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1.
J Transl Med ; 17(1): 298, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31470866

RESUMO

BACKGROUND: BRAF mutations occur in 2-4% non-small cell lung cancer (NSCLC) patients and can be categorized into three functional classes based on signaling mechanism and kinase activity: RAS-independent kinase-activating V600 monomers (class 1), RAS-independent kinase-activating dimers (class 2) and RAS-dependent kinase-inactivating heterodimers (class 3). The association between functional classes and clinical features in Chinese NSCLC patients remains unexplored. Our multi-center study aimed to survey the BRAF mutation rate and analyze the associated clinical features in this population. METHODS: Capture-based sequencing data of either plasma or tissue samples obtained from 8405 Chinese stage I-IV NSCLC patients were retrospectively analyzed. RESULTS: BRAF mutations were detected in 238 patients, revealing an overall mutation rate of 2.8%. Among them, 32%, 21% and 13% had BRAF mutant class 1, 2 and 3 respectively. The remaining 34% had other BRAF mutations. V600 (32%) and G469 (13%) were the two most predominant BRAF mutations. Patients with class 2 and 3 mutations were more likely to have concurrent KRAS mutations (P = 0.001). Collectively, BRAF mutations, including non-class 1-3 mutations, were more likely to occur in males (P < 0.01). However, females were more likely to harbor class 1 mutations (P < 0.02). We also compared the overall survival (OS) of first-line chemotherapy-treated advanced-stage patients and revealed comparable OS among the three groups. CONCLUSION: Our study revealed a 2.8% BRAF mutation rate in Chinese NSCLC patients. Our data also showed a male predominance when all BRAF mutations were considered collectively, and a female predominance for class 1 mutations. Furthermore, BRAF V600E is less likely to have concurrent KRAS mutations comparing to the other two classes.

2.
Int J Biol Macromol ; 140: 1167-1174, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31472208

RESUMO

Low-cost natural polymer lignin has been widely used to remove heavy metal ions from polluted water. But it still has some shortcomings, such as poor removal performance, and weak selective adsorption. Thus, in this study, the lignin prepared by Mannich reaction with black liquor was activated with triethylenetetramine (TETA) to achieve a novel adsorbent with high adsorption rates and a strong selectivity for specific oxygen-containing anions. The adsorption capacity of activated lignin (a-CL) on three oxygen-containing anions (i.e. As(V), P(V) and Cr(VI)) was investigated systematically. The adsorption mechanism of a-CL was elucidated theoretically by the density functional theory (DFT) method. Under the same conditions, the selectivity toward oxygen-containing anions by a-CL followed P(V) < Cr(VI) < As(V). Both FT-IR and DFT simulation results revealed that the hydrogen bond between HAsO42- and N dominated the remarkable selectivity of As (V), yielding a maximum adsorption capacity as high as 62.5 mg g-1. Moreover, the adsorption was very fast with a calculated large adsorption kinetic constant. The removal of As(V) reached 100% within 60 min. The As(V) adsorption kinetics and the adsorption isotherms followed the pseudo-second-order and the Langmuir model. This study provides a way for highly selecting removal of As(VI) from polluted water with the lignin.

3.
Eur J Pharm Sci ; : 105058, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31472255

RESUMO

The biofilm formation of Pseudomonas aeruginosa (P. aeruginosa) is regulated by a phenomenon of quorum sensing (QS). With 5-hydroxyl-3,4-halogenated-5H-furan-2-ones as beginning, analogs bearing alkyl chains, vinyl bromide, or aromatic rings were designed and synthesized. The minimum inhibitory concentration (MIC) of the compounds against P. aeruginosa was assayed and the biofilm inhibition ratio was determined at different concentrations lower than the MIC. C-5 aromatic substituted furanones showed remarkable biofilm formation as well as inhibition of virulence factor production in P. aeruginosa. Fluorescence report analysis identified the QS regulatory mechanism of the most active compound 29. This study provides us a novel candidate for combating drug resistant bacteria strains by merely inhibiting biofilm formation. Without suppressing the regular life cycle of the bacteria, bacterial resistance mechanisms may not be activated.

