Demethyleneberberine Alleviates Pulmonary Fibrosis through Disruption of USP11 Deubiquitinating GREM1.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal and chronic interstitial lung disease. Intricate pathogenesis of pulmonary fibrosis and only two approved medications with side effects and high cost bring us the challenge of fully understanding this lethal disease and urgency to find more safe and low-cost therapeutic alternatives. PURPOSE: Demethyleneberberine (DMB) has been demonstrated to have various anti-inflammatory, antioxidant, antifibrosis and anti-cancer bioactivities. The objective of this study was to evaluate the effect of DMB on pulmonary fibrosis and investigate the mechanism. METHODS: Bleomycin (BLM)-induced pulmonary fibrosis was established in mice to evaluate the antifibrotic effect of DMB in vivo. A549 and MRC5 cells were used to evaluate the effect of DMB on epithelial-mesenchymal transition (EMT) and fibroblast-myofibroblast transition (FMT) in vitro. High throughput sequencing, biotin-avidin system and site-directed mutagenesis were applied to explore the mechanism of DMB in alleviating pulmonary fibrosis. RESULTS: DMB alleviated BLM-induced pulmonary fibrosis in vivo by improving the survival state of mice, significantly reducing pulmonary collagen deposition and oxidative stress and improving lung tissue morphology. Meanwhile, DMB was demonstrated to inhibit epithelial-mesenchymal transition (EMT) and fibroblast-myofibroblast transition (FMT) in vitro. High throughput sequencing analysis indicated that GREM1, a highly upregulated profibrotic mediator in IPF and BLM-induced pulmonary fibrosis, was significantly downregulated by DMB. Furthermore, USP11 was revealed to be involved in the deubiquitination of GREM1 in this study and DMB promoted the ubiquitination and degradation of GREM1 by inhibiting USP11. Remarkably, DMB was demonstrated to selectively bind to the Met776 residue of USP11, leading to disruption of USP11 deubiquitinating GREM1. In addition, DMB presented an equivalent antifibrotic effect at a lower dose compared with pirfenidone and showed no obvious toxicity or side effects. CONCLUSIONS: This study revealed that USP11/GREM1 could be a potential target for IPF management and identified that DMB could promote GREM1 degradation by inhibiting USP11, thereby alleviating pulmonary fibrosis.

Enrichment and detection of VEGF165 in blood samples on a microfluidic chip integrated with multifunctional units.

In this paper, a multifunctional microfluidic chip integrated with a centrifugal separation zone, aqueous two-phase system (ATPS) mixing zone and enrichment detection zone was proposed and fabricated. An automatic and efficient separation and quantitative analysis method for vascular endothelial growth factor 165 (VEGF165) in whole blood samples was established with the designed microfluidic chip. A blood sample was divided into blood cells and plasma in the centrifugation zone. In the ATPS mixing zone, plasma was mixed with PEG/KH2PO4 aqueous two-phase solution containing Apt-Au NP nanoprobes. In the enrichment detection zone, the mixture was separated on CN140 modified with a ZnO NP-anti VEGF165 nanostructure. The VEGF165 captured by Apt-Au NPs was distributed in the PEG phase, concentrated at the front of CN140 and combined with anti-VEGF165 to form a sandwich structure. The sensitive detection of VEGF165 was achieved through fluorescence resonance energy transfer between rhodamine B and Au NPs on the nanoprobe. Under the optimized rotation program, capillary and centrifugal forces propelled the fluid in the whole process of pretreatment and detection. The detection linear range was between 1 pg mL-1 and 50 ng mL-1, the detection limit of VEGF165 in blood was 0.22 pg mL-1 and the enrichment efficiency was 983. It was illustrated that a convenient and reliable way for detection of tumor markers based on the multifunctional microfluidic chip was provided and it has a potential value for early screening and prognosis of clinical cancer.

Suppression of hepatocellular carcinoma progression by long noncoding RNA apolipoprotein C1 pseudogene via the regulation of the microRNA-106b-PTEN axis.

