Journal: Microbiology Spectrum

Article Title: Immunological and Pathological Peculiarity of Severe Acute Respiratory Syndrome Coronavirus 2 Beta Variant

doi: 10.1128/spectrum.02371-22

Figure Lengend Snippet: Comparison of in vitro infectivities and in vivo lethalities of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) wild-type (WT) strain and beta (β) variant. (a) Schematic of SARS-CoV-2 genome. Mutations in the β variant and WT strain are shown. (b) Plaque sizes. Vero E6 cells were infected with serial dilutions (dil.) of the SARS-CoV-2 WT strain or β variant. Plaque areas were calculated. The mean plaque size per well is shown on the left. Representative plaques are shown on the right. Bars represent means ± SDs from three independent experiments. * * , P < 0.01 (Student’s t test). (c to f) Eight-week-old male K18-hACE2 mice (six mice per group) were infected intranasally with 2 × 10 3 PFU of the WT strain or β variant of SARS-CoV-2. (c) Body weight. (d) Survival rate. (e and f) Individual clinical scores. Clinical status was scored according to the following grading scale: 0, healthy; 1, ruffled fur but active; 2, ruffled fur with reduced activity; 3, ruffled fur, inactive, and hunched; 4, imminent death; and 5, dead. Body weight symbols represent means ± SEM. * , P < 0.05; * * , P < 0.01; *** * , P < 0.0001 (two-way analysis of variance with Tukey’s multiple-comparison test). (g) Starting day of weight loss postinfection. (h) Day of symptom onset postinfection. (i) Number of days from the start of weight loss to death. (j) Number of days from symptom onset to death. * * , P < 0.01; *** * , P < 0.0001 (Student’s t test).

Article Snippet: Sections were incubated at 4°C overnight with SARS-CoV/SARS-CoV-2 nucleocapsid (no. 40143-R001; Sino Biological) or anti-neutrophil marker antibody (no. sc-71674; Cell Signaling Technology).

Techniques: In Vitro, In Vivo, Variant Assay, Infection, Activity Assay

Journal: Microbiology Spectrum

Article Title: Immunological and Pathological Peculiarity of Severe Acute Respiratory Syndrome Coronavirus 2 Beta Variant

doi: 10.1128/spectrum.02371-22

Figure Lengend Snippet: Viral load and pathology in various tissues of K18-hACE2 mice after SARS-CoV-2 infection. (a) Eight-week-old male K18-hACE2 mice (five mice per group) were infected intranasally with 2 × 10 3 PFU of SARS-CoV-2 WT (blue symbols) or β variant (B.1.351) (red symbols). Body weight was monitored. Mice were sacrificed at 4 and 6 dpi. Serum and tissue samples were collected. Viral load, pathology, multiple-organ damage, and cytokine and transcriptome profiles were analyzed. Uninfected mice (−) (black symbols) were used as controls. (b) Number of infectious virus particles in the lung and (c) mean plaque size per well. Bars represent means ± SD from two independent experiments. * , P < 0.05; * * , P < 0.01; *** * , P < 0.0001 (WT strain or β variant versus mock [two-way analysis of variance [ANOVA] with Sidak’s multiple-comparison test]; WT strain versus β variant [Student’s t test]). (d) Viral RNA levels by RT-qPCR in the lungs, brain, spleen, kidneys, and heart from two independent experiments. * , P < 0.05; * * , P < 0.01; ** * , P < 0.001; *** * , P < 0.0001 (WT strain or β variant over time [two-way ANOVA with Bonferroni correction]; WT strain versus β variant [Student’s t test]). ND, not detected. (e and f) Histopathological examination of the lungs by hematoxylin and eosin staining (four mice per group). (e) Representative images of low-power (left) (scale bars, 200 μm) and high-power (right) magnification (scale bars, 100 μm). (f) Histopathological scores. Bars present means ± SD from four independent experiments. (g to i) Multiple-organ damage evaluated by measuring serum markers in WT- and β variant-infected mice at 6 dpi. (g) Alanine aminotransferase (ALT). (h) Aspartate aminotransferase (AST). (i) Blood urea nitrogen (BUN). * , P < 0.05; * * , P < 0.01; ** * , P < 0.001; *** * , P < 0.0001. NS, not significant. (One-way ANOVA with Tukey’s multiple-comparison test was performed.)

