Journal: Viruses

Article Title: Cinnamaldehyde Inhibits the Replication of Porcine Reproductive and Respiratory Syndrome Virus Type 2 In Vitro

doi: 10.3390/v17040506

Figure Lengend Snippet: Cinnamaldehyde suppresses PRRSV infection in Marc-145 cells. ( A , B ) The effect of different concentrations (40, 80, 160 μM) of CA on PRRSV (MOI = 1) replication in Marc-145 cells was investigated by RT-qPCR ( A ) and a viral titer assay ( B ). ( C – F ) Western blotting ( C ) and an immunofluorescence assay ( E ) were used to detect the expression of PRRSV N protein at 24 hpi in the presence of CA (40, 80, 160 μM). Statistical analysis of Western blotting ( D ) and the IFA ( F ). Bar = 100 µm. The data are the results of three independent experiments (means ± SD). Significant differences are denoted by * p < 0.05 and ** p < 0.01.

Article Snippet: Subsequently, reverse transcription was conducted with the HiScript ® ⅢRT SuperMix for qPCR (+gDNA wiper) Vazyme Code: R323-01 100 rxns (Vazyme, China), based on the manufacturer’s instructions.

Techniques: Infection, Quantitative RT-PCR, Titer Assay, Western Blot, Immunofluorescence, Expressing

Journal: Viruses

Article Title: Cinnamaldehyde Inhibits the Replication of Porcine Reproductive and Respiratory Syndrome Virus Type 2 In Vitro

doi: 10.3390/v17040506

Figure Lengend Snippet: Cinnamaldehyde suppresses PRRSV infection in PAM cells. ( A ) The cytotoxicity of CA on PAM cells was evaluated using the CCK-8 assay. The non-toxic concentration of CA was determined to be 80 μM. ( B , C ) The effect of different concentrations (40, 80 μM) of CA on PRRSV (MOI = 1) replication in PAM cells was investigated by RT-qPCR ( B ) and a viral titer assay ( C ). ( D , E ) An immunofluorescence assay ( D ) was used to detect the expression of PRRSV N protein at 24 hpi in the presence of CA (40, 80 μM). Statistical analysis of the IFA ( E ). Bar = 100 µm. The data are the results of three independent experiments (means ± SD). Significant differences are denoted by * p < 0.05 and ** p < 0.01.

Article Snippet: Subsequently, reverse transcription was conducted with the HiScript ® ⅢRT SuperMix for qPCR (+gDNA wiper) Vazyme Code: R323-01 100 rxns (Vazyme, China), based on the manufacturer’s instructions.

Techniques: Infection, CCK-8 Assay, Concentration Assay, Quantitative RT-PCR, Titer Assay, Immunofluorescence, Expressing

Journal: Viruses

Article Title: Cinnamaldehyde Inhibits the Replication of Porcine Reproductive and Respiratory Syndrome Virus Type 2 In Vitro

doi: 10.3390/v17040506

Figure Lengend Snippet: Cinnamaldehyde exerts anti-PRRSV ability in different treatment modes. ( A ) The diagram shows three different administration treatment modes. Pre-treatment: Marc-145 cells were pre-treated with CA at 160 μM at different times (2, 4, 6, 8 h) and then infected with PRRSV for another 2 h. Co-treatment: Marc-145 cells were infected with PRRSV and incubated with CA (160 μM) for 2 h. Post-treatment: Marc-145 cells were infected with PRRSV for 2 h, then treated with CA at 160 μM at different times (2, 4, 6, 8 h). Samples were harvested at 24 hpi. ( B ) RT-qPCR was used to determine viral ORF7 expression. ( C ) The expression of PRRSV N protein was detected by an IFA. ( D ) Statistical analysis of the IFA. ( E ) Cell supernatants were collected for the TCID 50 assay. Bar = 100 µm. The data are the results of three independent experiments (means ± SD). Significant differences are denoted by * p < 0.05 and ** p < 0.01.

Article Snippet: Subsequently, reverse transcription was conducted with the HiScript ® ⅢRT SuperMix for qPCR (+gDNA wiper) Vazyme Code: R323-01 100 rxns (Vazyme, China), based on the manufacturer’s instructions.

Techniques: Infection, Incubation, Quantitative RT-PCR, Expressing

Journal: Viruses

Article Title: Cinnamaldehyde Inhibits the Replication of Porcine Reproductive and Respiratory Syndrome Virus Type 2 In Vitro

doi: 10.3390/v17040506

Figure Lengend Snippet: Cinnamaldehyde blocks the binding, entry, replication, and release of PRRSV. ( A ) A schematic diagram of PRRSV binding, entry, replication, and release. ( B ) A viral binding assay was performed. PRRSV was inoculated in the presence or absence of CA at 4 °C for 2 h. RT-qPCR was used to detect viral ORF7 expression. ( C ) A viral entry assay was performed. After virus binding, cells were transferred to 37 °C for another 2 h. RT-qPCR was used to analyze viral ORF7 expression. ( D ) Viral replication assays were performed. Cells were infected with PRRSV for 4 h (including binding and entry) and then treated with CA for another 6, 12, and 24 h. Cells were collected for RT-qPCR analysis. ( E ) A viral release assay was performed. Marc-145 cells were infected with PRRSV for 24 h. Then, cells were treated with CA for another 8 h. Cell supernatants were collected for the TCID 50 assay. The data are the results of three independent experiments (means ± SD). Significant differences are denoted by * p < 0.05 and ** p < 0.01.

