Popescu Adrian Florin Spac Gladiola Tantaru
In various drug delivery systems, solid lipid nanoparticles are dominantly lipid-based nanocarriers. Amiodarone hydrochloride is an antiarrhythmic agent used to treat severe rhythm disturbances. It has variable and hard-to-predict absorption in the gastrointestinal tract because of its low solubility and high permeability. The aims of this study were to improve its solubility by encapsulating amiodarone into solid lipid nanoparticles using two excipients—Compritol® 888 ATO (pellets) (C888) as a lipid matrix and Transcutol® (T) as a surfactant. Six types of amiodarone-loaded solid lipid nanoparticles (AMD-SLNs) were obtained using a hot homogenization technique followed by ultrasonication with varying sonication parameters. AMD-SLNs were characterized by their size distribution, polydispersity index, zeta potential, entrapment efficiency, and drug loading. Based on the initial evaluation of the entrapment efficiency, only three solid lipid nanoparticle formulations (P1, P3, and P5) were further tested. They were evaluated through scanning electron microscopy, Fourier-transform infrared spectrometry, near-infrared spectrometry, thermogravimetry, differential scanning calorimetry, and in vitro dissolution tests. The P5 formulation showed optimum pharmaco-technical properties, and it had the greatest potential to be used in oral pharmaceutical products for the controlled delivery of amiodarone.
]]>Methods and Protocols doi: 10.3390/mps6050096
Authors: Nontaporn Rattanachak Sattaporn Weawsiangsang Robert A. Baldock Theerasak Jaifoo Touchkanin Jongjitvimol Jirapas Jongjitwimol
The rise of multidrug resistance of Pseudomonas aeruginosa highlights an increased need for selective and precise antimicrobial treatment. Drug efflux pumps are one of the major mechanisms of antimicrobial resistance found in many bacteria, including P. aeruginosa. Detection of efflux genes using a polymerase chain reaction (PCR)-based system would enable resistance detection and aid clinical decision making. Therefore, we aimed to develop and optimize a novel method herein referred to as “effluxR detection assay” using multiplex digital PCR (mdPCR) for detection of mex efflux pump genes in P. aeruginosa strains. The annealing/extension temperatures and gDNA concentrations were optimized to amplify mexB, mexD, and mexY using the multiplex quantitative PCR (mqPCR) system. We established the optimal mqPCR conditions for the assay (Ta of 59 °C with gDNA concentrations at or above 0.5 ng/µL). Using these conditions, we were able to successfully detect the presence of these genes in a quantity-dependent manner. The limit of detection for mex genes using the effluxR detection assay with mdPCR was 0.001 ng/µL (7.04–34.81 copies/µL). Moreover, using blind sample testing, we show that effluxR detection assay had 100% sensitivity and specificity for detecting mex genes in P. aeruginosa. In conclusion, the effluxR detection assay, using mdPCR, is able to identify the presence of multiple mex genes in P. aeruginosa that may aid clinical laboratory decisions and further epidemiological studies.
]]>Methods and Protocols doi: 10.3390/mps6050095
Authors: Ana Castiñeira-Landeira Lua Vazquez Thierry Dagnac Maria Celeiro María Llompart
Hydroalcoholic gels or hand sanitisers have become essential products to prevent and mitigate the transmission of COVID-19. Depending on their use, they can be classified as cosmetics (cleaning the skin) or biocides (with antimicrobial effects). The aim of this work was to determine sixty personal care products frequently found in cosmetic formulations, including fragrance allergens, synthetic musks, preservatives and plasticisers, in hydroalcoholic gels and evaluate their compliance with the current regulation. A simple and fast analytical methodology based on solid-phase microextraction followed by gas chromatography–tandem mass spectrometry (SPME-GC-MS/MS) was validated and applied to 67 real samples. Among the 60 target compounds, 47 of them were found in the analysed hand sanitisers, highlighting the high number of fragrance allergens (up to 23) at concentrations of up to 32,458 μg g−1. Most of the samples did not comply with the labelling requirements of the EU Regulation No 1223/2009, and some of them even contained compounds banned in cosmetic products such as plasticisers. Method sustainability was also evaluated using the metric tool AGREEPrep, demonstrating its greenness.
]]>Methods and Protocols doi: 10.3390/mps6050094
Authors: Irida Papapostolou Florian Bochen Christine Peinelt Maria Constanza Maldifassi
Although 2D in vitro cancer cell cultures have been used for decades as a first line-of-research tool to investigate antitumoral drugs and treatments, their use presents many drawbacks, including the poor resemblance of such cultures to the characteristics of in vivo tumors. To mitigate these drawbacks, 3D culture models have emerged as a more representative alternative. Cancer cells cultured as 3D structures have the advantage of resembling solid tumors in their architecture and in their resistance to chemotherapeutic drugs, in part because of restrained drug penetration. Additionally, these 3D structures create a more physiological environment for the study of immune cell invasion and migration, comparable to solid tumors. In this paper, we describe a fast and cost-effective step-by-step protocol for the generation of 3D spheres using ultra-low-attachment (ULA) multiwell plates, which can be incorporated into the normal workflow of any laboratory. Using this protocol, spheroids of different human cancer cell lines can be obtained and can then be characterized on the basis of their morphology, viability, and expression of specific markers.
]]>Methods and Protocols doi: 10.3390/mps6050093
Authors: Thasmin Shahjahan Bilal Javed Vinayak Sharma Furong Tian
The aim of this research is to define optimal conditions to improve the stability of gold and silver nanoparticles’ anti-zearalenone antibody conjugates for their utilisation in lateral flow immunochromatographic assay (LFIA). The Turkevich–Frens method was used to synthesise gold nanoparticles (AuNPs), which were between 10 and 110 nm in diameter. Silver nanoparticles (AgNPs) with a size distribution of 2.5 to 100 nm were synthesised using sodium borohydride as a reducing agent. The onset of AuNP and AgNP aggregation occurred at 150 mM and 80 mM NaCl concentrations, respectively. Stable Au and Ag nanoparticle–antibody conjugates were achieved at 1.2 mM of K2CO3 concentration, which corresponds to the pH value of ≈7. Lastly, the highest degree of conjugation between Au and Ag nanoparticles and anti-zearalenone antibodies was at 4 and 6 µg/mL of antibody concentrations. The optimisation of the conjugation conditions can contribute to better stability of nanoparticles and their antibody conjugate and can improve the reproducibility of results of bioreporter molecules in biosensing lateral flow devices.
]]>Methods and Protocols doi: 10.3390/mps6050092
Authors: Quinn LaFave Shalini P. Etukuri Chaney L. Courtney Neha Kothari Trevor W. Rife Christopher A. Saski
Recent advances in phenotyping techniques have substantially improved the ability to mitigate type-II errors typically associated with high variance in phenotyping data sets. In particular, the implementation of automated techniques such as the High-Volume Instrument (HVI) and the Advanced Fiber Information System (AFIS) have significantly enhanced the reproducibility and standardization of various fiber quality measurements in cotton. However, micronaire is not a direct measure of either maturity or fineness, lending to limitations. AFIS only provides a calculated form of fiber diameter, not a direct measure, justifying the need for a visual-based reference method. Obtaining direct measurements of individual fibers through cross-sectional analysis and electron microscopy is a widely accepted standard but is time-consuming and requires the use of hazardous chemicals and specialized equipment. In this study, we present a simplified fiber histology and image acquisition technique that is both rapid and reproducible. We also introduce an automated image analysis program that utilizes machine learning to differentiate good fibers from bad and to subsequently collect critical phenotypic measurements. These methods have the potential to improve the efficiency of cotton fiber phenotyping, allowing for greater precision in unravelling the genetic architecture of critical traits such as fiber diameter, shape, areas of the secondary cell wall/lumen, and others, ultimately leading to larger genetic gains in fiber quality and improvements in cotton.
]]>Methods and Protocols doi: 10.3390/mps6050091
Authors: Alexandra M. Bodnaruc Miryam Duquet Denis Prud’homme Isabelle Giroux
The aim of the proposed scoping review is to describe and summarize studies assessing the associations between diet-related variables and depression in peri- and post-menopausal women. Studies examining the associations between diet-related variables and mental health indicators in women undergoing menopausal transition or in the post-menopausal period will be systematically retrieved via Medline, EMBASE, PsycINFO, Web of Science, and Scopus databases. All articles identified through the database searches will be imported into Covidence. Following the removal of duplicates, two authors will independently perform title and abstract screening, as well as full-text assessment against eligibility criteria. Data will be extracted using tables developed for observational and experimental studies. The methodological quality of randomized trials, cohort and cross-sectional studies, and case–control studies, will be assessed using the Cochrane risk-of-bias (RoB-2) tool, the NHLBI Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies, and the NHLBI Quality Assessment Tool for Case–Control studies, respectively. Data extraction tables will be used to produce two tables summarizing the main characteristics and findings of the studies included in the review. In the proposed review, we will systematically identify and summarize the currently available evidence on the association between diet-related variables and depression in peri- and post-menopausal women. To our knowledge, this is the first review focusing on this subgroup of the population. Protocol registration: osf.io/b89r6.
