High-density Vero cell perfusion culture in BioBLU 5p Single-Use Vessels

Vero cells are anchorage-dependent cells that are widely used as a platform for viral vaccine production. In stirred-tank bioreactors, they are ordinarily grown on microcarriers. Fibra-Cel® disks are a promising alternative attachment matrix with a high surface-to-volume ratio. They provide a three-dimensional environment that protects cells from damaging shear forces, helping to achieve high cell densities. In this study, we cultivated Vero cells in Eppendorf BioBLU 5p Single-Use Vessels pre-packed with Fibra-Cel. The process was controlled with a BioFlo® 320 bioprocess control station. We cultivated the cells in perfusion mode, which ensures a consistent supply of nutrients and the removal of toxic byproducts. We achieved the very high Vero cell density of approximately 43 million cells per mL, demonstrating great potential for Vero-cell-based vaccine production using Fibra-Cel packed-bed vessels

Cell culture scale-up in BioBLU® c rigid-wall, single-use bioreactors

For cultivation of mammalian cells in biopharmaceutical research and manufacturing, single-use technology possesses several advantages to autoclavable material. Bioreactor scalability is critical to streamlining the adaptation of culture volumes during process development and manufacturing. We analyzed BioBLU Single-Use Vessels of different sizes (maximum working volumes of 0.25 L, 3.75 L, and 40 L) that are of geometrically similar stirred-tank design. We identified a scalable tip speed zone and an overlapping range of kLa values, which cover most mammalian cell culture needs. Using computational fluid dynamics simulations we determined the power numbers of the BioBLU bioreactors. Based on these data we scaled up a mAb production process in CHO cells from 250 mL to 3.75 L to 40 L by keeping constant P/V values (impeller power consumption per liquid volume) among the differently sized vessels. Similar cell growth curves and mAb production profiles were achieved at all three scales. In summary, this study demonstrates the excellent scalability of the single-use bioreactors tested

Astragaloside IV inhibits pathological functions of gastric cancer-associated fibroblasts through regulation of HOXA6/ZBTB12 axis

[email protected], [email protected] Cancer-associated fibroblasts (CAFs) play critical roles in the tumor microenvironment and exert tumor-promoting or tumor-retarding effects on cancer development. Astragaloside IV has been suggested to rescue the pathological impact of CAFs in gastric cancer. This study aimed to investigate the potential mechanism of astragaloside IV in the regulation of CAF pathological functions in gastric cancer development. Homeobox A6 (HOXA6), and Zinc Finger and BTB Domain Containing 12 (ZBTB12) are highly expressed in gastric CAFs compared with normal fibroblasts (NFs) based on the GSE62740 dataset. We found that astragaloside IV-stimulated CAFs suppressed cell growth, migration, and invasiveness of gastric cancer cells. HOXA6 and ZBTB12 were downregulated after astragaloside IV treatment in CAFs. Further analysis revealed that HOXA6 or ZBTB12 knockdown in CAFs also exerted inhibitory effects on the malignant phenotypes of gastric cells. Additionally, HOXA6 or ZBTB12 overexpression in CAFs enhanced gastric cancer cell malignancy, which was reversed after astragaloside IV treatment. Moreover, based on the hTFtarget database, ZBTB12 is a target gene that may be transcriptionally regulated by HOXA6. The binding between HOXA6 and ZBTB12 promoter in 293T cells and CAFs was further confirmed. HOXA6 silencing also induced the downregulation of ZBTB12 mRNA and protein in CAFs. Astragaloside IV was demonstrated to regulate the expression of ZBTB12 by mediating the transcriptional activity of HOXA6. Our findings shed light on the therapeutic value of astragaloside IV for gastric cancer

