Methods and Protocols最新文献

{"title":"Optimization of Methodologies to Study Freeze/Thaw Processes in Drug Substance Bottles.","authors":"Sarah S Peláez, Hanns-Christian Mahler, Jörg Huwyler, Andrea Allmendinger","doi":"10.3390/mps7050068","DOIUrl":"10.3390/mps7050068","url":null,"abstract":"<p><p>Biological drug substance (DS) is often frozen to enhance storage stability, prolong shelf life, and increase flexibility during manufacturing. However, the freezing and thawing (F/T) of bulk DS at the manufacturing scale can impact product quality as a result of various critical conditions, including cryo-concentration during freezing, which are influenced, among other things, by product-independent process parameters (e.g., container type, fill level, F/T equipment, and protocols). In this article, we report the optimization of two major methodologies to study product-independent process parameters in DS bottles at the manufacturing scale, namely the recording of temperature profiles and liquid sampling after thawing to quantify the concentration gradients in the solution. We report experimentally justified measuring positions for temperature recordings, especially for the selection of the last point to freeze position, and highlight the implementation of camera-assisted inspection to determine the last point to thaw and the actual thawing time. In particular, we provide, for the first time, a detailed description of the technical implementation of these two measuring set-ups. Based on the reported case studies, we recommend choosing relevant measuring positions as a result of initial equipment characterization, resulting in a resource-conscious study set-up.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 5","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11417747/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291455","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":"CO₂-Free On-Stage Incubator for Live Cell Imaging of Cholangiocarcinoma Cell Migration on Microfluidic Device.","authors":"Shahab Ud Din, Puey Ounjai, Arthit Chairoungdua, Werasak Surareungchai","doi":"10.3390/mps7050069","DOIUrl":"10.3390/mps7050069","url":null,"abstract":"<p><p>Long-term live cell imaging requires sophisticated and fully automated commercial-stage incubators equipped with specified inverted microscopes to regulate temperature, CO₂ content, and humidity. In this study, we present a CO₂-free on-stage incubator specifically designed for use across various cell culture platforms, enabling live cell imaging applications. A simple and transparent incubator was fabricated from acrylic sheets to be easily placed on the stages of most inverted microscopes. We successfully performed live-cell imaging of cholangiocarcinoma (CCA) cells and HeLa cell dynamics in both 2D and 3D microenvironments over three days. We also analyzed directed cell migration under high serum induction within a microfluidic device. Interesting phenomena such as \"whole-colony migration\", \"novel type of collective cell migration\" and \"colony formation during cell and colony migration\" are reported here for the first time, to the best of our knowledge. These phenomena may improve our understanding of the nature of cell migration and cancer metastasis.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 5","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11417791/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291448","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":"Comparison of Antigen Retrieval Methods for Immunohistochemical Analysis of Cartilage Matrix Glycoproteins Using Cartilage Intermediate Layer Protein 2 (CILP-2) as an Example.","authors":"Taavi Torga, Siim Suutre, Kalle Kisand, Marina Aunapuu, Andres Arend","doi":"10.3390/mps7050067","DOIUrl":"10.3390/mps7050067","url":null,"abstract":"<p><p>The aim of this study was to compare different antigen retrieval methods to improve the outcome of immunohistochemistry (IHC) performed on osteoarthritic (OA) cartilage obtained from total knee replacement operation. A voluminous and dense extracellular matrix of articular cartilage inhibits antibody penetration, and therefore, proteins present at low concentrations and masked during fixation may need antigen retrieval to enhance an IHC outcome. We focused on the IHC detection of a minor but diagnostically promising cartilage glycoprotein, CILP-2 (cartilage intermediate layer protein 2), to demonstrate the effect of four different protocols: (1) heat-induced epitope retrieval (HIER), (2) proteolytic-induced epitope retrieval applying proteinase K and hyaluronidase (PIER), (3) HIER combined with PIER, and (4) no antigen retrieval (control). A semi-quantitative staining assessment based on the CILP-2 staining extent was applied. Out of the tested antigen retrieval protocols, the best CILP-2 IHC staining results were achieved by PIER. Combining PIER with HIER did not improve CILP-2 staining in the given experimental setting. Rather the opposite, the application of heat reduced the positive effect of PIER on CILP-2 staining and resulted in the frequent detachment of sections from the slides. Our findings emphasize the need for proper adaptation of antigen retrieval protocols for IHC to maximize the quantitative evaluation of minor matrix proteins in OA articular cartilage samples.