Sidharth Beniwal, Ranjita K Bose, Anastasiia O Krushynska
Viscoelastic behavior can be beneficial in enhancing the unprecedented dynamics of polymer metamaterials or, in contrast, negatively impacting their wave control mechanisms. It is, therefore, crucial to properly characterize the viscoelastic properties of a polymer metamaterial at its working frequencies to understand viscoelastic effects. However, the viscoelasticity of polymers is a complex phenomenon, and the data on storage and loss moduli at ultrasonic frequencies are extremely limited, especially for additively manufactured polymers. This work presents a protocol to experimentally characterize the viscoelastic properties of additively manufactured polymers and to use them in the numerical analysis of polymer metamaterials. Specifically, the protocol includes the description of the manufacturing process, experimental procedures to measure the thermal, viscoelastic, and mechanical properties of additively manufactured polymers, and an approach to use these properties in finite-element simulations of the metamaterial dynamics. The numerical results are validated in ultrasonic transmission tests. To exemplify the protocol, the analysis is focused on acrylonitrile butadiene styrene (ABS) and aims at characterizing the dynamic behavior of a simple metamaterial made from it by using fused deposition modeling (FDM) three-dimensional (3D) printing. The proposed protocol will be helpful for many researchers to estimate viscous losses in 3D-printed polymer elastic metamaterials that will improve the understanding of material-property relations for viscoelastic metamaterials and eventually stimulate the use of 3D-printed polymer metamaterial parts in various applications.
{"title":"Characterizing Dissipative Elastic Metamaterials Produced by Additive Manufacturing.","authors":"Sidharth Beniwal, Ranjita K Bose, Anastasiia O Krushynska","doi":"10.3791/66898","DOIUrl":"https://doi.org/10.3791/66898","url":null,"abstract":"<p><p>Viscoelastic behavior can be beneficial in enhancing the unprecedented dynamics of polymer metamaterials or, in contrast, negatively impacting their wave control mechanisms. It is, therefore, crucial to properly characterize the viscoelastic properties of a polymer metamaterial at its working frequencies to understand viscoelastic effects. However, the viscoelasticity of polymers is a complex phenomenon, and the data on storage and loss moduli at ultrasonic frequencies are extremely limited, especially for additively manufactured polymers. This work presents a protocol to experimentally characterize the viscoelastic properties of additively manufactured polymers and to use them in the numerical analysis of polymer metamaterials. Specifically, the protocol includes the description of the manufacturing process, experimental procedures to measure the thermal, viscoelastic, and mechanical properties of additively manufactured polymers, and an approach to use these properties in finite-element simulations of the metamaterial dynamics. The numerical results are validated in ultrasonic transmission tests. To exemplify the protocol, the analysis is focused on acrylonitrile butadiene styrene (ABS) and aims at characterizing the dynamic behavior of a simple metamaterial made from it by using fused deposition modeling (FDM) three-dimensional (3D) printing. The proposed protocol will be helpful for many researchers to estimate viscous losses in 3D-printed polymer elastic metamaterials that will improve the understanding of material-property relations for viscoelastic metamaterials and eventually stimulate the use of 3D-printed polymer metamaterial parts in various applications.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141617457","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Due to their physiological similarities to humans, pigs are used as experimental models for ex vivo lung perfusion (EVLP). EVLP is a technique that perfuses lungs that are not suitable for transplantation via an extracorporeal circulation pump to improve their function and increase their viability. Existing EVLP protocols are differentiated by the type of perfusion solution and perfusion flow, which varies from 40%-100% of the estimated cardiac output (CO) according to the body surface area (BSA). Devices for measuring CO use simple physical principles and other mathematical models. Thermodilution in animal models continues to be the reference standard for estimating CO because of its simplicity and ease of reproduction. Therefore, the objective of this study was to reproduce the measurement of CO by thermodilution in pigs and compare its precision and accuracy with those obtained by the BSA, weight, and Fick's method, to establish perfusion flow during EVLP. In 23 pigs, a thermodilution catheter was placed in the right jugular vein, and the carotid artery on the same side was cannulated. Blood samples were obtained for gasometry, and CO was estimated by thermodilution, adjusted body surface area, Fick's principle, and per body weight. The CO obtained by the BSA was greater (p = 0.0001, ANOVA, Tukey) than that obtained by the other methods. We conclude that although the methods used in this study to estimate CO are reliable, there are significant differences between them; therefore, each method must be evaluated by the investigator to determine which meets the needs of the protocol.
