Journal: Journal of Virology

Article Title: Pathogenicity and landscape of differential gene expression in mice orally infected with clinical coxsackievirus A6 (CA6)

doi: 10.1128/jvi.01358-23

Figure Lengend Snippet: Clinical data of CA6-infected mice via intragastric gavage. (A) Clinical scores. (B) Body weight changes. (C) Survival rates. Viral load at different tissues was determined by qRT-PCR at 1, 3, 5, and 7 dpi, respectively (D–Q). (D) Blood. (E) Skeletal muscle. (F) Heart. (G) Lung. (H) Liver. (I) Stomach. (J) Small intestine. (K) Large intestine. (L) Brain. (M) Brain stem. (N) Spinal cord. (O) Thymus. (P) Spleen. (Q) Comparison on the viral loads of each tissue to skeletal muscle at each timepoints, and * represents a significant difference in viral load of each tissue compared to muscle. Error bars show the SEM. n = 6 at each timepoint. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. The dashed line represents the detection limit. Clinical scores were determined as follows: 0, healthy; 1, ruffled fur and hunchback appearance; 2, wasting and limb weakness; 3, single limb paralysis; 4, paralysis of both limbs; 5, moribund and death.

Article Snippet: In this experiment, clinical isolates of CA6 (ATCC VR-1801) were used and stored.

Techniques: Infection, Quantitative RT-PCR, Comparison

Journal: Journal of Virology

Article Title: Pathogenicity and landscape of differential gene expression in mice orally infected with clinical coxsackievirus A6 (CA6)

doi: 10.1128/jvi.01358-23

Figure Lengend Snippet: Fluctuations of neutrophils and monocytes in peripheral blood of mice after CA6 infection. Typical FCS results were represented (A),and statistical charts for monocytes (B)and neutrophils (C)were shown. Error bars show the SEM. n = 6 at each timepoints. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: In this experiment, clinical isolates of CA6 (ATCC VR-1801) were used and stored.

Techniques: Infection

Journal: Journal of Virology

Article Title: Pathogenicity and landscape of differential gene expression in mice orally infected with clinical coxsackievirus A6 (CA6)

doi: 10.1128/jvi.01358-23

Figure Lengend Snippet: Changes in inflammatory factors and dynamic of gene expression profiles in peripheral blood of mice after CA6 infection. (A) Proliferation of inflammatory factors (n = 6). (B) Venn diagram of common differentially expressed genes (DEGs) at different timepoints. (C) GO analysis of common DEGs (B). (D) KEGG analysis of common DEGs (B). (E and F) Venn diagram of commonly upregulated (E) and downregulated (F) DEGs. (G) Fuzzy C-means clustering of DEGs in blood at 3, 5, and 7 dpi. The value of B_ vs _NC is set to 1. Error bars show the SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: In this experiment, clinical isolates of CA6 (ATCC VR-1801) were used and stored.

Techniques: Gene Expression, Infection

Journal: Journal of Virology

Article Title: Pathogenicity and landscape of differential gene expression in mice orally infected with clinical coxsackievirus A6 (CA6)

doi: 10.1128/jvi.01358-23

Figure Lengend Snippet: Pathological changes and dynamic of gene expression profiles in mouse muscles after CA6 infection. (A) Pathological changes of muscles. The arrows represent typical lesion sites, 200×. (B) Histopathological scores of muscle tissues. (C) Venn diagram of common DEGs at different timepoints. (D) GO analysis of common DEGs (C). (E) KEGG analysis of common DEGs (C). (F–G) Venn diagram of commonly upregulated (F) and downregulated (G) DEGs. (H) Fuzzy C-means clustering of muscles at 3, 5, and 7 dpi, respectively. The value of M_vs_NC is set to 1. Histopathological scores were determined as follows: 0, normal (normal muscle fibers without inflammation); 1, minimal (occasionally visible inflammatory cell infiltration); 2, mild (scattered or focal inflammatory cell infiltration); 3, moderate (diffuse inflammatory cell infiltration and mild muscle fiber degeneration or necrosis); 4, severe (severe inflammatory cell infiltration and muscle fiber degeneration and necrosis). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: In this experiment, clinical isolates of CA6 (ATCC VR-1801) were used and stored.

