High Expression of LINC01268 is Positively Associated with Hepatocellular Carcinoma Progression via Regulating MAP3K7.

OBJECTIVE: As one of the most common neoplastic diseases, hepatocellular carcinoma (HCC) has a high morbidity and mortality, which seriously threatens human health and places a heavy burden on society and medical care. At present, effective early diagnosis, prognosis and treatment of HCC are limited. Altered gene expression patterns of lncRNA are associated with the occurrence, development and prognosis of various malignancies, including HCC. The aim of this study was to investigate the correlation between the expression of LINC01268 and HCC, and to elucidate the potential underlying molecular mechanism. METHODS: Expression level and localization of LINC01268 in human liver cancer cells and HCC tissues were investigated using RT-qPCR and fluorescent in situ hybridization (FISH), respectively. Correlation of expression levels of LINC01268 and MAP3K7 with differentiation and poor overall patient survival of HCC were analyzed using in house collected and publicly available HCC tissue data. RT-qPCR and Western blot were applied to inspect the effects of depletion and overexpression of LINC01268 on MAP3K7 expression. HCC cell proliferation and apoptosis were also investigated by simultaneous overexpression of LINC01268 and knockdown of MAP3K7, in order to delineate that MAP3K7 is a downstream effector of LINC01268. RESULTS: In this study, we identified that LINC01268 was highly expressed in HCC cell lines and tissues. High LINC01268 expression level was associated with lower HCC nodule number, moderate/poor differentiation and poor overall survival. Knockdown of LINC01268 inhibited the proliferation of HCC cells, which was enhanced by overexpression of LINC01268. Co-expression analysis implied an interaction between LINC01268 and MAP3K7. Similar to LINC01268, MAP3K7 was highly expressed in HCC cells, and positively correlated with moderate/poor differentiation as well as poor prognosis. Knockdown of LINC01268 in HCC cell lines led to reduction of MAP3K7 at both mRNA and protein levels. Phenotypic effects due to LINC01268 overexpression in HCC cells were reversed by knockdown of MAP3K7. CONCLUSION: Taken together, the abnormal high expression of LINC01268 is associated with HCC progression via regulating MAP3K7, suggesting LINC01268 as a novel marker for HCC prognosis and potentially a new therapeutic target.

Identification of Novel PhoP-PhoQ Regulated Genes That Contribute to Polymyxin B Tolerance in Pseudomonas aeruginosa.

Polymyxin B and E (colistin) are the last resorts to treat multidrug-resistant Gram-negative pathogens. Pseudomonas aeruginosa is intrinsically resistant to a variety of antibiotics. The PhoP-PhoQ two-component regulatory system contributes to the resistance to polymyxins by regulating an arnBCADTEF-pmrE operon that encodes lipopolysaccharide modification enzymes. To identify additional PhoP-regulated genes that contribute to the tolerance to polymyxin B, we performed a chromatin immunoprecipitation sequencing (ChIP-Seq) assay and found novel PhoP binding sites on the chromosome. We further verified that PhoP directly controls the expression of PA14_46900, PA14_50740 and PA14_52340, and the operons of PA14_11970-PA14_11960 and PA14_52350-PA14_52370. Our results demonstrated that mutation of PA14_46900 increased the bacterial binding and susceptibility to polymyxin B. Meanwhile, mutation of PA14_11960 (papP), PA14_11970 (mpl), PA14_50740 (slyB), PA14_52350 (ppgS), and PA14_52370 (ppgH) reduced the bacterial survival rates and increased ethidium bromide influx under polymyxin B or Sodium dodecyl sulfate (SDS) treatment, indicating roles of these genes in maintaining membrane integrity in response to the stresses. By 1-N-phenylnaphthylamine (NPN) and propidium iodide (PI) staining assay, we found that papP and slyB are involved in maintaining outer membrane integrity, and mpl and ppgS-ppgH are involved in maintaining inner membrane integrity. Overall, our results reveal novel PhoP-PhoQ regulated genes that contribute to polymyxin B tolerance.

Fis Contributes to Resistance of Pseudomonas aeruginosa to Ciprofloxacin by Regulating Pyocin Synthesis.

