Journal: Journal of bioscience and bioengineering

Article Title: Novel transgenic Chlamydomonas reinhardtii strain with retargetable genomic transgene integration using Cre-loxP system.

doi: 10.1016/j.jbiosc.2021.07.006

Figure Lengend Snippet: FIG. 5. Analysis of IFNa-4 expression in Chlamy/P[IFN] cells. (A) RT-PCR analysis. Lane M, molecular weight markers (FX174-HincII digests); lane W, distilled water as a negative control; lane Fo, Chlamy/S; FE, Chlamy/P[IFN]-FE (i); CA, Chlamy/P[IFN]-CA (ii). CLBP gene was used as an internal control. (B) Concentration of IFNa-4 secreted in the culture medium. The cell clones (1 106 cells/mL) were cultured for 6 days. IFN concentrations in the medium were measured every 2 days using a commercial IFN ELISA kit. (C) Evaluation of IFNa-4 production stability. Passages were repeated every 2e3 days in the absence of drug pressure. The Chlamy/P[IFN]-FE cells were seeded in a T-25 flask containing 10 mL of medium at a density of 1 106 cells/mL. The culture medium was replaced with a fresh one every 2e3 days, and the cells were re-seeded at the same density. The cell culture was repeated for 30 days. IFNa-4 concentration in the medium was measured at passages 1 (days 0e2), 6 (days 13e15), and 12 (days 28e30). IFNa-4 productivities relative to that at passage 1 are indicated. (D) Western blot analysis of IFNa-4. Lane N, parental CC-406; lane Fo, Chlamy/S; lane FE, Chlamy/P[IFN]-FE; lane CA, Chlamy/P[IFN]-CA; lane P, commercial IFN protein (1 mg/mL). Data are expressed as mean SD (n ¼ 3).

Article Snippet: Measurement of IFNa-4 concentration IFNa-4 concentration was determined by an enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (RayBio Canine IFN-alpha ELISA Kit; RayBiotech Life, Peachtree Corners, GA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Negative Control, Control, Concentration Assay, Clone Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Scientific Reports

Article Title: Exome sequencing identifies novel compound heterozygous IFNA4 and IFNA10 mutations as a cause of impaired function in Crohn’s disease patients

doi: 10.1038/srep10514

Figure Lengend Snippet: Genotype association study of the IFNA10 and IFNA4 variants in expanded CD cases versus controls.

Article Snippet: ELISAs were performed using the human IFNA4 and IFNA10 kit (for IFNA4, Catalog No. SEA175Hu; for IFNA10, Catalog No. SEG971Hu; USCN Life Science, Houston, TX, USA) according to the manufacturer’s instructions.

Techniques:

Journal: Scientific Reports

Article Title: Exome sequencing identifies novel compound heterozygous IFNA4 and IFNA10 mutations as a cause of impaired function in Crohn’s disease patients

doi: 10.1038/srep10514

Figure Lengend Snippet: Association of IFNA4 variant with responses to medical treatment in CD Patients.

Article Snippet: ELISAs were performed using the human IFNA4 and IFNA10 kit (for IFNA4, Catalog No. SEA175Hu; for IFNA10, Catalog No. SEG971Hu; USCN Life Science, Houston, TX, USA) according to the manufacturer’s instructions.

Techniques: Variant Assay

Journal: Scientific Reports

Article Title: Exome sequencing identifies novel compound heterozygous IFNA4 and IFNA10 mutations as a cause of impaired function in Crohn’s disease patients

doi: 10.1038/srep10514

Figure Lengend Snippet: Sequencing and functional analysis of the IFNA4 and IFNA10 variants. ( A ) Confirmation of the IFNA4 and IFNA10 heterozygous mutations in CD patients compared to healthy subjects (wild-type alleles) using Sanger sequencing. ( B ) The mutation site and amino acids alteration of the IFNA4 and IFNA10 variants. ( C-D ) Huh7 cells were transiently transfected with plasmids expressing IFNA4, IFNA10, or mutants and then infected with HCV; the replicon copies were calculated using real-time PCR and PLV as a control; The supernatants of the cell culture were analyzed using ELISA. ( E ) Real-time PCR analyzed the mRNA expressions of IFNA4, IFNA10, FOXP3, TGFβ, IL10, IL2, IL6 and IL17 in NCM460 cells after transfection of the siRNA targeting IFNA4 and IFNA10. The scramble siRNA sequences were used as a negative control. The results indicate the mean ± SD of three independent experiments (* P < 0.05).

