Development of high-producing CHO cell lines through target-designed strategy

Productivity and stability are critical for the protein drug producing cell lines for manufacturing. Given that the integration sites of gene of interest (GOI) could contribute remarkable effect on the productivity and stability of GOI expression, we intended to develop a targeting-designed approach to generate the high-producing cell lines in a time-saving and less labor-intensive method through targeting the active and stable regions. To identify the active and stable regions located in CHO genome, two approaches were applied in our experiments. Firstly, the integration sites of GOI in cell clones developed by random integration were identified by whole genome sequencing. Secondly, we developed transposon-mediated low copy integration to discover novel active region located in CHO genome. It is interesting that the productivity per integrated GOI in cell clones developed by transposon system was more than two times to that in cell clones developed by random integration (random integration: 20-40 mg/L/copy; transposon-mediated integration: 40-140mg/L/copy). In addition, about 80% of cell clones developed by transposon system maintained the stability of antibody titer after culturing for 60 generations. These results implied that the potential active and stable integration region in the cell clones developed by transposon system. The identified integration regions could be applied for target integration. In order to verify the expression activity and stability of the integration sites, we employed CRISPR/Cas9 to specifically integrate the antibody gene into CHO genome for expression. Our data showed the cell pool generated by knock-in of expression vector into the IS1 integration site present higher expression titer than cell pools generated by integration into other sites or random integration. We further cultured the single cell clones derived from this cell pool by Clonepix and limiting dilution. These single cell clones have high expression titer ranging from 254 to 804 mg/L in batch culture of after 6 Days. A single cell clone(376 mg/L in batch culture) can reached 2 g/L in fed-batch culture. The stability analysis showed this clone maintain stable expression of GOI after 60 generation. Here, we demonstrated the generation of stable cell line with high protein expression by CRISPR/Cas9 mediated target integration. This approach will cost less time and labor than traditional method

Mobile Edge Computing Platform Deployment in 4G LTE Networks: A Middlebox Approach

This paper has been presented at : USENIX Workshop on Hot Topics in Edge Computing (Hot Edge '18)Low-latency demands for cellular networks have at-tracted much attention. Mobile edge computing (MEC), which deploys a cloud computing platform at the edge closer to mobile users, has been introduced as an enabler of low-latency performance in 4G and 5G networks. In this paper, we propose an MEC platform deployment so-lution in 4G LTE networks using a middlebox approach. It is standard-compliant and transparent to existing cel-lular network components, so they need not be modified. The MEC middlebox sits on the S1 interface, which con-nects an LTE base station to its core network, and does traffic filtering, manipulation and forwarding. It enables the MEC service for mobile users by hosting application servers. Such middlebox approach can save deployment cost and be easy to install. It is different from other stud-ies that require modifications on base stations or/and core networks. We have confirmed its viability through a pro-totype based on the OpenAirInterface cellular platform.We thank our shepherd Weisong Shi for his help, and also thank the anonymous reviewers for their valuable comments on improving this paper. This work was partially supported by the Ministry of Science and Technology, Taiwan, under grant numbers 106-2622-8-009-017 and 106-2218-E-009-018, and by the H2020 collaborative Europe/Taiwan research project 5G-CORAL (grant number 761586)

Inactivation of the Osteopontin Gene Enhances Vascular Calcification of Matrix Gla Protein–deficient Mice: Evidence for Osteopontin as an Inducible Inhibitor of Vascular Calcification In Vivo

Osteopontin (OPN) is abundantly expressed in human calcified arteries. To examine the role of OPN in vascular calcification, OPN mutant mice were crossed with matrix Gla protein (MGP) mutant mice. Mice deficient in MGP alone (MGP−/− OPN+/+) showed calcification of their arteries as early as 2 weeks (wk) after birth (0.33 ± 0.01 mmol/g dry weight), and the expression of OPN in the calcified arteries was greatly up-regulated compared with MGP wild-types. OPN accumulated adjacent to the mineral and colocalized to surrounding cells in the calcified media. Cells synthesizing OPN lacked smooth muscle (SM) lineage markers, SM α-actin and SM22α. However, most of them were not macrophages. Importantly, mice deficient in both MGP and OPN had twice as much arterial calcification as MGP−/− OPN+/+ at 2 wk, and over 3 times as much at 4 wk, suggesting an inhibitory effect of OPN in vascular calcification. Moreover, these mice died significantly earlier (4.4 ± 0.2 wk) than MGP−/− OPN+/+ counterparts (6.6 ± 1.0 wk). The cause of death in these animals was found to be vascular rupture followed by hemorrhage, most likely due to enhanced calcification. These studies are the first to demonstrate a role for OPN as an inducible inhibitor of ectopic calcification in vivo

SARS Exposure and Emergency Department Workers

Of 193 emergency department workers exposed to severe acute respiratory syndrome (SARS), 9 (4.7%) were infected. Pneumonia developed in six workers, and assays showed anti-SARS immunoglobulin (Ig) M and IgG. The other three workers were IgM-positive and had lower IgG titers; in two, mild illness developed, and one remained asymptomatic

High-yield antibody production using targeted integration and engineering CHO host

To identify the high expression sites in the CHO cells, we employed NGS to analyze the integration sites of a high producing cell line (titer \u3e 3g/L). The pair-end reads with one read mapped to the vector and the other read mapped to the CHO reference genome are extracted to identify the integration sites. To test the expression activity of the integration sites, we employed CRISPR/Cas9 to specifically integrate the antibody gene into CHO genome for expression. Our data showed 4 integration sites are in the high producing cell line. Among the 4 integration site, one integration site was tested by CRISPR/Cas9 for target integration of antibody gene for expression. The target integrated cell pool present higher expression level (130 mg/L/copy) and less copy number when compared other integration sites. Through single-copy integration method, we can also achieve 60-150 mg/L/copy in a batch culture. About 80% of the single-copy cell clones were stable at generation 60. We have also applied the CHO-specific microarray transcriptomics technology to identify genes that contribute to high productivity. Transfection of our proprietary dual promoter vector J 1.0 resulting in 1.65 to 2.4 fold increase in the expression in engineered CHO DXB11 host. Through fed-batch process development, 3 – 5 g/L mAb productivity can be achieved through targeted integration and engineered CHO host

