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Endoglucanase secreted by the fungus Trichoderma atroviride is a kind of cellulase. An endoglucanase gene egII was cloned from T. atroviride AS3.3013 and expressed in Saccharomyces cerevisiae INVScI. The open reading frame of the egII gene was composed of 1257 bp, encoding 418 amino acids with a molecular weight of 44.23 kDa plus a signal peptide of 21 amino acids. Based on sequence similarity, TaEGII belonged to the glycosyl hydrolase family 5. Expression of the egII gene in T. atroviride AS3.3013 can be induced by microcrystalline cellulose (MCC), bran, carboxymethyl cellulose (CMC), rice straw, and corn stalk but is inhibited by glucose. A highly efficient integrated expression vector (pYPIGH-B includes a sequence of the α-mating factor signal peptide (MF-α)) was constructed. The enzymatic activity of the supernatant of recombinant yeast YPIGH-B3 was 1.29 times higher than that of YES2-egII, demonstrating that the MF-α can significantly improve the expression of the recombinant EGII in S. cerevisiae. The recombinant endoglucanase TaEGII produced by S. cerevisiae showed maximum activity at pH 5.0 and temperature 60 °C. Under these conditions, the Km and Kcat values for Avicel and raffinose hydrolysis were 1.22 × 10 -2  mg ml -1 , 9.09 × 10 -2  s -1 and 1.06 × 10 -2  mg ml -1 , 9.18 × 10 -2  s -1 , respectively. The enzymatic activity of recombinant TaEGII was stable when incubated from 40 to 60 °C for 1 h. It was stable in a wide range of pH (4.0-7.0) and sensitive to various metal ions. Transgenic yeast strain YPIGH-B3 might be applied to cellulosic ethanol production.

Purpose: To investigate the intestinal inflammatory response and the abundance of intestinal bacteria in rats with high-fat diet (HFD)-induced nonalcoholic fatty liver disease (NAFLD) and assess the intervention effects of taurine (TAU). Methods: Forty male Sprague-Dawley rats were randomly divided into five groups: group I, normal diet and normal saline gavage; group II, normal diet and TAU gavage; group III, HFD and normal saline gavage; group IV, HFD and TAU gavage (from the 1st week); group V, HFD and TAU gavage (from the 10th week). At the end of the 16th week, all the animals were sacrificed. Body weight, liver weight, liver function, and serum lipid levels were measured. The histopathologies of the liver and ileum were observed. The mRNA and protein expression levels of interleukin 17 (IL-17) and IL-10 in the ileum were detected by reverse transcription quantitative polymerase chain reaction (qPCR) and immunohistochemistry. Three types of bacteria were detected in intestinal feces using the 16S rDNA qPCR method. Results: The ileal IL-17 level in group III was significantly higher than those in the other four groups (P < 0.01). The ileal IL-10 mRNA levels in group IV was significantly higher than those in groups III and V (P < 0.05), and IL-10 protein MOD levels in group III was significantly lower than those in the other four groups (P < 0.01). The numbers of Lactobacillus in group III were significantly lower than those in the other four groups (P < 0.01 or P < 0.05). The numbers of Bifidobacteria in groups IV and V were significantly increased compared with that in group III (P < 0.05). Conclusion: TAU may down-regulate the expression of IL-17, up-regulate the expression of IL-10 and regulate the intestinal flora, and alleviate the liver and intestinal damage in rats with HFD-induced NAFLD. [ABSTRACT FROM AUTHOR]

We have identified that NUDT21 plays a vital role in MDS transformations, while the transcription factor RUNX1 is essential for normal hematopoiesis, which is a high expression in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), and we aim to explore the linkage between the two genes and new pathways for MDS transformation to AML. Prediction of RUNX1 expression levels and its relationship with NUDT21 in AML and MDS patients was performed using bioinformatics techniques and validated in patients. Using lentiviral packaging technology, NUDT21 knockdown and overexpression models were developed in AML and MDS cell lines. These models were validated using quantitative polymerase chain reaction (qPCR) and western blotting. The cell cycle, apoptosis, differentiation, and cytokines were examined by flow cytometry, CCK-8 analyzed proliferation, and the intracellular localization of NUDT21 and RUNX1 was examined by immunofluorescence. mRNA transcriptome sequencing was performed on THP-1, MUTZ-1, and Dapars analyzed SKM-1 cell lines and the sequencing data to observe the knockdown effect of NUDT21 on RUNX1. qPCR and western blot revealed a positive correlation between NUDT21 and RUNX1; both were located in the nucleus. Overexpression of NUDT21 reduced apoptosis, promoted cell proliferation, and possibly increased the invasive ability of cells. It also altered the APA site in the RUNX1 3'-UTRs region. NUDT21 regulates RUNX1 gene expression and promotes AML transformation in MDS through an APA mechanism. [ABSTRACT FROM AUTHOR]