Article Snippet: ELISAs were performed using the human IFNA4 and IFNA10 kit (for IFNA4, Catalog No. SEA175Hu; for IFNA10, Catalog No. SEG971Hu; USCN Life Science, Houston, TX, USA) according to the manufacturer’s instructions.

Techniques: Sequencing, Functional Assay, Mutagenesis, Transfection, Expressing, Infection, Real-time Polymerase Chain Reaction, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Scientific Reports

Article Title: Exome sequencing identifies novel compound heterozygous IFNA4 and IFNA10 mutations as a cause of impaired function in Crohn’s disease patients

doi: 10.1038/srep10514

Figure Lengend Snippet: ELISA analysis of serum IFNA4 and IFNA10 levels. A Wilcoxon two-sample test was performed to evaluate the differences between 47 CD patients and 95 healthy controls. Comparison of the serum IFNA4 level between CD patients and healthy controls ( A ) and between wild-type and mutant alleles in CD patients and healthy controls ( B ) N indicates normal healthy control. Comparison of the serum IFNA10 level between CD patients and healthy controls ( C ) and between wild-type and mutant alleles in CD patients and healthy controls ( D ). ( E-F ) Comparison of serum IFNA4 and IFNA10 between IFN single-subtype and dual-subtype variants in CD patients, respectively (* P < 0.05).

Article Snippet: ELISAs were performed using the human IFNA4 and IFNA10 kit (for IFNA4, Catalog No. SEA175Hu; for IFNA10, Catalog No. SEG971Hu; USCN Life Science, Houston, TX, USA) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Mutagenesis, Control

Journal: Scientific Reports

Article Title: Exome sequencing identifies novel compound heterozygous IFNA4 and IFNA10 mutations as a cause of impaired function in Crohn’s disease patients

doi: 10.1038/srep10514

Figure Lengend Snippet: IFNA4 and IFNA10 control DSS-induced acute colitis. ( A-B ) Left panel showed the experimental protocol applied to induce mice acute colitis and the administration of IFNA subtypes; and right panel showed the mice body weight curve of DSS-induced acute colitis between the study groups. ( C ) The study mice were monitored for bleeding scores by a quantitative analysis for the presence of rectal blood. ( D-E ) Colon sections were fixed in formalin and stained with HE; the sections were analyzed for the inflammation score. ( F ) Real-time PCR analyzed the mRNA expressions of CCL2, CCL5, CD70, CXCL10 and TNFSF10 in colon tissues on DSS model. The data are given as the mean ± SD (n = 10, * P < 0.05).

Article Snippet: ELISAs were performed using the human IFNA4 and IFNA10 kit (for IFNA4, Catalog No. SEA175Hu; for IFNA10, Catalog No. SEG971Hu; USCN Life Science, Houston, TX, USA) according to the manufacturer’s instructions.

Techniques: Control, Staining, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Exome sequencing identifies novel compound heterozygous IFNA4 and IFNA10 mutations as a cause of impaired function in Crohn’s disease patients

doi: 10.1038/srep10514

Figure Lengend Snippet: IFNA4 and IFNA10 inhibit acute colitis through induction of Treg cells. ( A-B ) FACS analysis of isolated cells from mice DSS-induce acute colitis groups, upper panels indicate first gate of lower panels. (C-D) Quantification analysis of upper FACS results, as CD4 + CD25 + cells in CD3 + and Foxp3 + cells in CD4 + T cells in the colon tissues, 5 mice in each group and analyzed the samples in triplicate, bars represent the mean ± SD.

Article Snippet: ELISAs were performed using the human IFNA4 and IFNA10 kit (for IFNA4, Catalog No. SEA175Hu; for IFNA10, Catalog No. SEG971Hu; USCN Life Science, Houston, TX, USA) according to the manufacturer’s instructions.

Techniques: Isolation