Chungbay6070

Here, we describe protocols for the preparation and dissociation of human fetal and pediatric intestinal tissue to a high-viability epithelial single-cell suspension. This epithelium-enriched single-cell suspension can then be used to generate single-cell RNA sequencing data as well as to create human intestinal organoids from both the fetal and pediatric intestine. Finally, this protocol details the dissociation of the intestinal organoids for use in single-cell analysis or passaging of organoids. For complete details on the use and execution of this protocol, please refer to Elmentaite et al. (2020).During adulthood, the activation of adult neural stem cells (NSCs) has been mostly studied ex vivo in post-mortem tissues or in vivo in anesthetized animals. This protocol presents an approach that allows for the long-term and minimally invasive investigation of adult NSC activation and physiology in freely behaving animals. Cepharanthine solubility dmso By combining specific NSC labeling and mini-endoscopic microscopy, live imaging of NSC division and Ca2+ activity can be performed continuously for 2-3 days and even up to several months. For complete details on the use and execution of this protocol, please refer to Gengatharan et al. (2021).Anti-PD-1/PD-L1 therapy shows long-term effects in many cancer types, but resistance and relapse remain the main limitations of this therapy. Here, we describe a protocol to evaluate the tumor response to immunotherapy in a mouse lung cancer model. The protocol includes the establishment of the lung cancer mouse model, anti-PD-1 treatment, tumor-infiltrating lymphocyte isolation, immunofluorescence, and flow cytometry analysis. This protocol can also be applied to other cancer types and immunotherapies. For complete details on the use and execution of this protocol, please refer to Yu et al. (2021).Organoid models have been shown to be valuable tools for studying epithelial-mesenchymal crosstalk during biological and pathological settings. Our data identified ACTA2+ PDGFRα+ repair-supportive mesenchymal cells as an important component of the conducting airway niche. Here, we provide a detailed protocol for culturing airway organoids, or bronchiolospheres, which provide an assessment of the ability of mesenchymal cells to support club-cell growth. For complete details on the use and execution of this protocol, please refer to Moiseenko et al. (2020).Here, we describe a protocol for a photoaffinity labeling probe strategy for target deconvolution in live cells. We made a chemical probe by incorporation of a photoreactive group to covalently cross-link with adjacent amino acid residues upon UV irradiation. Click chemistry-based enrichment captures labeled proteins for proteomic analysis. Here, we detail specifics for finding targets of LXRβ, but the protocol has potential for application to other targets. For complete details on the use and execution of this protocol, please refer to Seneviratne et al. (2020).We have outlined the approach of visualizing autophagy specifically in the epithelial follicle stem cells of the Drosophila ovary using the LysoTracker dye. The advantage of using this protocol is that it details several techniques, including ovary dissection, immunofluorescence, and western blotting, that positively identify autophagy changes in a very small population of cells. One of the limitations of this protocol is that it needs to be combined with other genetic manipulations and positive markers of the autophagy pathway. For complete details on the use and execution of this protocol, please refer to Singh et al., (2018).Here, we describe a rapid and versatile protocol to generate gapped DNA substrates for single-molecule (SM) analysis using optical tweezers via site-specific Cas9 nicking and force-induced melting. We provide examples of single-stranded (ss) DNA gaps of different length and position. We outline protocols to visualize these substrates by replication protein A-enhanced Green Fluorescent Protein (RPA-eGFP) and SYTOX Orange staining using commercially available optical tweezers (C-TRAP). Finally, we demonstrate the utility of these substrates for SM analysis of bidirectional growth of RAD-51-ssDNA filaments. For complete details on the use and execution of this protocol, please refer to Belan et al. (2021).The cooperativity of six cations (Ca2+, Mg2+, Zn2+, Al3+, Cr3+ and Fe3+), three pectins (sugar beet, high and low methyl esterified), three dispersed phases (medium chain triglycerides (MCT), orange oil and hexadecane), time (30 days) and pH (2.0 and 6.0) has been investigated in the formation and stability against coarsening of oil-in-water emulsions. Cations generally influenced emulsion stability in the following order (most stable) Ca2+ > Mg2+ > Al3+ > Cr3+ > Zn2+ > Fe3+ (least stable). This order largely coincided with that of the strength of pectin-cation interactions showing that the higher the affinity of cation for pectin the less stable the emulsion. More stable emulsions were formed with sugar beet pectin, which was also unresponsive to the presence of cations, followed by high- and then low-methyl esterified samples. At pH 2.0 all pectins showed their best emulsification performance whereas shifting pH to 6.0 severely impaired emulsification capacity and longer term stability against droplet growth. Smaller droplets were created with hexadecane under all conditions studied followed by MCT and orange oil in agreement with their aqueous solubilities. The present results advance our understanding of the stabilisation of emulsions using pectin and allow us to tailor their functionality for applications in food, pharmaceutical and biomedical industries.[This corrects the article DOI 10.1093/noajnl/vdab017.].