Background: Numerous researches have reported that long noncoding RNAs (lncRNAs) participate in tumor development and progression. LncRNA apolipoprotein C-I pseudogene 1 (APOC1P1), a pseudogene located in 19q13.2 between apolipoprotein C-I and apolipoprotein C-IV, is involved in a variety of diseases. However, the role of lncRNA APOC1P1 in hepatocellular carcinoma (HCC) remains unknown. Methods: Quantitative polymerase chain reaction (qPCR) was performed to examine the expression of APOC1P1, miR-106b, and PTEN (phosphatase and TENsin homolog deleted on chromosome 10) in HCC tissues, adjacent normal tissues, and specific cell lines (LO2, Bel-7407, HCCLM3, MHCC-97H, Hep G2, and Huh-7). Upregulation of APOC1P1 and downregulation of miR-106b were conducted via application of vector transfection and microRNA (miRNA) inhibitor. Bioinformatics analysis and luciferase reporter assay were used to verify the binding sites of APOC1P1, miR-106b, and PTEN. Cell proliferation and invasion were determined with Cell Counting Kit-8 (CCK-8) and Transwell experiments. Subcellular location analysis was used to determine the distribution of APOC1P1 in cells, and Western blotting was used to detect the expression of PTEN. Results: It was found that the expressions of APOC1P1 and PTEN were downregulated, while that of miR-106b was upregulated in HCC tissues and cells. Subcellular location analysis showed that APOC1P1 was localized in cytoplasm and competitively bound to miR-106b. APOC1P1 overexpression and miR-106b inhibition suppressed HCC cell proliferation and invasion. qPCR indicated the negative correlation between APOC1P1 expression and miR-106b expression in HCC tissues and a positive correlation between APOC1P1 and PTEN. Conclusions: Our findings suggested that the lncRNA APOC1P1 inhibits HCC progression by competitively binding to miR-106b, leading to elevated PTEN expression, inhibiting cell proliferation and invasion in HCC cells. These results provide new insights into the diagnosis and therapy of HCC.

Direct separation and enumeration of CTCs in viscous blood based on co-flow microchannel with tunable shear rate: a proof-of-principle study.

Circulating tumor cells (CTCs), which have extremely low density in whole blood, are an important indicator of primary tumor metastasis. Isolation and enumeration of these cells are critical for clinical applications. Separation of CTCs from massive blood cells without labeling and addition of synthetic polymers is challenging. Herein, a novel well-defined co-flow microfluidic device is presented and used to separate CTCs in viscous blood by applying both inertial and viscoelastic forces. Diluted blood without any synthetic polymer and buffer solution were used as viscoelastic fluid and Newtonian fluid, respectively, and they were co-flowed in the designed chip to form a sheath flow. The co-flow system provides the function of particle pre-focusing and creates a tunable shear rate region at the interface to adjust the migration of particles or cells from the sample solution to the buffer solution. Successful separation of CTCs from viscous blood was demonstrated and enumeration was also conducted by image recognition after separation. The statistical results indicated that a recovery rate of cancer cells greater than 87% was obtained using the developed method, which proved that the direct separation of CTCs from diluted blood can be achieved without the addition of any synthetic polymer to prepare viscoelastic fluid. This method holds great promise for the separation of cells in viscous biological fluid without either complicated channel structures or the addition of synthetic polymers.

Detection of VEGF165 in Whole Blood by Differential Pulse Voltammetry Based on a Centrifugal Microfluidic Chip.

