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Aim: To evaluate the value of combined detection of plasma cfDNA concentration and integrity in the early diagnosis of NSCLC. Methods: Real-time fluorescence quantitative PCR was used to determine the concentration and integrity of plasma cfDNA in 71 NSCLC patients and 53 healthy people. Results: Combined detection of plasma cfDNA concentration and integrity had higher diagnostic power in differentiating NSCLC patients with stage I/II from healthy people than detection of plasma cfDNA concentration alone or integrity alone. The AUC, sensitivity and specificity of the combined detection of plasma cfDNA concentration and integrity were 0.781, 0.62 and 0.85. Conclusion: Combined detection of plasma cfDNA concentration and integrity could improve the diagnostic value in NSCLC detection.
The discovery of cfDNA has opened up a wide range of new possibilities for the diagnosis of cancer. CfDNA provides a noninvasive diagnostic approach for early screening, early detection and monitoring of patients with cancer. Currently, the application of cfDNA in clinical practice for NSCLC patients has been widely reported, which mainly focused on DNA methylation detection, oncogenic driver gene mutation detection. However, few studies have evaluated the diagnostic value of combined detection of plasma cfDNA concentration and integrity for NSCLC patients. Our study suggests that the combination of plasma cfDNA concentration and integrity has higher AUC value in differentiating NSCLC patients from healthy individuals than plasma cfDNA concentration alone or integrity alone.
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Background: Circulating cell-free DNA (cfDNA) concentration and integrity as noninvasive biomarkers play an important role in cancer diagnosis, prognosis and therapy monitoring. However, few studies have been conducted on the combination of plasma cfDNA concentration, integrity and tumor markers (CEA, CA125, NSE and CYFRA21-1) for cancer detection. Thus, the purpose of this study was to investigate the diagnostic value of combining plasma cfDNA concentration, integrity and tumor markers in early detection of non-small cell lung cancer (NSCLC). Methods: Plasma cfDNA concentration from 50 healthy controls and 84 NSCLC patients were assessed by quantitative real-time PCR of ALU repeated sequence. Plasma cfDNA integrity was calculated as the ratio of long to short fragments (ALU115/60). Results: Plasma cfDNA concentration (ALU60 and ALU115) and integrity ALU115/60 were significantly higher in NSCLC patients with stage III/IV than in healthy controls (p = 0.0002, p < 0.0001, and p = 0.0093, respectively). The receiver operating characteristic (ROC) curve for discriminating NSCLC patients from healthy controls had an area under the curve (AUC) of 0.936 (95 % CI, 0.939-0.996). Moreover, the combination of plasma cfDNA concentration, integrity and tumor markers (CEA, CA125, NSE and CYFRA21-1) had higher diagnostic performance than either plasma cfDNA concentration alone, integrity alone or tumor markers alone, with sensitivity, specificity and AUC value of 94.05%, 90.00% and 0.968, respectively. These results demonstrated that the combination of plasma cfDNA concentration, integrity and tumor markers could significantly improve the diagnostic accuracy of NSCLC. Conclusion: Combination of plasma cfDNA concentration, integrity and tumor markers is a promising biomarker for early diagnosis of NSCLC.
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Azoles and organoselenium compounds are pharmacologically important scaffolds in medicinal chemistry and natural products. We developed an efficient regioselective electrochemical aminoselenation reaction of 1,3-dienes, azoles, and diselenide derivatives to access selenium-containing allylazoles skeletons. This protocol is more economical and environmentally friendly and features a broad substrate scope; pyrazole, triazole, and tetrazolium were all tolerated under the standard conditions, which could be applied to the expedient synthesis of bioactive molecules and in the pharmaceutical industry.
