Construction of human interleukin-26 lentivirus expression plasmid and gene knockout plasmid and their activity verification in HEK293T cells / 中国生物制品学杂志

@#Objective To construct a lentivirus-based expression plasmid and gene knockout plasmid of human interleukin(IL)-26 so as to lay a foundation of studying the function of IL-26 gene in cell signaling pathway and autophagy.Methods IL-26 gene sequence was amplified from human peripheral blood mononuclear cells by RT-PCR and cloned into pCDH-CMVMCS-EF1-copGFP eukaryotic expression vector to construct overexpression plasmid;Four knockout targets,Exon1sgRNA1,Exon1sgRNA2,Exon3sgRNA1 and Exon3sgRNA2,were designed based on the exon sequence of IL-26,and constructed into lentiCRISPRv2 vector by CRISPR/Cas9 technology to construct gene knockout plasmid. The overexpression plasmid and gene knockout plasmid were transiently transfected into HEK293T cells respectively,and the expression of IL-26 was verified by RT-qPCR and Western blot. In addition,amino acid sequence analysis,structure prediction and subcellular localization observation of IL-26 were performed.Results The results of restriction digestion,sequencing and bioinformatics analysis showed that IL-26 was 516 bp in length,encoding 171 amino acids. The IL-26 mRNA level and protein level of HEK293T cells transfected with IL-26 overexpression plasmid increased by 656. 789 times and 1. 978 times respectively with significant differences as compared with the normal control group(t = 17. 976 and 7. 859,P < 0. 000 1 and < 0. 001,respectively). With the transfection of 4 knockout targets Exon1sgRNA1,Exon1sgRNA2,Exon3sgRNA1 and Exon3sg-RNA2 into HEK293T cells,the expression of IL-26 decreased by 0. 930,0. 980,0. 523 3 and 0. 316 9 times,respectively,among which Exon3sgRNA2 significantly down-regulated the expression of IL-26(t = 7. 440,P < 0. 001). IL-26protein showed signal peptide structure and certain transmembrane function in the first 22 amino acids,which existed in cytoplasm.Conclusion IL-26overexpression and gene knockout plasmids were successfully constructed,which laid a foundation of the follow-up study of the function of IL-26.

LncRNA DANCR restrained the survival of mycobacterium tuberculosis H37Ra by sponging miR-1301-3p/miR-5194.

Tuberculosis is a worldwide contagion caused by Mycobacterium tuberculosis (MTB). MTB is characterized by intracellular parasitism and is semi-dormant inside host cells. The persistent inflammation caused by MTB can form a granuloma in lesion regions and intensify the latency of bacteria. In recent years, several studies have proven that long non-coding RNAs (lncRNAs) play critical roles in modulating autophagy. In our study, the Gene Expression Omnibus (GEO) databases were searched for lncRNAs that are associated with tuberculosis. We found that lncRNA differentiation antagonizing non-protein coding RNA (DANCR) increased in the peripheral blood samples collected from 54 pulmonary tuberculosis patients compared to 23 healthy donors. By constructing DANCR overexpression cells, we analyzed the possible cellular function of DANCR. After analyzing our experiments, it was found that the data revealed that upregulation of DANCR facilitated the expression of signal transducer and activator of transcription 3, autophagy-related 4D cysteine peptides, autophagy-related 5, Ras homolog enriched in the brain, and microtubule-associated protein 1A/1B light chain 3 (STAT3, ATG4D, ATG5, RHEB, and LC3, respectively) by sponging miR-1301-3p and miR-5194. Immunofluorescence analysis indicated that DANCR played a positive role in both autophagosome formation and fusion of autolysosomes in macrophages. The colony-forming unit (CFU) assay data also showed that the cells overexpressing DANCR were more efficient in eliminating the intracellular H37Ra strain. Consequently, these data suggest that DANCR restrained intracellular survival of M. tuberculosis by promoting autophagy via miR-1301-3p and miR-5194.

Atmospheric environment monitoring technology and equipment in China: A review and outlook.

