Journal: International Journal of Biological Sciences

Article Title: The Different Immune Responses by Age Are due to the Ability of the Fetal Immune System to Secrete Primal Immunoglobulins Responding to Unexperienced Antigens

doi: 10.7150/ijbs.67203

Figure Lengend Snippet: Characterization of NAb-secreting FSCs-derived EVs (NAbs-FSC-EVs). (A) A schematic diagram verifying whether NAbs are contained in NAbs-FSC-EVs secreted from NAbs-FSCs cultured under new ex-vivo culture conditions. (B) The results of NTA on NAbs-FSC-EVs and FSC Control -EVs secreted from FSCs Control cultured on a typical 2D plate. The concentrations, average sizes, and distribution of FSC Control -EVs and NAbs-FSC-EVs are almost similar. There is no significant difference in EV secretion characteristics according to the culture conditions. (C) SEM (left) and TEM (right) images of FSC Control -EVs and NAbs-FSC-EVs, respectively. There are no significant differences in shapes and size distributions in both EVs according to the culture conditions. (D) The results of flow cytometry analysis to compare the expression patterns of exosome markers (CD9, CD63, and CD81) in FSC Control -EVs and NAbs-FSC-EVs. The expression aspects that CD63 and CD81 are expressed at higher levels than CD9 are similar in both EVs. (E) The results of co-expression analysis of exosome markers (CD9, CD63, CD81, and syntenin) in FSC Control -EVs and NAbs-FSC-EVs using ExoView®. They show expression patterns very similar to the flow cytometry results in Figure D, and these common expression patterns appear to be a unique characteristic of FSCs-derived EVs. (F) The results of flow cytometry analysis comparing the presence of IgG and NAbs (IgG3 and IgM) in FSC Control -EVs and NAbs-FSC-EVs. (G) The results of exosomal cargo analysis to compare the content of NAbs in FSC Control -EVs and NAbs-FSC-EVs using ExoView®. Similar to the flow cytometry results in Figure F, they show that NAbs-FSC-EVs contain higher levels of NAbs than FSC Control -EVs. (H) The results of analyzing the patterns of NAbs content in both EVs captured by each exosomal marker using Exoview®. (I) The results of protein antibody microarray analysis comparing relative expression levels of B cell-specific proteins related to the promotion of Ig transcription induced in FSC Control -EVs and NAbs-FSC-EVs. They show that B cell-specific proteins related to the promotion of Ig transcription are induced in NAbs-FSC-EVs at higher levels than FSC Control -EVs. (J) The results of analyzing the concentrations of IgG3 and IgM contained in both EVs obtained from the subculture of FSCs Control and NAbs-FSCs using each ELISA kit. IgG3 and IgM were not detected in all FSC Control -EVs, but the contents of IgG3 and IgM in NAbs-FSC-EVs gradually increased as the subculture progressed. Error bars represent standard deviation. *, P <0.05; **, P<0.01; ***, P<0.001; Student's t-test; N.D., Not Detected.

Article Snippet: The concentration of sHLA-G (shedding HLA-G1 and HLA-G5) was detected by Human HLA-G ELISA Kit (LSBio, Seattle, WA, USA) using MEM-G/9 Ab according to the manufacturer's instruction.

Techniques: Derivative Assay, Cell Culture, Ex Vivo, Control, Flow Cytometry, Expressing, Marker, Microarray, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International Journal of Biological Sciences

Article Title: The Different Immune Responses by Age Are due to the Ability of the Fetal Immune System to Secrete Primal Immunoglobulins Responding to Unexperienced Antigens

doi: 10.7150/ijbs.67203

Figure Lengend Snippet: Comparison of secretion characteristics of cytokines, HLA-G, and anti-viral proteins in FSCs established in various culture conditions. (A) Various FSCs established in different culture conditions to characterize the self-defense mechanism formed by NAbs-FSCs. (B) Cytokine analysis results of EVs from FSCs obtained under each culture condition using human cytokine array. The secretion levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) in FSC Control -EVs, itFSC-EVs, and NAbs-FSC-EVs were similar, but the secretion levels of Th1 cytokines (IFN-γ and IL-2), showing increased expression in asymptomatic infected person to COVID-19, were the highest in NAbs-FSC-EVs. (C) The results of flow cytometry analysis to compare the expression aspects of membrane-bound HLA-G1 and intracellular soluble HLA-G5/G6 in each FSC using 87G (detecting β2m of HLA-G1 and HLA-G5 isoforms) and 2A12 (detecting intron 4 of soluble HLA-G5 and HLA-G6 isoforms) Abs. Similar to the previous study results , all HLA-G expressions were increased in NAbs-FSCs and itFSCs compared to FSCs Control . In addition, the expression of HLA-G in NAbs-FSCs was also higher than in itFSCs due to the effect of increased IFN-γ stimulation. (D) sHLA-G (shedding HLA-G1 and HLA-G5) and HLA-G5/6 concentration analysis results of EVs from FSCs obtained under each culture condition through ELISA analysis using MEM-G/9 (detecting β2m of HLA-G1 and HLA-G5 isoforms) and 5A6G7 (detecting intron 4 of soluble HLA-G5 and HLA-G6 isoforms) Abs, respectively. Similar to the previous study results , all HLA-G concentrations were higher in itFSC-EVs and NAbs-FSC-EVs than in FSC Control -EVs. However, unlike HLA-G5/6 concentrations, sHLA-G concentration was higher in NAbs-FSC-EVs than itFSC-EVs. (E) A comparison of the concentrations of interferon-induced proteins with anti-viral functions in NAbs-FSC-EVs and itFSC-EVs. IFITM3 protein was expressed at a higher level in NAbs-FSC-EVs than itFSC-EVs using SET100 protein antibody microarray, and LY6E protein was contained at a higher level in NAbs-FSC-EVs than itFSC-EVs according to ESI-Q-TOF MS/MS analysis. (F) A comparison of absolute quantification of another anti-viral protein (TRPML2) in NAbs-FSC-EVs and itFSC-EVs using ESI-Q-TOF MS/MS. Error bars represent standard deviation. *, P <0.05; **, P <0.01;***, P<0.001; ****, P<0.0001; Student's t-test.