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Leukocytes have an essential role in patient clinical trajectories and progression. Traditional methods of leukocyte enrichment have many significant limitations for current applications. It is demonstrated a novel 3D printing leukocyte sorting accumulator that combines with centrifugation to ensure label-free initial leukocyte enrichment based on cell density and size. The internal structure of leukocyte sorting accumulator (revealed here in a new design, leukocyte sorting accumulator-3, upgraded from earlier models), optimizes localization of the buffy coat fraction and the length of the period allocated for a second centrifugation step to deliver a higher recovery of buffy coats than earlier models. Established methodological parameters were evaluated for reliability by calculating leukocyte recovery rates and erythrocyte depletion rates by both pushing and pulling methods of cell displacement. Results indicate that leukocyte sorting accumulator-3 achieves a mean leukocytes recovery fraction of 96.2 ± 2.38% by the pushing method of layer displacement. By the pulling method, the leukocyte sorting accumulator-3 yield a mean leukocytes recovery fraction of 94.4 ± 0.8%. New procedures for preliminary enrichment of leukocytes from peripheral blood that avoid cellular damage, as well as avert metabolic and phase cycle intervention, are required as the first step in many modern clinical and basic research assays.
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Procedimientos de Reducción del Leucocitos/métodos , Leucocitos/citología , Impresión Tridimensional/instrumentación , Capa Leucocitaria de la Sangre/clasificación , Capa Leucocitaria de la Sangre/citología , Centrifugación/instrumentación , Centrifugación/métodos , Humanos , Procedimientos de Reducción del Leucocitos/instrumentación , Leucocitos/clasificaciónRESUMEN
Aim: To explore the role of urine cell-free DNA (ucfDNA) concentration and integrity indexes as potential biomarkers for lung cancer diagnosis. Materials & methods: Quantitative real-time PCR targeting Arthrobacter luteus (ALU) repeats at three size fragments (ALU-60, 115 and 247 bp) was performed in 55 lung cancer patients and 35 healthy individuals. Results: ucfDNA concentration and integrity indexes were significantly higher in lung cancer patients than in healthy controls. The area under the receiver operating characteristic curve for differentiating patients with stage I/II from healthy controls by ALU fragments concentration were 0.856, 0.909 and 0.932, respectively. In addition, the ucfDNA integrity indexes in patients with lymph node metastasis were significantly higher than in patients with non-metastatic. Conclusion: ucfDNA concentration and integrity indexes could serve as promising biomarkers for lung cancer diagnosis.
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Ácidos Nucleicos Libres de Células/orina , Detección Precoz del Cáncer/métodos , Neoplasias Pulmonares/diagnóstico , Adulto , Anciano , Arthrobacter , Biomarcadores de Tumor , Femenino , Humanos , Neoplasias Pulmonares/patología , Metástasis Linfática , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Curva ROC , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurines. Mutations in the TPMT gene can affect drug activity, which may have adverse effects in humans. Thus, genotyping can help elucidate genetic determinants of drug response to thiopurines and optimize the selection of drug therapies for individual patients, effectively avoiding palindromia during maintenance treatment caused by insufficient dosing and the serious side effects caused by excessive doses. The current available detection methods used for TPMT*3B and TPMT*3C are complex, costly and timeconsuming. Therefore, innovative detection methods for TPMT genotyping are urgently required. The aim of the present study was to establish and optimize a simple, specific and timesaving TPMT genotyping method. Using the principles of Webbased AlleleSpecific PCR and competitive realtime fluorescent allelespecific PCR (CRASPCR), two pairs of Scorpion primers were designed for the detection of TPMT*3B and *3C, respectively, and a mutation in TPMT*3A was inferred based on data from TPMT*3B and *3C. In total, 226 samples from volunteers living in Chongqing were used for CRASPCR to detect TPMT*3 mutations. Results showed that nine (3.98%) were mutant (MT) heterozygotes and none were MT homozygotes for TPMT*3C, and no TPMT*3A and TPMT*3B mutations were found. Three TPMT*3C MT heterozygotes were randomly selected for DNA sequencing, and CRASPCR results were consistent with the sequencing results. In conclusion, in order to improve simplicity, specificity and efficiency, the present study established and optimized CRASPCR assays for commonly found mutant alleles of TPMT*3A (G460A and A719G), TPMT*3B (G460A), and TPMT*3C (A719G).