Techniques: Gene Expression, Muscles, Infection

Journal: Journal of Virology

Article Title: Pathogenicity and landscape of differential gene expression in mice orally infected with clinical coxsackievirus A6 (CA6)

doi: 10.1128/jvi.01358-23

Figure Lengend Snippet: Pathological changes and dynamic of gene expression profiles in mouse brain after CA6 infection. (A) Pathological changes in brain. 200×. (B) Venn diagram of common DEGs at different timepoints. (C) GO analysis of common DEGs (B). (D) KEGG analysis of common DEGs (B). (E and F) Venn diagram of commonly upregulated (E) and downregulated (F) DEGs. (G) Fuzzy C-means clustering of brain at 3, 5, and 7 dpi, respectively. The value of Br_vs_NC is set to 1.

Article Snippet: In this experiment, clinical isolates of CA6 (ATCC VR-1801) were used and stored.

Techniques: Gene Expression, Infection

Journal: Journal of Virology

Article Title: Pathogenicity and landscape of differential gene expression in mice orally infected with clinical coxsackievirus A6 (CA6)

doi: 10.1128/jvi.01358-23

Figure Lengend Snippet: Dynamic of gene expression profiles in mouse spleen and thymus after CA6 infection. (A) Venn diagram of common DEGs at different timepoints. (B) GO analysis of common DEGs (A). (C) KEGG analysis of common DEGs (A). (D and E) Venn diagram of commonly upregulated (D) and downregulated DEGs (E). (F) Fuzzy C-means clustering of spleen at 3, 5, and 7 dpi, respectively. (G) Venn diagram of common DEGs at different timepoints. (H) GO analysis of common DEGs (G). (I) KEGG analysis of common DEGs (G). (J and K) Venn diagram of commonly upregulated (J) and downregulated DEGs (K). (L) Fuzzy C-means clustering of thymus at 3, 5, and 7 dpi, respectively. The values of S_vs_NC and T_vs_NC are set to 1.

Article Snippet: In this experiment, clinical isolates of CA6 (ATCC VR-1801) were used and stored.

Techniques: Gene Expression, Infection

Journal: Journal of Virology

Article Title: Pathogenicity and landscape of differential gene expression in mice orally infected with clinical coxsackievirus A6 (CA6)

doi: 10.1128/jvi.01358-23

Figure Lengend Snippet: Pathological changes in the heart, lungs, stomach, large intestine, small intestine, liver, and spinal cord of mice infected with CA6 virus at 1, 3, 5, and 7 dpi. 200×. The arrows represent typical lesion sites.

Article Snippet: In this experiment, clinical isolates of CA6 (ATCC VR-1801) were used and stored.

Techniques: Infection, Virus

Journal: Nature Communications

Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6

doi: 10.1038/s41467-020-15334-5

Figure Lengend Snippet: a , b Overall structure of EiCsm6 homodimer bound to cFA6, shown in two orientations ( a : side view of the EiCsm6 homodimer; b : view of the cFA6 binding site at the CARF domain interface). The EiCsm6 homodimer is shown in cartoon representation (with protomers colored light and dark blue). cFA6 is depicted in sphere format. c Zoom-in view of the cA6 binding site at the CARF domain interface of EiCsm6, showing a 2m F O –D F C composite omit map, contoured at 1.0 σ and displayed within a radius of 2.2 Å around cFA6.

Article Snippet: Briefly, cA6, cFA6 (both synthesized by Biolog Life Science Institute GmbH & Co. KG), or supernatants from cA6 degradation reactions were incubated with EiCsm6 or its mutants in 10 mM HEPES pH 7.5 and 50 mM KCl in a total volume of 100 μl.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6

doi: 10.1038/s41467-020-15334-5

Figure Lengend Snippet: a – c Protein-cFA6 interactions. EiCsm6 is depicted in cartoon representation; cFA6 is shown as sticks. Hydrogen-bonding interactions are represented as black dotted lines. d Fluorogenic RNase activity assay of CARF domain mutants of EiCsm6 (0.5 nM) in the presence of 10 nM cyclic-hexa-AMP (cA6). AU, arbitrary units. e Growth curves (optical density at a wavelength of 600 nm) of Staphylococcus aureus strains harboring pTarget and pCRISPR plasmids. The pCRISPR plasmid expresses wild-type or CARF domain mutants of EiCsm6 and the Staphylococcus epidermidis Cas10–Csm effector complex containing an inactivating point mutation in the HD domain of Cas10. Data points in d , e represent the mean of three replicates; error bars represent the standard error of the mean (s.e.m.). Source data are provided as a Source Data file.

