A strategy of novel molecular hydrogen-producing antioxidative auxiliary system improves virus production in cell bioreactor.

In the increasing demand for virus vaccines, large-scale production of safe, efficient, and economical viral antigens has become a significant challenge. High-cell-density manufacturing processes are the most commonly used to produce vaccine antigens and protein drugs. However, the cellular stress response in large-scale cell culture may directly affect host cell growth and metabolism, reducing antigen production and increasing production costs. This study provided a novel strategy of the antioxidant auxiliary system (AAS) to supply molecular hydrogen (H2) into the cell culture media via proton exchange membrane (PEM) electrolysis. Integrated with a high-density cell bioreactor, the AAS aims to alleviate cellular stress response and increase viral vaccine production. In the results, the AAS stably maintained H2 concentration in media even in the high-air exposure tiding cell bioreactor. H2 treatment was shown safe to cell culture and effectively alleviated oxidative stress. In two established virus cultures models, bovine epidemic fever virus (BEFV) and porcine circovirus virus type 2 (PCV-2), were employed to verify the efficacy of AAS. The virus yield was increased by 3.7 and 2.5 folds in BEFV and PCV-2 respectively. In conclusion, the AAS-connected bioreactor effectively alleviated cellular oxidative stress and enhanced virus production in high-density cell culture.

A Senescence-Like Cellular Response Inhibits Bovine Ephemeral Fever Virus Proliferation.

During industrial-scale production of viruses for vaccine manufacturing, anti-viral response of host cells can dampen maximal viral antigen yield. In addition to interferon responses, many other cellular responses, such as the AMPK signaling pathway or senescence-like response may inhibit or slow down virus amplification in the cell culture system. In this study, we first performed a Gene Set Enrichment Analysis of the whole-genome mRNA transcriptome and found a senescence-like cellular response in BHK-21 cells when infected with bovine ephemeral fever virus (BEFV). To demonstrate that this senescence-like state may reduce virus growth, BHK-21 subclones showing varying degrees of a senescence-like state were infected with BEFV. The results showed that the BHK-21 subclones showing high senescence staining could inhibit BEFV replication while low senescence-staining subclones are permissive to virus replication. Using a different approach, a senescence-like state was induced in BHK-21 using a small molecule, camptothecin (CPT), and BEFV susceptibility were examined. The results showed that CPT-treated BHK-21 is more resistant to virus infection. Overall, these results indicate that a senescence-like response may be at play in BHK-21 upon virus infection. Furthermore, cell clone selection and modulating treatments using small molecules may be tools in countering anti-viral responses.

Diagnostic Performance of PI-RADS v2, Proposed Adjusted PI-RADS v2 and Biparametric Magnetic Resonance Imaging for Prostate Cancer Detection: A Preliminary Study.

PURPOSE: To evaluate the diagnostic performance of PI-RADS v2, proposed adjustments to PI-RADS v2 (PA PI-RADS v2) and biparametric magnetic resonance imaging (MRI) for prostate cancer detection. METHODS: A retrospective cohort of 224 patients with suspected prostate cancer was included from January 2016 to November 2018. All the patients underwent a multi-parametric MR scan before biopsy. Two radiologists independently evaluated the MR examinations using PI-RADS v2, PA PI-RADS v2, and a biparametric MRI protocol, respectively. Receiver operating characteristic (ROC) curves for the three different protocols were drawn. RESULTS: In total, 90 out of 224 cases (40.18%) were pathologically diagnosed as prostate cancer. The area under the ROC curves (AUC) for diagnosing prostate cancers by biparametric MRI, PI-RADS v2, and PA PI-RADS v2 were 0.938, 0.935, and 0.934, respectively. For cancers in the peripheral zone (PZ), the diagnostic sensitivity was 97.1% for PI-RADS v2/PA PI-RADS v2 and 96.2% for biparametric MRI. Moreover, the specificity was 84.0% for biparametric MRI and 58.0% for PI-RADS v2/PA PI-RADS v2. For cancers in the transition zone (TZ), the diagnostic sensitivity was 93.4% for PA PI-RADS v2 and 88.2% for biparametric MRI/PI-RADS v2. Furthermore, the specificity was 95.4% for biparametric MRI/PI-RADS v2 and 78.0% for PA PI-RADS v2. CONCLUSIONS: The overall diagnostic performance of the three protocols showed minimal differences. For lesions assessed as being category 3 using the biparametric MRI protocol, PI-RADS v2, or PA PI-RADS v2, it was thought prostate cancer detection could be improved. Attention should be paid to false positive results when PI-RADS v2 or PA PI-RADS v2 are used.

Development of a quick dot blot assay for the titering of bovine ephemeral fever virus.

BACKGROUND: Bovine ephemeral fever virus (BEFV) causes fever and muscle stiffness in cattle, resulting in negative economic impact for cattle and dairy farms. During the manufacturing process of inactivated vaccine for virus control, it is important to determine the virus titer, but traditional methods such as plaque assay and TCID50 assay require days of waiting time. We sought to develop a quick dot blot assay for BEFV titering. RESULTS: Three different kinds of BEFV antigens were prepared to raise primary antibodies for BEFV detection in dot blot assays: 1) purified BEFV particles, 2) Escherichia coli (E. coli)-expressed BEFV G1 region, and 3) E. coli-expressed BEFV N protein. Results showed that antibodies raised against purified BEFV particles can detect BEFV particles, but it also showed a high background level from the proteins of BHK-21 cells. Antibodies raised against E.coli-expressed BEFV G1 region could not detect BEFV particles in dot blot assays. Finally, antibodies raised against E.coli-expressed BEFV N protein detected BEFV particles with a high signal-to-noise ratio in dot blot assays. CONCLUSIONS: E.coli-expressed N protein is a suitable antigen for the production of antiserum that can detect BEFV particles with a high signal-to-noise ratio. A dot blot assay kit using this antiserum can be developed as a quick and economical way for BEFV titering.

Biocompatible testing and physical properties of curdlan-grafted poly(vinyl alcohol) scaffold for bone tissue engineering.

Curdlan was grafted to poly(vinyl alcohol) (PVA) to form a porous scaffold. The grafted PVA-curdlan 3D scaffold was then examined by Fourier transform infrared spectroscopy (FT-IR). Grafting increased the water absorbency of the scaffold by 280%. Scanning electron microscopic (SEM) observations of the material revealed that the 3D scaffold was highly porous when it was fabricated using a homogenizer at 300rpm. Compression testing revealed that, increasing the amount of curdlan increased the strength of the 3D scaffold to 8-16×10-3MPa. Over 28days, various enzymes degraded the 3D scaffold, causing a weight loss of up to 20-40%. In vivo tests revealed favorable cell proliferation and growth in a 3D scaffold.