Deep learning-assisted sensitive detection of fentanyl using a bubbling-microchip.

Deep learning-enabled smartphone-based image processing has significant advantages in the development of point-of-care diagnostics. Conventionally, most deep-learning applications require task specific large scale expertly annotated datasets. Therefore, these algorithms are oftentimes limited only to applications that have large retrospective datasets available for network development. Here, we report the possibility of utilizing adversarial neural networks to overcome this challenge by expanding the utility of non-specific data for the development of deep learning models. As a clinical model, we report the detection of fentanyl, a small molecular weight drug that is a type of opioid, at the point-of-care using a deep-learning empowered smartphone assay. We used the catalytic property of platinum nanoparticles (PtNPs) in a smartphone-enabled microchip bubbling assay to achieve high analytical sensitivity (detecting fentanyl at concentrations as low as 0.23 ng mL-1 in phosphate buffered saline (PBS), 0.43 ng mL-1 in human serum and 0.64 ng mL-1 in artificial human urine). Image-based inferences were made by our adversarial-based SPyDERMAN network that was developed using a limited dataset of 104 smartphone images of microchips with bubble signals from tests performed with known fentanyl concentrations and using our retrospective library of 17 573 non-specific bubbling-microchip images. The accuracy (± standard error of mean) of the developed system in determining the presence of fentanyl, when using a cutoff concentration of 1 ng mL-1, was 93 ± 0% in human serum (n = 100) and 95.3 ± 1.5% in artificial human urine (n = 100).

Self-management behaviors in adults with chronic hepatitis B: A structural equation model.

BACKGROUND: Chronic hepatitis B is a serious and chronic health problem, requiring self-management to control the disease and related complications. OBJECTIVES: To develop a structural model to identify how social support, self-efficacy and disease knowledge contribute to their self-management behaviors in adults with chronic hepatitis B. DESIGN: A cross-sectional study. SETTINGS: Hepatology units in two hospitals in Chongqing, China. PARTICIPANTS: A total of 306 patients with chronic hepatitis B were recruited. METHODS: Data were collected using Social Support Rating Scale, Self-Efficacy for Managing Chronic Disease, Hepatitis B Knowledge Questionnaire and Chronic Hepatitis B Self-Management Scale. Structural equation model was applied to analyze the data. RESULTS: The final model showed good model fit. Social support directly influenced self-management behaviors (ß = 0.19, p < 0.01), and indirectly influenced self-management behaviors (ß = 0.20, p < 0.01) through self-efficacy. Self-efficacy directly influenced self-management behaviors (ß = 0.37, p < 0.05). Disease knowledge indirectly influenced self-management behaviors (ß = 0.12, p < 0.05) through self-efficacy. CONCLUSIONS: Our findings indicated that social support, self-efficacy and disease knowledge directly or indirectly affected self-management behaviors in adults with chronic hepatitis B. This provides a theoretical basis for developing self-management interventions for patients with chronic hepatitis B, which may lead to health improvements in this population.

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To investigate the swimming ability of two Schizothorax species in the Yalung River and provide basic parameters for the studies on fish behavior and the design of fish passage, we exa-mined the induced velocity, critical swimming speed, and burst swimming speed in Schizothorax dolichonema and Schizothorax prenanti with incremental velocity method and the durable swimming speed in S. dolichonema with fixed velocity method. The results showed that the induced velocity of both species increased first and then plateaued with the increases of body length, with the maximum values being lower than 0.2 m·s-1. The critical swimming speed and burst swimming speed of S. dolichonema were (0.81±0.20) and (1.49±0.26) m·s-1, respectively, while the relative critical swimming speed and the relative burst swimming speed were (4.90±1.73) and (9.77±1.72) BL·s-1 (BL: body length), respectively. For S. prenanti, the critical swimming speed and burst swimming speed were (0.73±0.24) and (1.17±0.39) m·s-1, respectively, while the relative critical swimming speed was (6.88±2.82) BL·s-1, and the relative burst swimming speed was (11.75±2.77) BL·s-1. The swimming duration of S. dolichonema was negatively correlated with the flow velocity of 0.7-1.5 m·s-1, and the relationship between fatigue time (T) and flow velocity (V) was fitted into lgT=-2.52V+5.59. The relationship between expected fishway length (d) and the tolerable maximum average flow velocity (Vf max) was accordingly derived to be Vf max=-0.17lnd+1.74. Taken together, the fishway targeting S. dolichonema and S. prenanti was recommended to generate the in-channel velocity larger than 0.2 m·s-1, while the velocity at the entrance and verticle slot should be 0.73-1.67 m·s-1, and the main-flow velocity in rest pools should be 0.2-0.7 m·s-1.

