Our findings indicate that TSCs do not readily undergo SCT differentiation and closely resemble cells at the base of the cell columns from where EVT derives. In contrast, organoids are similar to VCT and undergo spontaneous SCT differentiation. A defining feature of human trophoblast is that VCT and SCT are human leukocyte antigen (HLA) null, whereas EVT expresses HLA-C, -G and -E molecules. We find that trophoblast organoids retain these in vivo characteristics. In contrast, TSCs express classical HLA-A and HLA-B molecules, and maintain their expression after EVT differentiation, with upregulation of HLA-G. Furthermore, HLA expression in TSCs differs when grown in 3D rather than in 2D, suggesting that mechanical cues are important. Our results can be used to select the most suitable model for the study of trophoblast development, function and pathology.Two recently developed models, trophoblast organoids and trophoblast stem cells (TSCs), are useful tools to further the understanding of human placental development. Both differentiate from villous cytotrophoblast (VCT) to either extravillous trophoblast (EVT) or syncytiotrophoblast (SCT). Here, we compare the transcriptomes and miRNA profiles of these models to identify which trophoblast they resemble  Summary: Characterization of trophoblast organoids and trophoblast stem cells as exciting models of human placentation enables the selection of the most suitable system to address specific research questions. The placenta has many biological functions that support the developing fetus. Trophoblasts, the main cell type of the placenta, arise from the trophectoderm of the implanting blastocyst. After implantation, trophoblast differentiates along two main pathways: villous and extravillous. Placental villi are surrounded by a layer of villous cytotrophoblast (VCT), which fuses to form a multi-nucleated layer of syncytiotrophoblast (SCT). This syncytium is in direct contact with maternal blood and is the principal site of nutrient and oxygen exchange. Where villi make contact with the maternal decidua at the tips of anchoring villi, VCT proliferates to form cell columns. A possible location for trophoblast progenitors is at the base of these cell columns (the ‘column niche’), marked by ITGA2/NOTCH1 expression . At theiin vitro in a two-dimensional (2D) stem cell model (in vivo with: (1) expression of typical trophoblast markers; (2) a distinctive profile of human leukocyte antigen (HLA) class I molecules; (3) expression of microRNAs (miRNAs) from the chromosome 19 cluster (C19MC); and (4) methylation of the ELF5 promoter . For all flow cytometric experiments, JEG-3 and JAR choriocarcinoma cells were used as controls for HLA expression for EVT and VCT, respectively  . In contrast, TSCs  .HLA class I genes are polymorphic and have common shared epitopes; it is therefore difficult to work out exactly which allotypes are expressed. By using W6/32, we were able to define whether the trophoblast sample resembles VCT (HLA-null). If there was expression with W6/32, it was then necessary to use locus-specific antibodies for HLA-G, -E and -C (EVT profile) to determine which of these HLA class I genes are expressed. We also tissue typed the individual samples to determine which HLA-A or HLA-B alleles are present and then used HLA-A, -B allele/group-specific antibodies to determine whether they are expressed (ectively . The embectively . The speig. S1A) . Furtherst, TSCs  displayer HLA-G) D. This H. Four out of the five TSC lines tested carried an HLA-A*02 allele, and all five had HLA-B alleles encoding the Bw6 epitope. Specific antibodies for HLA-A2 (BB7.2) and HLA-B (Bw6) and an antibody specific for all HLA-C and -E allotypes (DT9) were chosen for analysis by flow cytometry. JEG-3 cells were used as a positive control for pan-HLA class I (W6/32), HLA-C and -E (DT9) and HLA-G (MEMG-9) expression. We found that TSCs expressed HLA-A, -B and -C, but not -G . Out of the TSC lines tested, most exhibited HLA-A (three out of four) and HLA-B expression (four out of five) . One TSC line (CT30) displayed lower expression of W6/32 and was the only line that showed no indication of HLA-A or -B expression , we next asked which allotypes of HLA class I genes were expressed. HLA-A, -B, -C and/or -E allotypes could be expressed by TSCs because they are W6/32+, MEMG-9−. The HLA-A and HLA-B alleles present in the TSC lines were determined by HLA DNA genotyping . Our reasoning was that, because TOs fully recapitulate the in vivo HLA expression profile, culturing TSCs in 3D might alter their HLA expression. The expression of W6/32 decreased significantly when TSCs were cultured in 3D compared with 2D . This same decrease in W6/32 expression occurred when TSCs were cultured in 3D in their original TSC media, although the cultures could not be passaged long term . To test the reverse conditions, TOs were transferred into TSC medium or into 2D culture conditions; however, they were unable to survive long term in either media (TSC or TO) or in 3D in TSC medium . Following single cell dispersal, TSC-3D generated more organoids/per cell compared with TO, although individual organoid sizes were similar .We next cultured TSCs using conditions for TOs as an additional comparison (n=4 donors); (2) TOs differentiated to EVT ; (3) TSCs ; (4) TSCs cultured in 3D in TO medium ; (5) TSCs differentiated to EVT ; and (6) TSCs differentiated to SCT . All six groups showed different morphologies in culture  on six different groups: (1) TOs ; or (3) the top-250 most-differentially expressed genes (DEGs) between samples . Log2 normalized expression values were used for all heatmaps. Hierarchical clustering based upon trophoblast markers indicated a similar pairing as found in the PCA  were also high in both TSC-EVT and TO-EVT. TO-EVT did still contain some syncytium, and some off-target differentiation in TSC-EVT was likely. TOs and TSC-SCT were closely aligned and showed the highest expression of SCT markers. TSC-2D and TSC-3D clustered closely and resembled trophoblast and the niche in the cell columns. Interestingly, when only using the top-250 DEGs, the hierarchical clustering indicated that organoids were most like each other, and TSCs all clustered separately . Transcripts for HLA-A and HLA-B were significantly reduced in TSC-3D (HLA-A P=0.028 and HLA-B P=0.048) and TOs (HLA-A P=0.018 and HLA-B P=0.008) compared with TSC-2D ; and (2) those involved in binding, transport and peptide loading     known markers specific for trophoblast subtypes found in vivo   . TranscrFig. S1) . CIITA, P<0.05, fold change >2] . Many of the biological processes upregulated in TSC-3D were related to extracellular matrix remodeling and cell-cell interactions (Table S8). Conversely, terms upregulated in TOs were related to transport, metabolism and hormone signaling (Table S8). A PCA using the top-2000 most-variable genes between TOs and TSC-3D revealed that multiple SCT markers  accounted for the separation between the two groups  and were significantly upregulated in TOs (Table S9). To further address whether TSC-3D can form SCT, as TOs do, we stained cultures with Hematoxylin and Eosin (H&E)  and tested culture supernatants with a human chorionic gonadotropin (hCG-β) ELISA  B, perforβ) ELISA D. These Table S10). Terms related to cell shape, cell adhesion, polarity and development were highly represented. Downregulated terms (i.e. upregulated in TOs) are detailed in Table S11. Again, common parent terms were related to metabolic, catabolic and transport processes. Next, we asked whether these TSC-3D more closely recapitulate the column niche rather than villous trophoblast. In vivo, ITGA2 and NOTCH1 are characteristically expressed in the column niche . Previously, NOTCH1 was shown to reduce TP63 expression through the downregulation of IRF6 or IRF7 (A gene ontology (GO) semantic analysis was performed to visualize the functional similarities of the enriched pathways upregulated in TSC-3D compared with TOs . However, by drawing a gate on two negative controls (isotype control and HLA-negative JAR cells) and one HLA-positive control (JEG-3 cells), TSCs did express HLA class I molecules. This highlights the importance of choosing appropriate controls for HLA analysis, particularly because trophoblast displays a unique HLA profile. Another group also reported expression of HLA class I molecules (determined by W6/32) in TSCs  upregulated expression when cultured in 2D and downregulated expression in 3D. However, we did not find a complete absence of HLA expression in TSCs in any situation. It is known that 2D culture conditions do not always recapitulate, or maintain, all features of their in vivo equivalents as well as 3D culture systems  would fuse into SCT. TSCs, with high expression of EPCAM and