ev d68  (Biken Inc)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Comparative immunogenic and structural analysis of virus-like particle and inactivated whole-virion vaccines against enterovirus D68

doi: 10.1016/j.omtn.2026.102957

Figure Lengend Snippet: Preparation and characterization of IWV and VLP (A) Schematic representation of the construction of plasmids encoding EV-D68 P1 and 3CD. (B) Workflow for the expression and purification of VLP. (C–F) SDS-PAGE analysis of purified IWV and VLP. (C and D) VLP expressed in (C) Expi293F cells and (D) ExpiCHO-S cells purified with sucrose. The Hsp and Hsc identified by mass spectrometry are labeled in red lines. (E) VLP was expressed in ExpiCHO-S cells and further purified using both sucrose and iodixanol (OptiPrep) gradients. (F) IWV was purified by sucrose gradient ultracentrifugation. (G) IWV and VLP particle size distribution was measured by dynamic light scattering. (H) Representative negative-stain TEM images of IWV and VLP. Scale bars, 100 nm. (I) The thermal stability of the non-inactivated virus, IWV, and VLP was assessed using differential scanning fluorimetry (DSF). Data represent the mean of four independent measurements ( n = 4) for each sample.

Article Snippet: The use of EV-D68 was reviewed and approved by the Institutional Review Board of the Research Institute for Microbial Diseases, The University of Osaka (protocol number: BIKEN-00184-004).

Techniques: Expressing, Purification, SDS Page, Mass Spectrometry, Labeling, Staining, Virus

Journal: Molecular Therapy. Nucleic Acids

Article Title: Comparative immunogenic and structural analysis of virus-like particle and inactivated whole-virion vaccines against enterovirus D68

doi: 10.1016/j.omtn.2026.102957

Figure Lengend Snippet: Epitope specificities of IgG induced by IWV and VLP vaccines (A) Localization of neutralizing antigenic sites I–IV and corresponding epitope peptide sequences. Neutralizing antigenic sites I–IV were mapped onto a single icosahedral asymmetric unit of the EV-D68 MO strain capsid, based on the previously reported cryo-EM structure (PDB: 6CSG ). VP1, VP2, and VP3 are shown in gray, pink, and cyan, respectively. Neutralizing antigenic sites I, II, III, and IV are highlighted in yellow, green, blue, and magenta, respectively. The accompanying table lists the sequences of epitope peptides spanning the antigenic sites; residues constituting the neutralizing antigenic sites are indicated in red. Molecular graphics were generated using UCSF ChimeraX v1.9. (B) Plasma IgG levels specific to epitope peptides following boost immunization with either IWV or VLP. (C) Plasma IgG reactivity to mutant VLPs following boost immunization with IWV or wild-type VLP. Then, 1,000-fold diluted plasma samples were used. Details of the mutations in each mutant are summarized in the table on the right. (B–C) n = 5 per group. Data are presented as mean ± SD. (B) Statistical comparisons were performed using 50-fold diluted plasma samples. (B) “ns” indicates not significant. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001, as determined by Tukey’s test. (C) ∗ p < 0.05 and ∗∗∗∗ p < 0.0001, as determined by Dunnett’s multiple comparison test.

Article Snippet: The use of EV-D68 was reviewed and approved by the Institutional Review Board of the Research Institute for Microbial Diseases, The University of Osaka (protocol number: BIKEN-00184-004).

Techniques: Vaccines, Cryo-EM Sample Prep, Generated, Clinical Proteomics, Mutagenesis, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: Comparative immunogenic and structural analysis of virus-like particle and inactivated whole-virion vaccines against enterovirus D68

