Table1_Molecular Diagnosis and Prenatal Phenotype Analysis of Eight Fetuses With Ciliopathies.XLSX

Human ciliopathies are hereditary conditions caused by variants in ciliary-associated genes. Ciliopathies are often characterized by multiple system defects. However, it is not easy to make a definite diagnosis in the prenatal period only based on the imageology. In this report, eight new prenatal cases from five unrelated families diagnosed with ciliopathies were systematically examined. The clinical manifestations of these fetuses showed such prenatal diagnostic features as occipital encephalocele, and polydactyly and polycystic kidneys. Situs inversus caused by CPLANE1 variant was first reported. In Family 1 and Family 3, homozygous variants of CPLANE1 and NPHP4 caused by consanguineous marriage and uniparental disomy were detected by whole-exome sequencing, respectively. In Family 2, Family 4 and Family 5, compound heterozygotes of TMEM67 and DYNC2H1 including two novel missense variants and one novel nonsense variant were identified. The distribution of pathogenic missense variants along TMEM67 gene mainly clustered in the extracellular cysteine rich region, extracellular area with unknown structure, and the transmembrane regions. Genotype-phenotype relationship between CPLANE1 and TMEM67 genes was concluded. This report describes new clinical manifestations and novel variants in CPLANE1, TMEM67, NPHP4, and DYNC2H1.</p

Cell viability assay.

Viability was determined with a MTT assay at 48 h postinfection. The cell viability of each group is expressed relative to the normal RD cell control, which was defined as 100% survival. The data shown represent the means ± SD of three independent experiments (**p < 0.01).</p

Transfection efficiency of siRNA in RD cells.

<p>(A) Cellular distribution of BLOCK-iT Fluorescent Oligo in transfected RD cells. RD cells were transfected with different concentrations of BLOCK-iT Fluorescent Oligo and 2 μl of Lipofectamine 2000. At 24 h after transfection, the cells were observed under a fluorescence microscope. (B) RD cells transfected with BLOCK-iT Fluorescent Oligo were quantified with flow cytometry. The cells were assayed in three independent experiments.</p

RNAi inhibits viral RNA.

<p>RD cells were treated with each siRNA and then infected with strain EV71-2006-52-9 at an MOI of 0.01. At 24 h postinfection, the viral RNA was extracted and analyzed with real-time RT–PCR. The data shown represent the means ± SD of three independent experiments (**p < 0.01).</p

RNAi inhibits viral protein.

<p>RD cells transfected with siRNA were infected with strain EV71-2006-52-9 at an MOI of 0.01. At 36 h postinfection, total protein was extracted and analyzed with western blotting. β-Actin was used as the internal loading control. The protein measurements were made in three independent experiments.</p

Interferon pathway was not activated by siRNA.

The levels of IFN-α (A) and IFN-β (B) were determined by measuring the absorbance at 490 nm. The data shown represent the means ± SD of three independent experiments.</p

Table_1_Differential Expression and Prognostic Correlation of Immune Related Factors Between Right and Left Side Colorectal Cancer.docx

