Journal: eBioMedicine

Article Title: Autoantibodies against type I interferons in patients with zoonotic H7N9 influenza: an observational case–control study

doi: 10.1016/j.ebiom.2026.106387

Figure Lengend Snippet: Autoantibodies binding to IFNα 2 , IFNβ 1b and IFNω in patients with H7N9 infection and healthy controls. (A) Age and sex distribution of the three study groups. For each age group, the number of individuals positive for autoantibodies neutralising at least one tested IFN-I (IFNα 2 , IFNβ 1b , IFNω) at low concentrations is indicated in red. M, male; F, female; nAb+, positive for IFN-I-neutralising autoantibodies. (B) Detection of IgG autoantibodies binding to IFNα 2 , IFNβ 1b or IFNω in serum samples by multiplex bead-based assay. Samples with a Z-score >7 were considered positive for IFN-I-binding autoantibodies. Measurements were performed without technical replicates because of limited sample availability. (C) Prevalence of IFN-I-binding autoantibodies by IFN type and study group. IFNα 2 ± IFNβ 1b ± IFNω, positive for autoantibodies binding to at least one of the tested IFN-I; IFNα 2 + IFNω, positive for autoantibodies binding to both IFNα 2 and IFNω.

Article Snippet: Serum was diluted 1:50 in DMEM supplemented with 10% FCS and penicillin/streptomycin, and pre-incubated for 1 h at room temperature with one of the following IFN concentrations: (i) IFNα 2 (Novus Biologicals, NBP2-34971) at 10 ng/ml or 0.5 ng/ml; (ii) IFNβ 1b (PBL Assay Science, 11420-1) at 1 ng/ml or 0.25 ng/ml; (iii) IFN-ω (Novus Biologicals, NBP2-35893) at 10 ng/ml or 0.2 ng/ml.

Techniques: Binding Assay, Infection, Multiplex Assay, Bead-based Assay

Journal: eBioMedicine

Article Title: Autoantibodies against type I interferons in patients with zoonotic H7N9 influenza: an observational case–control study

doi: 10.1016/j.ebiom.2026.106387

Figure Lengend Snippet: Autoantibodies neutralising IFN-I in patients with H7N9 infection and healthy controls. (A) Luciferase-based reporter assay to assess the capacity of autoantibody positive sera to neutralise IFNα 2 (10 or 0.5 ng/ml), IFNβ 1b (1 or 0.25 ng/ml) or IFNω (10 or 0.2 ng/ml). Each sample was tested in biological duplicates and the mean values are shown. Samples were classified as neutralising if the mean of the relative luciferase activities was below 25% (dotted line) of the mean of the negative pool (four autoantibody-negative control sera). All sera positive for IFN-I-binding autoantibodies were tested; numbers are indicated above the graphs. Lines connect measurements of neutralising activity from the same serum sample at low and high IFN concentrations. (B) Prevalence of autoantibodies neutralising low IFN concentrations (IFNα 2 : 0.5 ng/ml, IFNβ 1b : 0.25 ng/ml; IFNω: 0.2 ng/ml) by IFN type and study group. IFNα 2 ± IFNβ 1b ± IFNω, positive for autoantibodies neutralising at least one tested IFN-I; IFNα 2 + IFNω, positive for autoantibodies neutralising both IFNα 2 and IFNω. (C) Area-proportional Venn diagrams illustrating the absolute numbers of samples with autoantibodies neutralising high and low concentrations of IFNα 2 (10 or 0.5 ng/ml), IFNβ 1b (1 or 0.25 ng/ml) or IFNω (10 or 0.2 ng/ml). Venn diagrams were created with BioVenn ( https://www.biovenn.nl/index.php ).

Article Snippet: Serum was diluted 1:50 in DMEM supplemented with 10% FCS and penicillin/streptomycin, and pre-incubated for 1 h at room temperature with one of the following IFN concentrations: (i) IFNα 2 (Novus Biologicals, NBP2-34971) at 10 ng/ml or 0.5 ng/ml; (ii) IFNβ 1b (PBL Assay Science, 11420-1) at 1 ng/ml or 0.25 ng/ml; (iii) IFN-ω (Novus Biologicals, NBP2-35893) at 10 ng/ml or 0.2 ng/ml.

