Submonolayer biolasers for ultrasensitive biomarker detection.

Biomarker detection is key to identifying health risks. However, designing sensitive and single-use biosensors for early diagnosis remains a major challenge. Here, we report submonolayer lasers on optical fibers as ultrasensitive and disposable biosensors. Telecom optical fibers serve as distributed optical microcavities with high Q-factor, great repeatability, and ultralow cost, which enables whispering-gallery laser emission to detect biomarkers. It is found that the sensing performance strongly depends on the number of gain molecules. The submonolayer lasers obtained a six-order-of-magnitude improvement in the lower limit of detection (LOD) when compared to saturated monolayer lasers. We further achieve an ultrasensitive immunoassay for a Parkinson's disease biomarker, alpha-synuclein (α-syn), with a lower LOD of 0.32 pM in serum, which is three orders of magnitude lower than the α-syn concentration in the serum of Parkinson's disease patients. Our demonstration of submonolayer biolaser offers great potentials in high-throughput clinical diagnosis with ultimate sensitivity.

Induction of Immune Responses and Phosphatidylserine Exposure by TLR9 Activation Results in a Cooperative Antitumor Effect with a Phosphatidylserine-targeting Prodrug.

Head and neck cancer is a major cancer type, with high motility rates that reduce the quality of life of patients. Herein, we investigated the effectiveness and mechanism of a combination therapy involving TLR9 activator (CpG-2722) and phosphatidylserine (PS)-targeting prodrug of SN38 (BPRDP056) in a syngeneic orthotopic head and neck cancer animal model. The results showed a cooperative antitumor effect of CpG-2722 and BPRDP056 owing to their distinct and complementary antitumor functions. CpG-2722 induced antitumor immune responses, including dendritic cell maturation, cytokine production, and immune cell accumulation in tumors, whereas BPRDP056 directly exerted cytotoxicity toward cancer cells. We also discovered a novel function and mechanism of TLR9 activation, which increased PS exposure on cancer cells, thereby attracting more BPRDP056 to the tumor site for cancer cell killing. Killed cells expose more PS in tumor for BPRDP056 targeting. Tumor antigens released from the dead cells were taken up by antigen-presenting cells, which enhanced the CpG-272-promoted T cell-mediated tumor-killing effect. These form a positive feed-forward antitumor effect between the actions of CpG-2722 and BPRDP056. Thus, the study findings suggest a novel strategy of utilizing the PS-inducing function of TLR9 agonists to develop combinational cancer treatments using PS-targeting drugs.

TLR9 and STING agonists cooperatively boost the immune response to SARS-CoV-2 RBD vaccine through an increased germinal center B cell response and reshaped T helper responses.

Vaccines are a powerful medical intervention for preventing epidemic diseases. Efficient inactivated or protein vaccines typically rely on an effective adjuvant to elicit an immune response and boost vaccine activity. In this study, we investigated the adjuvant activities of combinations of Toll-like receptor 9 (TLR9) and stimulator of interferon genes (STING) agonists in a SARS-CoV-2 receptor binding domain protein vaccine. Adjuvants formulated with a TLR9 agonist, CpG-2722, with various cyclic dinucleotides (CDNs) that are STING agonists increased germinal center B cell response and elicited humoral immune responses in immunized mice. An adjuvant containing CpG-2722 and 2'3'-c-di-AM(PS)2 effectively boosted the immune response to both intramuscularly and intranasally administrated vaccines. Vaccines adjuvanted with CpG-2722 or 2'3'-c-di-AM(PS)2 alone were capable of inducing an immune response, but a cooperative adjuvant effect was observed when both were combined. CpG-2722 induced antigen-dependent T helper (Th)1 and Th17 responses, while 2'3'-c-di-AM(PS)2 induced a Th2 response. The combination of CpG-2722 and 2'3'-c-di-AM(PS)2 generated a distinct antigen-dependent Th response profile characterized by higher Th1 and Th17, but lower Th2 responses. In dendritic cells, CpG-2722 and 2'3'-c-di-AM(PS)2 showed a cooperative effect on inducing expression of molecules critical for T cell activation. CpG-2722 and 2'3'-c-di-AM(PS)2 have distinct cytokine inducing profiles in different cell populations. The combination of these two agonists enhanced the expression of cytokines for Th1 and Th17 responses and suppressed the expression of cytokines for Th2 response in these cells. Thus, the antigen-dependent Th responses observed in the animals immunized with different vaccines were shaped by the antigen-independent cytokine-inducing profiles of their adjuvant. The expanded targeting cell populations, the increased germinal center B cell response, and reshaped T helper responses are the molecular bases for the cooperative adjuvant effect of the combination of TLR9 and STING agonists.

