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Methodological considerations on how to identify human hematopoietic stem cells.
No full textRecently, human CD34+ hematopoietic stem cells (HSCs) have been purified to a frequency of approximately 1 in 3 cells, a population denoted as CD34+CD38-CD45RA-CD90±EPCR+ HSCs. This work aimed to evaluate the methodology for CD34+ HSC isolation, exploring differences in antibody clones, conjugates, source of cells and additional cell surface antigens (integrin-α6, CLEC9A and GPRC5C) to enhance the purity of these EPCR+ HSCs. We are emphasizing here the importance of experimental planning and antibody panel selection concerning the isolation of these human HSCs from multiple sources and providing important notes on the pitfalls of the reagents used for such purposes. Our results should enable a better reproducibility of results between labs, as well as further pursue work towards improving the enrichment of human HSCs
Enantioselective OTUD7B fragment discovery through chemoproteomics screening and high-throughput optimisation.
No full textDeubiquitinating enzymes (DUBs) are key regulators of cellular homoeostasis, and their dysregulation is associated with several human diseases. The ovarian tumour protease (OTU) family of DUBs are biochemically well-characterised and of therapeutic interest, yet only a few tool compounds exist to study their cellular function and therapeutic potential. Here we present a chemoproteomics fragment screening platform for identifying novel DUB-specific hit matter, that combines activity-based protein profiling with high-throughput chemistry direct-to-biology optimisation to enable rapid elaboration of initial fragment hits against OTU DUBs. Applying these approaches, we identify an enantioselective covalent fragment for OTUD7B, and validate it using chemoproteomics and biochemical DUB activity assays
Towards an integrated approach for understanding glia in amyotrophic lateral sclerosis.
No full textSubstantial advances in technology are permitting a high resolution understanding of the salience of glia, and have helped us to transcend decades of predominantly neuron-centric research. In particular, recent advances in 'omic' technologies have enabled unique insights into glial biology, shedding light on the cellular and molecular aspects of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Here, we review studies using omic techniques to attempt to understand the role of glia in ALS across different model systems and post mortem tissue. We also address caveats that should be considered when interpreting such studies, and how some of these may be mitigated through either using a multi-omic approach and/or careful low throughput, high fidelity orthogonal validation with particular emphasis on functional validation. Finally, we consider emerging technologies and their potential relevance in deepening our understanding of glia in ALS
Language Teacher Emotional Experiences: A Systematic Review
No full textThis handbook synthesizes accumulated research evidence about the main areas of language teacher education. It systematically applies research synthesis to the field, providing coherent, systematic insights into various aspects of language teaching.
Each chapter compares research conducted between 2010–2020 within a specialized area of teacher education. The chapters discuss the theoretical and research underpinnings of each area, describing the purposes, methods and findings of the research, including impacts of teacher education on teacher gains and teaching effectiveness.
Areas addressed in this handbook include: teacher identity, motivation, demotivation and burnout, reflective practice, action research, Content and Language Integrated Learning (CLIL) teacher education, English Medium Instruction (EMI) teacher education, self-efficacy, assessment literacy, language awareness, Technological Pedagogical Content Knowledge (TPACK), supervision and mentoring, and nativeness/non-nativeness.
This handbook is an invaluable resource for teacher educators, student/Preservice teachers, inservice teachers, graduate students of Teaching English to speakers of other languages (TESOL) and Applied Linguistics, and teacher education researchers.</p
Generation of a human induced pluripotent stem cell line (CRICKi021-A) from a patient with Ullrich congenital muscular dystrophy carrying a pathogenic mutation in the COL6A1 gene.