Article Snippet: Briefly, cA6, cFA6 (both synthesized by Biolog Life Science Institute GmbH & Co. KG), or supernatants from cA6 degradation reactions were incubated with EiCsm6 or its mutants in 10 mM HEPES pH 7.5 and 50 mM KCl in a total volume of 100 μl.

Techniques: Activity Assay, Plasmid Preparation, Mutagenesis

Journal: Nature Communications

Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6

doi: 10.1038/s41467-020-15334-5

Figure Lengend Snippet: a Zoom-in view of nucleotides A1 and A6 of cFA6 bound to the EiCsm6 CARF domain. Residues implicated in the cleavage are represented as sticks; hydrogen-bonding interactions are depicted as black dotted lines. The red arrows represent the proposed reaction mechanism of cA6 cleavage involving nucleophilic attack of the 2’-hydroxyl group of A6 onto the phosphodiester group connecting A6 and A1. b RNase activity assay comparing the activity of WT and T11A EiCsm6 proteins in the presence of 500 nM cA6. The black arrows represent spike-ins of 0.8 pmol of RNaseAlert substrate at 5 min intervals. AU, arbitrary units. c Growth curves (optical density at a wavelength of 600 nm) of Staphylococcus aureus strains harboring pTarget and pCRISPR plasmids. The pCRISPR plasmid expresses wild-type or CARF domain mutants of EiCsm6 and the Staphylococcus epidermidis Cas10–Csm effector complex containing an inactivating point mutation in the Csm3 subunit (D32A; dCsm3). Data points in b , c represent the mean of three replicates; error bars represent the standard error of the mean (s.e.m.). Source data are provided as a Source Data file.

Article Snippet: Briefly, cA6, cFA6 (both synthesized by Biolog Life Science Institute GmbH & Co. KG), or supernatants from cA6 degradation reactions were incubated with EiCsm6 or its mutants in 10 mM HEPES pH 7.5 and 50 mM KCl in a total volume of 100 μl.

Techniques: Activity Assay, Plasmid Preparation, Mutagenesis

Journal: Nature Communications

Article Title: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6

doi: 10.1038/s41467-020-15334-5

Figure Lengend Snippet: a Zoom-in view of the HEPN domain ribonuclease active site in EiCsm6. The conserved R372, N373, H377 residues of the HEPN catalytic motif, and additionally R274, are represented as sticks. Electron density, interpreted as a sulfate ion (depicted as 2m F O –D F C composite omit map, contoured at 1.0 σ and displayed within a radius of 2.2 Å), is located within hydrogen bonding distance of H377, likely mimicking the scissile phosphate group of an RNA substrate. b Schematic model of the cA6-mediated allosteric activation mechanism of Csm6 enzymes.

Article Snippet: Briefly, cA6, cFA6 (both synthesized by Biolog Life Science Institute GmbH & Co. KG), or supernatants from cA6 degradation reactions were incubated with EiCsm6 or its mutants in 10 mM HEPES pH 7.5 and 50 mM KCl in a total volume of 100 μl.