Predicting the Severity and Prognosis of Trismus after Intensity-Modulated Radiation Therapy for Oral Cancer Patients by Magnetic Resonance Imaging

To develop magnetic resonance imaging (MRI) indicators to predict trismus outcome for post-operative oral cavity cancer patients who received adjuvant intensity-modulated radiation therapy (IMRT), 22 patients with oral cancer treated with IMRT were studied over a two-year period. Signal abnormality scores (SA scores) were computed from Likert-type ratings of the abnormalities of nine masticator structures and compared with the Mann-Whitney U-test and Kruskal–Wallis one-way ANOVA test between groups. Seventeen patients (77.3%) experienced different degrees of trismus during the two-year follow-up period. The SA score correlated with the trismus grade (r = 0.52, p<0.005). Patients having progressive trismus had higher mean doses of radiation to multiple structures, including the masticator and lateral pterygoid muscles, and the parotid gland (p<0.05). In addition, this group also had higher SA-masticator muscle dose product at 6 months and SA scores at 12 months (p<0.05). At the optimum cut-off points of 0.38 for the propensity score, the sensitivity was 100% and the specificity was 93% for predicting the prognosis of the trismus patients. The SA score, as determined using MRI, can reflect the radiation injury and correlate to trismus severity. Together with the radiation dose, it could serve as a useful biomarker to predict the outcome and guide the management of trismus following radiation therapy

The trend of susceptibilities to amphotericin B and fluconazole of Candida species from 1999 to 2002 in Taiwan

BACKGROUND: Candida species have various degrees of susceptibility to common antifungal drugs. The extent of resistance to amphotericin B and fluconazole of Candida glabrata isolates causing candidemia has been reported. Active surveillance may help us to monitor the trend of susceptibility to antifungal drugs and to determine if there is an emerging co-resistance to both drugs of Candida species, specifically, of C. glabrata in Taiwan. METHODS: The susceptibilities to amphotericin B and fluconazole of Candida species collected in 1999 and 2002 of the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) were determined by the microdilution method. RESULTS: The antifungal susceptibilities of 342 and 456 isolates collected from 11 hospitals participating in both TSARY 1999 and TSARY 2002, respectively, have been determined. The resistance rate to amphotericin B has increased from 0.3% in the TSARY1999 to 2.2% in the TSARY 2002. In contrast, the resistance rate to fluconazole has decreased from 8.8% to 2.2%. Nevertheless, significantly more C. glabrata isolates were not susceptible to fluconazole in the TSARY 2002 (47.4%) than that in the TSARY 1999 (20.8%). There were 9.8% and 11% of C. glabrata isolates having susceptible-dose dependent and resistant phenotype to fluconazole in the TSARY 1999, verse 45.3% and 2.1% in the TSARY 2002. CONCLUSION: There was an increase of resistance rate to amphotericin B in C. glabrata. On the other hand, although the resistance rate to fluconazole has decreased, almost half of C. glabrata isolates were not susceptible to this drug. Hence, continuous monitoring the emerging of co-resistance to both amphotericin B and fluconazole of Candida species, specifically, of C. glabrata, will be an important early-warning system

Detection of SARS-associated Coronavirus in Throat Wash and Saliva in Early Diagnosis

Early detection of SARS-CoV in throat wash and saliva suggests that these specimens are ideal for SARS diagnosis

Gas Sensors Based on Semiconducting Metal Oxide One-Dimensional Nanostructures

This article provides a comprehensive review of recent (2008 and 2009) progress in gas sensors based on semiconducting metal oxide one-dimensional (1D) nanostructures. During last few years, gas sensors based on semiconducting oxide 1D nanostructures have been widely investigated. Additionally, modified or doped oxide nanowires/nanobelts have also been synthesized and used for gas sensor applications. Moreover, novel device structures such as electronic noses and low power consumption self-heated gas sensors have been invented and their gas sensing performance has also been evaluated. Finally, we also point out some challenges for future investigation and practical application

Antifungal susceptibility profiles and drug resistance mechanisms of clinical Candida duobushaemulonii isolates from China

Candida duobushaemulonii, type II Candida haemulonii complex, is closely related to Candida auris and capable of causing invasive and non-invasive infections in humans. Eleven strains of C. duobushaemulonii were collected from China Hospital Invasive Fungal Surveillance Net (CHIF-NET) and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), VITEK 2 Yeast Identification Card (YST), and internal transcribed spacer (ITS) sequencing. Whole genome sequencing of C. duobushaemulonii was done to determine their genotypes. Furthermore, C. duobushaemulonii strains were tested by Sensititre YeastOne™ and Clinical and Laboratory Institute (CLSI) broth microdilution panel for antifungal susceptibility. Three C. duobushaemulonii could not be identified by VITEK 2. All 11 isolates had high minimum inhibitory concentrations (MICs) to amphotericin B more than 2 μg/ml. One isolate showed a high MIC value of ≥64 μg/ml to 5-flucytosine. All isolates were wild type (WT) for triazoles and echinocandins. FUR1 variation may result in C. duobushaemulonii with high MIC to 5-flucytosine. Candida duobushaemulonii mainly infects patients with weakened immunity, and the amphotericin B resistance of these isolates might represent a challenge to clinical treatment