No full textUllrich congenital muscular dystrophy (UCMD) represents the most severe subtype of collagen VI-related dystrophies (COL6-RDs), a spectrum of rare extracellular matrix disorders affecting skeletal muscle and connective tissue. Here, we generated an induced pluripotent stem cell (iPSC) line (CRICKi021-A) from a UCMD patient with de novo dominant-negative mutation in COL6A1 gene by reprogramming dermal fibroblasts using a non-integrating mRNA-based protocol. The resulting human iPSCs displayed normal morphology, expressed pluripotency-associated markers and differentiated into the three germ layers. This new COL6A1-mutant iPSC line can be employed for disease modelling and for investigating potential therapies for COL6-RDs
Local Value in the Television Archive: The Media Archive for Central England
No full textBritish television has in many ways been defined by competing visions of locality. Unlike American television, where local TV has often described small television stations, spatially bound to a city, suburb, or rural community, the ‘local’ in British television has largely been subsumed within the category of ‘the regional.’ When British television emerged in the 1930s, it was expressly national: centralized in London and operated by the BBC. In the 1950s, as transmitters were installed throughout the UK, concern grew over this centralization. With the arrival of commercial television in 1955, efforts were made to increase regional representation—to give voice to places, stories, and perspectives outside Britain’s cultural and economic center. However, UK’s broadcast regions did not emerge organically. They were instead determined by technology—their boundaries fixed by the geographical reach of transmitters—and by politics—so Wales, Scotland, and Northern Ireland could become regions just like the North or the Midlands. As such, the communities represented by these regions were vast, varied, and artificial. They included large cities, industrial towns, and rural farmlands. The programmes produced in these spaces were similarly diverse. Dramas, comedies, news, and magazine shows, set in distinct locations, representing unique communities, and broadcast to varying audiences all emerged under the somewhat clunky rubric of the ‘region.’ Different iterations of the local thus haunt these programmes, forming a fraught and almost accidental undercurrent of regional television.In this chapter, I seek to uncover the local within British regional television by turning to the archive, in particular the regional media archive—an institutional body explicitly concerned with collecting, preserving, and making accessible moving image material tied to specific territorial boundaries. Taking the Media Archive of Central England (MACE), whose remit concerns the moving image material of the Midlands, as my central case study, I explore the ways through which the regional media archive deconstructs the category of the broadcast region and articulates an alternative appraisal of televisual value, one in which locality is central. While regional television has included some of the UK’s most notable series, it has also embodied programming that falls outside the realm of national memory and popular consumption. It is these more niche programmes—the magazine series, quiz shows, and local news broadcasts rooted in the counties, cities, and towns that created them—that find their home in the regional media archive. By analyzing MACE’s collection, archival logic, and access initiatives, I examine how place and locality became central in the archive’s presentation of these often overlooked television programmes. They emerge as key components of their collecting practices and public engagements, breaking down the often artificial boundaries of regional television to return a more granular sense of place to the television housed within their walls. By removing television from its original broadcast context and repositioning it within the archive, I argue that MACE, and the regional media archive more generally, can serve as a space for the recovery of the ‘local’ in British television history.</p
Timely TGFβ signalling inhibition induces notochord.
No full textThe formation of the vertebrate body involves the coordinated production of trunk tissues from progenitors located in the posterior of the embryo. Although in vitro models using pluripotent stem cells replicate aspects of this process1-10, they lack crucial components, most notably the notochord-a defining feature of chordates that patterns surrounding tissues11. Consequently, cell types dependent on notochord signals are absent from current models of human trunk formation. Here we performed single-cell transcriptomic analysis of chick embryos to map molecularly distinct progenitor populations and their spatial organization. Guided by this map, we investigated how differentiating human pluripotent stem cells develop a stereotypical spatial organization of trunk cell types. We found that YAP inactivation in conjunction with FGF-mediated MAPK signalling facilitated WNT pathway activation and induced expression of TBXT (also known as BRA). In addition, timely inhibition of WNT-induced NODAL and BMP signalling regulated the proportions of different tissue types, including notochordal cells. This enabled us to create a three-dimensional model of human trunk development that undergoes morphogenetic movements, producing elongated structures with a notochord and ventral neural and mesodermal tissues. Our findings provide insights into the mechanisms underlying vertebrate notochord formation and establish a more comprehensive in vitro model of human trunk development. This paves the way for future studies of tissue patterning in a physiologically relevant environment
HSC70 coordinates COP9 signalosome and SCF ubiquitin ligase activity to enable a prompt stress response.
No full textThe SCF (SKP1/CUL1/F-box protein) ubiquitin ligase complex plays a protective role against external stress, such as ultraviolet irradiation. The emergence of substrates activates SCF through neddylation, the covalent attachment of ubiquitin-like protein NEDD8 to CUL1. After substrate degradation, SCF is inactivated through deneddylation by COP9-signalosome (CSN), a solo enzyme that can deneddylate SCF. How the activity of CSN and SCF is coordinated within the cell is not fully understood. Here, we find that heat-shock cognate 70 (HSC70) chaperone coordinates SCF and CSN activation dependent on the neddylation status and substrate availability. Under basal conditions and low substrate availability, HCS70 directly enhances CSN deneddylation activity, thereby reducing SCF activity. Under SCF-activated conditions, HSC70 interacts with neddylated SCF and enhances its ubiquitination activity. The alternative interaction between HSC70 and CSN or neddylated SCF is regulated by the presence or absence of SCF substrates. The knockdown of HSC70 decreases SCF-mediated substrate ubiquitination, resulting in vulnerability against ultraviolet irradiation. Our work demonstrates the pivotal role of HSC70 in the alternative activation of CSN deneddylation and SCF substrate ubiquitination, which enables a prompt stress response