Determine the Function of a Specific Immunoglobulin Domain of Titin in Zebrafish

Mutations in genes encoding cardiac ion channels, transcription factors, and myocardial structural proteins have been identified in patients with early-onset atrial fibrillation (EOAF). However, the underlying mechanisms by which structural proteins give rise to atrial fibrillation (AF) remain unclear. Titin (TTN) is a multi-domain protein essential for sarcomere assembly during heart development and the restoration of normal sarcomere length after cardiomyocyte contraction. Previous work on a truncated TTN suggested that disruption of the sarcomeric organization may lead to EOAF. However, it remains unknown what role a specific TTN domain might play in abnormal sarcomere development and/or maturation. Our collaborator identified a TTN missense mutation (T31115I) in a patient with EOAF and showed that it co-segregates in the patient’s family for three generations. To elucidate whether the T31115I mutant causes AF and what the underlying mechanisms might be, I adopted two CRISPR-Cas9-mediated approaches: 1) homology-directed repair to introduce this mutation into the zebrafish ortholog, ttna; and 2) generation of two mutant ttna alleles, ttnaΔ6 and ttnaΔ9, with 6 or 9 amino acids deleted, respectively, within a highly conserved region containing the site of T31115I mutation. ttnaΔ9/Δ9 zebrafish embryos demonstrated inflow tract edema, pericardial edema, blood clots, atrial enlargement, and reduced cardiac function, as indicated by lower ventricular contraction and cardiac output. These data suggest that the specific region deleted in ttnaΔ9 is important for proper cardiac function. As the highly conserved region contains the EOAF-associated TTN point mutation, further investigation of this region may not only reveal more biological pathways that involve titin, but also provide more insights into the pathogenesis of EOAF

A Small Internal Deletion in Titin Yields Ion Channel-Dependent Cardiomyopathy and Atrial Fibrillation

Genetic variants may lead to cardiac developmental defects, and the most common type of arrhythmia, Atrial fibrillation (AF), has been shown to be associated occasionally with cardiac developmental defects. However, the mechanisms contributing to this association are unclear. One of the major sarcomere structural proteins, titin, may help reveal these mechanisms because of its roles in both cardiac development and regulating cardiac function. In a screening of CRISPR/Cas9-mediated mutagenesis in zebrafish, I identified a small internal in-frame deletion of 9 amino acids in the Immunoglobin-like domain 139 (Ig-139) of the zebrafish titin A-band region (ttnaΔ9). Compared to previous studies focusing on titin truncating variants that remove large regions and multiple domains, our mutant titin with the small internal deletion may reveal a role of this specific region (Ig-139 domain) in the pathogenesis of atrial fibrillation. Briefly, ttnaΔ9/Δ9 zebrafish exhibit a transient ventricular function defect during development, followed by AF and cardiomyopathy in the adult. Meanwhile, atria exhibit enlargement and reduced contraction in both embryos and adults compared to wild-type clutchmates. How does this transient ventricular defect contribute to atrial fibrillation in adults? I hypothesized that atrial natriuretic peptide (ANP) upregulation plays a role in this mechanism because: (1) ANP secretion is upregulated in response to cardiac dysfunctions and (2) previous studies showed ANP overexpression leads to AF through increased potassium slow rectifier channel current (IKs). I found ttnaΔ9/Δ9 embryos show ANP upregulation and ANP-dependent IKs channel remodeling indicated by the downregulation of the IKs channel regulatory subunit kcne1. Specific blocking of the IKs channel with a chemical inhibitor in both the ttnaΔ9/Δ9 zebrafish model and TTNΔ9/Δ9 human iPSC-derived atrial cardiomyocytes reduced arrhythmia and improved atrial contraction, suggesting that the ANP-dependent IKs channel remodeling and increased activity contribute to the pathogenesis of AF and cardiomyopathy in the mutant adult. In conclusion, ttnaΔ9/Δ9 zebrafish exhibit a phenotype suggesting that subtle and transient cardiovascular defects during development may lead to a higher risk of atrial fibrillation in adults. The Δ9 mutation in the Ig-139 domain of titin A-band triggers an ANP-regulated IKs channel remodeling, which may be part of the mechanism and provide potential treatment targets for developmental defects-related adult cardiac diseases

Strength analysis and optimization of alkali activated slag backfills through response surface methodology

The significant difference in water-to-binder ratio, activator type and concentration between alkali-activated slag (AAS) paste/mortar/concrete and AAS-based cemented paste backfill (AAS-CPB) means that previous results related to the properties and mix optimization of AAS materials cannot be directly translated to AAS-CPB. This study statistically identifies the effect of key influential variables such as silicate modulus, slag fineness and activator concentration on 3- and 28 day unconfined compressive strength (UCS) of AAS-CPB by central composite design (CCD) established in response surface methodology (RSM). In this study, the prominence of independent variables and their relations are investigated by using ANOVA (analysis of variance) having a significant level of 0.05. ANOVA results certify that there is a strong link between the level of variable contribution on UCS performance of AAS-CPB and curing age. Obviously, silicate modulus and activator concentration are the most major variables influencing UCS at 3 and 28 days, respectively. Increased fineness of slag and higher pH of pore solution enhance 3 day UCS, but restrain the further hydration of unreacted slag and subsequent the gain in strength at advanced curing ages. The combination of independent variables of silicate modulus (0.295), slag fineness (12630.2), activator concentration (0.45) gives the optimum responses.National Natural Science Foundation of China (NSFC) 51804063 Fundamental Research Funds for the Central Universities N210104