Article Snippet: Sections were incubated at 4°C overnight with SARS-CoV/SARS-CoV-2 nucleocapsid (no. 40143-R001; Sino Biological) or anti-neutrophil marker antibody (no. sc-71674; Cell Signaling Technology).

Techniques: Infection, Variant Assay, Quantitative RT-PCR, Staining

Journal: Microbiology Spectrum

Article Title: Immunological and Pathological Peculiarity of Severe Acute Respiratory Syndrome Coronavirus 2 Beta Variant

doi: 10.1128/spectrum.02371-22

Figure Lengend Snippet: Chemokine and cytokine profiles after SARS-CoV-2 WT or β variant infection. Cytokine levels in the lungs (a and d), brain (b and e), and serum (c and f) from mice infected with SARS-CoV-2 WT (blue symbols) or β variant (red symbols) (five mice per group; two independent experiments) are shown. Uninfected mice (−) (black symbols) were used as controls. For serum analysis, samples collected before infection (pre) were used as controls. Sixteen cytokines were analyzed using a multiplex cytokine assay. The fold change of each cytokine was calculated compared to uninfected controls and plotted in heat maps (a to c) or on graphs (d to f). CXCL2 levels were additionally measured in the lungs by enzyme-linked immunosorbent assay. (a to c) Relative expression levels of chemokines, proinflammatory cytokines, and anti-inflammatory cytokines. (d to f) Relative expression levels of early-response (CCL2/3, CXCL1/10, IL-6, and TNF-α) and late-response (IL-1β/2/10 and IFN-γ) cytokines (see Fig. S4a to d for the actual expression level of each cytokine and statistics).

Article Snippet: Sections were incubated at 4°C overnight with SARS-CoV/SARS-CoV-2 nucleocapsid (no. 40143-R001; Sino Biological) or anti-neutrophil marker antibody (no. sc-71674; Cell Signaling Technology).

Techniques: Variant Assay, Infection, Multiplex Assay, Cytokine Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Microbiology Spectrum

Article Title: Immunological and Pathological Peculiarity of Severe Acute Respiratory Syndrome Coronavirus 2 Beta Variant

doi: 10.1128/spectrum.02371-22

Figure Lengend Snippet: Transcriptome profiling of the lungs of K18-hACE2 mice infected with SARS-CoV-2 WT strain or β variant. (a to d) Whole-transcriptome profiling of lung homogenates was performed by microarray analysis (five mice per group). Uninfected mice were used as controls. (a) Volcano plots of differentially expressed genes (DEGs) in the lungs of virus-infected mice versus uninfected mice at 4 and 6 dpi. Up- and downregulated genes are represented by dark yellow and blue dots, respectively (fold change ≥ 3 [blue vertical dashed lines]; P < 0.05 [red horizontal dashed lines]). (b) Venn diagrams showing the overlap of DEGs between different time points or virus strains compared to uninfected controls. Numbers in parentheses indicate the total number of DEGs. (c) Heat map of significantly upregulated genes annotated with the Gene Ontology (GO) term “positive regulation of cytokine production” (GO:0001819). (d) Heat map of genes involved in inflammasome activation. (e and f) Western blot analysis of the indicated proteins in the lungs of K18-hACE2 mice infected with the β variant of SARS-CoV-2. (e) Representative images (three mice per group). The full-length intact form of CXCR2 is indicated by the arrow. (f) The ratio of intact CXCR2 to β-actin was determined by densitometric analysis. * * , P < 0.01 (one-way analysis of variance with Tukey’s multiple-comparison test). (g to i) Neutrophil infiltration into lung lesions. Immunohistochemistry was performed on lung sections from K18-hACE2 mice infected with the WT strain or β variant of SARS-CoV-2 at 6 dpi using an anti-SARS-CoV-2 nucleocapsid (N) protein antibody (left) and a neutrophil marker (right). (g) Representative images (five mice per group) (scale bars, 100 μm). (h and i) SARS-CoV-2 N protein expression (h) and neutrophil infiltration (i) relative to the total lesion area for each of 20 acquired images per group. ** * , P < 0.001; *** * , P < 0.0001 (one-way analysis of variance with Tukey’s multiple-comparison test). (j) Schematic of the NLRP3 inflammasome signaling pathway.