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 5","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-08-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11417793/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291450","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":"Nanoparticle Lysis of <i>Cryptosporidium</i> Oocysts.","authors":"Ameya Vaidya, Claire Bankier, Helinor Johnston, Helen Bridle","doi":"10.3390/mps7050066","DOIUrl":"10.3390/mps7050066","url":null,"abstract":"<p><p>The extraction of DNA from <i>Cryptosporidium</i> oocysts is challenging due to the robust oocyst wall. Nanoparticles have been applied to disinfect <i>Cryptosporidium</i> oocysts; here, we demonstrate the utilisation of nanoparticles to disrupt the oocyst wall to enable sporozoite lysis and detection via PCR. Both silver and zinc oxide nanoparticles are investigated under different conditions and compared to existing techniques. Zinc oxide nanoparticles are shown to be as effective as freeze-thaw methods, suggesting that a nanoparticle lysis approach offers a viable alternative to existing methods.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 5","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11417895/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142291454","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":"Parasitological Examination of the Digestive System of Wild Boar from a Practical Point of View-Endoparasitological Sampling under Field Conditions.","authors":"Csaba Farkas, Alexandra Juhász, Balázs Fekete, Borisz Egri","doi":"10.3390/mps7040065","DOIUrl":"10.3390/mps7040065","url":null,"abstract":"<p><p>From 2015 to 2023, we conducted a comprehensive study in the 11,893-hectare hunting area managed by the Marcal-Bitvaközi Hunting Company, characterised by its substantial wild boar population. The research was carried out across various settings, including a free-range wild boar garden during large-scale hunts and free-living areas during individual hunts. We examined 216 wild boars in total, with 173 individuals from free-living areas and 43 from free-range areas. Throughout the sample collection process, we encountered numerous technical challenges that are infrequently detailed in the professional literature, often mentioned only tangentially. This oversight in existing publications neglects the significance of addressing field sampling difficulties, which are crucial for ensuring the precision and accuracy of research. This paper details the equipment requirements, sampling methodologies, and practical solutions to streamline fieldwork. While our primary focus was on endoparasitic infections of the stomach and small intestine, the described methodologies and findings are broadly applicable to research involving all internal organs.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 4","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11356853/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142080916","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":"Full Validation and Application to Clinical Research of a High-Performance Liquid Chromatography Method for the Assessment of Urinary 3-Indoxyl Sulfate in Pediatric Patients with Hematopoietic Stem Cell Transplant.","authors":"Christian Ezequiel Olivetti, María Florencia Fernández, Jana Stojanova, Silvina Ruvinsky, Andrea Mangano, Paula Schaiquevich","doi":"10.3390/mps7040064","DOIUrl":"10.3390/mps7040064","url":null,"abstract":"<p><p>3-indoxyl sulfate (3-IS) results from a hepatic transformation of indole, a tryptophan degradation product produced by commensal gut bacteria. The metabolite has shown promise as a biomarker of dysbiosis and clinical outcomes following hematopoietic stem cell transplant (HSCT) in adults. Nonetheless, there is a paucity of data regarding microbiome health and outcomes in the pediatric HSCT setting. We developed and thoroughly validated an affordable high-performance liquid chromatography/fluorescence detector (HPLC-FLD) method to quantify 3-IS in urine for use in the pediatric setting. Chromatographic separation was achieved on a C18 column (250 × 4.6 mm × 5 μm) with a mobile phase consisting of pH 4.0 acetic acid-triethylamine buffer and acetonitrile (88:12, <i>v</i>/<i>v</i>), eluted isocratically at 1 mL/min. 3-IS fluorescence detection was set at excitation/emission of 280 and 375, respectively. The method was fully validated according to FDA-specified limits including selectivity, linearity (0.10 to 10.00 mg/L, r² > 0.997), intra- and inter-day accuracy, and precision. 3-IS stability was confirmed after three freeze-thaw cycles, for short- and medium-term on a benchtop and at 4 °C and for long-term up to 60 days at -20 °C. The validated method was used to quantify 3-IS in urine samples from HSCT pediatric patients.