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Obtaining sequential and conformational information on proteins is vital to understand their functions. Although the nanopore-based electrical detection can sense single molecule (SM) protein and distinguish among different amino acids, this approach still faces difficulties in slowing down protein translocation and improving ionic current signal-to-noise ratio. Here, we observe the unfolding and multistep sequential translocation of SM cytochrome c (cyt c) through a surface enhanced Raman scattering (SERS) active conical gold nanopore. High bias voltage unfolds SM protein causing more exposure of amino acid residues to the nanopore, which slows down the protein translocation. Specific SERS traces of different SM cyt c segments are then recorded sequentially when they pass through the hotspot inside the gold nanopore. This study shows that the combination of SM SERS with a nanopore can provide a direct insight into protein segments and expedite the development of nanopore toward SM protein sequencing.
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Nanoporos , Proteínas , Nanotecnología , Oro/química , AminoácidosRESUMEN
Direct structural and dynamic characterization of protein conformers in solution is highly desirable but currently impractical. Herein, we developed a single molecule gold plasmonic nanopore system for observation of protein allostery, enabling us to monitor translocation dynamics and conformation transition of proteins by ion current detection and SERS spectrum measurement, respectively. Allosteric transition of calmodulin (CaM) was elaborately probed by the nanopore system. Two conformers of CaM were well-resolved at a single-molecule level using both the ion current blockage signal and the SERS spectra. The collected SERS spectra provided structural evidence to confirm the interaction between CaM and the gold plasmonic nanopore, which was responsible for the different translocation behaviors of the two conformers. SERS spectra revealed the amino acid residues involved in the conformational change of CaM upon calcium binding. The results demonstrated that the excellent spectral characterization furnishes a single-molecule nanopore technique with an advanced capability of direct structure analysis.
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Oro , Nanoporos , Oro/química , Espectrometría Raman/métodos , Proteínas , AminoácidosRESUMEN
We explored the application of two-dimensional covalent organic frameworks (2D COFs) in single molecule DNA analysis. Two ultrathin COF nanosheets were exfoliated with pore sizes of 1.1 nm (COF-1.1) and 1.3 nm (COF-1.3) and covered closely on a quartz nanopipette with an orifice of 20 ± 5 nm. COF nanopores exhibited high size selectivity for fluorescent dyes and DNA molecules. The transport of long (calf thymus DNA) and short (DNA-80) DNA molecules through the COF nanopores was studied. Because of the strong interaction between DNA bases and the organic backbones of COFs, the DNA-80 was transported through the COF-1.1 nanopore at a speed of 270 µs/base, which is the slowest speed ever observed compared with 2D inorganic nanomaterials. This study shows that the COF nanosheet can work individually as a nanopore monomer with controllable pore size like its biological counterparts.
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Estructuras Metalorgánicas , Nanoporos , ADN , Colorantes FluorescentesRESUMEN
Ultrathin nanosheets of two-dimensional covalent organic frameworks covered a quartz nanopipette and then acted as a nanopore device for single-molecule DNA sensing. Our results showed that a single DNA homopolymer as short as 6 bases could be detected. The dwell times of 30-mer DNA homopolymers were obviously longer than the times of 10- or 6-mer ones. For different bases, poly(dA)6 showed the slowest transport speed (â¼595 µs/base) compared with cytosine (â¼355 µs/base) in poly(dC)6 and thymine (â¼220 µs/base) in poly(dT)6. Such translocation speeds are the slowest ever reported in two-dimensional material-based nanopores. Poly(dA)6 also showed the biggest current blockade (94.74 pA) compared with poly(dC)6 (79.54 pA) and poly(dT)6 (71.41 pA). However, the present difference in blockade current was not big enough to distinguish the four DNA bases. Our study exhibits the shortest single DNA molecules that can be detected by COF nanopores at the present stage and lights the way for DNA sequencing based on solid-state nanopores.
