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81.
Birthe Tegtmeyer Gabrielle Vieyres Daniel Todt Chris Lauber Corinne Ginkel Michael Engelmann Maike Herrmann Christian K. Pfaller Florian W. R. Vondran Ruth Broering Ehsan Vafadarnejad Antoine-Emmanuel Saliba Christina Puff Wolfgang Baumgrtner Csaba Miskey Zoltn Ivics Eike Steinmann Thomas Pietschmann Richard J. P. Brown 《Journal of virology》2021,95(10)
82.
Joost Groot Yizhou Zhou Eric Marshall Patrick Cullen Thomas Carlile Dongdong Lin Chong-Feng Xu Justin Crisafulli Chao Sun Fergal Casey Baohong Zhang Christina Alves 《Biotechnology journal》2021,16(8):2000548
In recent years, High-Throughput Sequencing (HTS) based methods to detect mutations in biotherapeutic transgene products have become a key quality step deployed during the development of manufacturing cell line clones. Previously we reported on a higher throughput, rapid mutation detection method based on amplicon sequencing (targeting transgene RNA) and detailed its implementation to facilitate cell line clone selection. By gaining experience with our assay in a diverse set of cell line development programs, we improved the computational analysis as well as experimental protocols. Here we report on these improvements as well as on a comprehensive benchmarking of our assay. We evaluated assay performance by mixing amplicon samples of a verified mutated antibody clone with a non-mutated antibody clone to generate spike-in mutations from ∼60% down to ∼0.3% frequencies. We subsequently tested the effect of 16 different sample and HTS library preparation protocols on the assay's ability to quantify mutations and on the occurrence of false-positive background error mutations (artifacts). Our evaluation confirmed assay robustness, established a high confidence limit of detection of ∼0.6%, and identified protocols that reduce error levels thereby significantly reducing a source of false positives that bottlenecked the identification of low-level true mutations. 相似文献
83.
Tarah A. Word Ann P. Quick Christina Y. Miyake Mayra K. Shak Xiaolu Pan Jean J. Kim Hugh D. Allen Martha Sibrian-Vazquez Robert M. Strongin Andrew P. Landstrom Xander H. T. Wehrens 《Journal of cellular and molecular medicine》2021,25(13):6115-6124
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac arrhythmia syndrome that often leads to sudden cardiac death. The most common form of CPVT is caused by autosomal-dominant variants in the cardiac ryanodine receptor type-2 (RYR2) gene. Mutations in RYR2 promote calcium (Ca2+) leak from the sarcoplasmic reticulum (SR), triggering lethal arrhythmias. Recently, it was demonstrated that tetracaine derivative EL20 specifically inhibits mutant RyR2, normalizes Ca2+ handling and suppresses arrhythmias in a CPVT mouse model. The objective of this study was to determine whether EL20 normalizes SR Ca2+ handling and arrhythmic events in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from a CPVT patient. Blood samples from a child carrying RyR2 variant RyR2 variant Arg-176-Glu (R176Q) and a mutation-negative relative were reprogrammed into iPSCs using a Sendai virus system. iPSC-CMs were derived using the StemdiffTM kit. Confocal Ca2+ imaging was used to quantify RyR2 activity in the absence and presence of EL20. iPSC-CMs harbouring the R176Q variant demonstrated spontaneous SR Ca2+ release events, whereas administration of EL20 diminished these abnormal events at low nanomolar concentrations (IC50 = 82 nM). Importantly, treatment with EL20 did not have any adverse effects on systolic Ca2+ handling in control iPSC-CMs. Our results show for the first time that tetracaine derivative EL20 normalized SR Ca2+ handling and suppresses arrhythmogenic activity in iPSC-CMs derived from a CPVT patient. Hence, this study confirms that this RyR2-inhibitor represents a promising therapeutic candidate for treatment of CPVT. 相似文献
84.
Paraskevi Tsingeli Maria Christina Papadatou Despoina Psillaki Vasileios Tragoulias Nikolaos Groumas Yannis Dionyssiotis 《Journal of musculoskeletal & neuronal interactions》2021,21(2):326
Von Hippel Lindau (VHL) is a hereditary multiple neoplasia syndrome. We report a case series of two siblings with Von Hippel Lindau (VHL) disease admitted to the rehabilitation department after surgical excision of Central Nervous System (CNS) haemangioblastomas. These clinical cases present rehabilitation challenges in VHL disease. We present a 39-year-old brother and his 45-year-old sister, with the diagnosis of incomplete spinal cord injury (SCI) associated with VHL syndrome lesions. The female patient was diagnosed with chronic motor incomplete cervical SCI and the male patient with acute motor incomplete thoracic SCI. Our target was to increase their functionality and improve their quality of life. Both underwent a comprehensive inpatient rehabilitation program. Programs were individualized as the female patient was admitted 15 years after her spinal cord surgical intervention, while the male patient’s admission was after 4 months of his surgery. 相似文献
85.
