Jove-Journal of Visualized Experiments最新文献

{"title":"NMR 15N Relaxation Experiments for the Investigation of Picosecond to Nanoseconds Structural Dynamics of Proteins.","authors":"Tobias Stief, Katharina Vormann, Nils-Alexander Lakomek","doi":"10.3791/67088","DOIUrl":"10.3791/67088","url":null,"abstract":"<p><p>Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide groups of the protein backbone or the methyl groups in side chains are used as reporters of structural dynamics in proteins. A structural dynamics study of the protein backbone of globular proteins on ¹⁵N labeled and fully protonated samples usually works well for proteins with a molecular weight of up to 50 kDa. When side chain deuteration in combination with transverse relaxation optimized spectroscopy (TROSY) is applied, this limit can be extended up to 200 kDa for globular proteins and up to 1 MDa when the focus is on the side chains. When intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs) are investigated, these weight limitations do not apply but can go well beyond. The reason is that IDPs or IDRs, characterized by high internal flexibility, are frequently dynamically decoupled. Various NMR methods offer atomic-resolution insights into structural protein dynamics across a wide range of time scales, from picoseconds up to hours. Standard ¹⁵N relaxation measurements overview a protein's internal flexibility and characterize the protein backbone dynamics experienced on the fast pico- to nanosecond timescale. This article presents a hands-on protocol for setting up and recording NMR ¹⁵N R1, R2, and heteronuclear Overhauser effect (hetNOE) experiments. We show exemplary data and explain how to interpret them simply qualitatively before any more sophisticated analysis.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142647852","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}

{"title":"A Rat Model of Compound Acne.","authors":"Lin Chen, Qi Jin, Tian Xia, Xin Zhang, Ruiyi Cai, Xinyi Wu, Yue-Han Song, Guang-Zhong Zhang","doi":"10.3791/65859","DOIUrl":"10.3791/65859","url":null,"abstract":"<p><p>Acne vulgaris is a prevalent chronic skin condition characterized by the presence of comedones, papules, and pustules on the face, neck, and chest. To simulate the inflammation of acne vulgaris, this protocol details an approach to establish a compound acne rodent model by inducing acne inflammation in rat ears using oleic acid and Cutibacterium acnes (C. acnes). Rats were randomly divided into four groups: the normal control group (NC), ears treated with oleic acid group (OA), ears treated with C. acnes group (C. acnes), ears treated with oleic acid and C. acnes (OA + C. acnes). To mimic excessive sebum production, oleic acid was smeared on the ears of rats in OA and OA + C. acnes groups for 25 days. From days 21 to 25, C. acnes suspension was injected intradermally into the ears of rats in the C. acnes and OA + C. acnes groups to aggravate the acne inflammation. Ear thickness was measured weekly as a gauge of inflammation severity. Gross observation, hematoxylin and eosin staining, and immunohistochemistry (IHC) were conducted and the results showed that the ears of the OA group and the OA + C. acnes group were thickened and indurated, accompanied by erythema and the presence of comedones. Additionally, papules were observed in C. acnes and OA + C. acnes groups. The histopathology exhibited hyperkeratinization and expanded infundibulum of the hair follicles in OA and OA + C. acnes groups. Infiltration of inflammatory cells and abscesses were found in the dermis of C. acnes and OA + C. acnes groups. The IHC results confirmed increased levels of tumor necrosis factor (TNF)-α in the dermis of C. acnes and OA + C. acnes groups. All the above results collectively indicated the successful establishment of the compound acne rodent model.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142649396","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}

{"title":"An Automated Squint Method for Time-syncing Behavior and Brain Dynamics in Mouse Pain Studies.","authors":"Nathan McCutcheon, Micah S Johnson, Brandon Rea, Mahnoor Ghumman, Levi Sowers, Rainbo Hultman","doi":"10.3791/67136","DOIUrl":"10.3791/67136","url":null,"abstract":"<p><p>Spontaneous pain has been challenging to track in real time and quantify in a way that prevents human bias. This is especially true for metrics of head pain, as in disorders such as migraine. Eye squint has emerged as a continuous variable metric that can be measured over time and is effective for predicting pain states in such assays. This paper provides a protocol for the use of DeepLabCut (DLC) to automate and quantify eye squint (Euclidean distance between eyelids) in restrained mice with freely rotating head motions. This protocol enables unbiased quantification of eye squint to be paired with and compared directly against mechanistic measures such as neurophysiology. We provide an assessment of AI training parameters necessary for achieving success as defined by discriminating squint and non-squint periods. We demonstrate an ability to reliably track and differentiate squint in a CGRP-induced migraine-like phenotype at a sub second resolution.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142649398","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}