The Src-ZNRF1 axis controls TLR3 trafficking and interferon responses to limit lung barrier damage.

Type I interferons are important antiviral cytokines, but prolonged interferon production is detrimental to the host. The TLR3-driven immune response is crucial for mammalian antiviral immunity, and its intracellular localization determines induction of type I interferons; however, the mechanism terminating TLR3 signaling remains obscure. Here, we show that the E3 ubiquitin ligase ZNRF1 controls TLR3 sorting into multivesicular bodies/lysosomes to terminate signaling and type I interferon production. Mechanistically, c-Src kinase activated by TLR3 engagement phosphorylates ZNRF1 at tyrosine 103, which mediates K63-linked ubiquitination of TLR3 at lysine 813 and promotes TLR3 lysosomal trafficking and degradation. ZNRF1-deficient mice and cells are resistant to infection by encephalomyocarditis virus and SARS-CoV-2 because of enhanced type I interferon production. However, Znrf1-/- mice have exacerbated lung barrier damage triggered by antiviral immunity, leading to enhanced susceptibility to respiratory bacterial superinfections. Our study highlights the c-Src-ZNRF1 axis as a negative feedback mechanism controlling TLR3 trafficking and the termination of TLR3 signaling.

Therapeutic efficacy of humanized monoclonal antibodies targeting dengue virus nonstructural protein 1 in the mouse model.

Dengue virus (DENV) which infects about 390 million people per year in tropical and subtropical areas manifests various disease symptoms, ranging from fever to life-threatening hemorrhage and even shock. To date, there is still no effective treatment for DENV disease, but only supportive care. DENV nonstructural protein 1 (NS1) has been shown to play a key role in disease pathogenesis. Recent studies have shown that anti-DENV NS1 antibody can provide disease protection by blocking the DENV-induced disruption of endothelial integrity. We previously demonstrated that anti-NS1 monoclonal antibody (mAb) protected mice from all four serotypes of DENV challenge. Here, we generated humanized anti-NS1 mAbs and transferred them to mice after DENV infection. The results showed that DENV-induced prolonged bleeding time and skin hemorrhage were reduced, even several days after DENV challenge. Mechanistic studies showed the ability of humanized anti-NS1 mAbs to inhibit NS1-induced vascular hyperpermeability and to elicit Fcγ-dependent complement-mediated cytolysis as well as antibody-dependent cellular cytotoxicity of cells infected with four serotypes of DENV. These results highlight humanized anti-NS1 mAb as a potential therapeutic agent in DENV infection.

A novel chimeric dengue vaccine candidate composed of consensus envelope protein domain III fused to C-terminal-modified NS1 protein.

