Plasma cytokine levels as markers of pathogenesis and treatment response in patients with non-tuberculous mycobacterial pulmonary disease.

We investigated the value of plasma cytokine levels as markers of pathogenesis and treatment response in patients with non-tuberculous mycobacteria (NTM) pulmonary disease. Plasma cytokine levels were measured and compared among patients with NTM pulmonary disease (n=111), tuberculosis (TB) patients (n=50), and healthy individuals (n=40). Changes during treatment were monitored at 3 and 6 months after treatment. According to the treatment response, NTM patients were classified as 'resistance' or 'sensitivity' responders. The results revealed that five out of twelve cytokines exhibited significantly higher levels in NTM patients compared to controls. Among these, interleukin (IL)-6 demonstrated the strongest discriminating capacity for NTM. Furthermore, when combined with IL-1ß, they efficiently distinguished between NTM drug-resistant and drug-sensitive patients, as well as between NTM and TB groups. Additionally, IL-6 levels initially rose and then decreased in the NTM drug-resistant group during the six months of treatment, similar to the behavior of IL-1ß in the NTM drug-sensitive group. Subgroup analyses of the sensitive group with differential treatment responses revealed an increase in IL-10 levels in the six-month treatment responders. A high IL-6/IL-10 ratio was associated with increased disease severity of NTM and TB. Collectively, combinations of various plasma cytokines, specifically IL-1ß, IL-6, and IL-10, effectively distinguished NTM patients with varying mycobacterial burdens, with IL-6 and IL-10 emerging as potential biomarkers for early treatment response. The combination of IL-6 and IL-1ß demonstrated the highest discriminatory value for distinguishing between NTM-resistant and NTM-sensitive groups as well as between NTM and TB groups.

Transport Properties of Aqueous Methane Solutions and Blocking Behavior of Intelligent-Responsive Temporary Plugging Agent in a Switchable Nano-channel: A Dissipative Particle Dynamics Simulation Study.

Intelligent-responsive temporary plugging agents (TPAs) have great potential in the field of oil and gas resource extraction due to their self-adaptability to the environment. However, the transport mechanism of oil and gas molecules, such as aqueous methane solution in intelligent-responsive TPA-modified nano-channels and the blocking behavior of TPA, have not been verified yet. In this work, dissipative particle dynamics simulations (DPD) are conducted to investigate the velocity distribution and the force characteristics of aqueous methane solutions under different driving velocities, as well as the blocking effect of TPA to the flow of solution. Simulation results indicate that aqueous methane solution primarily concentrates in the uncovered area of the TPA and diffuses into the TPA-covered area when the nano-channel is closed. The velocity distribution of the flowing solution in the open nano-channel follows a subparabolic pattern. Methane molecules in the closed nano-channel show sharp oscillations in velocity and force profiles, which can be mitigated by increasing the methane concentration. The designed TPA effectively blocks fluid flow but its head and tail are vulnerable to the shear forces from the fluid. This study enhances the understanding of the nanoflows in the wellbores during the extraction of oil and natural gas resources.

A method for rapid nanobody screening with no bias of the library diversity.

Nanobody, referred to the variable domain of heavy-chain-only antibodies, has several advantages such as small size and feasible Escherichia coli expression, making them promising for scientific research and therapies. Conventional nanobody screening and expression methods often suffer from the need for subcloning into expression vectors and amplification-induced diversity loss. Here, we developed an integrated method for simultaneous screening and expression. Nanobody libraries were cloned and secretly expressed in the culture medium. Target-specific nanobodies were isolated through 1-3 rounds of dilution and regrowth following the Poisson distribution. This ensured no dismissal of positive clones, with populations of positive clones increasing over 10-fold in each dilution round. Ultimately, we isolated 5 nanobodies against death domain receptor 5 and 5 against Pyrococcus furiosus DNA polymerase directly from their immunized libraries. Notably, our approach enables nanobody screening without specialized instruments, demonstrating broad applicability in routine monoclonal nanobody production for diverse biomedical applications.

The macrophage polarization by miRNAs and its potential role in the treatment of tumor and inflammation (Review).