Article Snippet: Sections were incubated at 4°C overnight with SARS-CoV/SARS-CoV-2 nucleocapsid (no. 40143-R001; Sino Biological) or anti-neutrophil marker antibody (no. sc-71674; Cell Signaling Technology).

Techniques: Infection, Variant Assay, Microarray, Activation Assay, Western Blot, Immunohistochemistry, Marker, Expressing

Journal: Microbiology Spectrum

Article Title: Immunological and Pathological Peculiarity of Severe Acute Respiratory Syndrome Coronavirus 2 Beta Variant

doi: 10.1128/spectrum.02371-22

Figure Lengend Snippet: Delayed virus-induced death of K18-hACE2 mice by proinflammatory cytokine blockade. (a) Eight-week-old male K18-hACE2 mice (five mice per group) were infected intranasally (IN) with 2 × 10 3 PFU of SARS-CoV-2 β variant. Blocking antibodies (Ab) against CCL2 (5 mg/kg) were administered intraperitoneally (IP) at 1, 3, and 6 dpi. An isotype Ab was used as a negative control. (b) Body weight. (c) Survival rate. Body weight symbols represent means ± SEM. * , P < 0.05; * * , P < 0.01 (two-way analysis of variance with Tukey’s multiple-comparison test [body weight]; log-rank [Mantel-Cox] test [survival rate]).

Article Snippet: Sections were incubated at 4°C overnight with SARS-CoV/SARS-CoV-2 nucleocapsid (no. 40143-R001; Sino Biological) or anti-neutrophil marker antibody (no. sc-71674; Cell Signaling Technology).

Techniques: Infection, Variant Assay, Blocking Assay, Negative Control

Journal: Human Cell

Article Title: Hsa_circ_0000437 promotes the progression of rheumatic valvular heart disease by activating the mitogen-activated protein kinase signaling pathways after sponging let-7f-5p and targeting RAS-like proto-oncogene B

doi: 10.1007/s13577-025-01331-7

Figure Lengend Snippet: hsa_circ_0000437/let-7f-5p/RALB axis promotes inflammatory transformation during RVHD progression by activating the MAPK signaling pathway. Western blotting assay of MAPK14, ERK, JNK, and RALB proteins in hVICs cells transfected with si-hsa_circ_0000437 ( A ), pcDNA3.1-hsa_circ_0000437 ( B ), let-7f-5p mimic and pcDNA3.1-hsa_circ_0000437 ( C ), si-RALB, pcDNA3.1-hsa_circ_0000437 and let-7f-5p inhibitor ( D ). Numbers indicate the relative expression of proteins, and GAPDH serves as an internal reference. E Diagram of the regulatory mechanism of hsa_circ_0000437/let-7f-5p/RALB axis promoting the occurrence and development of RVHD, the target hsa_circ_0000437 was identified by microarray and confirmed in the cytoplasm that RALB could promote inflammatory transformation during RVHD by activating MAPK signaling pathway. RALB was also inhibited by let-7f-5p and enhanced by ceRNA hsa_circ_0000437 (some elements were created using BioRender.com). n = 3, n stands for biological replicates

Article Snippet: MAPK14 (Cat No. 14064–1-AP), extracellular regulated protein kinases (ERK) (Cat No. 11257–1-AP), c-Jun N-terminal kinase (JNK) (Cat No. 51153–1-AP), RAS-like proto-oncogene B (RALB) (Cat No. 12340–1-AP), and goat anti-rabbit IgG and goat anti-mouse IgG secondary antibody were purchased from a subsidiary of Proteintech Wuhan Sanying Biotechnology Co., LTD (China).

