Chalkophomycin Biosynthesis Revealing Unique Enzyme Architecture for a Hybrid Nonribosomal Peptide Synthetase and Polyketide Synthase.

Chalkophomycin is a novel chalkophore with antibiotic activities isolated from Streptomyces sp. CB00271, while its potential in studying cellular copper homeostasis makes it an important probe and drug lead. The constellation of N-hydroxylpyrrole, 2H-oxazoline, diazeniumdiolate, and methoxypyrrolinone functional groups into one compact molecular architecture capable of coordinating cupric ions draws interest to unprecedented enzymology responsible for chalkophomycin biosynthesis. To elucidate the biosynthetic machinery for chalkophomycin production, the chm biosynthetic gene cluster from S. sp. CB00271 was identified, and its involvement in chalkophomycin biosynthesis was confirmed by gene replacement. The chm cluster was localized to a ~31 kb DNA region, consisting of 19 open reading frames that encode five nonribosomal peptide synthetases (ChmHIJLO), one modular polyketide synthase (ChmP), six tailoring enzymes (ChmFGMNQR), two regulatory proteins (ChmAB), and four resistance proteins (ChmA'CDE). A model for chalkophomycin biosynthesis is proposed based on functional assignments from sequence analysis and structure modelling, and is further supported by analogy to over 100 chm-type gene clusters in public databases. Our studies thus set the stage to fully investigate chalkophomycin biosynthesis and to engineer chalkophomycin analogues through a synthetic biology approach.

Exogenous gibberellin delays maturation in persimmon fruit through transcriptional activators and repressors.

As the harvest season of most fruit is concentrated, fruit maturation manipulation is essential for the fresh fruit industry to prolong sales time. Gibberellin (GA), an important phytohormone necessary for plant growth and development, has also shown a substantial regulatory effect on fruit maturation; however, its regulatory mechanisms remain inconclusive. In this research, preharvest GA3 treatment effectively delayed fruit maturation in several persimmon (Diospyros kaki) cultivars. Among the proteins encoded by differentially expressed genes, 2 transcriptional activators (NAC TRANSCRIPTION FACTOR DkNAC24 and ETHYLENE RESPONSIVE FACTOR DkERF38) and a repressor (MYB-LIKE TRANSCRIPTION FACTOR DkMYB22) were direct regulators of GERANYLGERANYL DIPHOSPHATE SYNTHASE DkGGPS1, LYSINE HISTIDINE TRANSPORTER DkLHT1, and FRUCTOSE-BISPHOSPHATE ALDOLASE DkFBA1, respectively, resulting in the inhibition of carotenoid synthesis, outward transport of an ethylene precursor, and consumption of fructose and glucose. Thus, the present study not only provides a practical method to prolong the persimmon fruit maturation period in various cultivars but also provides insights into the regulatory mechanisms of GA on multiple aspects of fruit quality formation at the transcriptional regulation level.

Full-length transcriptome profiling for fruit development in Diospyros oleifera using nanopore sequencing.

OBJECTIVES: Diospyros oleifera, one of the most economically important Diospyros species, is an ideal model for studying the fruit development of persimmon. While, the lack of whole-transcriptome has hindered the complex transcriptional regulation mechanisms of sugar and tannin during fruit development. DATA DESCRIPTION: We applied Oxford Nanopore Technologies to six developmental stage of fruit from D. oleifera for use in transcriptome sequencing. As a result of full-length transcriptome sequencing, 55.87 Gb of clean data were generated. After mapping onto the reference genome of D. oleifera, 51,588 full-length collapsing transcripts, including 2,727 new gene loci and 43,223 transcripts, were obtained. Comprehensively annotated, 38,086 of new transcripts were functional annotation, and 972 lncRNAs, 7,159 AS events were predicted. Here, we released the transcriptome database of D. oleifera at different stage of fruit development,which will provide a fundamention of to investigatethe transcript structure, variants and evolution of persimmon.

Integrated Metabolomic-Transcriptomic Analysis Reveals Diverse Resource of Functional Ingredients From Persimmon Leaves of Different Varieties.