Accurate monitoring of the atmospheric environment and its evolution are important for understanding the sources, chemical mechanisms, and transport processes of air pollution and carbon emissions in China, and for regulatory and control purposes. This study gives an overview of atmospheric environment monitoring technology and equipment in China and summarizes the major achievements obtained in recent years. China has made great progress in the development of atmospheric environment monitoring technology and equipment with decades of effort. The manufacturing level of atmospheric environment monitoring equipment and the quality of products have steadily improved, and a technical & production system that can meet the requirements of routine monitoring activities has been initiated. It is expected that domestic atmospheric environment monitoring technology and equipment will be able to meet future demands for routine monitoring activities in China and provide scientific assistance for addressing air pollution problems.

Retrieval of tropospheric ozone profiles using ground-based MAX-DOAS.

In recent years, surface ozone concentrations have increased in many cities in China. Ground-based multi-axis differential optical absorption spectroscopy (MAX-DOAS) is a powerful technique for retrieving the profiles of tropospheric trace gases, such as NO2, SO2, and HCHO. However, since the difficulties in deducting the effects of stratospheric ozone, there are few studies on the retrieval of tropospheric ozone profiles using MAX-DOAS measurements. Here, we developed an accurate inversion method to retrieve tropospheric ozone concentrations during the PRIDE-GBA Campaign, wherein the ozone differential slant column densities (DSCDs) were retrieved in QDOAS software using the "time-interpolated zenith spectrum" as the reference spectrum. The tropospheric DSCDs (DSCDstrop) were then calculated by subtracting the simulated stratospheric DSCDs (DSCDsstr, simulated from the SCIATRAN model) from the DSCDs. Tropospheric ozone profiles were retrieved from the DSCDstrop using the optimal estimation method (OEM). The results showed that high values of tropospheric ozone were mainly distributed below 1 km, which is consistent with lidar measurements. In addition, the observed surface ozone concentrations were highly correlated with the in-situ measurements, with correlation coefficients (R) of 0.75 and 0.81, respectively. Combined with the retrieved NO2 and HCHO profiles using the MAX-DOAS measurements, we found that the planetary boundary layer ozone pollution of HeShan Observatory during the PRIDE-GBA Campaign are controlled by the NOx-limited regime. The results of this study indicate that the MAX-DOAS technique has the potential to retrieve tropospheric ozone profiles with high temporal and spatial resolution.

Spermidine Promotes Nb CAR-T Mediated Cytotoxicity to Lymphoma Cells Through Elevating Proliferation and Memory.

Purpose: Due to the natural advantages of spermidine in immunity, we investigated the effects of spermidine pretreatment on nanobody-based CAR-T cells (Nb CAR-T) mediated cytotoxicity and potential mechanism. Patients and Methods: The optimal concentration of spermidine was determined by detecting its impact on viability and proliferation of T cells. The phenotypic characteristic of CAR-T cells, which were treated with spermidine for 4 days, was examined by flow cytometry. The expansion ability of CAR-T cells was monitored in being cocultured with tumor cells. Additionally, CAR-T cells were stimulated by lymphoma cells to test its cytotoxicity in vitro, and the supernatant in co-culture models were collected to test the cytokine production. Furthermore, xenograft models were constructed to detect the anti-tumor activity of CAR-T cells in vivo. Results: The optimal concentration of spermidine acting on T cells was 5µM. The antigen-dependent proliferation of spermidine pretreatment CD19 CAR-T cells or Nb CAR-T cells was increased compared to control. Central memory T cells(TCM) dominated the CAR-T cell population in the presence of spermidine. When spermidine pretreatment CAR-T cells were stimulated with Daudi cells, the secretion of IL-2 and IFN-γ has been significantly enhanced. The ability of CAR-T cells to lysis Daudi cells was enhanced with the help of spermidine, even at higher tumor loads. Pre-treated Nb CAR-T cells with spermidine were able to control tumor cells in vivo, and therefore prolong mice survival. Conclusion: Our results revealed that spermidine could promote Nb CAR-T mediated cytotoxicity to lymphomas cells through enhancing memory and proliferation, and provided a meaningful approach to strengthen the anti-tumor effect of CAR-T cells.