A talented endophytic bacteria strain YINM00001, which showed strong antimicrobial activity and multiple antibiotic resistances, was isolated from a Chinese medicinal herb Peperomia dindygulensis Miq. Phylogenetic analysis based on 16S rRNA gene sequences demonstrated that strain was closely related to Streptomyces anulatus NRRL B-2000T (99.93%). The complete genome of strain YINM00001 was sequenced. The RAxML phylogenomic tree also revealed that strain YINM00001 was steadily clustered on a branch with strain Streptomyces anulatus NRRL B-2000T under the 100 bootstrap values. The complete genome of strain YINM00001 consists of an 8,372,992 bp linear chromosome (71.72 mol% GC content) and a 317,781 bp circular plasmid (69.14 mol% GC content). Genome mining and OSMAC approach were carried out to investigate the biosynthetic potential of producing secondary metabolites. Fifty-two putative biosynthetic gene clusters of secondary metabolites were found, including the putative cycloheximide, dinactin, warkmycin, and anthracimycin biosynthetic gene clusters which consist with the strong antifungal and antibacterial activities exhibited by strain YINM00001. Two new compounds, peperodione (1) and peperophthalene (2), and 17 known compounds were isolated from different fermentation broth. Large amounts and high diversity of antimicrobial and/or anticancer compounds cycloheximide, dinactin, anthracimycin, and their analogs had been found as predicted before, which highlights strain YINM00001 as an ideal candidate for further biosynthetic studies and production improvement of these valuable compounds. Meanwhile, several gene clusters that were highly conserved in several sequenced actinomycetes but significantly different from known gene clusters might be silent under proceeding fermentation conditions. Further studies, such as heterologous expression and genetic modification, are needed to explore more novel compounds from this talented endophytic Streptomyces strain. [ABSTRACT FROM AUTHOR]

The ATP-binding cassette (ABC) transporters have crucial roles in various biological processes such as growth, development and immune defense in eukaryotes. However, the roles of ABC transporters in the immune system of crustaceans remain elusive. In this study, 38 ABC genes were systematically identified and characterized in Penaeus vannamei. Bioinformation analysis revealed that PvABC genes were categorized into ABC A-H eight subfamilies with 17 full-transporters, 11 half transporters and 10 soluble proteins, and multiple immunity-related cis-elements were found in gene promoter regions. Expression analysis showed that most PvABC genes were widely and highly expressed in immune-related tissues and responded to the stimulation of Vibrio parahaemolyticus. To investigate whether PvABC genes mediated innate immunity, PvABCC5 , PvABCF1 and PvABCB4 were selected for dsRNA interference experiment. Knockdown of PvABCF1 and PvABCC5 not PvABCB4 increased the cumulative mortality of P. vannamei and bacterial loads in hepatopancreas after infection with V. parahaemolyticus. Further analysis showed that the PvABCF1 and PvABCC5 knockdown decreased expression levels of NF-κB pathway genes and antimicrobial peptides (AMPs). Collectively, these findings indicated that PvABCF1 and PvABCC5 might restrict V. parahaemolyticus challenge by positively regulating NF-κB pathway and then promoting the expression of AMPs, which would contribute to overall understand the function of ABC genes in innate immunity of invertebrates. • A total of 38 ABC genes were systematically identified and characterized in P. vannamei genome. • Most PvABC genes were ubiquitously expressed in all examined tissues with high expression in hemocytes, gill, hepatopancreas and intestine. • The mRNA expression of selected PvABC genes was obviously altered after V. parahemolyticus challenge. • PvABCC5 and PvABCF1 knockdown increased shrimp mortality and the bacteria loads in the hepatopancreas upon V. parahemolyticus challenge. • PvABCC5 and PvABCF1 might restrict V. parahaemolyticus challenge by positively regulating NF-κB pathway, and then promoting the expression of AMPs in P. vannamei. [ABSTRACT FROM AUTHOR]