Direct aperture optimization using an inverse form of back-projection

Direct aperture optimization (DAO) has been used to produce high dosimetric quality intensity-modulated radiotherapy (IMRT) treatment plans with fast treatment delivery by directly modeling the multileaf collimator segment shapes and weights. To improve plan quality and reduce treatment time for our in-house treatment planning system, we implemented a new DAO approach without using a global objective function (GFO). An index concept is introduced as an inverse form of back-projection used in the CT multiplicative algebraic reconstruction technique (MART). The index, introduced for IMRT optimization in this work, is analogous to the multiplicand in MART. The index is defined as the ratio of the optima over the current. It is assigned to each voxel and beamlet to optimize the fluence map. The indices for beamlets and segments are used to optimize multileaf collimator (MLC) segment shapes and segment weights, respectively. Preliminary data show that without sacrificing dosimetric quality, the implementation of the DAO reduced average IMRT treatment time from 13 min to 8 min for the prostate, and from 15 min to 9 min for the head and neck using our in-house treatment planning system PlanUNC. The DAO approach has also shown promise in optimizing rotational IMRT with burst mode in a head and neck test case

Intact RNA structurome reveals mRNA structure-mediated regulation of miRNA cleavage in vivo

[EN] MicroRNA (miRNA)-mediated cleavage is involved in numerous essential cellular pathways. miRNAs recognize target RNAs via sequence complementarity. In addition to complementarity, in vitro and in silico studies have suggested that RNA structuremay influence the accessibility of mRNAs to miRNA-induced silencing complexes (miRISCs), thereby affecting RNA silencing. However, the regulatory mechanism of mRNA structure in miRNA cleavage remains elusive. We investigated the role of in vivo RNA secondary structure in miRNA cleavage by developing the new CAP-STRUCTURE-seq method to capture the intact mRNA structurome in Arabidopsis thaliana. This approach revealed that miRNA target sites were not structurally accessible for miRISC binding prior to cleavage in vivo. Instead, we found that the unfolding of the target site structure plays a key role in miRISC activity in vivo. We found that the single-strandedness of the two nucleotides immediately downstream of the target site, named Target Adjacent nucleotideMotif, can promotemiRNA cleavage but not miRNA binding, thus decoupling target site binding from cleavage. Our findings demonstrate that mRNA structure in vivo can modulate miRNA cleavage, providing evidence of mRNA structure-dependent regulation of biological processes.Biotechnology and Biological Sciences Research Council [BB/L025000/1]; the NorwichResearch Park Science Links Seed Fund; and European Commission Horizon 2020 European Research Council, Starting Grant [680324]. Funding for open access charge: Biotechnology and Biological Sciences Research Council [BB/L025000/1]; the Norwich Research Park Science Links Seed Fund; and European Commission Horizon 2020 European Research Council, Starting Grant [680324].Yang, M.; Woolfenden, HC.; Zhang, Y.; Fang, X.; Liu, Q.; Vigh, ML.; Cheema, J.... (2020). 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Dosimetric effect due to the motion during deep inspiration breath hold for left-sided breast cancer radiotherapy

Deep inspiration breath-hold (DIBH) radiotherapy for left-sided breast cancer can reduce cardiac exposure and internal motion. We modified our in-house treatment planning system (TPS) to retrospectively analyze breath-hold motion log files to calculate the dosimetric effect of the motion during breath hold. Thirty left-sided supine DIBH breast patients treated using AlignRT were studied. Breath-hold motion was recorded — three translational and three rotational displacements of the treatment surface — the Real Time Deltas (RTD). The corresponding delivered dose was estimated using the beam-on portions of the RTDs. Each motion was used to calculate dose, and the final estimated dose was the equally weighted average of the multiple resultant doses. Ten of thirty patients had internal mammary nodes (IMN) purposefully included in the tangential fields, and we evaluated the percentage of IMN covered by 40 Gy. The planned and delivered heart mean dose, lungs V20 (volume of the lungs receiving > 20 Gy), percentage of IMN covered by 40 Gy, and IMN mean dose were compared. The averaged mean and standard deviation of the beam-on portions of the absolute RTDs were 0.81 ± 1.29 mm, 0.68 ± 0.85mm, 0.76 ± 0.85 mm, 0.96° ± 0.49°, 0.93° ± 0.43°, and 1.03° ± 0.50°, for vertical, longitudinal, lateral, yaw, roll, and pitch, respectively. The averaged planned and delivered mean heart dose were 99 and 101 cGy. Lungs V20 were 6.59% and 6.74%. IMN 40 Gy coverage was 83% and 77%, and mean IMN dose was 4642 and 4518 cGy. The averaged mean motion during DIBH was smaller than 1 mm and 1°, which reflects the relative reproducibility of the patient breath hold. On average, the mean heart dose and lungs V20 were reasonably close to what have been planned. IMN 40 Gy coverage might be modestly reduced for certain cases