]]>Methods and Protocols doi: 10.3390/mps6050090
Authors: Hiroshi Ueda Hee-Jin Jeong
Development of a rapid detection method for deoxycholic acid (DCA) is crucial for its diagnosis in the early stages of inflammation and cancer. In this study, we expressed a soluble recombinant anti-DCA single-chain variable fragment (scFv) in Escherichia coli. To convert scFv into a Quenchbody (Q-body), we labeled scFv using commercially available maleimide-linked fluorophores. The TAMRA-C5-maleimide-conjugated Q-body showed the highest response within a few minutes of DCA addition, indicating its applicability as a wash-free immunoassay probe for onsite DCA detection.
]]>Methods and Protocols doi: 10.3390/mps6050089
Authors: Mira Loock Luiza Berenguer Antunes Rhiannon T Heslop Antonio Alfonso De Lauri Andressa Brito Lira Igor Cestari
Saccharomyces cerevisiae is a powerful system for the expression of genome-wide or combinatorial libraries for diverse types of screening. However, expressing large libraries in yeast requires high-efficiency transformation and controlled expression. Transformation of yeast using electroporation methods is more efficient than chemical methods; however, protocols described for electroporation require large amounts of linearized plasmid DNA and often yield approximately 106 cfu/µg of plasmid DNA. We optimized the electroporation of yeast cells for the expression of whole-genome libraries to yield up to 108 cfu/µg plasmid DNA. The protocol generates sufficient transformants for 10–100× coverage of diverse genome libraries with small amounts of genomic libraries (0.1 µg of DNA per reaction) and provides guidance on calculations to estimate library size coverage and transformation efficiency. It describes the preparation of electrocompetent yeast cells with lithium acetate and dithiothreitol conditioning step and the transformation of cells by electroporation with carrier DNA. We validated the protocol using three yeast surface display libraries and demonstrated using nanopore sequencing that libraries’ size and diversity are preserved. Moreover, expression analysis confirmed library functionality and the method’s efficacy. Hence, this protocol yields a sufficient representation of the genome of interest for downstream screening purposes while limiting the amount of the genomic library required.
]]>Methods and Protocols doi: 10.3390/mps6050088
Authors: Lauren J. Klein John Benaiah Ayete-Nyampong Annette M. Williams Lori A. Harding Samuel A. Oppong Sari Acra Michael R. DeBaun Aamer Imdad
In pregnancies complicated by sickle cell disease (SCD), the maternal-fetal dyad is at high risk for mortality and morbidity. In healthy pregnancies, maternal nutritional status is a critical factor for the healthy growth and development of the fetus. However, there are no reviews of the current research on the nutritional status of pregnant women with SCD and pregnancy outcomes. First, we aim to assess the burden of malnutrition in pregnant women with SCD. Next, we aim to systematically evaluate if pregnant women with SCD who have poor nutritional status are at increased risk for adverse birth outcomes compared to pregnant women with sickle cell disease and normal nutritional status. We will systematically search multiple electronic databases. Our exposure is pregnant women with SCD and poor nutritional status. The primary outcomes of interest include low birth weight (categorical) and birth weight z-scores (continuous). We will also evaluate maternal and perinatal outcomes as secondary outcomes. We will evaluate the risk of bias and overall certainty of evidence with Risk of Bias in Non-randomized Studies—of Interventions (ROBINS-I), and the overall evidence will be assessed using Grading of Recommendation Assessment, Development, and Evaluation (GRADE) criteria. We will pool findings with a meta-analysis if sufficient homogeneity exists among studies. Findings will be published in a peer-reviewed journal and disseminated to SCD advocacy groups. PROSPERO registration number: CRD42023429412.
]]>Methods and Protocols doi: 10.3390/mps6050087
Authors: Stefan Balko Evan Kerr Edward Buchel Sarvesh Logsetty Afshin Raouf
The scratch assay is an in vitro assay that allows for high-throughput quantification of wound closure by keratinocytes and fibroblasts with relative ease. However, this assay is amenable to experimental variables, which can result in false-positive and false-negative data, making the interpretation of such data difficult. Also, data variability decreases the sensitivity of the scratch assay. Here, we identify important sources of data variation in the scratch assay and provide rational mitigation strategies that enable robust and highly reproducible quantification of scratch width and area, and ultimately the scratch closure rates. By eliminating these sources of variability, the sensitivity of the scratch assay is enhanced, thereby allowing for identification of dependent variables with wide-ranging impacts on wound closure in a robust and standardized manner.
]]>Methods and Protocols doi: 10.3390/mps6050086
Authors: Reinhard Vonthein Benito Baldauf Stefan Borov Ernest Lau Marzia Giaccardi Roberto Cemin Ojan Assadian Philippe Chévalier Kerstin Bode Hendrik Bonnemeier
Background: Cardiac implantable electronic device (CIED) placement comes with certain complications. CIED infection is a severe adverse event related to CIED placement. In randomised controlled trials, the preoperative intravenous administration of antibiotics and the adjunctive use of an antibiotic mesh envelope resulted in significant reduction in infections related to cardiac implantable electronic devices. The adjunctive use of taurolidine for this purpose is relatively novel and not considered in the guidelines. The required evidence may consist of a set of clinical studies. Methods: The European TauroPaceTM registry (ETPR) prospectively evaluates every consecutive invasive procedure involving any CIED with adjunct TauroPace™ use in the contributing centres. As the estimation of the infection rate needs to be defensible, only interventions registered prior to the procedure will be followed-up. The endpoint is a major cardiac implantable electronic device infection according to the novel CIED infection criteria (1). Secondary endpoints comprise all-cause mortality, complications, adverse events of all grades, and major CIED infections during all follow-up examinations. The follow-up times are three months, twelve months, and eventually 36 months, as acute, subacute, and long-term CIED infections are of interest. Results: As the rate of CIED infections is expected to be very low, this registry is a multicentre, international project that will run for several years. Several reports are planned. The analyses will be included in the case number calculations for future randomised controlled trials. Conclusions: The ETPR will accumulate large case numbers to estimate small event rates more precisely; we intend to follow up on participants for years to reveal possible late effects.
]]>Methods and Protocols doi: 10.3390/mps6050085
Authors: Mukhethwa Londani Olanrewaju Oladimeji
Tobacco use, particularly the initiation of smoking during adolescence and young adulthood, represents a significant public health concern in South Africa. The influence of socio-cultural factors, marketing strategies of the tobacco industry, and accessibility of tobacco products have all been implicated in this context. This systematic review and meta-analysis protocol aims to scrutinise the body of literature on this issue, providing a comprehensive understanding of the patterns and determinants of tobacco use among South African adolescents and young adults, with an eye towards informing more effective policy interventions. The available literature for studies on tobacco use will be systematically searched and reviewed. Five international scholarly databases, namely PubMed, MEDLINE, EMBASE, Global Health, and Scopus, will be searched. Peer-reviewed studies will be included if they are conducted in South Africa or South African provinces and if they include the prevalence of tobacco use among adolescents and young adults aged between 12 and 24 years. The results of such an analysis can guide future policy designs, enabling them to be more targeted and thus more effective. The findings can also have implications for shaping global tobacco control strategies, given the transferability of successful interventions across different populations and cultural contexts. This protocol has been registered in the PROSPERO database (ID: CRD42023428369).