Bacterial intestinal inflammation is a common disease of aquatic animal in high-density aquaculture. This study aims to construct a model of intestinal inflammation in northern snakehead, and analyze the intestinal transcriptome after Aeromonas hydrophila (A. hydrophila) infection by high-throughput sequencing technologies (RNA-seq), in order to obtain candidate genes and regulatory pathways that may participate in inflammatory response. Northern snakehead [(13.05 ± 2.39) g] were infected through anal for 24 h with different concentrations of A. hydrophila (1.0 × 10−4, 1.0 × 10−5, 1.0 × 10−6, 1.0 × 10−7 and 1.0 × 10−8 CFU/mL). Intestinal histology observation showed that infection with A. hydrophila caused obvious inflammatory responses in intestine, especially in hindgut. The results also indicated that intestinal permeability related parameters (D-LA and Na+-K+-ATP), immune-related parameters (C3, LZM, AKP, MPO, IL-1β and TNF-α) and immune-related gene expression (il-1β and tnf-α) were significantly changed compared to the CK group. In this process, high concentration of A. hydrophila infection caused the death of snakehead. On the basis of the above evaluation, we successfully constructed a model of intestinal inflammation induced by A. hydrophila in northern snakehead, and the optimal dosage of A. hydrophila was 1.0 × 10−6 CFU/mL. Subsequently, the constructed model was analyzed on the Illumina Genome Analyser II platform for intestinal transcriptome. Results showed that a total of 370,492,420 clean reads, 145,236 transcripts and 89,431 unigenes were obtained. Moreover, a total of 7566 differentially expressed genes (DEGs) were found, including 4486 upregulated genes and 3080 downregulated genes. Notably, the key pathways [Toll-like receptors (TLRs) and NOD-like receptors (NLRs)] and genes [ tlr1 , tlr2 , tlr5 , nf-κb , tnf-α , il-1β , il-10 , nod1 ] related to inflammation were discovered. These results revealed the response of northern snakehead to A. hydrophila at the transcriptional level, which provide insights for the regulatory mechanisms of intestinal inflammation in northern snakehead. • A model of intestinal inflammation in northern snakehead was constructed by Aeromonas hydrophila. • The optimal dosage of Aeromonas hydrophila for inducing intestinal inflammation in northern snakehead was 1.0 × 10−6 CFU/mL. • The key pathways and genes related to inflammation were discovered by high-throughput sequencing technologies. [ABSTRACT FROM AUTHOR]

Downregulation of PDZK1 by TGF-β1 via p38 MAPK and PI3K/AKT pathways promotes EMT upon oxidative stress. Inhibition of p38/JNK MAPK and PI3K/AKT pathways upregulated PDZK1 expression and attenuated renal fibrosis. [Display omitted] Transforming growth factor-beta 1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) of renal tubular cells promotes renal fibrosis and the progression of chronic kidney disease (CKD). PDZ domain-containing 1 (PDZK1) is highly expressed in renal tubular epithelial cells; however, its role in TGF-β1-induced EMT remains poorly understood. The present study showed that PDZK1 expression was extremely downregulated in fibrotic mouse kidneys and its negative correlation with TGF-β1 expression and the degree of renal fibrosis. In addition, TGF-β1 downregulated the mRNA expression of PDZK1 in a time- and concentration-dependent manner in vitro. The downregulation of PDZK1 exacerbated TGF-β1-induced EMT upon oxidative stress, while the overexpression of PDZK1 had the converse effect. Subsequent investigations demonstrated that TGF-β1 downregulated PDZK1 expression via p38 MAPK or PI3K/AKT signaling in vitro , but independently of ERK/JNK MAPK signaling. Meanwhile, inhibition of the p38/JNK MAPK or PI3K/AKT signaling using chemical inhibitors restored the PDZK1 expression, mitigated renal fibrosis, and elevated renal levels of endogenous antioxidants carnitine and ergothioneine in adenine-induced CKD mice. These findings provide the first evidence suggesting a negative correlation between PDZK1 and renal fibrosis, and identifying PDZK1 as a novel suppressor of renal fibrosis in CKD through ameliorating oxidant stress. [ABSTRACT FROM AUTHOR]