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Estructuras Metalorgánicas , Nanoporos , ADN , Nanotecnología , Poli A , Análisis de Secuencia de ADN/métodosRESUMEN
DNA and amino acids are important biomolecules in living organisms. Probing such biomolecules with structural characters can provide valuable information for life study. Here, gold plasmonic nanopores (GPNs) with high SERS activity (a local enhancement factor higher than 109) are synthesized at the tip of a glass nanopipette. An electric field drives individual molecules to translocate through the GPNs, which enables in situ collection of the surface-enhanced Raman scattering (SERS). Nonresonant biomolecules, including nucleobases, amino acids, and oligonucleotides (DNA), with single nucleobase differences can be distinguished. The intensity of SERS is tunable by modulating the affinity between DNA and the GPNs. The present study shows the feasibility of applying a plasmonic nanopore to DNA and protein detection, which may also provide an easy way for tracking single molecule translocation by developing a well-defined single plasmonic nanopore.
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Aminoácidos/química , ADN/química , Oro/química , Nanoporos , Espectrometría Raman , Propiedades de SuperficieRESUMEN
Single cell analysis is essential for understanding the heterogeneity, behaviors of cells, and diversity of target analyte in different subcellular regions. Nucleolin (NCL) is a multifunctional protein that is markedly overexpressed in most of the cancer cells. The variant expression levels of NCL in subcellular regions have a marked influence on cancer proliferation and treatments. However, the specificity of available methods to identify the cancer biomarkers is limited because of the high level of subcellular matrix effect. Herein, we proposed a novel technique to increase both the molecular and spectral specificity of cancer diagnosis by using aptamers affinity based portable nanopipette with distinctive surface-enhanced Raman scattering (SERS) activities. The aptamers-functionalized gold-coated nanopipette was used to capture target, while p-mercaptobenzonitrile (MBN) and complementary DNA modified Ag nanoparticles (AgNPs) worked as Raman reporter to produce SERS signal. The SERS signal of Raman nanotag was lost upon NCL capturing via modified DNA aptamers on nanoprobe, which further helped to verify the specificity of nanoprobe. For proof of concept, NCL protein was specifically extracted from different cell lines by aptamers modified SERS active nanoprobe. The nanoprobes manifested specifically good affinity for NCL with a dissociation constant Kd of 36 nM and provided a 1000-fold higher specificity against other competing proteins. Furthermore, the Raman reporter moiety has a vibrational frequency in the spectroscopically silent region (1800-2300 cm-1) with a negligible matrix effect from cell analysis. The subcellular localization and spatial distribution of NCL were successfully achieved in various types of cells, including MCF-7A, HeLa, and MCF-10A cells. This type of probing technique for single cell analysis could lead to the development of a new perspective in cancer diagnosis and treatment at the cellular level.
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Biomarcadores de Tumor/análisis , Nanopartículas del Metal/química , Sondas Moleculares/química , Nanotecnología , Fosfoproteínas/análisis , Proteínas de Unión al ARN/análisis , Plata/química , Análisis de la Célula Individual , Células HeLa , Humanos , Células MCF-7 , Espectrometría Raman , Propiedades de Superficie , Células Tumorales Cultivadas , NucleolinaRESUMEN
This study is to establish the UPLC fingerprint of red ginseng. The separation was performed on a Waters Acquity BEH C18 column (2.1 mm × 50 mm,1.7 µm), with the mobile phase consisting of acetonitrile and water for gradient elution. The detection wavelength was set at 203 nm. The UPLC fingerprint of red ginseng was established by using sample chromatography of 22 different purchase areas and 26 common peaks were found. Compared with the reference substances, 11 of the common peaks were identified as ginsenosides Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rh1, ginsenoside Rg2, ginsenoside Rb1, 20(S)-ginsenoside F1, ginsenoside Rb2, ginsenoside Rb3, 20(S)-ginsenoside Rg3 and 20(R)-ginsenoside Rg3, respectively. It is worth noting that 20(S)-ginsenoside Rg3 and 20(R)-ginsenoside Rg3 are the characteristic ingredients of red ginseng, and they could be used not only for distinguishing red ginseng and ginseng, but also for process controlling of the preparation of red ginseng. The similarity was analyzed with' Similarity Evaluation System for Chromatographic Fingerprint of Chinese Materia Medica, and the similarity of 18 batches samples is up to 0.9. Compared to the literature methods, the method is simple, time-saving,specific for the separation of ginsenosides from red ginseng. So, this method could be used for the species identification and quality control of ginseng, red ginseng and American ginseng, and it will alsoprovide a theoretical basis of raising quality standards of the above mentioned Chinese herb medicines.