]]>Methods and Protocols doi: 10.3390/mps6050084
Authors: Yazan Ranneh Abdulmannan Fadel Abdah Md Akim Iskandar Idris Bolaji Lilian Ilesanmi-Oyelere Leila Cheikh Ismail
Introduction: Metabolic endotoxemia (ME) is the main cause of sub-clinical chronic inflammation, which subsequently triggers the onset of several chronic diseases. However, recent reports have indicated that dietary fiber (DF) contributes significantly to ameliorating ME and inflammation. This protocol aims to provide an outline of all procedures in synthesizing the available data on the effect of DF against ME. Methods: Following the PRISMA 2020 guidelines for preparing protocols, this protocol was registered in the International Prospective Registry of Systematic Reviews (PROSPERO) with registration number (CRD42023417833). In this review, we specifically focused on the inclusion of clinical trials that met the following criteria: they were published or available as preprints, employed random, quasi-random, or cross-over designs, and were exclusively documented in the English language. Clinical medical subject headings (MeSH) as search terms were used on prominent databases such as MEDLINE, COCHRANE library, PubMed, World Health Organization International Clinical Trials Registry Platforms, and US National Institutes of Health Ongoing Trials Register Clinicaltrials.gov. Results and discussion: This protocol will guide the exploration of articles that report changes in ME biomarkers in subjects supplemented with DF. The findings of this protocol will ensure a comprehensive evaluation of available evidence, provide a quantitative summary, identify patterns and trends, enhance statistical power, and address heterogeneity, which collectively will clarify the optimal types, doses, and duration of DF interventions for managing ME and low-grade inflammation. Ethics and dissemination: The quantitative data of clinical trials will be collected, and a meta-analysis will be performed using RevMan V.5.3 software. Therefore, no ethical approval is required.
]]>Methods and Protocols doi: 10.3390/mps6050083
Authors: Jai K. Das Amira Khan Farhana Tabassum Zahra Ali Padhani Atif Habib Mushtaq Mirani Abdu R. Rahman Zahid Ali Khan Arjumand Rizvi Imran Ahmed Zulfiqar Bhutta
Poliomyelitis is a condition of great concern and is endemic in only two countries of the world: Pakistan and Afghanistan. Community mobilization plays a vital role in raising awareness and can help reduce polio vaccine refusals. The objective of this study will be to decrease polio vaccine refusals and zero-dose vaccines by motivating behavior change through the provision of conditional–collective–community-based incentives (C3Is) based on a reduction in polio vaccine refusals. The project will adopt a pretest/post-test quasi-experimental design with two intervention high-risk union councils (HRUCs) and two control union councils (UCs) of peri-urban (Karachi) and rural (Bannu) settings in Pakistan. A participatory community engagement and demand creation strategy with trust-building community mobilization with C3Is, to reduce vaccine refusals and improve polio immunization coverage in two HRUCs, will be used. These UCs will be divided into clusters based on the polio program framework and community groups will be formed in each cluster. These community groups will carry out awareness activities and will be given serial targets to reduce vaccine refusals and those who qualify will be provided C3Is. The project intends to create a replicable model that the government can integrate within health systems for long-term sustainability until the goal of eradication of poliovirus is achieved. The evaluation will be carried out by an independent data collection and analysis team at baseline and endline (after 12 months of intervention). The trial is registered with linicalTrials.gov with number NCT05721274.
]]>Methods and Protocols doi: 10.3390/mps6050082
Authors: Tanya Román Gerardo Acosta Beatriz G. de la Torre Constanza Cárdenas Fanny Guzmán Fernando Albericio
Used in solid-phase peptide synthesis (SPPS) for peptides with an acid termination, the 2-chlorotrityl chloride (2-CTC) resin is highly susceptible to moisture, leading to reduced resin loading and lower synthetic yields. It is therefore recommended that the resin be activated with thionyl chloride (SOCl2) before peptide assembly. Here we present an optimized procedure for resin activation that minimizes the use of SOCl2 as the activation reagent and reduces the activation time. Additionally, we demonstrate the feasibility of reusing the 2-CTC resin when following the activation protocol, achieving comparable results to the first usage of the resin. Moreover, we achieved different degrees of resin activation by varying the amount of SOCl2. For instance, the use of 2% SOCl2 in anhydrous dichloromethane (DCM) allowed up to 44% activation of the resin, thereby making it suitable for the synthesis of longer peptides. Alternatively, employing 25% SOCl2 in anhydrous DCM resulted in up to 80% activation with a reaction time of only 5 min in both cases.
]]>Methods and Protocols doi: 10.3390/mps6050081
Authors: Wenchao Zhu Mubasher Hussain Jing Gao Runqian Mao Xincheng An
The black soldier fly (BSF) is well known for its ability to biologically convert organic waste into insect biomass, including protein and oil, which can be utilised as animal feed. Since raw BSF products, such as BSF powder, are difficult to differentiate from other biological raw materials, therefore new analytical approaches are required. In this study, we have developed a new and fast method based on loop-mediated isothermal AMPlification (LAMP) reaction that can diagnose black soldier fly larvae and BSF byproducts with high accuracy, specificity and sensitivity. Species-specific primers for BSF were designed based on targeting the mitochondrial cytochrome C oxidase I (COI) gene. The assay was able to detect as low as 820 fg/L of BSF DNA in 60 min at 65 °C, which was a hundredfold higher than the detection limit of classical polymerase chain reaction and did not show cross-reactivity. In conclusion, the LAMP assay demonstrated excellent sensitivity and specificity to detect BSF and BSF byproducts, with a sampling-to-result identification time of 60 min.
]]>Methods and Protocols doi: 10.3390/mps6050080
Authors: Kangfu Chen Zongjie Wang
Advancements in single-cell-related technologies have opened new possibilities for analyzing rare cells, such as circulating tumor cells (CTCs) and rare immune cells. Among these techniques, single-cell proteomics, particularly single-cell mass spectrometric analysis (scMS), has gained significant attention due to its ability to directly measure transcripts without the need for specific reagents. However, the success of single-cell proteomics relies heavily on efficient sample preparation, as protein loss in low-concentration samples can profoundly impact the analysis. To address this challenge, an effective handling system for rare cells is essential for single-cell proteomic analysis. Herein, we propose a microfluidics-based method that offers highly efficient isolation, detection, and collection of rare cells (e.g., CTCs). The detailed fabrication process of the micropillar array-based microfluidic device is presented, along with its application for CTC isolation, identification, and collection for subsequent proteomic analysis.
]]>Methods and Protocols doi: 10.3390/mps6050079
Authors: Alexander G. Litov Egor V. Okhezin Ivan S. Kholodilov Alexandra E. Polienko Galina G. Karganova
The recently discovered Jingmenvirus group includes viruses with a segmented genome, RNA of a positive polarity, and several proteins with distant homology to the proteins of the members of the genus Orthoflavivirus. Some Jingmenvirus group members, namely the Alongshan virus (ALSV) and Jingmen tick virus, are reported to be tick-borne human pathogens that can cause a wide variety of symptoms. The ALSV is widely distributed in Eurasia, yet no reliable assay that can detect it exists. We describe a qPCR system for ALSV detection. Our data showed that this system can detect as little as 104 copies of the ALSV in a sample. The system showed no amplification of the common tick-borne viruses circulating in Eurasia, i.e., the Yanggou tick virus—which is another Jingmenvirus group member—or some known members of the genus Orthoflavivirus. The qPCR system was tested and had no nonspecific signal for the Ixodes ricinus, I. persulcatus, Dermacentor reticulatus, D. marginatus, Haemaphysalis concinna, and H. japonica ticks. The qPCR system had no nonspecific signal for human and sheep serum as well. Overall, the qPCR system described here can be used for reliable and quantitative ALSV detection.
]]>Methods and Protocols doi: 10.3390/mps6050078
Authors: Alexandra M. Bodnaruc Coralie Vincent Carolina Soto Miryam Duquet Denis Prud’homme Isabelle Giroux
Our objectives are to perform (1) an umbrella review on diet and depression, (2) a systematic review update on dietary patterns and depression, and (3) updated meta-analyses using studies from the previous two objectives. Systematic reviews examining the relationships between diet and depression and primary studies on the relationship between dietary patterns and depression will be systematically retrieved via several databases. All articles identified through the database searches will be imported into Covidence. Following duplicates removal, two authors will independently perform title and abstract screening and full-text assessment against eligibility criteria. Data will be extracted using tables developed for both systematic reviews and primary studies. The methodological quality of systematic reviews will be assessed using the AMSTAR-2 tool. The risk of bias in randomized trials, cohort and cross-sectional studies, as well as case-control studies, will be assessed with the Cochrane risk-of-bias (RoB-2) tool, the NHLBI Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies, and the NHLBI Quality Assessment Tool for Case-Control studies, respectively. For each dietary variable, data extracted will be used to produce: (1) a summary of systematic reviews’ characteristics and results table, (2) a summary of the primary studies characteristics table, (3) a qualitative summary of results from the primary studies table, and (4) a quantitative summary of results in the form of forest plots. The certainty of evidence will be assessed using the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) approach. Upon completion, this systematic review will be the most comprehensive and up-to-date synthesis of currently available evidence on the relationships between diet and depression. It will serve as a key reference to guide future research and as a resource for health professionals in the fields of nutrition and psychiatry. PROSPERO CRD42022343253.