ITGA2, closely recapitulated the column niche . Being aware of all of the advantages and limitations of these newly derived models will greatly enhance the field of trophoblast research and lead to a better in vitro representation of the in vivo situation.In conclusion, the two models of first-trimester trophoblast differed in their expression of HLA and other genes. Our findings will help researchers choose the most-appropriate model to study trophoblast development, function and response to insults. The exact pathways controlling TP63 expression and the molecular switches vital for VCT-to-cell column conversion could be unraveled using TSCs because they closely resemble the column niche . With thecretion . The mosecretion . HoweverAll tissue samples used for this study were obtained with written informed consent from all participants in accordance with the guidelines in The Declaration of Helsinki 2000. Tissue samples used for our study were obtained from Addenbrooke's Hospital (6-11 weeks gestation) under ethical approval from the Cambridge Local Research Ethics Committee (04/Q0108/23), which is now incorporated into the overarching ethics permission given to the Centre for Trophoblast Research biobank for the ‘Biology of the Human Uterus in Pregnancy and Disease Tissue Bank’ at the University of Cambridge under ethical approval from the East of England–Cambridge Central Research Ethics Committee (17/EE/0151).N-acetyl-L cysteine 1.25 mM , L-glutamine 2 mM , recombinant human EGF 50 ng/ml , CHIR99021 1.5 μM , recombinant human R-spondin-1 80 ng/ml , recombinant human FGF-2 100 ng/ml , recombinant human HGF 50 ng/ml , A83-01 500 nM , prostaglandin E2 2.5 μM , and Y-27632 2 μM ].In total, seven different patient-derived lines were used in this study. We have previously published a detailed protocol of the derivation, maintenance and differentiation of human TO cultures . For traFive different patient-derived TSC lines were used in this study  and were cultured according to the previously published protocol  with the−1 glutamine, 10 units ml−1 penicillin, 100 µg ml−1 streptomycin and 2 mg ml−1 gentamicin . Culture medium was replaced every 2-3 days. Approximately 4-6 days after plating, cells were removed from tissue culture flasks with TrypLE (Gibco) to be either passaged at a ratio of 1:3 or used as a control for flow cytometry experiments.Samples of decidual tissue were used for the isolation of decidual stromal cells . Cells w−1 glutamine, 10 units ml−1 penicillin, 100 µg ml−1 streptomycin and 2 mg ml−1 gentamicin. Culture medium was replaced every 2-3 days. Approximately 4-6 days after plating, cells were removed from tissue culture flasks with TrypLE to be either passaged at a ratio of 1:3 or used as a control for flow cytometry experiments.Choriocarcinoma cell lines, JEG-3 and JAR, and embryonal carcinoma line 2102Ep (used only as control cell lines for experiments in this study) were purchased from the American Type Culture Collection (ATCC) in 2015. Cells were expanded and frozen stocks were immediately made within a few passages of receipt of these cell lines. Early passage numbers were thawed for this study. Cells were cultured in RMPI-1640 (Thermo Fisher Scientific) supplemented with 10% (v/v) FBS, 2 mM lTable S12) were replaced with equivalent concentrations of isotype-matched mouse or rabbit IgG for controls. For HLA staining of first-trimester placenta, frozen placental sections were fixed in acetone prior to staining. Images were captured with an EVOS M5000 Imaging System (Thermo Fisher Scientific).Organoids were formalin fixed and embedded as previously described . ImmunohTSCs were plated on 35-mm ibidi μ-dishes and differentiated to either EVT or SCT. Once differentiation was complete, the cells were incubated with W6/32-488 conjugated antibody  for 30 min at 37°C. Cells were washed twice with PBS and culture media was replaced before imaging. Images were captured with a Zeiss LSM 700 Confocal Laser Scanning Microscope with ZEN Microscope Software.For ELISA, 48 h conditioned media was collected from TO and TSC-3D cultures and stored at −20°C until use. hCG-β ELISA  was performed following the manufacturer's instructions. Samples were run in duplicate alongside nonconditioned media controls and hCG-β standards. The concentration of hCG-β was calculated from the line formula of the standard plots in Microsoft Office Excel.Table S12). LIVE/DEAD Fixable Far Red Dead Cell Stain  was used for live/dead discrimination. Data were acquired using the Cytek Development DxP8 (488/637/561) or the Attune NxT. Data were analyzed in FlowJo (Tree Star) and all compensation was applied digitally after acquisition.Single cell suspensions of TOs and TO-EVT were prepared as previously described . TSC-3D Total cell extracts were prepared in radioimmunoprecipitation assay buffer , containing a protease inhibitor cocktail , and incubated at 4°C for 1 h. Western blotting was performed as previously described . In brieThe QIAamp DNA Blood Mini kit  was used to extract genomic DNA from TOs, TSC lines and JEG-3/JAR cell lines for HLA tissue typing. DNA quality and concentration were determined in a Nanodrop ND-1000 Spectrophotometer.Table S1. Details of the World Health Organization (WHO) nomenclature for factors of the HLA system, and Bw6 epitope determination can be found at www.hla.alleles.org.The DNA for HLA genotyping was processed via the workflows of a European Federation for Immunogenetics (EFI)-accredited Clinical Histocompatibility Laboratory. Low-resolution typing of the HLA-A, HLA-B and HLA-C genes was achieved with LABType kits (One Lambda), which rely on reaction patterns observed when sequence-specific DNA probes immobilized on fluorescent X-MAP polystyrene beads (Luminex) hybridize to biotin-labeled multiplexed gene-specific PCR amplicons. The hybrids were detected with a Liquichip 200 fluorimeter (Qiagen) and HLA allele assignment was performed using HLA Fusion software (One Lambda). Ultra-high-resolution typing of HLA-A, HLA-B and HLA-C was achieved with an ‘in-house’ third-generation sequencing pipeline using Single Molecule, Real-Time (SMRT) DNA sequencing technology (Pacific Biosciences) as previously described . Typing Total RNA was isolated using the miRNeasy isolation kit  with on-column DNase digestion . RNA quality was assessed on the Agilent 2100 bioanalyzer (Thermo Fisher Scientific). The RNA integrity number (RIN) of each tested sample was greater or equal to 8.https://nf-co.re/rnaseq) with the option ‘--aligner hisat2’. All scripts, with details of software versions, a pipeline usage report and expression count files, are freely available from https://github.com/CTR-BFX/Sheridan_Turco.mRNA and small RNA-seq was run on the NextSeq 500 (Illumina) using a 75-cycle high output, which generated approximately 400 million reads per run. Library preparation for bulk mRNA sequencing was performed using the Truseq stranded mRNA library kit (Illumina) following the manufacturer's protocol. Library preparation for small RNA-seq was performed by using the Bioo Nextflex protocol . Data were aligned to the GRCh38 human genome . The alignment and quality control were processed using the Nextflow  pipelineP-values <0.05 were identified. Significant DEGs from each comparison were selected based on the adjusted P-values <0.05; absolute log2FoldChange was greater than 1. For heatmaps, gene-level transcript expression values were derived by log2 normalized transformed values. ComplexHeatmap (v 2.6.2) was applied to generate the heatmap . Unbiased biological process (BP) GO-enriched pathway analysis was also performed for all up- and downregulated DEGs. Based on the number of BPs identified, GO semantic similarity was performed to cluster genes into different clusters based on their functional similarity, and used to measure the similarities among all, up- and downregulated GO BP terms to reduce redundancy. GO plots were drawn using R package ggplot2 (version 3.3.2) and enrichplot (v 1.10.2).GO and GO semantic similarity pathway analysis were performed using R package clusterProfiler (version 3.14.3) , GOsemSiE-MTAB-10429.Normalized read counts were used for the statistical analysis of the mRNA abundance of key genes. Raw sequencing reads are deposited in EMBL-EBI ArrayExpress with experimental code For sequencing reads from TSC-2D, TSC-3D and TO samples, adapters were first removed using Cutadapt v3.3  adapter using BoDifferential expression analyses between TSC-2D and TSC-3D and between TSC-2D and TOs were conducted using DESeq2 . DiffereE-MTAB-10438.Raw sequencing reads were deposited in EMBL-EBI ArrayExpress with experimental code n listed in the figure legends. All experiments involving TSCs had representation from both blastocyst-derived and first-trimester placenta-derived lines. Except in the case of RNA and small RNA-seq, each experiment was repeated a minimum of twice independently . Statistical analyses were performed by a paired two-tailed Student's t-test (t-test with Welch's correction (Fig. S4B.For all experiments, multiple patient lines were used, with the specific s t-test J and an rrection D. Statis


