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
Accelerating the Development of Extracellular Vesicles for Therapeutic Use
Guest Editors: W. Keith Jones, Simon Kaja
Historically, extracellular vesicles (EVs) were considered merely a cellular system to recycle or eliminate intracellular components. It is now recognized that EVs are critical for intercellular communication at an organismal level. EVs are a heterog… Show Moreenous group of membrane-bounded vesicles that differ in size, density, morphology and cargo. A subset of EVs, termed exosomes, has been the focus of intense scrutiny as mediators of immune response, stem cell effects and metastasis. EVs are known to elicit a diverse number of responses within recipient cells, including activation of signaling pathways and modulation of gene expression through the delivery of transcription factors, mRNAs, and miRNAs. EVs have been shown to contain systems for active aerobic respiration and can shuttle membrane receptors from cell to cell. Thus, EVs constitute a shared subcellular compartment that can connect cells in complicated and profound ways. The fact that some types of EVs have been shown to have low immunogenicity suggests that there may be advantages to EV therapeutics relative to cell transplantation, and opportunities for cell type-specific delivery of cargo exist. Current limitations that complicate interpretation of results are a lack of rigorous characterization and quality controls.
In this interdisciplinary methods collection, we will present the latest state-of-the art methods that enable and facilitate the development of EVs for therapeutic approaches. These will include methods for the isolation, characterization and modification of EV cargo and examples of the use of EVs as diagnostic markers and therapeutic agents in preclinical models. Show Less
In this interdisciplinary methods collection, we will present the latest state-of-the art methods that enable and facilitate the development of EVs for therapeutic approaches. These will include methods for the isolation, characterization and modification of EV cargo and examples of the use of EVs as diagnostic markers and therapeutic agents in preclinical models. Show Less
Responses of Livestock and Wildlife Species to Zoonotic Bacteria
Guest Editor: Catherine Vrentas
Due in large part to the immense potential of genetic manipulation in rodent systems, immunological investigations in mouse models have been used to reveal the intricacies of innate and acquired mammalian immune systems. Similarly, mice and rat… Show Mores provide excellent models of infection for a diversity of infectious bacterial diseases. However, pathogenesis and immune responses differ between rodent model systems and natural hosts of disease, such as livestock (pigs, cattle, sheep, goats, etc.), and wildlife species (deer, elk, bison, etc.). As one example, murine responses to brucellosis (caused by infection with Brucella sp. bacteria) differ from the responses of cattle and bison, both of which can become infected in the field. Research on veterinary species has many potential benefits, including utility in the development of vaccines and diagnostics for these animals, utility in better understanding immune responses through comparative immunology, and utility in understanding the transmission of zoonotic diseases. In this collection, manuscripts will present protocols and methodologies that have been adapted to livestock and wildlife systems. Necessary modifications may include validation of reagents for use in these non-model species, adaptations for the study of large animals, and optimization of parameters for procedures like immune cell cultivation from animal sources. Inclusion of in vivo components is not required. Show Less
Methods to Measure Functional Beta Cell Mass
Guest Editors: Jeffrey Tessem, Patrick Fueger
The beta cell plays an essential role in physiology. These cells, found in the pancreatic Islets of Langerhans, must sense elevated circulating blood glucose levels and secrete the hormone insulin to cause the organism to clear elevated glucose … Show Moreand maintain a state of normoglycemia. Beta cell function is dependent on insulin production and maturation, glucose uptake and metabolism, mitochondrial respiration, second messenger signaling, and membrane depolarization and vesicle exocytosis. Functional beta cell mass is also defined by the ability of beta cells to replicate while maintaining their mature glucose sensing phenotype, and the ability to survive damage that results in cell death. Therefore, functional beta cell mass is defined by both the ability to secrete insulin in response to elevated blood glucose and the overall beta cell proliferation and survival capacity. Both major forms of diabetes are characterized by impaired functional beta cell mass. An ultimate cure for either Type 1 or Type 2 diabetes will require improved functional beta cell mass. It is of utmost importance that cutting-edge tools and techniques be used to measure decreases in functional beta cell mass during the disease state and increases in potential therapeutic interventions. The goal of this collection is to compile novel protocols developed by experts to measure changes in functional beta cell mass using in vitro and in vivo models. Show Less
Materials and Methods for the Development of Optical and Ultrasound Phantoms
Guest Editor: Wenfeng Xia
Phantoms are widely used for performance evaluation of biomedical imaging systems, for training clinical practitioners, and for surgical planning. Materials that are suitable for phantom development are required to simulate specific physical properti… Show Morees of biological tissues. For optical imaging modalities such as diffuse optical tomography, optical coherence tomography and fluorescence imaging, these properties include optical absorption and scattering; and for ultrasound imaging modalities, properties such as acoustic attenuation, speed of sound and acoustic impedance are important. A range of phantom materials have been developed such as agar, gelatin, polyester resin, epoxy resin, silicone, polyurethane, Zerdine( TM), and condensed milk gel. Most of these materials are suitable for either optical or ultrasound imaging modalities. Recently, materials such as gel wax, poly(vinyl alcohol) gels, and polyvinyl chloride plastisol have been developed for hybrid modalities such as photoacoustics and acousto-optics owing to their favorable optical and acoustic properties. Patient-specific phantoms that are valuable for medical training and surgical planning have been developed using 3D printing technologies. This Methods Collection aims to highlight novel phantom materials which possess not only tunable optical and acoustic properties but also other desired properties such as good temporal stability, mechanical robustness, and self-healing. Also covered are novel methods for phantom fabrication, such as the production of phantoms with complex geometries using 3D printed molds or direct 3D printing of phantom materials. Additionally, the development of dynamic and functional phantoms such as phantoms that mimic blood flow, oxygen saturation, and thermodynamic properties is within the scope of this collection.