由于猪的生理结构与人类相似,因此被用作体外肺灌注(EVLP)的实验模型。EVLP是一种通过体外循环泵对不适合移植的肺进行灌注以改善其功能和提高其存活率的技术。现有的 EVLP 方案根据灌注液和灌注流量的类型进行区分,根据体表面积(BSA)的不同,灌注流量为估计心输出量(CO)的 40%-100% 不等。测量 CO 的设备使用简单的物理原理和其他数学模型。动物模型中的热稀释法因其简单和易于复制,仍是估算 CO 的参考标准。因此,本研究的目的是在猪身上再现通过热稀释法测量一氧化碳的方法,并将其精度和准确度与通过 BSA、体重和菲克法获得的精度和准确度进行比较,以确定 EVLP 期间的灌注流量。在 23 头猪的右颈静脉中插入热稀释导管,并在同侧颈动脉上插管。采集血液样本用于气体测量,并通过热稀释、调整体表面积、菲克原理和单位体重估算一氧化碳。用 BSA 得出的一氧化碳浓度(P = 0.0001,方差分析,Tukey)高于用其他方法得出的浓度(P = 0.0001,方差分析,Tukey)。我们的结论是,虽然本研究中用于估算 CO 的方法都很可靠,但它们之间存在显著差异;因此,研究人员必须对每种方法进行评估,以确定哪种方法符合方案的需要。
{"title":"Determination of Cardiac Output in a Porcine Model for Ex Vivo Pulmonary Perfusion.","authors":"J Raúl Olmos-Zuñiga, Mariana Silva-Martínez, Claudia Hernández-Jiménez, Rogelio Jasso-Victoria, Matilde Baltazares-Lipp","doi":"10.3791/66798","DOIUrl":"https://doi.org/10.3791/66798","url":null,"abstract":"<p><p>Due to their physiological similarities to humans, pigs are used as experimental models for ex vivo lung perfusion (EVLP). EVLP is a technique that perfuses lungs that are not suitable for transplantation via an extracorporeal circulation pump to improve their function and increase their viability. Existing EVLP protocols are differentiated by the type of perfusion solution and perfusion flow, which varies from 40%-100% of the estimated cardiac output (CO) according to the body surface area (BSA). Devices for measuring CO use simple physical principles and other mathematical models. Thermodilution in animal models continues to be the reference standard for estimating CO because of its simplicity and ease of reproduction. Therefore, the objective of this study was to reproduce the measurement of CO by thermodilution in pigs and compare its precision and accuracy with those obtained by the BSA, weight, and Fick's method, to establish perfusion flow during EVLP. In 23 pigs, a thermodilution catheter was placed in the right jugular vein, and the carotid artery on the same side was cannulated. Blood samples were obtained for gasometry, and CO was estimated by thermodilution, adjusted body surface area, Fick's principle, and per body weight. The CO obtained by the BSA was greater (p = 0.0001, ANOVA, Tukey) than that obtained by the other methods. We conclude that although the methods used in this study to estimate CO are reliable, there are significant differences between them; therefore, each method must be evaluated by the investigator to determine which meets the needs of the protocol.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141617458","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Carolyn Bomidi, Xi-Lei Zeng, Victoria Poplaski, Cristian Coarfa, Mary K Estes, Sarah E Blutt
Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid cultures provide ex vivo model systems that bridge the gap between animal models and clinical studies. The application of single cell transcriptomics to human intestinal organoid (HIO) models is revealing previously unrecognized cell biology, biochemistry, and physiology of the GI tract. The advanced single cell transcriptomics platforms use microfluidic partitioning and barcoding to generate cDNA libraries. These barcoded cDNAs can be easily sequenced by next generation sequencing platforms and used by various visualization tools to generate maps. Here, we describe methods to culture and differentiate human small intestinal HIOs in different formats and procedures for isolating viable cells from these formats that are suitable for use in single-cell transcriptional profiling platforms. These protocols and procedures facilitate the use of small intestinal HIOs to obtain an increased understanding of the cellular response of human intestinal epithelium at the transcriptional level in the context of a variety of different environments.