4.
PLoS One ; 14(9): e0221048, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31498791

RESUMO

Enterovirus 71 (EV71) induces apoptosis to promote viral particle release. Earlier work showed that EV71 utilizes its 3C protease to induce apoptosis in a caspase-3-dependent pathway, though the mechanism is unknown. However, work from Vagner, Holcik and colleagues showed that host protein heterogeneous ribonucleoprotein A1 (hnRNP A1) binds the IRES of cellular apoptotic peptidase activating factor 1 (apaf-1) mRNA to repress its translation. In this work, we show that apaf-1 expression is essential for EV71-induced apoptosis. EV71 infection or ectopic expression of 3C protease cleaves hnRNP A1, which abolishes its binding to the apaf-1 IRES. This allows IRES-dependent synthesis of apaf-1, activation of caspase-3, and apoptosis. Thus, we reveal a novel mechanism that EV71 utilizes for virus release via a 3C protease-hnRNP A1-apaf-1-caspase-3-apoptosis axis.

5.
Math Biosci Eng ; 16(5): 4788-4801, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31499690

RESUMO

Image watermarking focuses on hiding secret data into the cover image imperceptibly to protect the copyright of the original image. In this paper, we propose a new framework of robust digital watermarking for color images using combined embedding techniques of Discrete Fourier Transform (DFT) and Dual Tree Complex Wavelet Transform (DTCWT). The cover image is first divided into Y, U and V channels. The Y channel is then transformed by DFT and partitioned into the ring shapes. With an embedding key, we generate pseudo-random patterns to represent the watermark. These patterns are also transformed and partitioned. The watermark represented by the selection of patterns is then embedded into the rings of the DFT coefficients. We further embed a rectification watermark into the U channel, in which DTCWT is applied to achieve a capability of geometric distortion resilience. On the recipient's side, the detection and extraction of watermark can be successfully done. Compared with previous schemes, the proposed method is better on preserving the image quality. Meanwhile, the robustness against typical attacks is also stronger.

6.
J Biomed Sci ; 26(1): 65, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31481071

RESUMO

Enterovirus A71 (EV-A71) is an important emerging virus posing a threat to children under five years old. EV-A71 infection in infants or young children can cause hand-foot-and-mouth disease, herpangina, or severe neurological complications. However, there are still no effective antivirals for treatment of these infections. In this review, we summarize the antiviral compounds developed to date based on various targets of the EV-A71 life cycle. Moreover, development of a vaccine would be the most effective approach to prevent EV-A71 infection. Therefore, we also summarize the development and clinical progress of various candidate EV-A71 vaccines, including inactivated whole virus, recombinant VP1 protein, synthetic peptides, viral-like particles, and live attenuated vaccines.

7.
Int J Biol Macromol ; 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31493451

RESUMO

Human papilloma virus (HPV) affects predominantly the genital area, which includes vagina, cervix, penis, vulva scrotum, rectum and anus. Among 100 types of HPV, 14 types are considered to cause the risky cancer. The gene HPV-16 E7 is responsible for the development of cancer with the infected women. Earlier identification of this gene sequence avoids the cancer progression. The targeted HPV-16 E7 sequence was sandwiched by capture and reporter sequences on the carbodiimidazole-modified interdigitated electrode (IDE) surface. Target sequence at 100 fM was paired to the capture sequence immobilized on IDE sensing surface. To this surface, different concentrations of reporter sequence with and without gold rod (GNR) were evaluated. In both cases the detections were attained 1 aM by the reporter sequence pairing and with GNR increments in current were found. This enhancement was found to be 1000 folds, considering the condition was revealed in the absence of reporter. This sandwich detection strategy of capture-target-reporter sequences for HPV-16 detection on the IDE sensing surface helps to diagnose the association of cervical cancer.

