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Lin Mogensen posted an update 6 months ago
Background information There have been several studies to understand the influence of stiffness of the culture substrates for different types of adherent cells. It is generally accepted that cell proliferation, spreading and focal adhesions increase with higher matrix stiffness. selleck compound However, what remains unclear is whether this kind of cell behaviour may be reverted by culturing on soft substrates those cell lines that were originally selected or primed on stiff surfaces. Results Here, we studied the influence of substrate softness on proliferation, adhesion and morphology of the highly proliferative hepatic C9 cell line. We cultured C9 cells on soft and stiff polydimethylsiloxane (PDMS) substrates prepared with two different elastic moduli in the range of 10 and 200 kPa, respectively. Lower cell proliferation was observed on substrates with lower stiffness without affecting cell viability. The proliferation rate of C9 cell line along with extracellular growth-regulated kinase (ERK) phosphorylation was decreased l proliferation rate contrary to the effect observed in short periods of cell culture. In contrast to previous reports, cell death was not observed during these experiments, discarding a cell selection mechanism and suggesting soft cell adaptation may be limited in time in C9 cells.Recurrence of positive SARS CoV-2 PCR has been described in patients discharged from hospital after 2 consecutive negative PCR. We discuss possible explanations including false negative, reactivation and re-infection and propose different strategy to solve this issue. Prolonged SARS-CoV-2 RNA shedding and recurrence of viral RNA shedding in asymptomatic patients remain unknown. Transmission of SARS-CoV-2 by asymptomatic carriers had been documented. Considering the significance of this ongoing global public health emergency, it is necessary to carry out large studies to better understand the issue of potential SARS-CoV-2 recurrence in COVID-19 patients. This article is protected by copyright. All rights reserved.Homologous recombination over large genomic regions is difficult to achieve due to low efficiencies. Here, we report the successful engineering of a humanized mTert allele, hmTert, in the mouse genome by replacing an 18.1-kb genomic region around the mTert gene with a recombinant fragment of over 45.5 kb, using homologous recombination facilitated by the Crispr/Cas9 technology, in mouse embryonic stem cells (mESCs). In our experiments, with DNA double-strand breaks (DSBs) generated by Crispr/Cas9 system, the homologous recombination efficiency was up to 11% and 16% in two mESC lines TC1 and v6.5, respectively. Overall, we obtained a total of 27 mESC clones with heterozygous hmTert/mTert alleles and three clones with homozygous hmTert alleles. DSBs induced by Crispr/Cas9 cleavages also caused high rates of genomic DNA deletions and mutations at single-guide RNA target sites. Our results indicated that the Crispr/Cas9 system significantly increased the efficiency of homologous recombination-mediated gene editing over a large genomic region in mammalian cells, and also caused frequent mutations at unedited target sites. Overall, this strategy provides an efficient and feasible way for manipulating large chromosomal regions.Azithromycin (AZM) is a broad-spectrum antibiotic widely used to treat infections. AZM has also been shown to have anti-inflammatory and immunomodulatory functions unrelated to its antibacterial activity which contribute to the effectiveness of this drug in chronic respiratory diseases. The mechanisms behind these beneficial effects are not yet fully elucidated. We have previously shown that AZM enhances barrier integrity of bronchial epithelial cells and directs them towards epidermal differentiation. In this study, we analyzed the effect of AZM pre-treatment of bronchial and alveolar derived cell lines while mechanically stressed in a Cyclical Pressure Air-liquid interface Device (CPAD), that mimics the disruption of the epithelial barrier with increased inflammatory response in lung tissue, associated with ventilator-induced lung injury (VILI). Immunostainings along with imaging the cells in the electron microscope, show that barrier integrity of the epithelium is compromised by cyclically stressing the cells but maintained when cells were AZM pre-treated. Lamellar body formations were also revealed in AZM pre-treated cells possibly further supporting the barrier enhancing effects. RNA sequencing shows that the inflammatory response is attenuated by AZM pre-treatment before cyclical stress. Expression of YKL-40, an emerging inflammatory marker is shown to increase due to cyclical stress and by AZM treatment. These data suggest that AZM has barrier protective and immunomodulatory effects, attenuating the inflammatory response during mechanical stress and might therefore be lung protective during mechanical ventilation.The Patient-Oriented Eczema Measure (POEM, 0-28 points) is a self-assessed, repeatable measurement tool for measuring atopic dermatitis (AD) severity. How-ever, whether POEM score is influenced by allergic comorbidities and whether POEM’s severity banding is applicable in web-based surveys for AD remain unclear. A web-based questionnaire survey was conducted in 329 patients with AD. POEM, self-reported severity of AD, and comorbidity of allergic diseases including asthma, pollen rhinitis, allergic conjunctivitis, and food allergy were assessed. POEM scores were not affected by a history of comorbid allergic diseases. The severity banding for POEM scores on the web-based survey was as follows clear/almost clear = 0, mild = 1-8, moderate = 9-21, and severe/very severe = 22-28, which was comparable to previous banding. These results suggest that POEM is useful for determining AD severity, even in web-based surveys. Patients with POEM scores above 9 points may be grouped into moderate, severe, and very severe AD.The approval of BRAF and MEK inhibitors has significantly improved treatment outcomes for patients with BRAF-mutated metastatic melanoma. The 3 first-line targeted therapy trials have provided similar results, and thus the identification of predictive biomarkers may generate a more precise basis for clinical decision-making. Elevated baseline lactate dehydrogenase (LDH) has already been determined as a strong prognostic factor. Therefore, this indirect analysis compared subgroups with elevated baseline LDH across the pivotal targeted therapy trials co-BRIM, COMBI-v and COLUMBUS part 1. The Bucher method was used to compare progression-free survival, objective response rate and overall survival indirectly. The results show a non-significant risk reduction for progression in the subgroup with elevated baseline LDH receiving vemurafenib plus cobimetinib compared with dabrafenib plus trametinib and encorafenib plus binimetinib. Although an indirect comparison, these data might provide some guidance for treatment recommendations in melanoma patients with elevated LDH.
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