Techniques: Infection, Luciferase, Reporter Assay, Negative Control, Binding Assay, Activity Assay

Journal: eBioMedicine

Article Title: Autoantibodies against type I interferons in patients with zoonotic H7N9 influenza: an observational case–control study

doi: 10.1016/j.ebiom.2026.106387

Figure Lengend Snippet: Association between the presence of IFN-I-neutralising autoantibodies and H7N9 infection. (A) The association between age, sex and IFN-I-neutralising autoantibodies in patients with H7N9 infection or in the two control groups combined (poultry workers + close contacts) was assessed using Firth's penalised logistic regression. Predicted probabilities for the presence of autoantibodies with 95% confidence intervals (CIs, shaded areas around the curve) are shown across participant age for men and women. To visualise the modelled probabilities in relation to the underlying data, we overlaid sex-specific age density distributions beneath the predicted probability curves. (B) Odds ratios (OR) with 95% CIs for the presence of autoantibodies neutralising low IFN concentrations in patients compared to healthy controls, adjusted for age and sex, determined by Firth’s penalised logistic regression models. See also for the results of the logistic regression analyses and for unadjusted estimates. IFNα 2 ± IFNω ± IFNβ 1b , positive for autoantibodies neutralising at least one of the tested IFN-I; IFNα 2 ± IFNω, positive for autoantibodies neutralising IFNα 2 and/or IFNω; ∗∗∗∗, p < 0.0001 (Firth’s penalised logistic regression).

Article Snippet: Serum was diluted 1:50 in DMEM supplemented with 10% FCS and penicillin/streptomycin, and pre-incubated for 1 h at room temperature with one of the following IFN concentrations: (i) IFNα 2 (Novus Biologicals, NBP2-34971) at 10 ng/ml or 0.5 ng/ml; (ii) IFNβ 1b (PBL Assay Science, 11420-1) at 1 ng/ml or 0.25 ng/ml; (iii) IFN-ω (Novus Biologicals, NBP2-35893) at 10 ng/ml or 0.2 ng/ml.

Techniques: Infection, Control

Journal: eBioMedicine

Article Title: Autoantibodies against type I interferons in patients with zoonotic H7N9 influenza: an observational case–control study

doi: 10.1016/j.ebiom.2026.106387

Figure Lengend Snippet: Neutralising sera block the antiviral effect of IFNα 2 in cell culture infected with IAV. Antiviral activity of IFNα 2 (5 ng/ml) against IAV (PR8-GFP, MOI 1) alone or in the presence of serially diluted IFN-I-neutralising sera (n = 19), autoantibody-negative sera (n = 4), or a monoclonal anti-IFNα 2 antibody in A549 cells. Infection rates (GFP + /DAPI + cells) at 7 h post-infection were normalised to untreated, infected cells. The dotted line indicates the reduction of infected cells after IFN treatment alone. If possible, the mean of two independent experiments is shown. Sufficient material was available for 12 out of 19 samples.

Article Snippet: Serum was diluted 1:50 in DMEM supplemented with 10% FCS and penicillin/streptomycin, and pre-incubated for 1 h at room temperature with one of the following IFN concentrations: (i) IFNα 2 (Novus Biologicals, NBP2-34971) at 10 ng/ml or 0.5 ng/ml; (ii) IFNβ 1b (PBL Assay Science, 11420-1) at 1 ng/ml or 0.25 ng/ml; (iii) IFN-ω (Novus Biologicals, NBP2-35893) at 10 ng/ml or 0.2 ng/ml.

Techniques: Blocking Assay, Cell Culture, Infection, Activity Assay

Journal: Nucleic Acids Research

Article Title: Generation of precise and accurate engineered circRNAs using enzymatic ligation

doi: 10.1093/nar/gkag405

Figure Lengend Snippet: Evaluation of circularization efficiency and accuracy by different ligases. ( A ) Schematic depicting requirements and features of DNA ligase, RNA ligase 1, and RNA ligase 2. ( B ) Workflow of circRNA generation using enzymatic ligation and RNase R based purification which can be improved by addition of poly(A) tails to linear RNAs. ( C ) 3% urea–PAGE showed that all ligases were able to circularize 5′-monophosphate RNAs. Boxed bands depict the circRNAs that run slower than their linear counterparts. Contaminating RNAs of lower and higher size than circular or linear RNA were also observed suggesting that poly(A) tailing and RNase R treatments were insufficient to degrade them. Efficiency of ligation was calculated as percentage yields of RNAs remaining after all treatments divided by input RNA for each ligation reaction. CircRNAs derived from modified transcription templates (mod) had higher efficiencies particularly for DNA ligase and RNA ligase 2 than those derived from unmodified templates (unmod). RNA ligase 2 had the highest circularization efficiency, especially with circRNAs derived from mod templates in presence of an RNA splint. Representative data are from a mean of n = 3 technical replicates with SEM; (*) P ≤.05 (unpaired t -test). ( D ) Sanger sequencing of ligation junctions showed accurate sequences with circRNAs derived from modified transcription templates using all ligases. CircRNAs made with RNA Ligase 1 had errors with the linear RNAs from unmodified templates which were corrected with the use of modified templates.