To obtain transcriptomic insights into branchial responses to salinity challenge in Anabas testudineus, this study employed RNA sequencing (RNA‐Seq) to analyse the gill transcriptome of A. testudineus exposed to seawater (SW) for 6 days compared with the freshwater (FW) control group. A combined FW and SW gill transcriptome was de novo assembled from 169.9 million 101 bp paired‐end reads. In silico validation employing 17 A. testudineus Sanger full‐length coding sequences showed that 15/17 of them had greater than 80% of their sequences aligned to the de novo assembled contigs where 5/17 had their full‐length (100%) aligned and 9/17 had greater than 90% of their sequences aligned. The combined FW and SW gill transcriptome was mapped to 13,780 unique human identifiers at E‐value ≤1.0E‐20 while 952 and 886 identifiers were determined as up and down‐regulated by 1.5 fold, respectively, in the gills of A. testudineus in SW when compared with FW. These genes were found to be associated with at least 23 biological processes. A larger proportion of genes encoding enzymes and transporters associated with molecular transport, energy production, metabolisms were up‐regulated, while a larger proportion of genes encoding transmembrane receptors, G‐protein coupled receptors, kinases and transcription regulators associated with cell cycle, growth, development, signalling, morphology and gene expression were relatively lower in the gills of A. testudineus in SW when compared with FW. High correlation (R = 0.99) was observed between RNA‐Seq data and real‐time quantitative PCR validation for 13 selected genes. The transcriptomic sequence information will facilitate development of molecular resources and tools while the findings will provide insights for future studies into branchial iono‐osmoregulation and related cellular processes in A. testudineus. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effect and potential mechanisms of rutaecarpine (Rut) in a rat artery balloon-injury model.Methods: The intimal hyperplasia model was established by rubbing the endothelia with a balloon catheter in the common carotid artery (CCA) of rats. Fifty rats were randomly divided into five groups, ie. sham, model, Rut (25, 50 and 75 mg/kg) with 10 rats of each group. The rats were treated with or without Rut (25, 50, 75 mg/kg) by intragastric administration for 14 consecutive days following injury. The morphological changes of the intima were evaluated by hematoxylin-eosin staining. The expressions of proliferating cell nuclear antigen (PCNA) and smooth muscle (SM) α-actin in the ateries were assayed by immunohistochemical staining. The mRNA expressions of c-myc, extracellular signal-regulated kinase 2 (ERK2), MAPK phosphatase-1 (MKP-1) and endothelial nitric oxide synthase (eNOS) were determined by real-time reverse transcription-polymerase chain reaction. The protein expressions of MKP-1 and phosphorylated ERK2 (p-ERK2) were examined by Western blotting. The plasma contents of nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP) were also determined.Results: Compared with the model group, Rut treatment significantly decreased intimal thickening and ameliorated endothelial injury (P<0.05 or P<0.01). The positive expression rate of PCNA was decreased, while the expression rate of SM α-actin obviously increased in the vascular wall after Rut (50 and 75 mg/kg) administration (P<0.05 or P<0.01). Furthermore, the mRNA expressions of c-myc, ERK2 and PCNA were downregulated while the expressions of eNOS and MKP-1 were upregulated (P<0.05 or P<0.01). The protein expressions of MKP-1 and the phosphorylation of ERK2 were upregulated and downregulated after Rut (50 and 75 mg/kg) administration (P<0.05 or P<0.01), respectively. In addition, Rut dramatically reversed balloon injury-induced decrease of NO and cGMP in the plasma (P<0.05 or P<0.01).Conclusion: Rut could inhibit the balloon injury-induced carotid intimal hyperplasia in rats, possibly mediated by promotion of NO production and inhibiting ERK2 signal transduction pathways. [ABSTRACT FROM AUTHOR]