{"title":"Using Human Intestinal Organoids to Understand the Small Intestine Epithelium at the Single Cell Transcriptional Level.","authors":"Carolyn Bomidi, Xi-Lei Zeng, Victoria Poplaski, Cristian Coarfa, Mary K Estes, Sarah E Blutt","doi":"10.3791/66749","DOIUrl":"10.3791/66749","url":null,"abstract":"<p><p>Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid cultures provide ex vivo model systems that bridge the gap between animal models and clinical studies. The application of single cell transcriptomics to human intestinal organoid (HIO) models is revealing previously unrecognized cell biology, biochemistry, and physiology of the GI tract. The advanced single cell transcriptomics platforms use microfluidic partitioning and barcoding to generate cDNA libraries. These barcoded cDNAs can be easily sequenced by next generation sequencing platforms and used by various visualization tools to generate maps. Here, we describe methods to culture and differentiate human small intestinal HIOs in different formats and procedures for isolating viable cells from these formats that are suitable for use in single-cell transcriptional profiling platforms. These protocols and procedures facilitate the use of small intestinal HIOs to obtain an increased understanding of the cellular response of human intestinal epithelium at the transcriptional level in the context of a variety of different environments.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141617429","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, including biopharmaceuticals. Glycan structures exhibit significant heterogeneity, varying with production cell types, culture conditions, and purification methods. Consequently, monitoring and evaluating the glycan structures of recombinant proteins is vital, particularly in biopharmaceutical production. The lectin microarray, a technique complementary to mass spectrometry, boasts high sensitivity and ease of use. However, it typically requires more than a day to yield results. To adapt it to non-glycoscience research or drug product process development, an automated, high-throughput alternative is needed. Therefore, the world's first fully automated lectin-based glycan profiling system was developed, utilizing the "bead array in a single tip (BIST)" technology concept. This system allows for the preparation and storage of lectin-immobilized beads in units of 1,000, with customizable parallel insertion orders for various purposes. This article presents a practical protocol for research involving "glyco-qualified" recombinant proteins. After testing their reactivity against 12 polyacrylamide-glycan conjugates, 15 lectins were selected to increase the system's versatility. In addition, the sample labeling process was optimized by switching from Cy3 to biotin, reducing the overall processing time by 30 min. For immediate data qualification, lectin-binding signals are displayed as a dotcode on the top monitor. The system's reliability was confirmed through day-to-day reproducibility tests, repeatability tests, and long-term storage tests, with a coefficient of variation of <10%. This user-friendly and rapid glyco-analyzer has potential applications in the quality monitoring of endogenous glycoproteins for biomarker evaluation and validation. This method facilitates analysis for those new to glycoscience, thereby broadening its practical utility.
{"title":"Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System.","authors":"Sayaka Fuseya, Ayaka Ono, Hiromi Ootani, Saho Mizukado, Tomomi Obayashi, Nana Tanaka, Hiroko Shimazaki, Kenji Kajiyama, Moe Ashitomi, Shiori Yasuda, Takenori Miyabe, Kazuhiro Nakamura, Osamu Segawa, Kazumi Sawakami, Atsushi Kuno","doi":"10.3791/66571","DOIUrl":"https://doi.org/10.3791/66571","url":null,"abstract":"<p><p>Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, including biopharmaceuticals. Glycan structures exhibit significant heterogeneity, varying with production cell types, culture conditions, and purification methods. Consequently, monitoring and evaluating the glycan structures of recombinant proteins is vital, particularly in biopharmaceutical production. The lectin microarray, a technique complementary to mass spectrometry, boasts high sensitivity and ease of use. However, it typically requires more than a day to yield results. To adapt it to non-glycoscience research or drug product process development, an automated, high-throughput alternative is needed. Therefore, the world's first fully automated lectin-based glycan profiling system was developed, utilizing the \"bead array in a single tip (BIST)\" technology concept. This system allows for the preparation and storage of lectin-immobilized beads in units of 1,000, with customizable parallel insertion orders for various purposes. This article presents a practical protocol for research involving \"glyco-qualified\" recombinant proteins. After testing their reactivity against 12 polyacrylamide-glycan conjugates, 15 lectins were selected to increase the system's versatility. In addition, the sample labeling process was optimized by switching from Cy3 to biotin, reducing the overall processing time by 30 min. For immediate data qualification, lectin-binding signals are displayed as a dotcode on the top monitor. The system's reliability was confirmed through day-to-day reproducibility tests, repeatability tests, and long-term storage tests, with a coefficient of variation of <10%. This user-friendly and rapid glyco-analyzer has potential applications in the quality monitoring of endogenous glycoproteins for biomarker evaluation and validation. This method facilitates analysis for those new to glycoscience, thereby broadening its practical utility.