Methods Collections
Protein-Protein Interaction Methods
Guest Editors: Alastair Barr, Michael Overduin
The study of protein-protein interactions is key to understanding the network of molecular interactions underlying biological effects, and defining how individual proteins perform their functional role within a cell. Detailed understanding of t… Show Morehese interactions often provides insight into aberrant signaling in disease. This collection brings together the wide range of techniques that have been developed to study interactions using in vitro, cell-based and in vivo approaches. Each technique has its own strengths, for example enabling systematic interrogation of the complete ‘interactome’, providing detailed atomic level information about a single interaction or insight into the sub-cellular localization of interacting proteins. Some techniques are easily adaptable to high-throughput screening of libraries or pulling out novel interacting partners while others have a more limited capacity. Readouts such as microscopy and mass-spectrometry are employed in protein-protein interaction techniques and some provide quantitative information on the affinity of an interaction.
Examples of methods to be included in this collection are: co-immunoprecipitation and antibody interference; pull-down assays using fusion proteins or modular protein domains; proximity-dependent labelling techniques (e.g. BioID); far western and receptor affinity probes; affinity purification coupled with mass-spectrometry; two-hybrid technologies (yeast and mammalian); fluorescence microscopy (e.g. immunofluoresence, FRET, super-resolution microscopy); protein complementation assays (e.g. BiFC); biophysical approaches (X-ray crystallography, NMR, AUC, SPR) and microarray based techniques (i.e. tissue and protein arrays). Show Less
Examples of methods to be included in this collection are: co-immunoprecipitation and antibody interference; pull-down assays using fusion proteins or modular protein domains; proximity-dependent labelling techniques (e.g. BioID); far western and receptor affinity probes; affinity purification coupled with mass-spectrometry; two-hybrid technologies (yeast and mammalian); fluorescence microscopy (e.g. immunofluoresence, FRET, super-resolution microscopy); protein complementation assays (e.g. BiFC); biophysical approaches (X-ray crystallography, NMR, AUC, SPR) and microarray based techniques (i.e. tissue and protein arrays). Show Less
RNA/DNA In Situ Hybridization techniques
Guest Editor: Irinna Papangeli
In Situ Hybridization (ISH) provides a powerful tool for localized detection of RNA/DNA sequences, widely applied in both research and clinical contexts. This technique takes advantage of nucleic acid probes that are complementary to target RNA or DN… Show MoreA sequences, in order to localize the target sequences on chromosomes, in tissues, cells, organs or organisms. RNA ISH can be used to identify deregulated expression of mRNA, lncRNA, snRNA or miRNA transcripts, which can manifest as expression mis-localization or expression levels’ changes. DNA ISH is useful for distinguishing gene alleles, identifying chromosomal abnormalities/rearrangements, as well as detecting viral, bacterial or fungal genes commonly seen in infectious diseases.
This Methods Collection aims at creating a comprehensive collection of the most current protocols of RNA/DNA ISH; understanding the applicability, advantages and limitations of this technique will allow researchers to utilize it to its full potential. Examples of ISH protocols that can be included in this collection are: chromogenic or fluorescent ISH, whole mount, section or cell ISH, as well as assays that combine ISH with other techniques such as protein immunohistochemistry or PCR amplification. Show Less
This Methods Collection aims at creating a comprehensive collection of the most current protocols of RNA/DNA ISH; understanding the applicability, advantages and limitations of this technique will allow researchers to utilize it to its full potential. Examples of ISH protocols that can be included in this collection are: chromogenic or fluorescent ISH, whole mount, section or cell ISH, as well as assays that combine ISH with other techniques such as protein immunohistochemistry or PCR amplification. Show Less
Minimally Invasive Pancreatic Surgery
Guest Editors: Marc Besselink, Mohammed Abu Hilal, Ugo Boggi, Safi Dokmak, Amer Zureikat, Maarten Korrel, Frederique Vissers, Sanne Lof
Minimally invasive pancreatic surgery is on the rise worldwide. Minimally invasive distal pancreatectomy (MIDP) has been reported to be superior to open distal pancreatectomy (ODP) in terms of blood loss and hospital stay. The oncological safety of M… Show MoreIDP for pancreatic cancer, however, remains uncertain and randomized controlled trials are needed (Riviere et al, Cochrane Database Syst Rev 2016). Interestingly, a recent study comparing open and minimally invasive pancreatoduodenectomy (MIPD), found similar morbidity and hospital stay, but higher rates of postoperative pancreatic fistula in the MIPD group (Klompmaker et al, Ann Surg 2018). These results may be related to the steep learning curve in MIPD suggesting the need of careful patients selection, access to a high number of patients and intense training for a safe implementation of this procedure.