The use of liquid biopsy is of potential high importance for children with high grade (HGG) and diffuse midline gliomas (DMG), particularly where surgical procedures are limited, and invasive biopsy sampling not without risk. To date, however, the evidence that detection of cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA) could provide useful information for these patients has been limited, or contradictory.

We optimized droplet digital PCR (ddPCR) assays for the detection of common somatic mutations observed in pediatric HGG/DMG, and applied them to liquid biopsies from plasma, serum, cerebrospinal fluid (CSF), and cystic fluid collected from 32 patients.

Although detectable in all biomaterial types, ctDNA presented at significantly higher levels in CSF compared to plasma and/or serum. When applied to a cohort of 127 plasma specimens from 41 patients collected from 2011 to 2018 as part of a randomized clinical trial in pediatric non-brainstem HGG/DMG, ctDNA profiling by ddPCR was of limited use due to the small volumes (mean = 0.49 mL) available. In anecdotal cases where sufficient material was available, cfDNA concentration correlated with disease progression in two examples each of poor response in

_K27M-mutant DMG, and longer survival times in hemispheric

_V600E-mutant cases.

Tumor-specific DNA alterations are more readily detected in CSF than plasma. Although we demonstrate the potential of the approach to assessing tumor burden, our results highlight the necessity for adequate sample collection and approach to improve detection if plasma samples are to be used.

Tumor-specific DNA alterations are more readily detected in CSF than plasma. Although we demonstrate the potential of the approach to assessing tumor burden, our results highlight the necessity for adequate sample collection and approach to improve detection if plasma samples are to be used.Annotating the functions of gene products is a mainstay in biology. A variety of databases have been established to record functional knowledge at the gene level. However, functional annotations at the isoform resolution are in great demand in many biological applications. Although critical information in biological processes such as protein-protein interactions (PPIs) is often used to study gene functions, it does not directly help differentiate the functions of isoforms, as the 'proteins' in the existing PPIs generally refer to 'genes'. On the other hand, the prediction of isoform functions and prediction of isoform-isoform interactions, though inherently intertwined, have so far been treated as independent computational problems in the literature. Here, we present FINER, a unified framework to jointly predict isoform functions and refine PPIs from the gene level to the isoform level, enabling both tasks to benefit from each other. Extensive computational experiments on human tissue-specific data demonstrate that FINER is able to gain at least 5.16% in AUC and 15.1% in AUPRC for functional prediction across multiple tissues by refining noisy PPIs, resulting in significant improvement over the state-of-the-art methods. Some in-depth analyses reveal consistency between FINER's predictions and the tissue specificity as well as subcellular localization of isoforms.The importance of cell type-specific gene expression in disease-relevant tissues is increasingly recognized in genetic studies of complex diseases. However, most gene expression studies are conducted on bulk tissues, without examining cell type-specific expression profiles. Several computational methods are available for cell type deconvolution (i.e. inference of cellular composition) from bulk RNA-Seq data, but few of them impute cell type-specific expression profiles. We hypothesize that with external prior information such as single cell RNA-seq and population-wide expression profiles, it can be computationally tractable to estimate both cellular composition and cell type-specific expression from bulk RNA-Seq data. Here we introduce CellR, which addresses cross-individual gene expression variations to adjust the weights of cell-specific gene markers. It then transforms the deconvolution problem into a linear programming model while taking into account inter/intra cellular correlations and uses a multi-variate stochastic search algorithm to estimate the cell type-specific expression profiles. Analyses on several complex diseases such as schizophrenia, Alzheimer's disease, Huntington's disease and type 2 diabetes validated the efficiency of CellR, while revealing how specific cell types contribute to different diseases. In summary, CellR compares favorably against competing approaches, enabling cell type-specific re-analysis of gene expression data on bulk tissues in complex diseases.

The use of soft tissue fillers for facial rejuvenation is increasing rapidly and the complications, unfortunately, follow the same path. Blindness caused by intravascular filler injections is a rare but devastating complication. Knowledge of the individual arterial anatomy may aid the injector in avoiding injecting into an artery and thus to prevent blindness.

To evaluate if the use of magnetic resonance angiography (MRA) may visualize the arterial facial anatomy in a contrast- and radiation-free way and study the individual arterial variations using an augmented reality (AR) image.

The individual arterial anatomy of the 3 terminal branches of the ophthalmic artery (supraorbital [SO]; supratrochlear [STr]; and dorsal nasal [DN] arteries) of 20 volunteers was studied by a 3-Tesla MRI, combining infrared (IR) facial warming and 3-dimensional time-of-flight multiple overlapping thin slab acquisition MRA. The resulting visualization of the facial arteries was shown on the patient's face through AR technology.