]]>Methods and Protocols doi: 10.3390/mps6050077
Authors: Lamonielli F. Michaliski Laura P. Ióca Leandro S. Oliveira Camila M. Crnkovic Mirelle Takaki Vitor F. Freire Roberto G. S. Berlinck
Fungi are well-known producers of chemically diverse and biologically active secondary metabolites. However, their production yields through fermentation may hamper structural analysis and biological activity downstream investigations. Herein, a systematic experimental design that varies multiple cultivation parameters, followed by chemometrics analysis on HPLC-UV-MS or UHPLC-HRMS/MS data, is presented to enhance the production yield of fungal natural products. The overall procedure typically requires 3–4 months of work when first developed, and up to 3 months as a routine procedure.
]]>Methods and Protocols doi: 10.3390/mps6050076
Authors: Jonathan Sinclair Stephanie Dillon Robert Allan Johanne Brooks-Warburton Terun Desai Charlotte Lawson Lindsay Bottoms
Ulcerative colitis, characterized by its relapsing and remissive nature, negatively affects perception, body image, and overall quality of life. The associated financial burden underscores the need for alternative treatment approaches with fewer side effects, alongside pharmaceutical interventions. Montmorency tart cherries, rich in anthocyanins, have emerged as a potential natural anti-inflammatory agent for ulcerative colitis. This manuscript outlines the study protocol for a randomized placebo-controlled trial investigating the effects of Montmorency tart cherry in individuals with ulcerative colitis. The trial aims to recruit 40 participants with mild to moderate disease activity randomly assign them to either a Montmorency tart cherry or placebo group. The intervention will span 6 weeks, with baseline and 6-week assessments. The primary outcome measure is the Inflammatory Bowel Disease Quality of Life Questionnaire. Secondary outcomes include other health-related questionnaires and biological indices. Statistical analysis will adhere to an intention-to-treat approach using linear mixed effect models. Ethical approval has been obtained from the University of Hertfordshire (cLMS/SF/UH/05240), and the trial has been registered as a clinical trial (NCT05486507). The trial findings will be disseminated through a peer-reviewed publication in a scientific journal.
]]>Methods and Protocols doi: 10.3390/mps6050075
Authors: Amjad Iqbal Yaodong Yang
Procuring high-grade RNA from mature coconut tissues is a tricky and labor-intensive process due to the intricate scaffold of polysaccharides, polyphenols, lipids, and proteins that form firm complexes with nucleic acids. However, we have effectively developed a novel method for the first time, letting the retrieval of high-grade RNA from the roots, endosperm, and mesocarp of mature coconut trees take place. In this method, we exploited dichloromethane as a replacement to phenol/chloroform for RNA recovery from mature coconut tissues. The amount of high-grade RNA acquired from the roots of mature coconut trees was 120.7 µg/g, with an A260/280 ratio of 1.95. Similarly, the mature coconut mesocarp yielded 134.6 µg/g FW of quality RNA with A260/280 ratio of 1.98, whereas the mature coconut endosperm produced 120.4 µg/g FW of quality RNA with A260/280 ratio of 2.01. Furthermore, the RNA isolation using the dichloromethane method exhibited excellent performance in downstream experiments, particularly in RT-PCR for cDNA production and amplification. On the contrary, the RNA plant kit, TRIZOL, and Cetyl Trimethyl Ammonium Bromide (CTAB) methods were unsuccessful in isolating substantial quantities of RNA with exceptional purities from the mentioned coconut tissues. In view of these findings, we conclude that the newly developed method will be pivotal in effectively extracting RNA with high purity from mature coconut tissues.
]]>Methods and Protocols doi: 10.3390/mps6050074
Authors: Javier Albornoz-Guerrero Olga Barceló Sonia García-Merino Guillermo García-Pérez-de-Sevilla Igor Cigarroa Rafael Zapata-Lamana
Background: Childhood obesity has tripled, reaching critical levels of malnutrition. This factor is directly associated with a poorer health-related quality of life of the child and adolescent population. This article presents the study protocol of the project “Strong schoolchildren with a healthy lifestyle” (EF-Salud), which seeks to analyze the effects of a multicomponent program based on muscle strength exercises, sleep nutritional recommendations, and the use of screens in Chilean educational centers with extremely cold weather. Methods: The study protocol of a randomized controlled trial with a pre- and post-test conducted according to the CONSORT statement is reported. The total sample (n = 144) will be schoolchildren from six different school years, four of which will perform an intervention and two control. Intervention group 1 (from two different school years) will receive a muscular strength exercise program in the classroom once a day from Monday to Friday for six months and nutritional, sleep, and use of screens recommendations once a week. Intervention group 2 (from two different school years) will receive a program of nutritional, sleep, and use of screens recommendations once per week for six months. The control group (from two different school years) will carry out their usual school day in relation to physical education classes. Before and after the intervention, the investigators will evaluate the cardiovascular risk, physical condition, and lifestyle related to sleep and use of screens. Expected results: The schoolchildren in intervention group 1 will obtain significant results in increased strength, decreased cardiovascular risk, improved sleep habits, and fewer hours of screen use compared to the other two groups.
]]>Methods and Protocols doi: 10.3390/mps6050073
Authors: Olga Puzankova Vera Gavrilova Roman Chernyshev Ivan Kolbin Alexey Igolkin Alexandr Sprygin Ilya Chvala Ali Mazloum
Isolation of African swine fever virus (ASFV) is a critical step towards the identification, titration, characterization, and even modification of the virus. Therefore, it is important to identify a suitable cell line that supports the efficient replication of ASFV for these purposes. This should be achieved even when starting with a low virus load, as in the case of isolating the virus from field samples. This article presents a detailed protocol on the preparation of porcine bone marrow primary (PBMP) cell culture, which has a high sensitivity towards ASFV, resulting in high viral yields with a minimal risk of bacterial contamination.
]]>Methods and Protocols doi: 10.3390/mps6040072
Authors: Ida Eriksson Linda Vainikka Hans Lennart Persson Karin Öllinger
Loss of lysosomal membrane integrity results in leakage of lysosomal hydrolases to the cytosol which might harm cell function and induce cell death. Destabilization of lysosomes often precede apoptotic or necrotic cell death and occur during both physiological and pathological conditions. The weak base acridine orange readily enters cells and accumulates in the acidic environment of lysosomes. Vital staining with acridine orange is a well-proven technique to observe lysosomal destabilization using fluorescence microscopy and flow cytometry. These analyses are, however, time consuming and only adapted for discrete time points, which make them unsuitable for large-scale approaches. Therefore, we have developed a time-saving, high-throughput microplate reader-based method to follow destabilization of the lysosomal membrane in real-time using acridine orange. This protocol can easily be adopted for patient samples since the number of cells per sample is low and the time for analysis is short.
]]>Methods and Protocols doi: 10.3390/mps6040071
Authors: Hee Sam Na Yuri Song Yeuni Yu Jin Chung
Recent advances in genomic technologies have enabled more in-depth study of the oral microbiome. In this study, we compared the amplicons generated by primers targeting different sites of the 16S rRNA gene found in the Human Oral Microbiome Database (HOMD). Six sets of primer targeting V1–V2, V1–V3, V3–V4, V4–V5, V5–V7 and V6–V8 regions of 16S rRNA were tested via in silico simulation. Primers targeting the V1–V2, V3–V4, and V4–V5 regions generated more than 90% of the original input sequences. Primers targeting the V1–V2 and V1–V3 regions exhibited a low number of mismatches and unclassified sequences at the taxonomic level, but there were notable discrepancies at the species level. Phylogenetic tree comparisons showed primers targeting the V1–V2 and V3–V4 regions showed performances similar to primers targeting the whole 16s RNA region in terms of separating total oral microbiomes and periodontopathogens. In an analysis of clinical oral samples, V1–V2 primers showed superior performance for identifying more taxa and had better resolution sensitivity for Streptococcus than V3–V4 primers. In conclusion, primers targeting the V1–V2 region of 16S rRNA showed the best performance for oral microbiome studies. In addition, the study demonstrates the need for careful PCR primer selections.
