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Journal articleDesai SR, Sivarasan N, Johannson KA, et al., 2023,
High-resolution CT phenotypes in pulmonary sarcoidosis: a multinational Delphi consensus study.
, Lancet Respir MedOne view of sarcoidosis is that the term covers many different diseases. However, no classification framework exists for the future exploration of pathogenetic pathways, genetic or trigger predilections, patterns of lung function impairment, or treatment separations, or for the development of diagnostic algorithms or relevant outcome measures. We aimed to establish agreement on high-resolution CT (HRCT) phenotypic separations in sarcoidosis to anchor future CT research through a multinational two-round Delphi consensus process. Delphi participants included members of the Fleischner Society and the World Association of Sarcoidosis and other Granulomatous Disorders, as well as members' nominees. 146 individuals (98 chest physicians, 48 thoracic radiologists) from 28 countries took part, 144 of whom completed both Delphi rounds. After rating of 35 Delphi statements on a five-point Likert scale, consensus was achieved for 22 (63%) statements. There was 97% agreement on the existence of distinct HRCT phenotypes, with seven HRCT phenotypes that were categorised by participants as non-fibrotic or likely to be fibrotic. The international consensus reached in this Delphi exercise justifies the formulation of a CT classification as a basis for the possible definition of separate diseases. Further refinement of phenotypes with rapidly achievable CT studies is now needed to underpin the development of a formal classification of sarcoidosis.
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Journal articleYe L, Shi X, He Y, et al., 2023,
A novel botybirnavirus with a unique satellite dsRNA causes latent infection in Didymella theifolia isolated from tea plants
, Microbiology Spectrum, Vol: 11, ISSN: 2165-0497The unique, recently discovered fungus Didymella theifolia specifically infects local varieties of tea plant Camellia sinensis in China, and therefore, the characterization of its mycoviruses is important. Three double-stranded (ds) RNAs (1, 2, and 3, with 6,338, 5,910, and 727 bp in size, respectively) were identified in the avirulent D. theifolia strain CJP4-1, which exhibits normal growth and morphology. Characterization of these double-stranded RNAs (dsRNAs) revealed that the two largest elements are the genomic components of a novel botybirnavirus, tentatively named Didymella theifolia botybirnavirus 1 (DtBRV1). Conversely, dsRNA3 shares no detectable similarity with sequences deposited in public databases but has high similarity with the 5′-terminal regions of dsRNAs 1 and 2 and contains a duplicated region encoding a putative small peptide. All three dsRNAs are encapsidated in isometric virions ca. 40 nm in diameter, supporting the notion that dsRNA3 is a DtBRV1 satellite. SDS-polyacrylamide gel electrophoresis in combination with peptide mass fingerprint analysis revealed that the DtBRV1 capsid protein consists of polypeptides encoded by the 5′-terminal regions of both genomic components dsRNA1 and dsRNA2. Vertical transmission of DtBRV1 through conidia is efficient, while its horizontal transmission from CJP4-1 to other strains was not detected. DtBRV1, with or without dsRNA3, has no obvious effects on fungal growth and virulence, as illustrated following transfection of the virulent D. theifolia strain JYC1-6. In summary, DtBRV1 exhibits unique molecular traits and contributes to our understanding of mycovirus diversity.
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Journal articleGhani L, Kim S, Ehsan M, et al., 2023,
Melamine-cored glucosides for membrane protein solubilization and stabilization: importance of water-mediated intermolecular hydrogen bonding in detergent performance
, Chemical Science, Vol: 14, Pages: 13014-13024, ISSN: 2041-6520Membrane proteins play essential roles in a number of biological processes, and their structures are important in elucidating such processes at the molecular level and also for rational drug design and development. Membrane protein structure determination is notoriously challenging compared to that of soluble proteins, due largely to the inherent instability of their structures in non-lipid environments. Micelles formed by conventional detergents have been widely used for membrane protein manipulation, but they are suboptimal for long-term stability of membrane proteins, making downstream characterization difficult. Hence, there is an unmet need for the development of new amphipathic agents with enhanced efficacy for membrane protein stabilization. In this study, we designed and synthesized a set of glucoside amphiphiles with a melamine core, denoted melamine-cored glucosides (MGs). When evaluated with four membrane proteins (two transporters and two G protein-coupled receptors), MG-C11 conferred notably enhanced stability compared to the commonly used detergents, DDM and LMNG. These promising findings are mainly attributed to a unique feature of the MGs, i.e., the ability to form dynamic water-mediated hydrogen-bond networks between detergent molecules, as supported by molecular dynamics simulations. Thus, MG-C11 is the first example of a non-peptide amphiphile capable of forming intermolecular hydrogen bonds within a protein-detergent complex environment. Detergent micelles formed via a hydrogen-bond network could represent the next generation of highly effective membrane-mimetic systems useful for membrane protein structural studies.
