Implications of MDCK cell heterogeneity in cell-based influenza vaccine production

Influenza is a global public health issue that causes serious illness with high mortality rate. Currently, Madin-Darby canine kidney (MDCK) cell culture-based influenza vaccine production moving up to the front as an inexorable trend for the substitution of egg-based vaccine production, owing to its high degree of flexibility and scalability. However, MDCK cells are a continuous cell line and comprise a heterogeneous pool of non-clonal cells that differ in morphological as well as functional features in influenza virus production. The impurity of cell population may lead to fugacious tendency in virus production, and long-term culture may bring potential risk of unstable viral production or vaccine quality as cells in MDCK subclonal population may encounter unexpected manifestation of chromosomal rearrangement, loss of the virus susceptibility, or reduction of the virus partials packaging capability during the culture. Although many details of the influenza virus life cycle have already been unraveled, little is known about the ability of subclones in virus infection, intracellular replication, and virus release during viral vaccine production process. With the widely utilizing of omics-based approaches and progressively accumulating of omics database, transcriptome profile analysis will be a powerful strategy to explore the mechanism of cell heterogeneity, providing great significance for the development of robust virus producing cell line and robust virus production process. This work aims to explore a deeper understanding on the MDCK cell heterogeneity used in influenza virus production. For this purpose, a MDCK cell line that has been extensively used in industrial production was subcloned and examined for the influenza virus productivity. The virus productivity spread over a wide range of more than 300-fold among different clones, which revealed large variations in their ability to produce progeny viruses. The high and low producer as well as parent cell population were expanded to explore the intracellular virus propagation process, and the expression levels of all the annotated genes were quantified across the different subclones using RNA-seq. The RT-qPCR results showed that the influenza virus RNA synthesis and virus release differed dramatically among subclones during a synchronized single-cycle infection. Pathway analysis performed on the genes indicated that most of the genes are not differentially expressed, but a few key cellular metabolic pathways are differentially expressed among the subclones, especially the genes related to the virus infection, replication and release. These results spurs further hypothesis to improve our mechanistic understanding of cell line stability and virus propagation process, which will have significant impact on rationalizing cell line development of viral vaccine producing mammalian cells

Semi-Supervised First-Person Activity Recognition in Body-Worn Video

Body-worn cameras are now commonly used for logging daily life, sports, and law enforcement activities, creating a large volume of archived footage. This paper studies the problem of classifying frames of footage according to the activity of the camera-wearer with an emphasis on application to real-world police body-worn video. Real-world datasets pose a different set of challenges from existing egocentric vision datasets: the amount of footage of different activities is unbalanced, the data contains personally identifiable information, and in practice it is difficult to provide substantial training footage for a supervised approach. We address these challenges by extracting features based exclusively on motion information then segmenting the video footage using a semi-supervised classification algorithm. On publicly available datasets, our method achieves results comparable to, if not better than, supervised and/or deep learning methods using a fraction of the training data. It also shows promising results on real-world police body-worn video

Mutation analysis using the restriction site mutation (RSM) assay

The restriction site mutation RSM assay see Steingrimsdottir et al. H. Steingrimsdottir, D. Beare, J. Cole, J.F.M. Leal, Ž. Ž w T. Kostic, J. Lopez-Barea, G. Dorado, A.R. Lehmann, Development of new molecular procedures for the detection of genetic alteration in man, Mutat. Res. 353 1996 pp. 109–121 for a review has been developed as a genotypic mutation Ž . x . detection system capable of identifying mutations occurring in restriction enzyme sites of genomic DNA. Here we will report the steps taken to overcome some of the initial problems of the assay, namely the lack of quantitative data and limited sensitivity, the aim being to achieve a methodology suitable for the study of low dose chemical exposures. Quantitative data was achieved in the RSM assay by the inclusion of an internal standard molecule in the PCR amplification stage, thus allowing the calculation of both spontaneous and induced mutation frequencies. The sensitivity of the assay was increased through the discovery that intron sequences of genomic DNA accumulated more mutations in vivo compared to the exons, presumably due to differential selective pressure within genes G.J.S. Jenkins, I.deG. Mitchell, J.M. Parry, Enhanced w restriction site mutation RSM analysis of 1,2-dimethylhydrazine-induced mutations, using endogenous Ž . p53 intron sequences, Mutagenesis 12 1997 pp. 117–123 . This increased sensitivity was examined by applying the RSM assay to Ž . x analyse the persistence of N-ethyl-N-nitrosourea ENU -induced mutations in mice testes. Germ line mutations were sought Ž . in testes DNA 3, 10 and 100 days after ENU treatment. Mutations were detected in exons and especially intron regions, the intron mutations were more persistent, still being detected 100 days post-chemical treatment. Assignment of these mutations as ENU induced was complicated in some cases where the spontaneous mutation level was high. This theme of mutation persistence was further investigated by studying the presence of 4-nitroquinoline-1-oxide 4-NQO -induced DNA mutations Ž . in vitro. This study also analysed the relationship between DNA adduct formation and DNA mutation induction by the concurrent RSM analysis and 32P post-labelling analysis of 4-NQO treated human fibroblasts. The results demonstrated that early DNA mutations detected 4 days post-treatment by the RSM assay were probably ex vivo mutations induced by Taq polymerase misincorporation of 4-NQO adducted DNA, due to the maximum levels of 4-NQO adducts being present at this time point. A later mutational peak, after the adduct level had declined, was assumed to be due to DNA sequence changes produced in the fibroblasts by the in vivo processing of DNA adducts