Techniques: Transformation Assay, Western Blot, Transfection, Expressing, Microarray

Journal: Frontiers in Pharmacology

Article Title: A senescence-associated signature refines the classification of different modification patterns and characterization of tumor immune microenvironment infiltration in triple-negative breast cancer

doi: 10.3389/fphar.2023.1191910

Figure Lengend Snippet: The hub gene FAM3b and its prognostic value in TNBC. (A) Number of trees showing the importance proportion of SASP regulator genes. (B) FAM3B expression in paired tumor and normal tissues in BRCA from the TCGA database. (C) FAM3B expression according to the molecular subtypes of breast cancer. (D) UMAP plot of intratumoral immune cells and FAM3B showing the correlation between infiltration of different immune cells and FAM3B expression in the Alex, EMTAB8107, GSE150660 and GSE161529 datasets. (E,F) The impact of FAM3B on OS and DFS in breast cancer tissue microarray using Kaplan-Meier analysis.

Article Snippet: IHC staining of FAM3B protein expression in the tissue microarray was performed by incubation with rabbit polyclonal antibodies against human FAM3B antibody (27131-1-AP, Proteintech, 1:200) overnight, followed by incubation with goat monoclonal antibody against rabbit antibody (111-035-003, JACKSON, 1:1,000) for 1 h at room temperature.

Techniques: Expressing, Microarray

Journal: Stem Cell Research & Therapy

Article Title: Oxidant therapy improves adipogenic differentiation of adipose-derived stem cells in human wound healing

doi: 10.1186/s13287-021-02336-3

Figure Lengend Snippet: Characterization of the macrophage activation model. a THP1 cell treatment protocol for in vitro polarization of macrophages. b Morphology (phase contrast, scale bar 100μm) of THP1-derived macrophages on day of CM harvest. c Heatmap of log2 cytokine protein levels in differentially activated macrophages. The dendrogram represents the result from a complete linkage hierarchical clustering of mean-centered expression levels based on Euclidian similarity. d mRNA-expression levels of macrophage-selective markers (M (IFNG/LPS) : CD80, IL1B, and M (IL4/IL13) : CD200R, TGM2). Data were normalized to the 18S rRNA reference gene and shown as mean –ΔCt. Asterisks indicate p values < 0.05 (*), p <0.01 (**), and p <0.001 (***). Statistical significance ( n =5) was determined by using one-way ANOVA and Dunnett’s test for multiple comparison

Article Snippet: Macrophage-CM was loaded onto RayBio® Human Cytokine G5 Antibody Microarray Glass Chip (RayBiotech, Norcross, GA) which facilitates the detection of 80 targets.

Techniques: Activation Assay, In Vitro, Derivative Assay, Expressing

Journal: Mediators of Inflammation

Article Title: LR12 Promotes Liver Repair by Improving the Resolution of Inflammation and Liver Regeneration in Mice with Thioacetamide- (TAA-) Induced Acute Liver Failure

doi: 10.1155/2021/2327721

Figure Lengend Snippet: LR12 promoted hepatocyte regeneration via CCL20 secreted by macrophages. (a) The cytokine protein microarray of the supernatant from macrophages. (b) ELISA of CCL20 in the supernatant from macrophages. (c) The mRNA level of CCL20 in macrophages. (d) CLSM showing F4/80 and CCL20 staining in the liver tissues. (e) Western blot analysis of PCNA in LO2 cells stimulated with CCL20. Data were presented as the mean ± standard deviation (SD). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 versus control group.

Article Snippet: The RayBio Human Cytokine Antibody Array Kit (QAH-TH17-1-1 microarray; RayBiotech, Norcross, GA, USA) was utilized to identify the cytokines based on instructions provided by the manufacturer.

Techniques: Microarray, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Standard Deviation

Journal: Cell Death & Disease

Article Title: Single-cell sequencing resolves the landscape of immune cells and regulatory mechanisms in HIV-infected immune non-responders

doi: 10.1038/s41419-022-05225-6

Figure Lengend Snippet: A Heatmap of significantly expressed cytokine between INRs and IRs using cytokine antibody microarray. B , C , D , and E Concentrations (pg/ml) of IL-4, IL-7, IL-15, and MCP-1 in the plasma samples obtained from INR and IR patients. F GO and KEGG analyses were conducted using DAVID for upregulated cytokine in plasma from INRs compared with IRs.

Article Snippet: The AAH-BLG-507 cytokine antibody microarray (Raybiotech, Norcross, GA, USA) was used to detect cytokines in plasma samples derived from IR and INR subjects.

Techniques: Microarray