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Ginsenósidos/análisis , Panax/química , Cromatografía Líquida de Alta Presión , Medicamentos Herbarios Chinos/química , Control de CalidadRESUMEN
Using reversed-phase high performance liquid chromatography, nine ginsenosides were simultaneously separated on an Ultimateî°C18 column with high-resolution and high purity of each chromatographic peak. Adopting the QAMS quality evaluation model for traditional Chinese medicines, ginsenoside Rb1 was used as the internal reference substance, and the relative correction factors (RCFs) and the relative retention values (RTRs) of ginsenosides Rg1, Re, Rf, Rb1, Rc, Rb2, Rb3, Rd and 20 (S)-ginsenoside Rg3 to ginsenoside Rb1 were calculated individually. Through a series of methodology evaluations, and positioned by the red ginseng reference chromatograph and RTVs, nine ginsenosides in red ginseng were simultaneously assayed only by quantitative determined ginsenoside Rb1.
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Ginsenósidos/análisis , Panax/química , Cromatografía Líquida de Alta Presión , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVE: To study the efficacy of Huai Qi Huang granules in the treatment of childhood primary nephrotic syndrome. METHODS: Between July 2009 and December 2011, patients who were admitted and diagnosed for the first time as childhood primary nephrotic syndrome were randomized into a treatment group (Huai Qi Huang granules plus glucocorticoid; n=23) and a control group (glucocorticoid alone; n=19) for a prospective study. The two groups were compared for regression time of edema, time to urinary protein clearance, relapse rate, incidence of infection, dosage of glucocorticoid, and humoral and cellular immunological indicators. RESULTS: There were no significant differences in regression time of edema, time to urinary protein clearance, and relapse rate between the treatment and control groups (P>0.05). The treatment group had significantly lower incidence of infection and daily dose of glucocorticoid (at month 6) than the control group (P<0.05). Humoral and cellular immunological indicators showed no significant differences between the two groups (P>0.05). No Huai Qi Huang-related adverse events were observed in this study. CONCLUSIONS: Huai Qi Huang granules treatment can reduce the dose of glucocorticoid and the incidence of infection in children with primary nephrotic syndrome and has a favourable safety.
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Astragalus propinquus , Medicamentos Herbarios Chinos/uso terapéutico , Síndrome Nefrótico/tratamiento farmacológico , Niño , Preescolar , Femenino , Glucocorticoides/uso terapéutico , Humanos , Lactante , Masculino , Estudios ProspectivosRESUMEN
The Donnan potential is successfully isolated from ion pair potential on a ferrocene-labeled polyelectrolyte (DNA) monolayer. The isolated Donnan potential shifts negatively upon the increase in NaClO4 concentration with a slope of -58.8 mV/decade. With the salt concentration grown up to 1 M, the stretched DNA chains in low salt concentration are found to experience a gradual conformation relaxing process. At salt concentrations higher than 2 M, Donnan breakdown occurs where only the ion pair effect modulates the apparent potential. The apparent formal potential also shows strong dependence on solution pH, which reveals that the charge density in the polyelectrolyte monolayer plays an important role in the establishment of Donnan equilibrium.
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Polímeros/química , Electroquímica , Concentración de Iones de Hidrógeno , Percloratos/química , Compuestos de Sodio/químicaRESUMEN
Simultaneous detection and structural characterization of protein variants on a single platform are highly desirable but technically challenging. Herein, we present a single-molecule spectral system based on a gold plasmonic nanopore for analyzing two peptides and their single-point mutated variants. The gold plasmonic nanopore enabled the high-throughput acquisition of surface-enhanced Raman scattering (SERS) spectra at the single-molecule level by electrically driving analytes into hot spots. Furthermore, a statistical method based on Boolean operations was developed to extract prominent features from fluctuated single-molecule SERS spectra. The effects of the single-amino acid substitutions on both the intramolecular interactions and the peptide conformations were directly characterized by the nanopore system, and the results agreed with the predictions by AlphaFold2. This study highlights the mutual benefits of spectroscopy and nanopore technology, whereby the gold plasmonic nanopore offers a powerful tool for the structural analysis of single-molecule proteins.