There is an urgent need for a safe and effective vaccine against dengue virus (DENV) which infects about 390 million humans per year. In the present study we combined modifications of two DENV proteins, the nonstructural protein 1 (NS1) and the envelope (E) protein, to produce a DENV vaccine candidate with enhanced features. One of these modified proteins was a C-terminal-deleted fragment of NS1 called ΔC NS1 which we have shown previously to be protective without the potentially harmful effects of cross-reactive epitopes common to surface antigens on platelets and endothelial cells. The other modified protein was an envelope protein domain III (cEDIII) containing a consensus amino acid sequence among the four serotypes of DENV, which induces neutralizing antibody against all four DENV serotypes. The cEDIII and ΔC NS1 were expressed as a fusion protein cEDIII-ΔC NS1 and its protective effects against DENV were evaluated in a mouse model. C3H/HeN mice were immunized three times with cEDIII-ΔC NS1 fusion protein mixed with alum as adjuvant. Sera collected from cEDIII-ΔC NS1-immunized mice neutralized four serotypes of DENV and also caused complement-mediated cytolysis of HMEC-1 cells infected with each of the four different DENV serotypes. Mice immunized with cEDIII-ΔC NS1 and challenged with DENV showed reduced serum virus titer, soluble NS1 and bleeding time, compared with mice infected with DENV alone. The results reveal that antibodies induced by cEDIII-ΔC NS1 not only show anti-viral efficacy by in vitro assays but also provide protective effects against DENV infection in a mouse model. The cEDIII-ΔC NS1 thus represents a novel, effective DENV vaccine candidate.

Sharpening up tumor microenvironment to enhance the efficacy of immune checkpoint blockade on head and neck cancer using a CpG-oligodeoxynucleotide.

Head and neck cancers are a type of life-threatening cancers characterized by an immunosuppressive tumor microenvironment. Only less than 20% of the patients respond to immune checkpoint blockade therapy, indicating the need for a strategy to increase the efficacy of immunotherapy for this type of cancers. Previously, we identified a type B CpG-oligodeoxynucleotide (CpG-ODN) called CpG-2722, which has the universal activity of eliciting an immune response in grouper, mouse, and human cells. In this study, we further characterized and compared its cytokine-inducing profiles with different types of CpG-ODNs. The antitumor effect of CpG-2722 was further investigated alone and in combination with an immune checkpoint inhibitor in a newly developed syngeneic orthotopic head and neck cancer animal model. Along with other inflammatory cytokines, CpG-2722 induces the gene expressions of interleukin-12 and different types of interferons, which are critical for the antitumor response. Both CpG-2722 and anti-programmed death (PD)-1 alone suppressed tumor growth. Their tumor suppression efficacies were further enhanced when CpG-2722 and anti-PD-1 were used in combination. Mechanistically, CpG-2722 shaped a tumor microenvironment that is favorable for the action of anti-PD-1, which included promoting the expression of different cytokines such as IL-12, IFN-ß, and IFN-γ, and increasing the presence of plasmacytoid dendritic cells, M1 macrophages, and CD8 positive T cells. Overall, CpG-2722 provided a priming effect for CD8 positive T cells by sharpening the tumor microenvironment, whereas anti-PD-1 released the brake for their tumor-killing effect, resulting in an enhanced efficacy of the combined CpG-2722 and anti-PD-1.

An Improved Speciation Method Combining IC with ICPOES and Its Application to Iodide and Iodate Diffusion Behavior in Compacted Bentonite Clay.

An accurate and effective method combining ion chromatography (IC) and inductively coupled plasma optical emission spectrometry (ICP-OES) was applied in this work to qualitatively and quantitatively analyze individual and co-existing iodide (I-) and iodate (IO3-) at various concentrations. More specifically, a very strong linear relationship for the peak area for the co-existing I- and IO3- ions was reached, and a high resolution value between two peaks was observed, which proves the effectiveness of our combined IC-ICP-OES method at analyzing iodine species. We observed lower accessible porosity for the diffusion of both I- and IO3- in samples of bentonite clay using IC-ICP-OES detection methods, where the effective diffusion coefficient varied based on the anion exclusion effect and the size of the diffusing molecules. In fact, the distribution coefficients (Kd) of both I- and IO3- were close to 0, which indicates that there was no adsorption on bentonite clay. This finding can be explained by the fact that no change in speciation took place during the diffusion of I- and IO3- ions in bentonite clay. Our IC-ICP-OES method can be used to estimate the diffusion coefficients of various iodine species in natural environments.