For the rapid and sensitive detection of vascular endothelial growth factor 165 (VEGF165) in clinical blood samples, a microfluidic sensing chip that integrates a centrifugal separation pretreatment unit and a composite nanosensing film was proposed in this paper. An efficient sensing strategy and method was established. The blood sample was first separated and extracted by centrifugal force on the centrifugal microfluidic chip within 5 min after injection. The separated plasma can be automatically transferred through the designed microchannels to the detection area integrated electrodes for subsequent differential pulse voltammetric detection. The Au NPs/MCH/Apt2 sensing film was constructed on the surface of the Au working electrode. A sandwich sensing strategy based on "double aptamers" and "nanoprobe" for VEGF165 detection was established, by which the synthetic Apt1/PThi/Au NP nanoprobe was applied to capture VEGF165 in plasma and bind to the sensing film. By this method, the detection limit of VEGF165 in whole blood was 0.67 pg/mL and the linear range was between 1 pg and 10 ng, which met the needs of clinical VEGF165 detection. It was illustrated that the proposed methodology based on the centrifugal microfluidic chip had potential application prospects in the development of the point-of-care testing fields.

Detection of prostate specific antigen in whole blood by microfluidic chip integrated with dielectrophoretic separation and electrochemical sensing.

The efficient detection of cancer markers has faced many challenges, such as severe interference, complicated and time-consuming operation, low sensitivity and so on. In this paper, a microfluidic chip integrated with electrodes for dielectrophoretic (DEP) separation, microchannels for electrochemical nanoprobes binding and differential pulse voltammetry (DPV) detection was proposed for the sensitive and rapid detection of prostate specific antigen (PSA) in whole blood. The functional units, which could realize cell separation, PSA derivatization (binding of electrochemical nanoprobes), capture and detection, were integrated on the microfluidic chip. The well-designed V-shaped interdigital electrode arrays provided DEP separation for blood cells with efficiency as high as 98%. Particularly, DEP effect significantly improved the sensitivity of PSA detection and reduced the detection limit by two orders of magnitude. In order to achieve sensitive detection of PSA, binding of electrochemical nanoprobes and then DPV detection was selected and integrated following the DEP separation. A sandwich structure based on electrochemical nanoprobes and dual-aptamers for on-chip DPV detection was proposed, which included self-synthesized electrochemical nanoprobes bovine serum albumin/detection aptamer 2/polythionine@gold nanoparticles (BSA/Apt2/PThi@Au NPs), target PSA, and sensing interface 6-mercaptohexanol/capture aptamer 1/gold nanoparticles on gold electrode (MCH/Apt1/Au NPs/Au). The method of quantitative detection of PSA in whole blood was then established. The excellent performance of the microfluidic chip allowed the determination of PSA in whole blood in the range of 1 pg/mL ∼10 ng/mL with an ultralow limit of detection of 0.25 pg/mL, which was better than the results obtained by conventional methods.

Identification of genetic variations associated with drug resistance in non-small cell lung cancer patients undergoing systemic treatment.

Non-small cell lung cancer (NSCLC) is characterized by relatively rapid response to systemic treatments yet inevitable resistance and predisposed to distant metastasis. We thus aimed at performing sequencing analysis to determine genomic events and underlying mechanisms concerning drug resistance in NSCLC. We performed targeted sequencing of 40 medication-relevant genes on plasma samples from 98 NSCLC patients and analyzed impact of genetic alterations on clinical presentation as well as response to systemic treatments. Profiling of multi-omics data from 1024 NSCLC tissues in public datasets was carried out for comparison and validation of identified molecular events implicated in resistance. A genetic association of CYP2D6 deletion with drug resistance was identified through circulating tumor DNA (ctDNA) profiling and response assessment. FCGR3A amplification was potentially involved in resistance to EGFR inhibitors. We further verified our findings in tissue samples and focused on potential resistance mechanisms, which uncovered that depleted CYP2D6 affected a set of genes involved in EMT, oncogenic signaling as well as inflammatory pathways. Tumor microenvironment analysis revealed that NSCLC with CYP2D6 loss manifested increased levels of immunomodulatory gene expressions, PD-L1 expression, relatively high mutational burden and lymphocyte infiltration. DNA methylation alterations were also found to be correlated with mRNA expressions and copy numbers of CYP2D6. Finally, MEK inhibitors were identified by CMap as the prospective therapeutic drugs for CYP2D6 deletion. These analyses identified novel resistance mechanisms to systemic NSCLC treatments and had significant implications for the development of new treatment strategies.