]]>Methods and Protocols doi: 10.3390/mps6040070
Authors: Md Lokman Hossain Minh Nguyen Leah Benington Lee Yong Lim Katherine Hammer Dhanushka Hettiarachchi Cornelia Locher
The aim of this study was to assess the release profile of components in five different honeys (a New Zealand Manuka and two Western Australian honeys, a Jarrah honey and a Coastal Peppermint honey) and their corresponding honey-loaded gel formulations using a custom-designed Franz-type diffusion cell in combination with High-Performance Thin-Layer Chromatography (HPTLC). To validate the suitability of the customised setup, release data using this new approach were compared with data obtained using a commercial Franz cell apparatus, which is an established analytical tool to monitor the release of active ingredients from topical semisolid products. The release profiles of active compounds from pure honey and honey-loaded formulations were found to be comparable in both types of Franz cells. For example, when released either from pure honey or its corresponding pre-gel formulation, the percentage release of two Jarrah honey constituents, represented by distinct bands at RF 0.21 and 0.53 and as analysed by HPTLC, was not significantly different (p = 0.9986) at 12 h with over 99% of these honey constituents being released in both apparatus. Compared to the commercial Franz diffusion cell, the customised Franz cell offers several advantages, including easy and convenient sample application, the requirement of only small sample quantities, a large diffusion surface area, an ability to analyse 20 samples in a single experiment, and lower cost compared to purchasing a commercial Franz cell. Thus, the newly developed approach coupled with HPTLC is conducive to monitor the release profile of minor honey constituents from pure honeys and honey-loaded semisolid formulations and might also be applicable to other complex natural-product-based products.
]]>Methods and Protocols doi: 10.3390/mps6040069
Authors: Annisa Nurkhasanah Titouan Fardad Ceferino Carrera Widiastuti Setyaningsih Miguel Palma
This study aimed to determine the optimal UAE conditions for extracting anthocyanins from pigmented corn using the Box–Behnken design (BBD). Six anthocyanins were identified in the samples and were used as response variables to evaluate the effects of the following working variables: extraction solvent pH (2–7), temperature (10–70 °C), solvent composition (0–50% methanol in water), and ultrasound power (20–80%). The extraction time (5–25 min) was evaluated for complete recovery. Response surface methodology suggested optimal conditions, specifically 36% methanol in water with pH 7 at 70 °C using 73% ultrasound power for 10 min. The method was validated with a high level of accuracy (>90% of recovery) and high precision (CV < 5% for both repeatability and intermediate precision). Finally, the proposed analytical extraction method was successfully applied to determine anthocyanins that covered a wide concentration range (36.47–551.92 mg kg−1) in several pigmented corn samples revealing potential varieties providing more health benefits.
]]>Methods and Protocols doi: 10.3390/mps6040068
Authors: Christopher Steer Tshepo Rasekaba Kylie Owen Darren Jayasuriya Mira Kapur Kim Young Nicole Webb Irene Blackberry
Geriatric assessment (GA) is fundamental to optimising cancer care in older adults, yet implementing comprehensive GA tools in real-world clinical settings remains a challenge. This study aims to assess the feasibility and acceptability of integrating information from patient-derived photographs (PhotoVoice) into enhanced supportive care (ESC) for older adults with cancer. A feasibility randomised controlled trial will be conducted at a regional cancer care centre in Australia. Participants aged 70 and above will be randomised into two groups: PhotoVoice plus ESC or usual care (ESC) alone. In the PhotoVoice group, participants will provide four photographs for deduction of representations of different aspects of their lives using photo-elicitation techniques. ESC will be conducted for both groups, incorporating PhotoVoice analysis in the intervention group. PhotoVoice may improve patient-centred care outcomes, including enhanced communication, shared decision making, and identification of patient priorities and barriers. Findings will provide insights into implementing PhotoVoice in geriatric assessment and guide future trials in cancer among older adults.
]]>Methods and Protocols doi: 10.3390/mps6040067
Authors: Chiara Adembri Andrea Ungar Iacopo Cappellini Salvatore Mario Romano
(1) Background: Oncological demolitive–reconstructive surgeries in the head and neck region cause significant stress on patients’ biohumoural, cardiac, and vascular systems, leading to disturbances in macrocirculatory and microcirculatory parameters. Traditional monitoring addresses the symptoms, but not the underlying cause. Microcirculatory assessments complement macrocirculatory monitoring, and bladder-catheter-based technology offers a better representation of central microcirculation. Flap reconstruction surgeries involve demolitive and reconstructive phases, requiring optimal tissue perfusion. The literature lacks a consensus on macro–microcirculation coupling, and there is no agreement on the use of vasopressors during head and neck surgeries. Evidence-based guidelines are lacking, resulting in variations in vasopressor administration. (2) Methods: This is a 12-month observational, prospective study conducted in a single center. It aims to evaluate the impact of macro–microcirculation coupling on clinical complications in head and neck surgery. All consecutive patients undergoing oncologic surgery requiring flap reconstruction and meeting the inclusion criteria will be enrolled. The study will utilize standard hemodynamic monitoring and bladder catheterization for measuring urine output and temperature. (3) Conclusions: The study aims to evaluate the coupling of macro- and microcirculation in head and neck surgeries, assess hemodynamic parameters and microcirculatory changes, and investigate their association with postoperative complications. The results can enhance patient care and surgical outcomes.
]]>Methods and Protocols doi: 10.3390/mps6040066
Authors: Zainab L. Rai Morenike Magbagbeola Katie Doyle Lukas Lindenroth George Dwyer Amir Gander Agostino Stilli Danail Stoyanov Brian R. Davidson
Background: Machine perfusion (MP) is increasingly used for human transplant organ preservation. The use of MP for research purposes is another opportunity for this technology. The porcine pancreas and liver are similar in anatomical size and function to their human counterparts, making them an excellent resource for research, but they have some important differences from human organs which can influence their research use. In this paper, we describe a technique developed and tested for the retrieval of porcine organs for use in research on perfused viable organs. Methods: Whole-organ porcine pancreata and livers were harvested at a commercial abattoir, following standard slaughterhouse processes. The standard slaughterhouse process involved a thoracotomy and mid-line laparotomy, and all the thoracoabdominal organs were removed. The pancreas, fixed in the retroperitoneum, was carefully dissected from its attachments to the surrounding structures, and tissue planes between the pancreas, spleen, duodenum, and colon were meticulously identified and dissected. Vessel exposure and division: The aorta, portal vein (PV), hepatic vein (HV), and hepatic artery (HA) were dissected and isolated, preserving the input and output channels for the liver and pancreas. A distal 3 cm of the aorta was preserved and divided and served as the input for the pancreas perfusions. The liver, PV, HV, and HA were preserved and divided to preserve the physiological channels of the input (PV and HA) and output (HV) for the liver perfusions. The porcine hepatic and pancreas anatomy shares significant resemblance with the gross anatomy found in humans, and this was taken into consideration when designing the perfusion circuitry. The porcine pancreas and spleen shared a common blood supply, with branches arising from the splenic artery. The organs were flushed with cold, heparinised normal saline and transported in a temperature-regulated receptacle maintained at a core temperature between 4 and 8 °C, in line with the standards of static cold storage (SCS), to a dedicated perfusion lab and perfused using our novel perfusion machine with autologous, heparinised porcine blood, also collected at the abattoir.
]]>Methods and Protocols doi: 10.3390/mps6040065
Authors: Melika Shayegh Chase Sorenson Jackson Downey Summer Lin Yuxin Jiang Praneeti Sodhi Victoria Sullivan Katherine M. Howard Karl Kingsley
One protocol in healthcare facilities and dental offices due to the COVID-19 pandemic for reducing the amount of detectable oral SARS-CoV-2 has been gargling with mouthwash for 60 s. This protocol lasts longer than the daily routine for most patients and may have unexpected benefits in reducing oral microbes as a result. This project evaluated the prevalence of the newly identified oral pathogen Scardovia wiggsiae before and after this procedure to determine any measurable effects. Using an approved protocol, n = 36 pre-mouthwash patient samples, n = 36 matched post-mouthwash samples, and n = 36 matched recall samples were identified (total sample number n = 108). DNA was isolated from each sample (pre-, post-mouthwash, and recall). Screening using qPCR and validated primers revealed n = 10/36 or 27.8% tested positive for Scardovia among the pre-mouthwash (Sample A) isolates with n = 3/36 or 8.3% testing positive among the post-mouthwash (Sample B) isolates. Screening of the recall (Sample C) samples has revealed n = 10/36, or 27.8% once again tested positive for Scardovia, demonstrating that this pathogen was found among a significant proportion of pediatric patient samples. Moreover, the COVID-19-related procedure of requiring sustained mouth washing prior to clinical treatment appears to reduce the levels of detectable Scardovia, at least initially. However, this study found no long-term effects using this isolated protocol.