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141617427","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Craig McBeth, David Brown, Pawel Pokorski, Lydia Lei, Vicki Stone
Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive oxygen species studies. The method utilizes Ortho-phthalaldehyde (OPA) to quantify the cellular concentration of glutathione(s). OPA conjugates with reduced glutathione (GSH) via sulfhydryl binding to subsequently form an isoindole, resulting in a highly fluorescent conjugate. To attain an accurate result of both oxidized glutathione (GSSG) and GSH, a combination of masking agents and reducing agents, which have been implemented in this protocol, are required. Treatments may also impact cellular viability. Hence, normalization via protein assay is presented in this multiparametric assay. The assay demonstrates a pseudo-linear detection range of 0.234 - 30µM (R2=0.9932±0.007 (N=12)) specific to GSH. The proposed assay also allows for the determination of oxidized glutathione with the addition of the masking agent N-ethylmaleimide to bind reduced glutathione, and the reducing agent tris(2-carboxyethyl) phosphine is introduced to cleave the disulfide bond in GSSG to produce two molecules of GSH. The assay is used in combination with a validated bicinchoninic acid assay for protein quantification and an adenylate kinase assay for cytotoxicity assessment.
{"title":"Rapid Quantification of Oxidized and Reduced Forms of Glutathione Using Ortho -phthalaldehyde in Cultured Mammalian Cells In Vitro.","authors":"Craig McBeth, David Brown, Pawel Pokorski, Lydia Lei, Vicki Stone","doi":"10.3791/66267","DOIUrl":"https://doi.org/10.3791/66267","url":null,"abstract":"<p><p>Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive oxygen species studies. The method utilizes Ortho-phthalaldehyde (OPA) to quantify the cellular concentration of glutathione(s). OPA conjugates with reduced glutathione (GSH) via sulfhydryl binding to subsequently form an isoindole, resulting in a highly fluorescent conjugate. To attain an accurate result of both oxidized glutathione (GSSG) and GSH, a combination of masking agents and reducing agents, which have been implemented in this protocol, are required. Treatments may also impact cellular viability. Hence, normalization via protein assay is presented in this multiparametric assay. The assay demonstrates a pseudo-linear detection range of 0.234 - 30µM (R<sup>2</sup>=0.9932±0.007 (N=12)) specific to GSH. The proposed assay also allows for the determination of oxidized glutathione with the addition of the masking agent N-ethylmaleimide to bind reduced glutathione, and the reducing agent tris(2-carboxyethyl) phosphine is introduced to cleave the disulfide bond in GSSG to produce two molecules of GSH. The assay is used in combination with a validated bicinchoninic acid assay for protein quantification and an adenylate kinase assay for cytotoxicity assessment.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141617428","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Kun Li, Jin Huang, Ahmad Alhaskawi, Bin Wang, Nianbin Ma, Chengjun Yao, Yanzhao Dong, QingFang Zhao, Xiaodi Zou, Hui Lu
Malnutrition is a common issue in critically ill patients, often stemming from illness, injury, or surgery. Prolonged fasting leads to intestinal issues, emphasizing the importance of early enteral nutrition, specifically through jejunal nutrition. While enteral nutrition is crucial, complications with current techniques exist. Nasojejunal (NJ) tubes are commonly used, with placement methods categorized as surgical or non-surgical. Non-surgical methods, including endoscopic guidance, have varying success rates, with endoscopic-assisted placement being the most successful but requiring specialized expertise and logistics. This study introduces a bedside, visualized method for NJ tube placement to enhance success rates and reduce patient discomfort in the intensive care unit (ICU). In this study involving 19 ICU patients, the method achieved an initial success rate of 94.74% with an average insertion time of 11.2 ± 6.4 min. This visualized method demonstrates efficiency and reduces the need for additional imaging, and the introduction of a miniaturized endoscope shows promise, enabling successful intubation at the bedside and minimizing patient discomfort. Adjustments to the guidewire lens and catheter are necessary but pose opportunities for future refinements.