The characteristics of monocyte/macrophage lineage are diversity and plasticity, mainly manifested by M1 and M2 subtypes in the body tissues, and playing different roles in the immunity. In the polarization process of macrophages, the classic molecular mechanism is related to sequential transcription factors. Whether in tumor or inflammatory local microenvironment, the pathological factors of the local microenvironment often affect the polarization of M1 and M2 macrophages, and participate in the occurrence and development of these pathological processes. In recent years, a growing number of research results demonstrated that non­coding RNA (ncRNA) also participates in the polarization process of macrophages, in addition to traditional cytokines and transcriptional regulation signal pathway molecules. Among numerous ncRNAs, microRNAs (miRNAs) have attracted more attention from scholars both domestically and internationally, and significant progress has been made in basic and clinical research. Therefore, for improved understanding of the molecular mechanism of miRNAs in macrophage polarization and analysis of the potential value of this regulatory pathway in tumor and inflammatory intervention therapy, a comprehensive review of the progress of relevant literature research was conducted and some viewpoints and perspectives were proposed.

Role of miR­let­7c­5p/c­myc signaling axis in the committed differentiation of leukemic THP­1 cells into monocytes/macrophages.

In a preliminary experiment, it was found that c-myc expression was decreased following the differentiation of THP-1 cells into monocytes/macrophages induced by phorbol 12-myristate 13 acetate (PMA) + lipopolysaccharide (LPS) + interferon (IFN)-γ. The expression of miR-let-7c-5p was then found to be elevated by cross-sectional analysis using TargetScan and PubMed and differential microarray analysis. The present study aimed to investigate the role of the miR-let-7c-5p/c-myc signaling axis in the committed differentiation of THP-1 leukemic cells into monocytes/macrophages induced by PMA + LPS + IFN-γ. Human THP-1 leukemic cells were induced to differentiate into monocytes/macrophages by PMA + LPS + IFN-γ. Following induction for 48 h, the growth density of the THP-1 cells was observed directly under an inverted microscope, cell proliferation was measured using Cell Counting Kit-8 assay and the cell cycle and the expression of differentiation-related antigens (CD11b and CD14) were measured using flow cytometry. The mRNA expression of miR-let-7c-5p and c-myc was detected using reverse transcription-quantitative PCR and the protein expression of c-myc was detected using western blot analysis. Dual luciferase reporter gene analysis was used to detect the targeted binding of miR-let-7c-5p on the 3'UTR of c-myc. The relative expression of miR-let-7c-5p and c-myc genes in THP-1 cells induced by PMA + LPS + IFN-γ was found to be up- and downregulated respectively, and expression of miR-let-7c-5p was negatively correlated with the expression of c-myc gene. Dual luciferase reporter gene assays confirmed that miR-let-7c-5p targeted the 3'UTR of c-myc and inhibited luciferase activity. Following transfection with miR-let-7c-5p mimics, the expression of c-myc was markedly downregulated and the proliferative ability of the THP-1 cells was decreased, while the expression rate of CD11b and CD14 was significantly increased. The rescue experiment revealed that the effects of miR-let-7c-5p mimics on the proliferation and differentiation of THP-1 cells were attenuated by transfection with c-myc overexpression vector. Together, the findings of the present study demonstrated that miR-let-7c-5p can target the 3'UTR region of c-myc and that the miR-let-7c-5p/c-myc signaling axis is one of the critical pathways involved in the directional differentiation of leukemic cells into monocytes/macrophages.

Enrichment and Delivery of Target Proteins into the Cell Cytosol via Outer Membrane Vesicles.

Advanced intracellular delivery of proteins has profound applications in both scientific investigations and therapies. However, existing strategies relying on various chemical and physical methods have drawbacks such as the requirement of high concentrations of in vitro prepared target proteins and difficulty in labeling target proteins. Developing new delivery systems integrating the enveloping and labeling of target proteins would bring great advantages for efficient protein transfections. Here, we enriched a high concentration (62 mg/mL) of several target proteins into the outer membrane vesicles (OMVs) of Escherichia coli to employ the native property of OMVs to deliver proteins into the cytosol of eukaryotic cells. The results revealed a high protein transfection efficiency from 90 to 97% for different cell lines. Moreover, the free penetration of molecules less than 600 Da across the membrane of OMVs allows direct labeling of target proteins within OMVs, facilitating the visualization of target proteins. Importantly, the nanobody delivered intracellularly by OMVs retains the biological activity of binding with its target, highlighting the advantages of OMVs as an emerging tool for efficient intracellular delivery of proteins.