African lungfishes are ammonotelic in water. They can aestivate for long periods on land during drought. During aestivation, the gills are covered with dried mucus and ammonia excretion ceases. In fishes, ammonia excretion through the gills involves Rhesus glycoproteins (RhGP/Rhgp). This study aimed to obtain the complete cDNA coding sequences of rhgp from the gills of Protopterus annectens, and to determine their branchial mRNA and protein expression levels during the induction, maintenance and arousal phases of aestivation. Three isoforms of rhgp (rhag, rhbg and rhcg) were obtained in the gills of P. annectens. Their complete cDNA coding sequences ranged between 1311 and 1398 bp, coding for 436 to 465 amino acids with estimated molecular masses between 46.8 and 50.9 kDa. Dendrogramic analyses indicated that Rhag was grouped closer to fishes, while Rhbg and Rhcg were grouped closer to tetrapods. During the induction phase, the protein abundance of Rhag, but not its transcript level, was down-regulated in the gills, suggesting that there could be a decrease in the release of ammonia from the erythrocytes to the plasma. Furthermore, the branchial transcript levels of rhbg and rhcg decreased significantly, in preparation for the subsequent shutdown of gill functions. During the maintenance phase, the branchial expression levels of rhag/Rhag, rhbg/Rhbg and rhcg/Rhcg decreased significantly, indicating that their transcription and translation were down-regulated. This could be part of an overall mechanism to shut down branchial functions and save metabolic energy used for transcription and translation. It could also be regarded as an adaptive response to stop ammonia excretion. During the arousal phase, it is essential for the lungfish to regain the ability to excrete ammonia. Indeed, the protein abundance of Rhag, Rhbg and Rhcg recovered to the corresponding control levels after 1 day or 3 days of recovery from 6 months of aestivation. [ABSTRACT FROM AUTHOR]

Vascular calcification is an active process of osteoblastic differentiation of vascular smooth muscle cells; however, its definite mechanism remains unknown. Vinpocetine, a derivative of the alkaloid vincamine, has been demonstrated to inhibit the high glucose-induced proliferation of vascular smooth muscle cells; however, it remains unknown whether vinpocetine can affect the osteoblastic differentiation of vascular smooth muscle cells. We hereby investigated the effect of vinpocetine on vascular calcification using a beta-glycerophosphate-induced cell model. Our results showed that vinpocetine significantly reduced the osteoblast-like phenotypes of vascular smooth muscle cells including ALP activity, osteocalcin, collagen type I, Runx2 and BMP-2 expression as well as the formation of mineralized nodule. Vinpocetine, binding to translocation protein, induced phosphorylation of extracellular signal-related kinase and Akt and thus inhibited the translocation of nuclear factor-kappa B into the nucleus. Silencing of translocator protein significantly attenuated the inhibitory effect of vinpocetine on osteoblastic differentiation of vascular smooth muscle cells. Taken together, vinpocetine may be a promising candidate for the clinical therapy of vascular calcification. [ABSTRACT FROM AUTHOR]

Purpose: Chemo-resistance is the leading cause of failure in cancer therapy, however, much remains to be understood about the intrinsic mechanisms. In the present study, we discovered the novel miR-4443 that regulated malignancy of breast cancer both in vitro and in vivo. Methods: We examined the expression of miR-4443 in MDA-MB-231/S and MDA-MB-231 Epirubicin-resistant cell lines with 76 breast cancer formalin-fixed paraffin-embedded tissues by real-time PCR. Also, we investigated the loss- and gain-functions of miR-4443 by MTT assay and flow cytometry. Furthermore, we detected miR-4443 mediated tissue inhibitor of metalloproteinase 2 expression in cells by TargetScan, RT-qPCR and western blot. Results: We identified the up-regulated expression of miR-4443 in Epi-resistant cell lines versus MDA-MB-231/S cell(Epi versus S) and in post-chemotherapy FFPE tissues, along with statistically differential expressions in PR(partial response) versus SD(stable disease)/PD(progressive disease) patients. Overexpression of miR-4443 increased the IC50 value of Epi for the target cells transfected, while inhibition of miR-4443 could restored sensitivity of the target cells to Epi. Besides, down-regulation of endogenous miR-4443 by miRNA-inhibitors significantly enhanced Epi-induced apoptosis while up-regulation of miR-4443 by miRNA-mimics lead to less Epi-induced apoptotic cells. Consequently, changes in TIMP2 mRNA and protein expression revealed that miR-4443 mimics suppressed expression of TIMP2 and induced migration in breast cancer cells. Furthermore, TIMP2 expression associated with better prognosis(HR = 0.721, 95%CI: 0.529–0.983). Conclusions: We revealed that miR-4443 induced malignancy of breast cancer mainly in chemo-resistance aspect for the very first time, providing a novel biomarker in breast cancer diagnosis and therapy. [ABSTRACT FROM AUTHOR]