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Sustitución de Aminoácidos , Oro , Nanoporos , Péptidos , Espectrometría Raman , Oro/química , Espectrometría Raman/métodos , Péptidos/química , Propiedades de SuperficieRESUMEN
OBJECTIVE: To establish an HPLC-DAD method for simultaneous determination of eight ginsenosides in Renshenshouwu capsules. METHOD: Ultimate C18 column (4.6 mm x 250 mm, 5 microm) was adopted for gradient elution, with acetonitrile and water as the mobile phase. The flow rate was 1 mL x min(-1), the column temperature was set at 30 degrees C, and the detection wavelength was 203 nm. RESULT: A good linearity was observed in the range of 0.242-12.1, 0.222-11.1, 0.251-25.1, 0. 245-24.5, 0.232-23.2, 0.232-23.2, 0.264-26.4, 0.244-24.4 microg for ginsenoside Rg1, Re, Rb1, Rc, Rb2, Rb3, Rd and 20(S)-ginsenoside Rg3, respectively, with the average recoveries of 102.7%, 103.2%, 101.6%, 101.2%, 102.0%, 100.7%, 101.9%, 102.0%, respectively. CONCLUSION: The method was so simple, accurate and effective that it could be used for quality control of the above eight components in Renshenshouwu capsules.
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Cromatografía Líquida de Alta Presión/métodos , Medicamentos Herbarios Chinos/análisis , Ginsenósidos/análisis , Cápsulas/análisis , Cromatografía Líquida de Alta Presión/instrumentaciónRESUMEN
The simultaneous and accurate detection of intracellular pH (pHi) and extracellular pH (pHe) is essential for studying the complex physiological activities of cancer cells and exploring pH-related therapeutic mechanisms. Here, we developed a super-long silver nanowire-based surface-enhanced Raman scattering (SERS) detection strategy for simultaneous sensing of pHi and pHe. A surface-roughened silver nanowire (AgNW) with a high aspect ratio is prepared at a nanoelectrode tip using a Cu-mediated oxidation process, which is then modified by pH-sensitive 4-mercaptobenzoic acid (4-MBA) to form 4-MBA@AgNW as a pH sensing probe. With the assistance of a 4D microcontroller, 4-MBA@AgNW is efficient in simultaneously detecting pHi and pHe in both 2D and 3D culture cancer cells by SERS, with minimal invasiveness, high sensitivity, and spatial resolution. Further investigation proves that the surface-roughened single AgNW can also be used in monitoring the dynamic variation of pHi and pHe of cancer cells upon stimulation with anticancer drugs or under a hypoxic environment.
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Nanopartículas del Metal , Nanocables , Plata , Espectrometría Raman/métodos , Compuestos de SulfhidriloRESUMEN
OBJECTIVE: To explore the role of autophagy in quercetin (Que)-induced apoptosis in human bladder carcinoma BIU-87 cells in vitro. METHODS: To determine the proliferative inhibition by MTT colorimetric assay after treating BIU-87 cells with quercetin at various concentrations. To identify autophagy and apoptosis in the BIU-87 cells after Que treatment by monodansylcadaverin (MDC) and Hoechst 33258 fluorescent staining, respectively. To examine the cytotoxic effect of Que and influence of autophagy on apoptosis by studying LDH leakage rate and flow cytometry, after blocking the autophagy with 3-methlyadenine (3-MA), a specific autophagy inhibitor. RESULTS: There was an obvious inhibitory effect of Que on the proliferation of BIU-87 cells in a time- and dose-dependent manner. The inhibition rate of BIU-87 cells after 200 µmol/L Que treatment for 72 hours was 89.2%. Autophagy and apoptosis were induced and detected in Que-treated BIU-87 cells and autophagy occurred earlier than apoptosis. The apoptosis peak became much higher after the autophagy was blocked. Whenever the autophagy was blocked before or after Que treatment, the Que-induced cytotoxicity in BIU-87 cells was enhanced. CONCLUSIONS: Quercetin significantly inhibits the proliferation of BIU-87 cells, and the autophagy is induced earlier than apoptosis. In the process of Que-induced apoptosis of BIU-87 cells, autophagy may play a protective role at the initiation phase, delay apoptosis and reduce the Que-induced death of BIU-87 cells.