Protein Conformational Exchanges Modulated by the Environment of Outer Membrane Vesicles.

Protein function, in many cases, is strongly coupled to the dynamics and conformational equilibria of the protein. The environment surrounding proteins is critical for their dynamics and can dramatically affect the conformational equilibria and subsequently the activities of proteins. However, it is unclear how protein conformational equilibria are modulated by their crowded native environments. Here we reveal that outer membrane vesicle (OMV) environments modulate the conformational exchanges of Im7 protein at its local frustrated sites and shift the conformation toward its ground state. Further experiments show both macromolecular crowding and quinary interactions with the periplasmic components stabilize the ground state of Im7. Our study highlights the key role that the OMV environment plays in the protein conformational equilibria and subsequently the conformation-related protein functions. Furthermore, the long-lasting nuclear magnetic resonance measurement time of proteins within OMVs indicates that they could serve as a promising system for investigating protein structures and dynamics in situ via nuclear magnetic spectroscopy.

Diverse Roles of ScSERF in Modifying the Fibril Growth of Amyloidogenic Proteins.

The aggregation of amyloidogenic proteins is often related to the occurrence of neurodegenerative diseases, including fused in sarcoma protein (FUS) in frontotemporal lobar degeneration and amyotrophic lateral sclerosis diseases. Recently, the SERF protein family has been reported to have a significant regulatory effect on amyloid formation, but it is still unclear about the detailed mechanisms of SERF acting on different amyloidogenic proteins. Herein, nuclear magnetic resonance (NMR) spectroscopy and fluorescence spectroscopy were used to explore interactions of ScSERF with three amyloidogenic proteins FUS-LC, FUS-Core, and α-Synuclein. NMR chemical shift perturbations reveal them sharing similar interaction sites on the N-terminal region of ScSERF. However, the amyloid formation of α-Synuclein protein is accelerated by ScSERF, while ScSERF inhibits fibrosis of FUS-Core and FUS-LC proteins. Both the primary nucleation and the total amount of fibrils produced are detained. Our results suggest a diverse role of ScSERF in regulating the fibril growth of amyloidogenic proteins.

An analysis of silybin meglumine tablets in the treatment of drug-induced liver injury as assessed for causality with the updated Roussel Uclaf Causality Assessment Method using a nationwide database.

AIM: Drug-induced liver injury (DILI) poses significant challenges to clinical practice. Currently, there is no recommended therapy to treat DILI; therefore, it is vital to explore new therapeutic agents. This study aimed to investigate the efficacy and safety of silybin meglumine tablets in treating DILI. METHODS: This study analysed 34 296 DILI cases assessed by the updated RUCAM from a nationwide database. A total of 301 patients with RUCAM scores ≥6 were directly enrolled in this study, while an additional 340 patients with RUCAM scores <6 who were adjudged as probable DILI by a panel of three hepatologists were also included in the analysis. The enrolled patients were divided into the silybin meglumine group and the control group. The propensity score matching (PSM) method was used to obtain comparable characteristics between the two groups. RESULTS: There were 129 cases in the silybin meglumine group and 512 cases in the control group. After applying PSM, 129 matched pairs were obtained. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) resumption rates in the silybin meglumine group were significantly higher than the control group (58.91% vs. 20.93%, P ≤ .0001 and 63.49% vs. 37.50%, P ≤ .0001). The univariate and multivariate logistic regression analysis revealed that grouping factor (odds raio [OR], 5.42; 95% confidenxe interval [CI], 3.12-9.39; P < .0001 and OR, 6.10; 95% CI, 2.98-12.48; P < .0001) and ALT levels (OR, 0.95; 95% CI, 0.93-0.98; P = .0015 and OR, 0.95; 95% CI, 0.92-0.99; P = .0157) were essential influencing factors for ALT normalization. CONCLUSIONS: Silybin meglumine tablets are safe and effective in DILI treatment. Large-scale and randomized controlled trials are required to further confirm their efficacy.