Factor for inversion stimulation (Fis) is a versatile DNA binding protein that plays an important role in coordinating bacterial global gene expression in response to growth phases and environmental stresses. Previously, we demonstrated that Fis regulates the type III secretion system (T3SS) in Pseudomonas aeruginosa In this study, we explored the role of Fis in the antibiotic resistance of P. aeruginosa and found that mutation of the fis gene increases the bacterial susceptibility to ciprofloxacin. We further demonstrated that genes related to pyocin biosynthesis are upregulated in the fis mutant. The pyocins are produced in response to genotoxic agents, including ciprofloxacin, and the release of pyocins results in lysis of the producer cell. Thus, pyocin biosynthesis genes sensitize P. aeruginosa to ciprofloxacin. We found that PrtN, the positive regulator of the pyocin biosynthesis genes, is upregulated in the fis mutant. Genetic experiments and electrophoretic mobility shift assays revealed that Fis directly binds to the promoter region of prtN and represses its expression. Therefore, our results revealed novel Fis-mediated regulation on pyocin production and bacterial resistance to ciprofloxacin in P. aeruginosaIMPORTANCEPseudomonas aeruginosa is an important opportunistic pathogenic bacterium that causes various acute and chronic infections in human, especially in patients with compromised immunity, cystic fibrosis (CF), and/or severe burn wounds. About 60% of cystic fibrosis patients have a chronic respiratory infection caused by P. aeruginosa The bacterium is intrinsically highly resistant to antibiotics, which greatly increases difficulties in clinical treatment. Therefore, it is critical to understand the mechanisms and the regulatory pathways that are involved in antibiotic resistance. In this study, we elucidated a novel regulatory pathway that controls the bacterial resistance to fluoroquinolone antibiotics, which enhances our understanding of how P. aeruginosa responds to ciprofloxacin.

High-efficiency protein delivery into transfection-recalcitrant cell types.

Intracellular delivery of functional proteins is of great interest for basic biological research as well as for clinical applications. Transfection is the most commonly used method, however, it is not applicable to large-scale manipulation and inefficient in important cell types implicated in biomedical applications, such as epithelial, immune and pluripotent stem cells. In this study, we explored a bacterial type III secretion system (Bac-T3SS)-mediated proteofection method to overcome these limitations. An attenuated Pseudomonas aeruginosa vector was constructed, which has features of low toxicity, high T3SS activity, and self-limiting growth. Compared to the method of transfection, the Bac-T3SS showed significantly higher efficiencies of Cre recombinase translocation and target site recombination for hard-to-transfect human cell lines. Furthermore, through the delivery of ß-lactamase in live animals, we demonstrated the feasibility and biosafety of in vivo application of the Bac-T3SS. This study provided an efficient and low-cost proteofection strategy for laboratory use as well as for application in large-scale cell manipulations.

Pseudomonas aeruginosa Polynucleotide Phosphorylase Contributes to Ciprofloxacin Resistance by Regulating PrtR.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes various acute and chronic infections. It is intrinsically resistant to a variety of antibiotics. However, production of pyocins during SOS response sensitizes P. aeruginosa to quinolone antibiotics by inducing cell lysis. The polynucleotide phosphorylase (PNPase) is a conserved phosphate-dependent 3'-5' exonuclease that plays an important role in bacterial response to environmental stresses and pathogenesis by influencing mRNA and small RNA stabilities. Previously, we demonstrated that PNPase controls the type III and type VI secretion systems in P. aeruginosa. In this study, we found that mutation of the PNPase coding gene (pnp) increases the bacterial resistance to ciprofloxacin. Gene expression analyses revealed that the expression of pyocin biosynthesis genes is decreased in the pnp mutant. PrtR, a negative regulator of pyocin biosynthesis genes, is upregulated in the pnp mutant. We further demonstrated that PNPase represses the expression of PrtR on the post-transcriptional level. A fragment containing 43 nucleotides of the 5' untranslated region was found to be involved in the PNPase mediated regulation of PrtR. Overall, our results reveled a novel layer of regulation on the pyocin biosynthesis by the PNPase in P. aeruginosa.

Identification of novel genes that promote persister formation by repressing transcription and cell division in Pseudomonas aeruginosa.