Techniques: Activation Assay

Journal: Journal of Virology

Article Title: Platelet factor 4-derived C15 peptide broadly inhibits enteroviruses by disrupting viral attachment

doi: 10.1128/jvi.01859-24

Figure Lengend Snippet: PF4 inhibits the replication of CA6 and EVD68. ( A and B ) HEK-293T cells transfected with cDNA-PF4-Myc or the VR1012 expression vector serving as a control. After 12 h, the cells were infected with CA6 (MOI = 0.5) or EVD68 (MOI = 1). At the indicated time points, viral loads in both the cells and the supernatant (concentrated 100-fold) were assessed using Western blot analysis with an anti-VP1 antibody, while tubulin was used as a loading control. ( C and D ) Viral loads in the supernatant were evaluated by measuring VP1 gene mRNA levels using reverse transcription quantitative PCR (RT-qPCR). ( E and H ) PF4-His proteins expressed in Escherichia coli were added to the cell supernatant at the indicated concentration, and the cells were simultaneously infected with CA6 or EVD68. Cell lysates and supernatants (concentrated 100-fold) were collected at 48 h. Western blot was performed to detect the viral VP1 protein in the cells and supernatant, with tubulin serving as a loading control. ( F and I ) Intracellular mRNA levels of VP1 were determined by RT-qPCR, using GAPDH as a control. ( G and J ) 50% tissue culture infective dose (TCID 50 ) assays were performed using supernatants collected from infected rhabdomyosarcoma cells at 48 h, containing various concentrations of PF4-His. All data are derived from three independent experiments and are presented as mean ± SD. Statistical significance was determined using one-way analysis of variance. *** P < 0.001.

Article Snippet: The following antibodies were used in this study: EV71-VP1 (GeneTex, catalog #GTX132339); CA6-VP1 (GeneTex, catalog #GTX132346); and EVD68-VP1 (GeneTex, catalog #GTX132313); antibodies to CA16-VP1 are laboratory-made, and the specific methods were described previously ( ); PF4 antibody (Proteintech, #21157–1-AP); anti-HA polyclonal Ab (Invitrogen, catalog #71–5500); and anti-Myc (Proteintech, #16286–1-AP).

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Infection, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Concentration Assay, Derivative Assay

Journal: Journal of Virology

Article Title: Platelet factor 4-derived C15 peptide broadly inhibits enteroviruses by disrupting viral attachment

doi: 10.1128/jvi.01859-24

Figure Lengend Snippet: PF4 lacking 15 amino acids at the C-terminus loses its anti-viral activity. ( A ) Schematic representation of the PF4 gene. ( B ) Prediction of the three-dimensional (3D) structure of the PF4 protein using AlphaFold2. ( C and D ) HEK-293T cells were transfected with varying concentrations of cDNA-PF4-Myc, with the VR1012 expression vector as a control. Twelve hours later, the cells were infected with CA6 or EVD68. Western blot analysis was performed to detect the expression levels of various proteins using the corresponding antibodies. ( E and F ) RT-qPCR was used to measure the mRNA levels of VP1 of CA6 or EVD68 in cells transfected with different concentrations of cDNA-PF4-Myc. GAPDH was used as a normalization control. ( G and H ) TCID 50 assays were performed using supernatants collected from infected rhabdomyosarcoma cells at 48 h, containing various concentrations of PF4-Myc. All data are derived from three independent experiments and are presented as mean ± SD. Statistical significance was determined using one-way analysis of variance. ** P < 0.001. ns, no significance.

Article Snippet: The following antibodies were used in this study: EV71-VP1 (GeneTex, catalog #GTX132339); CA6-VP1 (GeneTex, catalog #GTX132346); and EVD68-VP1 (GeneTex, catalog #GTX132313); antibodies to CA16-VP1 are laboratory-made, and the specific methods were described previously ( ); PF4 antibody (Proteintech, #21157–1-AP); anti-HA polyclonal Ab (Invitrogen, catalog #71–5500); and anti-Myc (Proteintech, #16286–1-AP).

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Control, Infection, Western Blot, Quantitative RT-PCR, Derivative Assay

Journal: Journal of Virology

Article Title: Platelet factor 4-derived C15 peptide broadly inhibits enteroviruses by disrupting viral attachment