Persimmon leaves are used for making persimmon leaf tea or as functional ingredients due to their enrichment in flavonoids, the beneficial mineral contents, and favorable flavors contributed by volatile aroma compounds. The varieties/cultivars had a significant influence on the quality and flavor of persimmon leaf tea. In this study, the integrated metabolomic-transcriptomic analysis was conducted to investigate the potential in flavonoid biosynthesis, mineral absorption, and degradation of aromatic compounds from tender leaves of "Diospyros kaki. Heishi" (HS), "Diospyros kaki Thunb. Nishimurawase" (NM), and "Diospyros kaki Thunb. Taifu" (TF), using rootstock "Diospyros Lotus Linn" (DL) as the control. The metabolomic analysis showed that 382, 391, and 368 metabolites were differentially accumulated in the comparison of DL vs. HS, DL vs. NM, and DL vs. TF, respectively, and 229 common metabolites were obtained by comparative analysis. By RNA sequencing, 182,008 unigenes with 652 bp of mean length were annotated and 2,598, 3,503, and 3,333 differentially expressed genes (DEGs) were detected from the comparison of DL vs. HS, DL vs. NM, and DL vs. TF, respectively. After the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, 6, 6, and 3 DEGs [with | log2(fold change)| ≥ 1 simultaneously in the three comparisons] involved in flavonoid biosynthesis, mineral absorption, and degradation of aromatic compounds, respectively, were selected for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) validation and the consistent trends of the relative expression level of each DEG with RNA sequencing (RNA-seq) data were observed. Based on the transcriptomic analysis and qRT-PCR validation, it was observed that the leaves of HS, NM, and TF had the greatest level of mineral absorption, flavonoid biosynthesis, and degradation of aromatic compounds, respectively. In addition, a positive correlation between the 15 DEGs and their metabolites was observed by the conjoint analysis. Thus, the tender leaves of HS, NM, and TF could be recommended for the production of persimmon leaf tea rich in mineral elements, flavonoid, and aroma compounds, respectively.

Undescribed benzophenone and xanthones from cave-derived Streptomyces sp. CB09001.

A new benzophenone huanglongmycin (HLM) D (1) and two new monomeric xanthones huanglongmycin E (2) and F (3), together with four known aromatic polyketides aloesaponarin II (4) and the previously isolated huanglongmycin A-C (5-7) were obtained from cave-derived Streptomyces sp. CB09001. The structures of 1-3 were established based on 1D, 2D NMR and HRMS data. Compounds 1-7 may be biosynthesized by a type II huanglongmycin polyketide synthase based on gene inactivation of hlmG encoding KSɑ in hlm gene cluster and their plausible biosynthetic mechanism was proposed.

Characterization of Chalkophomycin, a Copper(II) Metallophore with an Unprecedented Molecular Architecture.

Metals play essential roles in life by coordination with small molecules, proteins, and nucleic acids. Although the coordination of copper ions in many proteins and methanobactins is known, the coordination chemistry of Cu(II) in natural products and their biological functions remain underexplored. Herein, we report the discovery of a Cu(II)-binding natural product, chalkophomycin (CHM, 1), from Streptomyces sp. CB00271, featuring an asymmetric square-coordination system of a bidentate diazeniumdiolate and a conjugated 1H-pyrrole 1-oxide-oxazoline. The structure of 1 may inspire the synthesis of Cu(II) chelators against neurodegenerative diseases or Cu(II)-based antitumor therapeutics.

Characterization of the complete chloroplast genome sequence of Diospyros kaki cv. Luotiantianshi.

Diospyros kaki cv. Luotiantianshi is a rare germplasm of Diospyros Linn in the world. In this study, we generated the complete chloroplast (cp) genome of D. kaki cv. Luotiantianshi. The complete cp genome was 157,773 bp in length, containing a large single copy region (LSC) of 87,066 bp, a small single copy region (SSC) of 18,529 bp, and two inverted repeat (IR) regions of 26,089 bp. The new sequence has a total of 131 genes, including 86 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Further, phylogenetic analysis showed that the D. kaki cv. Luotiantianshi has a close relationship with Diospyros kaki. This study provides important information for future evolution, genetic and molecular biology studies of Diospyros.