]]>Methods and Protocols doi: 10.3390/mps6040064
Authors: Christopher Foster Todd Jensen Christine Finck Courtney K. Rowe
Urethral healing is plagued by strictures, impacting quality of life and medical costs. Various growth factors (GFs) have shown promise as therapeutic approaches to improve healing, but there is no protocol for in vitro comparison between GFs. This study focuses the development of a biomimetic in vitro urothelial healing assay designed to mimic early in vivo healing, followed by an evaluation of urothelial cell growth in response to GFs. Methods: Wound-healing assays were developed with human urothelial cells and used to compared six GFs (EGF, FGF-2, IGF-1, PDGF, TGF-β1, and VEGF) at three concentrations (1 ng/mL, 10 ng/mL, and 100 ng/mL) over a 48 h period. A commercial GF-containing medium (EGF, TGF-α, KGF, and Extract P) and a GF-free medium were used as controls. Results: There was a statistically significant increase in cell growth for IGF-1 at 10 and 100 ng/mL compared to both controls (p < 0.05). There was a statistically significant increase in cell growth for EGF at all concentrations compared to the GF-free medium control (p < 0.05). Conclusion: This study shows the development of a clinically relevant wound-healing assay to evaluate urothelial cell growth. It is the first to compare GFs for future use in reconstructive techniques to improve urethral healing.
]]>Methods and Protocols doi: 10.3390/mps6040063
Authors: Shan Zhang Chao Wang
As an essential characterization, size distribution is an important indicator for the synthesis, optimization, and application of nanoparticles. Electron microscopes such as transmission electron microscopes (TEMs) are commonly utilized to collect size information on nanoparticles. However, the current popular statistical method of manually measuring large particles one by one, using a ruler tool in the corresponding image analysis software is time-consuming and can introduce manual errors. Moreover, it is difficult to determine the measurement interval for irregularly shaped nanoparticles. Therefore, it is necessary to use an efficient and standard method to perform size distribution analysis of nanoparticles. In this work, we use basic ImageJ software (1.53 t) to analyze the size of typical silica nanoparticles in a TEM image and use Origin software to process the data, to obtain its accurate distribution quickly. Using it as a template, we believe that this work can provide a paradigm for the standardized analysis of nanoparticle size.
]]>Methods and Protocols doi: 10.3390/mps6040062
Authors: Yirlis Yadeth Pineda-Rodriguez Marcelo F. Pompelli Alfredo Jarma-Orozco Novisel Veitía Rodríguez Luis Alfonso Rodriguez-Paez
Limnospira maxima is a remarkable organism showing great potential as a versatile and sustainable food source, offering a powerful solution to address the pressing issues of malnutrition and undernourishment worldwide. L. maxima contains high amounts of proteins, vitamins, minerals, and essential fatty acids. It can be grown in both bioreactors and open systems; however, before considering industrial production, optimization studies of the cultivation must be conducted to obtain knowledge about the ideal environmental conditions. Additionally, for the molecular typing of L. maxima strains and their industrial scaling, high-quality and large quantity DNA extraction is required. Notwithstanding, DNA extraction from L. maxima can be challenging due to the low amount of DNA in cells and the presence of difficult-to-remove substances such as polysaccharides and polyphenols. In this study, the quality and quantity of DNA extracted from two types of L. maxima samples (Limnospira maxima strain SISCA accession GenBank: OR195505.1) were evaluated using three commercially available DNA extraction kits and two types of input biological material. The results showed that Pbact-P kit had the highest quantity and quality of DNA, while CTAB-P allowed for a higher quantity and quality of RNA, making them optimal protocols for nucleic acid extraction to improve PCR, rt-PCR, and genome sequencing of L. maxima compared with other extraction methods.
]]>Methods and Protocols doi: 10.3390/mps6040061
Authors: Ella-Erika Söderlund Heikki Kyröläinen Outi M. Laitinen-Vapaavuori Heli K. Hyytiäinen
The number of dogs and, with it, dog sports are growing in popularity, and the training of dogs begins at an early age. Although fitness testing is an imperative part of purposeful training and sports, to our knowledge, no objective field tests are available for measuring young dogs’ endurance fitness. The aim of this study is to describe a simple, easy-to-repeat, and inexpensive way to test training intervention effects on endurance fitness in young Labrador Retrievers. Healthy client-owned 16-week-old Labrador Retrievers will be recruited and divided into test and control groups. The test group will have an eight-week training program followed by a four-week detraining period, while the control group will live a normal puppy life. All dogs will be tested for endurance fitness four times at four-week intervals: at baseline, one month later, two months later at the end of the training period, and one month after ending the training program. Each of the four testing sessions will be identical and will consist of four measurements of heart rate (HR) and blood lactate (BL): at baseline, after trotting 1000 m, after sprinting 200 m, and at recovery 5–8 min after the sprint. The training-induced changes in endurance fitness are evaluated by changes in HR and heart rate recovery times (HRR), BL, and running times.
]]>Methods and Protocols doi: 10.3390/mps6040060
Authors: Vasiliki Keramari Sophia Karastogianni Stella Girousi
The detection of toxic heavy metal ions, especially cadmium (Cd), lead (Pb), zinc (Zn), and copper (Cu), is a global problem due to ongoing pollution incidents and continuous anthropogenic and industrial activities. Therefore, it is important to develop effective detection techniques to determine the levels of pollution from heavy metal ions in various media. Electrochemical techniques, more specifically voltammetry, due to its properties, is a promising method for the simultaneous detection of heavy metal ions. This review examines the current trends related to electrode formation and analysis techniques used. In addition, there is a reference to advanced detection methods based on the nanoparticles that have been developed so far, as well as formation with bismuth and the emerging technique of screen-printed electrodes. Finally, the advantages of using these methods are highlighted, while a discussion is presented on the benefits arising from nanotechnology, as it gives researchers new ideas for integrating these technologies into devices that can be used anywhere at any time. Reference is also made to the speciation of metals and how it affects their toxicity, as it is an important subject of research.
]]>Methods and Protocols doi: 10.3390/mps6040059
Authors: Tomoyuki Kawase Katsuya Suzuki Masami Kamimura Tomoharu Mochizuki Takashi Ushiki
Platelet polyphosphate (polyP) can be conveniently quantified by exploiting a recent methodological breakthrough using 4′,6-diamidino-2-phenylindole (DAPI). However, the preservation of these biological samples has not yet been standardized. In a preliminary study, potential protocols were screened, while accepted protocols were further tested in this study. Pure-platelet-rich plasma (P-PRP) samples and washed platelet suspensions were prepared using blood obtained from non-smoking healthy male donors and were fixed with ThromboFix for 20–24 h at 4 °C. Mass polyP levels were determined using a fluorometer at wavelengths of 425 and 525 nm. Platelet polyP levels were normalized to platelet counts. Statistical analyses were performed using non-parametric tests. Platelet polyP levels significantly decreased by 20% after 7 days in the platelet suspension maintained under fixed conditions at 4 °C (control). In contrast, the platelet polyP levels in both the P-PRP and washed platelet suspensions were maintained without a significant reduction for up to 6 weeks by removing ThromboFix after fixation and subsequent freezing in pure water at −80 °C. Fluorometric polyP quantification often interferes with the low specificity of DAPI binding and the wavelength used. Our validated protocols will enable long-term preservation and high-throughput polyP quantification and can be applied to relatively large cohort studies.
]]>Methods and Protocols doi: 10.3390/mps6030058
Authors: Amanjyot Singh Sainbhi Nuray Vakitbilir Alwyn Gomez Kevin Y. Stein Logan Froese Frederick A. Zeiler
The ability of cerebral vessels to maintain a fairly constant cerebral blood flow is referred to as cerebral autoregulation (CA). Using near-infrared spectroscopy (NIRS) paired with arterial blood pressure (ABP) monitoring, continuous CA can be assessed non-invasively. Recent advances in NIRS technology can help improve the understanding of continuously assessed CA in humans with high spatial and temporal resolutions. We describe a study protocol for creating a new wearable and portable imaging system that derives CA maps of the entire brain with high sampling rates at each point. The first objective is to evaluate the CA mapping system’s performance during various perturbations using a block-trial design in 50 healthy volunteers. The second objective is to explore the impact of age and sex on regional disparities in CA using static recording and perturbation testing in 200 healthy volunteers. Using entirely non-invasive NIRS and ABP systems, we hope to prove the feasibility of deriving CA maps of the entire brain with high spatial and temporal resolutions. The development of this imaging system could potentially revolutionize the way we monitor brain physiology in humans since it would allow for an entirely non-invasive continuous assessment of regional differences in CA and improve our understanding of the impact of the aging process on cerebral vessel function.