Effects of marine environments on methane hydrate formation in clay nanopores: A molecular dynamics study.

In nature, CH4 hydrates are mainly buried in marine sediments. The complex marine environments on the seafloor continuously affect hydrate formation. Herein, systematic molecular simulations have been performed to investigate CH4 hydrate formation in clay nanopore, mainly affected by several marine environmental factors, including seawater salinity, pressure and temperature. Simulation results show that these factors exert different effects on hydrate formation in the nanopore and the outside bulk solutions by affecting the mass transfer and phase separation inside and outside of the nanopore. Specifically, high salinity hinders the diffusion of CH4 molecules from nanopores into the outside bulk solutions, promoting hydrate formation in nanopore and inhibiting hydrate formation in bulk solution; salinity has a dual effect on hydrate formation in the whole system by changing the local CH4 concentration via the formation of the hydration of salt ions. High pressure favors the diffusion of CH4 molecules from nanopore into outside bulk solutions, promoting hydrate formation in bulk solution and inhibiting hydrate formation in nanopore; high pressure promotes hydrate formation at the nanopore throats by increasing CH4 concentration and reducing ion concentration therein. In contrast, temperature significantly affects hydrate formation in the system by causing phase separation, i.e. high temperature promotes the aggregation of CH4 molecules to form nanobubbles and inhibits hydrate formation. Under high temperature conditions, the nanobubble in the nanopore gradually decomposes, while the nanobubble in the outside bulk solution grows an extra-large cylindrical nanobubble. These molecular insights into the formation behavior of CH4 hydrates in clay nanopores are helpful for understanding the formation process of natural gas hydrates in marine sediments and the development and utilization of CH4 hydrates.

miR-let-7c-3p targeting on Egr-1 contributes to the committed differentiation of leukemia cells into monocyte/macrophages.

In preliminary experiments, it was found that the expression of early growth response-1 (Egr-1) was upregulated during the committed differentiation of leukemia cells into monocytes/macrophages. The cross-analysis of gene chip detection and database prediction indicated that Egr-1 was associated with upstream microRNA (miR)-let-7c-3p, thus the present study focused on the role of the miR-let-7c-3p/Egr-1 signaling axis in the committed differentiation of leukemia cells into monocytes/macrophages. Phorbol 12-myristate 13-acetate (PMA) was used to induce the directed differentiation of human K562 leukemia cells into monocytes/macrophages and the differentiation of K562 leukemia cells was determined by cell morphology observation and expression of differentiation antigens CD11b and CD14 by flow cytometry. The expression levels of Egr-1 and miR-let-7c-3p were detected by reverse transcription-quantitative PCR and the protein expression of Egr-1 was detected by western blotting. The effect of Egr-1 on the differentiation of K562 cells was detected by short interfering (si)RNA interference assay. A dual-luciferase reporter assay was used to detect target binding of miR-let-7c-3p on the 3'UTR of Egr-1. Cell transfection of miR-let-7c-3p mimics and inhibitors was used to modulate the expression of miR-let-7c-3p, as indicated by RT-qPCR assays. Western blotting was also used to examine the effect of miR-let-7c-3p on Egr-1 expression. The PMA-induced differentiation of K562 cells was transfected with miR-let-7c-3p and the expression of differentiation antigen was detected by flow cytometry. A differentiation model of K562 leukemia cells into monocytes/macrophages was induced by PMA, which was indicated by morphological observations and upregulation of CD11b and CD14 antigens. The gene or protein expression of Egr-1 was significantly higher compared with that of the control group, while the expression of miR-let-7c-3p was significantly lower compared with that of the control group. siRNA interference experiments showed that the expression of cell differentiation antigen CD14 in the 100 µg/ml PMA + si-Egr-1 group was significantly lower compared with that in the 100 µg/ml PMA + si-ctrl group. The dual luciferase reporter gene results showed that the luciferase activity of the co-transfected mimic and Egr-1 WT groups was significantly lower than that of the NC control group, while the luciferase activity of the co-transfected mimic and Egr-1 MUT groups was comparable to that of the NC control group. Therefore, the dual-luciferase reporter gene assay confirmed that miR-let-7c-3p can target Egr-1. Western blotting showed that the expression of Egr-1 following transfection with miR-let-7c-3p inhibitor was significantly higher compared with that of the negative control and the expression of Egr-1 after transfection with miR-let-7c-3p mimic was significantly lower than that of the negative control. Following exposure to PMA, the expressions of CD11b and CD14 in the miR-let-7c-3p inhibitor group were significantly higher than those in the miR-let-7c-3p NC group, as indicated by CD11b and CD14 respectively. In conclusion, miR-let-7c-3p could bind to the 3'UTR of Egr-1 and negatively regulated Egr-1 expression. The miR-let-7c-3p/Egr-1 signaling axis was closely associated with the committed differentiation of K562 cells from leukemia cells to monocytes/macrophages.