HBV-Pol is crucial for HBV-mediated inhibition of inflammasome activation and IL-1ß production.

BACKGROUND & AIMS: Hepatitis B virus (HBV) infection is the most critical factor underlying liver cirrhosis and hepatocellular carcinoma worldwide. IL-1ß and IL-18, generated by activation of the inflammasome/caspase-1 signaling pathway, play important roles in the control and clearance of HBV. However, the specific relationship between the inflammasome response and IFN-α resistance or viral persistence is yet to be established. METHODS: Blood samples of patients and supernatant fractions of HBV cell lines were collected for analysis and the effects on inflammasome activation and IL-1ß production evaluated via enzyme-linked immunosorbent assay (ELISA), western blot, quantitative RT-PCR and immunofluorescence. RESULTS: IL-1ß and IL-18 levels produced in sera of IFN-α non-responders were significantly lower than those of responders and normal donors. Additionally, expression of IL-1ß and inflammasome components was decreased in peripheral blood mononuclear cells (PBMC) of non-responders, compared with those of responders. In vitro experiments on HepG2, HepG2.2.15 and HepAD38 cell lines showed that HBV induces a significant decrease in IL-1ß production through inhibiting activation of the NF-κB signaling and inflammasome/caspase-1 pathways. And hepatitis B virus polymerase (HBV-Pol) appeared crucial for these inhibitory effects of HBV. CONCLUSION: IL-1ß production is suppressed in HBV carriers and IFN-α non-responders. HBV induces a significant decrease in IL-1ß production through inhibiting the NF-κB signaling and inflammasome pathways, for which HBV-Pol is a crucial requirement. Trial approval number: 20 173 402.

Factors for self-management activities among rural patients with chronic hepatitis B: A cross-sectional study.

AIMS AND OBJECTIVES: To assess the self-management activities among rural patients with chronic hepatitis B (CHB), and the influence of psychosocial and demographic factors on their self-management activities. BACKGROUND: Chronic hepatitis B is a serious public health concern. Rural patients may have limited access to healthcare services. Although self-management is important for controlling chronic hepatitis B, few studies focus on the self-management activities among rural patients with chronic hepatitis B. Understanding self-management activities and related factors in this population are important to design and implement appropriate intervention strategies. DESIGN: A cross-sectional study. METHODS: From June-December 2017, totally 236 rural patients with chronic hepatitis B were recruited from hepatology department in two hospitals in Chongqing, China. The questionnaire included demographic characteristics, Chronic Hepatitis B Self-Management Scale, Self-Efficacy for Managing Chronic Disease, and Social Support Rating Scale. The study followed the STROBE checklist. RESULTS: Rural patients with chronic hepatitis B reported poor self-management activities for the score indexes of symptom management (57.36%), lifestyle management (54.89%), psychosocial coping (54.84%) and disease information management (53.11%) were all below 60%. Self-efficacy, objective support, subjective support, gender, education level and marital status showed significant effect on self-management activities. CONCLUSION: Rural patients with chronic hepatitis B were found to perform insufficient self-management activities. Self-efficacy, social support, gender, education level and marital status were identified to influence their self-management activities. RELEVANCE TO CLINICAL PRACTICE: Self-management activities should be promoted among rural patients with chronic hepatitis B. The factors that were identified in this study should be addressed when developing interventions to promote the performance of self-management activities for rural patients with chronic hepatitis B.

