Jove-Journal of Visualized Experiments最新文献_第10页

Individualized Reconstitution of Human Milk Microbiota: A Feasible Approach in Real-World Settings.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-02-07 DOI: 10.3791/67769

Leonardo Gonçalves Gomes, Lucas de Figueiredo Soveral, Izadora Borgmann Frizzo, Thaise Brancher Soncini, Lívia Budziarek Eslabão, Daiana Silva Marcos Maniero, Isis Maia Apolinário de Mello, Jussara Kasuko Palmeiro, Thaís Cristine Marques Sincero, Oscar Bruna-Romero, Maria Marlene de Souza Pires, Carlos Rodrigo Zárate-Bladés

{"title":"Individualized Reconstitution of Human Milk Microbiota: A Feasible Approach in Real-World Settings.","authors":"Leonardo Gonçalves Gomes, Lucas de Figueiredo Soveral, Izadora Borgmann Frizzo, Thaise Brancher Soncini, Lívia Budziarek Eslabão, Daiana Silva Marcos Maniero, Isis Maia Apolinário de Mello, Jussara Kasuko Palmeiro, Thaís Cristine Marques Sincero, Oscar Bruna-Romero, Maria Marlene de Souza Pires, Carlos Rodrigo Zárate-Bladés","doi":"10.3791/67769","DOIUrl":"https://doi.org/10.3791/67769","url":null,"abstract":"Mother's own milk (MOM) is the most complete nutritional resource for newborns. In cases where mothers are unable to produce sufficient milk or cannot breastfeed, the preferred alternative is pasteurized donor human milk (PDM), which is routinely provided by human milk banks. PDM offers a superior range of nutritional and immunological elements compared to any commercially available formula. However, to ensure biosafety, PDM undergoes pasteurization, a process that inactivates commensal microbiota and reduces certain bioactive compounds. This study presents a protocol designed to restore the microbiota of PDM using MOM as a microbial source, adapting the approach to a real-world clinical setting. The protocol was implemented in a clinical trial conducted at a maternity hospital and its associated human milk bank, with the aim of providing personalized donor milk to preterm infants whose mothers cannot produce sufficient milk. The methodology involves inoculating PDM with 10% of MOM, followed by incubation at 37 °C for 4 h. Microbiological analysis demonstrated successful bacterial growth in the inoculated milk (IM) post incubation, with the microbiota profile of the reconstituted milk (RM) closely resembling that of MOM, indicating effective microbiota restoration. These results suggest that the reconstitution protocol is feasible for implementation in neonatal care, with the potential to enhance the nutritional and immunological quality of PDM, thereby supporting the health and development of non-breastfed newborns.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143494445","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}

引用次数: 0

Visualizing Intracellular Sialylation with Click Chemistry and Expansion Microscopy.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-02-07 DOI: 10.3791/67479

Yannick Masson, Aude Sivery, Corentin Spriet, Anthony Treizebre, Christophe Biot, Cedric Lion

{"title":"Visualizing Intracellular Sialylation with Click Chemistry and Expansion Microscopy.","authors":"Yannick Masson, Aude Sivery, Corentin Spriet, Anthony Treizebre, Christophe Biot, Cedric Lion","doi":"10.3791/67479","DOIUrl":"https://doi.org/10.3791/67479","url":null,"abstract":"Metabolic labeling techniques allow the incorporation of bioorthogonal reporters into glycans, enabling the targeted bioconjugation of molecular dyes within cells through click and bioorthogonal chemistry. Metabolic oligosaccharide engineering (MOE) has attracted considerable interest due to the essential role of glycosylation in numerous biological processes that involve molecular recognition and its impact on pathologies ranging from cancer to genetic disorders to viral and bacterial infections. Although MOE is better known for the detection of cell surface glycoconjugates, it is also a very important methodology for the study of intracellular glycans in physiological and pathological contexts. Such studies greatly benefit from high spatial resolution. However, super-resolution microscopy is not readily available in most laboratories and poses challenges for daily implementation. Expansion microscopy is a recent alternative that enhances the resolution of microscopy by physically enlarging biological specimens labeled with fluorescent markers. By embedding the sample in a swellable gel and causing it to expand isotropically through chemical treatment, subcellular structures can be visualized with enhanced precision and resolution without the need for super-resolution techniques. In this work, we illustrate the capacity of expansion microscopy to visualize intracellular sialylated glycans through the combined use of MOE and click chemistry. Specifically, we propose a procedure for bioorthogonal labeling and expansion microscopy that employs a reporter targeting sialylation, which may be associated with immunofluorescence for co-localization studies. This protocol enables localization studies of sialoconjugate biosynthesis, intracellular trafficking, and recycling.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143494292","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}