The Opposite Roles of White Light in Regulating Germination of Fresh and Aged Seed in Tobacco.

Light is one of the important environmental factors for seeds to evaluate whether the natural environment is appropriate for germination and subsequent seedlings emergence. The mechanism of light-mediated germination is mainly concerned with fresh seeds (FS) of model plants but is poorly understood in aged seeds. Here, the effects of light on germination of FS and naturally aged seeds (NAS) in tobacco and their relationship with plant hormones gibberellins (GA) and abscisic acid (ABA) were investigated. The results demonstrated that light promoted and inhibited the germination of FS and NAS, respectively. GA and ABA were involved in the germination control of NAS, as well as in FS. However, light suppressed GA signal and stimulated ABA signal in NAS, whereas it stimulated GA signal and suppressed ABA signal in FS. In addition, light stimulated the GA accumulation and reduction in ABA in FS while inhibiting the increase in GA level in NAS. Together, the present study demonstrates that light has opposite effects on the germination of FS and NAS, which are closely related to the metabolism and/or signaling of plant hormones ABA and GA.

Unraveling hydrogen sulfide-promoted lateral root development and growth in mangrove plant Kandelia obovata: insight into regulatory mechanism by TMT-based quantitative proteomic approaches.

Mangroves are the main intertidal ecosystems with varieties of root types along the tropical and subtropical coastlines around the world. The typical characteristics of mangrove habitats, including the abundant organic matter and nutrients, as well as the strong reductive environment, are favor for the production of hydrogen sulfide (H2S). H2S, as a pivotal signaling molecule, has been evidenced in a wide variety of plant physiological and developmental processes. However, whether H2S functions in the mangrove root system establishment is not clear yet. Here, we reported the possible role of H2S in regulation of Kandelia obovata root development and growth by tandem mass tag (TMT)-based quantitative proteomic approaches coupled with bioinformatic methods. The results showed that H2S could induce the root morphogenesis of K. obovata in a dose-dependent manner. The proteomic results successfully identified 8075 proteins, and 697 were determined as differentially expressed proteins. Based on the functional enrichment analysis, we demonstrated that H2S could promote the lateral root development and growth by predominantly regulating the proteins associated with carbohydrate metabolism, sulfur metabolism, glutathione metabolism and other antioxidant associated proteins. In addition, transcriptional regulation and brassinosteroid signal transduction associated proteins also act as important roles in lateral root development. The protein-protein interaction analysis further unravels a complicated regulation network of carbohydrate metabolism, cellular redox homeostasis, protein metabolism, secondary metabolism, and amino acid metabolism in H2S-promoted root development and growth of K. obovata. Overall, our results revealed that H2S could contribute to the morphogenesis of the unique root system of mangrove plant K. obovata, and play a positive role in the adaption of mangrove plants to intertidal habitats.

Physiological and Proteomic Analyses of Two Acanthus Species to Tidal Flooding Stress.

The mangrove plant Acanthus ilicifolius and its relative, Acanthus mollis, have been previously proved to possess diverse pharmacological effects. Therefore, evaluating the differentially expressed proteins of these species under tidal flooding stress is essential to fully exploit and benefit from their medicinal values. The roots of A. ilicifolius and A. mollis were exposed to 6 h of flooding stress per day for 10 days. The dry weight, hydrogen peroxide (H2O2) content, anatomical characteristics, carbon and energy levels, and two-dimensional electrophoresis coupled with MALDI-TOF/TOF MS technology were used to reveal the divergent flooding resistant strategies. A. ilicifolius performed better under tidal flooding stress, which was reflected in the integrity of the morphological structure, more efficient use of carbon and energy, and a higher percentage of up-regulated proteins associated with carbon and energy metabolism. A. mollis could not survive in flooding conditions for a long time, as revealed by disrupting cell structures of the roots, less efficient use of carbon and energy, and a higher percentage of down-regulated proteins associated with carbon and energy metabolism. Energy provision and flux balance played a role in the flooding tolerance of A. ilicifolius and A. mollis.