{"title":"A Minimally Invasive, Visualized Method for Nasojejunal Tube Placement.","authors":"Kun Li, Jin Huang, Ahmad Alhaskawi, Bin Wang, Nianbin Ma, Chengjun Yao, Yanzhao Dong, QingFang Zhao, Xiaodi Zou, Hui Lu","doi":"10.3791/66551","DOIUrl":"https://doi.org/10.3791/66551","url":null,"abstract":"<p><p>Malnutrition is a common issue in critically ill patients, often stemming from illness, injury, or surgery. Prolonged fasting leads to intestinal issues, emphasizing the importance of early enteral nutrition, specifically through jejunal nutrition. While enteral nutrition is crucial, complications with current techniques exist. Nasojejunal (NJ) tubes are commonly used, with placement methods categorized as surgical or non-surgical. Non-surgical methods, including endoscopic guidance, have varying success rates, with endoscopic-assisted placement being the most successful but requiring specialized expertise and logistics. This study introduces a bedside, visualized method for NJ tube placement to enhance success rates and reduce patient discomfort in the intensive care unit (ICU). In this study involving 19 ICU patients, the method achieved an initial success rate of 94.74% with an average insertion time of 11.2 ± 6.4 min. This visualized method demonstrates efficiency and reduces the need for additional imaging, and the introduction of a miniaturized endoscope shows promise, enabling successful intubation at the bedside and minimizing patient discomfort. Adjustments to the guidewire lens and catheter are necessary but pose opportunities for future refinements.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141617453","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Quantum dots, also known as semiconductor nanocrystals, are novel fluorescent labels for biological imaging and sensing. However, quantum dot-antibody conjugates with small dimensions (~10 nm), prepared through laborious purification procedures, exhibit limited sensitivity in detecting certain trace disease markers using lateral flow immunoassay strips. Herein, we present a method for the preparation of quantum dot nanobeads (QDNB) using a one-step emulsion evaporation method. Using the as-prepared QDNB, a fluorescent lateral flow immunoassay was fabricated to detect disease biomarkers using C-reactive protein (CRP) as an example. Unlike single quantum dot nanoparticles, quantum dot nanobead-antibody conjugates are more sensitive as immunoassay labels due to signal amplification by encapsulating hundreds of quantum dots in one polymer composite nanobead. Moreover, the larger size of QDNBs facilitates easier centrifugation separation when conjugating QDNBs with antibodies. The fluorescent lateral flow immunoassay based on QDNBs was fabricated, and the CRP concentration in the sample was measured in 15 min. The test results can be qualitatively assessed under UV light illumination and quantitatively measured using a fluorescent reader within 15 min.
{"title":"Fluorescent Lateral Flow Immunoassay Based on Quantum Dots Nanobeads.","authors":"Lingzhi Fan, Yue Luo, Wannian Yan, Huanxing Han, Pengfei Zhang","doi":"10.3791/67000","DOIUrl":"https://doi.org/10.3791/67000","url":null,"abstract":"<p><p>Quantum dots, also known as semiconductor nanocrystals, are novel fluorescent labels for biological imaging and sensing. However, quantum dot-antibody conjugates with small dimensions (~10 nm), prepared through laborious purification procedures, exhibit limited sensitivity in detecting certain trace disease markers using lateral flow immunoassay strips. Herein, we present a method for the preparation of quantum dot nanobeads (QDNB) using a one-step emulsion evaporation method. Using the as-prepared QDNB, a fluorescent lateral flow immunoassay was fabricated to detect disease biomarkers using C-reactive protein (CRP) as an example. Unlike single quantum dot nanoparticles, quantum dot nanobead-antibody conjugates are more sensitive as immunoassay labels due to signal amplification by encapsulating hundreds of quantum dots in one polymer composite nanobead. Moreover, the larger size of QDNBs facilitates easier centrifugation separation when conjugating QDNBs with antibodies. The fluorescent lateral flow immunoassay based on QDNBs was fabricated, and the CRP concentration in the sample was measured in 15 min. The test results can be qualitatively assessed under UV light illumination and quantitatively measured using a fluorescent reader within 15 min.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141617460","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Karin Tourle, Amber Rucinski, Alexander Grainger, Ioannis J Limnios, Macarena Gonzalez Ruiz, Jonathan K H Tan
The spleen is an immune organ that plays a key role in blood-borne immune responses. The anatomical or functional loss of this tissue increases susceptibility to severe blood infections and sepsis. Auto-transplantation of spleen slices has been used clinically to replace lost tissue and restore immune function. However, the mechanism driving robust and immunologically functional spleen tissue regeneration has not been fully elucidated. Here, we aim to develop a method for aggregating and encapsulating spleen cells within a semi-solid matrix in order to investigate the cellular requirements for spleen tissue formation. Basement membrane matrix encapsulated cell constructs are amenable to both in vitro tissue culture of three-dimensional organoids as well as transplantation under the kidney capsule to directly assess in vivo tissue formation. By manipulating the input cells for aggregation and encapsulation, we demonstrate that graft-derived PDGFRβ+MAdCAM-1- neonatal stromal cells are required for spleen tissue regeneration under animal transplantation models.