Study of an Arctic blowing snow-induced bromine explosion event in Ny-Ålesund, Svalbard.

Bromine explosion events (BEEs) are important processes that influence the atmospheric oxidation capacity, especially in the polar troposphere during spring. Although sea ice surface is thought to be a significant bromine source, bromine release mechanisms remain unclear. High-resolution ground-based observations of reactive bromine, such as BrO, are important for assessing the potential impacts on tropospheric ozone and evaluating chemical models. However, previous model studies paid little attention to Svalbard, which is surrounded by both open ocean and sea ice. In this paper, we present continuous BrO slant column densities and vertical column densities derived by Multi-Axis Differential Optical Absorption Spectroscopy deployed at Ny-Ålesund (78.92°N, 11.93°E) in March 2017. We focused on one BEE in mid-March, during which the vertical column densities of BrO surged from 4.26 × 1013 molecular cm-2 to the peak at 1.23 × 1014 molecular cm-2 on March 17, surface ozone depleted from a background level of 46.25 parts per billion by volume (ppbv) to 13.9 ppbv. This case study indicates that the BEE was strongly associated with blowing snow induced by the cyclone systems that approached Svalbard from March 14 to 18. By considering meteorological conditions, sea ice coverage, and airmass trajectory history, we demonstrate that sea salt aerosols (SSAs) from blowing snow on sea ice, rather than from open ocean, are attributed to the occurrence of this BEE. Model results from a parallelized-tropospheric offline model of chemistry and transport (p-TOMCAT) indicate that this BEE was mainly triggered by a blowing snow event associated with a low-pressure cyclone system. The concentration of blowing-snow-sourced SSAs surged to peak when the airmass pass across the sea-ice-covered area under high wind speed, which is a critical factor in the process of bromine explosion observed in Ny-Ålesund. Due to the coarse resolution, the possible delayed timing of bromine release from SSA and the model-data discrepancies still exist.

Potential and functional prediction of six circular RNAs as diagnostic markers for colorectal cancer.

Background: Circular RNAs (circRNAs) have been discovered in colorectal cancer (CRC), but there are few reports on the expression distribution and functional mining analysis of circRNAs. Methods: Differentially expressed circRNAs in CRC tissues and adjacent normal tissues were screened and identified by microarray and qRT-PCR. ROC curves of the six circRNAs were analyzed. A series of bioinformatics analyses on differentially expressed circRNAs were performed. Results: A total of 207 up-regulated and 357 down-regulated circRNAs in CRC were screened, and three top up-regulated and down-regulated circRNAs were chosen to be verified in 33 pairs of CRCs by qRT-PCR. 6 circRNAs showed high diagnostic values (AUC = 0.6860, AUC = 0.8127, AUC = 0.7502, AUC = 0.9945, AUC = 0.9642, AUC = 0.9486 for hsa_circRNA_100833, hsa_circRNA_103828, hsa_circRNA_103831 and hsa_circRNA_103752, hsa_circRNA_071106, hsa_circRNA_102293). A circRNA-miRNA-mRNA regulatory network (cirReNET) including six candidate circRNAs, 19 miRNAs and 210 mRNA was constructed, and the functions of the cirReNET were predicted and displayed via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on these mRNAs and protein-protein interaction (PPI) network of the hub genes acquired by string and CytoHubba. Conclusion: A cirReNET containing potential diagnostic and predictive indicators of CRCs and several critical circRNA-miRNA-mRNA regulatory axes (cirReAXEs) in CRC were mined, and may provide a novel route to study the mechanism and clinical targets of CRC.

First observation of tropospheric nitrogen dioxide from the Environmental Trace Gases Monitoring Instrument onboard the GaoFen-5 satellite.