Functional activity of anti-LINGO-1 antibody opicinumab requires target engagement at a secondary binding site.

LINGO-1 is a membrane protein of the central nervous system (CNS) that suppresses myelination of axons. Preclinical studies have revealed that blockade of LINGO-1 function leads to CNS repair in demyelinating animal models. The anti-LINGO-1 antibody Li81 (opicinumab), which blocks LINGO-1 function and shows robust remyelinating activity in animal models, is currently being investigated in a Phase 2 clinical trial as a potential treatment for individuals with relapsing forms of multiple sclerosis (AFFINITY: clinical trial.gov number NCT03222973). Li81 has the unusual feature that it contains two LINGO-1 binding sites: a classical site utilizing its complementarity-determining regions and a cryptic secondary site involving Li81 light chain framework residues that recruits a second LINGO-1 molecule only after engagement of the primary binding site. Concurrent binding at both sites leads to formation of a 2:2 complex of LINGO-1 with the Li81 antigen-binding fragment, and higher order complexes with intact Li81 antibody. To elucidate the role of the secondary binding site, we designed a series of Li81 variant constructs that eliminate it while retaining the classic site contacts. These Li81 mutants retained the high affinity binding to LINGO-1, but lost the antibody-induced oligodendrocyte progenitor cell (OPC) differentiation activity and myelination activity in OPC- dorsal root ganglion neuron cocultures seen with Li81. The mutations also attenuate antibody-induced internalization of LINGO-1 on cultured cortical neurons, OPCs, and cells over-expressing LINGO-1. Together these studies reveal that engagement at both LINGO-1 binding sites of Li81 is critical for robust functional activity of the antibody.

The persimmon (Diospyros oleifera Cheng) genome provides new insights into the inheritance of astringency and ancestral evolution.

Persimmon (Diospyros kaki) is an oriental perennial woody fruit tree whose popular fruit is produced and consumed worldwide. The persimmon fruit is unique because of the hyperaccumulation of proanthocyanidins during fruit development, causing the mature fruit of most cultivars to have an astringent taste. In this study, we obtained a chromosome-scale genome assembly for 'Youshi' (Diospyros oleifera, 2n = 2x = 30), the diploid species of persimmon, by integrating Illumina sequencing, single-molecule real-time sequencing, and high-throughput chromosome conformation capture techniques. The assembled D. oleifera genome consisted of 849.53 Mb, 94.14% (799.71 Mb) of which was assigned to 15 pseudochromosomes, and is the first assembled genome for any member of the Ebenaceae. Comparative genomic analysis revealed that the D. oleifera genome underwent an ancient γ whole-genome duplication event. We studied the potential genetic basis for astringency development (proanthocyanidin biosynthesis) and removal (proanthocyanidin insolublization). Proanthocyanidin biosynthesis genes were mainly distributed on chromosome 1, and the clustering of these genes is responsible for the genetic stability of astringency heredity. Genome-based RNA-seq identified deastringency genes, and promoter analysis showed that most of their promoters contained large numbers of low oxygen-responsive motifs, which is consistent with the efficient industrial application of high CO2 treatment to remove astringency. Using the D. oleifera genome as the reference, SLAF-seq indicated that 'Youshi' is one of the ancestors of the cultivated persimmon (2n = 6x = 90). Our study provides significant insights into the genetic basis of persimmon evolution and the development and removal astringency, and it will facilitate the improvement of the breeding of persimmon fruit.

A Prodomain Fragment from the Proteolytic Activation of Growth Differentiation Factor 11 Remains Associated with the Mature Growth Factor and Keeps It Soluble.

