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Malaria cases reported by county laboratories were further tested in the provincial laboratory in Guizhou province by using PCR test and microscopy. The consistency between PCR and microscopic results in the provincial laboratory was set as the basis for evaluation of microscopic results in county laboratories. In 89 samples, 24 were identified by PCR to be positive for malaria, among which 15 were infected with P. falciparum, 7 with P. vivax, and 2 with P. ovale; all were imported cases. And 21 samples had consistent identifications by PCR test and microscopic examination in the provincial laboratory. The total coincidence rate between county and provincial laboratories was 79.8%(67/84), and the undetected and error rates in county laboratories were 9.5%(2/21) and 23.8% (15/63), respectively. The Kappa value between county and provicial diagnosis was 0.6, being at the medium-to-high level of consistency.
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Malaria , China , Humanos , Microscopía , Reacción en Cadena de la PolimerasaRESUMEN
OBJECTIVE: To establish a single-tube single-run multiplex PCR technique that can detect single or mixed samples with four species of Plasmodium. METHODS: Folding primers were designed based on the fast nested PCR. The reaction component concentrations were optimized and the primers were selected based on the annealing temperature. The established single-tube single-run folding-primer multiplex PCR (FP-PCR) was tested for its sensitivity and specificity to detect single-species and mixed samples with P. vivax, P. falciparum, P. ovale (including P. ovale wallikeri) and/or P. malariae. RESULTS: In all the seven experimental repeats, FP-PCR successfully detected single-species infection for all the four species, with the detection limit reaching or close to 1 parasite/µl blood. For mixed infections with 2-4 species at different densities with the highest being 100 times of the lowest, FP-PCR identified all the species in each combination in 57 out of 84 tests. Further, in 10 dried blood samples on filter paper from healthy subjects, no FP-PCR amplification was found, except weak formation of dimers. CONCLUSION: FP-PCR is a simple and sensitive method for detecting both single-species and mixed infections with human Plasmodium, and can be applied for malaria diagnosis, screening and monitoring.
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Reacción en Cadena de la Polimerasa Multiplex , Plasmodium , Coinfección , Cartilla de ADN , HumanosRESUMEN
Fractional crystallization plays a critical role in generating the differentiated continental crust on Earth. However, whether efficient crystal-melt separation can occur in viscous felsic magmas remains a long-standing debate because of the difficulty in discriminating between differentiated melts and complementary cumulates. Here, we found large (~1 per mil) potassium isotopic variation in 54 strongly peraluminous high-silica (silicon dioxide >70 weight %) leucogranites from the Himalayan orogen, with potassium isotopes correlated with trace elemental proxies (e.g., strontium, rubidium/strontium, and europium anomaly) for plagioclase crystallization. Quantitative modeling requires up to ~60 to 90% fractional crystallization to account for the progressively light potassium isotopic composition of the fractionated leucogranites, while plagioclase accumulation results in enrichment of heavy potassium isotopes in cumulate leucogranites. Our findings strongly support fractional crystallization of high-silica magmas and highlight the great potential of potassium isotopes in studying felsic magma differentiation.
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Endothelial dysfunction in atherosclerotic cardiovascular diseases has become one of the main characteristics in patients with diabetes mellitus, which is usually caused by abnormal inflammation and oxidative stress response. Presently, we focused on the role of Notoginsenoside R1 (NR1), a major component isolated from Panax notoginseng, in endothelial dysfunction caused by high glucose (HG). Human umbilical vein endothelial cells (HUVECs) were treated with HG and then dealt with NR1. Cell counting kit-8 assay and 5-bromo-2'-dexoyuridine assay were conducted to examine cell proliferation and viability. Flow cytometry was used to measure apoptosis. The angiogenesis of HUVECs was determined by tube formation assay. Moreover, the expressions of miR-147a, inflammatory cytokines (TNF-α, IL-6, and IL-10) and oxidative stress markers malondialdehyde, superoxide dismutase, and glutathione peroxidase were measured. The protein levels of MyD88/TRAF6/NF-κB axis, Bax, Bcl2, and Caspase3 were detected by Western blot. Furthermore, gain and loss of functional assays of miR-147a were performed to verify the role of miR-147a in NR1-mediated effects. Our data confirmed that NR1 (at 10-40 µM) reduces HG-induced HUVECs proliferation and viability inhibition, mitigates apoptosis, and enhances tube formation ability. Meanwhile, NR1 inhibited oxidative stress and inflammatory response and blocked the activation of the MyD88/TRAF6/NF-κB pathway induced by HG. In addition, NR1 promoted the expression of miR-147a, which targeted MyD88. Overexpression of miR-147a markedly inactivated MyD88/TRAF6/NF-κB pathway, while the miR-147a inhibitors reversed NR1-mediated protective effect in HG-induced HUVECs through activating MyD88/TRAF6/NF-κB pathway. In conclusion, NR1 relieves HG-induced endothelial cell injury by downregulating the MyD88/TRAF6/NF-κB pathway via upregulating miR-147a.