Toll-Like Receptor 21 of Chicken and Duck Recognize a Broad Array of Immunostimulatory CpG-oligodeoxynucleotide Sequences.

CpG-oligodeoxynucleotides (CpG-ODNs) mimicking the function of microbial CpG-dideoxynucleotides containing DNA (CpG-DNA) are potent immune stimuli. The immunostimulatory activity and the species-specific activities of a CpG-ODN depend on its nucleotide sequence properties, including CpG-hexamer motif types, spacing between motifs, nucleotide sequence, and length. Toll-like receptor (TLR) 9 is the cellular receptor for CpG-ODNs in mammalian species, while TLR21 is the receptor in avian species. Mammalian cells lack TLR21, and avian cells lack TLR9; however, both TLRs are expressed in fish cells. While nucleotide sequence properties required for a CpG-ODN to strongly activate mammalian TLR9 and its species-specific activities to different mammalian TLR9s are better studied, CpG-ODN activation of TLR21 is not yet well investigated. Here we characterized chicken and duck TLR21s and investigated their activation by CpG-ODNs. Chicken and duck TLR21s contain 972 and 976 amino acid residues, respectively, and differ from TLR9s as they do not have an undefined region in their ectodomain. Cell-based TLR21 activation assays were established to investigate TLR21 activation by different CpG-ODNs. Unlike grouper TLR21, which was preferentially activated by CpG-ODN with a GTCGTT hexamer motif, chicken and duck TLR21s do not distinguish among different CpG-hexamer motifs. Additionally, these two poultry TLR21s were activated by CpG-ODNs with lengths ranging from 15 to 31 nucleotides and with different spacing between CpG-hexamer motifs. These suggested that compared to mammalian TLR9 and grouper TLR21, chicken and duck TLR21s have a broad CpG-ODN sequence recognition profile. Thus, they could also recognize a wide array of DNA-associated molecular patterns from microbes. Moreover, CpG-ODNs are being investigated as antimicrobial agents and as vaccine adjuvants for different species. This study revealed that there are more optimized CpG-ODNs that can be used in poultry farming as anti-infection agents compared to CpG-ODN choices available for other species.

Epigenetic Silencing of Ubiquitin Specific Protease 4 by Snail1 Contributes to Macrophage-Dependent Inflammation and Therapeutic Resistance in Lung Cancer.

There is a positive feedback loop driving tumorigenesis and tumor growth through coordinated regulation of epigenetics, inflammation, and stemness. Nevertheless, the molecular mechanism linking these processes is not well understood. In this study, we analyzed the correlation of de-ubiquitinases (DUBs) expression with survival data from the OncoLnc database. Among the DUBs analyzed, ubiquitin specific protease 4 (USP4) had the lowest negative Cox coefficient. Low expression of USP4 was associated with poor survival among lung cancer patients and was inversely correlated with expression of stemness and inflammation markers. Expression of USP4 were reduced at more advanced stages of lung cancer. Mechanistically, expression of USP4 was downregulated in snail1-overexpressing and stemness-enriched lung cancer cells. Snail1 was induced in lung cancer cells by interaction with macrophages, and epigenetically suppressed USP4 expression by promoter methylation. Stable knockdown of USP4 in lung cancer cells enhanced inflammatory responses, stemness properties, chemotherapy resistance, and the expression of molecules allowing escape from immunosurveillance. Further, mice injected with USP4 knockdown lung cancer cells demonstrated enhanced tumorigenesis and tumor growth. These results reveal that the Snail1-mediated suppression of USP4 is a potential mechanism to orchestrate epigenetic regulation, inflammation and stemness for macrophage-promoted tumor progression.

ERK Activation Modulates Cancer Stemness and Motility of a Novel Mouse Oral Squamous Cell Carcinoma Cell Line.