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 4","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11357030/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142080915","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":"A Mathematical Model of Pressure Ulcer Formation to Facilitate Prevention and Management.","authors":"Ioannis G Violaris, Konstantinos Kalafatakis, Nikolaos Giannakeas, Alexandros T Tzallas, Markos Tsipouras","doi":"10.3390/mps7040062","DOIUrl":"10.3390/mps7040062","url":null,"abstract":"<p><p>Pressure ulcers are a frequent issue involving localized damage to the skin and underlying tissues, commonly arising from prolonged hospitalization and immobilization. This paper introduces a mathematical model designed to elucidate the mechanics behind pressure ulcer formation, aiming to predict its occurrence and assist in its prevention. Utilizing differential geometry and elasticity theory, the model represents human skin and simulates its deformation under pressure. Additionally, a system of ordinary differential equations is employed to predict the outcomes of these deformations, estimating the cellular death rate in skin tissues and underlying layers. The model also incorporates changes in blood flow resulting from alterations in skin geometry. This comprehensive approach provides new insights into the optimal bed surfaces required to prevent pressure ulcers and offers a general predictive method to aid healthcare personnel in making informed decisions for at-risk patients. Compared to existing models in the literature, our model delivers a more thorough prediction method that aligns well with current data. It can forecast the time required for an immobilized individual to develop an ulcer in various body parts, considering different initial health conditions and treatment strategies.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 4","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11356783/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142080889","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":"Evaluation of an Italian Population-Based Programme for Risk Assessment and Genetic Counselling and Testing for BRCA1/2-Related Hereditary Breast and Ovarian Cancer after 10 Years of Operation: An Observational Study Protocol.","authors":"Stefano Ferretti, Priscilla Sassoli de Bianchi, Debora Canuti, Cinzia Campari, Laura Cortesi, Valentina Arcangeli, Elena Barbieri, Cecilia D'Aloia, Rita Danesi, Pierandrea De Iaco, Margherita De Lillo, Laura Lombardo, Gabriella Moretti, Antonino Musolino, Dante Palli, Caterina Palmonari, Mila Ravegnani, Alfredo Tafà, Alessandra Tononi, Daniela Turchetti, Claudio Zamagni, Valentina Zampiga, Lauro Bucchi, The Hboc Study Group","doi":"10.3390/mps7040063","DOIUrl":"10.3390/mps7040063","url":null,"abstract":"<p><p>Hereditary breast/ovarian cancer (HBOC) syndrome is caused by the inheritance of monoallelic germline BRCA1/2 gene mutations. If BRCA1/2 mutation carriers are identified before the disease develops, effective actions against HBOC can be taken, including intensive screening, risk-reducing mastectomy and salpingo-oophorectomy, and risk-reducing medications. The Italian National Prevention Plan mandates the creation of regional BRCA genetic testing programmes. So far, however, only informal data have been reported on their implementation. We have designed a study aimed at evaluating the results of a population-based programme for risk assessment and genetic counselling and testing for BRCA1/2-related HBOC that is underway in the Emilia-Romagna region (northern Italy). The programme-which is entirely free-includes basic screening with an estimate of the likelihood of carrying a BRCA1/2 mutation using a familial risk assessment tool, a closer examination of women with suspected risk increase, an assessment of the need for further genetic counselling and, if needed, genetic testing and risk-reducing interventions. In this paper, the design of the programme and the protocol of the study are presented. The study has an observational, historical cohort design. Eligible are the women found to be at an increased risk of HBOC (profile 3 women). The main objectives are (i) to determine the precision of the programme in measuring the level of risk of HBOC for profile 3 women; (ii) to determine the characteristics of profile 3 women and their association with the risk management strategy chosen; (iii) to compare the age at onset, histologic type, tumour stage, molecular subtype, and prognosis of breast/ovarian cancers observed in the cohort of profile 3 women with the features of sporadic cancers observed in the general female population; (iv) to determine the level and the determinants of adherence to recommendations; and (v) to determine the appropriateness and timing of risk-reducing surgery and medications. Investigating the quality and results of the programme is necessary because the best practices in risk assessment and genetic counselling and testing for BRCA1/2-related cancer and the challenges they encounter should be identified and shared. The study has the potential to provide sound empirical evidence for the factors affecting the effectiveness of this type of service.