{"title":"Using Motion Capture Technology in the Instrumented Timed Up and Go Test to Detect the Risk of Falling in Aged Adults.","authors":"Xiaoping Cai, Hongyi Zhao, Xinjun Shan, Yonghua Huang, Fangyuan Wei","doi":"10.3791/66025","DOIUrl":"https://doi.org/10.3791/66025","url":null,"abstract":"<p><p>Despite efforts made by medicine and technology, the incidence of falls in older adults is still increasing. Therefore, early detection of the falling risk is becoming increasingly important for falling prevention. The Timed Up and Go (TUG) test is a well-accepted tool to assess mobility and can be used in predicting future fall risk in aged adults. In clinical practice, the total time to complete the test is the main outcome measure of the TUG test. Owing to its simplicity and generality, the traditional TUG test has been considered a global test for movement analysis. However, recently, researchers have attempted to split the TUG test into subcomponents and have updated its score system for further investigations. The instrumented Time Up and Go (iTUG) test, which is a new modification of the traditional TUG test, has been reported to be a sensitive tool for predicting movement disorders and the risk of falls in older adults. The goal of the present study was to analyze the iTUG test subcomponents using motion capture technology, and to determine which iTUG test subtasks are related to the potential risk of future falls.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 212","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142630471","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}

{"title":"Microscopic Varicocelectomy under Local Anesthesia as the Treatment of Varicocele.","authors":"Gaoyue Zhang, Jianying Li, Zhiming Xu, Jianhui Li, Shanwen Chen","doi":"10.3791/66269","DOIUrl":"https://doi.org/10.3791/66269","url":null,"abstract":"<p><p>Varicocele, characterized by the dilation of veins within the spermatic cord, is a prevalent disease in males, accounting for 35%-40% of infertility cases among men of reproductive age. This condition may disrupt normal neuroregulation, blood supply, and temperature regulation in the testicles, thereby impacting sperm count, quality (morphology, fragmentation rate), and functionality (motility). It may even induce symptoms such as pain and testicular atrophy. The international standard treatment method is microscopic varicocelectomy, typically performed under general anesthesia. This requires patients to fast before surgery and necessitates recovery and fasting care postoperatively. The arterial blood pressure drop caused by anesthesia may obscure the fluctuation of the spermatic artery, making the precise intraoperative isolation and protection of the spermatic artery more challenging. Therefore, this study proposes and implements microscopic varicocelectomy under local anesthesia. This method facilitates rapid and precise identification of the fluctuating spermatic artery, reducing the risk of spermatic artery damage. It also provides more humane care for patients, reduces economic burden, and offers a new perspective on the treatment of varicocele.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 212","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142630444","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}