doi: 10.1016/j.omtn.2026.102957

Figure Lengend Snippet: Cryo-EM structure of EV-D68 MO strain VLP (A) Cryo-EM density maps showing the overall structures of the mature virion (PDB: 6CSG ), empty particle (PDB: 6CRU ), and VLP of the EV-D68 MO strain. A schematic illustration of the viral particle is shown in the left-most. The 5-, 3-, and 2-fold symmetry axes are indicated by a pentagon, triangle, and circle, respectively. (B) Ribbon representations of the mature virion (PDB: 6CSG ), empty particle (PDB: 6CRU ), and VLP structures around the 2-fold axis. The 3-fold and 2-fold axes are denoted by a triangle and circle, respectively. (C) Structural comparison of the icosahedral asymmetric units of the VLP with those of the mature virion (left, PDB: 6CSG ) and the empty particle (middle, PDB: 6CRU ). VP1, VP0, and VP3 of the VLP were superimposed onto the corresponding subunits of the mature virion and the empty particle. Representative structures are shown based on superposition via VP1. All structures are depicted as ribbon models. The table on the right summarizes the number of pruned atom pairs and root-mean-square deviation (RMSD) values for each superposition.

Article Snippet: The use of EV-D68 was reviewed and approved by the Institutional Review Board of the Research Institute for Microbial Diseases, The University of Osaka (protocol number: BIKEN-00184-004).

Techniques: Cryo-EM Sample Prep, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: Comparative immunogenic and structural analysis of virus-like particle and inactivated whole-virion vaccines against enterovirus D68

doi: 10.1016/j.omtn.2026.102957

Figure Lengend Snippet: Preparation and characterization of IWV and VLP (A) Schematic representation of the construction of plasmids encoding EV-D68 P1 and 3CD. (B) Workflow for the expression and purification of VLP. (C–F) SDS-PAGE analysis of purified IWV and VLP. (C and D) VLP expressed in (C) Expi293F cells and (D) ExpiCHO-S cells purified with sucrose. The Hsp and Hsc identified by mass spectrometry are labeled in red lines. (E) VLP was expressed in ExpiCHO-S cells and further purified using both sucrose and iodixanol (OptiPrep) gradients. (F) IWV was purified by sucrose gradient ultracentrifugation. (G) IWV and VLP particle size distribution was measured by dynamic light scattering. (H) Representative negative-stain TEM images of IWV and VLP. Scale bars, 100 nm. (I) The thermal stability of the non-inactivated virus, IWV, and VLP was assessed using differential scanning fluorimetry (DSF). Data represent the mean of four independent measurements ( n = 4) for each sample.

Article Snippet: The cryo-EM map of the EV-D68 MO strain VLP has been deposited in the Electron Microscopy DataBank with accession code EMD-65634.

Techniques: Expressing, Purification, SDS Page, Mass Spectrometry, Labeling, Staining, Virus

Journal: Molecular Therapy. Nucleic Acids

Article Title: Comparative immunogenic and structural analysis of virus-like particle and inactivated whole-virion vaccines against enterovirus D68

doi: 10.1016/j.omtn.2026.102957

Figure Lengend Snippet: Epitope specificities of IgG induced by IWV and VLP vaccines (A) Localization of neutralizing antigenic sites I–IV and corresponding epitope peptide sequences. Neutralizing antigenic sites I–IV were mapped onto a single icosahedral asymmetric unit of the EV-D68 MO strain capsid, based on the previously reported cryo-EM structure (PDB: 6CSG ). VP1, VP2, and VP3 are shown in gray, pink, and cyan, respectively. Neutralizing antigenic sites I, II, III, and IV are highlighted in yellow, green, blue, and magenta, respectively. The accompanying table lists the sequences of epitope peptides spanning the antigenic sites; residues constituting the neutralizing antigenic sites are indicated in red. Molecular graphics were generated using UCSF ChimeraX v1.9. (B) Plasma IgG levels specific to epitope peptides following boost immunization with either IWV or VLP. (C) Plasma IgG reactivity to mutant VLPs following boost immunization with IWV or wild-type VLP. Then, 1,000-fold diluted plasma samples were used. Details of the mutations in each mutant are summarized in the table on the right. (B–C) n = 5 per group. Data are presented as mean ± SD. (B) Statistical comparisons were performed using 50-fold diluted plasma samples. (B) “ns” indicates not significant. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001, as determined by Tukey’s test. (C) ∗ p < 0.05 and ∗∗∗∗ p < 0.0001, as determined by Dunnett’s multiple comparison test.

Article Snippet: The cryo-EM map of the EV-D68 MO strain VLP has been deposited in the Electron Microscopy DataBank with accession code EMD-65634.