引用次数: 0

A Data-Driven Approach to Quantifying Immune States in Sepsis.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-02-07 DOI: 10.3791/68074

Shan Li, Tengxiao Liang, Fangliang Xing, Shangshang Jiang

{"title":"A Data-Driven Approach to Quantifying Immune States in Sepsis.","authors":"Shan Li, Tengxiao Liang, Fangliang Xing, Shangshang Jiang","doi":"10.3791/68074","DOIUrl":"https://doi.org/10.3791/68074","url":null,"abstract":"In sepsis, understanding the interplay among white blood cells, lymphocytes, and neutrophils is crucial for assessing the immune condition and optimizing treatment strategies. Blood samples were collected from 512 patients diagnosed with sepsis and 205 healthy controls, totaling 717 samples. Data visualization analysis and three-dimensional numerical fitting were performed to establish a mathematical model describing the relationships among white blood cells, lymphocytes, and neutrophils. Self-organizing feature map (SOFM) was employed to automatically cluster the sepsis sample data in the three-dimensional space represented by the model, yielding different immune states. Analysis revealed that white blood cell, lymphocyte, and neutrophil counts are constrained within a three-dimensional plane, as described by the equation: WBC = 1.098 × Neutrophils + 1.046 × Lymphocytes + 0.1645, yielding a prediction error (RMSE) of 1%. This equation is universally applicable to all samples despite differences in their spatial distributions. SOFM clustering identified nine distinct immune states within the sepsis patient population, representing different levels of immune status, oscillation periods, and recovery stages. The proposed mathematical model, represented by the equation above, reveals a basic constraint boundary on the immune cell populations in both sepsis patients and healthy controls. Furthermore, the SOFM clustering approach provides a comprehensive overview of the distinct immune states observed within this constraint boundary in sepsis patients. This study lays the foundation for future work on quantifying and categorizing the immune condition in sepsis, which may ultimately contribute to the development of more objective diagnostic and treatment strategies.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143494436","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}

引用次数: 0

Prediction of Red Blood Cell Antibody Significance Using the Monocyte-Macrophage Assay. 利用单核细胞-巨噬细胞检测法预测红细胞抗体的重要性

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-02-07 DOI: 10.3791/67877

Lindsay K Hillis, Selena Cen, Clemence Salou, Yeniley Ruiz Noa, Donald R Branch

{"title":"Prediction of Red Blood Cell Antibody Significance Using the Monocyte-Macrophage Assay.","authors":"Lindsay K Hillis, Selena Cen, Clemence Salou, Yeniley Ruiz Noa, Donald R Branch","doi":"10.3791/67877","DOIUrl":"https://doi.org/10.3791/67877","url":null,"abstract":"Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, and foreign pathogens. The phagocytic capacity of monocytes versus macrophages is a concept that is not well understood. Here, we aim to examine a difference in the phagocytosis of monocytes versus macrophages, specifically M1/M2 macrophages, against various opsonized red cells using a modified and updated version of the established monocyte monolayer assay (MMA). Peripheral blood mononuclear cells (PBMCs) were isolated from donor buffy coats. Using purified monocytes, inflammatory M1 and anti-inflammatory M2 macrophages were produced by in vitro culture and polarization. M1/M2 cells were harvested and used in an MMA-like assay, which we refer to as the M-MA, to decipher clinically significant phagocytosis of various red cell antibodies. A phagocytic index (PI) > 5 was deemed clinically significant phagocytosis with the use of monocytes. A phagocytic index (PI) > 12 was deemed clinically significant phagocytosis with the use of M1/M2 macrophages. M2 macrophages demonstrate an increased ability to phagocytose opsonized RBCs compared to monocytes and M1s. The same weak antibody (anti-S) yields significant phagocytosis with only M2 macrophages (PI=43) but not M1s (PI=2) or monocytes (PI=0), and this was demonstrated repeatedly using various antibodies. The use of M2 macrophages instead of monocytes may allow for more accurate results as these cells are more phagocytic, offering further clinical relevance to the assay. Further studies with different antibodies to red blood cells, including validation of the monocyte-macrophage assay (M-MA) with antibodies having known clinical significance, may show the M-MA more useful to help predict clinically significant red cell alloantibodies and transfusion reactions. This method will advance the field of transfusion medicine and immunology.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143494467","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}