OBJECTIVES: Bacterial persisters are a small subpopulation of cells that are highly tolerant of antibiotics and contribute to chronic and recalcitrant infections. Global gene expression in Pseudomonas aeruginosa persister cells and genes contributing to persister formation remain largely unknown. The objective of this study was to examine the gene expression profiles of the persister cells and those that regained growth in fresh medium, as well as to identify novel genes related to persister formation. METHODS: P. aeruginosa persister cells and those that regrew in fresh medium were collected and subjected to RNA sequencing analysis. Genes up-regulated in the persister cells were overexpressed to evaluate their roles in persister formation. The functions of the persister-contributing genes were assessed with pulse-chase assay, affinity chromatography, fluorescence and electron microscopy, as well as a light-scattering assay. RESULTS: An operon containing PA2282-PA2287 was up-regulated in the persister cells and down-regulated in the regrowing cells. PA2285 and PA2287 play key roles in persister formation. PA2285 and PA2287 were found to bind to RpoC and FtsZ, which are involved in transcription and cell division, respectively. Pulse-chase assays demonstrated inhibitory effects of PA2285 and PA2287 on the overall transcription. Meanwhile, light-scattering and microscopy assays demonstrated that PA2285 and PA2287 interfere with cell division by inhibiting FtsZ aggregation. PA2285 and PA2287 are conserved in pseudomonads and their homologous genes in Pseudomonas putida contribute to persister formation. CONCLUSIONS: PA2285 and PA2287 are novel bifunctional proteins that contribute to persister formation in P. aeruginosa.

Prophylactic treatment of L-Arg improves malaria outcomes by regulating host immune responses during Plasmodium yoelii 17XL infection.

L-arginine (L-Arg), the precursor of nitric oxide (NO), plays multiple, important roles in nutrient metabolism and immune regulation. Hypoargininemia is one of the distinctive features of malaria patients in endemic areas. To understand the immunoregulatory function of L-Arg in malaria, we investigated the effects of L-Arg, pre- or/and post-treatment, on the cellular/humoral immune response during Plasmodium yoelii 17XL (P.y17XL) infection in DBA/2 mice. Populations of splenic CD4+T-bet+IFN-γ+ T cells (Th1), F4/80+ macrophages, CD4+GATA-3+IL-4+ T cells (Th2), B220+CD138+ plasmacytes and antibody-producing cells (IgG+/IgG1+-plasma cells) were assessed by flow cytometry. Pro-inflammatory cytokines and antibodies (IgG and IgG1) were quantified by immunoassays. We found that treatment with L-Arg significantly decreased parasitemia and shortened disease duration. Prophylactic treatment with L-Arg promotes an enhanced Th1 cell response during the early stages of P.y17XL infection, and treatment with L-Arg in the course of infection facilitates the later humoral immune response. Our findings suggest that treatment with L-Arg may decrease parasite burden and control the host's susceptibility to parasite synchronously by regulating host immune responses against P.y17XL, producing better outcomes for malaria infection. This implies that the supplementation of L-Arg may be a promising adjunctive therapy to reduce malaria-associated mortality in endemic areas.

Norepinephrine inhibits the cytotoxicity of NK92­MI cells via the ß2­adrenoceptor/cAMP/PKA/p­CREB signaling pathway.

Norepinephrine (NE) can regulate natural killer (NK) cell activity, but the mechanism remains unclear. In the present study the roles of adrenergic receptors (ARs) in inhibiting NK92­MI cells­mediated cytotoxicity by NE were investigated. To examine the effect of NE on NK92­MI cytotoxicity, a lactate dehydrogenase­release cytotoxicity assay was used to determine the cytotoxicity of NK92­MI cells against K562 cells. To evaluate the possible function of the α, ß1 and ß2 AR in mediating NE­induced effects, NK92­MI cells were pre­incubated with phenol­amine, CGP20712A and ICI118551 prior to stimulation by NE. To evaluate the role of cyclic adenosine monophosphate (cAMP)­protein kinase A (PKA) signaling pathway in the inhibitory effect on cytotoxicity of NK92­MI cell by NE, NK92­MI cells were pre­incubated with PKA inhibitor Rp­8­Br­cAMP prior to stimulation by NE. It was demonstrated that NE decreased cytotoxicity and downregulated the expression of perforin, granzyme B and interferon (IFN)­Î³ of NK92­MI cells in a dose­dependent manner. Blocking NE functional receptors by ARs antagonists, particularly of ß2 AR antagonist, suppressed the inhibitory effect of NE on cytotoxicity and expression of perforin, granzyme B, IFN­Î³ of NK92­MI cells significantly. Blockade of ß2 AR in NE treated NK92­MI cells resulted in a reduction of the expression of phosphorylated (p)­cAMP­responsive element­binding protein (CREB) and intracellular cAMP concentration. Inhibiting the activity of PKA by Rp­8­Br­cAMP in NE treated NK92­MI cells resulted in increased cytotoxicity. The results of the present study suggest that NE can inhibit cytotoxicity and expression of perforin, granzyme B, IFN­Î³ of NK92­MI cell mainly via the ß2­AR/cAMP/PKA/p­CREB signaling pathway.