Then different ratios of dodecyl dimethyl betaine (BS-12)-modified bentonite (BS-B) were loaded on the surfaces of biochar (BC) and MBC to prepare BS-B-loaded BC and MBC composites, called BS-B/BC and BS-B/MBC, respectively. The physicochemical and structural properties of the composites were characterized by scanning electron microscopy, Fourier transform infrared spectrometry, thermogravimetric analysis, specific surface area (SBET) analysis, and vibrating sample magnetometry, and the adsorption efficiencies of BS-B/BC and BS-B/MBC to Cu2+ and tetracycline (TC) were studied. The following results were obtained. (1) Compared with BS-B/BC, BS-B/MBC had decreased pH and cation exchange capacity (CEC) and increased SBET. The pH, CEC, and SBET of BS-B/BC and BS-B/MBC decreased with the increase in the BS-12 proportion of BS-B. The surface of BS-B/MBC became rough after Fe3O4 loading. (2) The residual rate of BS-B/MBC was higher than that of BS-B/BC after high-temperature combustion, and the residual rate decreased with the increase in the BS-12 proportion of BS-B. The 2D infrared spectra showed that Fe3O4 and BS-12 were modified on the surface of BS-B/MBC. MBC and BS-B/MBC had splendid magnetism and could be separated by external magnetic field. (3) Compared with unmagnetized ones, the adsorption effects of Cu2+ and TC on different BS-B/MBCs improved, and the average adsorption rate reached the largest value of 91.92% and 97.76%, respectively. Cu2+ and TC adsorptions were spontaneous, endothermic, and entropy-increasing processes. The pH and SBET of the material had a great influence on Cu2+ and TC adsorptions, respectively, than CEC.To realize simultaneous adsorption of heavy metal and antibiotic pollutants by a BC-based recyclable material, Fe Heavy metal and antibiotics in livestock farm wastewater has become one of the focuses of research . The met2+ and methylene blue are 30.19 and 46.60 mg/g, respectively  are eluted for the recycling of the MBC  were 1.3–1.8 and 4.0–8.3 times higher than those on unmodified soil sample, respectively (Bentonite (B) has strong adsorption capacity, ion exchange capacity and expansibility as a good pollution remediation material . Currentectively . The magectively . TherefoAlternanthera philoxeroides BC and BS-12-modified B (BS-B) were prepared by oxygen-limiting high-temperature pyrolysis and wet method, respectively, and MBC was prepared by co-precipitation method to verify the absorption effect of BS-B-loaded MBC (BS-B/MBC) on heavy metal and antibiotic pollution. Then, BS-B-loaded BC (BS-B/BC) and BS-B/MBC were prepared by separately loading BS-B on the surfaces of BC and MBC, and the properties and structure of the composite materials were characterized. In addition, the isothermal adsorption and thermodynamic characteristics of Cu2+ and tetracycline (TC) on the composite materials were studied to provide a reference for the application of BC-based composite materials in sewage treatment.3·6H2O, FeSO4·7H2O and NaOH were all purchased from Chengdu Kelong Chemical Reagent Factory, Chengdu City, Sichuan Province, China. TC was purchased from Sigma  and had a purity of 99.9%. Cu2+ solution was used as pollutant, and the solution was prepared by using CuSO4·5H2O  purchased from Chengdu Kelon Chemical Reagent Factory. Dodecyl dimethyl betaine was used as the amphoteric modifier, which abbreviated as BS-12 . The B used was sodium B, which purchased from Henan Xinyang Bentonite Produce Company and purified by washing method before use . The bas2O), dried, grinded and sieved through 60-mesh nylon sieve to obtain BS-B. The amount use of BS-12 modifier was defined by BS-B was prepared by wet method . BS-12 sWBS stands for the mass (g) of BS-12; m stands for the mass (g) of bentonite; CEC stands for the cation exchange capacity of the bentonite (mmol/kg); MBS stands for the relative molar mass of BS-12 (g/mol); RBS stands for the modification ratio (50% or 100%) of BS-12; CBS stands for the content (mass fraction) of BS-12.A. philoxeroides was washed with dH2O, dried to constant weight under 60 °C, grinded and sieved through a 200-mesh nylon sieve, and fired for 8 h under 400 °C by oxygen-limiting high-temperature pyrolysis to obtain BC. Co-precipitation method was used to prepare MBC , and characterized by scanning electron microscopy (SEM), thermogravimetry (TG), Fourier transform infrared (FT-IR) spectroscopy, and vibration sample magnetometry (VSM).BS-B samples with BS-12 proportions of 0%, 50%, and 100% were prepared and named as B (bentonite only), 50BS-B, and 100BS-B, respectively. BC and MBC were used as the control (CK). BC, B/BC, 50BS-B/BC, 100BS-B/BC, MBC, B/MBC, 50BS-B/MBC, and 100BS-B/MBC  were analyzed for the pH, CEC, and Brunaer–Emmett–Telle specific surface area  under the following conditions: temperature range 25–900 °C; sample quality, 10–15 mg; heating rate, 10 °C/min; N2 atmosphere. FT-IR analysis was performed on a Nicolet 5DX type Fourier transform infrared spectrometer, and the 2D FTIR spectra were analyzed by 2DShige software. Magnetic curves were determined by Lakeshore 665 VSM method.pH value was determined using a HQ411D table pH meter . CEC was determined by sodium acetate–ammonium acetate method. S2+ (TC) solutions under different concentration gradients, shaken at room temperature for 12 h (200 r/min), and centrifuged at 4,800 r/min for 15 min. The supernatant was collected to determine the Cu2+ (TC) concentration, and the actual adsorption amount of the test material was calculated by subtraction  of each composite material were separately packed in 50 mL plastic centrifuge tubes, added with 20 mL of Cutraction . The exp2+ and TC was calculated using The equilibrium adsorption amount of CuC0 (mmol/L) and Ce (mmol/L) are the initial and equilibrium concentrations of Cu2+ (or TC) in the solution, respectively. V (mL) is the volume of Cu2+ (or TC) solution added. W0 (g) is the weight of the tested material. q (mmol/kg) is the equilibrium adsorption amount of Cu2+ (or TC) on the tested material.AR (%) of Cu2+ and TC was calculated using The adsorption rate The Langmuir isotherm was selected on the basis of the adsorption isotherm trend and the isothermal equation qm indicates the maximum adsorption amount of Cu2+ (or TC) on the different materials, mmol/kg; b represents the apparent equilibrium constant of the Cu2+ (or TC) adsorption, which can be used to measure the affinity of adsorption.b in the Langmuir model is equivalent to the apparent adsorption constant of equilibrium constant, and the thermodynamic parameter calculated by b = K or Ka is called the apparent thermodynamic parameters; Parameter G is the standard free energy change (kJ/mol), R is a constant (8.3145 J/mol/K), T is the adsorption temperature , ∆H is the enthalpy of adsorption process (kJ/mol), and ∆S is the entropy change of adsorption process (J/mol/K).p < 0.05 were considered significant . The maximum adsorption capacity (qm) of different materials changed from 135.07 mmol/kg to 322.00 mmol/kg. Compared with unmagnetized ones, the magnetized materials had higher qm for Cu2+. 100BS-B/MBC had the best adsorption effect on Cu2+; its qm reached 322.00 mmol/kg, and its average adsorption rate (ARe) was 91.92%. The values of the adsorption constant (b) of magnetized materials for Cu2+ were smaller than those of unmagnetized ones, which indicates that magnetization reduced the adsorption affinity of BC materials to Cu2+. This outcome might be due to the fact that Fe3O4 particles block the pores of BC materials, which results in the reduction of exchangeable cation sites on the surface. The b values of BS-B/BC and BS-B/MBC were larger than those of BC and MBC, which indicates that the adsorption affinity to Cu2+ became greater after BS-B loading. B had a larger CEC and could have an ion exchange reaction with more Cu2+, and B loading could promote Cu2+ adsorption. The value of b was the largest when BC was loaded with B but decreased with the increasing in BS-12 modification rate on BS-B when BC was loaded with BS-B. This result indicates that BS-12 formed an organic coating on the surface of BC and reduced its adsorption affinity for Cu2+.G) values for the Cu2+ adsorption of each test material were less than 0, which indicates that the adsorption was a spontaneous reaction. The adsorption enthalpy change (ΔH) of each tested material to Cu2+ was greater than 0, which indicates that the adsorption was an endothermic reaction, and