Time series analysis and prediction model of percentage of influenza-like illness （ILI） cases in Shanghai

ObjectiveTo predict the incidence trend of influenza-like illness proportion （ILI%） in Shanghai using the seasonal autoregressive integrated moving average model （SARIMA）， and to provide an important reference for timely prevention and control measures.MethodsTime series analysis was performed on ILI% surveillance data of Shanghai Municipal Center for Disease Control and Prevention from the 15th week of 2015 to the 52nd week of 2019， and a prediction model was established. Seasonal autoregressive integrated moving average （SARIMA） model was established using data from the foregoing 212 weeks， and prediction effect of the model was evaluated using data from the latter 36 weeks.ResultsFrom the 15th week of 2015 to the 52nd week of 2019， the average ILI% in Shanghai was 1.494%， showing an obvious epidemic peak. SARIMA（1，0，0） （2，0，0） 52 was finally modeled. The residual of the model was white noise sequence， and the true values were all within the 95% confidence interval of the predicted values.ConclusionSARIMA（1，0，0） （2，0，0） 52 can be used for the medium term prediction of ILI% in Shanghai， and can play an early warning role for the epidemic and outbreak of influenza in Shanghai

Heterologous Booster Immunization Based on Inactivated SARS-CoV-2 Vaccine Enhances Humoral Immunity and Promotes BCR Repertoire Development

Recent studies have indicated that sequentially administering SARS-CoV-2 vaccines can result in increased antibody and cellular immune responses. In this study, we compared homologous and heterologous immunization strategies following two doses of inactivated vaccines in a mouse model. Our research demonstrates that heterologous sequential immunization resulted in more immune responses displayed in the lymph node germinal center, which induced a greater number of antibody-secreting cells (ASCs), resulting in enhanced humoral and cellular immune responses and increased cross-protection against five variant strains. In further single B-cell analysis, the above findings were supported by the presence of unique B-cell receptor (BCR) repertoires and diversity in CDR3 sequence profiles elicited by a heterologous booster immunization strategy

Immunological Study of Combined Administration of SARS-CoV-2 DNA Vaccine and Inactivated Vaccine

Objective: We constructed two DNA vaccines containing the receptor-binding domain (RBD) genes of multiple SARS-CoV-2 variants and used them in combination with inactivated vaccines in a variety of different protocols to explore potential novel immunization strategies against SARS-CoV-2 variants. Methods: Two DNA vaccine candidates with different signal peptides (namely, secreted and membrane signal peptides) and RBD protein genes of different SARS-CoV-2 strains (Wuhan-Hu-1, B.1.351, B.1.617.2, C.37) were used. Four different combinations of DNA and inactivated vaccines were tested, namely, Group A: three doses of DNA vaccine; B: three doses of DNA vaccine and one dose of inactivated vaccine; C: two doses of inactivated vaccine and one dose of DNA vaccine; and D: coadministration of DNA and inactivated vaccines in two doses. Subgroups were grouped according to the signal peptide used (subgroup 1 contained secreted signal peptides, and subgroup 2 contained membrane signal peptides). The in vitro expression of the DNA vaccines, the humoral and cellular immunity responses of the immunized mice, the immune cell population changes in local lymph nodes, and proinflammatory cytokine levels in serum samples were evaluated. Results: The antibody responses and cellular immunity in Group A were weak for all SARS-CoV-2 strains; for Group B, there was a great enhancement of neutralizing antibody (Nab) titers against the B.1.617.2 variant strain. Group C showed a significant increase in antibody responses (NAb titers against the Wuhan-Hu-1 strain were 768 and 1154 for Group C1 and Group C2, respectively, versus 576) and cellular immune responses, especially for variant B.1.617.2 (3240 (p p < 0.05) for Group C1 and Group C2, versus 450); Group D showed an improvement in immunogenicity. Group C induced higher levels of multiple cytokines. Conclusion: The DNA vaccine candidates we constructed, administered as boosters, could enhance the humoral and cellular immune responses of inactivated vaccines against COVID-19, especially for B.1.617.2

HSV-1 Infection of Epithelial Dendritic Cells Is a Critical Strategy for Interfering with Antiviral Immunity

Herpes simplex virus type 1 (HSV-1), an α subgroup member of the human herpesvirus family, infects cells via the binding of its various envelope glycoproteins to cellular membrane receptors, one of which is herpes virus entry mediator (HVEM), expressed on dendritic cells. Here, HVEM gene-deficient mice were used to investigate the immunologic effect elicited by the HSV-1 infection of dendritic cells. Dendritic cells expressing the surface marker CD11c showed an abnormal biological phenotype, including the altered transcription of various immune signaling molecules and inflammatory factors associated with innate immunity after viral replication. Furthermore, the viral infection of dendritic cells interfered with dendritic cell function in the lymph nodes, where these cells normally play roles in activating the T-cell response. Additionally, the mild clinicopathological manifestations observed during the acute phase of HSV-1 infection were associated with viral replication in dendritic cells