8.
Nat Commun ; 10(1): 3544, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391463

RESUMO

Simultaneous measurement of proteins and mRNA in single cells enables quantitative understanding and modeling of cellular functions. Here, we present an automated microfluidic system for multi-parameter and ultra-sensitive protein/mRNA measurements in single cells. Our technology improves the sensitivity of digital proximity ligation assay by up to 55-fold, with a detection limit of 2277 proteins per cell and with detection efficiency of as few as 29 protein molecules. Our measurements using this system reveal higher mRNA/protein correlation in single mammalian cells than previous estimates. Furthermore, time-lapse imaging of herpes simplex virus 1 infected epithelial cells enabled by our device shows that expression of ICP4 -a major transcription factor regulating hundreds of viral genes- is only partially correlated with viral protein counts, suggesting that many cells go through abortive infection. These results highlight the importance of high-sensitivity protein/mRNA quantification for understanding fundamental molecular mechanisms in individual cells.

9.
Pediatr Res ; 2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31450232

RESUMO

BACKGROUND: Genetic defects account for a substantial proportion of pediatric cholestasis. This study explored the molecular findings in a large cohort of Chinese patients with inherited cholestasis. METHODS: Between January 2012 and June 2016, 809 Chinese pediatric patients with suspected inherited intrahepatic cholestasis were evaluated by Sanger sequencing and/or panel sequencing. RESULTS: Of the 809 patients, 273 (33.7%) obtained a genetic diagnosis. The rate of positive genetic diagnosis in patients with disease onset at 0-3 month of age was higher than that in patients with disease onset at 4 month of age or later. There were 17 distinct genetic defects diagnosed. The top 4 resulted from mutations in SLC25A13 (44.3%), JAG1 (24.5%), ABCB11 (11.0%), and ATP8B1 (5.9%). All 17 genetic disorders were diagnosed in patients with disease onset at 0-3 months of age; but only 5 were diagnosed in patients with disease onset beyond 4 months of age. A total of 217 distinct pathogenic variants, including 41 novel variants, were identified. Ten recurrent mutations were detected in SLC25A13, ATP8B1, and CYP27A1. They accounted for 48.2% of the total 477 mutant alleles. CONCLUSIONS: There were 17 distinct genetic disorders diagnosed in Chinese pediatric patients with inherited cholestasis.

10.
Ying Yong Sheng Tai Xue Bao ; 30(7): 2309-2319, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31418234

RESUMO

We estimated grassland NPP using CASA model in the Loess Plateau during 2000-2015 and further analyzed the spatiotemporal dynamics, stability and persistence of grassland NPP. The driving factors of grassland NPP were analyzed from four aspects, i.e., vegetation types, topographic factors, climate change, and human activities. The results showed that the average NPP was 202.93 g C·m-2·a-1. The grassland NPP showed an increasing trend with an average increase rate of 2.43 g C·m-2·a-1. The distribution of NPP in grassland had obvious spatial heterogeneity, which was generally high in the south and low in the north. 91.2% of the total grassland area showed an increasing trend, mainly distributed in most areas of Shaanxi Province, Longdong and Longzhong areas of Gansu Province, and most parts of Qinghai Province. The regions with a stable growth condition of grassland NPP mainly located in the south of Ordos, northern Shaanxi, and Gansu. The future change trend of grassland NPP would be consistent with that of the past in most areas. The grassland NPP would continue to increase in most areas of Shaanxi Province, Longzhong and Longdong areas in Gansu Province. The average NPP of slope grassland was 703.37 g C·m-2·a-1, while that of alpine and subalpine grassland was 57.28 g C·m-2·a-1. The grassland NPP was higher in high altitude area and relative low in plain and hilly area. The increased precipitation promoted the increase of grassland NPP during the study period. Human activities such as improvement of overgrazing and returning cropland to grassland also played an important role in the increase of grassland NPP in the Loess Plateau.