Association of Epstein-Barr virus infection with peripheral immune parameters and clinical outcome in advanced nasopharyngeal carcinoma.

The purpose of this study was to investigate the association of Epstein-Barr virus (EBV) with peripheral blood immune cell counts and clinical outcomes in advanced nasopharyngeal carcinoma (NPC) patients. In a retrospective design, 146 patients with NPC at stage IV were enrolled in this study. The association of EBV status with peripheral blood immune cell counts, distant metastases, and long-term survival in patients with advanced NPC were determined. Eighty-seven (59.6%) of all patients were positive for EBV. Compared with patients with normal NK cell count, patients with lower NK cell count showed a significantly lower EBV viral load (median: 614.0 vs. 2190.0 copies/mL, P = 0.024). EBV-positive patients showed a significantly higher incidence of liver metastasis than EBV-negative patients (32.6% vs. 23.7%, P = 0.021). Multi-variant regression analysis showed that EBV infection was independently associated with liver metastasis (OR: 2.33, P = 0.043). EBV positive patients showed a significantly worse PFS (P = 0.001) and OS (P = 0.001) than EBV negative patients. Multivariate Cox regression analysis revealed that EBV infection was independently associated with a worse PFS (HR: 1.94, P = 0.003), and OS (HR: 2.12, P = 0.014) in advanced NPC. In conclusion, EBV infection is associated with a high risk of liver metastasis and is also an independent negative predictor for PFS and OS in patients with advanced NPC. EBV infection is associated with lower CD8% and higher NK%, while lower NK cell count is associated with lower EBV viral load.

An Electrochemical Immunosensor Based on a Self-Assembled Monolayer Modified Electrode for Label-Free Detection of α-Synuclein.

This research demonstrated the development of a simple, cost-effective, and label-free immunosensor for the detection of α-synuclein (α-Syn) based on a cystamine (CYS) self-assembled monolayer (SAM) decorated fluorine-doped tin oxide (FTO) electrode. CYS-SAM was formed onto the FTO electrode by the adsorption of CYS molecules through the head sulfur groups. The free amine (-NH2) groups at the tail of the CYS-SAM enabled the immobilization of anti-α-Syn-antibody, which concurrently allowed the formation of immunocomplex by covalent bonding with α-Syn-antigen. The variation of the concentrations of the attached α-Syn at the immunosensor probe induced the alternation of the current and the charge transfer resistance (Rct) for the redox response of [Fe(CN)6]3-/4-, which displayed a linear dynamic range from 10 to 1000 ng/mL with a low detection limit (S/N = 3) of ca. 3.62 and 1.13 ng/mL in differential pulse voltammetry (DPV) and electrochemical impedance spectra (EIS) measurements, respectively. The immunosensor displayed good reproducibility, anti-interference ability, and good recoveries of α-Syn detection in diluted human serum samples. The proposed immunosensor is a promising platform to detect α-Syn for the early diagnose of Parkinson's disease, which can be extended for the determination of other biologically important biomarkers.

Peptide PD29 treats bleomycin-induced pulmonary fibrosis by inhibiting the TGF-ß/smad signaling pathway.