Techniques: Vaccines, Cryo-EM Sample Prep, Generated, Clinical Proteomics, Mutagenesis, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: Comparative immunogenic and structural analysis of virus-like particle and inactivated whole-virion vaccines against enterovirus D68

doi: 10.1016/j.omtn.2026.102957

Figure Lengend Snippet: Cryo-EM structure of EV-D68 MO strain VLP (A) Cryo-EM density maps showing the overall structures of the mature virion (PDB: 6CSG ), empty particle (PDB: 6CRU ), and VLP of the EV-D68 MO strain. A schematic illustration of the viral particle is shown in the left-most. The 5-, 3-, and 2-fold symmetry axes are indicated by a pentagon, triangle, and circle, respectively. (B) Ribbon representations of the mature virion (PDB: 6CSG ), empty particle (PDB: 6CRU ), and VLP structures around the 2-fold axis. The 3-fold and 2-fold axes are denoted by a triangle and circle, respectively. (C) Structural comparison of the icosahedral asymmetric units of the VLP with those of the mature virion (left, PDB: 6CSG ) and the empty particle (middle, PDB: 6CRU ). VP1, VP0, and VP3 of the VLP were superimposed onto the corresponding subunits of the mature virion and the empty particle. Representative structures are shown based on superposition via VP1. All structures are depicted as ribbon models. The table on the right summarizes the number of pruned atom pairs and root-mean-square deviation (RMSD) values for each superposition.

Article Snippet: The cryo-EM map of the EV-D68 MO strain VLP has been deposited in the Electron Microscopy DataBank with accession code EMD-65634.

Techniques: Cryo-EM Sample Prep, Comparison

Journal: Lancet Regional Health - Americas

Article Title: Enterovirus D68 in United States wastewater: a longitudinal surveillance study integrating climatic, demographic, and clinical data

doi: 10.1016/j.lana.2026.101446

Figure Lengend Snippet: EV-D68 RNA in wastewater at national and state levels, 2023–2025. Smoothed, PMMoV-normalized EV-D68 RNA concentration for (a) United States, (b) California, and (c) Pennsylvania. The shaded band denotes the EV-D68 activity duration; the vertical line marks the center of the EV-D68 season. For the national series (a), duration was defined as the period when concentrations exceeded a threshold equal to a baseline plus three standard deviations, where the baseline and standard deviation were estimated from the quiet tail (lowest tertile) of the data distribution. For state series (b–c), duration was defined as the continuous run containing the center of season during which values were detectable, with a minimum of at least two consecutive weeks with EV-D68 RNA concentration above 0. Dates with day-of-month annotate the start and end of the duration and the season center in each state. ∗EV-D68 RNA concentration refers to the smoothed, PMMoV-normalized concentration, calculated using a five-day centered trimmed moving average. For each five-day window, the highest and lowest daily values were excluded, and the remaining three were averaged.

Article Snippet: Most EV-D68 infections cause mild or no symptoms and therefore rarely prompt clinical testing, and EV-D68 specific diagnostics are not routinely performed.

Techniques: Concentration Assay, Activity Assay, Standard Deviation

Journal: Lancet Regional Health - Americas

Article Title: Enterovirus D68 in United States wastewater: a longitudinal surveillance study integrating climatic, demographic, and clinical data

doi: 10.1016/j.lana.2026.101446

Figure Lengend Snippet: State-level timing and duration of EV-D68 activity in U.S. wastewater, with WWTP-level geographic gradients. (a) Map shows, for each state, the calendar date of the EV-D68 seasonal center estimated from the state-aggregated weekly series (PMMoV-normalized, 5-day–trimmed), after first aggregating across WWTPs within state and week. Colors run from earlier (Jun ’24) to later (Nov ’24). (b) Map shows the length of the detectable activity window (displayed in months) around the seasonal center of the state, computed on the same state-aggregated weekly series by identifying consecutive weeks with detectable signal (runs of at least two weeks above the non-detect threshold) and converting weeks to months. Gray states do not have wastewater data available. Alaska and Hawaii are shown out of scale for layout. Panels c–f are weighted bivariate regressions displaying gradients between longitude or latitude and EV-D68 center of season (C and d) or activity duration (e and f). Each point is a WWTP within the United States (n = 146) Clinton, Iowa had W = 0 (no detections after normalisation and trimming) and therefore was excluded. Point size denotes the plant weight WW, defined as the sum across weeks of PMMoV-normalised, 5-day–trimmed EV-D68 concentrations, with non-detects set to 0. Larger circles indicate greater cumulative signal and therefore greater influence on the weighted least-squares fits. Lines show weighted least-squares fits with 95% CIs.