We established the NHRI-HN1 cell line from a mouse tongue tumor induced by 4-nitroquinoline 1-oxide (4-NQO)/arecoline, with further selection for cell stemness via in vitro sphere culture, to evaluate potential immunotherapies for oral squamous cell carcinoma (OSCC) in East and Southeast Asia. In vivo and in vitro phenotypic characterization, including tumor growth, immune modulator administration, gene expression, morphology, migration, invasion, and sphere formation assays, were conducted. NHRI-HN1 cells are capable of generating orthotopic tumors in syngeneic mice. Interestingly, immune stimulation via CpG oligodeoxynucleotide (CpG-ODN) dramatically reduced the tumor growth in NHRI-HN1 cell-injected syngeneic mice. The pathways enriched in genes that were differentially expressed in NHRI-HN1 cells when compared to non-tumorigenic cells were similar to those that were identified when comparing human OSCC and non-tumorous tissues. NHRI-HN1 cells have characteristics of epithelial-mesenchymal transition (EMT), including enhanced migration and invasion. NHRI-HN1 cells showed aggressive cell growth and sphere formation. The blockage of extracellular signal-regulated kinase (ERK) activation suppressed cell migration and reduced stemness characteristics in NHRI-HN1 cells, similar to human OSCC cell lines. Our data suggest that NHRI-HN1 cells, showing tumorigenic characteristics of EMT, cancer stemness, and ERK activation, are sufficient in modeling human OSCC and also competent for use in investigating oral cancer immunotherapies.

Comparative Proteomic Analysis Reveals the Regulatory Effects of H2S on Salt Tolerance of Mangrove Plant Kandelia obovata.

As a dominant mangrove species, Kandelia obovata is distributed in an intertidal marsh with an active H2S release. Whether H2S participates in the salt tolerance of mangrove plants is still ambiguous, although increasing evidence has demonstrated that H2S functions in plant responses to multiple abiotic stresses. In this study, NaHS was used as an H2S donor to investigate the regulatory mechanism of H2S on the salt tolerance of K. obovata seedlings by using a combined physiological and proteomic analysis. The results showed that the reduction in photosynthesis (Pn) caused by 400 mM of NaCl was recovered by the addition of NaHS (200 µM). Furthermore, the application of H2S enhanced the quantum efficiency of photosystem II (PSII) and the membrane lipid stability, implying that H2S is beneficial to the survival of K. obovata seedlings under high salinity. We further identified 37 differentially expressed proteins by proteomic approaches under salinity and NaHS treatments. Among them, the proteins that are related to photosynthesis, primary metabolism, stress response and hormone biosynthesis were primarily enriched. The physiological and proteomic results highlighted that exogenous H2S up-regulated photosynthesis and energy metabolism to help K. obovata to cope with high salinity. Specifically, H2S increased photosynthetic electron transfer, chlorophyll biosynthesis and carbon fixation in K. obovata leaves under salt stress. Furthermore, the abundances of other proteins related to the metabolic pathway, such as antioxidation (ascorbic acid peroxidase (APX), copper/zinc superoxide dismutase (CSD2), and pancreatic and duodenal homeobox 1 (PDX1)), protein synthesis (heat-shock protein (HSP), chaperonin family protein (Cpn) 20), nitrogen metabolism (glutamine synthetase 1 and 2 (GS2), GS1:1), glycolysis (phosphoglycerate kinase (PGK) and triosephosphate isomerase (TPI)), and the ascorbate-glutathione (AsA-GSH) cycle were increased by H2S under high salinity. These findings provide new insights into the roles of H2S in the adaptations of the K. obovata mangrove plant to high salinity environments.

Hydrogen plasma-treated MoSe2 nanosheets enhance the efficiency and stability of organic photovoltaics.