Article Snippet: The concentration of sHLA-G (shedding HLA-G1 and HLA-G5) was detected by Human HLA-G ELISA Kit (LSBio, Seattle, WA, USA) using MEM-G/9 Ab according to the manufacturer's instruction.

Techniques: Comparison, Control, Expressing, Infection, Flow Cytometry, Membrane, Concentration Assay, Enzyme-linked Immunosorbent Assay, Microarray, Tandem Mass Spectroscopy, Quantitative Proteomics, Standard Deviation

Journal: Cancer Science

Article Title: Adoptive cell therapy using tumor‐infiltrating lymphocytes for melanoma refractory to immune‐checkpoint inhibitors

doi: 10.1111/cas.15009

Figure Lengend Snippet: Total mutation burden, transcriptome signature (ssGSEA score), and quantitative real‐time polymerase chain reaction analysis of pretreatment melanoma cells and expanded TILs. A, To evaluate their mutation status, exome sequencing was performed on the melanoma cells used for TIL manufacturing. The numbers of mutations and the mutated genes in each tumor are shown in the upper columns. Transcriptome signatures based on single‐sample gene set enrichment analysis (ssGSEA) of the tumor cells are shown in the lower columns. B, ssGSEA scores of gene sets related to tumor phenotype (based on microarray data) in the tumors of the three melanoma patients. The cut‐off score was set at 4,000. C, D, Quantitative RT‐PCR analysis of the expression levels of chemokines in the primary tumors (C) and of cytotoxic factors in the expanded TIL products (D). The results are fold‐changes in gene expression normalized to the endogenous reference gene; error bars are standard deviations

Article Snippet: Total RNA from isolated melanoma cells was subjected to microarray analysis using an Agilent SurePrint G3 Human GE v3 8x60K Microarray (DNA Chip Research Inc.).

Techniques: Mutagenesis, Real-time Polymerase Chain Reaction, Sequencing, Microarray, Quantitative RT-PCR, Expressing

Journal: PLoS ONE

Article Title: Adenomatous polyposis coli-binding protein end-binding 1 promotes hepatocellular carcinoma growth and metastasis

doi: 10.1371/journal.pone.0239462

Figure Lengend Snippet: (a and b) RNA microarray analysis to identify genes differentially expressed in the presence of EB1. Comparison of gene expression levels in EB1-KO HuH7 cells infected with control and EB1-expressing lentiviruses identified a number of genes upregulated by EB1 re-expression.

Article Snippet: After properly converting the extracted RNA to the cRNA labeled with Cy3 as recommended by the manufacturers, 0.6 μg of the cRNA was fragmented and hybridized at 65°C for 17 h to an Agilent SurePrint G3 Human GE v3 8x60K Microarray (Design ID: 072363) containing a total of 58,201 probes excluding control probes.

Techniques: Microarray, Expressing, Infection

Journal: Life

Article Title: NSD1 Mutations in Sotos Syndrome Induce Differential Expression of Long Noncoding RNAs, miR646 and Genes Controlling the G2/M Checkpoint

doi: 10.3390/life12070988

Figure Lengend Snippet: Real-time PCR validation of differentially expressed genes. ( a ) Quantitative expression measurements of five noncoding RNAs (i.e., lnc-C15orf57 , MIR646 , lnc-C2orf84-1 , Inc-C00665 and lnc-C20orf197-3 ) and ( b ) seven mRNAs (i.e., NOS3 , ZNF883 , CD19 , SFN , GSC , NDRG2 and SORBS1 ). The fold change represents the average difference in expression level of the respective genes between the SoS fibroblasts and the controls. The black bars depict the fold changes detected with the microarray, whereas the white bars show the average fold change with respect to the triplicate real-time PCR assessments. The fold changes indicating a downregulated expression are represented with negative values. The error bars represent the standard error of the mean. Significant levels were at p < 0.05. ( c ) Compared to healthy controls, five noncoding RNAs ( lin-C005, Inc-C20orf97 , MIR646HG , lnc-C15orf57 and Inc-C20orf84-1 ) and ( d ) seven mRNAs ( NOS3 , ZNF883 , CD19 , SFN , NDRG2 , SORBS1 and GSC ) were selected. The black bars depict healthy control (Ctr), and the white bars represent the Sotos syndrome patients (SoS pts). The results were consistent with the findings obtained from the microarray analysis. Data are presented as the mean ± standard deviation. * p < 0.05, ** p < 0.01.

Article Snippet: Briefly, cyanine-3-CTP-labeled cRNA was hybridized onto SurePrint G3 microarray chips (Design ID: G4851C), which contained 50,599 probes for 32,776 human mRNAs and 17,438 human lncRNAs; these were derived from authoritative databases, including RefSeq, Ensemble, GenBank and the Broad Institute.

Techniques: Real-time Polymerase Chain Reaction, Biomarker Discovery, Expressing, Microarray, Control, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Identification of potential biomarkers and immune infiltration characteristics in recurrent implantation failure using bioinformatics analysis

doi: 10.3389/fimmu.2023.992765

Figure Lengend Snippet: Baseline Characteristics.

Article Snippet: Techniques , GPL16043 platform , Agilent-039494 SurePrint G3 Human GE v2 8x60K Microarray 039381 , GPL15789 platform , 10X Genomics Chromium platform.

Techniques: Microarray, Control