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Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Autofagia/fisiología , Quercetina/farmacología , Neoplasias de la Vejiga Urinaria/patología , Adenina/análogos & derivados , Adenina/farmacología , Antioxidantes/administración & dosificación , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , L-Lactato Deshidrogenasa/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Quercetina/administración & dosificaciónRESUMEN
OBJECTIVE: To investigate the effect of different chromatographic conditions on QAMS relative correlation factors (RCF) and relative retention values (RT(R)). METHOD: C18 columns were used with methyl alcohol-0.4% phosphoric acid water (85: 15) as the mobile phase. The detection wavelength was 254 nm, the column temperature was 30 degrees C, and the flow rate was 1.0 mL x min(-1). The five anthraquinones in Rhei Radix et Rhizoma were selected to be the objects of study. The RCF and RT(R) among aloe-emodin, rhein, chrysophanol, physcion and emodin were determined under different chromatographic conditions. RESULT: Their RCFs showed no significant difference. CONCLUSION: The RCFs among anthraquinones established by QAMS can be used as a constant in content determination of traditional Chinese medicines/patent traditional Chinese medicines.
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Cromatografía Líquida de Alta Presión/métodos , Rheum/químicaRESUMEN
Tree-based and deep learning methods can automatically generate useful features. Not only can it enhance the original feature representation, but it can also learn to generate new features. This paper develops a strategy based on Light Gradient Boosting Machine (LightGBM or LGB) and Gated Recurrent Unit (GRU) to generate features to improve the expression ability of limited features. Moreover, a SARIMA-GRU prediction model considering the weekly periodicity is introduced. First, LightGBM is used to learn features and enhance the original features representation; secondly, GRU neural network is used to generate features; finally, the result ensemble is used as the input for prediction. Moreover, the SARIMA-GRU model is constructed for predicting. The GRU prediction consequences are revised by the SARIMA model that a better prediction can be obtained. The experiment was carried out with the data collected by Ride-hailing in Chengdu, and four predicted indicators and two performance indexes are utilized to evaluate the model. The results validate that the model proposed has significant improvements in the accuracy and performance of each component.
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ETHNOPHARMACOLOGICAL RELEVANCE: Jieduquyuziyin prescription (JP) is a traditional Chinese medicine (TCM) formula. According to both TCM theory and more than a decade of clinical practice, JP has been testified to be effective for systemic lupus erythematosus (SLE) treatment as an approved hospital prescription in China. AIM OF THE STUDY: To determine the effect of JP on the treatment of SLE by glucocorticoid (GC) and to further examine the molecular mechanisms. MATERIALS AND METHODS: We conducted in vivo experiments to estimate the effect of JP on hepatic gluconeogenesis in MRL/lpr mice treated with GC. Additionally, isoproterenol (ISO) induced hepatic gluconeogenesis model and GC-treated MRL/lpr mouse hepatocytes were carried out in vitro experiments to verify the effect of JP on gluconeogenesis. RESULTS: The results showed that JP combined with GC could effectively alleviate the lupus symptoms in MRL/lpr mice and improve the pathological changes of the kidney and liver. And the combination of JP reduced the side effects caused by GC, which was related to the inhibition of GC-induced hepatic gluconeogenesis in MRL/lpr mice. Specifically, JP up-regulated the expression of glucocorticoid receptor (GR) α, phosphoinositide-3-kinase (PI3K) and Akt restrained by GC to reduce the production of forkhead box O1 (FoxO1), peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), and the gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). In vivo, the use of JP either alone or with GC could reduce spleen enlargement, high levels of serum antibodies, aggravated urine protein and renal pathological damage in MRL/lpr mice. Furthermore, the glucose content was reduced in the liver of MRL/lpr mice treated with JP, and the liver damage and steatosis were also alleviated. In vitro, the expressions of PI3K and Akt increased and the expressions of FoxO1, PGC-1α, PEPCK and G6Pase decreased after JP treatment in ISO-treated hepatocytes. Compared with MRL/MP mice, we found that JP could significantly inhibit the expression of gluconeogenesis in the hepatocytes of MRL/lpr mice induced by GC to a greater extent. CONCLUSIONS: The therapeutic effect of JP on GC-induced is likely related to hepatic gluconeogenesis, which provides a new perspective to reveal the positive role of JP in SLE.