Pulmonary fibrosis (PF) is an end-stage change in lung disease characterized by fibroblast proliferation, massive extracellular matrix (ECM) aggregation with inflammatory damage, and severe structural deterioration. PD29 is a 29-amino acid peptide which has the potential to alleviate PF pathogenesis via three mechanisms: anti-angiogenesis, inhibition of matrix metalloproteinase activities, and inhibition of integrins. In this study, fibrotic lung injuries were induced in SD rats by a single intratracheal instillation of 5 mg/kg bleomycin (BLM). Then, these rats were administered 7.5, 5, or 2.5 mg/kg PD29 daily for 30 days. BLM induced-syndromes including structure distortion, excessive deposition of ECM, excessive inflammatory infiltration, and pro-inflammatory cytokine release were used to evaluate the protective effect of PD-29. Oxidative stress damage in lung tissues was attenuated by PD29 in a dose-dependent manner. The expression of TGF-ß1 and the phosphorylation of Smad-2/-3-its downstream targets-were enhanced by BLM and weakened by PD29. In vitro, PD29 inhibited TGF-ß1-induced epithelial-mesenchymal transition (EMT) and transformation in A549 cells and mouse primary fibroblasts into myofibroblasts. In summary, PD29 reversed EMT and transformation of fibroblasts into myofibroblasts in vitro and prevented PF in vivo possibly by suppressing the TGF-ß1/Smad pathway.

Natural Killer Cells may Exert Antidepressant-like Effects in Mice by Controlling the Release of Inflammatory Factors.

Depression or stress is reportedly related to the overflow of inflammatory factors in the body and T cells were reported to play important roles in balancing the release of inflammatory factors through vagus nerve circuit. However, few works have been conducted to find if natural killer (NK) cells can also exert the similar function in the reported vagus nerve circuit as T cells and if there was any relationship between depression and this function. In the present study, the behavioral tests on BALB/c mice indicated that the depressant-like symptoms could be improved and simultaneously the concentrations of inflammatory factors in peripheral blood could be reduced significantly by adoptively transferring NK cells into stressed BALB/c mice. The results revealed that NK cells could control the release of inflammatory factors secreted by macrophages and ß2-AR (ß2-adrenergic receptor) on the NK cells were of great importance. Behavioral tests on NCG mice indicated that the antidepressant-like effects of NK cells notably declined after adoptively transferring NK cells with ß2-AR deficiency or with ChAT (choline acetyltransferase) deficiency into stressed NCG mice. Simultaneously, the anti-inflammatory effects also declined significantly both in vivo and in vitro, which indicated that the antidepressant-like property of NK cells may be related to its ability of controlling the release of inflammatory factors. Taken together, we find that NK cells may balance the release of inflammatory factors in our body by transporting the information between the terminal vagal branches and macrophages, which is the mechanism that NK cells may exert antidepressant-like effects.

Sensitivity of non-small cell lung cancer to erlotinib is regulated by the Notch/miR-223/FBXW7 pathway.

Recent evidence supports a role for microRNA-223 (miR-223) in modulating tumor cell sensitivity to chemotherapeutic drugs; however, its role in cellular resistance to the effects of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) used in treatment of non-small cell lung cancer (NSCLC) remains to be elucidated. The levels of miR-223 in parental cell line (HCC827) and erlotinib resistant HCC827 cell line (HCC827/ER) were detected by qRT-PCR. HCC827/ER cells were treated with MK-2206 to block the Akt signaling pathway or RO4929097 to block the Notch signaling pathway, and then transfected with an miR-223 inhibitor or interference expression plasmid of F-Box/WD repeat-containing protein 7 (FBXW7) or insulin-like growth factor 1 receptor (IGF1R). HCC827 cells were transfected with miR-223 mimics. Next, CCK-8, colony formation, and flow cytometric apoptosis assays were used to assess cell resistance to erlotinib. When compared with its expression in HCC827 cells, miR-223 expression was significantly up-regulated in HCC827/ER cells. Blocking either the Akt or Notch signaling pathway and reducing miR-223 expression resulted in decreased resistance in HCC827/ER cells. Conversely, increasing miR-223 expression induced cell resistance to erlotinib in HCC827 cells. miR-223 enhanced resistance to erlotinib by down-regulating FBXW7 expression. Reducing FBXW7 expression lowered resistance to erlotinib in HCC827/ER cells, while interference with expression of IGF1R produced no significant effect. This study demonstrated that NSCLC cells can up-regulate their levels of miR-223 expression via the Akt and Notch signaling pathways. miR-223 may serve as an important regulator of erlotinib sensitivity in NSCLC cells by targeting FBXW7.