Genome-Wide Association and Functional Studies Identify SCML4 and THSD7A as Novel Susceptibility Genes for Coronary Artery Disease.

OBJECTIVE: The genetic contribution to coronary artery disease (CAD) remains largely unclear. We combined genetic screening with functional characterizations to identify novel loci and candidate genes for CAD. APPROACH AND RESULTS: We performed genome-wide screening followed by multicenter validation in 8 cohorts consisting of 21 828 participants of Han ethnicity and identified 3 novel intragenic SNPs (single nucleotide polymorphisms), rs9486729 (SCML4 [Scm polycomb group protein-like 4]; odds ratio, 1.25; 95% CI, 1.17-1.34; P=3.51×10-11), rs17165136 (THSD7A [thrombospondin type 1 domain-containing 7A]; odds ratio 1.28; 95% CI, 1.21-1.35; P<1.00×10-25), and rs852787 (DAB1 [disabled-1]; odds ratio, 1.29; 95% CI, 1.21-1.38; P=2.02×10-14), associated with CAD with genome-wide significance. The risk allele of rs9486729 and protective allele of rs17165136 were associated with the decreased expression of their host genes, SCML4 and THSD7A, respectively, whereas rs852787 did not have transcriptional effects on any gene. Knockdown of SCML4 activated endothelial cells by increasing the expression of IL-6, E-selectin, and ICAM and weakened their antiapoptotic activity, whereas the knockdown of THSD7A had little effect on these endothelial cell functions but attenuated monocyte adhesion via decreasing the expression of ICAM, L-selectin, and ITGB2. We further showed that inhibiting the expression of SCML4 exacerbated endothelial dysfunction and vascular remodeling in a rat model with partial carotid ligation. CONCLUSIONS: We identify 3 novel loci associated with CAD and show that 2 genes, SCML4 and THSD7A, make functional contributions to atherosclerosis. How rs852787 and its host gene DAB1 are linked to CAD needs further studies.

Serum TGF-ß1 and SMAD3 levels are closely associated with coronary artery disease.

BACKGROUND: Coronary artery disease (CAD) is one of the most common diseases leading to mortality and morbidity worldwide. There is considerable debate on whether serum transforming growth factor ß1 (TGF-ß1) levels are associated with long-term major adverse cardiovascular events in patients with CAD, and to date, no study has specifically addressed levels in patients with different degrees of CAD severity. METHODS: Serum TGF-ß1 and mothers against decapentaplegic homolog 3 (SMAD3) concentrations were evaluated in 279 patients with CAD and 268 controls without CAD. The clinical and biochemical characteristics of all subjects were also determined and analyzed. RESULTS: TGF-ß1 and SMAD3 concentrations in CAD patients were significantly higher than those in the controls. The serum TGF-ß1 level in acute myocardial infarction (AMI) was significantly higher than that in both stable angina pectoris (SAP) and unstable angina pectoris (UAP) (p < 0.05), while there was no marked difference between levels in SAP and UAP (p > 0.05). SMAD3 levels showed no obvious difference among AMI, SAP, and UAP. TGF-ß1 and SMAD3 are potential biomarkers for CAD, and may be more accurate than Lpa, ApoA1, uric acid, BUN, or triglycerides (TG). CONCLUSIONS: Serum TGF-ß1 and SMAD3 levels are closely associated with CAD, and may become useful biomarkers for diagnosis and risk stratification.

Circulating microRNA-19a as a potential novel biomarker for diagnosis of acute myocardial infarction.

Acute myocardial infarction (AMI) is a serious cardiovascular disease. Investigating new susceptibility genes for effective methods of early diagnosis of AMI is important. In the current study, peripheral blood miR-19a levels were detected by real-time polymerase chain reaction. Significant differences and logistic correlation analyses were carried out by grouping of disease types and stratification of risk factors. Receiver-operator characteristic curve analysis was used to compare the current common clinical biochemical markers and evaluate the sensitivity and specificity of miR-19a for diagnosing AMI. Circulating miR-19a expression in the AMI group was higher than that in controls. The diagnostic effect of circulating miR-19a levels was superior to current clinical biochemical indices, such as CK, CK-MB, MYO, hs-TnI, and BNP. Our results show that there is a close association of circulating miR-19a levels with susceptibility to AMI. Circulating miR-19a levels could be a candidate diagnostic biomarker for AMI.

Magnetic field-assisted fabrication of quasi-bilayered, multi-responsive and patternable actuators.