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Cartilla de ADN/genética , Técnicas de Genotipaje/métodos , Metiltransferasas/genética , Mutación , Adulto , Animales , Femenino , Voluntarios Sanos , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Adulto JovenRESUMEN
BACKGROUND: While blood-derived cell-free DNA has been shown to be a candidate biomarker able to provide diagnostic and prognostic insight in cancer patients, little is known regarding the potential application of urine cell-free DNA (ucfDNA) in diagnosis of cancer. Thus, the aim of this study was to investigate ucfDNA concentration and integrity index as potential biomarkers for early detection of non-small-cell lung cancer (NSCLC). METHODS: Urine samples were collected from 35 healthy controls and 55 NSCLC patients at various tumor node metastasis (TNM) stages. Two long interspersed nuclear element 1 (LINE1) fragments (LINE1-97 and 266 bp) were quantified via quantitative real-time PCR (qPCR). DNA integrity index was calculated as the ratio of LINE1-266/LINE-97. RESULTS: LINE1 fragments concentrations of ucfDNA (LINE1-97, 266 bp) were significantly higher in NSCLC patients with stage III/IV than in stage I/II and in healthy controls. The receiver operating characteristic (ROC) curves for discriminating patients with stage III/IV from healthy controls had areas under the curves (AUC) of 0.84 and 0.886, respectively. Moreover, ucfDNA integrity LINE1-266/97 was significantly higher in patients with stage III/IV than in stage I/II and in healthy controls. The AUC of ROC curve for discriminating patients with stage III/IV from healthy controls was 0.800. Furthermore, LINE1-266 fragment concentration was significantly higher in lymph node metastasis (LNM)-positive patients relative to LNM-negative patients. The ROC curve for discriminating LNM-positive from LNM-negative patients had an AUC of 0.822. CONCLUSION: UcfDNA could serve as a promising biomarker for early detection of NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Ácidos Nucleicos Libres de Células/orina , Neoplasias Pulmonares/diagnóstico , Adulto , Anciano , Biomarcadores de Tumor/orina , Detección Precoz del Cáncer , Femenino , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Masculino , Persona de Mediana Edad , Curva ROCRESUMEN
Epidermal growth factor receptor (EGFR) mutations are associated with response of tyrosine kinase inhibitors (TKIs) for patients with advanced non-small cell lung cancer (NSCLC). However, the existing methods for detection of samples having rare mutations(i.e. ~0.01%) have limits in terms of specificity, time consumption or cost. In the current study, novel wild-type blocking (WTB) oligonucleotides modified with phosphorothioate or inverted dT at the 5'-termini were designed to precisely detect 11 common deletion mutations in exon 19 of EGFR gene (E19del) using a WTB-PCR assay. And internal competitive leptin amplifications were further applied to enhance the specificity of the WTB-PCR system. Our results showed that WTB-PCR could completely block amplification of wild-type EGFR when 200 ng of DNA was used as template. Furthermore, the current WTB-PCR assay facilitated the detection of E19del mutations with a selectivity of 0.01% and sensitivity as low as a single copy. And, the results showed that the current WTB-PCR system exceeded detection limits afforded by the ARMS-PCR assay. In conclusion, the current WTB-PCR strategy represents a simple and cost-effective method to precisely detect various low-abundance deletion mutations.