doi: 10.1128/jvi.01859-24

Figure Lengend Snippet: C15 inhibits the replication of multiple EVs. ( A ) Cell viability of rhabdomyosarcoma (RD) cells after 48 h treatment with different concentrations of C15 was assessed using the Cell Counting Kit-8 assay ( n = 3). ( B, E, H, and K ) RD cells were infected with indicated enterovirus (at various MOIs) and treated with different concentrations of C15 for 48 h. Western blot analysis was conducted to detect the VP1 protein levels in cell lysates and supernatants (concentrated 100-fold), with tubulin used as a loading control. ( C, F, I, and L ) Intracellular mRNA levels of the VP1 gene from different enterovirus strains were quantified by RT-qPCR, with GAPDH as a normalization control. All data are from three independent experiments and are presented as mean ± SD. The EC 50 values were determined using GraphPad Prism version 9 with a variable slope (four parameters). ( D, G, J, and M ) TCID 50 assays were performed using supernatants collected from infected RD cells at 48 h, containing various concentrations of C15. All data are from three independent experiments and are presented as mean ± SD. Statistical significance was assessed using one-way analysis of variance. *** P < 0.001. ( N, O, and P ) Intracellular mRNA levels of different enterovirus strains were quantified by RT-qPCR, with GAPDH as a normalization control. All data are from three independent experiments and are presented as mean ± SD. The EC 50 values were determined using GraphPad Prism version 9 with a variable slope (four parameters).

Article Snippet: The following antibodies were used in this study: EV71-VP1 (GeneTex, catalog #GTX132339); CA6-VP1 (GeneTex, catalog #GTX132346); and EVD68-VP1 (GeneTex, catalog #GTX132313); antibodies to CA16-VP1 are laboratory-made, and the specific methods were described previously ( ); PF4 antibody (Proteintech, #21157–1-AP); anti-HA polyclonal Ab (Invitrogen, catalog #71–5500); and anti-Myc (Proteintech, #16286–1-AP).

Techniques: Cell Counting, Infection, Western Blot, Control, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Platelet factor 4-derived C15 peptide broadly inhibits enteroviruses by disrupting viral attachment

doi: 10.1128/jvi.01859-24

Figure Lengend Snippet: C15 inhibits viral entry into cells by affecting virus binding to cells. ( A and B ) A time-of-addition assay with C15 was performed. RD cells were treated with C15 at different stages: pretreatment, incubation treatment, or post-entry of CA6 (MOI = 0.05) and EVD68 (MOI = 0.1). The inhibitory effect was assessed by measuring intracellular vRNA concentrations at 12 h post-infection ( n = 3). ( C and D ) CA6 and EVD68 were incubated with C15 in an Eppendorf (EP) tube for 4 h at 4°C. The mixture was then concentrated 1:100 and concentrated using a 100 kDa protein concentrator tube, with three exchanges to remove excess C15. The final virus solution was re-suspended in cell culture medium and used to infect RD cells. After 48 h, cells were collected and analyzed by RT-qPCR. ( E ) RD cells in six-well plates were infected with CA6 (MOI = 0.5) and EVD68 viruses containing C15 (100 and 1,000 nM) at 4°C for 1 h. The cells were then washed three times with cold phosphate-buffered saline, lysed, and vRNA levels were measured by RT-qPCR. ( F ) EVD68 virus bound to the surface of HeLa cells was detected by immunofluorescence. Multicellular (flat image) and single cell (stereoscopic 3D image) are shown. Bars of flat image are 100 μm, and bars for 3D image are as follows: length, 58 µm; width, 58 µm; height, 12 µm. The histogram shows the fluorescence ratio. All data are from three independent experiments and are presented as mean ± SD. Statistical significance was assessed using one-way analysis of variance. *** P < 0.001. ns, no significance.

Article Snippet: The following antibodies were used in this study: EV71-VP1 (GeneTex, catalog #GTX132339); CA6-VP1 (GeneTex, catalog #GTX132346); and EVD68-VP1 (GeneTex, catalog #GTX132313); antibodies to CA16-VP1 are laboratory-made, and the specific methods were described previously ( ); PF4 antibody (Proteintech, #21157–1-AP); anti-HA polyclonal Ab (Invitrogen, catalog #71–5500); and anti-Myc (Proteintech, #16286–1-AP).