Article Snippet: Most EV-D68 infections cause mild or no symptoms and therefore rarely prompt clinical testing, and EV-D68 specific diagnostics are not routinely performed.

Techniques: Activity Assay

Journal: Lancet Regional Health - Americas

Article Title: Enterovirus D68 in United States wastewater: a longitudinal surveillance study integrating climatic, demographic, and clinical data

doi: 10.1016/j.lana.2026.101446

Figure Lengend Snippet: EV-D68 center of season as a function of mean environmental conditions across WWTPs (n = 146). For each plant, the x-axis is the mean of the indicated variable (daily values aggregated to weekly means over the EV-D68 activity window); the y-axis is the calendar date of the season center. Lines are univariate weighted least-squares fits with 95% CIs; weights are proportional to each plant's cumulative EV-D68 signal, with non-detects set to 0. Point sizes are proportional to the weights and the weighted R 2 is shown on each panel.

Article Snippet: Most EV-D68 infections cause mild or no symptoms and therefore rarely prompt clinical testing, and EV-D68 specific diagnostics are not routinely performed.

Techniques: Activity Assay

Journal: Lancet Regional Health - Americas

Article Title: Enterovirus D68 in United States wastewater: a longitudinal surveillance study integrating climatic, demographic, and clinical data

doi: 10.1016/j.lana.2026.101446

Figure Lengend Snippet: Duration of EV-D68 circulation by wastewater treatment plant (WWTP)-level characteristics. Each panel shows the distribution of EV-D68 circulation duration (in weeks) across categories of a single WWTP–level determinant. Points represent individual WWTPs and boxplots show the median, interquartile range (IQR), and whiskers at 1.5 × IQR. Airport presence is dichotomized as absent and present. Hospital and nursing home coverage are split using the dataset medians: 2 hospitals and 8 nursing homes within each WWTP catchment area, respectively. Urbanicity is defined by the proportion of the WWTP catchment area classified as urban (≤50% vs >50%). The proportions of children aged ≤5 years, adults aged ≥65 years, crowded households, birth rate (per capita), childcare density (per km 2 ), and population density (per km 2 ) are grouped into within-study tertiles (“low”, “middle”, “high”). Sample sizes (n) shown in each panel indicate the number of WWTPs included in each comparison group. Kruskal–Wallis test was used to assess overall differences, and pairwise comparisons used Wilcoxon rank-sum tests with Bonferroni adjustment. Horizontal connector bars indicate statistically significant differences; asterisks denote adjusted p-values (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Article Snippet: Most EV-D68 infections cause mild or no symptoms and therefore rarely prompt clinical testing, and EV-D68 specific diagnostics are not routinely performed.

Techniques: Comparison

Journal: Lancet Regional Health - Americas

Article Title: Enterovirus D68 in United States wastewater: a longitudinal surveillance study integrating climatic, demographic, and clinical data

doi: 10.1016/j.lana.2026.101446

Figure Lengend Snippet: National and within-state correlations between wastewater EV-D68 RNA concentrations and clinical diagnoses. (a–d) National time-series comparing wastewater EV-D68 RNA concentrations (black lines, right axis) with proportions of clinical diagnoses (bars, left axis) for wheezing in children ≤5 years (A), wheezing in adults ≥65 years (b), enterovirus-specific encounters (c), and acute flaccid myelitis (AFM) (d). (e–h) State-level Spearman correlations between wastewater EV-D68 and AFM (e), enterovirus-specific encounters (f), wheezing ≤5 years (g), and wheezing ≥65 years (h). Points represent correlation coefficients, with colors denoting effect size and significance (adjusted p < 0.05, Bonferroni). Correlations at the state level were performed using data filtered to the EV-D68 seasonal duration defined by wastewater and extended ±2 weeks for clinical diagnoses, and ±2 months for AFM.

