Thorpehirsch5432

Mouse embryonic stem cells (mESCs) and mouse epiblast-like cells (mEpiLCs) recapitulate in vitro the epiblast first cell lineage decision, providing a powerful tool to investigate the mechanisms underlying the pluripotent state transition. Here, we describe a defined and robust protocol to transiently induce mEpiLCs from mESCs, together with a concise overview for their unbiased characterization for subsequent downstream applications.Advances in induced pluripotent stem cell (iPSC) technology provide a renewable source of cells for tissue regeneration and therefore hold great promise for cell replacement therapy. However, immune rejection of allograft due to human leukocyte antigen (HLA) mismatching remains a major challenge. Considerable efforts have been devoted to overcoming the immunogenicity of allograft transplantation. One of the approaches is an elimination of HLA molecules on the surface of allogeneic cells using genome editing technology to generate universal stem cells. Here, we present a simple and effective genome editing approach to knockout the β-2-immunoglobulin (B2M) gene, which encodes B2M protein that forms a heterodimer with HLA class I proteins, in induced pluripotent stem cells (iPSCs) leading to HLA class I (HLA-I) depletion. We also describe detailed procedures for validation of the B2M-knockout iPSCs using flow cytometry, and genotypic analysis for potential off-target regions. Our protocol is also applicable for knocking out other genes in iPSCs and other cell types.Neuronal differentiation is an intricate and a complex process which involves crosstalk among various signaling pathways, growth factors, transcription factors, and epigenetic modifiers. During different stages of neuronal development, there are various histone modifiers which drive the expression of lineage-specific genes. Polycomb group proteins are one of the histone modifiers that control transcriptional repression of specific genes in development, differentiation, and functionality of various tissues. Chromatin immunoprecipitation (ChIP) is a widely used technique to investigate the interaction of proteins and DNA; ChIP combined with quantitative real-time PCR (qPCR) gives a quantitative data about the occupancy of specific protein on a particular stretch of DNA, and this can help us investigate how a protein regulates expression of a specific gene. In this chapter, we describe a protocol for ChIP coupled to qPCR during early neuronal differentiation to identify the specific genomic targets regulated by components of Polycomb repressive complex 1.Prediabetes is defined as a condition of abnormal glucose metabolism, characterised by plasma glucose above normal range but not as high as required for the diagnosis of diabetes mellitus (DM). It represents a heterogeneous entity of intermediate glucose metabolism, including impaired fasting glucose, impaired glucose tolerance, and borderline glycated haemoglobin. Prediabetes is being increasingly recognised as an important metabolic state not only predisposing to a higher probability of future progression to DM, but also to an increased risk of different micro- and macrovascular complications. The recently proposed sub-phenotyping of individuals at increased risk of type 2 DM, which distinguishes six different clusters, offers the opportunity for the improvement in screening, prevention, and treatment algorithms. Such progress should also enable more efficient and cost-effective strategies aimed at decreasing the disease burden associated with prediabetes.Recent advances in genome editing, especially CRISPR-Cas nucleases, have revolutionized both laboratory research and clinical therapeutics. CRISPR-Cas nucleases, together with the DNA damage repair pathway in cells, enable both genetic diversification by classical non-homologous end joining (c-NHEJ) and precise genome modification by homology-based repair (HBR). Genome editing in zygotes is a convenient way to edit the germline, paving the way for animal disease model generation, as well as human embryo genome editing therapy for some life-threatening and incurable diseases. HBR efficiency is highly dependent on the DNA donor that is utilized as a repair template. Here, we review recent progress in improving CRISPR-Cas nuclease-induced HBR in mammalian embryos by designing a suitable DNA donor. Moreover, we want to provide a guide for producing animal disease models and correcting genetic mutations through CRISPR-Cas nuclease-induced HBR in mammalian embryos. Finally, we discuss recent developments in precise genome-modification technology based on the CRISPR-Cas system.In this chapter, we review methods for video-based heart monitoring, from classical signal processing approaches to modern deep learning methods. In addition, we propose a new method for learning an optimal filter that can overcome many of the problems that can affect classical approaches, such as light reflection and subject's movements, at a fraction of the training cost of deep learning approaches. Following the usual procedures for region of interest extraction and tracking, robust skin color estimation and signal pre-processing, we introduce a least-squares error optimal filter, learnt using an established training dataset to estimate the photoplethysmographic (PPG) signal more accurately from the measured color changes over time. Selleck AG-1478 This method not only improves the accuracy of heart rate measurement but also resulted in the extraction of a cleaner pulse signal, which could be integrated into many other useful applications such as human biometric recognition or recognition of emotional state. The method was tested on the DEAP dataset and showed improved performance over the best previous classical method on that dataset. The results obtained show that our proposed contact-free heart rate measurement method has significantly improved on existing methods.Recent years have seen an explosion of interest in digitising museum collections. Among the objects of interest are anatomical and pathological specimens found in medical museums. As researchers increasingly produce digital replicas of these preparations, ways of integrating these resources into the medical curriculum must be explored. This article takes a medical humanities approach to this topical question, comparing the historic use of anatomical specimens to modern intentions, and exploring the potential for using digital anatomy to help integrate humanities into the curriculum. The use of anatomical specimens by William Hunter (1718-1783), whose collection is now kept at the Hunterian in the University of Glasgow, provides a key historic focus. The teaching intentions for his private courses of anatomy are examined, to investigate how specimens were used in an eighteenth-century "curriculum". The motivations behind digitisation and the use of digital anatomy in modern curriculums are then examined and compared.