Assuntos
Pradaria , Modelos Teóricos , China , Mudança Climática , Atividades Humanas
11.
Talanta ; 205: 120083, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31450430

RESUMO

In vitro synthesis of ATP catalyzed by the ATP-synthase requires membrane vesicles, in which the ATP-synthase is present within the bilayer membrane. Inverted vesicle prepared from Gram negative cells (e.g., Escherichia coli or Pseudomonas putida) can be readily obtained and used for in vitro ATP-synthesis. Up to now, quantification of ATP synthesized by membrane vesicles has been mostly analyzed via bioluminescence-based assays. Alternatively, vesicle respiration and the associated ATP level can be determined using biosensors, which not only provide high selectivity, but allow ATP measurements without the sample being illuminated. Here, we present a microbiosensor for ATP in combination with scanning electrochemical microscopy (SECM) using an innovative two-compartment electrochemical cell for the determination of ATP levels at E.coli or P. putida inverted vesicles. For a protein concentration of 22 mg/ml, a total amount of 0.29 ±â€¯0.03 µM/µl ATP per vesicle was determined in case of E.coli; in turn, P. putida derived vesicles yielded 0.48 ±â€¯0.02 µM/µl ATP per vesicle at a total protein concentration of 25.2 mg/ml. Inhibition experiments with Venturicidin A clearly revealed that the respiratory chain enzyme complex responsible for ATP generation is effectively involved.

12.
Nat Prod Res ; : 1-7, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31274005

RESUMO

Two new phthalide derivatives, (-)-3-carboxypropyl-7-hydroxyphthalide (1) and (-)-3-carboxypropyl-7-hydroxyphthalide methyl ester (2), were isolated from the endophytic fungus Penicillium vulpinum isolated from the Chinese medicinal plant Sophora tonkinensis. Their structures were elucidated using spectroscopic methods, mainly on 1D and 2D NMR. Compound 1 exhibited medium antibacterial activities against Bacillus subtilis, Shigella dysenteriae and Enterobacter areogenes with MIC values of 12.5-25 µg/mL, and 2 showed a medium inhibition to E. areogenes with MIC value of 12.5 µg/mL.

13.
Chem Commun (Camb) ; 55(61): 9011-9014, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31290869

RESUMO

For effective hydrogen generation with remarkable durability, carbon nanotubes (CNTs) grown on Ni nanofibers and their post hydroxylation treatment engendered active Ni nanofiber catalysts an efficient decomposition of hydrous hydrazine with a turnover frequency (TOF) of 19.4 h-1 and an activation energy down to 51.05 KJ mol-1.

14.
J Trop Pediatr ; 2019 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-31302704

RESUMO

To identify whether lymphocyte hydrogen sulfide production is a potential biomarker for predicting coronary artery lesions (CAL) in children with Kawasaki disease (KD). Eighty-six children with KD, 33 normal children and 43 children with fever from June 2016 to January 2019 in Shaanxi Provincial People's Hospital were enrolled. Of 86 KD patients, 16 patients exhibited CAL. Lymphocyte hydrogen sulfide production was significantly greater in KD patients (13.7 ± 2.7) nmol/min/108 lymphocytes than in the controls (9.26 ± 3.33) nmol/min/108 lymphocytes and the fever group (8.21 ± 2.77) nmol/min/108 lymphocytes. The lymphocyte hydrogen sulfide production was greater in CAL patients than the non-CAL patients [(16.24 ± 1.81) vs. (13.12 ± 2.58), p < 0.001]. Receiver operating characteristic curve indicated when the lymphocyte hydrogen sulfide production was >15.285 nmol/min/108 lymphocytes, the sensitivity and specificity for predicting CAL at convalescence were 87.5% and 82.9%, respectively. Lymphocyte hydrogen sulfide production in the acute period is a potentially useful biomarker for predicting CAL in KD children.