Quasi-bilayered actuators composed of Fe3O4-decorated graphene oxide and polyvinylidene fluoride have been fabricated in a magnetic field. The actuators could stably respond to multiple stimuli including infrared light, acetone vapour and a magnetic field. The actuator is also patternable because of the magnetism-induced spatial distribution of fillers in the matrix.

Improved expression of secretory and trimeric proteins in mammalian cells via the introduction of a new trimer motif and a mutant of the tPA signal sequence.

Ideal immunogenicity in antigens is a prerequisite to eliciting a sufficiently strong immune and memory response via either DNA or protein vaccines. To improve immunogenicity, efforts have focused on high-level expression of target proteins and on maintaining their natural conformations. In the present work, two trimer motifs (MTQ and MTI) were designed and introduced into a plasmid vector with the tissue plasminogen activator signal peptide (tPA-SP). Next, we examined the efficacy and the efficiency of the two motifs as well as the introduction of tPA-SP and its mutant forms, 22P/A and 22P/G, in facilitating the secretory expression of trimeric proteins in mammalian cells. We found that both trimer motifs could produce the target protein in a trimeric form at a high level. Introduction of tPA-SP 22P/A markedly increased the secretory expression level. The combination of the trimer motif, MTQ, and the signal peptide, 22P/A, may serve as a universal mammalian vector for producing trimeric proteins in vaccine development.

DNA adjuvant Amiloride conjunct long immunization interval promote higher antibody responses to HIV-1 gp41 and gp140 immunogens.

Recent studies have revealed that the interface of gp120 and gp41 and some parts of gp41 are also critical epitopes for elicitation of broadly neutralizing antibodies. Therefore, potential trimeric gp41 or gp140 immunogen candidates are needed. Previously, we developed a trimer motif MTQ and demonstrated that it could help formation of trimeric gp120 and gp140 proteins. In the present study, we immunized Balb/c mice using trimeric gp41-expressing plasmid for prime and monomeric gp41 or trimeric gp140 protein as well as a mutant (Q577A) for boost. The antibody responses in the context of regimens with various immunization intervals and DNA adjuvants including praziquantel (PZQ), cimetidine (CIM), and amiloride (AML) were evaluated. We found that these three adjuvants were not enough to elicit remarkable specific Abs after gp41 DNA immunization, while AML could significantly promote humoral immune responses after protein boosts. Long immunization interval could induce the specific binding Abs earlier and higher and maintain a high level of Abs in the following 27 weeks after final protein boost. Moreover, two times of protein boosts with DNA adjuvant and a longer time interval achieved a higher titer of specific Abs than three times of protein boosts with a shorter time interval. Q577A mutant was benefit for trimeric gp140 boost in the production of binding Abs but harmful to inducing neutralizing Abs, while this mutant in monomeric gp41 presented the opposite trend which may be associated with the immunogen structures. This study highlights the significance of DNA adjuvant Amiloride and long immunization interval in promoting antibody responses and provides new insights into effective HIV immunization regimen design in the future.

Divergent Primary Immune Responses Induced by Human Immunodeficiency Virus-1 gp120 and Hepatitis B Surface Antigen Determine Antibody Recall Responses.

The development of a vaccine based on human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) that elicits potent protective antibodies against infection has been challenging. Recently, we compared the antibody production patterns of HIV-1 Env gp120 and hepatitis B virus surface antigen (HBsAg) to provide insights into how we may improve the protective efficacy of Env-based immunogens. Our previous study showed that HIV Env and HBsAg display different mechanisms of antibody elicitation and that T cells facilitate the responses to repeated immunizations. Here, to elucidate the detailed roles of primary immunization in immune memory response formation and antibody production, we immunized C57BL/6 mice with each antigen and evaluated the development of T follicular helper (Tfh) cells, germinal centers, and the memory responses involved in prime and boost immunizations. We found that after prime immunization, compared with HBsAg, gp120 induced higher frequencies of Tfh cells and programmed death (PD)-1+ T cells, greater major histocompatibility complex II expression on B cells, comparable activated B cells, but weaker germinal center (GC) reactions and memory B cell responses in the draining lymph nodes, accompanied by slower antibody recall responses and poor immune memory responses. The above results suggested that more PD-1+ T cells arising in primary immunization may serve as major contributors to the slow antibody recall response elicited by HIV-1 Env.

Comparison of the patterns of antibody recall responses to HIV-1 gp120 and hepatitis B surface antigen in immunized mice.

