Cell-derived microparticles for cell therapy, cargo delivery, and applications in CHO-cell biotechnology

Mammalian cells release into the extracellular environment microparticles (MPs; less than 1 micron) under some stress or activation process. MPs result from direct budding off the plasma membrane, and are important in intercellular communication by transferring RNA, proteins, and lipids between cells. Cells endow their MPs with signaling or functional molecules, to target specific cell types. They enrich their MPs in specific miRNAs, piRNAs, and long ncRNAs to program or reprogram target cells towards functional differentiation or specific cellular actions. Cells also use MPs to get rid of “death molecules”, and/or promote cell survival and “renewal” of target cells. We will discuss the characterization and potential applications of MPs from two biological systems: megakaryocytic MPs (MkMPs) derived from human hematopoietic stem and progenitor cells (HSPCs) and CHO-cell MPs (ChocMPs). Megakaryocytes (Mks) derive from the differentiation of HSPCs in the bone marrow, and as they mature, they achieve high-ploidy status. Platelets are produced from polyploid Mks under the action of biomechanical forces. We showed that mature Mks also shed MkMPs, whose generation is dramatically enhanced by shear flow. Co-culture of MkMPs with HSPCs promotes HSPC differentiation to Mks without exogenous thrombopoietin (the growth factor that stimulates Mk production from HSPCs), thus identifying a novel and previously unexplored physiological role for MkMPs. We show that MkMPs target HSPCs with exquisite specificity, and discuss mechanisms by which MkMPs target and act upon HSPCs. We argue for using MkMPs for regenerative-medicine applications, notably for the treatment of thrombocytopenia, as well as in as vectors for delivering nucleic-acid and protein “cargo” to HSPCs. The mechanism and kinetics of ChocMP generation and their biological role remain unexplored. We will discuss the first characterization of ChocMPs by examining the kinetics and mechanism of formation, their RNA content and efforts to identify their functional role. CHO cells produce ChocMPs from the very beginning of the culture, with different kinetics for attached versus suspension CHO cells. E.g., in suspension culture, ChocMPs concentration decreases with culture progression, thus suggesting that ChocMPs are fused into or endocytosed by CHO cells. In attached CHO cells, ChocMP generation is associated with “star- or bubble-studded” cellular images, characteristic of tumor-cell MP generation, thus providing first clues as to of the role of ChocMPs, since tumor-cell MPs have rather unique make up and role. ChocMP generation is promoted by stationary-phase conditions (notably, serum starvation), and biomechanical forces. Based on paradigms from other cells, we envision using ChocMPs as a means to predict culture fate, identify the role of small RNAs they are enriched in, and enhance culture performance

RGD Peptide-Grafted Graphene Oxide as a New Biomimetic Nanointerface for Impedance-Monitoring Cell Behaviors

A new biomimetic nanointerface was constructed by facile grafting the bioactive arginylglycylaspartic acid (RGD) peptide on the graphene oxide (GO) surface through carbodiimide and N-hydroxysuccinimide coupling amidation reaction. The formed RGD-GO nanocomposites own unique two-dimensional structure and desirable electrochemical performance. The linked RGD peptides could improve GO’s biocompatibility and support the adhesion and proliferation of human periodontal ligament fibroblasts (HPLFs) on RGD-GO biofilm surface. Furthermore the biologically active RGD-GO nanocomposites were demonstrated as a potential biomimetic nanointerface for monitoring cell biobehaviors by electrochemical impedance spectroscopy (EIS). By analysis of the data obtained from equivalent circuit-fitting impedance spectroscopy, the information related to cell membrane capacitance, cell-cell gap resistance, and cell-electrode interface gap resistance in the process of cell adhesion and proliferation could be obtained. Besides, this proposed impedance-based cell sensor could be used to assess the inhibition effect of the lipopolysaccharide (LPS) on the HPLFs proliferation. Findings from this work suggested that RGD peptide functionalized GO nanomaterials may be not only applied in dental tissue engineering but also used as a sensor interface for electrochemical detection and analysis of cell behaviors in vitro

Monitoring and analysis of blasting vibration in tunnel excavation of nuclear power plant