increasing temperature was conducive to the occurrence of adsorption. The entropy change (∆S) of each test material was greater than 0, which indicates that the disorder of the system in the Cu2+ adsorption process was increased by the tested materials.At 20 and 40 °C, the Gibbs free energy (Δp < 0.01). The qm for TC adsorption changed from 118.60 to 602.83 mmol/kg, B/MBC had the best qm for TC adsorption, and 50BS-B/MBC had the largest ARe of 97.76%. Compared with the unmagnetized materials, the magnetized materials could reach the TC adsorption equilibrium quickly and had higher qm values for TC adsorption. After B and BS-B loading, the b values of the materials for TC adsorption were larger than those of BC and MBC. At 20 and 40 °C, all test materials for TC adsorption had ΔG < 0, which indicates that the adsorption was a spontaneous reaction; ΔH > 0, which indicates that the adsorption was an endothermic reaction; and ∆S > 0, which indicates that the disorder of the system increased in the TC adsorption process.qm and ARe values of each test material for Cu2+ and TC were linearly fitted to the physicochemical properties of the material, and the fitting results are shown in qm and ARe for Cu2+ and TC adsorptions; pH and SBET had positive correlations with the qm and ARe for TC adsorption but had negative correlations with the qm and ARe for Cu2+ adsorption; pH had a negative correlation with the ARe for TC adsorption. Moderate correlations between qm and pH, and between ARe and SBET were observed in Cu2+ adsorption (r > 0.5); and the other indexes maintained a low degree of correlation. qm and SBET in TC adsorption were moderately correlated, and the other indexes maintained a low degree of correlation. The results indicate that the pH and SBET of the material have a greater influence on Cu2+ and TC adsorptions respectively, than CEC.The 2+ and TC on BS-B/MBC. The SBET, pore structure, and functional groups of BC determined its adsorption capacity for Cu2+ and TC, but the adsorption capacity of original BC was limited. Solid–liquid separation could be realized under the action of external magnetic field by magnetizing the material with the magnetic medium. Fe3O4 loading increased the SBET and effective adsorption sites on the surface of BC. MBC and BS-B/MBC had good magnetic separation performances and could be separated by applying a magnetic field.2+ and TC. When MBC was loaded with BS-B, BS-B/MBC had a strong affinity for heavy metal ions and could effectively fix Cu2+, and which increases the adsorption capacity for Cu2+. The adsorption mechanism of Cu2+ comes from ion exchange, complexation, and electrostatic attraction by BC and from complexation and electrostatic attraction by BS-B (2+ and TC adsorptions were compared (2+ adsorption, which indicates that C=O and C–H on the surface of 100BS-B/MBC were involved in the Cu2+ and TC adsorption processes. The appearance of O–H bonds on 100BS-B/MBC after TC adsorption showed that hydroxyl group participates in the TC adsorption process. Additionally, the movement of the peak to a higher wave number means that the energy required for vibration is lower, which indicates that the group is more stable.B had strong adsorption capacity and ion exchange capacity, and amphoteric modification could remarkably improve its adsorption capacity for heavy metals and organic pollutants . Therefo by BS-B . TC has  by BS-B . When th by BS-B . The rap by BS-B . Moreovecompared . The resBET. BC had a smooth surface, but the surface smoothness of BS-B/BC increased with the increase in BS-12 modification on BS-B. After Fe3O4 was loaded, a large number of Fe3O4 particles adhered to the surface of BS-B/MBC, and its surface became rough. Compared with unmagnetizatic ones, the magnetized materials had higher weight loss rates. FTIR and VSM analyses proved that BS-12 and Fe3O4 were modified to the surface of BS-B/MBC. MBC and BS-B/MC had good magnetic separation performances. BS-B/MBC had a good adsorption effects on Cu2+ and TC, and its ARe was more than 80%. Cu2+ and TC adsorptions were spontaneous, endothermic, and entropy-adding reactions. The pH and SBET of the material had a great influence on Cu2+ and TC adsorptions, respectively.After magnetization, BC had reduced pH and CEC and increased S10.7717/peerj.13030/supp-1Supplemental Information 1Click here for additional data file.


Aedes albopictus. However, the IOL CHIKV of the most recent outbreaks during 2016–2020 in India, Pakistan, Bangladesh, the Maldives, Myanmar, Thailand, and Kenya lacked E1-A226V but carried E1-K211E and E2-V264A. Recent CHIKV genome sequences of the Maldives and Thailand were determined, and their phylogenetic relationships were further investigated together with IOL sequences reported in 2004–2020 in the database. The results showed that the ancestral IOLs diverged to a sub-lineage E1-K211E/E2-V264A, probably in India around 2008, and caused sporadic outbreaks in India during 2010–2015 and in Kenya in 2016. The massive expansion of this new sub-lineage occurred after the acquisition of E1-I317V in other neighboring and remote regions in 2014–2020. Additionally, the phylogenetic tree indicated that independent clades formed according to the geographical regions and introduction timing. The present results using all available partial or full sequences of the recent CHIKVs emphasized the dynamics of the IOL sub-lineages in the Indian subcontinent, Southeast Asia, and Eastern Africa.The Indian Ocean Lineage (IOL) of the chikungunya virus (CHIKV) East/Central/South African (ECSA) genotype, which originated in Kenya, spread to the Indian ocean and the Indian subcontinent, and then expanded through Southeast Asia in the previous decade. It carried an adaptive mutation E1-A226V, which enhances CHIKV replication in  Alphavirus, family Togaviridae, and causes fever, arthralgia, and rash in humans. The viral genome is a single positive-stranded RNA of 11–12 kb in length consisting of 5′ and 3′ untranslated regions (UTRs) and two open reading frames (ORFs) of nonstructural proteins nsP1-4 and structural polyproteins capsid, E3, E2, 6K, and E1. The first CHIKV was isolated during an outbreak in Tanzania in 1953. Nowadays, CHIKV is phylogenetically classified into three major genotypes: East/Central/South/African (ECSA), West African (WA), and Asian, named after the location where these genotypes were first recognized [Aedes aegypti [Aedes albopictus [Chikungunya virus (CHIKV) is a member of the genus However, IOL CHIKV of the most recent outbreaks during 2016–2017 in India, Pakistan, and Bangladesh lacked E1-A226V. Instead, they carried two novel mutations, a lysine-to-glutamic acid mutation at position 211 within the E1 glycoprotein (E1-K211E) and a valine-to-alanine mutation at position 264 within the E2 glycoprotein (E2-V264A) ,6. We prhttps://www.viprbrc.org) (accessed on 1 August 2021) [To understand the diversity and evolution of the current IOL sub-lineage E1-K211E/E2-V264A, IOL sequences with these mutations were collected in the Virus Pathogen Resource  . In addist 2021)  and thosSixteen existing, previously published CHIKV real-time RT-PCR-positive sera were obtained from Indira Gandhi Memorial Hospital, the Maldives in 2019 ,15 and 1http://www.ddbj.nig.ac.jp) (accessed on 13 December 2021) with the access number LC664141-LC664166  were used to isolate virus strains in C6/36 cells in order to increase the number of the virus using the previously described protocol [LC664166 . Newly g were useLC664166 .The CHIKV sequences were collected using the ViPR database by filtering the geography and time option as the retrieval date of August 2021 . GenbankThe dataset of 271 CDS sequences of IOL CHIKV was trimmed to two datasets of the nonstructural polyprotein ORF (7473 bp) and structural polyprotein ORF (3744 bp). These datasets were used for selection pressure analysis implemented in HyPhy using four methods: a mixed-effects model of evolution (MEME), fast, unconstrained Bayesian approximation (FUBAR), single-likelihood ancestor counting (SLAC), and fixed-effect likelihood (FEL) for the site-specific selection.A total of 26 CHIKV sequences of nearly the whole genome, including 16 Maldives strains from the 2019 outbreak and 10 Thailand strains from the 2020 outbreak, were generated . CHIKV gTo determine the distribution of novel mutations of E1-K211E and E2-V264A, CHIKV nucleotide sequences available in the ViPR database retrieved on 1 August 2021 were investigated. Seven hundred and sixty-five IOL CHIKV sequences covering at least the E2 to E1 region were analyzed. Among them, 427 showed the presence of E1-K211E and E2-V264A . In Tabl2 = 0.91)  . The tim = 0.91) . The treAedes albopictus. Co-circulation of the IOL E1-A226V variant with E1-226A virus was primarily observed in the Indian