The Environmental Trace Gases Monitoring Instrument (EMI) is the first Chinese satellite-borne UV-Vis spectrometer aiming to measure the distribution of atmospheric trace gases on a global scale. The EMI instrument onboard the GaoFen-5 satellite was launched on 9 May 2018. In this paper, we present the tropospheric nitrogen dioxide (NO2) vertical column density (VCD) retrieval algorithm dedicated to EMI measurement. We report the first successful retrieval of tropospheric NO2 VCD from the EMI instrument. Our retrieval improved the original EMI NO2 prototype algorithm by modifying the settings of the spectral fit and air mass factor calculations to account for the on-orbit instrumental performance changes. The retrieved EMI NO2 VCDs generally show good spatiotemporal agreement with the satellite-borne Ozone Monitoring Instrument and TROPOspheric Monitoring Instrument (correlation coefficient R of ~0.9, bias < 50%). A comparison with ground-based MAX-DOAS (Multi-Axis Differential Optical Absorption Spectroscopy) observations also shows good correlation with an R of 0.82. The results indicate that the EMI NO2 retrieval algorithm derives reliable and precise results, and this algorithm can feasibly produce stable operational products that can contribute to global air pollution monitoring.

[A case of neuromyelitis optica spectrum disorders complicated with systemic lupus erythematosus and thymoma].

A 53-year-old male patient presented with hypopsia of his right eye for 2 months and lower extremities weakness for 8 days. Thoracic MRI demonstrated a lesion at T3 level appearing as hyperintense on T2-weighted images with non-enhancement by contrast medium and demyelinating lesion was considered. Aquaporin-4-Ab was positive and the antibody titer was 1:320 in serum. The diagnosis of neuromyelitis optica spectrum disorders was made. In addition, systemic lupus erythematosus and thymoma coexisted in this patient. After methylprednisolone impact treatment, plasma exchange and immunosuppressive therapy, the right vision and lower extremities weakness of the patient were improved.

[Bidirectional Reflectance Distribution Function of Space-Borne Quartz Volume Diffuser].

Quartz Volume Diffuser(QVD) is used in the observing system of Space-Borne differential optical absorption spectrometer. The precision of observed solar spectrum directly influences the accuracy of the gas retrievals. Therefore the QVD is required for well Lambert feature to ensure the accuracy of full field solar spectrum, and it can provide uniformity source in the observing view of the instrument. Using bidirectional reflectance distribution function(BRDF) measurement instrument, adopting the powder pressboard of F4(polytetrafluoroethylene(PTFE)), QVD's BRDF is measured by choosing the relative measurement method. Four kinds of QVD's BRDF is obtained in the range of 180～880 nm, the observing view of -70°ï½ž+70°. Two kinds of QVD which has a well Lambert feature are selected by analyzing the QVD's BRDF. The diffuse sunlight measured by QVD and F4 is compared, which show that QVD has well scattering properties with regard to solar spectrum and can be selected as the measuring diffuser. That supports for next Ultraviolet irradiation measurement, atomic oxygen erosion measurement and comparison measurement.

Study on the Non-Uniformity Calibration of Space-Born Differential Optical Absorption Spectrometer.

Space-borne differential optical absorption spectrometer is a nadir viewing wide field imaging spectrometer, which adopts two-dimensional CCD detector arrays. The pixel response non-uniformity exists in each column of spatial dimension, which will introduce high-frequency instrument-related spectral structures in the measurement data. However, the non-uniformity calibration of space-born imaging spectrometer is difficulty due to two factors: the spectral smile effect and the large field of view. For this reason, a method of non-uniformity calibration is discussed in detail. The result shows that the spectral smile effect and non-uniformity of full FOV image are corrected effectively, and high-frequency instrument-related structures in the measurement data are removed.

Effect of AO/UV/RD exposure on spaceborne diffusers: a comparative experiment.