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Ginsenósidos/farmacología , Glucosa/toxicidad , Inflamación/prevención & control , MicroARNs/fisiología , Estrés Oxidativo/efectos de los fármacos , Células Cultivadas , Ginsenósidos/uso terapéutico , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/fisiología , FN-kappa B/fisiología , Neovascularización Fisiológica/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
OBJECTIVE: To compare the usefulness of Tag-primer nested/multiplex PCR (UT-PCR) method with microscopy in malaria diagnosis and surveillance. METHODS: 400 blood smears and blood samples on filter paper were taken from febrile patients which were initially diagnosed as malaria or suspected malaria during surveillance in mixed endemic areas of Plasmodium falciparum (Pf) and P. vivax (Pv) in Hainan and Yunnan provinces and a malaria-controlled area in Guangxi Zhuang Autonomous Region. The initial test results of both UT-PCR and microscopy for the 400 samples were compared under double blind condition. Blood smears with discrepant results between the two methods were retested by an experienced microscopist, and also repeated by UT-PCR for 2-3 times. The sensitivity and specificity of the two methods were evaluated following the Tjitra's method. RESULTS: Among the 400 blood samples, 234 were found plasmodium-positive by microscopy with 125 Pf and 109 Pv; 235 were positive by UT-PCR including 124 Pf, 109 Pv and 2 mixed infection. Altogether, the coincidence between the two methods stood for 92.5 % (370/400), including 154 negatives and 216 positives (Pv 117, Pf 99). 25 samples with discrepancy from the initial detections were retested, which covered 11 microscopy negative and PCR positive, 10 microscopy positive and PCR negative, 3 microscopy Pf positive and PCR Pv positive, 1 microscopy Pv positive but PCR mixed infection. 15 of the 25 samples showed same UT-PCR results, 7 "false positives" and 3 "false negatives" . Therefore, the sensitivity and specificity of UT-PCR was 99.6% and 98.8% respectively. CONCLUSION: As a diagnosis method, UT-PCR is useful for confirmation of malaria diagnosis and differentiation of Plasmodium species, also for improving the effectiveness and quality of malaria surveillance.
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Malaria/diagnóstico , Reacción en Cadena de la Polimerasa Multiplex , Diagnóstico Diferencial , Humanos , Malaria Falciparum/diagnóstico , Malaria Vivax/diagnóstico , Microscopía , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: To improve the sensitivity, specificity and stability of the Tag-primer nested/multiplex PCR for malaria diagnosis. METHODS: Filter paper blood samples were collected from 30 non-malaria fever patients and 20 infectious disease patients (common cold, influenza, typhoid, hepatitis, etc.). Four ml blood each taken from one falciparum malaria patient and one vivax malaria patient was serially diluted. Healthy blood sample was used as negative control. Improved direct heating method was used to prepare DNA template. The cytochrome oxidase gene (coxI) located in mitochondrion was selected as target gene. Relevant web resources and software (PUBMED, NCBI-BLAST, Mfold server and Primer Premier 5.0) were employed to design and optimize Tag-primer nested/multiplex PCR (UT-PCR) which was used to test all blood samples. RESULTS: A 611 bp band and a 255 bp band were seen in serially diluted infected blood samples (1,000, 100, 10 and 1 parasite/microl) from P.f and P.v patient tested by UT-PCR. The detection limit of either P. falciparum or P. vivax reached 1 parasite/microl, and the tested blood samples of non-malaria fever patients, patients with other infectious diseases and healthy persons were all negative. Consistent results of each sample in more than 3 duplicated tests were obtained. CONCLUSION: The optimized Tag-primer nested/multiplex PCR shows high sensitivity, specificity and stability in malaria diagnosis.
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ADN Protozoario/genética , Malaria Falciparum/diagnóstico , Malaria Vivax/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Animales , Cartilla de ADN , ADN Protozoario/sangre , Complejo IV de Transporte de Electrones/genética , Humanos , Malaria Falciparum/sangre , Malaria Falciparum/parasitología , Malaria Vivax/sangre , Malaria Vivax/parasitología , Plasmodium falciparum/genética , Plasmodium vivax/genética , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: To establish a sensitive, simple to use and low noise nested/multiplex PCR for simultaneously detection of Plasmodium falciparum (Pf) and Plasmodium vivax (P.v). METHODS: The tag primer amplification technique, software Primer Premier 5.0, NCBI-BLAST web resources and the matrix test were used to optimize the nested/multiplex PCR for detection of P.f and P.v with filter paper blood samples taken from malaria patients diagnosed by microscopy, and the results of the optimized nested/multiplex PCR and microscopy were evaluated. RESULTS: The sensitivity of the optimized PCR, determined by the examination of imitative filter paper blood samples, was about 1-2 parasites / microl for P.f and 5-10 parasites / microl for P.v. Primer-dimer and other PCR noise were removed. When 71 field filter paper blood samples taken from microscopically diagnosed patients (24 P.f, 47 P.v) were examined, the concordance between the optimized PCR and microscopy was 875% for Pf and 100% for P.v. CONCLUSION: The nested/multiplex PCR optimized by tag primer amplification technique is simple, with low noise and being able to detect Pf and P.v simultaneously. It is more sensitive in detecting cases with low parasitaemia and more accurate in identifying Plasmodium species than microscopy.
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Malaria/diagnóstico , Plasmodium falciparum/aislamiento & purificación , Plasmodium vivax/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Animales , Cartilla de ADN , ADN Protozoario/sangre , Humanos , Malaria/parasitología , Plasmodium falciparum/genética , Plasmodium vivax/genética , Sensibilidad y EspecificidadRESUMEN
Alpinia zerumbet 'Variegata' is an aromatic medicinal plant, its foliage producing an intense, unique fragrant odor. This study identified 46 volatile compounds in the leaf tissue of this plant using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). The major compounds included 1, 8-cineole (43.5%), p-cymene (14.7%), humulene (5.5%), camphor (5.3%), linalool (4.7%), (E)-methyl cinnamate (3.8%), gamma-cadinene (3.3%), humulene oxide II (2.1%) and a-terpineol (1.5%). The majority of the volatiles were terpenoids of which oxygenated monoterpenes were the most abundant, accounting for 57.2% of the total volatiles. Alcohols made up the largest (52.8%) and aldehydes the smallest (0.2%) portions of the volatiles. Many bioactive compounds were present in the volatiles.