The citation for the representative image is: Maneas, E., Xia, W., Ogunlade, O., Fonseca, M., Nikitichev, D.I., David, A.L., West, S.J., Ourselin, S., Hebden, J.C., Vercauteren, T. and Desjardins, A.E., 2018. Gel wax-based tissue-mimicking phantoms for multispectral photoacoustic imaging. Biomedical optics express, 9(3), pp.1151-1163. Show Less
The citation for the representative image is: Maneas, E., Xia, W., Ogunlade, O., Fonseca, M., Nikitichev, D.I., David, A.L., West, S.J., Ourselin, S., Hebden, J.C., Vercauteren, T. and Desjardins, A.E., 2018. Gel wax-based tissue-mimicking phantoms for multispectral photoacoustic imaging. Biomedical optics express, 9(3), pp.1151-1163. Show Less
Techniques for the Detection of Early Stage Prostate Cancer
Guest Editors: Fubo Wang, Huan Xu
This Methods Collection focuses on the early detection and diagnosis of prostate cancer (PCa). PCa is the second most frequently diagnosed cancer overall and is the fifth leading cause of cancer mortality in men. Prognosis is relatively positive if t… Show Morehe disease is diagnosed at an early stage and a radical prostatectomy in conjunction with androgen-deprivation therapy (ADT) can be performed. Measurement of blood levels of prostate-specific antigen (PSA) and MRI are currently the most widely used early stage PCa detection methods, however, early diagnosis is still incredibly challenging. Currently, biopsy is the gold standard for early stage PCa detection, however, it is an invasive technique that can result in complications.
Recently, an increasingly large array of PCa markers have been discovered and are being assayed using novel detection methods. Specifically, plasma levels of extracellular microRNAs, circulating exosomes, as well as circulating cancer cells can be measured as prognostic biomarkers for PCa. Plasma levels are typically assayed using either a plasmon sensor chip or flow cytometry, both of which yield extremely precise marker quantitation. Additionally, MRI in conjunction with hyperpolarized [1-13C] pyruvate detects PCa and other cancers with high accuracy. This Methods Collection aims to serve as a central repository of the most significant advances in PCa early detection. Show Less
Recently, an increasingly large array of PCa markers have been discovered and are being assayed using novel detection methods. Specifically, plasma levels of extracellular microRNAs, circulating exosomes, as well as circulating cancer cells can be measured as prognostic biomarkers for PCa. Plasma levels are typically assayed using either a plasmon sensor chip or flow cytometry, both of which yield extremely precise marker quantitation. Additionally, MRI in conjunction with hyperpolarized [1-13C] pyruvate detects PCa and other cancers with high accuracy. This Methods Collection aims to serve as a central repository of the most significant advances in PCa early detection. Show Less
Viral and Non-Viral Delivery Methods of CRISPR/Cas9 Toolbox
Guest Editors: Alessandra Recchia, Daniela Benati
CRISPR/Cas genome editing systems represent one of the most robust platforms in basic biomedical research and therapeutic applications. To date, the strategies of CRISPR/Cas9 delivery are primarily physical/chemical approaches such as transfecti… Show Moreon, electroporation, hydrodynamic injection or viral vectors such as lentivirus, adenovirus, or adeno-associated virus. Despite the several available molecular formats of Cas9/gRNA and molecular components includeing plasmid, mRNA, and ribonucleoprotein for physical/chemical delivery, they often are more successful in vitro than in vivo. To deliver Cas9/gRNA components in vivo, various viral and non-viral methods have been developed. Viral vectors carrying the Cas9/gRNA are used extensively because of their high efficiency of gene delivery and long-term transgene expression. However, their intrinsic drawbacks including risk of carcinogenesis, limitation of insertion size and immune responses severely limit their application. As a low toxicity alternative, non-viral methods including lipid nanoparticles, gold nanoparticles, and nanoparticles conjugated to penetrating peptide (CPP) offer appealing possibilities in CRISPR/Cas9 delivery. Current CRISPR/Cas9 delivery systems have shown relatively low gene delivery efficiency and transgene expression. However, with the rapid development of novel biomaterials in recent years, efficient delivery of gene payloads to pass the multiple barriers present under physiological conditions and promote transgene expression can be achieved. The goal of this Methods Collection is to showcase standardized protocols for delivering Cas9/sgRNA components to target cells both in vitro and in vivo. Show Less
Methods for the Assessment of Mitochondrial Bioenergetics and Functions
Guest Editor: Ryan J. Mailloux
Mitochondria are the main source of ATP in most, if not all, mammalian cells. However, these dynamic organelles fulfill a myriad of other cell functions including providing antioxidant support and reactive oxygen species to the cell, serving as an im… Show Moreportant site for the biosynthesis of various biomolecules, and have been documented to serve as a signaling platform, regulating a range of cellular functions in response to different physiological cues. As a consequence, mitochondria are integral for modulating a myriad of physiological functions ranging from locomotion to immune cell function and memory. Defects in mitochondrial function have also been linked to a number of human pathologies transforming this double-membraned organelle into a valuable target for gene or pharmacological therapy. It is, therefore, of the utmost importance that cutting-edge tools and techniques are used to understand the relationship between basic mitochondrial functions and their role in physiology and disease. The goal of this collection is to bring together a rich panoply of methods developed by experts to study mitochondrial functions using in vitro, cell-based, and in vivo approaches. Of interest in this collection are the novel protocols used to measure mitochondrial bioenergetics and functions and how findings can be applied to understand the role of defective mitochondria in the pathogenesis of human diseases. 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