Effects of the combination of loxoprofen sodium and sodium hyaluronate on osteoarthritis and knee function

Purpose: To determine the treatment efficacy of the combination of loxoprofen sodium and sodium hyaluronate in osteoarthritis (OA), and its role in knee joint function. Methods: 98 patients with OA admitted to Guang'an People's Hospital, Sichuan, China were allocated into control group (CNG, given loxoprofen sodium n, = 51) and study group (SG, given loxoprofen sodium and sodium hyaluronate, n = 47). Both groups were compared in terms of the levels of inflammatory factor, Lysholm, VAS, WOMAC scores, treatment effects, serum MDA, NO, SOD levels, adverse effects, and blood rheology indices. Results: The study group had higher SOD levels, and higher BALP and BGP than CNG (p < 0.05). SG had lower TRACP-5b and blood rheological indices than CNG (p < 0.05). The difference in the incidence of adverse reactions was not statistically significant between the two groups (p > 0.05). Conclusion: The combination of loxoprofen sodium and sodium hyaluronate effectively improves the function and blood rheological indices of knee joints. It reduces the occurrence of adverse reactions and the level of pain in patients with OA, and improves OA prognosis. However further clinical trials are required prior to application in clinical practice

Association between tumour infiltrating lymphocytes, histotype and clinical outcome in epithelial ovarian cancer.

BACKGROUND: There is evidence that some ovarian tumours evoke an immune response, which can be assessed by tumour infiltrating lymphocytes (TILs). To facilitate adoption of TILs as a clinical biomarker, a standardised method for their H&E visual evaluation has been validated in breast cancer. METHODS: We sought to investigate the prognostic significance of TILs in a study of 953 invasive epithelial ovarian cancer tumour samples, both primary and metastatic, from 707 patients from the prospective population-based SEARCH study. TILs were analysed using a standardised method based on H&E staining producing a percentage score for stromal and intratumoral compartments. We used Cox regression to estimate hazard ratios of the association between TILs and survival. RESULTS: The extent of stromal and intra-tumoral TILs were correlated in the primary tumours (n = 679, Spearman's rank correlation = 0.60, P < 0.001) with a similar correlation in secondary tumours (n = 224, Spearman's rank correlation = 0.62, P < 0.001). There was a weak correlation between stromal TIL levels in primary and secondary tumour samples (Spearman's rank correlation = 0.29, P < 0.001) and intra-tumoral TIL levels in primary and secondary tumour samples (Spearman's rank correlation = 0.19, P = 0.0094). The extent of stromal TILs differed between histotypes (Pearson chi2 (12d.f.) 54.1, P < 0.0001) with higher levels of stromal infiltration in the high-grade serous and endometriod cases. A significant association was observed for higher intratumoral TIL levels and a favourable prognosis (HR 0.74 95% CI 0.55-1.00 p = 0.047). CONCLUSION: This study is the largest collection of epithelial ovarian tumour samples evaluated for TILs. We have shown that stromal and intratumoral TIL levels are correlated and that their levels correlate with clinical variables such as tumour histological subtype. We have also shown that increased levels of both intratumoral and stromal TILs are associated with a better prognosis; however, this is only statistically significant for intratumoral TILs. This study suggests that a clinically useful immune prognostic indicator in epithelial ovarian cancer could be developed using this technique