{"title":"Preclinical Positron Emission Tomography with Body Conforming Animal Molds for Cloud-Based Automated Image Analysis in Mice.","authors":"Edward Cabral, Mary Katherine Montgomery, Meaghan Berg, Lisa Kathryn Manzuk, Anand Giddabasappa, Ziyue Karen Jiang","doi":"10.3791/67370","DOIUrl":"https://doi.org/10.3791/67370","url":null,"abstract":"<p><p>Positron Emission Tomography (PET) is a molecular imaging modality that can be used to investigate a multitude of pharmacological questions, such as biomarker modulation, receptor occupancy, and biodistribution of compounds of interest. In biodistribution studies, experimental subjects are often longitudinally imaged after receiving the test article. The images are then analyzed to derive the compound's distribution profile in various organs at different timepoints. This constitutes a crucial step in drug development to understand the distribution and potentially binding profile of an investigative compound. Standard/manual methods of PET imaging-based biodistribution analyses, however, are labor-intensive and time-consuming and are often associated with high inter-operator variability. Further, it is challenging to keep the animals' positions consistent across different timepoints. To address these shortcomings, a series of mouse Body Conforming Animal Molds (BCAMs) were used to enable rigid and consistent positioning of animals during PET/CT imaging acquisition. Further, a Software-as-a-Service (SaaS) platform consisting of a cloud-based Organ Probability Map (OPM) and an artificial intelligence-powered segmentation tool were employed to enable reliable and automated quantitation of in vivo PET imaging data. The workflow presented here includes (1) prepping mice for imaging with the BCAMs, including the proper implantation of subcutaneous tumors to be compatible with the molds, (2) acquiring PET/CT images with BCAMs using the G8 scanner, and 3) performing automated organ segmentation and biodistribution analysis using the cloud-based SaaS. [¹⁸F]FDG was used as an exemplar tracer here, but other biomarkers and/or radio-labeled compounds can be readily adapted into the workflow. This procedure can be executed accurately and effectively with minimal training, and the automated PET data analysis yielded satisfactory results consistent with the manual method.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 212","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142630445","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}

{"title":"Reconstructing Terrestrial Paleoclimate and Paleoecology with Fossil Leaves Using Digital Leaf Physiognomy and Leaf Mass Per Area.","authors":"Alexander J Lowe, Andrew G Flynn, Matthew J Butrim, Aly Baumgartner, Dana L Royer, Daniel J Peppe","doi":"10.3791/66838","DOIUrl":"https://doi.org/10.3791/66838","url":null,"abstract":"<p><p>Climate and environment strongly influence the size, shape, and toothiness (physiognomy) of plants' leaves. These relationships, particularly in woody non-monocotyledonous angiosperms, have been used to develop leaf-based proxies for paleoclimate and paleoecology that have been applied to reconstruct ancient terrestrial ecosystems for the last ~120 million years of Earth's history. Additionally, given that these relationships have been documented in living plants, they are important for understanding aspects of plant evolution and how plants respond to climatic and environmental changes. To conduct these types of analyses on modern and fossil plants, leaf physiognomy must be measured accurately using a reproducible methodology. This protocol describes a computer-based method for measuring and analyzing a variety of leaf physiognomic variables in modern and fossil leaves. This method allows for the measurement of leaf physiognomic traits, in particular variables related to leaf serrations, leaf area, leaf dissection, and linearity that are used in the digital leaf physiognomy proxy for reconstructing paleoclimate, as well as petiole width and leaf area, which are used for reconstructing leaf mass per area, a paleoecological proxy. Because this digital leaf trait measurement method can be applied to fossil and living plants, it is not limited to applications related to reconstructing paleoclimate and paleoecology. It can also be used to explore leaf traits that may be informative for understanding the function of leaf morphology, leaf development, phylogenetic relationships of leaf traits, and plant evolution.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 212","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142630449","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}

{"title":"Separation of Avian Preovulatory Follicle Granulosa and Theca Cell Layers for Downstream Applications.","authors":"Ashley E Kramer, Kathryn M Ellwood, Erin M Brannick, Aditya Dutta","doi":"10.3791/67344","DOIUrl":"https://doi.org/10.3791/67344","url":null,"abstract":"<p><p>Layer hens (egg-laying chickens) and broiler breeders (breeding stock for meat-producing chickens) are crucial to the world's food supply as a reliable source of protein. They are also an emerging animal model for the study of human reproductive disease. As the field of poultry research develops, the health and function of the layer hen and broiler breeder ovary will be an important point of study for both agricultural and biomedical researchers. One of the challenges presented by this emerging interest is the need for replicable techniques that all researchers can employ in ovarian specimen collection. In particular, a detailed visual process must be established to define the proper separation of the specialized granulosa and theca cell layers from hen follicles to achieve agreement and consistency among researchers. This study describes the extraction of preovulatory follicles and ovary tissue in white leghorn hens of prime reproductive age. The separation of these follicles is performed under cold, liquid conditions to congeal the yolk for easier manipulation and to prevent the follicle's own weight from tearing apart cell layers during the separation process. Once the separation is complete, the desired cell layers can be further digested for tissue culture approaches or can be cryopreserved for genomic and proteomic analyses.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 212","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142630450","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}