Hepatitis B core antigen can regulate NLRP3 inflammasome pathway in HepG2 cells.

Hepatitis B virus (HBV) has four open reading frames (ORFs) of which ORF C is consists of the pre Core and Core genes encodes the Hepatitis B core antigen (HBcAg) and Hepatitis B e antigen (HBeAg). Studies have shown that HBeAg significantly inhibits the NLRP3 inflammasome activation and interleukin-1ß (IL-1ß) production. However, the role of HBcAg and ORF C proteins (in this paper, ORF C proteins = HBcAg + HBeAg) were remain unclear. Our study aims to assess whether HBcAg and ORF C proteins can affect the NLRP3 inflammasome pathway. Vectors expressing ORF C proteins and HBcAg were designed and transfected into HepG2 cells. And then, cells were stimulated with lipopolysaccharide (LPS). Activation of the NLRP3 inflammasome and the levels of IL-1ß and IL-18 were evaluated by Western blot analysis, quantitative reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and immunofluorescence. The expression of NLRP3 and IL-1ß peaked when HepG2 cells were stimulated with 1000 ng/mL LPS for 18 to 24 hours. HBcAg, but not ORF C proteins, promoted LPS-induced NLRP3 inflammasome activation and IL-1ß production. These findings provide a novel mechanism on how the HBV causes liver inflammation and may provide insights into the search for new therapeutic strategies.

HEV ORF3 downregulatesCD14 and CD64 to impair macrophages phagocytosis through inhibiting JAK/STAT pathway.

Hepatitis E virus (HEV) could induce chronic hepatitis and liver failure with high mortality through unknown mechanisms. The previous study showed that the HEV open reading frames 3 (ORF3) could inhibit macrophages inflammatory response. Impaired macrophages phagocytosis was also found in patients infected with HEV and its nucleic acids could be detected in macrophages. To elucidate the role of HEV ORF3 on phagocytosis, the phagocytosis activation was measured by phagocytosis test, flow cytometry, and phalloidin staining. Meanwhile, the expression of key phagocytic receptors and the activation of transduction pathway were investigated by using reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. Results of phagocytosis test showed that the HEV ORF3 could significantly impair the absorption capacity of latex beads. Furthermore, results of RT-qPCR and Western blot analysis showed that the expression of CD14 and CD64 decreased. Afterward, the present study showed that the activation of Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway was inhibited by HEV ORF3 and downregulation of CD14 and CD64 could be reversed by interferon γ, one activator of the JAK1/STAT1 signaling pathway. In conclusion, HEV ORF3 could significantly impair the phagocytosis of macrophage by downregulating expression of CD14 and CD64, which may function by inhibiting the activation of the JAK1/STAT1 signaling pathway.

HEV ORF3 downregulates TLR7 to inhibit the generation of type I interferon via impairment of multiple signaling pathways.

Hepatitis E is the most common type of acute hepatitis prevalent worldwide. The open reading frame 3 protein of HEV (HEV ORF3) is proposed to create a favorable environment for viral replication and pathogenesis. However, the mechanisms by which HEV overcomes the effects of host immunity, particularly the role of ORF3, remain to be established. Expression of IFNα and IFNß in supernatant and cell samples was examined via ELISA and quantitative RT-PCR. The protein levels of specific signaling factors in cells overexpressing HEV ORF3 were examined via western blot. Analyses of cells transfected with vectors expressing ORF3 demonstrated that HEV ORF3 significantly impairs the generation of endogenous type I interferon through downregulating TLR3 and TLR7 as well as their corresponding downstream signaling pathways. Moreover, inhibition of NFκB, JAK/STAT and JNK/MAPK signaling pathways contributed significantly to suppression of increased levels of TLR7. Levels of p-P65, p-STAT1 and p-JNK were markedly impaired in ORF3-expressing cells, even upon treatment with the respective agonists. HEV ORF3 inhibits the production of endogenous type I interferon through downregulation of TLR3 and TLR7. Furthermore, suppression of TLR7 is achieved through impairment of multiple signaling pathways, including NFκB, JAK/STAT and JNK/MAPK.

