Methods and Protocols最新文献

{"title":"Scale-Up of the Fermentation Process for the Production and Purification of Serratiopeptidase Using Silkworm Pupae as a Substrate.","authors":"Jhon Jairo Melchor-Moncada, Alejandra García-Barco, Augusto Zuluaga-Vélez, Luz Angela Veloza, Juan Carlos Sepúlveda-Arias","doi":"10.3390/mps7020019","DOIUrl":"10.3390/mps7020019","url":null,"abstract":"<p><p>Serratiopeptidase, a bacterial metalloprotease known for its pain-relieving and anti-inflammatory properties, can be produced through fermentation with <i>S. marcescens</i>. 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 k_La 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.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 2","pages":""},"PeriodicalIF":2.4,"publicationDate":"2024-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10961818/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140207252","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Housing European Ground Squirrels (<i>Spermophilus citellus</i>) for an Ex Situ Conservation Program.","authors":"Boróka Bárdos, Vilmos Altbacker, Henrietta Kinga Török, István Nagy","doi":"10.3390/mps7020018","DOIUrl":"10.3390/mps7020018","url":null,"abstract":"<p><p>European ground squirrel (<i>Spermophilus citellus</i>) 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.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 2","pages":""},"PeriodicalIF":2.4,"publicationDate":"2024-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10885073/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139931972","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Crafting a Rigorous, Clinically Relevant Large Animal Model of Chronic Myocardial Ischemia: What Have We Learned in 20 Years?","authors":"Christopher R Stone, Dwight D Harris, Mark Broadwin, Meghamsh Kanuparthy, Sharif A Sabe, Cynthia Xu, Jun Feng, M Ruhul Abid, Frank W Sellke","doi":"10.3390/mps7010017","DOIUrl":"10.3390/mps7010017","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 1","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10891802/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139931970","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Pilot Study: Safety and Performance Validation of an Ingestible Medical Device for Collecting Small Intestinal Liquid in Healthy Volunteers.","authors":"Alexandre Tronel, Anne-Sophie Silvent, Elena Buelow, Joris Giai, Corentin Leroy, Marion Proust, Donald Martin, Audrey Le Gouellec, Thomas Soranzo, Nicolas Mathieu","doi":"10.3390/mps7010015","DOIUrl":"10.3390/mps7010015","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 1","pages":""},"PeriodicalIF":2.4,"publicationDate":"2024-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10892249/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139931971","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Making the Most of Lateral Flow Immunochromatographic Tests: An Efficient Protocol to Recover DNA.","authors":"Sara C Zapico, Gabriela Roca","doi":"10.3390/mps7010008","DOIUrl":"10.3390/mps7010008","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 1","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10801598/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139512995","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mapping m6A Sites on HIV-1 RNA Using Oligonucleotide LC-MS/MS","authors":"Alice Baek, Asif Rayhan, Ga-Eun Lee, Sarah Golconda, H. Yu, Shihyoung Kim, P. Limbach, Balasubrahmanyam Addepalli, Sanggu Kim","doi":"10.3390/mps7010007","DOIUrl":"https://doi.org/10.3390/mps7010007","url":null,"abstract":"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.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 4","pages":""},"PeriodicalIF":2.4,"publicationDate":"2024-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139439522","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimal Selection of Sampling Points within Sewer Networks for Wastewater-Based Epidemiology Applications","authors":"Yao Yao, Yibo Zhu, Regina Nogueira, Frank Klawonn, Markus Wallner","doi":"10.3390/mps7010006","DOIUrl":"https://doi.org/10.3390/mps7010006","url":null,"abstract":"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.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"35 5","pages":""},"PeriodicalIF":2.4,"publicationDate":"2024-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139383979","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Introducing Artificial Intelligence in Interpretation of Foetal Cardiotocography: Medical Dataset Curation and Preliminary Coding—An Interdisciplinary Project","authors":"J. L. Aeberhard, A. Radan, Ramin Abolfazl Soltani, K. Strahm, S. Schneider, Adriana Carrié, Mathieu Lemay, Jens Krauss, Ricard Delgado-Gonzalo, Daniel Surbek","doi":"10.3390/mps7010005","DOIUrl":"https://doi.org/10.3390/mps7010005","url":null,"abstract":"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.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"23 5","pages":""},"PeriodicalIF":2.4,"publicationDate":"2024-01-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139385685","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Scoping Review Protocol of Social Determinants of HIV/TB Coinfections in Sub-Saharan Africa","authors":"Lucas Banda, Olanrewaju Oladimeji","doi":"10.3390/mps7010004","DOIUrl":"https://doi.org/10.3390/mps7010004","url":null,"abstract":"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.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"67 38","pages":""},"PeriodicalIF":2.4,"publicationDate":"2024-01-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139385505","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Generation of Viral Particles with Brain Cell-Specific Tropism by Pseudotyping HIV-1 with the Zika Virus E Protein","authors":"Hai Dang Ngo, Jan Patrick Formanski, Vivien Grunwald, Birco Schwalbe, Michael Schreiber","doi":"10.3390/mps7010003","DOIUrl":"https://doi.org/10.3390/mps7010003","url":null,"abstract":"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.","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"67 8","pages":""},"PeriodicalIF":2.4,"publicationDate":"2023-12-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139150253","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}