Techniques: Virus, Binding Assay, Incubation, Infection, Cell Culture, Quantitative RT-PCR, Saline, Immunofluorescence, Fluorescence

Journal: Journal of Virology

Article Title: Platelet factor 4-derived C15 peptide broadly inhibits enteroviruses by disrupting viral attachment

doi: 10.1128/jvi.01859-24

Figure Lengend Snippet: PF4 interacts with the VP3 proteins of CA6 and EVD68. ( A and B ) HEK293T cells were transfected with cDNA-PF4-Myc or PF4△C15-Myc. The cell lysates were incubated with protein G containing Myc antibody, then CA6 and EVD68 suspensions were added for 4 h, followed by co-IP analysis. VR1012 expression vector served as a control. ( C and D ) Co-IP analysis of the interaction between CA6/EVD68-VP1/2/3-HA and PF4-Myc in HEK293T cells transfected with the indicated plasmids. VR1012 expression vector was used as a control. ( E and F ) Co-IP analysis of the interaction between CA6/EVD68-VP3-HA and PF4-Myc or PF4ΔC15-Myc in HEK293T cells transfected with the indicated plasmids. VR1012 empty vector served as a control. ( G and H )Co-IP analysis of the interaction between CA6/EVD68-VP3-HA and GFP-C15 in HEK293T cells transfected with the indicated plasmids. p-EGFP expression vector was used as a control. ( I )Docking result showing the interaction between C15 and EVD68-VP3. Blue, C15; gray, EVD68-VP3; purple, EVD68-VP3 interaction region (155–170). ( J and K ) Co-IP analysis of the interaction between CA6/EVD68-VP3-HA (wild type or Δ155–170) and PF4-Myc in HEK293T cells transfected with the indicated plasmids. VR1012 expression vector was used as a control.

Article Snippet: The following antibodies were used in this study: EV71-VP1 (GeneTex, catalog #GTX132339); CA6-VP1 (GeneTex, catalog #GTX132346); and EVD68-VP1 (GeneTex, catalog #GTX132313); antibodies to CA16-VP1 are laboratory-made, and the specific methods were described previously ( ); PF4 antibody (Proteintech, #21157–1-AP); anti-HA polyclonal Ab (Invitrogen, catalog #71–5500); and anti-Myc (Proteintech, #16286–1-AP).

Techniques: Transfection, Incubation, Co-Immunoprecipitation Assay, Expressing, Plasmid Preparation, Control

Journal: Journal of Virology

Article Title: Platelet factor 4-derived C15 peptide broadly inhibits enteroviruses by disrupting viral attachment

doi: 10.1128/jvi.01859-24

Figure Lengend Snippet: The interaction between C15 and the VP3 proteins of CA6 and EVD68 via a direct ionic interaction. ( A and D ) HEK-293T cells were transfected with cDNA-PF4-Myc or its mutants, with the VR1012 empty vector serving as a control. After 12 h, the cells were infected with CA6 (MOI = 0.5) and EVD68 (MOI = 1). Viral productions were assessed using Western blot analysis with an anti-VP1 antibody at 48 h, while tubulin was used as a loading control. ( B and E ) Viral mRNA levels in the cells were evaluated by quantifying VP1 mRNA levels using RT-qPCR. ( C and F ) Co-IP analysis of the interaction between CA6/EVD68-VP3-HA and PF4-Myc or its mutants in HEK293T cells transfected with the indicated plasmids. VR1012 expression carriers were used as a control. ( G and H ) Co-IP analysis was conducted to investigate the interaction between CA6/EVD68-VP3-HA or their D156A mutants and PF4-Myc in HEK293T cells transfected with the indicated plasmids. The VR1012 expression vector served as a control. ( I and J ) The binding ability of peptide C15 and its mutants to CA6 and EVD68 was assessed by ELISA and RT-qPCR analysis ( n = 3). The relative RNA copy number of the virus binding to peptides was normalized to the binding of the virus to C15 and its mutants. ( K and L ) At a concentration of 100 nM, peptide C15 effectively inhibited the replication of CA6 and EVD68, whereas its mutant did not exhibit such inhibitory effects. Three independent experiments are presented as mean ± SD. Statistical significance was assessed using one-way analysis of variance. ** P < 0.01, *** P < 0.001. ns, no significance.