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 4","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11357531/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142080914","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":"The Sri Lanka Mother and Newborn Growth (S-MaNGro) Cohort: Protocol of a Nationwide Prospective Study.","authors":"Malshani Lakshika Pathirathna, Megumi Haruna, Satoshi Sasaki, Kaori Yonezawa, Yuriko Usui, Yasuhiro Hagiwara","doi":"10.3390/mps7040061","DOIUrl":"10.3390/mps7040061","url":null,"abstract":"<p><p>Perinatal cohort studies with a prospective longitudinal design are critical for determining the effects of early-life exposures on offspring's health outcomes. The Sri Lanka Mother and Newborn Growth cohort study aims to investigate the impact of maternal nutritional and psychosocial factors on newborns' birth weight in the Sri Lankan context. This paper presents the methodology of participant recruitment, follow-ups, an overview of measurements, and planned data analyses. This study included a nationally representative sample of Sri Lankan pregnant women recruited in their first trimester of pregnancy. Follow-up assessments were conducted once during the second and third trimesters of pregnancy and after the baby's birth, prospectively tracking the women's dietary intake, mental health, hemoglobin concentrations, and gestational weight gain data. Once the participants delivered their babies, the data on gestational age, sex of the newborn, birth weight, length and occipitofrontal circumference at birth, and mode of delivery were collected. Between August 2022 and August 2023, we recruited 2000 first-trimester pregnant women to the cohort and continued to follow up with them until the baby's birth. The response rates were 90.4%, 81.4%, and 75.2% in the first, second, and third follow-ups. We plan to analyze the data in July 2024. We expect this study to provide valuable insights into various early-life exposures affecting neonatal birth weight. The study's findings will serve as a valuable information resource for a broader scientific community, enabling the development of effective policies to prevent low-birth-weight deliveries in low-resource settings.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 4","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11356845/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142080919","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":"Phytochemical Composition and Antimicrobial and Antibiofilm Effect of <i>Myrciaria cauliflora</i> Hydroethanolic Extract against <i>Staphylococcus aureus</i> and <i>Acinetobacter baumannii</i>.","authors":"Luciane Dias de Oliveira, Ana Luisa Monteiro Ribeiro, Sthéfani de Oliveira Dias, Geovani Moreira da Cruz, Raquel Teles de Menezes, Lara Steffany de Carvalho, Mariana Gadelho Gimenez Diamantino, Thaís Cristine Pereira, Maria Cristina Marcucci, Amjad Abu Hasna","doi":"10.3390/mps7040060","DOIUrl":"10.3390/mps7040060","url":null,"abstract":"<p><p><i>Staphylococcus aureus</i> and <i>Acinetobacter baumannii</i> are opportunistic pathogens, and both are involved in different oral infections. This work aimed to analyze the phytochemical composition of <i>Myrciaria cauliflora</i> hydroethanolic extract and to evaluate its antimicrobial and antibiofilm action against <i>Staphylococcus aureus</i> (ATCC 6538) and <i>Acinetobacter baumannii</i> (ATCC 19606; multi-resistant clinical strains 58004, 50098, 566006, and H557). <i>Myrciaria cauliflora</i> hydroethanolic extract was prepared, and the content of soluble solids, flavonoids, and phenols was quantified. High-performance liquid chromatography (HPLC) was performed later. The minimum inhibitory concentration was determined using the broth microdilution method according to the Clinical and Laboratory Standards Institute, standard M7-A6, and subsequently, its minimum bactericidal concentration was determined. Then, the most effective concentrations were analyzed against biofilms. Statistical analysis was performed using the ANOVA method with Tukey's test. The soluble solids content in the prepared hydroethanolic extract of <i>M. cauliflora</i> was 2.22%. Additionally, the total flavonoid content, measured using the quercetin standard curve, was 0.040 mg/mL. Furthermore, the total phenol content, determined using the gallic acid standard curve, was 0.729 mg/mL. HPLC analysis presented peaks of gallic acid (11.80 m), p-coumaric acid (12.09 m), cinnamic acid derivative (19.02 m), and ellagic acid (29.83 m). The extract demonstrated antimicrobial and antibiofilm action against all tested strains. However, the most effective antibacterial concentration against all the tested bacteria was 5.55 mg/mL. Therefore, these chemical components justify that <i>M. cauliflora</i> hydroethanolic extract is effective in reducing biofilm formation in <i>S. aureus</i> (standard strain) and <i>A. baumannii</i> (standard and clinical strains).</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"7 4","pages":""},"PeriodicalIF":2.3,"publicationDate":"2024-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11357044/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142080917","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}