Article Snippet: Five micrograms of RNA were poly(A)-tailed using 5 units of Escherichia coli poly(A) polymerase (NEB #M0276L) for 30 min at 37°C.

Techniques: Ligation, Purification, Derivative Assay, Modification, Sequencing

Journal: Nucleic Acids Research

Article Title: Generation of precise and accurate engineered circRNAs using enzymatic ligation

doi: 10.1093/nar/gkag405

Figure Lengend Snippet: Purification of circRNAs and extending the RNA ligase 2 (RL2)-dependent circularization method to other RNAs. ( A ) CircRNAs synthesized with RNA ligase 2 using DNA splint were purified using three different approaches: from 3% urea–PAGE using crush and soak method, or from EX E-gels either using the crush and soak method or using column-based kit. As a control, linear RNAs were also extracted using the same methods. CircRNAs extracted from 3% urea–PAGE or EX E-gel using a crush and soak method had more intact circRNAs with less nicking compared to those extracted from EX E-gel using column-based kits. Linear RNAs on the other hand remained intact with each of the approaches. ( B ) Schematic of RNase-H based circularity confirmation assay that uses a short ssDNA probe which cleaves intact circRNAs into a single linear band, while nicked circRNAs or linear RNAs are cut into two shorter bands. ( C ) RNase-H based assay confirmed circularity of EGFP-IRES circRNAs. Linear RNAs were cleaved into two shorter bands of expected sizes while circRNAs derived from modified DNA templates were linearized to the size of full-length linear precursor. ( D, E ) 5′-monophosphate linear precursors of human immunodeficiency virus (HIV) and mCherry were ligated using RNA ligase 2 and respective DNA splints. For circHIV, urea–PAGE purification of circRNAs derived from modified templates had the highest yields with the least contaminating RNAs. Yields of mCherry circRNAs were much higher with polyA + RNase R approach on RNAs from modified template ligated using RNA ligase 2, however urea–PAGE showed higher and lower sized undesired RNAs. Representative data are from a mean of n = 3 technical replicates with SEM. ( F ) Sanger sequencing confirmed accuracy of circRNAs. Clean chromatograms were observed for ligation junctions of both HlV and mCherry circRNAs derived from modified DNA templates purified either through poly(A) tailing and RNase R treatment or from urea–PAGE purification.

Article Snippet: Five micrograms of RNA were poly(A)-tailed using 5 units of Escherichia coli poly(A) polymerase (NEB #M0276L) for 30 min at 37°C.

Techniques: Purification, Synthesized, Control, Rnase H Assay, Derivative Assay, Modification, Virus, Sequencing, Ligation

Journal: iScience

Article Title: ISGylation is disrupted by UBA7 gene variants identified in individuals with neurodevelopmental disorder phenotypes

doi: 10.1016/j.isci.2026.115454

Figure Lengend Snippet: UBA7 variants from individuals with neurodevelopmental disorders alter protein structure (A) Pedigrees of the families studied in this article indicate the affected individuals 1, 2, and 3. A legend is provided for the symbols used. (B) Diagram of UBA7 protein domains and their functions. The location of the identified variants in the structure is indicated. IAD: inactive adenylation domain, AAD: active adenylation domain, FCCH: first catalytic cysteine half-domain, SCCH: second catalytic cysteine half-domain, UFD: ubiquitin-fold domain. (C) Alpha-fold models of UBA7 protein resulting from UBA7 wild type, or p.Trp311∗ and p.Lys709Serfs∗45 variants. The Red helix represents the added residues resulting from the p.Lys709Serfs∗45 frameshift variant. (D) Structure of wild-type UBA7 in complex with UBE2L6 and ISG15 (left; EMB-16891; pdb: 8OIF ). The top-view (middle) and zoomed areas (right) highlight the adenylation pocket and location of the p.Val548Leu mutation shown in red.

Article Snippet: NEB 5-alpha Competent E. coli (DH5α) , New England Biolabs , Cat# C2987.

Techniques: Ubiquitin Proteomics, Variant Assay, Mutagenesis