Growth differentiation factor 11 (GDF11), a member of the transforming growth factor ß (TGF-ß) family, plays diverse roles in mammalian development. It is synthesized as a large, inactive precursor protein containing a prodomain, pro-GDF11, and exists as a homodimer. Activation requires two proteolytic processing steps that release the prodomains and transform latent pro-GDF11 into active mature GDF11. In studying proteolytic activation in vitro, we discovered that a 6-kDa prodomain peptide containing residues 60-114, PDP60-114, remained associated with the mature growth factor. Whereas the full-length prodomain of GDF11 is a functional antagonist, PDP60-114 had no impact on activity. The specific activity of the GDF11/PDP60-114 complex (EC50 = 1 nM) in a SMAD2/3 reporter assay was identical to that of mature GDF11 alone. PDP60-114 improved the solubility of mature GDF11 at neutral pH. As the growth factor normally aggregates/precipitates at neutral pH, PDP60-114 can be used as a solubility-enhancing formulation. Expression of two engineered constructs with PDP60-114 genetically fused to the mature domain of GDF11 through a 2x or 3x G4S linker produced soluble monomeric products that could be dimerized through redox reactions. The construct with a 3x G4S linker retained 10% activity (EC50 = 10 nM), whereas the construct connected with a 2x G4S linker could only be activated (EC50 = 2 nM) by protease treatment. Complex formation with PDP60-114 represents a new strategy for stabilizing GDF11 in an active state that may translate to other members of the TGF-ß family that form latent pro/mature domain complexes.

Effect of mesenchymal stem cells on Sjögren-like mice and the microRNA expression profiles of splenic CD4+ T cells.

Mesenchymal stem cells (MSCs) serve immuno-regulatory functions and offer a promising novel treatment for certain autoimmune diseases. The present study investigated the therapeutic effect of mice bone marrow (BM)-MSCs on mice with relatively late stage of Sjögren-like disease and the impact of BM-MSCs on the microRNA (miRNA) expression profiles of splenic CD4+ T cells. Female NOD/Ltj mice were randomized into two groups: The disease group (n=8) and the MSC-treated group (n=8). Female ICR mice served as the healthy control group (n=8). The MSC-treated group received an injection of MSCs when they were 26 weeks old. Water intake, blood glucose and salivary flow rate were measured and submandibular glands were resected and stained with hematoxylin and eosin to calculate the focus score. The concentrations of interleukin (IL)-2, IL-6, hepatocyte growth factor, interferon γ, IL-10, prostaglandin E2, transforming growth factor ß1 and tumor necrosis factor-α in serum were measured using ELISA. The expression of miRNAs in splenic CD4+ T cells were measured using deep sequencing. The results demonstrated that treatment with BM-MSCs prevented a decline in the salivary flow rate and lymphocyte infiltration in the salivary glands of NOD mice, indicating that MSC-treatment had a therapeutic effect on NOD mice with relatively late stage of Sjögren-like disease. ELISA and deep sequencing results showed that the three groups of mice had different serum concentrations of cytokines/growth factors and different miRNA expression profiles of splenic CD4+ T cells. This implies that the alteration in serum levels of cytokines/growth factors and miRNA expression profiles of splenic CD4+ T cells may explain the therapeutic effect MSCs have on Sjögren's syndrome.

Determination of the Disulfide Structure of Murine Meteorin, a Neurotrophic Factor, by LC-MS and Electron Transfer Dissociation-High-Energy Collisional Dissociation Analysis of Proteolytic Fragments.

Meteorin and Cometin (Meteorin-like) are secreted proteins belonging to a newly discovered growth factor family. Both proteins play important roles in neural development and may have potential as therapeutic targets or agents. Meteorin and Cometin are homologues and contain ten evolutionarily conserved Cys residues across a wide variety of species. However, the status of the Cys residues has remained unknown. Here, we have successfully determined the disulfide structure for murine Meteorin by LC-MS analysis of fragments generated by trypsin plus endoprotease-Asp-N. For proteolytic fragments linked by more than one disulfide bond, we used electron transfer dissociation (ETD) to partially dissociate disulfide bonds followed by high-energy collisional dissociation (HCD) to determine disulfide linkages. Our analysis revealed that the ten Cys residues in murine Meteorin form five disulfide bonds with Cys7 (C1) linked to Cys28 (C2), Cys59 (C3) to Cys95 (C4), Cys148 (C5) to Cys219 (C8), Cys151 (C6) to Cys243 (C9), and Cys161 (C7) to Cys266 (C10). Since the ten Cys residues are highly conserved in Meteorin and Cometin, it is likely that the disulfide linkages are also conserved. This disulfide structure information should facilitate structure-function relationship studies on this new class of neurotrophic factors and also assist in evaluation of their therapeutic potentials.