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Medicamentos Herbarios Chinos/uso terapéutico , Gluconeogénesis/efectos de los fármacos , Hígado/efectos de los fármacos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Femenino , Glucocorticoides , Humanos , Hígado/metabolismo , Ratones , Ratones Endogámicos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Fosfatidilinositol 3-Quinasas/genética , Fitoterapia , Proteínas Proto-Oncogénicas c-akt/genéticaRESUMEN
Establishing a systematic molecular information analysis strategy for cell culture models is of great significance for drug development and tissue engineering technologies. Here, we fabricated single silver nanowires with high surface-enhanced Raman scattering activity to extract SERS spectra in situ from two-dimensional (2D) and three-dimensional (3D) cell culture models. The silver nanowires were super long, flexible and thin enough to penetrate through multiple cells. A single silver nanowire was used in combination with a four-dimensional microcontroller as a cell endoscope for spectrally analyzing the components in cell culture models. Then, we adopted a machine learning algorithm to analyze the obtained spectra. Our results show that the abundance of proteins differs significantly between the 2D and 3D models, and that nucleic acid-rich and protein-rich regions can be distinguished with satisfactory accuracy.
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Nanocables , Ácidos Nucleicos , Plata , Técnicas de Cultivo Tridimensional de Células , Espectrometría Raman/métodos , Imagen MolecularRESUMEN
BACKGROUND: Atherosclerosis is one of the main causes of coronary artery ostial lesions seen clinically. Secondary coronary artery ostial lesions are rare, and cases reported previously were associated with syphilitic vasculitis and aortic dissection. Here, we report three rare cases of secondary coronary ostial lesions. Due to their rareness, these lesions can easily be neglected, which may lead to misdiagnosis and missed diagnosis. CASE SUMMARY: We present three patients with acute myocardial infarction and unstable angina caused by secondary coronary artery ostial lesions. In Case 1, coronary angiography (CAG) revealed 90% stenosis of the left main coronary ostium. Chest contrast computed tomography (CT) suggested thymic carcinoma invading the left main coronary ostium. Coronary artery bypass grafting and tumor resection were performed. In Case 2, echocardiography revealed a sinus of Valsalva aneurysm (SVA)-like dilatation. CAG showed a right coronary sinus giant aneurysm and complete obstruction of the right coronary artery (RCA) ostium. Aortic contrast CT confirmed these findings. The Bentall procedure was performed. In Case 3, CT CAG identified an anomalous origin of the right coronary artery (AORCA) from the left sinus of Valsalva coursing between the aorta and pulmonary trunk, causing severe RCA ostium stenosis by compression. Surgical correction of the AORCA was performed. CONCLUSION: The cases reported here suggest that we should consider other causes of coronary ostial lesions other than atherosclerosis.
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BACKGROUND: Coronovirus disease 2019 (COVID-19) has spread rapidly across the globe. People of all ages are susceptible to COVID-19. However, literature reports on pediatric patients are limited. METHODS: To improve the recognition of COVID-19 infection in children, we retrospectively reviewed two confirmed pediatric cases from two family clusters. Both clinical features and laboratory examination results of the children and their family members were described. RESULTS: The two confirmed children only presented with mild respiratory or gastrointestinal symptoms. Both of them had normal chest CT images. After general and symptomatic treatments, both children recovered quickly. Both families had travel histories to Hubei Province. CONCLUSIONS: Pediatric patients with COVID-19 are mostly owing to family cluster or with a close contact history. Infected children have relatively milder clinical symptoms than infected adults. We should attach importance to early recognition, early diagnosis, and early treatment of infected children.