BackgroundGrowing evidence suggests that colorectal cancer (CRC) should be considered a heterogeneous disease. The right side (RCC) and left side (LCC) colorectal cancer have different clinical characteristics and immune landscapes. The aim of this study was to analyze differential expression and prognostic correlation of immune-related factors between RCC and LCC.MethodsThe gene expression profile and clinical characteristics of CRC patients were retrieved from The Cancer Genome Atlas data portal (n=525). Using a deconvolution algorithm, immune cell infiltration in RCC and LCC based on the RNA-seq data was analyzed. Differentially expressed genes (DEGs) were obtained by performing differential gene expression analysis. Immune-related DEGs were derived by the intersection with immune-related factors downloaded from the IMMPORT database. To further validate the findings, we applied immunohistochemical (IHC) staining of a CRC tissue microarray (TMA). The distribution of immune cells in RCC and LCC and changes in the expression of immune molecules on their membranes were verified. The expression levels of circulating cytokines were measured by flow cytometry to detect the cytokines secreted by immune cells in RCC and LCC. Furthermore, to reveal the prognostic value of differential immune factors on RCC and LCC patients, survival analysis based on mRNA levels using TCGA cohort and survival analysis using protein levels was performed using our CRC patients.ResultsThe infiltration of immune cells differed between RCC and LCC, the infiltration degree of macrophages M0 was significantly higher in LCC, while the infiltration degree of differentiated macrophages M1 and M2, CD4+ T and CD8+ T cells was significantly higher in RCC. The expression of related molecules by immune cells also differed between RCC and LCC. The expression of 7 genes in RCC was higher than that in LCC, which were CCR5, CD209, CD8A, HCK, HLA-DPB1, HLA-DQA1, HLA-DRA, respectively. Meanwhile, the expression of 2 genes in LCC was higher than in RCC, which were IL-34 and PROCR. Patients with RCC having high expression of HLA-DQA1 mRNA or proteins had better survival and LCC patients with high expression of IL 34 mRNA or protein had better survival.ConclusionsIn this study, we comprehensively compared differences in immune cells and regulating factors between left and right colorectal cancer. Different expression patterns and their effects on survival were identified. The analysis of immune-related factors may provide a theoretical basis for precise immunotherapy of RCC and LCC.</p

siRNAs protect cells against EV71-induced cytopathic effects.

<p>Morphological changes in RD cells were observed after infection. Cells were transfected with each siRNA at a final concentration of 60 nM and then infected with strain EV71-2006-52-9 at an MOI of 0.01. Micrographs were taken at 48 h postinfection under an inverted microscope. The tests were performed in three independent experiments.</p

Sequences of the synthesized siRNA molecules and their positions in the EV71 2A^pro genomic region.

<p>Sequences of the synthesized siRNA molecules and their positions in the EV71 2A^pro genomic region.</p

Table_3_Differential Expression and Prognostic Correlation of Immune Related Factors Between Right and Left Side Colorectal Cancer.xlsx

BackgroundGrowing evidence suggests that colorectal cancer (CRC) should be considered a heterogeneous disease. The right side (RCC) and left side (LCC) colorectal cancer have different clinical characteristics and immune landscapes. The aim of this study was to analyze differential expression and prognostic correlation of immune-related factors between RCC and LCC.MethodsThe gene expression profile and clinical characteristics of CRC patients were retrieved from The Cancer Genome Atlas data portal (n=525). Using a deconvolution algorithm, immune cell infiltration in RCC and LCC based on the RNA-seq data was analyzed. Differentially expressed genes (DEGs) were obtained by performing differential gene expression analysis. Immune-related DEGs were derived by the intersection with immune-related factors downloaded from the IMMPORT database. To further validate the findings, we applied immunohistochemical (IHC) staining of a CRC tissue microarray (TMA). The distribution of immune cells in RCC and LCC and changes in the expression of immune molecules on their membranes were verified. The expression levels of circulating cytokines were measured by flow cytometry to detect the cytokines secreted by immune cells in RCC and LCC. Furthermore, to reveal the prognostic value of differential immune factors on RCC and LCC patients, survival analysis based on mRNA levels using TCGA cohort and survival analysis using protein levels was performed using our CRC patients.ResultsThe infiltration of immune cells differed between RCC and LCC, the infiltration degree of macrophages M0 was significantly higher in LCC, while the infiltration degree of differentiated macrophages M1 and M2, CD4+ T and CD8+ T cells was significantly higher in RCC. The expression of related molecules by immune cells also differed between RCC and LCC. The expression of 7 genes in RCC was higher than that in LCC, which were CCR5, CD209, CD8A, HCK, HLA-DPB1, HLA-DQA1, HLA-DRA, respectively. Meanwhile, the expression of 2 genes in LCC was higher than in RCC, which were IL-34 and PROCR. Patients with RCC having high expression of HLA-DQA1 mRNA or proteins had better survival and LCC patients with high expression of IL 34 mRNA or protein had better survival.ConclusionsIn this study, we comprehensively compared differences in immune cells and regulating factors between left and right colorectal cancer. Different expression patterns and their effects on survival were identified. The analysis of immune-related factors may provide a theoretical basis for precise immunotherapy of RCC and LCC.</p