Article Snippet: The following antibodies were used in this study: EV71-VP1 (GeneTex, catalog #GTX132339); CA6-VP1 (GeneTex, catalog #GTX132346); and EVD68-VP1 (GeneTex, catalog #GTX132313); antibodies to CA16-VP1 are laboratory-made, and the specific methods were described previously ( ); PF4 antibody (Proteintech, #21157–1-AP); anti-HA polyclonal Ab (Invitrogen, catalog #71–5500); and anti-Myc (Proteintech, #16286–1-AP).

Techniques: Transfection, Plasmid Preparation, Control, Infection, Western Blot, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Expressing, Binding Assay, Enzyme-linked Immunosorbent Assay, Virus, Concentration Assay, Mutagenesis

Journal: Journal of Virology

Article Title: Platelet factor 4-derived C15 peptide broadly inhibits enteroviruses by disrupting viral attachment

doi: 10.1128/jvi.01859-24

Figure Lengend Snippet: PF4 protects neonatal mice from CA6 lethal challenge. ( A ) The CA6 virus (10 7 TCID 50 /mL) with varying concentrations of C15 (1 and 5 mg/kg) was intracranially injected into 1 day-old ICR mice (10 µL/mouse). Various indicators were observed over 7 days, and samples were collected for analysis. ( B ) Clinical scores and survival rates were monitored for 6 days post-infection. Clinical disease severity was categorized as follows: 0, healthy; 1, lethargy and inactivity; 2, wasting; 3, limb tremor and weakness; 4, hind-limb paralysis; and 5, moribund or dead. ( C ) Changes in body weight of the mice over a 7 day period were recorded. ( D ) Survival rates of the mice over 7 days were evaluated. ( E ) Copy numbers of CA6 in brain, lung, spinal skeletal muscle, and hind-limb muscle tissues of infected mice were assessed by RT-qPCR on day 4. ( F ) Representative images of hematoxylin and eosin-stained tissues from mice subjected to various treatments were captured on day 4. Magnification: ×200. Scale bars: 100 µm.

Article Snippet: The following antibodies were used in this study: EV71-VP1 (GeneTex, catalog #GTX132339); CA6-VP1 (GeneTex, catalog #GTX132346); and EVD68-VP1 (GeneTex, catalog #GTX132313); antibodies to CA16-VP1 are laboratory-made, and the specific methods were described previously ( ); PF4 antibody (Proteintech, #21157–1-AP); anti-HA polyclonal Ab (Invitrogen, catalog #71–5500); and anti-Myc (Proteintech, #16286–1-AP).

Techniques: Virus, Injection, Infection, Quantitative RT-PCR, Staining

Journal: Scientific Reports

Article Title: Carbonic Anhydrase 6 Gene Variation influences Oral Microbiota Composition and Caries Risk in Swedish adolescents

doi: 10.1038/s41598-018-36832-z

Figure Lengend Snippet: Characteristics of the 17-year-old participants all together and by S. mutans and caries status.

Article Snippet: CA6 protein concentration in saliva was measured by ELISA (CA6 ELISA Kit, OKCD01736, Nordic Biosite, Täby, Sweden) following the manufacturer’s instructions.

Techniques: Next-Generation Sequencing, Protein Concentration, Concentration Assay

Journal: Scientific Reports

Article Title: Carbonic Anhydrase 6 Gene Variation influences Oral Microbiota Composition and Caries Risk in Swedish adolescents

doi: 10.1038/s41598-018-36832-z

Figure Lengend Snippet: Haplotype map of CA6 locus. ( a ) The CA6 gene structure and distribution of selected and quality-controlled tag single nucleotide polymorphic sites (SNP). Graphics illustrates pairwise linkage disequilibrium (LD) between polymorphisms and each square display coefficient of linkage disequilibrium D′ value (%) between marker pairs. In the event of D′ = 100% where no recombination events are observed, the boxes are empty. ( b ) Haploblocks are estimated using Haploview as described in the method section. Uncommon haplotypes (observed in <1% of participants) are not shown. Genetically linked haploblocks are connected with (inherited together with >1% (thin line) or >10% (thick line) together) lines.

Article Snippet: CA6 protein concentration in saliva was measured by ELISA (CA6 ELISA Kit, OKCD01736, Nordic Biosite, Täby, Sweden) following the manufacturer’s instructions.