Ocean clade and the South Asian clade during 2005–2013  with an interval of June 2004–February 2005 (95% HPD: 2004.48–2005.10), then spread and circulated in India, Sri Lanka, and Bangladesh, subsequently descended to the new clades, and was distributed to wider geographic regions. The tMRCA for the Southeast Asian clade, including sequences from Singapore, Malaysia, Thailand, Laos, and Cambodia, was estimated to be July 2007 (2007.50) with an interval of February–November 2007 (95% HPD: 2007.16–2007.88), whereas that for the Italian clade was July 2006 (2006.52) with an interval of February 2006–January 2007 (95% HPD: 2006.14–2007.0). During the first emergence of the IOL, the remarkable adaptive mutation E1-A226V was identified as it enhanced IOL CHIKVs replication in 005–2013 . The cir005–2013 .Since 2010, the proportion of E1-K211E/E2-V264A in the background of E1-226A has increased year by year . As desc−3 substitutions/site/year (s/s/y). Besides the mutations E1-K211E/E2-V264A, this clade shared an additional amino acid substitution of E1-I317V specific to the recent IOL 2014-2020. The phylogenetic tree in The expansion of the IOL sub-lineage E1-K211E/E2-V264A was observed in recent outbreaks after 2015. This new IOL sub-lineage India and Pakistan strains circulating in 2016 spread rapidly to Bangladesh in 2017 and from Bangladesh to Myanmar, Thailand, and China in 2018–2020. Moreover, this IOL sub-lineage also spread from India to the Maldives and Kenya, Sudan, and Djibouti in 2014–2019. In −3 (2.86 × 10−3–4.51 × 10−3) s/s/y. The IE viruses had C-N79S and E2-A76T as the common amino acid substitutions among the clade. In addition, there were two distinct subclades within this clade with different geographic regions of their sampling site: (1) Eastern Africa subclade or (IE-a) and (2) Maldives subclade (IE-b) that originated in September 2013 (2013.74) with an interval of July 2013–January 2014 (95% HPD: 2013.52–2014.0) and May 2018 (2018.39) with an interval of January–October 2018 (95% HPD: 2018.06–2018.77), respectively. The IE-a subclade contained Kenyan isolates 2014–2018 together with viruses from neighboring countries, including Sudan in 2018 and Djibouti in 2019. IE-a viruses shared the mutation of E2-M74I. On the other hand, the IE-b subclade was limited to viruses only from the Maldives collected in 2019. The most related strain to IE-b was India strain 2019 (MW042255), suggesting that the new sub-lineage had spread from India to the Maldives, circulating widely to the south of the Indian subcontinent. Those sequences shared the unique amino acid mutation of nsP4-L500S.The Indian subcontinent/Eastern Africa clade (IE clade) contained viruses from India 2016–2019, Kenya 2014–2018, Sudan 2018, Djibouti 2019, and Maldives 2019. The tMRCA of this clade was estimated at January 2013 (2013.04) with an interval of June 2012–July 2013 (95% HPD: 2012.47–2013.58), with a substitution rate of 1.88 × 10−4 (3.11 × 10−4–9.51 × 10−4) s/s/y. The analysis suggested an expansion route of IOL sub-lineage -E1-K211E/E2-V264A with E1-I317V virus that reached the adjacent regions including Pakistan and Bangladesh in the 2016–2017 epidemic from India, especially northern India, particularly Delhi and Maharashtra, located in the south of Delhi. The phylogenetic tree indicated that the IS-a subclade arose in Pakistan around June 2015 (2015.44) with an interval of February–October 2015 (95% HPD: 2015.15–2015.78) and that the IS-b subclade emerged in Bangladesh in December 2015 (2015.97) with an interval of September 2015–March 2016 (95% HPD: 2015.74–2016.24). The IS-c subclade was composed of Italian viruses that arose in July 2016 (2016.56) with an interval of April–October 2016 (95% HPD: 2016.31–2016.83). Additionally, the IS-b subclade consisted of the descending strains circulating during 2018–2020 in Southeast Asia, including Thailand, Myanmar, and China (cities adjacent to Myanmar), forming a clade IS-d separate from the Bangladesh strains. The re-introduction of the IOL to these regions occurred around May 2017 (2017.34) with an interval of January–August 2017 (95% HPD: 2017.07–2017.63). Moreover, local transmission in China was observed in the IS-e subclade. In addition, the IS clade was defined by amino acid mutations of nsP2-E145D and nsP4-S55N, whereas the IS-d subclade was defined by nsP2-N495S and C-K73R.The Indian subcontinent/Southeast Asia clade (IS clade) was a large monophyletic clade comprising isolates from India, Pakistan, and Bangladesh in 2016–2017, and Thailand, Myanmar, and China (adjacent cities to Myanmar) in 2018–2020. The tMRCA was estimated at January 2015 (2015.02) with an interval of January 2014–July 2015 (95% HPD: 2014.08–2015.52) with a substitution rate of 6.04 × 10The dataset of 271 IOL CHIKV sequences was screened for positive selection using different individual site models. Seven codon sites were identified under positive selection by at least two methods . Of thesAedes albopictus was identified [Aedes albopictus was abundant, such as in Kerala in India in the 2007 outbreak [In the present study, IOL CHIKV sequences with time spanning from 2004 to the present were analyzed to investigate the evolution of the IOL for a better understanding of the current IOL genetic diversity. The first emergence of the IOL of CHIKV genotype ECSA was in late 2002, consistent with the previous studies . Based oentified . The IOLoutbreak . Consequoutbreak ,28,29,30Aedes aegypti by increasing infectivity, dissemination, and transmission in this mosquito species [Aedes aegypti was present in several areas over India instead of Aedes albopictus [After the initial major outbreaks, Indian IOL has retained E1-226A but gained additional mutations of E1-K211E and E2-V264A. As described above, the earlier presence of E1-K211E/E2-V264A IOL was reported in early 2010, particularly in Tamil Nadu, Andra Pradesh, and Delhi in India ,31. The  species . Aedes abopictus ,35. EvidAedes aegypti and Aedes albopictus coexisted. The mutation E1-K211E with E2-V264A and the E1-A226V with E2-L210Q or E2-K252Q enhanced virus fitness in Aedes aegypti and Aedes albopictus, respectively [In terms of the amino acid variation at 211 in E1 among CHIKV genotypes, an E1-K211E was observed in the IOL variant in 2006 in Kerala and Puducherry, India . On the ectively ,38. AccoAt first, the earliest E1-K211E/E2-V264A IOL strain sparsely circulated in Delhi, Mumbai, and Kerala in India since 2010 . Then, tAedes aegypti was predominant. Notably, the earliest Kenya strain collected in 2014 was related to Indian 2016 isolates (MK473627-MK473628) and India isolate 2014 (MW042254). This IOL variant was also detected in the ongoing outbreaks in nearby countries Sudan and Djibouti that occurred coincidently with the rise of the suspected CHIKV infections in other Eastern African countries such as Ethiopia [Shortly after its emergence in 2012, E1-K211E/E2-V264A/E1-I317V IOL separated into geographically associated clades. In 2013, this variant was introduced to coastal Kenya in Kilifi and Mombasa in 2014 . InteresEthiopia ,10,11,42Ethiopia .Regarding travel-associated CHIKV transmission to remote regions in the non-endemic area such as East Asia, imported cases were reported in Hong Kong returning from India in 2016 , in ShenAedes albopictus, the localized vector in Italy, showed that both had similar infection and transmission rates [Aedes aegypti was identified as the primary vector, and viral RNA was also found in another mosquito species, Culex quinquefasciatus [Aedes aegypti or other mosquito species needs to be clarified by further studies in the future.The functional role of the lineage-specific mutation E1-I317V is still less known. The mutation was certainly detected in circulating IOL from 2014 to the present, but there was no evidence of positive selection pressure on this mutation. The study of in vivo pathogenesis among CHIKV lineages showed that there was no difference in the swelling of mouse footpads between the Indian IOL 2010 strain with E1-317I and the Indian IOL 2016 strain with E1-317V . Likewison rates . In addiasciatus ,51. In Masciatus ,52. WhetThe present study characterized the diversity of IOL lineages, especially the current circulating strains. The limitations of this study are the availability of data and the difficulty inferring the origin of sequences from the database. Since collection probably placed a priority on outbreaks and symptomatic patients, fewer viral sequences were deposited within the inter-epidemic/non-outbreak period. The different sampling strategies or surveillances in each collection location would also generate biases. To better understand the dynamics of the IOL, a sampling schema over a wide range of geography, time, and transmission vectors diversity is important.