{"title":"Laparoscopic Non-Mesh Cerclage Pectopexy with Uterine Preservation for Pelvic Organ Prolapse.","authors":"Chaoxia Lyu, Willy Cecilia Cheon, Hextan Yuen Sheung Ngan, Yuzhen Wei, Wenju Zhang","doi":"10.3791/67239","DOIUrl":"https://doi.org/10.3791/67239","url":null,"abstract":"<p><p>Pelvic organ prolapse (POP) affects millions of women globally and carries a significant socioeconomic burden. Adequate apical support is essential for treating POP. Recent research has increasingly validated the efficacy and safety of laparoscopic pectopexy (LP) for addressing apical POP. However, the cost of synthetic mesh and associated complications restrict the widespread use of this technique. Our team previously published a study describing a novel, non-mesh procedure called laparoscopic non-mesh cerclage pectopexy (LNMCP), demonstrating successful outcomes with satisfactory objective and subjective success rates. Many patients express a preference for retaining their uterus during prolapse surgery due to considerations related to sexuality, partnership, and body image. The present research introduces a novel approach known as laparoscopic non-mesh cerclage pectopexy with uterine preservation (LNMCPUP) for POP, wherein the uterus is suspended to the iliopectineal ligament through the round ligament using permanent cerclage sutures. We successfully performed this procedure in 14 cases at our hospital, six of whom still had menstruation, and the remaining eight were postmenopausal, with a mean operation time of 54.43 min (± 10.18 min) and an average bleeding volume of 53.57 mL (± 48.77 mL). The mean follow-up duration was 19.71 ± 15.87 months. The objective success rate of LNMCPUP was 100%, with a subjective success rate of 92.86%. No significant complications were observed during or after surgery. LNMCPUP integrates cervical cerclage and shortening of the round ligament, as well as LP without using mesh, thereby eliminating the risk of mesh erosion and lowering healthcare costs. Moreover, this novel technique is relatively easy to master, making it accessible even in rural and underdeveloped areas where synthetic mesh is unavailable. Therefore, it is worthwhile to adopt LNMCPUP in POP patients who desire the preservation of their uterus.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 212","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142630442","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}

{"title":"Evaluation of the Effect of Growth Factors on Chlorophylls a and b Production from Microalgae.","authors":"Frizek Nathaniel Morales-Rivera, Sofia Torres-Momber, Julio César Jacuinde-Ruíz, Juan Cristóbal Camacho-Arriaga, Juan Carlos González-Hernández","doi":"10.3791/67208","DOIUrl":"https://doi.org/10.3791/67208","url":null,"abstract":"<p><p>Microalgae contain two main groups of pigments: chlorophylls and carotenoids. Chlorophyll is a green pigment that absorbs light energy and transforms it into chemical energy to facilitate the synthesis of organic compounds. This pigment serves as a valuable primary source for biotechnological input products in the food, pharmaceutical, and cosmetic industries due to its high antioxidant properties and coloring capabilities. The objective of this research was to evaluate the effect of growth factors (CO2 concentration, light color, and light intensity) through a Taguchi L4 experimental design on cell growth and the cellular content of chlorophyll a and b in Chlorella sorokiniana, followed by validation of the method using Haematococcus pluvialis microalgae as an additional study model. Cell growth was quantified using the optical density spectrophotometric technique at a wavelength of 550 nm. For the quantification of chlorophylls, a cell extract was obtained using a 90% pure acetone solution, and subsequently, the concentrations of chlorophylls a and b were quantified using spectrophotometric techniques at wavelengths of 647 nm and 664 nm, according to the method described by Jeffrey and Humphrey. The experimental results indicated that controlling conditions of low CO2 addition, purple light, and low light intensity increases both cell growth and the concentration of chlorophylls a and b within the cells. The implementation of this chlorophyll quantification method allows for quick, simple, and precise determination of chlorophyll content, as the wavelengths used are at the absorbance peaks of both types of chlorophylls, making this technique easily reproducible for any microalgae under study.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 212","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142630535","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}