Probing the light harvesting and charge rectification of bismuth nanoparticles behind the promoted photoreactivity onto Bi/BiOCl catalyst by (in-situ) electron microscopy

State-of-the-art electron microscopy has enabled us to investigate microstructural details down to sub-subångström and milli-electron-volt resolution level. The enhanced photoreactivity over bismuth hybridized BiOCl catalyst (Bi/BiOCl) has been reported recently, however, the mechanistic understandings of this improved photoreactivity especially the optical behavior of bismuth nanoparticles (Bi NPs) are still obscured and in debate. The optical absorption features of Bi NPs and the charge transfer characteristic between bismuth and BiOCl have been considered as the major physicochemical origin for the promoted photoreactivity. Based on the advanced (in-situ) electron microscopy of monochromated electron energy loss spectroscopy in scanning transmission electron microscopy imaging mode (Mono-STEM-EELS) along with related theoretical investigations, in this work, we for the first time distinguished and explained the optical absorption originated from the localized surface plasmon resonances (LSPR) effect and direct band gap transition in an individual bismuth nanoparticle as well as transportation of photogenerated carriers at the interface of Bi/BiOCl. These findings could provide better understandings about the origin of the improved photoreactivity of various bismuth-hybridized photocatalysts

Recombinant mycobacterium tuberculosis fusion protein for diagnosis of mycobacterium tuberculosis infection: a short-term economic evaluation

ObjectivesRecombinant Mycobacterium tuberculosis fusion protein (EC) was anticipated to be used for the scale-up of clinical application for diagnosis of Mycobacterium tuberculosis infection in China, but it lacked a head-to-head economic evaluation based on the Chinese population. This study aimed to estimate the cost-utility and the cost-effectiveness of both EC and tuberculin pure protein derivative (TB-PPD) for diagnosis of Mycobacterium tuberculosis infection in the short term.MethodsFrom a Chinese societal perspective, both cost-utility analysis and cost-effectiveness analysis were performed to evaluate the economics of EC and TB-PPD for a one-year period based on clinical trials and decision tree model, with quality-adjusted life years (QALYs) as the utility-measured primary outcome and diagnostic performance (including the misdiagnosis rate, the omission diagnostic rate, the number of patients correctly classified, and the number of tuberculosis cases avoided) as the effective-measured secondary outcome. One-way and probabilistic sensitivity analyses were performed to validate the robustness of the base-case analysis, and a scenario analysis was conducted to evaluate the difference in the charging method between EC and TB-PPD.ResultsThe base-case analysis showed that, compared with TB-PPD, EC was the dominant strategy with an incremental cost-utility ratio (ICUR) of saving 192,043.60 CNY per QALY gained, and with an incremental cost-effectiveness ratio (ICER) of saving 7,263.53 CNY per misdiagnosis rate reduction. In addition, there was no statistical difference in terms of the omission diagnostic rate, the number of patients correctly classified, and the number of tuberculosis cases avoided, and EC was a similar cost-saving strategy with a lower test cost (98.00 CNY) than that of TB-PPD (136.78 CNY). The sensitivity analysis showed the robustness of cost-utility and cost-effectiveness analysis, and the scenario analysis indicated cost-utility in EC and cost-effectiveness in TB-PPD.ConclusionThis economic evaluation from a societal perspective showed that, compared to TB-PPD, EC was likely to be a cost-utility and cost-effective intervention in the short term in China

Monetary incentives and peer referral in promoting digital network-based secondary distribution of HIV self-testing among men who have sex with men in China: study protocol for a three-arm randomized controlled trial.