In-Plane Anisotropic Molecular Orientation of Pentafluorene and Its Application to Linearly Polarized Electroluminescence.

By preparing parallelly aligned 1.9-µm-high SiO2 microfluidic channels on an indium tin oxide substrate surface, the solution flow direction during spin-coating was controlled to be parallel to the grating. Using this technique, a pentafluorene-4,4'-bis(N-carbazolyl)-1,1'-biphenyl (CBP) binary solution in chloroform was spin-coated to embed a 40-50 nm-thick 10 wt %-pentafluorene:CBP thin film in the channels. In-plane polarized photoluminescence measurements revealed that the pentafluorene molecules tended to orient along the grating, demonstrating that one-dimensional fluid flow can control the in-plane molecular orientation. Furthermore, the dependences of the photoluminescence anisotropy on the spin speed and substrate material suggest that the velocity of the solution flow and/or its gradient in the vertical direction greatly affects the resulting orientation. This indicates that the mechanism behind the molecular orientation is related to stress such as the shear force. The effect of the solution flow on the molecular orientation was demonstrated even in organic light-emitting diodes (OLEDs). Linearly polarized electroluminescence was obtained by applying the in-plane orientation to OLEDs, and it was found that the dichroic ratio of the electroluminescence orthogonal (x) and parallel (y) to the grating is x/y = 0.75.

Low-temperature direct heterogeneous bonding of polyether ether ketone and platinum.

Direct heterogeneous bonding between polyether ether ketone (PEEK) and Pt was realized at the temperatures lower than 150°C. In order to create sufficient bondability to diverse materials, the surface was modified by vacuum ultraviolet (VUV) irradiation, which formed hydrate bridges. For comparison, direct bonding between surfaces atomically cleaned via Ar fast atom bombardment (FAB) was conducted in a vacuum. The VUV irradiation was found to be effective for creating an ultrathin hydrate bridge layer from the residual water molecules in the chamber. Tight bonds were formed through dehydration of the hydrate bridges by heating at 150°C, which also contributed to enhancing interdiffusion across the interface. The VUV-modified surfaces showed bondability as good as that of the FAB-treated surfaces, and the VUV-modified samples had shear strengths at the same level as those of FAB-treated surfaces. This technology will be of practical use in the packaging of lightweight, flexible biomedical devices.

RNA editing of hepatitis B virus transcripts by activation-induced cytidine deaminase.

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. The mechanism by which AID triggers SHM and CSR has been explained by two distinct models. In the DNA deamination model, AID converts cytidine bases in DNA into uridine. The uridine is recognized by the DNA repair system, which produces DNA strand breakages and point mutations. In the alternative model, RNA edited by AID is responsible for triggering CSR and SHM. However, RNA deamination by AID has not been demonstrated. Here we found that C-to-T and G-to-A mutations accumulated in hepatitis B virus (HBV) nucleocapsid DNA when AID was expressed in HBV-replicating hepatic cell lines. AID expression caused C-to-T mutations in the nucleocapsid DNA of RNase H-defective HBV, which does not produce plus-strand viral DNA. Furthermore, the RT-PCR products of nucleocapsid viral RNA from AID-expressing cells exhibited significant C-to-T mutations, whereas viral RNAs outside the nucleocapsid did not accumulate C-to-U mutations. Moreover, AID was packaged within the nucleocapsid by forming a ribonucleoprotein complex with HBV RNA and the HBV polymerase protein. The encapsidation of the AID protein with viral RNA and DNA provides an efficient environment for evaluating AID's RNA and DNA deamination activities. A bona fide RNA-editing enzyme, apolipoprotein B mRNA editing catalytic polypeptide 1, induced a similar level of C-to-U mutations in nucleocapsid RNA as AID. Taken together, the results indicate that AID can deaminate the nucleocapsid RNA of HBV.