{"title":"Basement Membrane Matrix Encapsulated Cell Aggregation for Investigating Murine Spleen Tissue Formation.","authors":"Karin Tourle, Amber Rucinski, Alexander Grainger, Ioannis J Limnios, Macarena Gonzalez Ruiz, Jonathan K H Tan","doi":"10.3791/66682","DOIUrl":"https://doi.org/10.3791/66682","url":null,"abstract":"<p><p>The spleen is an immune organ that plays a key role in blood-borne immune responses. The anatomical or functional loss of this tissue increases susceptibility to severe blood infections and sepsis. Auto-transplantation of spleen slices has been used clinically to replace lost tissue and restore immune function. However, the mechanism driving robust and immunologically functional spleen tissue regeneration has not been fully elucidated. Here, we aim to develop a method for aggregating and encapsulating spleen cells within a semi-solid matrix in order to investigate the cellular requirements for spleen tissue formation. Basement membrane matrix encapsulated cell constructs are amenable to both in vitro tissue culture of three-dimensional organoids as well as transplantation under the kidney capsule to directly assess in vivo tissue formation. By manipulating the input cells for aggregation and encapsulation, we demonstrate that graft-derived PDGFRβ<sup>+</sup>MAdCAM-1<sup>-</sup> neonatal stromal cells are required for spleen tissue regeneration under animal transplantation models.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141617456","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sefa Ozturk, Cafer Ikbal Gulsever, Duran Sahin, Fatih Koksoy, Alperen Poyraz, Duygu Dolen
Pineal neoplasms have a significant impact on children although they are relatively uncommon. They account for approximately 3-11% of all childhood brain tumors, which is considerably higher than the <1% seen in adult brain tumors. These tumors can be divided into three main categories: germ cell tumors, parenchymal pineal tumors, and tumors arising from related anatomical structures. Obtaining an accurate and minimally invasive tissue diagnosis is crucial for selecting the most appropriate treatment regimen for patients with pineal gland tumors. This is due to the diverse treatment options available and the potential risks associated with complete resection. In cases where patients present with acute obstructive hydrocephalus caused by a pineal gland tumor, immediate treatment of the hydrocephalus is necessary. The urgency stems from the potential complications of hydrocephalus, including increased intracranial pressure and neurological deficits. To address these challenges, a minimally invasive endoscopic approach provides a valuable opportunity. This technique allows clinicians to promptly relieve hydrocephalus and obtain a histological diagnosis simultaneously. This dual benefit enables a more comprehensive understanding of the tumor and assists in determining the most effective treatment strategy for the patient.