Article Snippet: Subsequently, reverse transcription was conducted with the HiScript ® ⅢRT SuperMix for qPCR (+gDNA wiper) Vazyme Code: R323-01 100 rxns (Vazyme, China), based on the manufacturer’s instructions.

Techniques: Binding Assay, Quantitative RT-PCR, Expressing, Virus, Infection, Release Assay

Journal: JAMA Ophthalmology

Article Title: Biomarker Detection and Validation for Corneal Involvement in Patients With Acute Infectious Conjunctivitis

doi: 10.1001/jamaophthalmol.2024.2891

Figure Lengend Snippet: A, Each data point represents the mean area under the receiver operating characteristic curve (AUROC) of logistic regression models that left out the respective gene over 1000 randomized training and validation splits. The red dotted line represents the mean AUROC for all splits of the full model of all 13 genes. The blue dotted lines represent the 95% CIs of the full model. Error bars represent 95% CIs for the individual models. B, Validation of APOE in conjunctival samples of 48 patients with presumed acute infectious conjunctivitis using quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis. APOE expression was normalized to glyceraldehyde 3-phosphate dehydrogenase and expressed as Δ threshold cycle (Δ CT). P value was calculated using the Mann-Whitney U test.

Article Snippet: The Fisher exact test was used to determine the discriminability of APOE RT-qPCR with clinical findings of corneal involvement (GraphPad version 10).

Techniques: Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, MANN-WHITNEY

Journal: Journal of the American Heart Association

Article Title: Stroke Causes DNA Methylation at Ncx1 Heart Promoter in the Brain Via DNMT1/MeCP2/REST Epigenetic Complex

doi: 10.1161/jaha.123.030460

Figure Lengend Snippet: Figure 1. The epigenetic factors DNMT1 and MeCP2 and the transcriptional factor REST are involved in the negative modulation of Ncx1 mRNA and protein expression. A and B, qRT-PCR for DNMT1 and MECP2 transcripts in SH-SY5Y cells transfected for 48 hours with siDNMT1 or siMeCP2 at a final concentration of 50 nM. An siCTL was used as scrambled. *P≤0.05 vs siCTL by Student’s t test (n=3). C, Ncx1 mRNA and protein expression in SH-SY5Y cells transfected for 48 hours with siCTL, siDNMT1, siMeCP2, and siREST. *P≤0.05 vs siCTL by 1-way ANOVA analysis followed by Tukey’s post hoc test (n=3/4). D and E, qRT-PCR for DNMT1 and MECP2 transcripts in SH-SY5Y transfected for 48 hours with vector overexpressing DNMT1 or MeCP2. pcDNA3.1 vector was used as EV. *P≤0.05 vs EV by Student’s t test (n=3). F, Ncx1 mRNA and protein expression in SH-SY5Y cells transfected for 48 hour with EV, DNMT1, MeCP2, or REST plasmids. *P≤0.05 vs EV by 1-way ANOVA analysis followed by Tukey’s post hoc test (n=3/4). CTL indicates control; DNMT1, DNA-methyltransferase-1; EV, empty vector; MeCP2, methyl-CpG binding protein 2; NCX1, sodium/calcium exchanger 1; qRT-PCR, quantitative real time polymerase chain reaction; REST, repressor element 1-silencing transcription factor; si, small interfering; and siCTL, control siRNA.

Article Snippet: Chromatin lysates were incubated overnight with 3 μg of antibody for DNMT1 (Mouse mAb, cod: sc- 271 729 Santa Cruz Biotechnology, dilution 1:500), MeCP2 (Rabbit mAb, cod: 3456 Cell Signaling, dilution 1:1000), REST, HDAC1, and HDAC2.3 Normal rabbit IgG was used as negative control.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Concentration Assay, Plasmid Preparation, Control, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association