Effects of surface property of mixed clays on methane hydrate formation in nanopores: A molecular dynamics study.

HYPOTHESIS: Mixed clays (e.g. montmorillonite, illite and kaolinite) are ubiquitous in hydrate-bearing sediments under seafloor, and their surfaces inevitably affect the formation of natural gas hydrates therein. Nevertheless, the actual effects of clay surfaces on hydrate formation remain elusive. EXPERIMENTS: Systematic molecular dynamics simulations have been performed to investigate CH4 hydrate formation in mixed clay nanopores of montmorillonite, illite and kaolinite, to examine the effects of surface property and layer charges of mixed clays. FINDINGS: Simulation results indicate that the surfaces of mixed clays affect CH4 hydrate formation in the nanopores by changing the CH4 concentration (xCH4) and ion concentration (xions) in the middle region of the nanopores via surface adsorption for CH4, H2O and ions. Specifically, the surfaces of montmorillonite and illite, the siloxane and gibbsite surfaces of kaolinite show different affinities for adsorbing CH4, H2O and ions, which can significantly affect the xCH4 and xions in the interfacial and middle regions of the nanopores. Moreover, hydrate growth shows certain surface preference. These molecular insights into the effect of mixed clay surfaces on CH4 hydrate formation can help to understand the formation mechanism of natural gas hydrate in marine sediments.

Backbone resonance assignments and dynamics of S. cerevisiae SERF.

Abnormal protein aggregation and precipitation are associated with the perturbation of cellular function and underlie a variety of neurodegenerative diseases. S. cerevisiae SERF (ScSERF), a homolog of modifier of aggregation-4 (MOAG-4) and small EDRK-rich factor protein (SERF1a) is highly conserved and discovered as an enhancer of amyloid formation of Aß40 and α-synuclein both in vitro and in vivo. However, the detailed molecular mechanism whereby ScSERF and its homologs accelerate amyloid formation is not well known yet. Herein, we present the 1 H, 15 N and 13 C NMR assignments of the 68 amino acids long ScSERF. Although ScSERF displays a very high degree of disorder, secondary chemical shifts of Cα, Cß, 15 N{1 H}-NOE values and the residue-specific secondary structure propensity (SSP) scores indicate the segment spanning residues 36E-65 K has a strong helical propensity. This work sets the stage for further detailed structural and dynamic investigations of ScSERF and the molecular mechanism it utilizes in accelerating amyloid formation.

Creation of a quick and sensitive fluorescent immunosensor for detecting the mineralocorticoid steroid hormone aldosterone.

Aldosterone (ALD) is a steroid hormone secreted by the zona glomerulosa of the adrenal cortex that mainly acts on the kidney to regulate sodium ion and water reabsorption. Detection of ALD plays an important role in the diagnosis of primary aldosteronism in patients with hypertension. For the first time, the gene encoding the anti-ALD antibody, A2E11, was successfully cloned and analyzed using phage display technology. The antibody had an affinity of 2.5 nM against ALD, and after binding to ALD, it reached saturation within 5 s. Using this antibody, a Quenchbody (Q-body) was constructed by labeling the N-termini of heavy and light chains of the antigen-binding fragment of A2E11 with the fluorescent dye ATTO520 to detect ALD based on the principle of photoinduced electron transfer. The sensor detected ALD in 2 min, and the limit of detection was 24.1 pg/mL with a wide detection range from 24.1 pg/mL to 10 µg/mL and a half-maximal effective concentration of 42.3 ng/mL. At the highest concentration of ALD in the assay, the fluorescence intensity increased by 5.0-fold compared to the original fluorescence intensity of the Q-body solution. The Q-body could be applied to analyze 50% of human serum without a significant influence of the matrix. The recoveries of ALD in spiked serum samples with the Q-body assay were confirmed to range from 90.3% to 98.2%, suggesting their potential applications in the diagnosis of diseases, such as essential hypertension.