{"title":"Endoscopic Third Ventriculostomy and Pineal Biopsy from a Single Entry Point.","authors":"Sefa Ozturk, Cafer Ikbal Gulsever, Duran Sahin, Fatih Koksoy, Alperen Poyraz, Duygu Dolen","doi":"10.3791/66837","DOIUrl":"https://doi.org/10.3791/66837","url":null,"abstract":"<p><p>Pineal neoplasms have a significant impact on children although they are relatively uncommon. They account for approximately 3-11% of all childhood brain tumors, which is considerably higher than the <1% seen in adult brain tumors. These tumors can be divided into three main categories: germ cell tumors, parenchymal pineal tumors, and tumors arising from related anatomical structures. Obtaining an accurate and minimally invasive tissue diagnosis is crucial for selecting the most appropriate treatment regimen for patients with pineal gland tumors. This is due to the diverse treatment options available and the potential risks associated with complete resection. In cases where patients present with acute obstructive hydrocephalus caused by a pineal gland tumor, immediate treatment of the hydrocephalus is necessary. The urgency stems from the potential complications of hydrocephalus, including increased intracranial pressure and neurological deficits. To address these challenges, a minimally invasive endoscopic approach provides a valuable opportunity. This technique allows clinicians to promptly relieve hydrocephalus and obtain a histological diagnosis simultaneously. This dual benefit enables a more comprehensive understanding of the tumor and assists in determining the most effective treatment strategy for the patient.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141617459","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Inna Blokina, Egor Iluykov, Dmitry Myagkov, Dmitry Tuktarov, Sergey Popov, Timofey Inozemzev, Ivan Fedosov, Alexander Shirokov, Andrey Terskov, Alexander Dmitrenko, Arina Evsyukova, Daria Zlatogorskaya, Viktoria Adushkina, Matvey Tuzhilkin, Maria Manzhaeva, Valeria Krupnova, Alexander Dubrovsky, Inna Elizarova, Maria Tzoy, Oxana Semyachkina-Glushkovskaya
The meningeal lymphatic vessels (MLVs) play an important role in the removal of toxins from the brain. The development of innovative technologies for the stimulation of MLV functions is a promising direction in the progress of the treatment of various brain diseases associated with MLV abnormalities, including Alzheimer's and Parkinson's diseases, brain tumors, traumatic brain injuries, and intracranial hemorrhages. Sleep is a natural state when the brain's drainage processes are most active. Therefore, stimulation of the brain's drainage and MLVs during sleep may have the most pronounced therapeutic effects. However, such commercial technologies do not currently exist. This study presents a new portable technology of transcranial photobiomodulation (tPBM) under electroencephalographic (EEG) control of sleep designed to photo-stimulate removal of toxins (e.g., soluble amyloid beta (Aβ)) from the brain of aged BALB/c mice with the ability to compare the therapeutic effectiveness of different optical resources. The technology can be used in the natural condition of a home cage without anesthesia, maintaining the motor activity of mice. These data open up new prospects for developing non-invasive and clinically promising photo-technologies for the correction of age-related changes in the MLV functions and brain's drainage processes and for effectively cleansing brain tissues from metabolites and toxins. This technology is intended both for preclinical studies of the functions of the sleeping brain and for developing clinically relevant treatments for sleep-related brain diseases.
{"title":"Photobiomodulation under Electroencephalographic Controls of Sleep for Stimulation of Lymphatic Removal of Toxins from Mouse Brain.","authors":"Inna Blokina, Egor Iluykov, Dmitry Myagkov, Dmitry Tuktarov, Sergey Popov, Timofey Inozemzev, Ivan Fedosov, Alexander Shirokov, Andrey Terskov, Alexander Dmitrenko, Arina Evsyukova, Daria Zlatogorskaya, Viktoria Adushkina, Matvey Tuzhilkin, Maria Manzhaeva, Valeria Krupnova, Alexander Dubrovsky, Inna Elizarova, Maria Tzoy, Oxana Semyachkina-Glushkovskaya","doi":"10.3791/67035","DOIUrl":"https://doi.org/10.3791/67035","url":null,"abstract":"<p><p>The meningeal lymphatic vessels (MLVs) play an important role in the removal of toxins from the brain. The development of innovative technologies for the stimulation of MLV functions is a promising direction in the progress of the treatment of various brain diseases associated with MLV abnormalities, including Alzheimer's and Parkinson's diseases, brain tumors, traumatic brain injuries, and intracranial hemorrhages. Sleep is a natural state when the brain's drainage processes are most active. Therefore, stimulation of the brain's drainage and MLVs during sleep may have the most pronounced therapeutic effects. However, such commercial technologies do not currently exist. This study presents a new portable technology of transcranial photobiomodulation (tPBM) under electroencephalographic (EEG) control of sleep designed to photo-stimulate removal of toxins (e.g., soluble amyloid beta (Aβ)) from the brain of aged BALB/c mice with the ability to compare the therapeutic effectiveness of different optical resources. The technology can be used in the natural condition of a home cage without anesthesia, maintaining the motor activity of mice. These data open up new prospects for developing non-invasive and clinically promising photo-technologies for the correction of age-related changes in the MLV functions and brain's drainage processes and for effectively cleansing brain tissues from metabolites and toxins. This technology is intended both for preclinical studies of the functions of the sleeping brain and for developing clinically relevant treatments for sleep-related brain diseases.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141617464","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}