DUSP)12 is an atypical member of the protein tyrosine phosphatase family, which are overexpressed in multiple types of malignant tumors. This protein family protect cells from apoptosis and promotes the proliferation and motility of cells. However, the pathological role of DUSP12 in hepatocellular carcinoma (HCC) is incompletely understood.Dual specificity protein phosphatase RNA decreased the proliferation and migration of HCC cells. Besides, DUSP12 expression was positively associated with the infiltration of cluster of differentiation (CD)4+ T cells , macrophages, neutrophils and dendritic cells. DUSP12 expression was positively associated with immune-checkpoint moieties, and was downregulated in a C3 immune-subgroup of HCC .DUSP12 may have a critical role in the tumorigenesis, infiltration of immune cells, and prognosis of HCC.These data suggest that  Hepatocellular carcinoma (HCC) is the most common malignant tumor of the liver . HCC is DUSP)12 is an atypical member of the protein tyrosine phosphatase (PTP) family. DUSP12 regulates the proliferation, apoptosis, and migration of cells by dephosphorylating tyrosine and serine/threonine residues -1 and IL-6, and increase IL-10 expression. DUSP12-expressed hepatocytes are less inflamed and cause less hepatic steatosis than DUSP12-deleted hepatocytes  is an interactive Internet resource for analyzing cancer OMICS data. Ualcan was used to analyze transcription expression, prognosis, and the methylation of genes in The Cancer Genome Atlas (TCGA) datasets  was used to plot overall survival (OS) and disease-free survival (DFS) curves. Group cutoff was based on the median of gene expression and prognostic status of patients in the TCGA-LIHC dataset  was employed for OS (including 364 patients), DFS (including 316 patients), progression-free survival (PFS) (including 370 patients) and disease-specific survival (DSS) (including 262 patients) analysis using data from RNA-sequencing of a liver-cancer dataset  was used to validate gene expression in liver-cancer tissues and normal liver tissues at the protein level (The HPA database (in level .http://www.DepMap Broadinstitute.org/ccle/) was employed to analyze gene expression in HCC cells (The Cancer Cell Line Encyclopedia database (CC cells . RNA-exphttp://lifeome.net/database/hccdb/home.html) is an integrative molecular database of HCC with 15 datasets. Co-expressed genes were computed and displayed in HCC according the guidelines on the HCCDB website (GSE14520 were downloaded from HCCDB.The Hepatocellular Cancer Database (HCCDB)  is an online tool for analyzing the mutation characteristics of genes in a Liver Hepatocellular Carcinoma  dataset , the list of correlations was filtered for interactions with P <0.05 and correlation coefficient >0.2.The correlation of copy number variation (CNV) of genes with the abundance of tumor-infiltrating immune cells (TIICs) was displayed by an online tool in the TIMER database . In thishttp://cis.hku.hk/TISIDB/) was employed to analyze gene expression in patients with different immune subtypes of HCC (TISIDB (s of HCC .http://xena.ucsc.edu/). The CIBERSORT method was used within the R package (http://www.r-project.org/) .A total of 369 tumor samples extracted from the Genomic Data Commons (GDC)-TCGA-LIHC dataset were downloaded from UCSC.XENA (ct.org/) . After rhttp://metascape.org/) is a resource to aid the annotation and analyses of genes, which helps biologists make sense of one or multiple gene lists. Metascape was applied for analyses of protein–protein interaction (PPI) networks. Analyses of functional enrichment and enrichment of pathways were done using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and DisGeNET databases  database is utilized to evaluate the probabilities of dependency of DUSP12 in HCC cell lines with CERES score based on data from CRISPR  cohort  and cultivated in Dulbecco’s modified Eagle’s medium  with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Gibco) at 37 °C in an atmosphere of 5% CODUSP12  and horseradish peroxidase-conjugated secondary antibody . These actions were followed by exposure to enhanced chemiluminescence. The housekeep gene β-tubulin  was used as a loading control.Cell proteins were extracted by denaturing buffer and then quantified by a bicinchoninic acid protein assay . Protein lysates from the HCC cell line were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes , blocked, and then detected by primary antibody DUSP12 primer sequence  were: CCGGGTTGAGTGGCAACTGAAATTATCTCGAGATAATTTCAGTTGCCACTCAAGTTTTTG, and AATTCAAAAAGTTGAGTGGCAACTGAAATTATCTCGAGATAATTTCAGTTGCCACTCAAG for shDUSP12-1; CCGGGTGGATACCTCTAGTGCAATTCTCGAGAATTGCACTAGAGGTATCCACTTTTTG, and AATTCAAAAAGTGGATACCTCTAGTGCAATTCTCGAGAATTGCACTAGAGGTATCCAC for shDUSP12-2.Annealing and connection of short hairpin (sh)RNA were undertaken followed by construction into the modified plasmid pLKO.1. Well-constructed vectors were transinfected into HEK293T cells by lentivirus packaging plasmids psPAX and pMD2.0G. The shRNA sequences and Lentivirus-infected stable cells were seeded into 96-well plates and cultured in DMEM containing 10% fetal bovine serum . Then, cells were collected at different timepoints. The cell number in each well was counted by Cell Counting Kit 8 (CCK8). Absorbance at 450 nm was measured to determine of the number of viable cells.5 cells per well, five parallel wells). Then, 800 µL of DMEM containing 10% fetal bovine serum was added to the lower chamber and incubation allowed for 36 h at 37 °C. After removing the cells at the upper surface of the membrane, cells were passed through a filter and fixed with 4% paraformaldehyde, stained with 0.1% Crystal Violet solution and photographed using an inverted fluorescence microscope.Lentivirus-infected stable cells were seeded in the upper chamber of a Transwell chamber  in 200 µL of serum-free DMEM  stage-III, grade-3, P53-mutant group than that in a Caucasian, TNM stage-I/II, grade-1/2, P53-nonmutant group. The non-significant change in DUSP12 expression between the stage-IV/grade-4/N1 group compared with that in other groups may have been caused by the number of samples in the stage-IV/grade-4/N1 group being significantly less compared with that in other groups. The top positively and negatively correlated genes in TCGA dataset for DUSP12 were also downloaded from UALCAN database  (DUSP12 in liver-cancer cell lines (D: 3222) , 3B and ll lines .DUSP12 had shorter OS and DFS in the TCGA-LIHC dataset  dataset. The mutant frequency of DUSP12 in HCC was 33.0%  dataset  at procurement —>1 —>1 . Analyseatio)—>1 , 7C. Minatio)—>1 . MCODE 1DUSP12 expression with TIICs by TIMER database. DUSP12 expression was moderately  positively correlated with the abundance of infiltrating B cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells . The result suggested that DUSP12 expression was moderately  positively correlated with the abundance of infiltrating B cells and CD4+ T cells regardless of the method employed  subgroup had the highest DUSP12 expression, whereas the C3 (inflammatory) and C6 (TGF-β dominant) subgroups had lower expression of DUSP12 (DUSP12 had a higher ratio of T-regulatory cells (Tregs) and activated natural-killer cells compared with those with low expression of DUSP12  cohort. The CERES scores of cell lines ranged from −0.04 to −0.47 while mean value was equal to −0.21 (DUSP12 (shDUSP12) and a nonspecific shRNA (NC)  . CCK8 anh7 cells , 10C.DUSP12, a member of the PTP family, was different in HCC tissues and normal liver tissues in multiple datasets. In addition, the clinical features of HCC patients had a strong relationship with DUSP12 expression, including ethnicity, TNM stage, histology grade, and P53-mutant status. Survival analyses using the Kaplan–Meier method demonstrated that HCC patients with higher expression of DUSP12 had shorter survival than those with lower expression of DUSP12.We propose that expression of DUSP12 and 20 genes in liver-cancer samples in HCCDB was constructed. These genes, including ubiquitin-conjugating enzyme E2T (UBE2T), ILF2, SETDB1, CCT3, and UFC1, were engaged mainly in histone methylation, cullin RING ubiquitin ligase complexes, nuclear specks, and ubiquitin-like protein transferase activity. UBE2T has been demonstrated to promote the growth of HCC cells by regulating ubiquitination of P53, transition of the G2/M phase of the cell cycle, and the protein kinase B signaling pathway . Furthermore, we investigated the ratio of various types of immune cells in total TIICs between a DUSP12-high-expression group and DUSP12-low-expression group. We showed that the infiltrating abundance of Tregs was higher in DUSP12-high-expression HCC samples compared with that in DUSP12-low-expression HCC samples. Tregs can inhibit the anti-tumor effects of immune cells and facilitate immune evasion by liver-cancer cells  ICGC-LIRI-JP. (B) GSE14520. ****P < 0.0001.DUSP12 expression in HCC and normal liver tissues in ICGC-LIRI-JP and Click here for additional data file.10.7717/peerj.11929/supp-2Supplemental Information 2The top positively correlated genes in TCGA dataset for DUSP12 (Pearson correlation coefficient > = 0.3).Click here for additional data file.10.7717/peerj.11929/supp-3Supplemental Information 3Dependency of DUSP12 in HCC cell linesClick here for additional data file.10.7717/peerj.11929/supp-4Supplemental Information 4The top negatively correlated genes in TCGA dataset for DUSP12 (Pearson correlation coefficient < = −0.3).Click here for additional data file.10.7717/peerj.11929/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.11929/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.11929/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.11929/supp-8Supplemental Information 8Click here for additional data file.