]]>Methods and Protocols doi: 10.3390/mps6030057
Authors: Arseniy Pelevin Natalia Kurzina Vladislav Zavialov Anna Volnova
This article presents a low-cost and flexible software solution for acoustic startle response (ASR) test that can be used with a Spike2-based interface. ASR is a reflexive response to an unexpected, loud acoustic stimulus, and prepulse inhibition (PPI) is a phenomenon in which the startle response is reduced when preceded by a weak prestimulus of the same modality. Measuring PPI is important because changes in PPI have been observed in patients with various psychiatric and neurological disorders. Commercial ASR testing systems are expensive, and their closed source code affects their transparency and result reproducibility. The proposed software is easy to install and use. The Spike2 script is customizable and supports a wide range of PPI protocols. As an example of PPI recording, the article presents data obtained in female rats, both wild-type (WT) and dopamine transporter knockout (DAT-KO), showing the same tendency as the data obtained in males, with ASR on a single pulse higher than ASR on prepulse+pulse, and PPI reduced in DAT-KO rats compared to WT.
]]>Methods and Protocols doi: 10.3390/mps6030056
Authors: Nitikorn Noraphaiphipaksa Surangkana Katepun Thanapong Waitayawinyu Chaosuan Kanchanomai
Distal radius fractures (DRFs) are one of the most common fractures of the upper extremity system. To evaluate the performance of DRF treatments, the construct (i.e., a DRF fixed by an implant) was compressed at the distal radius in the axial direction to evaluate the compressive stiffness. In previous studies, various constructs of both cadaveric and synthetic radii have been proposed for biomechanical testing for DRF. Unfortunately, high deviations of the measured stiffness have been reported across the literature, which may relate to the inconsistency of applied mechanical actions (i.e., the tested radii may under various combinations including compression, bending, and shear). In the present study, a biomechanical apparatus and an experimental procedure were proposed for the biomechanical testing of radii under pure compression. After the biomechanical tests of synthetic radii, it was found that the standard deviation of stiffness was significantly lower than that in previous studies. Thus, the biomechanical apparatus and the experimental procedure were proven to be a practical method for the evaluation of radii stiffness.
]]>Methods and Protocols doi: 10.3390/mps6030055
Authors: Alexander Tishchenko Cliff Van Waesberghe Herman W. Favoreel
Protein phosphorylation is a ubiquitous post-translational modification that regulates a plethora of intracellular processes, making its analysis crucial for understanding intracellular dynamics. The commonly used methods, such as radioactive labeling and gel electrophoresis, do not provide information about subcellular localization. Immunofluorescence using phospho-specific antibodies and subsequent analysis via microscopy allows researchers to assess subcellular localization, but it typically lacks validation whether the observed fluorescent signal is phosphorylation specific. In this study, an on-slide dephosphorylation assay coupled with immunofluorescence staining using phospho-specific antibodies on fixed samples is proposed as a fast and simple approach to validate phosphorylated proteins in their native subcellular context. The assay was validated using antibodies against two different phosphorylated target proteins, connexin 43 phosphorylated at serine 373, and phosphorylated substrates of protein kinase A, with a dramatic reduction in the signal upon dephosphorylation. The proposed approach provides a convenient way to validate phosphorylated proteins without the need for additional sample preparation steps, reducing the time and effort required for analysis, while minimizing the risk of protein loss or alteration.
]]>Methods and Protocols doi: 10.3390/mps6030054
Authors: Asmaa Q. Ibrahim Mohammed S. Abdullah Mamoun Ahram Shtaywy Abdalla
Background: Vascular smooth muscle cells (VSMCs) and vascular endothelial cells are key participants in the pathogenesis of atherosclerosis. Human umbilical vein endothelial cells (HUVECs) and VSMCs are useful models to design therapeutic strategies for many cardiovascular diseases (CVDs). However, procuring a VSMC cell line by researchers, to model atherosclerosis, for example, is impeded by time and cost limitations, as well as by many other logistic problems in many countries. Results: This article describes a protocol for the quick and cheap isolation of VSMCs from human umbilical cords using a mechanical and enzymatic method. This VSMC protocol yields a confluent primary culture that could be obtained within 10 days and sub-cultured for 8–10 passages. The isolated cells are characterized by their morphology and the expression of mRNA of marker proteins analyzed by reverse transcription polymerase chain reaction (RT-qPCR). Conclusion: The protocol described herein for the isolation of VSMCs from human umbilical cords is easy and is time- and cost-efficient. Isolated cells are useful models for understanding the mechanisms underlying many pathophysiological conditions.
]]>Methods and Protocols doi: 10.3390/mps6030053
Authors: Johanna Romina Zuccoli Priscila Ayelén Pagnotta Viviana Alicia Melito Jimena Verónica Lavandera Victoria Estela Parera Ana María Buzaleh
The Multidrug Resistance protein (ABCB1, MDR1) is involved in the transport of xenobiotics and antiretroviral drugs. Some variants of the ABCB1 gene are of clinical importance; among them, exon 12 (c.1236C>T, rs1128503), 21 (c.2677G>T/A, rs2032582), and 26 (c.3435C>T, rs1045642) have a high incidence in Caucasians. Several protocols have been used for genotyping the exon 21 variants, such as allele-specific PCR-RFLP using adapted primer to generate a digestion site for several enzymes and automatic sequencing to detect the SNVs, TaqMan Allele Discrimination assay and High-Resolution Melter analysis (HRMA). The aim was to describe a new approach to genotype the three variants c.2677G>T/A for the exon 21 doing only one PCR with the corresponding primers and the digestion of the PCR product with two restriction enzymes: BrsI to identify A allele and BseYI to differentiate between G or T. An improvement of this methodology was also described. The proposal technique here described is demonstrated to be very efficient, easy, fast, reproducible, and cost-effective.
]]>Methods and Protocols doi: 10.3390/mps6030052
Authors: Fabian P. Stangl Laila Schneidewind Bernhard Kiss Jennifer Kranz Florian M. Wagenlehner Truls E. Bjerklund Johansen Béla Köves Jose Medina-Polo Ana Maria Tapia Zafer Tandogdu
Introduction: Patients with neurogenic lower urinary tract dysfunction (NLUTD) reliant on intermittent self-catheterization for bladder emptying are at an increased risk of recurrent urinary tract infections (rUTI). So far, the most common practice in the prevention of rUTIs is long-term low-dose antibiotic prophylaxis, phytotherapy, and immunomodulation, whereby antibiotic prophylaxis inevitably leads to the emergence of drug-resistant pathogens and difficulty in treating infections. Therefore, non-antibiotic alternatives in the prevention of rUTIs are urgently required. We aim to identify the comparative clinical effectiveness of a non-antibiotic prophylaxis regimen in the prevention of recurrent urinary tract infections in patients with neurogenic bladder dysfunction who practice intermittent self-catheterization. Methods and analysis: In this multi-centre, prospective longitudinal multi-arm observational study, a total of 785 patients practising intermittent self-catheterisation due to NLUTD will be included. After inclusion, non-antibiotic prophylaxis regimens will be instilled with either UroVaxom® (OM-89) standard regimen, StroVac® (bacterial lysate vaccine) standard regimen, Angocin®, D-mannose (oral dose 2 g), bladder irrigation with saline (once per day). The management protocols will be pre-defined, but the selection of the protocol will be at the clinicians’ discretion. Patients will be followed for 12 months from the onset of the prophylaxis protocol. The primary outcome is to identify the incidence of breakthrough infections. The secondary outcomes are adverse events associated with the prophylaxis regimens and the severity of breakthrough infections. Other outcomes include the exploration of change in susceptibility pattern via the optional rectal and perineal swab, as well as health-related quality of life over time (HRQoL), which will be measured in a random subgroup of 30 patients. Ethics and dissemination: Ethical approval for this study has been granted by the ethical review board of the University Medical Centre Rostock (A 2021-0238 from 28 October 2021). The results will be published in a peer-reviewed journal and presented at relevant meetings. Study registration number: German Clinical Trials Register: Number DRKS00029142.