Identification of key genes in non­alcoholic fatty liver disease progression based on bioinformatics analysis.

Due to economic development and lifestyle changes, the incidence of non­alcoholic fatty liver disease (NAFLD) has gradually increased in recent years. However, the pathogenesis of NAFLD is not yet fully understood. To identify candidate genes that contribute to the development and progression of NAFLD, two microarray datasets were downloaded from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) were identified and functional enrichment analyses were performed. A protein­protein interaction network was constructed and modules were extracted using the Search Tool for the Retrieval of Interacting Genes and Cytoscape. The enriched functions and pathways of the DEGs included 'cellular macromolecule biosynthetic process', 'cellular response to chemical stimulus', 'extracellular matrix organization', 'metabolic pathways', 'insulin resistance' and 'forkhead box protein O1 signaling pathway'. The DEGs, including type­1 angiotensin II receptor, formin­binding protein 1­like, RNA­binding protein with serine­rich domain 1, Ras­related C3 botulinum toxin substrate 1 and polyubiquitin­C, were identified using multiple bioinformatics methods and validated in vitro with reverse transcription­quantitative polymerase chain reaction analysis. In conclusion, five hub genes were identified in the present study, and they may aid in understanding of the molecular mechanisms underlying the development and progression of NAFLD.

Screening and identification of key biomarkers in hepatocellular carcinoma: Evidence from bioinformatic analysis.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Intense efforts have been made to elucidate the pathogeny, but the molecular mechanisms of HCC are still not well understood. To identify the candidate genes in the carcinogenesis and progression of HCC, microarray datasets GSE19665, GSE33006 and GSE41804 were downloaded from Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified, and function enrichment analyses were performed. The protein-protein interaction network (PPI) was constructed and the module analysis was performed using STRING and Cytoscape. A total of 273 DEGs were identified, consisting of 189 downregulated genes and 84 upregulated genes. The enriched functions and pathways of the DEGs include protein activation cascade, complement activation, carbohydrate binding, complement and coagulation cascades, mitotic cell cycle and oocyte meiosis. Sixteen hub genes were identified and biological process analysis revealed that these genes were mainly enriched in cell division, cell cycle and nuclear division. Survival analysis showed that BUB1, CDC20, KIF20A, RACGAP1 and CEP55 may be involved in the carcinogenesis, invasion or recurrence of HCC. In conclusion, DEGs and hub genes identified in the present study help us understand the molecular mechanisms underlying the carcinogenesis and progression of HCC, and provide candidate targets for diagnosis and treatment of HCC.

Gene expression profile after knockdown of USP18 in Hepg2.2.15 cells.

In our previous work, we found that the expression of ubiquitin-specific protease 18 (USP18), also known as UBP43, is associated with the efficiency of interferon alpha (IFN-α) treatment in patients with chronic hepatitis B (CHB). To elucidate the influence of USP18 on hepatitis B virus (HBV) replication and the mechanism of this activity, we silenced USP18 by introducing short hairpin RNA (shRNA) into Hepg2.2.15 cells. To identify the changed genes and pathways in Hepg2.2.15-shRNA-USP18 cells, we performed a microarray gene expression analysis to compare the Hepg2.2.15 stably expressing USP18-shRNA cells versus control cells using the Affymetrix Human Transcriptome Array (HTA) 2.0 microarrays. Microarray analysis indicated that genes involved in regulation of thyroid hormone signaling pathway, complement, and coagulation cascades, PERK-mediated unfolded protein response, and insulin-like growth factor-activated receptor activity were significantly altered after USP18 knockdown for 72 h. Furthermore, genes involved in hepatocyte proliferation, liver fibrosis, such as cell cycle regulatory gene CCND1, were also altered after USP18 knockdown in Hepg2.2.15 cells. In conclusion, USP18 is critical for regulating the replication of HBV in Hepg2.2.15 cells, which suggest that USP18 may be a candidate target for HBV treatment.