Since the D614G substitution in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, the variant strain has undergone a rapid expansion to become the most abundant strain worldwide. Therefore, this substitution may provide an advantage for viral spreading. To explore the mechanism, we analyzed 18 viral isolates containing S proteins with either G614 or D614 . The plaque assay showed a significantly higher virus titer in S-G614 than in S-D614 isolates. We further found increased cleavage of the S protein at the furin substrate site, a key event that promotes syncytium formation, in S-G614 isolates. The enhancement of the D614G substitution in the cleavage of the S protein and in syncytium formation has been validated in cells expressing S protein. The effect on the syncytium was abolished by furin inhibitor treatment and mutation of the furin cleavage site, suggesting its dependence on cleavage by furin. Our study pointed to the impact of the D614G substitution on syncytium formation through enhanced furin-mediated S cleavage, which might increase the transmissibility and infectivity of SARS-CoV-2 strains containing S-G614. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes a rapid accumulation of confirmed and fatal cases and poses a threat to public health around the world. A better understanding of the virus’s evolution and characterization of viral genetic variations usually provide valuable insights into the mechanisms linked to pathogenesis, antiviral drug resistance, and immune responses, which also impact the development of new vaccines, antiviral drugs, and diagnostic tests –3. There–4–Among the genetic variations that have evolved during the course of the SARS-CoV-2 outbreak, the D614G substitution in the spike (S) protein, which corresponds to a change of the A nucleotide at genome position 23403 to a G, has been identified as the signature of the A2a clade of SARS-CoV-2, the most prevalent clade , 8. This9–13–The host protease-mediated cleavage of envelope protein is involved in the maturation, pathogenesis, and infectivity of many viruses. S of SARS-CoV-2 also undergoes two proteolytic cleavages critical for membrane fusion and viral entry. One cleavage site is located at the S1 and S2 boundary, a polybasic motif recognized by furin and related proprotein convertases (PCs); the other is located at the S2′ site within the S2 domain, which is recognized by the TMPRSS2 protease . In our n = 10) had significantly higher virus titers than the S-D614-containing viruses (n = 8), as determined by a plaque assay . The results obtained with the virus isolates suggest a possible functional effect of the D614G substitution in increasing virus production.To investigate the effect of the D614G mutation on SARS-CoV-2 replication, we first compared the virus titers of 18 clinical SARS-CoV-2 isolates (NTU01 to NTU18) . (B) Northern blot analysis of viral RNA isolated from Vero E6 cells infected with SARS-CoV-2 strains (NTU01 to NTU18) at 48 h postinfection. Viral RNA was quantified by qRT-PCR targeting the E gene, as indicated below the Northern blot results. The ratio (percentage) of the E gene was normalized to the NTU01 level (100%).  Representative immunoblot of S protein extracted from the supernatants and cell lysates of S-D614-containing (lanes 1 to 4) or S-G614-containing (lanes 5 to 9) virus-infected Vero E6 cells at 48 h postinfection (MOI = 0.01). Full-length (FL) S proteins, cleaved S proteins, and nucleocapsid (N) proteins are marked as indicated. The ratio of cleaved S to full-length S protein for each SARS-CoV-2 strain is indicated below the immunoblot results. The ratios in the S-D614- and S-G614-containing viruses were compared and are presented as means ± SD in the right panel (P < 0.05*).  Immunoblot of S protein extracted from the cell lysates as described for panel C. The ratio of cleaved S to full-length S protein for each SARS-CoV-2 strain is indicated below the immunoblot results. The ratios were compared and are presented as means ± SD in the right panel (P < 0.05*). Download FIG S1, TIF file, 4.0 MB.Virus production was higher for S-G614-containing SARS-CoV-2 than for S-D614-containing SARS-CoV-2, which was associated with an increase in the cleavage of the S protein in Vero E6 cells. (A) Comparison of the virus titers (PFU per milliliter) (MOI = 0.01) in the supernatants of Vero E6 cells infected with SARS-CoV-2 strains NTU01 to NTU18 (8 strains expressing wild-type S-D614 and 10 strains expressing mutant S-G614) at 24 h (left) and 48 h (right) postinfection. Data are means ± SD . Download Table S1, DOCX file, 0.02 MB.SARS-CoV-2 genome (GISAID ID). The sequencing data and the accession codes of 18 virus isolates were deposited in GISAID ; the GISAID accession numbers for these 18 isolates are listed in Table S2, DOCX file, 0.04 MB.Summary of the mutations in SARS-CoV-2 isolates NTU01 to NTU18. SARS-CoV-2 genetic sequences, including NTU01 to NTU18 and the reference sequence (MN_908947), were aligned and compared. Nucleotide 23403, corresponding to aa 614 of the spike protein, is marked in red text, and the orange-shaded nucleotides indicate nucleotides different from the reference nucleotides. Sequence data were obtained from GISAID    , right. < 0.001) .10.1128/mBio.00587-21.2FIG S2P < 0.001***). S, spike; N, nucleocapsid. Download FIG S2, TIF file, 1.1 MB.The amount of S protein was significantly higher in S-G614-containing than in S-D614-containing SARS-CoV-2 viruses. The ratios of total (cleaved plus full-length) S/N (left) and cleaved S/N (right) for each SARS-CoV-2 isolate were derived from the immunoblot results for the supernatants of individual virus-infected Calu-3 cells, as shown in Copyright © 2021 Cheng et al.2021Cheng et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P < 0.001)  , right. < 0.001)  and D. TGenetic heterogeneity beyond the D614G substitution among different SARS-CoV-2 isolates might cause confusion regarding our observation. Therefore, we tested our hypothesis in cultured cells expressing only the S protein. Codon-optimized expression plasmids for wild-type S-D614 and the single-substitution mutant (S-G614) were individually transfected into Vero E6 cells, which were harvested at 24 h posttransfection for analysis. We first examined the syncytial phenotype via the observation of fused cells containing multiple nuclei visible by light microscopy. Compared with control cells transfected with only the vector, cells expressing the S-D614 protein exhibited a moderate syncytial phenotype, which was increased in cells expressing the mutant S-G614 protein . Immunob+ and ACE2– effector cells. Two ACE2+ target cell lines were selected: 293T cells stably transfected with human ACE2 (293T-hACE2) and Vero E6 cells (with endogenous ACE2 expression). Neither effector cells nor target cells alone developed syncytia due to the lack of the binding partner (ACE2 or the S protein). To quantitatively examine the fusion of effector and target cells, we developed a one-hybrid luciferase reporter assay. The pGAL4DBD-hAR-NTD plasmid was cotransfected with an individual S expression plasmid into the effector cells; the pGAL4/UAS-TK-Luc plasmid was transfected into the target