Vibration monitoring of blasting excavation of drainage tunnel in Lufeng Nuclear Power Plant is carried out and the data of blasting vibration is analyzed in this paper. The results show that: (1) The vertical vibration velocity of the rock mass is greater than the horizontal radial and horizontal tangential vibration velocity (2) The blasting vibration velocity of rock mass decreases with distance, which is affected by rock structure and explosive quantity. The monitoring research in this paper has guiding significance for vibration prediction and control in tunnel blasting excavation

Enablement by single-use technology of production of two billion vaccine doses of adenovirus-vectored vaccine in under a year

Manufacturing of the simian adenovirus-vectored vaccine ChAdOx1 nCoV-19 (AZD1222, Vaxzevria) has played an important role in control of the COVID-19 pandemic. More than two billion doses have been produced, with the majority both made and used in low and middle income countries. This has been enabled by a programme of early technology transfer to multiple drug substance production sites, occurring in parallel with process development. The University of Oxford was transferring technology to five sites by March 2020, and AstraZeneca subsequently extended the drug substance manufacturing network to 12 countries. This innovative approach was possible only as a result of single-use technology (SUT), and the presentation will provide a case study of the application of such technology to pandemic response. Pre-pandemic research in the Jenner Institute had developed a potentially scalable adenovirus manufacturing process, prioritising simplicity and end-to-end single-use product contact materials to facilitate future technology transfer. From February 2020, this was aggressively scaled up, reaching 200L by April and 1000L by June. In most respects the process scaled linearly, with small-scale work largely predictive of parameters which were successful at large scale. In parallel, process optimisations targeted further simplification. This simple SUT-based process has been the critical factor enabling distributed manufacturing of a novel product type in existing facilities. Several of the facilities contributing to the production (including those in LMICs and the one which has contributed most to global output) had no previous experience of viral vector manufacture, yet were able to rapidly and effectively on-board the process and. Use of shared SUT has also assisted product comparability across the network. SUT has thus played a pivotal role in both scale-up and scale-out, allowing manufacturing to reach a scale we believe to be unprecedented for any viral vector, and making a major contribution to equity of access to COVID-19 vaccines

Re-evaluation of the carcinogenic significance of hepatitis B virus integration in hepatocarcinogenesis

To examine the role of hepatitis B virus (HBV) integration in hepatocarcinogenesis, a systematic comparative study of both tumor and their corresponding non-tumor derived tissue has been conducted in a cohort of 60 HBV associated hepatocellular carcinoma (HCC) patients. By using Alu-polymerase chain reaction (PCR) and ligation-mediated PCR, 233 viral-host junctions mapped across all human chromosomes at random, no difference between tumor and non-tumor tissue was observed, with the exception of fragile sites (P = 0.0070). HBV insertions in close proximity to cancer related genes such as hTERT were found in this study, however overall they were rare events. No direct correlation between chromosome aberrations and the number of HBV integration events was found using a sensitive array-based comparative genomic hybridization (aCGH) assay. However, a positive correlation was observed between the status of several tumor suppressor genes (TP53, RB1, CDNK2A and TP73) and the number of chromosome aberrations (r = 0.6625, P = 0.0003). Examination of the viral genome revealed that 43% of inserts were in the preC/C region and 57% were in the HBV X gene. Strikingly, approximately 24% of the integrations examined had a breakpoint in a short 15 nt viral genome region (1820-1834 nt). As a consequence, all of the confirmed X gene insertions were C-terminal truncated, losing their growth-suppressive domain. However, the same pattern of X gene C-terminal truncation was found in both tumor and non-tumor derived samples. Furthermore, the integrated viral sequences in both groups had a similar low frequency of C1653T, T1753V and A1762T/G1764A mutations. The frequency and patterns of HBV insertions were similar between tumor and their adjacent non-tumor samples indicating that the majority of HBV DNA integration events are not associated with hepatocarcinogenesis

TiEV: The Tongji Intelligent Electric Vehicle in the Intelligent Vehicle Future Challenge of China