To date, we still lack an ideal strategy for designing envelope glycoprotein (Env) vaccines to elicit potent protective antibodies against HIV-1 infection. Since the human hepatitis B virus surface antigen (HBsAg) is representative of effective vaccines that can induce ideal humoral immune responses, knowledge of how it elicits antibody responses and T helper cells would be an useful reference for HIV vaccine development. We compared the characteristics of the HIV-1 Env gp120 trimer and HBsAg in antibody elicitation and induction of T follicular helper (Tfh) and memory B cells in immunized Balb/c mice. Using the strategy of protein prime-protein boost, we found that HIV-1 gp120 induced slower recall antibody responses but redundant non-specific IgG responses at early time after boosting compared to HBsAg. The higher frequency of PD-1hiCD4+ T cells and Tfh cells that appeared at the early time point after gp120 boosting is likely to limit the development of memory B cells, memory T cells, and specific antibody recall responses. These findings regarding the different features of HIV envelope and HBsAg in T helper cell responses may provide a direction to improve HIV envelope immunogenicity.

[Fluctuation in the resistance of Plasmodium falciparum to chloroquine in China].

OBJECTIVE: To investigate whether chloroquine-resistance of Plasmodium falciparum had changed after stopping or reducing the use of chloroquine as an antimalarial in Hainan and Yunnan provinces. METHODS: WHO standard in vitro microtest and 4-week in vivo test were used, assays were carried out in different time after stopping or reducing the use of chloroquine. RESULTS: In vitro test in Hainan indicated that the rate of chloroquine-resistant P. falciparum was 97.9% in 1981, and dropped to 26.7% in 1997 (P<0.01). The mean concentration of chloroquine for complete inhibition of schizont formation was 10.46 +/- 7.14 pmol/microl blood in 1981, decreased to 1.63 +/- 1.47 pmol/microl blood in 1997 (P<0.01). The proportion of samples taken from malaria cases that required high concentration (>6.4 pmol/microl blood) of chloroquine for complete inhibition of schizont formation was 83.3% in 1981 and only 6.7% in 1997 (P<0.01). In the 4-week in vivo test, the rate of chloroquine-resistant P. falciparum decreased from 84.2% in 1981 to 18.4% in 1997 (P<0.01). RIII cases accounted for 53.1% of the total resistant cases in 1981, and for 14.3% in 1997 (P<0.01). In vitro test in Yunnan revealed that the rate of chloroquine-resistant P. falciparum, the mean concentration of chloroquine for complete inhibition of schizont formation and the proportion of samples taken from malaria cases that required >6.4 pmol/microl blood of chloroquine for complete inhibition of schizont formation were 97.4%, 17.2 +/- 12.6 pmol/microl blood and 58.9% in 1981 respectively, and dropped to 70.4% (P< 0.01), 4.0 +/- 3.3 pmol/microl blood (P<0.01) and 16.6% (P<0.01) in 2002 respectively. CONCLUSION: The resistance of P. falciparum to chloroquine declined progressively after its use had been stopped or reduced in Hainan and Yunnan provinces.

[Study on treatment of multi-drug resistant falciparum malaria by using a combination of dihydroartemisinin and pyronaridine].

OBJECTIVE: To provide a combined medication scheme for the treatment of multi-drug resistant falciparum malaria. METHODS: Combined administration of dihydroartemisinin and pyronaridine was given to the 32 cases of falciparum malaria cases with multi-drug resistance. The indices for evaluation on day 14, 21, and 28 after treatment included the mean fever subsidence time, mean asexual form clearance time, mean recrudescence time of asexual form and recrudescence rate, proportion of gametocyte carriers, mean density of gametocytes and its mean clearance time, cure rate and rate of side-effects. A double blind clinical test was performed with standard schemes of dihydroartemisinin (20 cases) and pyronaridine (25 cases) as control. RESULTS: The mean fever subsidence time of treated patients by dihydroartemisinin/pyronaridine combination, dihydroartemisinin and pyronaridine was 35.7 +/- 24.7 h, 52.6 +/- 38.9 h and 35.8 +/- 16.5 h respectively, showing a significant difference between the combination group and dihydroartemisinin groups (P < 0.01). The mean asexual form clearance time was 23.8 +/- 10.1 h, 22.9 +/- 6.5 h and 49.4 +/- 20.3 h respectively, showing significantly faster in the combination group than the pyronaridine group (P < 0.01). The recrudescence rate was 0, 4.2% and 0 respectively. The proportion of gametocyte carriers was 20.0%, 16.7% and 60.9% respectively, with a significantly higher rate in the group of pyronaridine than the group of combination (P < 0.01). CONCLUSION: The combination of dihydroartemsinin and pyronaridine is an ideal medication scheme for the treatment of falciparum malaria cases with multi-drug resistance.