Techniques: Marker

Journal: Scientific Reports

Article Title: Carbonic Anhydrase 6 Gene Variation influences Oral Microbiota Composition and Caries Risk in Swedish adolescents

doi: 10.1038/s41598-018-36832-z

Figure Lengend Snippet: Associations between CA6 gene variation and having detectable S. mutans in saliva or tooth biofilm by NGS. The association between 27 CA6 SNPs and detectable S. mutans in tooth biofilm and saliva by NGS was estimated using a Chi 2 test under 3 models; genotype model (green, SNPs marked with G), dominant model (yellow, SNPs marked with D) and recessive model (purple, SNPs marked with R). For each model, the width of the bar connecting the model name to a SNP indicates the relative–log10 p-value testing the null hypothesis of no association with detectable S. mutans . The red bar highlights SNPs where the null hypothesis was rejected after adjustment of p-values for multiple testing (FDR < 0.25).

Article Snippet: CA6 protein concentration in saliva was measured by ELISA (CA6 ELISA Kit, OKCD01736, Nordic Biosite, Täby, Sweden) following the manufacturer’s instructions.

Techniques:

Journal: Scientific Reports

Article Title: Carbonic Anhydrase 6 Gene Variation influences Oral Microbiota Composition and Caries Risk in Swedish adolescents

doi: 10.1038/s41598-018-36832-z

Figure Lengend Snippet: CA6 SNP and haploblock associations with S. mutans detection in both saliva and tooth biofilm by NGS.

Article Snippet: CA6 protein concentration in saliva was measured by ELISA (CA6 ELISA Kit, OKCD01736, Nordic Biosite, Täby, Sweden) following the manufacturer’s instructions.

Techniques: Blocking Assay

Journal: Scientific Reports

Article Title: Carbonic Anhydrase 6 Gene Variation influences Oral Microbiota Composition and Caries Risk in Swedish adolescents

doi: 10.1038/s41598-018-36832-z

Figure Lengend Snippet: CA6 SNP and haploblock associations with detection of viable mutans streptococci by saliva culturing.

Article Snippet: CA6 protein concentration in saliva was measured by ELISA (CA6 ELISA Kit, OKCD01736, Nordic Biosite, Täby, Sweden) following the manufacturer’s instructions.

Techniques: Blocking Assay

Journal: Scientific Reports

Article Title: Carbonic Anhydrase 6 Gene Variation influences Oral Microbiota Composition and Caries Risk in Swedish adolescents

doi: 10.1038/s41598-018-36832-z

Figure Lengend Snippet: CA6 gene variation and tooth biofilm microbiota. ( a ) PLS scatter plot illustrating separation of participants carrying either the G or C of the rs12138897 SNP based on their tooth biofilm microbiota using the dominant SNP model (G/G + G/C vs. C/C); ( b ) PLS loading scatter plot of the X and Y weights (w* and c ) illustrating the relation between the microbiota species and the G (red) and C (green) genotype of CA6 . Red/green dots refer to taxa with a VIP-value > 1.5, and grey dots to taxa with a VIP value < 1.5. ( c ) Correlation coefficients (mean (95% CI; shown on the left x-axis) for taxa with a VIP value > 1.5 (shown on the right x-axis) in plot B. Red bars show association with G and green bars with C genotypes of CA6 . The dots on the lines represent median prevalence for each taxon and the stars (*) statistically significant difference between the two genotypes using univariate Chi 2 test and p-values < 0.05.

Article Snippet: CA6 protein concentration in saliva was measured by ELISA (CA6 ELISA Kit, OKCD01736, Nordic Biosite, Täby, Sweden) following the manufacturer’s instructions.

Techniques:

Journal: Scientific Reports

Article Title: Carbonic Anhydrase 6 Gene Variation influences Oral Microbiota Composition and Caries Risk in Swedish adolescents

doi: 10.1038/s41598-018-36832-z

Figure Lengend Snippet: CA6 SNP and haploblock associations with being caries-affected or caries-free.

Article Snippet: CA6 protein concentration in saliva was measured by ELISA (CA6 ELISA Kit, OKCD01736, Nordic Biosite, Täby, Sweden) following the manufacturer’s instructions.

Techniques: Blocking Assay