Investigating the Role of Artemin Glycosylation.

PURPOSE: Oligosaccharides play diverse and unpredictable functional roles when attached to proteins and are a largely unexplored scaffold for deconstructing and attributing novel functions to proteins during drug development. Here, the glycoprotein Artemin (ART) was carefully assessed by multiple analytical methods that allow us to provide a comprehensive understanding of how N-linked glycosylation impact the structural and functional properties of ART. METHODS: Modification of the N-linked glycan of ART was performed by incubation with various enzymes. Biological assays and systems were used to examine the relative activity and pharmacokinetic properties of ART as a function of glycosylation. In order to reveal the conformational impact of glycosylation on ART, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was employed in addition to differential scanning calorimetry. The colloidal stability of ART glycovariants was assessed by dynamic light scattering, viscometry, and solubility assays. RESULTS: No difference in pharmacokinetics or relative potency was revealed between glycosylated and nonglycosylated ART. Surprisingly, the HDX-MS data indicated that the glycan does not greatly influence the conformation and dynamics of the protein. In contrast, differences in thermal and colloidal stability clearly revealed a role of glycosylation in increasing the solubility and stability of ART. CONCLUSIONS: Our findings demonstrate how careful analysis using multiple advanced techniques can be used to identify and dissect the multiple potential functions of protein glycosylation and form a prerequisite for glycoengineering and drug development of glycoproteins.

Meteorin reverses hypersensitivity in rat models of neuropathic pain.

Neuropathic pain is caused by a lesion or disease to the somatosensory nervous system and current treatment merely reduces symptoms. Here, we investigate the potential therapeutic effect of the neurotrophic factor Meteorin on multiple signs of neuropathic pain in two distinct rat models. In a first study, two weeks of intermittent systemic administration of recombinant Meteorin led to a dose-dependent reversal of established mechanical and cold hypersensitivity in rats after photochemically-induced sciatic nerve injury. Moreover, analgesic efficacy lasted for at least one week after treatment cessation. In rats with a chronic constriction injury (CCI) of the sciatic nerve, five systemic injections of Meteorin over 9 days dose-dependently reversed established mechanical and thermal hypersensitivity as well as weight bearing deficits taken as a surrogate marker of spontaneous pain. The beneficial effects of systemic Meteorin were sustained for at least three weeks after treatment ended and no adverse side effects were observed. Pharmacokinetic analysis indicated that plasma Meteorin exposure correlated well with dosing and was no longer detectable after 24 hours. This pharmacokinetic profile combined with a delayed time of onset and prolonged duration of analgesic efficacy on multiple parameters suggests a disease-modifying mechanism rather than symptomatic pain relief. In sciatic nerve lesioned rats, delivery of recombinant Meteorin by intrathecal injection was also efficacious in reversing mechanical and cold hypersensitivity. Together, these data demonstrate that Meteorin represents a novel treatment strategy for the effective and long lasting relief from the debilitating consequences of neuropathic pain.

The regulatory role of microRNA-1187 in TNF-α-mediated hepatocyte apoptosis in acute liver failure.