TiEV is an autonomous driving platform implemented by Tongji University of China. The vehicle is drive-by-wire and is fully powered by electricity. We devised the software system of TiEV from scratch, which is capable of driving the vehicle autonomously in urban paths as well as on fast express roads. We describe our whole system, especially novel modules of probabilistic perception fusion, incremental mapping, the 1st and the 2nd planning and the overall safety concern. TiEV finished 2016 and 2017 Intelligent Vehicle Future Challenge of China held at Changshu. We show our experiences on the development of autonomous vehicles and future trends

Analysis on monitoring and controlling techniques about blasting vibration effect of open channel in Taishan nuclear power station

The blasting for bedrock excavation on land for open channel project has a great influence on lock gate in Taishan Nuclear Power Station, therefore, based on blasting vibration monitoring data, the attenuation law of blasting vibration signal has been studied through regression analysis of practical test data by Sadaovsk empirical formula and corresponding time-frequency characteristics was analyzed by Empirical Mode Decomposition based on Hilbert-Huang transform. As for those monitoring data, the results of blast vibration velocity for vertical direction are generally larger than horizontal radial and horizontal tangential direction in the near field of blasting source and the peak particle velocity of vertical direction is usually lower than horizontal radial and horizontal tangential direction in the far field of blasting source; at the same time, their main vibration frequency mostly vary from 10 Hz to 80 Hz which is much higher than natural frequency of lock gate and is beneficial to structural safety and stability of surrounding rock mass for reducing the probability of resonance. To ensure the safety of lock gate, it is of great significance to control maximum explosive weight per delay in advance for different distance from monitoring point to the explosion source according to Safety Regulations for Blasting (GB6722-2014), which shows the excellent effect on blasting damage control of the lock gate and surrounding rock mass. The results from the analysis can be for reference to similar blasting design and blasting construction

Human Hepatocytes with Drug Metabolic Function Induced from Fibroblasts by Lineage Reprogramming

SummaryObtaining fully functional cell types is a major challenge for drug discovery and regenerative medicine. Currently, a fundamental solution to this key problem is still lacking. Here, we show that functional human induced hepatocytes (hiHeps) can be generated from fibroblasts by overexpressing the hepatic fate conversion factors HNF1A, HNF4A, and HNF6 along with the maturation factors ATF5, PROX1, and CEBPA. hiHeps express a spectrum of phase I and II drug-metabolizing enzymes and phase III drug transporters. Importantly, the metabolic activities of CYP3A4, CYP1A2, CYP2B6, CYP2C9, and CYP2C19 are comparable between hiHeps and freshly isolated primary human hepatocytes. Transplanted hiHeps repopulate up to 30% of the livers of Tet-uPA/Rag2−/−/γc−/− mice and secrete more than 300 μg/ml human ALBUMIN in vivo. Our data demonstrate that human hepatocytes with drug metabolic function can be generated by lineage reprogramming, thus providing a cell resource for pharmaceutical applications

Searching for the nano-Hertz stochastic gravitational wave background with the Chinese Pulsar Timing Array Data Release I

Observing and timing a group of millisecond pulsars (MSPs) with high rotational stability enables the direct detection of gravitational waves (GWs). The GW signals can be identified from the spatial correlations encoded in the times-of-arrival of widely spaced pulsar-pairs. The Chinese Pulsar Timing Array (CPTA) is a collaboration aiming at the direct GW detection with observations carried out using Chinese radio telescopes. This short article serves as a `table of contents' for a forthcoming series of papers related to the CPTA Data Release 1 (CPTA DR1) which uses observations from the Five-hundred-meter Aperture Spherical radio Telescope (FAST). Here, after summarizing the time span and accuracy of CPTA DR1, we report the key results of our statistical inference finding a correlated signal with amplitude \log A_{\rm c}= -14.4 \,^{+1.0}_{-2.8} for spectral index in the range of $\alpha\in [-1.8, 1.5]$ assuming a GW background (GWB) induced quadrupolar correlation. The search for the Hellings-Downs (HD) correlation curve is also presented, where some evidence for the HD correlation has been found that a 4.6-$\sigma$ statistical significance is achieved using the discrete frequency method around the frequency of 14 nHz. We expect that the future International Pulsar Timing Array data analysis and the next CPTA data release will be more sensitive to the nHz GWB, which could verify the current results.Comment: 18 pages, 6 figures, submitted to "Research in astronomy and astrophysics" 22nd March 202