Isolation of a Monoclonal Antibody and its Derived Immunosensor for Rapid and Sensitive Detection of 17ß-Estradiol.

Estrogens are effective for stimulating several functions in living organisms and for regulating cancer development by promoting cell proliferation. Estradiol can disrupt the reproductive and endocrine systems, leading to the development of various diseases. In this study, the monoclonal antibody ESC9 was developed by immunizing mice with a 17ß-estradiol (E2) conjugate, preparing an antibody phage display library, and screening monoclonal antibodies from the prepared library. An antibody with the same sequence as that of ESC9 has not been reported previously. The equilibrium dissociation constant between ESC9 and E2 was found to be 43.3 nM. Additionally, we generated an ESC9-derived immunosensor named as the ESC9 Quenchbody (Q-body), which can rapidly and sensitively detect E2. The assay can be completed within 2 min with a limit of detection of 3.9 pg/ml and half-maximal effective concentration of 154.0 ng/ml. Serum E2 levels were measured using the ESC9 Q-body without pretreatment with serum and with a high recovery rate of 83.3-126.7%. The Q-body immunosensor shows potential for clinical applications based on its excellent detection speed and sensitivity.

Advances in the application of Let-7 microRNAs in the diagnosis, treatment and prognosis of leukemia.

The lethal-7 (Let-7) family of microRNAs (miRNAs) controls the process of development and differentiation, but is also related to the occurrence of tumors and a poor prognosis of patients with tumors. Thus, a more comprehensive exploration of its functions will provide further insights into these processes, and may promote the diagnosis and treatment of tumors. Leukemia is a type of progressive malignant disease, and its pathogenesis involves a variety of epigenetic factors. Amongst the several related epigenetic factors, the Let-7 miRNAs are an important family of molecules that play a crucial role in maintaining a variety of critical biological processes, including development, differentiation and proliferation. In the present study, the role of Let-7 as a tumor suppressor gene and oncogene is reviewed, and the complex regulatory functions of several Let-7 family members in different subtypes of leukemia are described. The current body of knowledge thus far indicates that Let-7 is not only a potential diagnostic and prognostic marker of leukemia, but also a potential therapeutic target for the treatment of affected patients, with particular potential when targeted by adjuvant treatments alongside traditional treatment to improve their survival rate.

ILT3 promotes tumor cell motility and angiogenesis in non-small cell lung cancer.

Immunoglobulin-like transcript (ILT) 3 is an immunosuppressive molecule that negatively regulates myeloid cell activation. ILT3 overexpression in tumor cells induces immune escape of solid tumors and facilitates invasion of monocytic acute myeloid leukemia cells. However, the expression and function of ILT3 in non-small cell lung cancer (NSCLC) cells remain elusive. Herein, we found that ILT3 was enriched in human NSCLC cells, and predicted advanced disease and poor overall survival. ILT3 overexpression enhanced the migration and invasion of NSCLC cells and tubule formation of human umbilical vein endothelial cells by upregulating and interacting with its ligand apolipoprotein E (ApoE) in vitro. Mechanistically, ILT3 recruited SHP2 and SHIP1, and subsequently activated ERK1/2 signaling mediating epithelial-mesenchymal transition (EMT) and increasing vascular endothelial growth factor (VEGF)-A expression in NSCLC cells, which are responsible for tumor cell motility and angiogenesis, respectively. Using murine metastasis models, we further confirmed ILT3 promoted NSCLC metastasis and explored the exact correlation of ILT3 with ApoE, EMT, and VEGF-A in vivo. These results unraveled novel mechanisms for ILT3-induced tumor progression and proposed ILT3 as a potential therapeutic target and prognostic biomarker for NSCLC patients.

LncRNA NEAT1 Regulates 5-Fu Sensitivity, Apoptosis and Invasion in Colorectal Cancer Through the MiR-150-5p/CPSF4 Axis.