In this paper we report the effect on the power conversion efficiency (PCE) and stability of photovoltaic devices after incorporating hydrogenated two-dimensional (2D) MoSe2 nanosheets into the active layer of bulk heterojunction (BHJ) organic photovoltaics (OPV). The surface properties of 2D MoSe2 nanosheets largely affect their dispersion in the active layer blend and, thus, influence the carrier mobility, PCE, and stability of corresponding devices. We treated MoSe2 nanosheets with hydrogen plasma and investigated their influence on the polymer packing and fullerene domain size of the active layer. For the optimized devices incorporating 37.5 wt% of untreated MoSe2, we obtained a champion PCE of 9.82%, compared with the champion reference PCE of approximately 9%. After incorporating the hydrogen plasma-treated MoSe2 nanosheets, we achieved a champion PCE of 10.44%-a relative increase of 16% over that of the reference device prepared without MoSe2 nanosheets. This PCE is the one of the highest ever reported for OPVs incorporating 2D materials. We attribute this large enhancement to the enhanced exciton generation and dissociation at the MoSe2-fullerene interface and, consequently, the balanced charge carrier mobility. The device incorporating the MoSe2 nanosheets maintained 70% of its initial PCE after heat-treatment at 100 °C for 1 h; in contrast, the PCE of the reference device decreased to 60% of its initial value-a relative increase in stability of 17% after incorporating these nanosheets. We also incorporated MoSe2 nanosheets (both with and without treatment) into a polymer donor (PBDTTBO)/small molecule (IT-4F) acceptor system. The champion PCEs reached 7.85 and 8.13% for the devices incorporating the MoSe2 nanosheets with and without plasma treatment, respectively-relative increases of 8 and 12%, respectively, over that of the reference. These results should encourage a push toward the implementation of transition metal dichalcogenides to enhance the performances of BHJ OPVs.

[Clinical effect and safety of clopidogrel combined with aspirin in antithrombotic therapy for children with Kawasaki disease complicated by small/medium-sized coronary artery aneurysms].

OBJECTIVE: To study the clinical effect and safety of clopidogrel combined with aspirin in antithrombotic therapy for children with Kawasaki disease (KD) complicated by coronary artery aneurysm (CAA). METHODS: A total of 77 KD children who were diagnosed with multiple small/medium-sized CAAs by echocardiography between January 2013 and June 2018 were enrolled. They were randomly divided into observation group with 38 children (treated with clopidogrel and aspirin) and control group with 39 children (treated with low-molecular-weight heparin and aspirin). All children were followed up regularly, and the first 3 months of the course of the disease was the observation period. The children were observed in terms of the change of the coronary artery and the incidence of complications. RESULTS: At month 3 of follow-up, among the children in the observation group, 6 had normal coronary artery, 11 had coronary artery retraction, 19 had stable coronary artery, and 2 progressed to giant coronary aneurysm; among the children in the control group, 7 had normal coronary artery, 12 had coronary artery retraction, 19 had stable coronary artery, and 1 progressed to giant coronary aneurysm; there was no significant difference in the change of the coronary artery between the two groups (P>0.05). There were 2 cases of epistaxis and 6 cases of skin ecchymosis in the observation group, and 1 case of epistaxis and 7 cases of petechiae and ecchymosis at the injection site in the control group, and no other serious bleeding events were observed in either group. CONCLUSIONS: Clopidogrel combined with low-dose aspirin is safe and effective in antithrombotic therapy for children with KD complicated by CAA.

A genomic extension to the sequence of HLA-A*02:13, identified using third-generation sequencing.

Pacific Biosciences' SMRT sequencing method was used to extend the sequence of HLA-A*02:13.

Identification of a new allele, HLA-DRB1*01:01:01:02 discovered using third-generation sequencing.

HLA-DRB1*01:01:01:02 is the first reported intronic variant of DRB1*01:01:01, differing by a single nucleotide substitution.

A novel allele, HLA-C*07:01:01:30 identified using third-generation sequencing.

The novel allele, HLA-C*07:01:01:30 differs by one nucleotide substitution with HLA-C*07:01:01:01 at gDNA 627.