]]>Methods and Protocols doi: 10.3390/mps6030051
Authors: Celina H. Shirazipour Rachel M. Ruggieri-Bacani Laura Lockshon Christopher Waring Aubrey Jarman Novalyn Cruz Catherine Bresee Angela J. Fong Pao-Hwa Lin Gillian Gresham Arash Asher Stephen J. Freedland
Background: Despite the known benefits of physical activity in cancer survivors, adherence to exercise guidelines remains low. Known barriers to adhering to guidelines include a lack of time and an unwillingness to return to treatment facilities. Virtual exercise programming could assist in mitigating these barriers. This protocol presents a single arm pilot study exploring the feasibility of personalized Zoom-delivered exercise training for breast and prostate cancer survivors. A secondary objective is to determine the preliminary efficacy of participation on body composition, estimated VO2max, hand grip, one repetition maximum leg press, resting heart rate, resting blood pressure, exercise self-efficacy, and intentions to remain active. Methods: Breast (n = 10) and prostate (n = 10) cancer survivors will participate in a 24-week feasibility study, including (1) 12 weeks of one-on-one virtual personal training with an exercise physiologist (EP) via Zoom, and (2) individual exercise for a 12-week follow-up period using recordings of Zoom sessions for guidance. Physical assessments and surveys will be implemented at baseline, 12 weeks, and at the end of the study (24 weeks from baseline). Conclusions: While virtual exercise programming became popularized during the pandemic, evidence is still required to understand whether it can successfully address barriers and promote participation.
]]>Methods and Protocols doi: 10.3390/mps6030050
Authors: Alice Rocha Teixeira Netto Marc Dieter Hrusa Karl-Ulrich Bartz-Schmidt Sven Schnichels José Hurst
In ophthalmic research, there is a strong need for in vitro corneal cell models. Here, we describe different protocols for the cultivation of primary corneal cells that were isolated from porcine eyes. This primary cell culture can be used to test new therapeutic options for corneal diseases, such as dry eye disease, traumatic injuries, or corneal infections, and to study limbal epithelial stem cell (LESC) expansion. Two different isolation methods were performed: the outgrowth and the collagenase method. To perform the outgrowth protocol, small explants of the corneal limbus were generated and incubated in culture flasks in an incubator for 4–5 weeks. Regarding the collagenase method, to extract corneal cells, porcine corneas were removed, cut into small pieces, and incubated with collagenase. After incubation and centrifugation, the cells were seeded in 6- or 12-well plates and incubated in an incubator for 2–3 weeks. The differences between corneal cell cultivation with fetal bovine serum (FBS) and without it are also discussed. Therefore, the main advantages of the outgrowth method are that it requires fewer porcine eyes, and it takes less time to be performed compared to the collagenase method. On the other hand, with the collagenase method, mature cells are obtained earlier, at about 2 to 3 weeks.
]]>Methods and Protocols doi: 10.3390/mps6030049
Authors: Dorelly Tanayra Martinez Del Carmen Pablo Saldaña Gutierrez Ramon Vila Coll Elena Iborra Ortega
In the past decades, we have witnessed tremendous developments in endovascular surgery. Nowadays, highly complex procedures are performed by minimally invasive means. A key point is equipment improvement. Modern C-arms provide advanced imaging capabilities, facilitating endovascular navigation with an adequate open surgical environment. Nevertheless, radiation exposure remains an issue of concern. This study aims to analyze radiation used during endovascular procedures according to complexity, comparing a mobile X-ray system with a hybrid room (fixed X-ray system). This is an observational and prospective study based on a cohort of non-randomized patients treated by endovascular procedures in a Vascular Surgery department using two imaging systems. The study is planned for a 3-year duration with a recruitment period of 30 months (beginning 20 July 2021) and a 1-month follow-up period for each patient. This is the first prospective study designed to describe the radiation dose according to the complexity of the procedure. Another strength of this study is that radiologic variables are obtained directly from the C-arm and no additional measurements are required for feasibility benefit. The results from this study will help us determine the level of radiation in different endovascular procedures, in view of their complexity.
]]>Methods and Protocols doi: 10.3390/mps6030048
Authors: Rondi Anderson Sojib Bin Zaman Mark Limmer
Introduction: Midwives have the potential to significantly contribute to health-delivery systems by providing sexual, reproductive, maternal, newborn, and adolescent health (SRMNAH) care. However, scant research finds barriers to understanding what midwives need to realize their full potential. There are gaps in the definition of a midwife and an understanding of effective means to support the implementation of midwifery care. Mentorship has been found to support systems and healthcare providers to improve care availability and quality. Objectives: We describe the methodology of an integrative review that aims to generate evidence of the impact of introducing midwives and also on-site facility mentoring to better understand facilitators and barriers to implementation of the quality and availability of SRMNAH services in low- and middle-income countries (LMICs). Methods: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines will be used to carry out the integrative review. Four electronic bibliographic databases, PubMed MEDLINE, EMBASE, Scopus, and CINAHL, will be used to identify eligible studies. All types of qualitative or quantitative studies will be considered. Eligible studies will be screened according to Population, Intervention, Comparison, and Outcome (PICO) inclusion criteria, and data will be extracted against a predetermined format. The aspects of health system strengthening in providing improved SRMNCH care will be examined in this review to generate evidence on how midwives and mentorship can improve routine care and health outcomes using the World Health Organization’s Six Building Blocks approach. The quality of the articles will be thematically analyzed in four areas: coherence and integrity, appropriateness for answering the question, relevance and focus, and overall assessment using the Gough weight-of-evidence framework. Expected results: The literature review will consider assessing both upstream health systems regulators and downstream effectors for implementing midwifery interventions. Within this building block framework, this research will report on the outcomes and experiences of introducing midwives and the effectiveness of mentoring midwives and other staff in midwives’ roles in improving care quality and health outcomes.
]]>Methods and Protocols doi: 10.3390/mps6030047
Authors: Daniel J. Phipps Kyra Hamilton
The effect of arbitrary stimulus selection is a persistent concern when employing implicit measures. The current study tests a data-driven multi-step procedure to create stimulus items using a combination of free-recall and survey data. Six sets of stimulus items were created, representing healthy food and high sugar items in children, adolescents, and adults. Selected items were highly representative of the target concepts, in frequent use, and of near equal length. Tests of the piloted items in two samples showed slightly higher implicit measure–behavior relations compared to a previously used measure, providing preliminary support for the value in empirically based stimulus selection. Further, the items reported as being the most associated with their target concepts differed notably from what one may expect from the guidelines or population consumption patterns, highlighting the importance of informed stimulus selection.
]]>Methods and Protocols doi: 10.3390/mps6030046
Authors: Aleksandr Udalov Lexman Kumar Anna N. Gaudette Ran Zhang Joao Salomao Sanjay Saigal Mehdi Nosrati Sean D. McAllister Pierre-Yves Desprez
The longitudinal monitoring of patient circulating tumor DNA (ctDNA) provides a powerful method for tracking the progression, remission, and recurrence of several types of cancer. Often, clinical and research approaches involve the manual review of individual liquid biopsy reports after sampling and genomic testing. Here, we describe a process developed to integrate techniques utilized in data science within a cancer research framework. Using data collection, an analysis that classifies genetic cancer mutations as pathogenic, and a patient matching methodology that identifies the same donor within all liquid biopsy reports, the manual work for research personnel is drastically reduced. Automated dashboards provide longitudinal views of patient data for research studies to investigate tumor progression and treatment efficacy via the identification of ctDNA variant allele frequencies over time.
]]>Methods and Protocols doi: 10.3390/mps6030045
Authors: Ana Salomé Pires Sveva Bollini Maria Filomena Botelho Ingrid Lang-Olip Peter Ponsaerts Carolina Balbi Anna Lange-Consiglio Mathilde Fénelon Slavko Mojsilović Ekaterine Berishvili Fausto Cremonesi Maria Gazouli Diana Bugarski Alexandra Gellhaus Halima Kerdjoudj Andreina Schoeberlein
The last 18 years have brought an increasing interest in the therapeutic use of perinatal derivatives (PnD). Preclinical studies used to assess the potential of PnD therapy include a broad range of study designs. The COST SPRINT Action (CA17116) aims to provide systematic and comprehensive reviews of preclinical studies for the understanding of the therapeutic potential and mechanisms of PnD in diseases and injuries that benefit from PnD therapy. Here we describe the publication search and data mining, extraction, and synthesis strategies employed to collect and prepare the published data selected for meta-analyses and reviews of the efficacy of PnD therapies for different diseases and injuries. A coordinated effort was made to prepare the data suitable to make statements for the treatment efficacy of the different types of PnD, routes, time points, and frequencies of administration, and the dosage based on clinically relevant effects resulting in clear increase, recovery or amelioration of the specific tissue or organ function. According to recently proposed guidelines, the harmonization of the nomenclature of PnD types will allow for the assessment of the most efficient treatments in various disease models. Experts within the COST SPRINT Action (CA17116), together with external collaborators, are doing the meta-analyses and reviews using the data prepared with the strategies presented here in the relevant disease or research fields. Our final aim is to provide standards to assess the safety and clinical benefit of PnD and to minimize redundancy in the use of animal models following the 3R principles for animal experimentation.
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