cells. The effector and target cells were then cocultured and harvested for reporter activity analysis. The expression of pGAL4/UAS-TK-Luc in the target cells was activated only upon formation of syncytia consisting of effector and target cells (mediated by the interaction of S and ACE2), which is driven by the transcriptional activator encoded by the pGAL4DBD-hAR-NTD plasmid in the effector cells.To confirm the effect of the D614G substitution on S and ACE2 binding-mediated cell syncytium formation, we established a luciferase-based reporter assay to quantitatively compare levels of syncytium induction by the S-G614 and S-D614 proteins  , 30. TheIn addition, our results showed quite interesting differences between the amounts of cleavage of S present in the supernatant (55.95% and 27.71%) and those in the lysates (64.96% and 39.13%). During biogenesis, the spike monomer travels from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC), where it forms a trimer and incorporates into viral particles, budding into the lumens of vesicles, which are then trafficking for release through exocytosis . The cleMoreover, we found a higher S/N ratio in S-G614 viruses than in S-D614 viruses . ConsistCurrently, S-G614-containing SARS-CoV-2 has expanded and is the dominant strain worldwide. As the enhancement of cleavage by furin/PCs is critical for its transmission, blockade of this mechanism by targeting furin/PC protease activity for inhibition might become a potential antiviral strategy to block transmission of this strain. As shown in our recent study, two furin/PC inhibitors, CMK and naphthofluorescein, can abolish S cleavage, virus production, and pathogenic syncytium formation of an S-G614-containing SARS-CoV-2 isolate (NTU03) . ConsistSputum specimens from SARS-CoV-2-infected patients were kept in viral transport medium. Virus (multiplicity of infection [MOI] = 0.02) isolated from the specimens was propagated in Vero E6 cells in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 μg/ml tosylsulfonyl phenylalanyl chloromethyl ketone-trypsin, which may enhance infectivity and viral isolation efficacy, as documented previously –36. The –5 cells/well) were seeded in 24-well tissue culture plates and maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics. After 24 h of incubation, SARS-CoV-2 was treated to the cell monolayer for 1 h at 37°C. After removal of the virus, the cell monolayer was washed once with phosphate-buffered saline (PBS), and the cells were maintained with medium containing 1% methylcellulose and incubated for 5 to 7 days. Subsequently, cells were fixed with 10% formaldehyde overnight and stained with 0.5% crystal violet in order to count the plaques. Virus titers are means from three independent experiments.The plaque assay was performed as previously described, with minor modifications . In brieCTCGGTACAATTCACGCCCTGATACAGCACGGC-3′) and S-D614G-R (5′-GCCGTGCTGTATCAGGGCGTGAATTGTACCGAG-3′). The primer set for R682A spike was 5′-ACGCTCCGGGCTCTTGCGGGAGAGTTTGTCTG-3′ and 5′-CAGACAAACTCTCCCGCAAGAGCCCGGAGCGT-3′.Humanized pUC57-2019-nCoV-S was a kind gift from Che Ma at the Institute of Genomics Research Center, Academia Sinica, Taiwan. The spike sequence was cloned into the pcDNA3.0-HA vector with the addition of a hemagglutinin (HA) tag at the spike protein C terminus via NheI and XbaI sites. A QuikChange II site-directed mutagenesis kit (Agilent) was used to generate the mutant D614G spike or R682A spike construct. The primer set for D614G spike was S-D614G-F . The furin inhibitor CMK  (Tocris Bioscience) was added to the medium at the indicated concentrations 2 h posttransfection. Cells were harvested 24 h posttransfection for subsequent Western blotting.293T cells stably expressing human ACE2 (293T-ACE2) were kindly provided by Mi-Hua Tao. The 293T, 293T-hACE2, and Vero E6 cells were maintained and grown at 37°C in DMEM  containing 10% FBS  in a 5% COA quantitative GAL4-based mammalian one-hybrid assay was established to assess cell-cell fusion activity. This reporter assay contains two plasmids . One is the reporter construct pGAL4/UAS-TK-Luc, which encodes firefly luciferase under the control of the GAL4 response element (upstream activation sequence [UAS]) and a thymidine kinase (TK) promoter. The other is the transcriptional activator construct pGAL4DBD-hAR-NTD, which consists of aa 1 to 560 of the AR transcriptional activation domain with the GAL4 DNA-binding domain (DBD) fused at its N terminus. The protein encoded by pGAL4DBD-hAR-NTD can bind to the UAS GAL4 response element of the pGAL4/UAS-TK-Luc reporter and activate the transcription of the luciferase reporter gene by the transcriptional activator of AR-NTD.5 293T cells were seeded in 12-well plates, and Vero E6 and 293T-hACE2 cells were seeded in 24-well plates (2 × 105/well) overnight. After 24 h, the 293T cells were cotransfected with the pGAL4DBD-hAR-NTD and D614-spike or G614-spike plasmid. Vero E6 cells and 293T-hACE2 cells were transfected with pGAL4/UAS-TK-Luc and pCMV-Renilla. Twenty-four hours posttransfection, 293T cells expressing the GAL4DBD-hAR-NTD protein and D614 spike or G614 spike protein were suspended in trypsin, and 1 × 105 cells were seeded on Vero E6 or 293T-hACE2 cells expressing the GAL4/UAS-TK-Luc protein. The cells were cocultured for 24 h and harvested with passive lysis buffer (PLB) for the dual-luciferase assay according to the manufacturer’s instructions (Promega).To detect cell-cell fusion activity, 293T-hACE2 and Vero E6 (which endogenously express ACE2) cells transfected with pGAL4/UAS-TK-Luc were prepared as target cells; 293T cells expressing the SARS-CoV-2 spike protein and pGAL4DBD-hAR-NTD were prepared as effector cells. In brief, 4 × 10Western blotting was performed as previously described . In brieAAGCTGGACTTCCCTATGGTGC-3′ and 5′-CCTTGGGTTTGTTCTGGACCACG-3′. The probes of porphobilinogen deaminase (PBGD) used as the internal control in Northern blotting were 5′-GGTGACCAGCACACTTTGGG-3′ and 5′-AGCCGGGTGTTGAGGTTTCC-3′.RNA was extracted with a NucleoSpin RNA kit (Macherey-Nagel) according to the manufacturer’s instructions. Northern blotting was performed as previously described . In brieACAGGTACGTTAATAGTTAATAGCGT-3′ and 5′-ATATTGCAGCAGTACGCACACA-3′. The RNA level of the E gene in the cells was determined relative to the internal control, the cellular PBGD gene, with the primer set 5′-GCATCGCTGAAAGGGCCTTCC-3′ and 5′-TCATCCTCAGGGCCATCTTCATGC-3′.The qRT-PCR was conducted by following the protocol described previously . In briet test. A P value of 0.05 or lower was used to indicate statistical significance .The virus titers quantified by plaque assays in triplicate are shown as means ± standard deviations (SD). Results from the reporter assay are shown as data representative of three independent experiments and are presented as means ± SD. Differences in data from the virus titer, qRT-PCR, and reporter assays between each indicated paired sample group were evaluated by Student's https://www.gisaid.org/CoV2020/). The accession codes are listed in The sequencing data for the 18 virus isolates used in the current study (hCoV-19/Taiwan/NTU01/2020 to hCoV-19/Taiwan/NTU18/2020) have been deposited in GISAID (