Article Snippet: Most EV-D68 infections cause mild or no symptoms and therefore rarely prompt clinical testing, and EV-D68 specific diagnostics are not routinely performed.

Techniques:

Journal: Genes & Diseases

Article Title: Salt-inducible kinase 1 is a key gene in suppressing EVD68-induced asthma by modulating antiviral immunity

doi: 10.1016/j.gendis.2025.101845

Figure Lengend Snippet: The common differentially expressed genes between EV-D68 infection and asthma. (A, B) Volcano plot of differentially expressed genes (DEGs) in the EV-D68-related GSE184488 dataset and asthma-related GSE143303 dataset. (C) Common DEGs in the GSE184488 dataset and the GSE143303 dataset were represented by Venn diagrams. (D) KEGG enrichment analysis of the 74 common DEGs identified in EVD68 infection and asthma. (E) GO enrichment analysis of the 74 common DEGs identified in EVD68 infection and asthma. BP, biological process; CC, cellular component; MF, molecular function.

Article Snippet: The EV-D68 (ATCC VR-1826), EV-A71, HSV-1, and VSV-GFP were kept in our laboratory.

Techniques: Infection

Journal: Genes & Diseases

Article Title: Salt-inducible kinase 1 is a key gene in suppressing EVD68-induced asthma by modulating antiviral immunity

doi: 10.1016/j.gendis.2025.101845

Figure Lengend Snippet: SIK1 expression is induced by EV-D68 infection in vivo . Eight-to-ten-week-old type I interferon receptor-deficient mice ( Ifna −/− ) were treated with EV-D68 (50 μL of 1 × 10 7 PFU/mL viral stock for each mouse) or an equal volume of phosphate-buffered saline through the intranasal route after anesthesia. Lungs were harvested 48 h after infection and then analyzed by hematoxylin-eosin staining, quantitative PCR, and western blotting. (A) Images showing lung inflammation 48 h after treatment with EV-D68 or mock phosphate-buffered saline, as visualized by hematoxylin-eosin staining (scale bar: 50 mm). (B–I) The indicated genes were detected by quantitative PCR and normalized to GAPDH expression. Values are from three independent experiments and expressed as mean ± standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (J) The protein expression level of SIK1 in the lungs of mice from the mock and EV-D68 groups was detected by western blotting.

Article Snippet: The EV-D68 (ATCC VR-1826), EV-A71, HSV-1, and VSV-GFP were kept in our laboratory.

Techniques: Expressing, Infection, In Vivo, Saline, Staining, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

Journal: Genes & Diseases

Article Title: Salt-inducible kinase 1 is a key gene in suppressing EVD68-induced asthma by modulating antiviral immunity