We analyzed the global genetic variation pattern of Capsella bursa‐pastoris (Brassicaceae) as expressed in allozymic (within‐locus) diversity and isozymic (between‐locus) diversity. Results are based on a global sampling of more than 20,000 C. bursa‐pastoris individuals randomly taken from 1,469 natural provenances in the native and introduced range, covering a broad spectrum of the species’ geographic distribution. We evaluated data for population genetic parameters and F‐statistics, and Mantel tests and AMOVA were performed. Geographical distribution patterns of alleles and multilocus genotypes are shown in maps and tables. Genetic diversity of introduced populations is only moderately reduced in comparison with native populations. Global population structure was analyzed with structure, and the obtained cluster affiliation was tested independently with classification approaches and macroclimatic data using species distribution modeling. Analyses revealed two main clusters: one distributed predominantly in warm arid to semiarid climate regions and the other predominantly in more temperate humid to semihumid climate regions. We observed admixture between the two lineages predominantly in regions with intermediate humidity in both the native and non‐native ranges. The genetically derived clusters are strongly supported in macroclimatic data space. The worldwide distribution patterns of genetic variation in the range of C. bursa‐pastoris can be explained by intensive intra‐ and intercontinental migration, but environmental filtering due to climate preadaption seems also involved. Multiple independent introductions of genotypes from different source regions are obvious. “Endemic” genotypes might be the outcome of admixture or of de novo mutation. We conclude that today's successfully established Capsella genotypes were preadapted and found matching niche conditions in the colonized range parts. 相似文献
86.
Intrapopulation foraging niche variation between phenotypes and genotypes of Spirit bear populations
Christina N. Service Travis Ingram Thomas E. Reimchen Chris T. Darimont 《Ecology and evolution》2021,11(10):5025
Foraging niche variation within a species can contribute to the maintenance of phenotypic diversity. The multiniche model posits that phenotypes occupying different niches can contribute to the maintenance of balanced polymorphisms. Using coastal populations of black bears (Ursus americanus kermodei) from British Columbia, Canada, we examined potential foraging niche divergence between phenotypes (black and white “Spirit” coat color) and between genotypes (black‐coated homozygote and heterozygous). We applied the Bayesian multivariate models, with biotracers of diet (δ13C and δ15N) together comprising the response variable, to draw inference about foraging niche variation. Variance–covariance matrices from multivariate linear mixed‐effect models were visualized as the Bayesian standard ellipses in δ13C and δ15N isotopic space to assess potential seasonal and annual niche variation between phenotypes and genotypes. We did not detect a difference in annual isotopic foraging niche area in comparisons between genotypes or phenotypes. Consistent with previous field experimental and isotopic analyses, however, we found that white phenotype Spirit bears were modestly more enriched in δ15N during the fall foraging season, though with our modest sample sizes these results were not significant. Although also not statistically significant, variation in isotopic niches between genotypes revealed that heterozygotes were moderately more enriched in δ13C along hair segments grown during fall foraging compared with black‐coated homozygotes. To the extent to which the pattern of elevated δ15N and δ13C may signal the consumption of salmon (Oncorhynchus spp.), as well as the influence of salmon consumption on reproductive fitness, these results suggest that black‐coated heterozygotes could have a minor selective advantage in the fall compared with black‐coated homozygotes. More broadly, our multivariate approach, coupled with knowledge of genetic variation underlying a polymorphic trait, provides new insight into the potential role of a multiniche mechanism in maintaining this rare morph of conservation priority in Canada''s Great Bear Rainforest and could offer new understanding into polymorphisms in other systems. 相似文献
87.
Oliver Spadiut Christian Leitner Tien-Chye Tan Roland Ludwig Christina Divne 《Biocatalysis and Biotransformation》2013,31(1-2):120-127
Site-directed mutagenesis was used to enhance the catalytic activity of pyranose 2-oxidase (P2Ox) from Trametes multicolor with different substrates. To this end, threonine at position 169 was replaced by glycine, alanine and serine, respectively. Using oxygen as electron acceptor the mutant T169G was equally active with d-glucose and d-galactose, whereas wild-type recombinant P2Ox only showed 5.2% relative activity with the latter substrate. When d-galactose was used as electron donor in saturating concentrations, T169G showed a 4.5-fold increase in its catalytic efficiency kcat/KM for the alternative electron acceptor 1,4-benzoquinone and a nine-fold increased kcat/KM value with the ferricenium ion compared with wt recP2Ox. Variant T169S showed an increase in its catalytic efficiency both with 1,4-benzoquinone (3.7 times) as well as with the ferricenium ion (1.4 times) when d-glucose was the substrate. 相似文献
88.