15.
J Nat Prod ; 82(8): 2067-2077, 2019 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-31310122

RESUMO

Fungal drug resistance is a major health threat, and reports of clinical resistance worldwide are becoming increasingly common. In a research program to discover new molecules to help overcome this problem, 14 new lanostane-type triterpenoids, gibbosicolids A-G (2-8) and gibbosic acids I-O (9-15), were isolated from the fruiting bodies of Ganoderma gibbosum, along with seven known triterpenoid derivatives. These compounds featured high levels of oxidation, epimerization, and γ-lactonization. Structures were elucidated by comprehensive spectroscopic analyses and HRMS data. Absolute configurations were assigned based on quantum chemical calculations, including calculated chemical shift with DP4+ analysis, coupling constants, and electronic circular dichroism (ECD) methods. Results show that the calculated NMR with DP4+ analysis could not reliably establish the overall spatial configuration of molecules possessing independent and free-rotational stereoclusters. All these compounds significantly increased the sensitivity of fluconazole (FLC)-resistant C. albicans to FLC. Compounds 2, 5, 9, 12, 16, 17, and 21 exhibited strong antifungal activity against FLC-resistant C. albicans when combined with FLC, with MIC50 values ranging from 3.8 to 8.8 µg/mL.

16.
Strabismus ; : 1-7, 2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31335253

RESUMO

Background/Aim: Strabismus surgery could achieve motor alignment and improve health-related quality of life (HRQOL) in adults with large angle exotropia, but whether it could improve binocularity and whether the binocularity is associated with HRQOL are unknown. The aim of this study is to evaluate the binocularity and HRQOL following successful surgical correction of childhood large angle exotropia in adults. Methods: Consecutive adults with childhood large angle exotropia (≥90 prism diopters) who underwent strabismus surgery in the Department of Ophthalmology and Vision Science, Eye and ENT Hospital, Shanghai Medical School, Fudan University were enrolled. Static and dynamic stereopsis tests were performed before surgery and 1 month after surgery. At the same time, Adult Strabismus-20 (AS-20), and Amblyopia and Strabismus questionnaire (A&SQ) were investigated. Results: A total of 29 adults achieved successful alignment: 18 intermittent exotropes (IXT) and 11 constant exotropes (CXT). One month after surgery, stereopsis was demonstrated better in all types of tests (p < .05 for all comparisons). Twelve patients gained normal stereopsis. More patients in IXT group gained normal stereopsis than in CXT group (x2 = 7.62 p = .008). All HRQOL scores showed statistically significant improvement after surgery (p < .05 for all comparisons). IXT group had more changes of the composite score (t = 2.24, p = .03) and the functional score (t = 2.92, p = .007) of AS-20, and the functional score (t = 3.06, p= .005) of A&SQ than CXT group. Only the change of the functional score of A&SQ was demonstrated a statistically significant correlation with the normal stereopsis (t = 2.67, p = .02). Conclusions: The stereopsis and HRQOL in adults with childhood large angle exotropia can be improved after successful surgical correction. More patients in IXT group gained normal stereopsis correlated with better functional aspects of HRQOL.

17.
Parasit Vectors ; 12(1): 358, 2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31337442

RESUMO

BACKGROUND: Excretory/secretory products (ESPs) released by parasites influence the development and functions of host dendritic cells (DCs). However, little is known about changes of DNA (hydroxy)methylation on DC development during Fasciola gigantica infection. The present study aimed to investigate whether F. gigantica ESPs (FgESPs) affects the development and functions of buffalo DCs through altering the DNA (hydroxy)methylation of DCs. METHODS: Buffalo DCs were prepared from peripheral blood mononuclear cells (PBMCs) and characterized using scanning and transmission electron microscopy (SEM/TEM) and quantitative reverse transcriptional PCR (qRT-RCR). DCs were treated with 200 µg/ml of FgESPs in vitro, following DNA extraction. The DNA methylome and hydroxymethylome were profiled based on (hydroxy)methylated DNA immunoprecipitation sequencing [(h)MeDIP-Seq] and bioinformatics analyses. qRT-RCR was also performed to assess the gene transcription levels of interest. RESULTS: FgESPs markedly suppressed DC maturation evidenced by morphological changes and downregulated gene expression of CD1a and MHC II. Totals of 5432 and 360 genes with significant changes in the 5-methylcytosine (5-mC) and the 5-hydroxymethylcytosine (5-hmC) levels, respectively, were identified in buffalo DCs in response to FgESPs challenge. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that these differentially expressed genes were highly enriched in pathways associated with immune response. Some cancer-related pathways were also indicated. There were 111 genes demonstrating changes in both 5-mC and 5-hmC levels, 12 of which were interconnected and enriched in 12 pathways. The transcription of hypermethylated genes TLR2, TLR4 and IL-12B were downregulated or in a decreasing trend, while the mRNA level of high-hydroxymethylated TNF gene was upregulated in buffalo DCs post-exposure to FgESPs in vitro. CONCLUSIONS: To our knowledge, the present study provides for the first time a unique genome-wide profile of DNA (hydroxy)methylation for DCs that interact with FgESPs, and suggests a possible mechanism of FgESPs in suppressing DC maturation and functions that are involved in TLR signaling.