In the current study, we aimed at elucidating the regulatory mechanisms through which microR-1187 (miR-1187) participates in hepatocyte apoptosis in acute liver failure (ALF). An ALF model was induced with D-galactosamine (D-GalN) plus lipopolysaccharide (LPS) in BALB/c mice. The hepatic miRNA expression profile was detected by microarray analysis and verified by quantitative real-time PCR (qRT-PCR). The possible underlying mechanism was investigated in vitro using an embryonic murine hepatocyte cell line (BNLCL2) and miR-1187 mimic. Caspase-8 protein was detected by Western blotting and cell apoptosis was assayed by flow cytometry. Hepatic miR-1187 was down-regulated in ALF mice based on microarray data (P<0.001) and verified by qRT-PCR (P<0.01). Target scan revealed that caspase-8 was the putative target of miR-1187. In an in vitro study, miR-1187 showed the highest up-regulation in BNLCL2 cells transfected with the miR-1187 mimic at a 50 nM concentration for 12 h compared with cells transfected with the non-specific mimic (P<0.001). miR-1187 was down-regulated (P<0.01) but caspase-8 mRNA (P<0.01) as well as protein (P<0.05) were up-regulated in the BNLCL2 cells treated with D-GalN/TNF. Furthermore, overexpressed miR-1187 reduced caspase-8 expression at both the mRNA and protein levels significantly (P<0.01 and P<0.05 respectively), and significantly attenuated the apoptotic rate of BNLCL2 cells (P<0.05). We show that miR-1187 regulates hepatocyte apoptosis by targeting caspase-8. miR-1187 may serve as a potential therapeutic target for the treatment of ALF.

Death receptor 6 negatively regulates oligodendrocyte survival, maturation and myelination.

Survival and differentiation of oligodendrocytes are important for the myelination of central nervous system (CNS) axons during development and crucial for myelin repair in CNS demyelinating diseases such as multiple sclerosis. Here we show that death receptor 6 (DR6) is a negative regulator of oligodendrocyte maturation. DR6 is expressed strongly in immature oligodendrocytes and weakly in mature myelin basic protein (MBP)-positive oligodendrocytes. Overexpression of DR6 in oligodendrocytes leads to caspase 3 (casp3) activation and cell death. Attenuation of DR6 function leads to enhanced oligodendrocyte maturation, myelination and downregulation of casp3. Treatment with a DR6 antagonist antibody promotes remyelination in both lysolecithin-induced demyelination and experimental autoimmune encephalomyelitis (EAE) models. Consistent with the DR6 antagoinst antibody studies, DR6-null mice show enhanced remyelination in both demyelination models. These studies reveal a pivotal role for DR6 signaling in immature oligodendrocyte maturation and myelination that may provide new therapeutic avenues for the treatment of demyelination disorders such as multiple sclerosis.

[The role of miRNA-122 expression during the acute liver failure in mice induced by D-GalN/LPS].

OBJECTIVE: To investigate the expression of miR-122 and its relationship with progression and development of acute liver failure in mice induced by D-GalN/LPS, and to explore new biomarker(s) for early diagnosis of acute liver failure. METHODS: BALB/C mice were randomly divided into four groups: the mice were given D-GalN (900 mg/kg body weight) and LPS (10 micog/kg body weight) intraperitoneally (i.p.) to construct the acute liver model; whereas the control groups were given D-GalN (900 mg/kg), LPS (10 microg/kg) and normal saline respectively. All biochemical and histological indexes were determined at 0, 1, 3, 5, 7 and 9 h respectively after administration. Real-time RT-PCR were used to detect the expression of miR-122 and pro-inflammatory cytokines, furthermore, the expression of miR-122 was verified by LNA (lock nucleic acid)-Northern-blot. ALT and AST levels were tested by biochemistry analyzer. Serum pro-inflammatory cytokine levels were tested by ELISA. RESULTS: The mortality rate was about 80% at 24h after D-GalN/LPS treatment, but no mortality was observed in the other three control groups. Liver special miRNA miR-122 was highly expressed in liver tissue of normal mice (ct is approximately equal to 14), it was up-regulated significantly (P = 0.013) at first hour after treatment then down-regulated according to the development of acute liver failure, the change was more obvious at 9 h (ct is approximately equal to 15, P = 0.002). ALT and AST levels increased obviously at 3h after treatment and reached peak at 7 hours then they were declined sharply. It was found that the expression of miR-122 was faster and more durable than ALT. Pro-inflammatory cytokines related to acute liver failure including TNFa and IL-6 were all up-regulated in serum as well as liver tissue (P less than 0.05). Correlation analysis showed that miR-122 had a negative correlation with ALT (correlation coefficients -0.505) and positive correlations with TNFa and IL-6 (correlation coefficients were 0.493 and 0.674 respectively). CONCLUSIONS: Liver-specific miR-122 supposed be a new marker molecule for early diagnosis of liver cells injury in the acute liver failure.