Paschalis-Thomas Doulias Chara Vlachou Christina Boudouri Panagiotis Kanavaros Kostas C. Siamopoulos 《Free radical research》2013,47(3):253-259
A small part of cellular iron, usually called ‘labile iron pool’ (LIP), is not securely stored and has the potential to catalyse the formation of highly reactive oxygen species. The present work estimated LIP levels in human white cells by using the analytical power of flow cytometry. The method relies essentially on already established principles but has the added value of monitoring LIP in different subpopulations of human blood cells concurrently in a single sample. Examination of 41 apparently healthy individuals revealed a positive correlation between LIP levels and the age of the donors (r=0.656, 0.572 and 0.702 for granulocytes, lymphocytes and monocytes, respectively, p<0.0001), indicating that cells of older individuals are prone to oxidations in conditions of oxidative stress. It is suggested that LIP estimation may represent a valuable tool in examinations searching for links between iron and a variety of oxidative stress-related pathological conditions. 相似文献
89.
Anne McHugh Paulette Bierzychudek Christina Greever Tessa Marzulla Richard Van Buskirk Greta Binford 《Journal of Insect Conservation》2013,17(6):1237-1253
The genetic structure of lineages can provide important information for delineating “evolutionarily significant units” (ESUs) for conservation, and for planning actions to protect and restore taxa threatened with extinction. Speyeria zerene hippolyta, the Oregon silverspot butterfly, is a U.S.A. federally threatened subspecies that is the focus of considerable conservation effort, but whose evolutionary relationships with other Speyeria taxa are not well-understood. We conducted a genetic analysis of nine Speyeria species and 25 subspecies from western U.S.A., using both mitochondrial and nuclear markers. Our goal was to determine whether such data supported (a) S. z. hippolyta’s designation as an ESU, and (b) the current morphologically-based taxonomy of Speyeria spp. Our data for S. z. hippolyta were equivocal; while nuclear markers resolved all these individuals into a single clade, mtDNA data suggested the existence of two clades. Aside from S. cybele, which was consistently supported as monophyletic, our data provided little support for most of the species currently recognized for western U.S. Speyeria, including S. zerene, and even less for the many subspecies designations. These genetic findings stand in contrast to the morphological differences recognized by experts, and suggest a relatively recent origin for many of these taxa. Two of 66 individuals screened for Wolbachia infection tested positive for this symbiont. Our results provide no persuasive evidence that S. z. hippolyta should lose its status as an ESU, but they have important implications for ongoing management actions such as population augmentation. 相似文献
90.
Global Analysis of Apicomplexan Protein S‐Acyl Transferases Reveals an Enzyme Essential for Invasion
Karine Frénal Chwen L. Tay Christina Mueller Ellen S. Bushell Yonggen Jia Arnault Graindorge Oliver Billker Julian C. Rayner Dominique Soldati‐Favre 《Traffic (Copenhagen, Denmark)》2013,14(8):895-911
The advent of techniques to study palmitoylation on a whole proteome scale has revealed that it is an important reversible modification that plays a role in regulating multiple biological processes. Palmitoylation can control the affinity of a protein for lipid membranes, which allows it to impact protein trafficking, stability, folding, signalling and interactions. The publication of the palmitome of the schizont stage of Plasmodium falciparum implicated a role for palmitoylation in host cell invasion, protein export and organelle biogenesis. However, nothing is known so far about the repertoire of protein S‐acyl transferases (PATs) that catalyse this modification in Apicomplexa. We undertook a comprehensive analysis of the repertoire of Asp‐His‐His‐Cys cysteine‐rich domain (DHHC‐CRD) PAT family in Toxoplasma gondii and Plasmodium berghei by assessing their localization and essentiality. Unlike functional redundancies reported in other eukaryotes, some apicomplexan‐specific DHHCs are essential for parasite growth, and several are targeted to organelles unique to this phylum. Of particular interest is DHHC7, which localizes to rhoptry organelles in all parasites tested, including the major human pathogen P. falciparum. TgDHHC7 interferes with the localization of the rhoptry palmitoylated protein TgARO and affects the apical positioning of the rhoptry organelles. This PAT has a major impact on T. gondii host cell invasion, but not on the parasite's ability to egress. 相似文献