Article Title: Stroke Causes DNA Methylation at Ncx1 Heart Promoter in the Brain Via DNMT1/MeCP2/REST Epigenetic Complex

doi: 10.1161/jaha.123.030460

Figure Lengend Snippet: Figure 4. DNMT1 and MeCP2 bind Ht, but not Br promoter sequence, in the temporoparietal cortex 24 hours after tMCAO. Chromatin immunoprecipitation with anti-DNMT1 (A), anti-MeCP2 (B) anti-REST (C), anti-HDAC1 (D), anti-HDAC2 (E), antibodies followed by qPCR of Ncx1 brain promoter (Br) (white columns) and Ncx1 heart promoter (Ht) (gray columns) in peri-ischemic cortex of mice euthanized 12 or 24 hours after tMCAO. IgG was used as negative control. *P≤0.05 vs Sham immunopreciptated with IgG antibody by 1-way ANOVA analysis followed by Tukey’s post hoc test (n=3). DNMT1 indicates DNA-methyltransferase-1; HDAC, histone deacetylase; IP, immunoprecipitation; MeCP2, methyl-CpG binding protein 2; NCX1, sodium/calcium exchanger 1; qRT-PCR, quantitative real time polymerase chain reaction; REST, repressor element 1-silencing transcription factor; and tMCAO, transient middle cerebral artery occlusion.

Article Snippet: Chromatin lysates were incubated overnight with 3 μg of antibody for DNMT1 (Mouse mAb, cod: sc- 271 729 Santa Cruz Biotechnology, dilution 1:500), MeCP2 (Rabbit mAb, cod: 3456 Cell Signaling, dilution 1:1000), REST, HDAC1, and HDAC2.3 Normal rabbit IgG was used as negative control.

Techniques: Sequencing, Chromatin Immunoprecipitation, Negative Control, Histone Deacetylase Assay, Immunoprecipitation, Binding Assay, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Cancers

Article Title: Synergistic Autophagy Effect of miR-212-3p in Zoledronic Acid-Treated In Vitro and Orthotopic In Vivo Models and in Patient-Derived Osteosarcoma Cells

doi: 10.3390/cancers11111812

Figure Lengend Snippet: Zoledronic acid (ZOL) induced autophagy by repressing the Akt/mTOR pathway in osteosarcoma (OS) cells and in an orthotopic in vivo model. ( a ) Immunoblotted cell lysates (30 μg) are shown with the corresponding antibodies. ( b ) ELISA was performed to quantify the level of phosphor-p70S6K at Thr389, phosphor-4EBP1 at Thr37/46, and p70S6K-4EBP1 in KHOS/NP, U2OS, and cells from a patient with OS after ZOL treatment. Each concentration was tested in quadruplicate, and each experiment was repeated two times. The data shown represent the combined mean ± SD from two independent experiments; * p < 0.05, ** p < 0.01, ns > 0.05. ( c ) p -Akt expression in the orthotopic model was examined by immunohistochemistry. Representative images are provided as indicated; * p < 0.05.

Article Snippet: Antibodies against Atg5, Beclin 1, LC3(A/B), p-mTOR (Ser2448), p-Akt (S473), p-P70S6K (Thr389), p-4EBP1 (Thr37/46), and p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Concentration Assay, Expressing, Immunohistochemistry

Journal: Cancers

Article Title: Synergistic Autophagy Effect of miR-212-3p in Zoledronic Acid-Treated In Vitro and Orthotopic In Vivo Models and in Patient-Derived Osteosarcoma Cells

doi: 10.3390/cancers11111812

Figure Lengend Snippet: The autophagic target relationship between miR-212-3p and zoledronic acid (ZOL) in an in vivo model. ( a ) Image of isolated tumors derived from osteosarcoma (OS) xenografts intratumorally treated with ZOL or miR- 212-3p mimics or inhibitor. ( b ) Representative PET/CT images of KHOS tumor-bearing mice after injection of [ 18 F]-[Fluorine-18(18F)]-fluorodeoxyglucose (FDG). The radioactivity of [ 18 F]-FDG in tumors is presented as the maximal value of SUV (mean ± S.D); ** p < 0.01. ( c ) miR-212-3p levels were analyzed by qRT-PCR in in vivo tissues treated with ZOL only; miR212-3p mimics only; ZOL + miR212-3p mimics; or ZOL + miR212-3p inhibitor. Values represent the means of three experiments ± SD; * p < 0.05, *** p < 0.001. ( d ) Tumors were excised and weighed at the end of the experiment (six weeks after tumor cell inoculation); * p < 0.05. ( e ) Mouse body weights were assessed at 14 days. ( f ) Beclin1 mRNA expression levels in mice receiving each treatment; * p < 0.05, ** p < 0.01, *** p < 0.001. ( g ) Hematoxylin and eosin (H&E) staining and LC3, Beclin1, and p -mTOR expression in tumor xenografts were examined by immunohistochemistry. * p < 0.05, ** p < 0.01, ns > 0.05.

Article Snippet: Antibodies against Atg5, Beclin 1, LC3(A/B), p-mTOR (Ser2448), p-Akt (S473), p-P70S6K (Thr389), p-4EBP1 (Thr37/46), and p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: In Vivo, Isolation, Derivative Assay, Positron Emission Tomography-Computed Tomography, Injection, Radioactivity, Quantitative RT-PCR, Expressing, Staining, Immunohistochemistry