BACKGROUND: Human immunodeficiency virus (HIV) testing is a crucial strategy for HIV prevention. HIV testing rates remain low among men who have sex with men (MSM) in China. Digital network-based secondary distribution is considered as an effective model to enhance HIV self-testing (HIVST) among key populations. Digital platforms provide opportunities for testers to apply for HIVST kits by themselves, and secondary distribution allows them to apply for multiple kits to deliver to their sexual partners or members within their social network. We describe a three-arm randomized controlled trial to examine the effect of monetary incentives and peer referral in promoting digital network-based secondary distribution of HIVST among MSM in China. METHODS: Three hundred MSM in China will be enrolled through a digital platform for data collection. The eligibility criteria include being biological male, 18 years of age or over, ever having had sex with another man, being able to apply for kits via the online platform, and being willing to provide personal telephone number for follow-up. Eligible participants will be randomly allocated into one of the three arms: standard secondary distribution arm, secondary distribution with monetary incentives arm, and secondary distribution with monetary incentives plus peer referral arm. Participants (defined as "index") will distribute actual HIV self-test kits to members within their social network (defined as "alter") or share referral links to encourage alters to apply HIV self-test kits by themselves. All index participants will be requested to complete a baseline survey and a 3-month follow-up survey. Both indexes and alters will complete a survey upon returning the results by taking a photo of the used kits with the unique identification number. DISCUSSION: HIV testing rates remain suboptimal among MSM in China. Innovative interventions are needed to further expand the uptake of HIV testing among key populations. The findings of the trial can provide scientific evidence and experience on promoting secondary distribution of HIVST to reach key populations who have not yet been covered by existing testing services. TRIAL REGISTRATION: The study was registered in the Chinese Clinical Trial Registry (ChiCTR1900025433) on 26, August 2019, http://www.chictr.org.cn/showproj.aspx?proj=42001. Prospectively registered

SProtP: A Web Server to Recognize Those Short-Lived Proteins Based on Sequence-Derived Features in Human Cells

Protein turnover metabolism plays important roles in cell cycle progression, signal transduction, and differentiation. Those proteins with short half-lives are involved in various regulatory processes. To better understand the regulation of cell process, it is important to study the key sequence-derived factors affecting short-lived protein degradation. Until now, most of protein half-lives are still unknown due to the difficulties of traditional experimental methods in measuring protein half-lives in human cells. To investigate the molecular determinants that affect short-lived proteins, a computational method was proposed in this work to recognize short-lived proteins based on sequence-derived features in human cells. In this study, we have systematically analyzed many features that perhaps correlated with short-lived protein degradation. It is found that a large fraction of proteins with signal peptides and transmembrane regions in human cells are of short half-lives. We have constructed an SVM-based classifier to recognize short-lived proteins, due to the fact that short-lived proteins play pivotal roles in the control of various cellular processes. By employing the SVM model on human dataset, we achieved 80.8% average sensitivity and 79.8% average specificity, respectively, on ten testing dataset (TE1-TE10). We also obtained 89.9%, 99% and 83.9% of average accuracy on an independent validation datasets iTE1, iTE2 and iTE3 respectively. The approach proposed in this paper provides a valuable alternative for recognizing the short-lived proteins in human cells, and is more accurate than the traditional N-end rule. Furthermore, the web server SProtP (http://reprod.njmu.edu.cn/sprotp) has been developed and is freely available for users