In this collection, laparoscopic and robot-assisted procedures (both MIDP and MIPD) performed by world-renowned pancreatic surgeons are shown. Show Less
In this collection, laparoscopic and robot-assisted procedures (both MIDP and MIPD) performed by world-renowned pancreatic surgeons are shown. Show Less
Techniques for high throughput lifespan measurement in invertebrate model systems
Guest Editor: Jonathan Millen
Recently, a great deal of effort has been focused on longevity and aging. In particular, new treatments for age-related diseases and the extension of the human healthspan are the subject of much this attention. A major inhibitor of progress towards b… Show Morereakthroughs in longevity treatments is slow and rigorous nature of lifespan research. A potential solution to this problem is to develop scalable high throughput systems that can track the lifespan of multiple organisms under multiple conditions with little to no human intervention. The development of high throughput assays in S. cerevisiae, C. elegans, and potentially other systems allow for the screening of 100 to 100's of thousands of chemical compounds at a time. It is vital that these methods are clear and accessible so that the gerontological community can develop the best defense against the ravages of aging and age-related disease. Show Less
Prostate Organoid Research Techniques and Methods: Three-Dimensional (3D) Culture, Spin-down, Dimpled Parafilm and Fusion Method
Guest Editors: Christina Jamieson, Sanghee Lee
Organoids are three-dimensional (3D) ex vivo cellular structures that are believed to recapitulate and maintain the in vivo conditions of cellular viability and responsiveness to therapeutic drugs. Matrigel or collagen have been utilized as an extrac… Show Moreellular matrix to enable patient-derived cells to form 3D/organoids including cyst-like structures and spheroids. 3D cultured organoids are considered to be excellent models to understand the mechanisms of tumorigenesis and to determine the effects of anticancer agents. Therefore, methods have been developed for a range of cancers such as brain, breast, lung and prostate, among others. Prostate, however, is somewhat unique in that current methods for culturing organoids have a lower success rate than for other cancers. Difficulty in establishing prostate organoid cultures may be driven in part by tumorigenic heterogeneity. Most prostate organoids are derived from patient-derived xenografts (PDXs) and are genomically representative of the patient’s prostate cancer. Therefore, PDX organoids models are invaluable for studying down-stream signaling of target molecules and the effects of anti-cancer agent treatment. Several groups have developed modified protocols that are optimized for their experimental objectives. The goal of this collection is to (1) provide several examples of optimized culture condition for patient-derived prostate cancer 3D organoids, (2) introduce follow-up methodologies for single cell methods such as RNA-FISH and flow cytometry, (3) share meticulous details of immunohistochemistry (IHC)/ immunofluorescent (IF) technique specific for organoids and (4) differential criteria to determine the effects of drug treatment on organoids. Show Less
New Techniques and Approaches for the Assessment or Intervention in Learning Disabilities and ADHD
Guest Editors: Celestino Rodríguez, Débora Areces, Marisol Cueli, Trinidad García
International classifications of mental disorders for the diagnosis of Learning Disabilities (LD) and Attention Deficit/Hyperactivity Disorders (ADHD) establish that they are not the result of other disabling conditions, although they might occur con… Show Morecurrently with other disabilities. Consequently, people who have a disability could also show limitations in learning process, professional or social development. These disorders are understood as intrinsic to the individual, assumed to be due to the dysfunction in the central nervous system, and may occur throughout the life cycle.
Taking into account the symptomatic complexity of these difficulties, as well as their high prevalence rates, it is necessary for professionals and researchers to have reliable and valid tools and methods for diagnosis and effective intervention design. These techniques would make it easier for professionals and researchers to develop their work in an appropriate and innovative way.
However, the main difficulty is the lack of correspondence between assessment and intervention proposals which is not always apparent. Assessment methods, tools, and diagnostic techniques are being currently developed, which provide objective information on the characteristics and needs of this population. This collection aims to bring together a wide range of techniques to provide insight into the assessment and intervention of LD and ADHD. Show Less
Taking into account the symptomatic complexity of these difficulties, as well as their high prevalence rates, it is necessary for professionals and researchers to have reliable and valid tools and methods for diagnosis and effective intervention design. These techniques would make it easier for professionals and researchers to develop their work in an appropriate and innovative way.