18.
Drug Dev Res ; 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31169945

RESUMO

The prevalence of multidrug resistance among clinically significant bacterial pathogens underlines a critical need for the development of new classes of antibacterial agents with novel structural scaffolds. Cajaninstilbene acid (CSA), which is isolated from pigeonpea leaves, has shown potent antibacterial activity. In this study, a series of 2-hydroxyl-4-methoxyl-3-(3-methylbut-2-en-1-yl)-6-(4-phenylbenzoylamino)benzoic acid derivatives derived from CSA were designed and synthesized, and their antibacterial activities were evaluated. Several synthesized compounds exhibit better antibacterial activity than CSA against Staphylococcus aureus, Staphylococcus epidermidis, and two strains of methicillin-resistant S. aureus. Meanwhile, the results of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assays illustrate the good selectivity between bacteria and normal cells of the most active compounds 6u and 6v. Furthermore, well combinations with bacterial RNA polymerase of 6u arising from docking study imply the possible mechanism of antibacterial activity of these synthetic compounds.

19.
Proteomics Clin Appl ; : e1800060, 2019 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-31162828

RESUMO

PURPOSE: This study is designed to screen serum proteins that may serve as biomarkers for gestational diabetes mellitus (GDM), and clarify the mechanisms of disease. EXPERIMENTAL DESIGN: By using isobaric tags for relative and absolute quantitation proteomics analysis, serum proteins levels were quantified in pregnant women who subsequently developed GDM (12-16 weeks), GDM patients (24-28 weeks), and their corresponding controls. The strategy of mixing samples is used in proteomic analysis. RESULTS: Thirty-one and 27 differentially expressed proteins are identified in the serum of pregnant women with developed GDM at 12-16 weeks and GDM patients during 24-28 weeks, respectively. Thirty eight and 28 proteins are identified in 24-28 weeks versus 12-16 weeks controls (24/12 CTR group), and 24-28 weeks GDM patients versus 12-16 weeks women with subsequently developed GDM (24/12 GDM group), respectively. Most of these proteins in the case and control subjects are associated with diabetes and maternal and perinatal short- and long-term complications. CONCLUSIONS AND CLINICAL RELEVANCE: The results highlight the roles of complement system and the blood clotting cascade in the pathogenesis of GDM. Differentially expressed proteins may serve as potential biomarkers for GDM prediction and diagnosis in the future.

20.
PLoS One ; 14(6): e0218076, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31173620

RESUMO

Previous studies have found that high social class individuals are more dishonest than low social class ones. However, the underlying mechanism of this phenomenon is still unclear. The "ignoring negative consequences" hypothesis suggests that belonging to a high social class makes individuals ignore the negative consequences of dishonesty, whereas the "self-focused" hypothesis suggests that belonging to a high social class makes individuals focus more on the self and self-interests. The present study aims to examine these two hypotheses by measuring participants' subjective social class, narcissism, fear of negative evaluation, and tendency to be dishonest. To this end, an online survey was conducted. Results provide evidence for the self-focused hypothesis by showing that subjective social class positively predicts the tendency to be dishonest, and narcissism plays a mediating role in this relationship.

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