Transcription of the <i>UL111A</i> gene and expression of transcription factors in PMA-treated and untreated THP-1 cells infected with HCMV.

<p>(A) Expression levels of the hcmvIL-10 and LAcmvIL-10 transcripts in differentiated and undifferentiated HCMV-infected THP-1 cells, analyzed by RT-PCR. (B) Quantitative analysis of the data in (A) from three independent experiments. RNA transcripts were normalized to cellular GAPDH. (C) Western blots showing the expression level of AML-1, GATA-1, and C/EBP β proteins in differentiated and undifferentiated HCMV-infected THP-1 cells. (D) Quantitative analysis of the data in (C) from three independent experiments. Proteins were normalized to cellular β-actin. *<i>P</i> < 0.05, <i>**P</i> < 0.01, ***<i>P</i> < 0.001.</p

Epigenetic modification status in the URR of <i>UL111A</i> from undifferentiated and differentiated HCMV-infected THP-1 cells.

<p>(A) ChIP analysis showing the association of the transcription factors binding sites with histone H3 acetylated at K9 position (H3 acetyl-K9) and dimethylated at K9 position (H3 dimethyl-K9). The lanes are designated as: “input”—PCR amplification of input DNA, “anti-acetyl”—PCR amplification of the chromatin DNA fragments precipitated by antibody against H3 acetyl-K9, “anti-dimethyl”—PCR-amplification of the chromatin DNA fragments precipitated by antibody against H3 dimethyl-K9, “isotype”—a negative control for non-specific reactions. (B) Quantitative analysis of the data in (A) from three independent ChIP experiments. The results were normalized to the input signal, which was set to 1. (C) Methylation status of the CpG sites in the URR of the <i>UL111A</i> gene in undifferentiated and differentiated infected THP-1 cells infected with HCMV. The methylation status of individual CpG dinucleotides is indicated as: ○ and ●, which represent unmethylated and methylated cytosine, respectively. (D) Quantitative analysis of the total methylation data in (C) from three independent BSP experiments. <i>**P</i> < 0.01.</p

AML-1 suppression affects the <i>UL111A</i> gene expression.

<p>(A) FACS analysis of transfection efficiency. The infection efficiency was measured by FACS analysis in HCMV-infected THP-1 cells transfected with LV-shRNA-AML1 or LV-shRNA-NC at 72 h post-transfection. (B) Western blot was detected in HCMV-infected THP-1 cells transfected with LV-shRNA-AML1 or LV-shRNA-NC. **<i>P</i><0.01 Verse LV-shRNA-NC. (C) HCMV-infected THP-1 cells stimulated with PMA were transfected with LV-shRNA-AML1 or LV-shRNA-NC for 72h. RT-PCR was taken to analyze the change of hcmvIL-10. *<i>P</i><0.05. Verse LV-shRNA-NC.</p

Primers used for ChIP-DNA PCR amplification.

<p>Primers used for ChIP-DNA PCR amplification.</p

Induction of viral transcription in infected THP-1 cells with PMA treatment.

<p>(A) Microscope image showing differentiation of HCMV-infected THP-1 cells into an adherent and macrophage-like phenotype following stimulation with PMA (100 ng/μl) (original magnification ×200). (B) Viral DNA loads over time in HCMV-infected THP-1 cells following PMA treatment. Each sample was assayed in triplicate by qPCR. (C) Expression of viral transcripts in PMA-treated THP-1 cells infected with HCMV. RT− represents the RNA without prior reverse transcription, employed as a negative control.</p

Identification of transcription factor binding sites in the URR of the <i>UL111A</i> gene.

<p>(A) Potential binding sites for the myeloid transcription factors AML-1, C/EBP β, and GATA-1 in the URR of the <i>UL111A</i> gene predicted using the MATCH program and TRANSFAC database. (B) ChIP analysis of three transcription factors binding to their corresponding sites. The lanes are designated as: “input”—PCR amplification of input DNA, “anti-TF”—PCR amplification of chromatin DNA fragments precipitated by antibodies against transcription factors, “isotype”—a control for non-specific reactions. (C) Comparison of transcription factor binding between undifferentiated and differentiated THP-1 cells. Quantitative analysis of the data is from three independent experiments. *** <i>P</i> < 0.001.</p

Primers used for mRNA RT-PCR amplification of HCMV mRNAs.

<p>Primers used for mRNA RT-PCR amplification of HCMV mRNAs.</p

Evolution of H6N6 viruses in China between 2014 and 2019 involves multiple reassortment events

H6N6 avian influenza viruses (AIVs) have been widely detected in wild birds, poultry, and even mammals. Recently, H6N6 viruses were reported to be involved in the generation of H5 and H7 subtype viruses. To investigate the emergence, evolutionary pattern, and potential for an epidemic of H6N6 viruses, the complete genomes of 198 H6N6 viruses were analyzed, including 168 H6N6 viruses deposited in the NCBI and GISAID databases from inception to January 2019 and 30 isolates collected from China between November 2014 and January 2019. Using phylogenetic analysis, the 198 strains of H6N6 viruses were identified as 98 genotypes. Molecular clock analysis indicated that the evolution of H6N6 viruses in China was constant and not interrupted by selective pressure. Notably, the laboratory isolates reassorted with six subtype viruses: H6N2, H5N6, H7N9, H5N2, H4N2, and H6N8, resulting in nine novel H6N6 reassortment events. These results suggested that H6N6 viruses can act as an intermediary in the evolution of H5N6, H6N6, and H7N9 viruses. Animal experiments demonstrated that the 10 representative H6N6 viruses showed low pathogenicity in chickens and were capable of infecting mice without prior adaptation. Our findings suggest that H6N6 viruses play an important role in the evolution of AIVs, and it is necessary to continuously monitor and evaluate the potential epidemic of the H6N6 subtype viruses.</p

Additional file 1 of Characterizing neuroinflammation and identifying prenatal diagnostic markers for neural tube defects through integrated multi-omics analysis

Additional file 1: Figure S1. Schematic diagram of data analysis and experiments validation in this study. NTDs, neural tube defects; CNS, central nervous system; RA, retinoic acid; GW, gestational week

Co-regulation of miRNAs to angiogenic factors.

<p>CNE cells were induced with or without DFOM (6–1), transfected with <i>miR-15b, miR-16, miR-20a</i>, or <i>miR-20b</i> (6–2) and transfected with Construct I or Construct II (6–3). Cell lysate was collected, and the expression of angiogenic factors uPAR (A), COX2 (B), c-MET (C), and PTN (D) were determined by Western Blotting.</p