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Journal articleDavies J, Matthews J, Dobra R, et al., 2023,
Levelling the playing field through the London Network of the UK Clinical Trials Accelerator Platform
, Contemporary Clinical Trials Communications, ISSN: 2451-8654 -
Journal articleBamezai S, Maresca di Serracapriola G, Morris F, et al., 2023,
Protein engineering in the computational age: An open source framework for exploring mutational landscapes in silico
, Engineering Biology, Vol: 7, Pages: 29-38, ISSN: 2398-6182The field of protein engineering has seen tremendous expansion in the last decade, with researchers developing novel proteins with specialised functionalities for a range of uses, from drug discovery to industrial biotechnology. The emergence of computational tools and high-throughput screening technology has substantially sped up the process of protein engineering. However, much of the expertise required to engage in such projects is still concentrated in the hands of a few specialised individuals, including computational biologists and structural biochemists. The international Genetically Engineered Machine (iGEM) competition represents a platform for undergraduate students to innovate in synthetic biology. Yet, due to their complexity, arduous protein engineering projects are hindered by the resources available and strict timelines of the competition. The authors highlight how the 2022 iGEM Team, 'Sporadicate', set out to develop InFinity 1.0, a computational framework for increased accessibility to effective protein engineering, hoping to increase awareness and accessibility to novel in silico tools.
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Journal articleBeattie JW, Rowland-Jones RC, Farys M, et al., 2023,
Application of Raman spectroscopy to dynamic binding capacity analysis
, Applied Spectroscopy, Vol: 77, Pages: 1393-1400, ISSN: 0003-7028Protein A affinity chromatography is a key step in isolation of biotherapeutics (BTs) containing fragment crystallizable regions, including monoclonal and bispecific antibodies. Dynamic binding capacity (DBC) analysis assesses how much BT will bind to a protein A column. DBC reduces with column usage, effectively reducing the amount of recovered product over time. Drug regulatory bodies mandate chromatography resin lifetime for BT isolation, through measurement of parameters including DBC, so this feature is carefully monitored in industrial purification pipelines. High-performance affinity chromatography (HPAC) is typically used to assess the concentration of BT, which when loaded to the column results in significant breakthrough of BT in the flowthrough. HPAC gives an accurate assessment of DBC and how this changes over time but only reports on protein concentration, requires calibration for each new BT analyzed, and can only be used offline. Here we utilized Raman spectroscopy and revealed that this approach is at least as effective as both HPAC and ultraviolet chromatogram methods at monitoring DBC of protein A resins. In addition to reporting on protein concentration, the chemical information in the Raman spectra provides information on aggregation status and protein structure, providing extra quality controls to industrial bioprocessing pipelines. In combination with partial least square (PLS) analysis, Raman spectroscopy can be used to determine the DBC of a BT without prior calibration. Here we performed Raman analysis offline in a 96-well plate format, however, it is feasible to perform this inline. This study demonstrates the power of Raman spectroscopy as a significantly improved approach to DBC monitoring in industrial pipelines.
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Journal articleThee S, Ekkelenkamp M, Shah A, 2023,
AMR-Lung: a European Clinical Research Collaboration on antimicrobial resistance in chronic lung disease.
, Eur Respir J, Vol: 62 -
Journal articleKoudstaal T, Funke-Chambour M, Kreuter M, et al., 2023,
Pulmonary fibrosis: from pathogenesis to clinical decision-making.
, Trends Mol Med, Vol: 29, Pages: 1076-1087Pulmonary fibrosis (PF) encompasses a spectrum of chronic lung diseases that progressively impact the interstitium, resulting in compromised gas exchange, breathlessness, diminished quality of life (QoL), and ultimately respiratory failure and mortality. Various diseases can cause PF, with their underlying causes primarily affecting the lung interstitium, leading to their referral as interstitial lung diseases (ILDs). The current understanding is that PF arises from abnormal wound healing processes triggered by various factors specific to each disease, leading to excessive inflammation and fibrosis. While significant progress has been made in understanding the molecular mechanisms of PF, its pathogenesis remains elusive. This review provides an in-depth exploration of the latest insights into PF pathophysiology, diagnosis, treatment, and future perspectives.
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Journal articleSchindler D, Walker RSK, Jiang S, et al., 2023,
Design, construction, and functional characterization of a tRNA neochromosome in yeast
, Cell, Vol: 186, Pages: 5237-5253.e22, ISSN: 0092-8674Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs.
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Journal articleShaw WM, Khalil AS, Ellis T, 2023,
A multiplex MoClo Toolkit for extensive and flexible engineering of Saccharomyces cerevisiae
, ACS Synthetic Biology, Vol: 12, Pages: 3393-3405, ISSN: 2161-5063Synthetic biology toolkits are one of the core foundations on which the field has been built, facilitating and accelerating efforts to reprogram cells and organisms for diverse biotechnological applications. The yeast Saccharomyces cerevisiae, an important model and industrial organism, has benefited from a wide range of toolkits. In particular, the MoClo Yeast Toolkit (YTK) enables the fast and straightforward construction of multigene plasmids from a library of highly characterized parts for programming new cellular behavior in a more predictable manner. While YTK has cultivated a strong parts ecosystem and excels in plasmid construction, it is limited in the extent and flexibility with which it can create new strains of yeast. Here, we describe a new and improved toolkit, the Multiplex Yeast Toolkit (MYT), that extends the capabilities of YTK and addresses strain engineering limitations. MYT provides a set of new integration vectors and selectable markers usable across common laboratory strains, as well as additional assembly cassettes to increase the number of transcriptional units in multigene constructs, CRISPR-Cas9 tools for highly efficient multiplexed vector integration, and three orthogonal and inducible promoter systems for conditional programming of gene expression. With these tools, we provide yeast synthetic biologists with a powerful platform to take their engineering ambitions to exciting new levels.
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