Cell identity is determined by the selective activation or silencing of specific genes via transcription factor binding and epigenetic modifications on the genome. Chromatin immunoprecipitation ...(ChIP) has been the standard technique for mapping the sites of transcription factor binding and histone modification. Recently, alternative methods to ChIP have been developed for addressing the increasing demands for low-input epigenomic profiling. Chromatin integration labeling (ChIL) followed by sequencing (ChIL-seq) has been demonstrated to be particularly useful for epigenomic profiling of low-input samples or even single cells because the technique amplifies the target genomic sequence before cell lysis. After labeling the target protein or modification in situ with an oligonucleotide-conjugated antibody (ChIL probe), the nearby genome sequence is amplified by Tn5 transposase-mediated transposition followed by T7 RNA polymerase-mediated transcription. ChIL-seq enables the detection of the antibody target localization under a fluorescence microscope and at the genomic level. Here we describe the detailed protocol of ChIL-seq with assessment methods for the key steps, including ChIL probe reaction, transposition, in situ transcription and sequencing library preparation. The protocol usually takes 3 d to prepare the sequencing library, including overnight incubations for the ChIL probe reaction and in situ transcription. The ChIL probe can be separately prepared and stored for several months, and its preparation and evaluation protocols are also documented in detail. An optional analysis for multiple targets (multitarget ChIL-seq) is also described. We anticipate that the protocol presented here will make the ChIL technique more widely accessible for analyzing precious samples and facilitate further applications.
Image-based cell profiling is a high-throughput strategy for the quantification of phenotypic differences among a variety of cell populations. It paves the way to studying biological systems on a ...large scale by using chemical and genetic perturbations. The general workflow for this technology involves image acquisition with high-throughput microscopy systems and subsequent image processing and analysis. Here, we introduce the steps required to create high-quality image-based (i.e., morphological) profiles from a collection of microscopy images. We recommend techniques that have proven useful in each stage of the data analysis process, on the basis of the experience of 20 laboratories worldwide that are refining their image-based cell-profiling methodologies in pursuit of biological discovery. The recommended techniques cover alternatives that may suit various biological goals, experimental designs, and laboratories' preferences.
Traditional Chinese medicines (TCMs) are gaining more and more attention all over the world, due to their specific theory and long historical clinical practice. But the uncontrollable quality is a ...bottleneck for its modernization and globalization. This paper reviewed the recent analytical methods in the quality control of TCMs, including screening strategies of bioactive markers from TCMs through biochromatographic methods, the traditional chromatographic methods, DNA methods, as well as the spectroscopic methods, including FT-IR, NIR and NMR. The comprehensive methods, such as fingerprint and multi-component quantification are emphasized; hyphenated techniques, like HPLC-MS, GC-MS, CE-MS, LC-NMR, chemometric methods, and combination of chemical and biological methods, such as biofingerprint, metabolic fingerprint are now more and more widely used in TCMs. In a few word, the analysis and quality control of TCMs are moving towards an integrative and comprehensive direction, in order to better address the inherent holistic nature of TCMs.
Background
Radical surgery provides the best chance of cure for adrenocortical carcinoma (ACC), but perioperative surgical care for these patients is yet to be standardized.
Methods
A working group ...appointed jointly by ENSAT and ESES used Delphi methodology to produce evidence‐based recommendations for the perioperative surgical care of patients with ACC. Papers were retrieved from electronic databases. Evidence and recommendations were classified according to the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system, and were discussed until consensus was reached within the group.
Results
Twenty‐five recommendations for the perioperative surgical care of patients with ACC were formulated. The quality of evidence is low owing to the rarity of the disease and the lack of prospective surgical trials. Multi‐institutional prospective cohort studies and prospective RCTs are urgently needed and should be strongly encouraged.
Conclusion
The present evidence‐based recommendations provide comprehensive advice on the optimal perioperative care for patients undergoing surgery for ACC.
The best evidence available
This review provides an overview of recent developments in "chemical virology." Viruses, as materials, provide unique nanoscale scaffolds that have relevance in chemical biology and nanotechnology, ...with diverse areas of applications. Some fundamental advantages of viruses, compared to synthetically programmed materials, include the highly precise spatial arrangement of their subunits into a diverse array of shapes and sizes and many available avenues for easy and reproducible modification. Here, we will first survey the broad distribution of viruses and various methods for producing virus-based nanoparticles, as well as engineering principles used to impart new functionalities. We will then examine the broad range of applications and implications of virus-based materials, focusing on the medical, biotechnology, and energy sectors. We anticipate that this field will continue to evolve and grow, with exciting new possibilities stemming from advancements in the rational design of virus-based nanomaterials.
Aptamers are single stranded DNA or RNA molecules that have been selected using in vitro techniques to bind target molecules with high affinity and selectivity, rivaling antibodies in many ways. In ...order to use aptamers in research and clinical applications, a thorough understanding of aptamer–target binding is necessary. In this article, we review methods for assessing aptamer–protein binding using separation based techniques such as dialysis, ultrafiltration, gel and capillary electrophoresis, and HPLC; as well as mixture based techniques such as fluorescence intensity and anisotropy, UV–vis absorption and circular dichroism, surface plasmon resonance, and isothermal titration calorimetry. For each method the principle, range of application and important features, such as sample consumption, experimental time and complexity, are summarized and compared.
We provide an overview of recent developments in big data analyses in the context of precision medicine and health informatics. With the advance in technologies capturing molecular and medical data, ...we entered the area of “Big Data” in biology and medicine. These data offer many opportunities to advance precision medicine. We outline key challenges in precision medicine and present recent advances in data integration‐based methods to uncover personalized information from big data produced by various omics studies. We survey recent integrative methods for disease subtyping, biomarkers discovery, and drug repurposing, and list the tools that are available to domain scientists. Given the ever‐growing nature of these big data, we highlight key issues that big data integration methods will face.
In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition ...of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.
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