Abrahamsenmcgowan1358

During the development of cell lines for therapeutic protein production, a vector harboring a product transgene is integrated into the genome. To ensure production stability and consistent product quality, single-cell cloning is then performed. Since cells derived from the same parental clone have the same transgene integration locus, the identity of the integration site can also be used to verify the clonality of a production cell line. In this study, we present a high-throughput pipeline for clonality verification through integration site analysis. Sequence capture of genomic fragments that contain both vector and host cell genome sequences was used followed by next-generation sequencing to sequence the relevant vector-genome junctions. A Python algorithm was then developed for integration site identification and validated using a cell line with known integration sites. Using this system, we identified the integration sites of the host vector for 31 clonal cell lines from five independent vector integration events while using one set of probes against common features of the host vector for transgene integration. Cell lines from the same lineage had common integration sites, and they were distinct from unrelated cell lines. The integration sites obtained for each clone as part of the analysis may also be used for clone selection, as the sites can have a profound effect on the transgene's transcript level and the stability of the resulting cell line. This method thus provides a rapid system for integration site identification and clonality verification. © 2020 American Institute of Chemical Engineers.BACKGROUND The etiology and prognosis of acute liver failure (ALF) remains unknown in a significant proportion of cases. Signs of autoimmunity may be present, but no consistent pattern has been observed. We aimed to analyse if pretransplant immunological findings, HLA haplotypes and clinical features among patients with unknown etiology differ from those of autoimmune or other known etiology. We also analysed whether such signs impact post-transplant biopsy findings or complications. METHODS All adult ALF patients undergoing liver transplantation (LT) in Finland during 1987-2015 were followed to 2016. Data were from the LT registry, pathology database and patient records. 124 patients were included in the analysis. Study subgroups were acute autoimmune hepatitis (AIH) (n=25), known non-AIH etiology (n=54), and unknown etiology (n=45). RESULTS The unknown etiology group differed from the known non-AIH group with regard to the following pretransplant autoimmunity-associated features positive pANCA (35% vs 8%; P=0.02), higher mean IgA (3.2±1.7 vs 2.1±1.4, P=0.006) and IgG (12.7±4.3 vs 8.5±3.6, P=0.001). AIH-associated HLA haplotypes B8, DR3 and B8DR3 were more common in the AIH group (40%, 44% and 36%) and in the unknown group (29%, 33% and 29%) than in the known non-AIH group (11%, 17% and 11%) or in the Finnish general population (17%, 18% and 8%). However, these findings had no association with protocol biopsies, extrahepatic autoimmune diseases or survival. Patients with ≥1 rejection episode had higher pretransplant IgA (3.7±2.3 vs 2.6±1.2, P=0.02) and IgG (16.4±10.2 vs 12.4±6.8, P=0.03) than those without rejections. CONCLUSIONS Autoimmunity-associated pretransplant laboratory findings and HLA haplotypes were common in ALF of unknown etiology, but showed minimal predictive value for post-transplant biopsy findings, clinical complications or survival. This article is protected by copyright. All rights reserved.We report a Brucella outbreak with seven cases in the Northern Region of Portugal in 2018-2019, associated with the consumption of fresh cheese. This outbreak has implications for risk assessment in Portuguese migrants related to this area, and it is an example of cooperation between public institutions, in a One Health based approach. © 2020 Blackwell Verlag GmbH.The presentation of pre-sliced specimens is a frequently used method in the laboratory teaching of cross-sectional anatomy. In the present study, a new teaching method based on a hands-on slicing activity was introduced into the teaching of brain, heart, and liver cross-sectional anatomy. A randomized, controlled trial was performed. A total of 182 third-year medical students were randomized into a control group taught with the prosection mode (pre-sliced organ viewing) and an experimental group taught with the dissection mode (hands-on organ slicing). These teaching methods were assessed by testing the students' knowledge of cross-sectional specimens and cross-sectional radiological images, and analyzing students' feedback. Using a specimen test on three organs (brain, heart, and liver), significant differences were observed in the mean scores of the control and experimental groups for brain 59.6% (±14.2) vs. 70.1% (±15.5), (P  less then  0.001, Cohen's d = 0.17); for heart 57.6% (±12.5) vs. 75.6% (±15.3), (P  less then  0.001, d = 0.30); and for liver 60.4% (±14.5) vs. 81.7% (±14.2), (P  less then  0.001, d = 0.46). In a cross-sectional radiological image test, better performance was also found in the experimental group (P  less then  0.001). The mean scores of the control vs. experimental groups were as follows for brain imaging 63.9% (±15.1) vs. 71.1% (±16.1); for heart imaging 64.7% (±14.5) vs. 75.2% (±15.5); and for liver imaging 61.1% (±15.5) vs. 81.2% (±14.6), respectively. The effect sizes (Cohen's d) were 0.05, 0.23, and 0.52, respectively. Students in the lower tertile benefited the most from the slicing experiences. Students' feedback was generally positive. Hands-on slicing activity can increase the effectiveness of anatomy teaching and increase students' ability to interpret radiological images. © 2020 American Association of Anatomists.Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) has been involved in several inflammation dependent diseases by mediating the chemotaxis of pro-inflammatory cells in response to allergy and other responses through PGD2 ligation. This CRTH2-PGD2 signaling pathway has become a target for treating allergic and type 2 inflammation dependent diseases, with many inhibitors developed to target the PGD2 binding pocket. One of such inhibitors is the ramatroban analog, CT-133, which exhibited therapeutic potency cigarette smoke-induced acute lung injury in patients. Nonetheless, the molecular mechanism and structural dynamics that accounts for its therapeutic prowess remain unclear. Employing computational tools, this study revealed that although the carboxylate moiety in CT-133 and the native agonist PGD2 aided in their stability within the CRTH2 binding pocket, the tetrahydrocarbazole group of CT-133 engaged in strong interactions with binding pocket residues which could have formed as the basis of the antagonistic advantage of CT-133. Tetrahydrocarbazole group interactions also enhanced the relative stability CT-133 within the binding pocket which consequently favored CT-133 binding affinity. CT-133 binding also induced an inactive or 'desensitized' state in the helix 8 of CRTH2 which could conversely favor the recruitment of arrestin. These revelations would aid in the speedy development of small molecule inhibitors of CRTH2 in the treatment of type 2 inflammation dependent diseases. ACH-0144471 © 2020 Wiley-VHCA AG, Zurich, Switzerland.Mitral valvulopathy presents as regurgitation, stenosis, or mixed disease and can occur in both native and prosthetic valves. Such disease develops in conjunction with pathophysiologic changes in the left atrium (LA) and drives changes in LA compliance, pressure, and thus clinical syndromes. With advances in the understanding and treatment of structural heart disease and in the setting of higher-risk patient populations, less-invasive transcatheter approaches have become increasingly commonplace in the treatment of mitral valve disease. Over time, transcatheter mitral valve interventions have evolved to include paravalvular leak closure, mitral valve repair, and mitral valve replacement. Parallel to this evolution, advances in invasive intracardiac pressure monitoring, particularly at the level of the LA, have also occurred. These advances emphasize the unique interplay between mitral valve disease and LA function; account for limitations of noninvasive assessment; and guide beneficial outcomes in each area of transcatheter mitral valve intervention. As a result, continuous transseptal LA pressure monitoring has developed into an indispensable instrument in successful percutaneous mitral valve intervention, complementing traditional noninvasive assessment. © 2020 Wiley Periodicals, Inc.Bioresorbable vascular scaffolds (BVSs) were designed to overcome the limitations of metallic stents. Absorb BVS has provided information about strut thickness, scaffold degradation, vessel wall coverage, and their influence on thrombosis. Clinical trials have shown higher rates of target vessel myocardial infarction and stent thrombosis with the absorb BVS than with second-generation drug-eluting stents. © 2020 Wiley Periodicals, Inc.We identified a probable new null HLA-C allele, C*0323N, which originated from C*03040102, but does not react with Cw3 antibodies. This allele was identified by sequence analysis, which indicated that a single G-to-A substitution at position 406 in exon 3 created a null allele under a new mechanism the mutation changes the position of the intron 2-exon 3 splice site to be further into exon 3, leading to a frameshift and a premature stop codon. Sequence analysis of cDNA confirmed the existence of the causative alternative acceptor splice site and the resultant deletion of 64 nucleotides in exon 3. Analysis of 220 blood or bone marrow donors in Japan with C*0323N demonstrated that Japanese HLA-C*0323N is on the haplotype A*2601∼C*0323N∼B*4002∼DRB1*0901. © 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.In order to determine six alkaloids (mass fraction) of nicotine, nornicotine, myosmine, anatabine, anabasine, and nicotyrine in tobacco and tobacco products quickly, accurately, and simultaneously, a novel method based on direct analysis of real-time model in situ ionization technique combined tandem mass spectrometry with a modified sample pretreatment was established, in which experimental parameters such as the type and amount of extraction solvent and injection rate were optimized, respectively. The samples of five commercial cigarettes and five kinds of tobacco leaves were analyzed by the established method, and the determined values were compared with those obtained using a gas chromatography with mass spectrometry method (1) Under optimized conditions (30 mL ultrapure water as extraction solvent and with extraction rate of 0.6 mm/s), analysis could be completed within 10 min. (2) The linear range of the method was 0.002-2000 μg/g with R 2 = 0.9957 , the recovery ranged from 86.8 to 105.6%, and the limit of detection and the limit of quantification were 0.004-0.835 μg/g and 0.013-2.787 μg/g, respectively. (3) The relative standard deviation between direct analysis of real-time method and the gas chromatography with mass spectrometry method was 0.34-8.83%. The established method is rapid, reliable, and suitable for the ultrafast determination of six alkaloids in tobacco and tobacco products. © 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.