Discovering antiviral restriction factors and pathways using genetic screens

Viral infections activate the powerful interferon (IFN) response that induces the expression of several hundred IFN stimulated genes (ISGs). The principal role of this extensive response is to create an unfavourable environment for virus replication and to limit spread; however, untangling the biological consequences of this large response is complicated. In addition to a seemingly high degree of redundancy, several ISGs are usually required in combination to limit infection as individual ISGs often have low to moderate antiviral activity. Furthermore, what ISG or combination of ISGs are antiviral for a given virus is usually not known. For these reasons, and since the function(s) of many ISGs remains unexplored, genome-wide approaches are well placed to investigate what aspects of this response result in an appropriate, virus-specific phenotype. This review discusses the advances screening approaches have provided for the study of host defence mechanisms, including clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9), ISG expression libraries and RNA interference (RNAi) technologies.

Sperm-borne miR-202 targets SEPT7 and regulates first cleavage of bovine embryos via cytoskeletal remodeling

In mammals, sperm-borne regulators can be transferred to oocytes during fertilization and have different effects on the formation of pronuclei, the first cleavage of zygotes, the development of preimplantation embryos and even the metabolism of individuals after birth. The regulatory role of sperm microRNAs (miRNAs) in the development of bovine preimplantation embryos has not been reported in detail. By constructing and screening miRNA expression libraries, we found that miR-202 was highly enriched in bovine sperm. As a target gene of miR-202, co-injection of SEPT7 siRNA can partially reverse the accelerated first cleavage of bovine embryos caused by miR-202 inhibitor. In addition, both a miR-202 mimic and SEPT7 siRNA delayed the first cleavage of somatic cell nuclear transfer (SCNT) embryos, suggesting that miR-202-SEPT7 mediates the delay of first cleavage of bovine embryos. By further exploring the relationship between miR-202 /SEPT7, HDAC6 and acetylated α-tubulin during embryonic development, we investigated how sperm-borne miR-202 regulated the first cleavage process of bovine embryos by SEPT7 and demonstrated the potential of sperm-borne miRNAs to improve the efficiency of SCNT.

Autoimmune antibodies correlate with immune checkpoint therapy-induced toxicities

Immune checkpoint (IC) therapy provides substantial benefits to cancer patients but can also cause distinctive toxicities termed immune-related adverse events (irAEs). Biomarkers to predict toxicities will be necessary to improve management of patients receiving IC therapy. We relied on serological analysis of recombinant cDNA expression libraries to evaluate plasma samples from patients treated with IC therapy and identified autoantibodies, both in pretreatment and on-treatment samples prior to the development of irAEs, which correlate with the development of immune-related hypophysitis (anti-GNAL and anti-ITM2B autoantibodies) and pneumonitis (anti-CD74 autoantibody). We developed an enzyme-linked immunosorbent assay and tested additional patient samples to confirm our initial findings. Collectively, our data suggest that autoantibodies may correlate with irAEs related to IC therapy, and specific autoantibodies may be detected early for the management of irAEs.

Auto-Antigens Targeted By Auto-Antibodies IgG on the Bone Marrow Hematopoietic Cells of the Patients with Immuno-Related Pancytopenia

Objective: To screen and identify the auto-antigens targeted by auto-antibodies IgG on the bone marrow hematopoietic cells of the patients with immuno-related pancytopenia(IRP). Background: Immune-related pancytopenia (IRP) is characterized by pancytopenia caused by autoantibody-mediated bone marrow destruction or suppression.. Our previous studies have demonstrated that DC2 increased in the bone marrow of IRP patients, which promote Th0 cells to polarize to Th2 cells and cause the over-function of B lymphocytes.These B cells produce auto-antibodies that mediated BM destruction.. But the antigens that induced DC2 elevated are not clear at present, our group found several target antigens by western blot method before, including G protein coupled receptor (GPCR) 156 variants, P chain of red blood cells with band three proteins, lactoferrin and WD repeat sequences of proteins.Autoimmune diseases often have several or even dozens of target antigens, so this experiment adopts the method of cDNA expression library of serological analysis (SEREX) to sceen antigens targeted by IRP autoantibodies and further verify their antigenicity. Materials and Methods: We established the recombinant cDNA expression libraries derived from K562 cells. We performed immunoblotting analysis of this library with sera from patients and normal healthy controls. The presumptive phage expressed autoantigen proteins were initially proposed based on the differential affinity to the sera antibodies of patients versus normal healthy controls,the plagues which were positive with IRP serum but negative with normal control serum were positive screened plagues. The insert fragment of each positive plague was sequenced. After the gene sequence confirmation, the sequence of FTL gene, TRMT112 gene and TRAPPC gene were amplified by polymerase chain reaction (PCR) then inserted into Peasy-E1 vector separately. Each recombinant plasmid was transformed into E.coli BL-21 and was identified by PCR and sequencing. After gene identification, the right recombinant plasmid was transformed into E.coli BL21 and the bacteria was induced by isopropyl-β-d-thiogalactoside (IPTG) to express. The expected protein was identified by Western blot and purified by ProteinIso Ni-NTA Resin. Then we coated ELISA plate with purified FTL protein and EPOR protein respectively, and detected the titer of the serum antibody of IRP patients and normal controls by indirect ELISA. Finally, the clinical index of 121 IRP patients were analysed. Results: 1.We established the recombinant cDNA expression libraries derived from K562 cells successfully. The library has good capacity, ligation efficiency and recombination efficiency. 2. We screend 11 positive plagues by immunoblotting, the seven of them were sequenced and blasted successfully. 3.3 fusion protein was expressed and confirmed by Western blot. The purified protein was obtained by ProteinIso Ni-NTA Resin. 4. The titer of antibody against the FTL and EPOR proteins was higher in untreated IRP than in recovered IRP patients and in normal control. Conclusion: We screened 7 proteins that maybe autoantigens of IRP by SEREX. Furthermore, we expressed and purified 3 fusion proteins. Finally we identified the antigenicity of the FTL and epor proteins. Disclosures No relevant conflicts of interest to declare.

Ten-year anniversary of CD3δ deficiency

CD3δ deficiency was first identified a decade ago by using differential expression libraries derived from thymocytes of patients with severe combined immunodeficiency. The phenotype was surprisingly severe unlike the mouse model or other human CD3 deficiencies known at that time.