引用次数: 0

Use of In Vivo Assembly for High-efficiency Plasmid Construction.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-02-07 DOI: 10.3791/67870

Hannah G Braun, Jenny-Lee Thomassin

引用次数: 0

The Isolation and Characterization of Low- and Normal- Density Neutrophils from Whole Blood. 从全血中分离低密度和正常密度中性粒细胞并确定其特征。

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-02-07 DOI: 10.3791/67805

Anjali S Yennemadi, Joseph Keane, Gina Leisching

{"title":"The Isolation and Characterization of Low- and Normal- Density Neutrophils from Whole Blood.","authors":"Anjali S Yennemadi, Joseph Keane, Gina Leisching","doi":"10.3791/67805","DOIUrl":"https://doi.org/10.3791/67805","url":null,"abstract":"Emerging research shows that the circulating neutrophil population in humans consists of diverse subtypes and should not be studied as a single population, as has been done historically. In particular, low-density and normal-density neutrophils (LDNs, NDNs) have been shown to have functionally and metabolically distinct profiles, a factor that must be considered when publishing neutrophil research. Here, we present a modified method for the untouched isolation and separation of LDNs and NDNs from whole blood. The density gradient medium (1.135 g/mL) is combined at 9:10 with 10x PBS. Specific density gradients of 55%, 70%, and 81% are subsequently made by combining the 100% density gradient medium with 1x phosphate-buffered saline (PBS). Neutrophils isolated from 12 mL of peripheral whole blood obtained from consented donors using a negative selection-based magnetic isolation kit are resuspended in the 55% fraction. A volume of 3 mL of the 81% and 70% fractions is layered into a 15 mL tube, followed by the 55% fraction containing total neutrophils. The density gradients are then centrifuged at 720 x g for 30 min. Two distinct bands are obtained at the 55%/70% interface (LDNs) and 70%/81% interface (NDNs). The cells are carefully pipetted into separate tubes and washed using PBS. The purity of the isolated fractions is determined using flow cytometry. Both LDNs and NDNs were defined as CD14lo CD15+ SSChi by flow cytometry. Isolation purity was calculated at ≥93% of viable cells for both types. This method provides a reliable and efficient approach for separating LDN and NDNs from peripheral blood, ensuring high purity and viability of the isolated cells. Enhancing the precision of neutrophil isolation facilitates more accurate downstream analyses of these distinct neutrophil subpopulations. These are critical for advancing our understanding of neutrophil heterogeneity and its implications in various physiological and pathological contexts.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143493749","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}

引用次数: 0

Improved Methodology for Studying Postnatal Osteogenesis via Intramembranous Ossification in a Murine Bone Marrow Injury Model.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-02-07 DOI: 10.3791/67727

Marta Stetsiv, Matthew Wan, Shagun Prabhu, Rosa Guzzo, Archana Sanjay

{"title":"Improved Methodology for Studying Postnatal Osteogenesis via Intramembranous Ossification in a Murine Bone Marrow Injury Model.","authors":"Marta Stetsiv, Matthew Wan, Shagun Prabhu, Rosa Guzzo, Archana Sanjay","doi":"10.3791/67727","DOIUrl":"10.3791/67727","url":null,"abstract":"Long bone injuries heal through either endochondral or intramembranous bone formation pathways. Unlike the endochondral pathway that requires a cartilage template, the process of intramembranous ossification involves the direct conversion of skeletal stem and progenitor cells (SSPCs) into bone-forming osteoblasts. There are limited surgical methods to model this process in experimental mice. Here, we have improved upon a bone marrow injury model in mice to facilitate the study of bone repair via intramembranous ossification and to assess postnatal regulators of osteogenesis. This method is highly reproducible and user-friendly, and it allows temporal assessment of new bone formation in a short period (3-7 days post-injury) using micro-computed tomography (µCT) and frozen section histology. Furthermore, the contributions of SSPCs and mature osteoblasts can be readily assessed using a combination of fluorescent reporter mice and this intramembranous bone marrow injury model. In clinical contexts, intramembranous bone formation is relevant for healing critical size defects, stabilized fractures, cortical defects, trauma from tumor resections, and joint replacements.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143494443","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}