doi: 10.1016/j.gendis.2025.101845

Figure Lengend Snippet: SIK1 shows antiviral effects in various viral infections. (A, B) A549 cells were infected with EV-D68 (MOI = 0.1 or 1) for 24 h. (A) Quantitative reverse transcription PCR (RT-qPCR) analysis of relative SIK1 mRNA expression. The results were normalized to GAPDH expression. (B) Western blotting analysis of SIK1 and EV-D68 VP1 protein expression. β-actin was used as the loading control. Values were from three independent experiments and expressed as mean ± standard deviation. (C, D) RD cells were infected with EV-A71 (MOI = 0.1 or 0.5) for 24 h. (C) RT-qPCR analysis of relative SIK1 mRNA expression. The results were normalized to GAPDH expression. (D) Western blotting analysis of SIK1 and EV-A71 VP1 protein expression. β-actin was used as the loading control. Values were from three independent experiments and expressed as mean ± standard deviation. (E, F) A549 cells were transfected with si-SIK1 or si-NC and then infected with EV-D68 (MOI = 0.5) for 24 h. (E) The relative viral RNA copy numbers were determined by RT-qPCR and normalized to GAPDH. (F) The protein expression levels of EV-D68 VP1 and SIK1 were detected by western blotting. β-actin was used as the loading control. Values were from three independent experiments and expressed as mean ± standard deviation. (G, H) A549 cells were transfected with plasmid pCDH-SIK1 or empty pCDH vector and then infected with EV-D68 (MOI = 0.5) for 24 h. The viral replication and protein expression level of EV-D68 VP1 and SIK1 were detected as described above. Values were from three independent experiments and expressed as mean ± standard deviation. (I, J) A549 cells were infected with VSV-GFP for 6 h (MOI = 0.1, 0.5) or HSV-1 for 24 h (MOI = 0.1, 0.2), and then the relative mRNA expression of SIK1 was analyzed by RT-qPCR. Values were from three independent experiments and expressed as mean ± standard deviation. (K, L) Box plots represent the normalized expression levels of SIK1 using Z-score normalization in GSE157103 (for CV-A6) and GSE243200 (for SARS-COV-2) datasets. SIK1 expression correlation was analyzed using Spearman's method. (M, N) A549 cells were transfected with si-SIK1 or si-NC and then infected with VSV-GFP (MOI = 0.5) for 12 h. The replication of VSV-GFP was visualized by immunofluorescence microscopy (scale bar: 50 μm), and VSV-GFP RNA synthesis was determined by RT-qPCR analysis. Values were from three independent experiments and expressed as mean ± standard deviation. (O, P) A549 cells were transfected with plasmid pCDH-SIK1 or empty pCDH vector and then infected with VSV-GFP (MOI = 0.5) for 12 h. To detect viral replication by quantitative PCR, viral titers were determined by plaque assay as described in the methods section, and viral RNA synthesis was determined by RT-qPCR analysis. Values were from three independent experiments and expressed as mean ± standard deviation. (Q, R) SIK1 was interfered with by si-SIK1 or overexpressed via transfection of plasmid pCDH-SIK1 in A549 cells, and then the cells were infected with HSV-1. Viral replication was determined by RT-qPCR. Values were from three independent experiments and expressed as mean ± standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; ns, non-significant.

Article Snippet: The EV-D68 (ATCC VR-1826), EV-A71, HSV-1, and VSV-GFP were kept in our laboratory.

Techniques: Infection, Reverse Transcription, Quantitative RT-PCR, Expressing, Western Blot, Control, Standard Deviation, Transfection, Plasmid Preparation, Immunofluorescence, Microscopy, Real-time Polymerase Chain Reaction, Plaque Assay

Journal: Genes & Diseases

Article Title: Salt-inducible kinase 1 is a key gene in suppressing EVD68-induced asthma by modulating antiviral immunity

doi: 10.1016/j.gendis.2025.101845

Figure Lengend Snippet: Metformin-mediated activation of SIK1 protects against EV-D68-driven asthma exacerbation in house dust mite (HDM)-sensitized mice. (A) C57BL/6 mice (6–8 weeks) were administered metformin at doses of 100 mg/kg or 250 mg/kg once daily via intraperitoneal injection on day 1 and day 2. On day 3, lung tissues were collected, and the protein level of SIK1 was determined by western blotting analysis. (B) Experimental timeline. C57BL/6 mice (6–8 weeks) were intranasally sensitized with 250 μg kg −1 HDM extract on day 0 and challenged daily with the same dose on days 7–11. On days 12–13, animals received EV-D68 (1 × 10 6 PFU/kg) or DMEM (vehicle) intranasally. Metformin (100 mg/kg, intraperitoneal) was administered once daily on days 12–14. Airway hyper-responsiveness measurements and broncho-alveolar lavage fluid (BALF) collection were performed on day 15; lung tissue was used for quantitative PCR analyses. (C) Airway responsiveness to increasing doses of methacholine. (D) Differential cell counts of BALF by Wright-Giemsa staining. (E – H) The indicated genes were detected by quantitative PCR and normalized to GAPDH expression. Values were from three independent experiments and expressed as mean ± standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: The EV-D68 (ATCC VR-1826), EV-A71, HSV-1, and VSV-GFP were kept in our laboratory.

Techniques: Activation Assay, Injection, Western Blot, Real-time Polymerase Chain Reaction, Staining, Expressing, Standard Deviation