Characterization of SHARPIN knockout Syrian hamsters developed using CRISPR/Cas9 system

Background : SHARPIN (SHANK- associated RH domain interactor) is a component ofthe linear ubiquitination complex that regulates the NF- κB signaling pathway. To betterunderstand the function of SHARPIN, we sought to establish a novel geneticallyengineered Syrian hamster with SHARPIN disruption using the CRISPR/Cas9 system.Methods : A single- guide ribonucleic acid targeting exon 1 of SHARPIN gene was designedand constructed. The zygotes generated by cytoplasmic injection of the Cas9/gRNA ribonucleoprotein were transferred into pseudopregnant hamsters. Neonatalmutants were identified by genotyping. SHARPIN protein expression was detectedusing Western blotting assay. Splenic, mesenteric lymph nodes (MLNs), and thymicweights were measured, and organ coefficients were calculated. Histopathologicalexamination of the spleen, liver, lung, small intestine, and esophagus was performedindependently by a pathologist. The expression of lymphocytic markers and cytokineswas evaluated using reverse transcriptase- quantitative polymerase chain reaction.Results : All the offspring harbored germline- transmitted SHARPIN mutations.Compared with wild- type hamsters, SHARPIN protein was undetectable in SHARPIN −/−hamsters. Spleen enlargement and splenic coefficient elevation were spotted inSHARPIN −/− hamsters, with the descent of MLNs and thymuses. Further, eosinophilinfiltration and structural alteration in spleens, livers, lungs, small intestines, and esophagiwere obvious after the deletion of SHARPIN. Notably, the expression of CD94 and CD22 was downregulated in the spleens of knockout (KO) animals. Nonetheless,the expression of CCR3, CCL11, Il4 , and Il13 was upregulated in the esophagi. Theexpression of NF- κB and phosphorylation of NF- κB and IκB protein significantly diminishedin SHARPIN −/− animals.Conclusions : A novel SHARPIN KO hamster was successfully established using theCRISPR/Cas9 system. Abnormal development of secondary lymphoid organs andeosinophil infiltration in multiple organs reveal its potential in delineating SHARPINfunction and chronic inflammation.

Immune checkpoint inhibitors in cancer therapy

In recent years immune checkpoint inhibitors have garnered attention as being one of the most promising types of immunotherapy on the horizon. There has been particular focus on the immune checkpoint molecules, cytotoxic Tlymphocyte antigen-4（CTLA-4） and programmed cell death protein 1（PD-1） which have been shown to have potent immunomodulatory effects through their function as negative regulators of T cell activation. CTLA-4, through engagement with its ligands B7-1（CD80） and B7-2（CD86）, plays a pivotal role in attenuating the activation of naive and memory T cells. In contrast, PD-1 is primarily involved in modulating T cell activity in peripheral tissues via its interaction with PD-L1 and PD-L2. The discovery of these negative regulators of the immune response was crucial in the development of checkpoint inhibitors. This shifted the focus from developing therapies that targeted activation of the host immune system against cancer to checkpoint inhibitors, which aimed to mediate tumor cell destruction through the removal of coinhibitory signals blocking anti-tumor T cell responses.

Discovery of Digenic Mutation, KCNH2 c.1898A > C and JUP c.916dupA, in a Chinese Family with Long QT Syndrome via Whole-Exome Sequencing

Long QT syndrome(LQTS),which is caused by an ion channel–related gene mutation,is a malignant heart disease with a clinical course of a high incidence of ventricular fi brillation and sudden cardiac death in the young.Mutations in KCNH2(which encodes potassium voltage-gated channel subfamily H member 2)are responsible for LQTS in many patients.Here we report the novel mutation c.1898A>C in KCNH2 in a Chinese family with LQTS through whole-exome sequencing.The c.916dupA mutation in JUP(which encodes junction plakoglobin)is also discovered.Mutations in JUP were found to be associated with arrhythmogenic right ventricular cardiomyopathy.The double mutation in the proband may help explain his severe clinical manifestations,such as sudden cardiac death at an early age.Sequencing for the proband’s family members revealed that the KCNH2 mutation descends from his paternal line,while the mutation in JUP came from his maternal line.The data provided in this study may help expand the spectrum of LQTS-related KCNH2 mutations and add support to the genetic diagnosis and counseling of families affected by malignant arrhythmias.

Identifi cation of Novel TTN Mutations in Three Chinese Familial Dilated Cardiomyopathy Pedigrees by Whole Exome Sequencing

Familial dilated cardiomyopathy(DCM)is associated with numerous genes,especially those of the sarcomere family.The titin gene(TTN)consists of 365 exons and encodes the largest sarcomere protein(titin)in our bodies.Titin is associated with many diseases,such as hypertrophic cardiomyopathy and DCM.Here we screened three Chinese families affected by DCM,and found that each harbors a stop-gain or splice site mutation in TTN(c.G20137T,c.G52522T,c.44610-2A>C).Assessment of the probands by electrocardiogram,B-mode echocardiography,and cardiac magnetic resonance imaging revealed impaired cardiac function,arrhythmia,or abnormal cardiac structure.In conclusion,using whole exome sequencing,we found three unreported TTN mutations associated with DCM.This has expanded the TTN mutation spectrum of Chinese DCM patients,especially in Henan,the most populous province.These data provide new genetic targets for the diagnosis and treatment of DCM,and will increase our understanding of the relationship between TTN mutation and DCM clinical symptoms.