引用次数: 0

Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-02-07 DOI: 10.3791/67964

Ainul Huda, Thomas J Vaden, Hua Bai, Riley T Rawls, Rebecca J Peppers, Charlotte F Monck, Henry D Holley, Allison N Castaneda, Lina Ni

{"title":"Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster.","authors":"Ainul Huda, Thomas J Vaden, Hua Bai, Riley T Rawls, Rebecca J Peppers, Charlotte F Monck, Henry D Holley, Allison N Castaneda, Lina Ni","doi":"10.3791/67964","DOIUrl":"10.3791/67964","url":null,"abstract":"Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study introduces easy-to-implement setups for applying optogenetic methods in Drosophila melanogaster. Two optogenetic tools, CsChrimson, a red-light-activated cation channel, and GtACR2, a blue-light-activated anion channel, were employed in four experimental approaches. Three of these approaches involve single-fly experiments: (1) a blue-light optogenetic thermotactic positional preference assay targeting temperature-sensitive heating cells, (2) a red-light optogenetic positional preference assay activating bitter sensing neurons, and (3) a proboscis extension response assay activating the sweet-sensing neurons. The fourth approach (4) is a fly maze setup to assess avoidance behaviors using multiple flies. The ability to manipulate neural activity temporally and spatially offers powerful insights into sensory processing and decision-making, underscoring the potential of optogenetics to advance our knowledge of neural function. These methods provide an accessible and robust framework for future research in neuroscience to enhance the understanding of specific neural pathways and their behavioral outcomes.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143494379","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}

引用次数: 0

Direct Cryosectioning of Drosophila Heads for Enhanced Brain Fluorescence Staining and Immunostaining.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-02-07 DOI: 10.3791/67791

John Watson, Jonathan R Roth, Girish C Melkani

{"title":"Direct Cryosectioning of Drosophila Heads for Enhanced Brain Fluorescence Staining and Immunostaining.","authors":"John Watson, Jonathan R Roth, Girish C Melkani","doi":"10.3791/67791","DOIUrl":"https://doi.org/10.3791/67791","url":null,"abstract":"Immunostaining Drosophila melanogaster brains is essential for exploring the mechanisms behind complex behaviors, neural circuits, and protein expression patterns. Traditional methods often involve challenges such as performing complex dissection, maintaining tissue integrity, and visualizing specific expression patterns during high-resolution imaging. We present an optimized protocol that combines cryosectioning with fluorescence staining and immunostaining. This method improves tissue preservation and signal clarity and reduces the need for laborious dissection for Drosophila brain imaging. The method entails rapid dissection, optimal fixation, cryoprotection, and cryosectioning, followed by fluorescent staining and immunostaining. The protocol significantly reduces tissue damage, enhances antibody penetration, and yields sharp, well-defined images. We demonstrate the effectiveness of this approach by visualizing specific neural populations and synaptic proteins with high fidelity. This versatile method allows for the analysis of various protein markers in the adult brain across multiple z-planes and can be adapted for other tissues and model organisms. The protocol provides a reliable and efficient tool for researchers conducting high-quality immunohistochemistry in Drosophila neurobiology studies. This method's detailed visualization facilitates comprehensive analysis of neuroanatomy, pathology, and protein localization, making it particularly valuable for neuroscience research.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143494451","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}

引用次数: 0

Robotics in Surgery: A Modular Robotic Platform Driven Gastric Wedge Resection. 机器人手术：模块化机器人平台驱动的胃楔切除术。

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-02-07 DOI: 10.3791/66826

Tim Fahlbusch, Albert Tafelmeier, Ilya Slobodkin, Waldemar Uhl, Orlin Belyaev

引用次数: 0