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Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Oligonucleótidos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Exones/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sistemas de Lectura/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Eliminación de Secuencia/genéticaRESUMEN
Prospective testing for variants in the thiopurine S-methyltransferase (TPMT) is considered a key process in the development of thiopurine therapy. This testing is done to avoid toxicity and side effects in the management of diverse immunological and malignant conditions. Real-time fluorescent PCR techniques using duplex-crossed allele-specific primers in a single tube (DCAS-PCR) were developed in this study to genotype the common loss-of-function TPMT*3B c.460Gâ¯>â¯A (rs1800460) and TPMT*3C c.719Aâ¯>â¯G (rs1142345) usually occurring in individuals of Chinese ethnicity. In this method, several integrated strategies were used to completely eliminate the non-specific amplification that is commonly presented in traditional allele-specific (AS) PCR. These strategies include using AS-primers (ASP) that both are artificially mismatched in the penultimate positions and phosphorothioate modifications in the 5'-termini positions. In the assay, an AS-blocker was used, locus-specific TaqMan (LST) probes were used and we used at least two fragments were simultaneously amplified in a single tube which satisfy the thermodynamic characteristics of DNA polymerase to eliminate non-specific amplification. In a group of 200 unselected subjects, the results showed that 8 samples were heterozygous of TPMT*3C, and all samples possessed wild-type TPMT*3B. There was no non-specific amplification, and the genotypes were 100% consistent with Sanger sequencing.
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Alelos , Metiltransferasas/genética , Reacción en Cadena de la Polimerasa/métodos , Cartilla de ADN , Polimorfismo de Nucleótido SimpleRESUMEN
Tuberculosis, caused by Mycobacterium tuberculosis infection, remains a global public health threat. The success of M. tuberculosis largely contributes to its manipulation of host cell fate. The role of M. tuberculosis PE/PPE family effectors in the host destiny was intensively explored. In this study, the role of PPE60 (Rv3478) was characterized by using Rv3478 recombinant M. smegmatis. PPE60 can promote host cell pyroptosis via caspases/NLRP3/gasdermin. The production of pro-inflammatory cytokines, such as IL-1ß, IL-6, IL-12p40 and TNF-α was altered by PPE60. We found that LUBAC was involved in PPE60-elicited NF-κB signaling by using Linear Ubiquitin Chain Assembly Complex (LUBAC)-specific inhibitor gliotoxin. The PPE60 recombinant M. smegmatis survival rate within macrophages is increased, as well as elevated resistance to stresses such as low pH, surface stresses and antibiotics exposure. For a first time it is firstly reported that M. tuberculosis effector PPE60 can modulate the host cell fate via LUBAC-mediated NF-κB signaling.
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Citocinas/biosíntesis , Macrófagos/inmunología , Mycobacterium tuberculosis/patogenicidad , FN-kappa B/inmunología , Tuberculosis/inmunología , Ubiquitina/inmunología , Antígenos Bacterianos , Proteínas Bacterianas/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Interacciones Huésped-Patógeno , Humanos , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/metabolismo , FN-kappa B/metabolismo , PPAR gamma/metabolismo , Piroptosis/inmunología , Transducción de Señal , Células THP-1 , Tuberculosis/metabolismo , Tuberculosis/microbiología , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitina/metabolismoRESUMEN
Aplastic anaemia (AA) is a life-threatening hematopoietic disorder characterized by hypoplasia and pancytopenia with increasing fat cells in the bone marrow (BM). The BM-derived mesenchymal stem cells (MSCs) from AA are more susceptible to be induced into adipogenic differentiation compared with that from control, which may be causatively associated with the fatty BM and defective hematopoiesis of AA. Here in this study, we first demonstrated that levamisole displayed a significant suppressive effect on the in vitro adipogenic differentiation of AA BM-MSCs. Mechanistic investigation revealed that levamisole could increase the expression of ZFP36L1 which was subsequently demonstrated to function as a negative regulator of adipogenic differentiation of AA BM-MSCs through lentivirus-mediated ZFP36L1 knock-down and overexpression assay. Peroxisome proliferator-activated receptor gamma coactivator 1 beta (PPARGC1B) whose 3'-untranslated region bears adenine-uridine-rich elements was verified as a direct downstream target of ZFP36L1, and knock-down of PPARGC1B impaired the adipogenesis of AA BM-MSCs. Collectively, our work demonstrated that ZFP36L1-mediated post-transcriptional control of PPARGC1B expression underlies the suppressive effect of levamisole on the adipogenic differentiation of AA BM-MSCs, which not only provides novel therapeutic targets for alleviating the BM fatty phenomenon of AA patients, but also lays the theoretical and experimental foundation for the clinical application of levamisole in AA therapy.