Identifi cation of Three FBN1 Mutations in Chinese Patients with Typical or Incomplete Marfan Syndrome by Whole-Exome Sequencing

Objective:The purpose of this work was to obtain the phenotypes and detect potential mutations in three Chinese patients with Marfan syndrome(MFS)or incomplete MFS phenotypes.Methods:Three unrelated patients with a defi nite or suspected clinical diagnosis of MFS and their family members were recruited for research.Genomic DNA was extracted from peripheral blood of these patients and their family members.All the exons were sequenced by next-generation sequencing and the variants were further validated by Sanger sequencing.The functional consequences of the mutations were analyzed with various genomic resources and bioinformatics tools.Results:Three FBN1 mutations were identifi ed in the three patients,including one novel mutation(2125G>A)and two previously reported mutations(4786C>T and 6325C>T).It was interesting to note that the parents of these patients were normal as assessed by clinical features or genetic testing,but all these mutations were detected in their offspring,except for the variant 6325C>T.We also found that a few young members of the family of probands(proband 1 and proband 2)have exhibited no manifestations of MFS so far,although they carry the same disease-causing mutation.Conclusions:We found three FBN1 mutations in three unrelated Chinese families with MFS by genome sequencing,and the relationship between genotypes and phenotypes in MFS patients needs further exploration.

Regulation of immune-related diseases by multiple factors: a meeting report of 2017 International Workshop of the Chinese Academy of Medical Sciences Initiative for Innovative Medicine on Tumor Immunology

Immune cells play key roles in cancer and chronic inflammatory disease. A better understanding of the mechanisms and risks will help develop novel target therapies. At the 2017 International Workshop of the Chinese Academy of Medical Sciences Initiative for Innovative Medicine on Tumor Immunology held in Beijing, China, on May 12, 2017, a number of speakers reported new findings and ongoing studies on immune-related diseases such as cancer, fibrotic disease, diabetes, and others. A considerably insightful overview was provided on cancer immunity, tumor microenvironments,and new immunotherapy for cancer. In addition, chronic inflammatory diseases were discussed. These findings may offer new insights into targeted immunotherapy.

Epithelial-to-mesenchymal transition in cancer： complexity and opportunities

The cell-biological program termed the epithelial-to-mesenchymal transition （EMT） plays an important role in both development and cancer progression. Depending on the contextual signals and intracellular gene circuits of a particular cell, this program can drive fully epithelial cells to enter into a series of phenotypic states arrayed along the epithelial-mesenehymal phenotypic axis. These cell states display distinctive cellular characteristics, including stemness, invasiveness, drug-resistance and the ability to form metastases at distant organs, and thereby contribute to cancer metastasis and relapse. Currently we still lack a coherent overview of the molecular and biochemical mechanisms inducing cells to enter various states along the epithelial-mesenchymal phenotypic spectrum. An improved understanding of the dynamic and plastic nature of the EMT program has the potential to yield novel therapies targeting this cellular program that may aid in the management of high-grade malignancies.

CD8^+ T cell response mediates the therapeutic effects of oncolytic adenovirus in an immunocompetent mouse model

The role of anti-tumor immune responses in oncolytic adenoviral therapy has not been well studied due to lack of efficacious tu- mor model in immunocompetent mice.Here,we evaluated the contributions of immune components to the therapeutic effects of oncolytic adenoviruse in an immunocompetent murine tumor model permissive for infection and replication of adenovirus.We found that CD8+T cells were critical mediator for antitumor efficacy by oncolytic adenovirus.Intratumoral viral therapy induced intensive infiltration of CD8+T cells in tumor,increased tumor-specific IFN-?(interferon-?)production and CTL(cytotoxic T lymphocyte)activity of lymphocytes,and generated a long-term tumor-specific immune memory.Boosting CD8+T cell responses by agonistic anti-4-1BB(cluster differentiation 137,CD137)antibody showed synergistic anticancer effects with oncolytic viro- therapy.Our results provide insight into antitumor mechanisms of oncolytic adenovirus in addition to their direct oncolytic effect.

How TP53 (almost) became an oncogene

Like the histories of many other genes,the discovery and characterization of p53 are associated with some false leads,dead ends,and major detours.The chronicle of p53,s early years is no exception.As is now well known,the protein was actually discovered independently by three groups-those of Arnie Levine,David Lane,and Lloyd Old.