Correction: Suppression of USP18 Potentiates the Anti-HBV Activity of Interferon Alpha in HepG2.2.15 Cells via JAK/STAT Signaling.

[This corrects the article DOI: 10.1371/journal.pone.0156496.].

Suppression of USP18 Potentiates the Anti-HBV Activity of Interferon Alpha in HepG2.2.15 Cells via JAK/STAT Signaling.

Ubiquitin-specific protease 18 (USP18, also known as UBP43) has both interferon stimulated gene 15 (ISG15) dependent and ISG15-independent functions. By silencing the expression of USP18 in HepG2.2.15 cells, we studied the effect of USP18 on the anti-HBV activity of IFN-F and demonstrated that knockdown of USP18 significantly Inhibited the HBV expression and increased the expression of ISGs. Levels of hepatitis B virus surface antigen (HBsAg), hepatitis B virus e antigen (HBeAg), HBV DNA and intracellular hepatitis B virus core antigen (HBcAg) were dramatically decreased with or without treatment of indicated dose of IFN-F. Suppression of USP18 activated the JAK/STAT signaling pathway as shown by the increased and prolonged expression of phosphorylated signal transducer and activator of transcription 1 (p-STAT1) in combination with enhanced expression of several interferon stimulated genes (ISGs). Our results indicated that USP18 modulates the anti-HBV activity of IFN-F via activation of the JAK/STAT signaling pathway in Hepg2.2.15 cells.

ORF3 of Hepatitis E Virus Inhibits the Expression of Proinflammatory Cytokines and Chemotactic Factors in LPS-Stimulated Human PMA-THP1 Cells by Inhibiting NF-κB Pathway.

Hepatitis E virus (HEV) is one of the primary causative agents of acute hepatitis. It is noteworthy that HEV can develop chronic infection and even lead to liver cirrhosis; however, the mechanism has not been revealed. In this study, the ELISA assay was used to detect protein levels, and we found that HEV open reading frame 3 (ORF3) protein inhibited the expression of proinflammatory cytokines (tumor necrosis factor-alpha [TNF-α], interleukin [IL]-1ß, IL-6, IL-8, IL-12p40, and IL-18) and chemotactic factors (nitric oxide [NO], interferon-inducible protein-10 (IP-10), macrophage inflammatory protein (MIP)-1α, monocyte chemoattractant protein-1 (MCP-1), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF)] in lipopolysaccharide (LPS)-stimulated human PMA-THP1 cells. Further study showed that mRNA and protein levels of pattern recognition receptors (PRRs), such as Toll-like receptor 4 (TLR4), TNF receptor-associated factor 6 (TRAF6), and nucleotide-binding oligomerization domain containing 2 (NOD2), decreased after infection of pLL3.7-ORF3 (pORF3); moreover, the inhibition produced corresponding upregulation of IκBα and downregulation of phosphorylated IκB kinase IKKɛ (p-IKKɛ) and phosphorylated nuclear factor (NF)-κB (p-NF-κB), but little variation was found in the concentration of IKKɛ and NF-κB. Taken together, our results demonstrated that HEV ORF3 attenuated LPS-induced cytokine production and chemotactic factors, predominantly by inhibiting various PRRs-mediated NF-κB signaling pathways. The anti-inflammatory properties might be of great importance to clarify the role and mechanism of macrophages in chronic HEV infection and cirrhosis.