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Adipocitos/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Anemia Aplásica/genética , Factor 1 de Respuesta al Butirato/genética , Proteínas Portadoras/genética , Levamisol/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Adipocitos/metabolismo , Adipocitos/patología , Adipogénesis/genética , Adolescente , Adulto , Anemia Aplásica/metabolismo , Anemia Aplásica/patología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Factor 1 de Respuesta al Butirato/agonistas , Factor 1 de Respuesta al Butirato/metabolismo , Proteínas Portadoras/metabolismo , Estudios de Casos y Controles , Diferenciación Celular , Femenino , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Persona de Mediana Edad , Cultivo Primario de Células , Proteínas de Unión al ARN , Transducción de SeñalRESUMEN
The ArsR family of transcriptional regulators are widespread among microorganisms and are involved in various important cellular events, such as metal ion homeostasis, biofilm formation, primary and secondary metabolism, symbiosis, response to adverse condition, and virulence. Its N-terminus contains a winged helix-turn-helix DNA-binding domain that can repress or activate transcription by binding to downstream target promoters. With the increasing number of members in this family identified over the past few decades, the ArsR family members have been intensively explored. In this review, we summarize the function of ArsR family of transcriptional regulators and the mechanisms of metal-regulated gene expression.
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Bacterias/metabolismo , Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Metales/metabolismo , Transactivadores , Bacterias/genéticaRESUMEN
Lipids and lipases/esterases are essential for Mycobacterium tuberculosis (Mtb) survival and persistence, even virulence. Mycobacterium tuberculosis H37Rv Rv1076 (LipU), a member of lipase family, is homologous to the human Hormone Sensitive Lipase (HSL) based on the presence of conserved motif 'GXSXG'. To define the enzymatic characteristics of rv1076, the gene was cloned, and expressed in Escherichia coli. The protein was purified for enzymatic characterization. LipU showed high specific activity for the hydrolysis of short carbon chain substrates with optimal activity at 40°C/pH 8.0 and stability at low temperature and near-neutral pH. The specific activity, Km and Vmax of LipU was calculated to 176.7U/mg, 1.73µM and 62.24µM/min respectively. Ionic detergents can inhibit its activity. The active-site residues of LipU were determined to be Ser140, Asp244 and His269 by site-directed mutagenesis. The upregulation of Mycobacterium tuberculosis rv1076 under nutritive stress implicates a role in starvation.
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Proteínas Bacterianas/metabolismo , Lipasa/metabolismo , Mycobacterium tuberculosis/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Dominio Catalítico , Clonación Molecular , Activación Enzimática/efectos de los fármacos , Inducción Enzimática , Estabilidad de Enzimas , Escherichia coli/genética , Esterasas/metabolismo , Concentración de Iones de Hidrógeno , Lipasa/genética , Lipasa/aislamiento & purificación , Mutagénesis Sitio-Dirigida , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium tuberculosis/genética , Estructura Secundaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Mycobacterium tuberculosis PE/PPE family proteins, named after the presence of conserved PE (Pro-Glu) and PPE (Pro-Pro-Glu) domains at N-terminal, are prevalent in M. tuberculosis genome. The function of most PE/PPE family proteins remains elusive. To characterize the function of PE_PGRS18, the encoding gene was heterologously expressed in M. smegmatis, a nonpathogenic mycobacterium. The recombinant PE_PGRS18 is cell wall associated. M. smegmatis PE_PGRS18 recombinant showed differential response to stresses and altered the production of host cytokines IL-6, IL-1ß, IL-12p40 and IL-10, as well as enhanced survival within macrophages largely via attenuating the apoptosis of macrophages. In summary, the study firstly unveiled the role of PE_PGRS18 in physiology and pathogenesis of mycobacterium.