Effect of ribavirin and interferon beta on miRNA profile in the hepatitis C virus subgenomic replicon-bearing Huh7 cells.

Hepatitis C virus (HCV) infection is still a major global health issue despite decades of research. The liver-specific microRNA-122 (miR-122) can stimulate HCV replication/translation in vitro, indicating that miR-122 contributes to pathogenesis of HCV. However, it remains controversial whether interferon (IFN) inhibits HCV via modulating miR-122 expression. The underlying mechanism of ribavirin (RBV) in enhancing IFN treatment for HCV patients has yet to be explored. We investigated the relationship between miR-122 expression and anti-HCV activity of IFN beta in combination with RBV in vitro, due to difficulty accessing an HCV animal model. Upregulation of ISG54 mRNA or cytostatic effect was detected in Huh7 and HCV replicon cell lines in response to IFN beta or RBV stimulation, respectively. It was found that IFN beta and/or RBV suppressed miR-122 expression marginally, with a synergetic anti-HCV effect between IFN beta and RBV. Marginal modification of other miRNAs was also observed in these cell lines, using miRNA array following IFN beta and RBV treatment. Taken together, our data suggest that miRNAs are not crucial in anti-HCV action, following IFN beta and/or RBV stimulation in vitro.

MicroRNA155 is induced in activated CD4(+) T cells of TNBS-induced colitis in mice.

AIM: To investigate the expression of microRNA155 (miRNA155) in trinitrobenzene sulphonic acid (TNBS)-induced colitis and the relationship between miRNA155 and tumor necrosis factor (TNF) expressions. METHODS: In TNBS colitis mice, miRNA155 and TNF mRNA expressions were measured in colons and CD4(+) T cells of draining lymph nodes (LNs). CD4(+) T cells were cultured in vitro with or without anti-CD3/CD28 antibody, and the expressions of miRNA155 and TNF mRNA in cells and TNF concentration in culture media were examined. RESULTS: miRNA155 and TNF mRNA expressions in colons and in cells of LNs were significantly increased in TNBS colitis compared with controls. In TNBS colitis, miRNA155 and TNF mRNA expressions in CD4(+) T cells of LNs and TNF concentration in CD4(+) T cells culture media increased compared with controls. When cultured with anti-CD3/CD28 antibody, miRNA155 and TNF mRNA expressions in CD4(+) T cells and TNF concentration in the CD4(+) T cells culture media were significantly higher than those cultured without anti-CD3/CD28 antibody. Following analysis using the Pearson's correlation coefficient, miRNA155 expression had a significant positive correlation with either TNF mRNA expression in CD4(+) T cells (r = 0.860, P < 0.05) or TNF concentration in CD4(+) T cells culture media (r = 0.892, P < 0.05). CONCLUSION: miRNA155 is induced in colons and activated CD4(+) T cells in TNBS colitis, and the levels of miRNA155 and TNF expressions have a significant positive correlation.

[Real-time quantification of microRNAs in Huh7 cells by stem-loop reverse transcriptase polymerase chain reaction].

OBJECTIVE: To establish a convenient realtime PCR which can detect microRNAs in the human hepatoma cell line, Huh7 cells. METHODS: Total RNAs in Huh7 cells were extracted. MicroRNA 122, 24 and 146a were assayed by microRNA array, and then verified by Northern blot. Stem-loop RT-PCR and poly(A)-tailed RT-PCR were used to detect the above microRNAs. Data were analyzed with Quantity One software and 7500 system software. RESULTS: Microarray signal intensity of microRNA 122, 24 and 146a in Huh7 cells was 2201.49, 410.20 and 4.70, whose relative expression was confirmed as 0.0383, 0.0249, 0.0001 through Northern blot. While the poly(A)-tailed RT-PCR might only measure microRNA 122, Stem-loop RT-PCR could detect microRNA 122, 24 and 146a, whose average dCt was 2.5, 5.8 and 12.1 in accordance with microRNA array and Northern blot. CONCLUSION: Stem-loop RT-PCR can specifically and sensitively quantity microRNA levels, regardless of their abundance.