However, the main difficulty is the lack of correspondence between assessment and intervention proposals which is not always apparent. Assessment methods, tools, and diagnostic techniques are being currently developed, which provide objective information on the characteristics and needs of this population. This collection aims to bring together a wide range of techniques to provide insight into the assessment and intervention of LD and ADHD. Show Less
Current Research Methods in Rabies Diagnosis, Prevention, Treatment, and Control
Guest Editor: Charles E. Rupprecht
Rabies is an acute progressive encephalitis caused by a lyssavirus. The case fatality of this zoonosis is the highest for any infectious disease. Although laboratory techniques on rabies have progressed greatly over the last century, typically such i… Show Morenformation falls in disparate locations and is often unavailable for easy access by those with the greatest need. Hence, the opportunity for a visual, methodological catalog is critical for basic research in pathobiology, immunology and molecular biology, as well for applied diagnostics, prophylaxis, therapy and intervention. The time is opportune because a global program is underway for the elimination of human rabies transmitted by dogs through mass canine vaccination, requiring multiple tests for risk mitigation and measurement of programmatic success. Advances have occurred in pathogen discovery, characterization, novel biologics and anti-virals. Based on in vitro and animal models, hope has arisen not only for immunization after viral exposure but for potential treatment after illness. Besides direct application to humans and domestic animals, efficacious vaccines may also be distributed to free-ranging wildlife en masse via vaccine-laden baits. The objective of this collection is to: share standardized protocols for detection, characterization and response; promote broader global access to existing methods; produce open networks for research collaborations anew; and ultimately create a pathway for an evidence-based approach for additional knowledge on this neglected disease. We trust this approach will encourage a myriad of scientists to participate in this global endeavor in a trans-disciplinary manner, towards renewed biomedical progress in human and veterinary science and conservation biology. Show Less
Comprehensive Guide to Inflammation Detection Methodologies
Guest Editor: Payel Sil
Inflammation is the natural response of the body to harmful stimuli and inflammatory symptoms are routinely used to diagnose ailments. However, acute and chronic inflammation display differing characteristics, which requires deeper exploration for ac… Show Morecurate identification. The tools that are currently available are often lacking in this aspect. With the rapid advancement in the field of immunotherapies, there has been a surge in interest in developing effective methodologies to gain a deeper insight into the state of inflammation.
Swelling, redness and pain are the primary indicators of inflammation. Various detection strategies utilize surface markers, cytokine release and migration patterns of cells as inflammation indicators. The effectiveness of these methods in both immune and non-immune cells is limited by the subtleties of cellular functioning. The signaling pathways and associated mechanisms that launch inflammatory responses are hard to correlate with physiological outcomes. Therefore, it is essential to discover as well as document novel techniques to measure and quantify markers of inflammation in a disease context. This methods collection aims to highlight the unique inflammatory characteristics of multiple cell types relevant to different diseases such as cancer, autoimmune disorders and infections. The collection is intended to be a repository of cutting edge methods developed by researchers to encourage knowledge transfer and innovation, promote cross-pollination of ideas between research teams and ultimately enhance the accuracy of disease diagnosis. Show Less
Swelling, redness and pain are the primary indicators of inflammation. Various detection strategies utilize surface markers, cytokine release and migration patterns of cells as inflammation indicators. The effectiveness of these methods in both immune and non-immune cells is limited by the subtleties of cellular functioning. The signaling pathways and associated mechanisms that launch inflammatory responses are hard to correlate with physiological outcomes. Therefore, it is essential to discover as well as document novel techniques to measure and quantify markers of inflammation in a disease context. This methods collection aims to highlight the unique inflammatory characteristics of multiple cell types relevant to different diseases such as cancer, autoimmune disorders and infections. The collection is intended to be a repository of cutting edge methods developed by researchers to encourage knowledge transfer and innovation, promote cross-pollination of ideas between research teams and ultimately enhance the accuracy of disease diagnosis. Show Less
Current methods in Astyanax mexicanus research
Guest Editors: Misty Rose Riddle, Clifford J. Tabin
The Mexican Tetra, Astyanax mexicanus is an excellent model organism to study the developmental and genetic basis of evolution. This species of fish consists of a river-dwelling (surface) population and a number of cave-dwelling po… Show Morepulations that evolved from surface fish trapped in caves. Cavefish have distinct morphology and physiology including: absence of eyes, reduced pigment, increased sensory structures, sensitive smell, reduced circadian rhythm, altered blood glucose regulation, and increased fat accumulation. Surface fish and all cavefish are interfertile allowing for genetic mapping studies that, in combination with a sequenced and annotated genome, have revealed the genetic changes leading to some of the unique cavefish traits. A. mexicanus have been bred in the lab for generations and numerous protocols exist for investigating their biology. The goal of this collection is to demonstrate standardized protocols for breeding, provide broader access to existing methods, and ultimately lower the threshold for researchers interested in working with this species.