The environmental measuring instrument (EMI) is a nadir-viewing wide-field imaging spectrometer, which adopts spaceborne diffusers in in-flight calibration systems, including an aluminum diffuser and a quartz volume diffuser. Spaceborne diffusers, are the key components of in-flight calibration systems, and are used to introduce sunlight into the EMI. Hemispheric reflectance and bidirectional reflectance distribution function were experimentally measured to analyze spaceborne diffuser performance. Radiation exposure experiments on atomic oxygen, UV, and radiation dose of the spaceborne diffusers were performed at ground level because the EMI works in low Earth orbit space environments. Effects of radiation exposure on spaceborne diffusers were discussed in detail. Protective methods were introduced to reduce the effects of the space environment, and an in-orbit monitoring method was also proposed.

[Spectral Calibration of Space-born Imaging Spectrometers Using Spectrum-Matching Technique].

Spectral calibration of space-born imaging spectrometers based on spectrum-matching technique is presented, which adopts atmospheric absorption lines as the matching lines, and chooses correlation coefficient method as the criteria. In order to simulation the onboard spectral calibration, the spectrum-matching technique is applied on the imaging spectrometers that after the vibration test. The vibration test is able to simulation the launching. The spectral resolution, center wavelength of two-dimensional pixel is determined during onboard spectral calibration. As the calibration results show, the spectral resolution of imaging spectrometers after the vibration test is 0.40 nm, it is the same comparing to the value before the vibration, the wavelength shifts 0.08 nm towards the long wave for the spectral pixels, and the spectral smile is determined for all spatial elements, which shifts towards the short wave direction, with the max smile value is 0.96 nm, the result is similar to that before the vibration. As a result, the spectrum-matching technique is tested and verified.

[Research on the NO2 mean concentration measurement with target differential optical absorption spectroscopy technology].

A new monitoring method of NO2 concentration near ground with the target difference absorption spectrum technology (Target DOAS) is introduced in the present paper. This method is based on the passive difference absorption spectrum technology. The instrument collects solar reflection spectrum of remote objectives, such as wall of building and mountain, and a specific reference spectrum is chosen to subtract the influence of trace gases from the target to atmospheric top, then integrated concentration of NO2 along the path between the target and instrument can be calculated through the differential absorption spectra inversion algorithm. Since the distance between the instrument and target is given, the mean concentration of NO2 can be derived. With developed Target DOAS instrument, NO2 concentration measurement was carried out in Hefei. And comparison was made between the target DOAS and long path difference absorption spectrometer. Good consistency was presented, proving the feasibility of this method.

[MAX-DOAS measurements of NO2 column densities and vertical distribution at Ny-Alesund, Arctic during summer].

The multi-axis differential optical absorption spectroscopy (MAX-DOAS), one of the remote sensing techniques for trace gases measurements, is sensitive to the lower atmosphere by eliminating the influence of stratosphere retrieved from zenith-sky spectroscopy. Ground-based MAX-DOAS measurements were carried out to observe NO2 at Ny-Alesund, Arctic from 5th Jul to 1st Aug 2011. The differential slant column densities (DSCDs) of NO2 at four off-axis angles showed typical pattern of tropospheric absorbers. Based on the assumption that NO2 was well mixed in 0-1 km of the troposphere, the mean mixing ratio of NO2 during the measurement period was 1.023E11 molec x cm(-3). The fluctuation of NO2 might be related to the fossil fuel combustions and the photochemical reactions. The vertical distribution of NO2 at 0-3 km showed that NO2 was mainly originated from boundary layer of sea surface.

[Spectral calibration for space-borne differential optical absorption spectrometer].

Space-borne differential optical absorption spectrometer is used for remote sensing of atmospheric trace gas global distribution. This instrument acquires high accuracy UV/Vis radiation scattered or reflected by air or earth surface, and can monitor distribution and variation of trace gases based on differential optical absorption spectrum algorithm. Spectral calibration is the premise and base of quantification of remote sensing data of the instrument, and the precision of calibration directly decides the level of development and application of the instrument. Considering the characteristic of large field, wide wavelength range, high spatial and spectral resolution of the space-borne differential optical absorption spectrometer, a spectral calibration method is presented, a calibration device was built, the equation of spectral calibration was calculated through peak searching and regression analysis, and finally the full field spectral calibration of the instrument was realized. The precision of spectral calibration was verified with Fraunhofer lines of solar light.