BACKGROUND: Colorectal cancer (CRC) is one of the most prevalent malignancies in the world. Long non-coding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) is involved in the development of many cancers. However, its role and mechanism in CRC progression still need further exploration. METHODS: The expression levels of lnc-NEAT1, microRNA-150-5p (miR-150-5p) and cleavage and polyadenylation specific factor 4 (CPSF4) were determined by quantitative real-time PCR (qRT-PCR). The sensitivity of cells to 5-fluorouracil (5-Fu) was measured by 3-(4,5-dimethyl-2 thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Cell apoptosis and invasion were evaluated by flow cytometry and transwell assays, respectively. Western blot (WB) analysis was used to assess the levels of resistance-related proteins and CPSF4 protein. Besides, dual-luciferase reporter assay was used to verify the interactions among lnc-NEAT1, miR-150-5p and CPSF4. Also, mice xenograft models were used to determine the effect of lnc-NEAT1 on CRC tumor growth in vivo. RESULTS: In CRC, the expression of lnc-NEAT1 was upregulated and miR-150-5p was downregulated, and the expression of both was negatively correlated. Silencing of lnc-NEAT1 promoted the 5-Fu sensitivity, enhanced the apoptosis and suppressed the invasion of CRC cells. MiR-150-5p could be sponged by lnc-NEAT1, and its inhibitors could partially reverse the effect of lnc-NEAT1 silencing on CRC progression. Besides, CPSF4 could be targeted by miR-150-5p, and its overexpression also could invert the effect of lnc-NEAT1 knockdown on CRC progression. Further, CPSF4 expression was regulated by lnc-NEAT1 and miR-150-5p. In addition, interference of lnc-NEAT1 reduced tumor volume and improved the sensitivity of CRC to 5-Fu in vivo. CONCLUSION: Lnc-NEAT1 acted as an oncogene in CRC through regulating CPSF4 expression by sponging miR-150-5p. The discovery of lnc-NEAT1/miR-150-5p/CPSF4 axis provided a novel approach for CRC genomic therapy strategy.

Tob2 Inhibits TLR-Induced Inflammatory Responses by Association with TRAF6 and MyD88.

Optimal activation of TLR pathways is crucial for the initiation of inflammatory responses and eliminating invading micro-organisms. However, excessive of TLR activation may lead to autoimmune and inflammatory diseases. Thus, TLR pathways should be tightly controlled. In this study, we identify Tob2, a Tob/BTG family member, as a suppressor of TLR pathways. Tob2 deficiency enhances TLR-induced NF-κB and MAPK activation and promotes the expression of proinflammatory cytokines in primary peritoneal macrophages of C57BL/6 mice. Furthermore, Tob2-defective C57BL/6 mice may be more susceptible to endotoxemic shock in vivo. Mechanistically, Tob2 interacts with TRAF6 and MyD88 and thus inhibits signaling from the MyD88-TRAF6 complex in primary peritoneal macrophages and HEK293T cells. Therefore, our results uncover a regulatory mechanism of TLR pathways and provide a potential target for the intervention of diseases with excessive TLR activation.

The Epstein-Barr virus-encoded G protein-coupled receptor BILF1 upregulates ICAM-1 through a mechanism involving the NF-қB pathway.

Although the Epstein-Barr virus (EBV) infection is usually asymptomatic, a primary encounter with the virus can cause mononucleosis. EBV infection is also strongly associated with lymphoma and epithelial cancers. The structure and infection mechanism of EBV have been well studied, but the EBV-encoded G protein-coupled receptor, BILF1, is not fully understood. Here, it was found that the EBV BILF1 was expressed early in the viral lytic cycle and its ectopic expression strikingly upregulated the ICAM-1 expression in Raji cells. The positive effect of BILF1 on the ICAM-1 promoter was observed and the BILF1 deficiency attenuated ICAM-1 promoter activity. Moreover, NF-κB binding sites were important for the regulation of ICAM-1 promoter by BILF1. Furthermore, BILF1 reduced the constitutive level of the IқB-a protein and increased the amount of nuclear NF-қB in Raji cells. In conclusion, this study determined that BILF1 upregulated ICAM-1 in a mechanism involving NF-қB.