Methods and Techniques for Micro and Electro-Propulsion for Cubesats and Smart Satellites
Guest Editors: Igor Levchenko, Angelo Cervone, Stéphane Mazouffre, Mitchel Walker, Shuyan Xu
Rapid development of information technologies and material science has formed a unique base for the space exploration renaissance. Miniaturization of electronic and energy generating devices, along with novel materials and innovative paradigms in roc… Show Moreket technology, have evoked a dawn of miniaturized spaceship capable of performing all and even more functions intrinsic to oldfashioned large satellites. A task of extensive exploration of a whole Solar system is now faced, and appropriate efforts already undertaken by setting a global satellite-based communication system and sending the scientific orbiters and landers to planets including Venus and Mars.
Like smartphones in our pockets, smart satellites replace the obsolete heavy equipment at orbit. On the other hand, one persistent problem hinders the further progress in modern, smart spaceship technology; namely, miniaturized satellites require miniature but efficient engines, which are a challenge now. Powerful chemical rocket engines are inherently inefficient upon miniaturization; thus, only electric propulsion technique which does not have any physical limitations on the exhaust velocity could be a base for efficient yet small thrusters capable of driving modern, miniaturized, smart satellites between orbits, make them interference with each other and so on.
In this collection we will see various methods and techniques for designing, testing and space application of quite various electric propulsion techniques, with a stress to those suitable for smart satellites. Hall-type thrusters, ion gridded thrusters and several other perspective types will be represented by their creators, thus forming a whole picture describing various types of electric and plasma types that different labs and research groups specialize on. On the other hand, each presentation will describe some specific type of thrusters and thus could be selfconsistent in some extent. Significant attention will be devoted to research groups already tested their systems in space. Show Less
Like smartphones in our pockets, smart satellites replace the obsolete heavy equipment at orbit. On the other hand, one persistent problem hinders the further progress in modern, smart spaceship technology; namely, miniaturized satellites require miniature but efficient engines, which are a challenge now. Powerful chemical rocket engines are inherently inefficient upon miniaturization; thus, only electric propulsion technique which does not have any physical limitations on the exhaust velocity could be a base for efficient yet small thrusters capable of driving modern, miniaturized, smart satellites between orbits, make them interference with each other and so on.
In this collection we will see various methods and techniques for designing, testing and space application of quite various electric propulsion techniques, with a stress to those suitable for smart satellites. Hall-type thrusters, ion gridded thrusters and several other perspective types will be represented by their creators, thus forming a whole picture describing various types of electric and plasma types that different labs and research groups specialize on. On the other hand, each presentation will describe some specific type of thrusters and thus could be selfconsistent in some extent. Significant attention will be devoted to research groups already tested their systems in space. 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
Current Methods in Surgical Treatment for Primary Colorectal Cancer
Guest Editors: Al-guo Lu, Wolfgang Erwin Thasler , Feng Hao
Surgery is currently the most appropriate choice of curative treatment for primary colorectal cancer. The current management is a complete mesocolic excision (CME) for colonic cancer or a total mesorectal excision (TME) of the rectum to remove … Show Morethe tumor as well as the surrounding lymphatic drainage basin of the affected colonic segment or rectum, including the major vascular pedicle.
The increasing awareness of postoperative morbidity of colorectal cancer, as well as the influence of quality of life after colorectal cancer surgery, has caused an increasing interest in various surgical approach including conventional open surgery, laparoscopic surgery, robotic surgery, and transanal endoscopic surgery.
This Methods Collection focuses on the surgical treatment of primary colorectal cancer. Show Less
The increasing awareness of postoperative morbidity of colorectal cancer, as well as the influence of quality of life after colorectal cancer surgery, has caused an increasing interest in various surgical approach including conventional open surgery, laparoscopic surgery, robotic surgery, and transanal endoscopic surgery.
This Methods Collection focuses on the surgical treatment of primary colorectal cancer. Show Less