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Methods and Techniques for the Development of Drug Delivery Systems
Guest Editor: Wing-Fu Lai
As far as pharmaceutical interventions are concerned, one of the important factors to be considered is drug bioavailability, which can be easily jeopardized by various factors (particularly the physiological barriers and blood clearance) after drug a… Show Moredministration. This has limited the sustainability of the drug effect and has hampered the therapeutic efficacy. Over the years, different drug carriers (e.g., liposomes, polymers, emulsions, hydrogels, and metal nanoparticles) have emerged. Since the turn of the last century, the versatility and tunability of these carriers have been further enhanced by advances in microfabrication technologies. Many of
these technologies have shown tremendous promise in modulating the pharmacokinetic parameters of diverse medicinal entities, ranging from protein drugs to therapeutic genes. As a multidisciplinary undertaking, drug delivery research may involve experimental techniques not only in materials chemistry but also in biomedical sciences. Such a multidisciplinary nature of the discipline may turn out to be one of the challenges to researchers who wish to enter this rapidly advancing field of research. Regarding this fact, this collection describes techniques and methods to synthesize and characterize different materials systems, and to evaluate the performance of these systems as drug carriers. Its objective is to streamline the research process undertaken by beginning researchers, and to increase the consistency and comparability of data collected from different laboratories during the development, characterization and evaluation of drug delivery systems. Show Less
these technologies have shown tremendous promise in modulating the pharmacokinetic parameters of diverse medicinal entities, ranging from protein drugs to therapeutic genes. As a multidisciplinary undertaking, drug delivery research may involve experimental techniques not only in materials chemistry but also in biomedical sciences. Such a multidisciplinary nature of the discipline may turn out to be one of the challenges to researchers who wish to enter this rapidly advancing field of research. Regarding this fact, this collection describes techniques and methods to synthesize and characterize different materials systems, and to evaluate the performance of these systems as drug carriers. Its objective is to streamline the research process undertaken by beginning researchers, and to increase the consistency and comparability of data collected from different laboratories during the development, characterization and evaluation of drug delivery systems. Show Less
Chloroplast research methods: probing the localization, topology, targeting and interactions of chloroplast proteins
Guest Editor: Paul Jarvis
Chloroplasts are the organelles that define plants. Along with many other metabolic, developmental and signalling functions, chloroplasts are responsible for photosynthesis – the process whereby sunlight energy is harnessed to power the cellula… Show Morer activities of life. Consequently, chloroplasts are essential, not only for plants but also for the myriad ecosystems that depend on plants, and for agriculture. Chloroplasts are composed of thousands of different proteins, most of which are nucleus-encoded and imported from the cytosol before being routed internally to one of six, clearly-distinct intraorganellar compartments. To achieve a more complete understanding of chloroplast development and functions, and to enable strategies through which chloroplasts may be manipulated to address global challenges linked to food security or bioenergy, it will be essential to determine the localization, topology, targeting and interactions of important chloroplast proteins. The methods presented here – which focus on the widely-used model plant Arabidopsis thaliana (thale cress), but may also be adapted and applied to other organisms – will be critically important as we pursue these goals. Show Less
Multi-modal Analytics in Energy Storage
Guest Editors: Partha P. Mukherjee, Leela M. R. Arava, George J. Nelson
Energy storage is a critical enabler for vehicle electrification and renewable energy integration into the electric grid. Primary challenges in lithium based intercalation (Li-ion) and conversion (e.g., Li-S) chemistries continue to remain in en… Show Moreergy and power density trade-off, life and safety. These energy storage systems consist of coupled physical processes at disparate spatio-temporal scales in porous electrodes, including species and charge transport in the solid and electrolyte phases, electrochemical reaction at active interfaces, heat generation and thermal transport, chemical reactions, and transport-mechanics manifestation. In recent years, there has been significant emphasis and progress in experimentally examining such coupled processes at different scales. This JoVE Methods Collection seeks contributions in multi-modal analytics, including but not limited to, electrochemical, spectroscopic, microscopic and tomographic analysis and diagnostics to examine coupled physicochemical processes in energy storage (e.g. Li-ion, and beyond Li-ion chemistries including Li-S, Li-air, Mg-ion, Na-ion, K-ion) systems. Show Less