Acquisition of resistance to docetaxel (Doc) is one of the most important problems in treatment of breast cancer patients, but the underlying mechanisms are still not fully understood. In present study, Doc-resistant MDA-MB-231 and MCF-7 breast cancer cell lines (MDA-MB-231/Doc and MCF-7/Doc) were successfully established in vitro by gradually increasing Doc concentration on the basis of parental MDA-MB-231 and MCF-7 cell lines (MDA-MB-231/S and MCF-7/S). The potential miRNAs relevant to the Doc resistance were screened by miRNA microarray. We selected 5 upregulated miRNAs (has-miR-3646, has-miR-3658, has-miR-4438, has-miR-1246, and has-miR-574-3p) from the results of microarray for RT-qPCR validation. The results showed that expression level of miR-3646 in MDA-MB-231/Doc cells was significantly higher than in MDA-MB-231/S cells. Compared to MCF-7/S cells, miR-3646 expression was up-regulated in MCF-7/Doc cells. Further studies revealed that transfection of miR-3646 mimics into MDA-MB-231/S or MCF-7/S cells remarkably increased their drug resistance, in contrast, transfection of miR-3646 inhibitors into MDA-MB-231/Doc or MCF-7/Doc cells resulted in significant reduction of the drug resistance. By the pathway enrichment analyses for miR-3646, we found that GSK-3β/β-catenin signaling pathway was a significant pathway, in which GSK-3β was an essential member. RT-qPCR and Western blot results demonstrated that miR-3646 could regulate GSK-3β mRNA and protein expressions. Furthermore, a marked increase of both nuclear and cytoplasmic β-catenin expressions (with phosphorylated-β-catenin decrease) was observed in MDA-MB-231/Doc cells compared with MDA-MB-231/S cells, and their expression were positively related to miR-3646 and negatively correlated with GSK-3β. Taken together, our results suggest that miR-3646-mediated Doc resistance of breast cancer cells maybe, at least in part, through suppressing expression of GSK-3β and resultantly activating GSK-3β/β-catenin signaling pathway. [ABSTRACT FROM AUTHOR]

This study was undertaken to isolate active secondary metabolites from immunostimulatory Bacillus Licheniformis XY-52 and evaluate their activities at 0%, 0.5%, 1.0%, and 2.0% doses supplementation with feed on immune response in common carp at weeks 1, 2, and 3. By applying chromatography techniques and successive recrystallization, two purified metabolites were obtained and identified by spectral data (mass spectrometry and nuclear magnetic resonance) as: Cyclo-(Phe–Tyr) and Cyclo-(Phe–Gly). The results revealed that humoral innate immune parameters (lysozyme activity, phagocytic activity and bactericidal activity) were significantly (P < 0.05) increased after feeding on the two active compounds-supplemented diet. Furthermore, administration of the two active compounds significantly (P < 0.05) up regulated IL-1β, Type 1 IFN, IFN g2b, IL10 and TNF-α gene expression. Heat shock protein 70 (HSP70) gene expression was significantly (P < 0.05) lower as compared to control group at the end of trial. Common carp fed with the two compounds had higher survival rates (69.3%) compared to the controls (32.0%) after challenged with the pathogen, Aeromonas hydrophila . The present study indicates that the two isolated active compounds could positively influence immune response and enhance disease resistance of common carp against A. hydrophila infection. [ABSTRACT FROM AUTHOR]

Argininosuccinate synthase (Ass) and argininosuccinate lyase (Asl) are involved in arginine synthesis for various purposes. The complete cDNA coding sequences of ass and asl from the liver of Protopterus annectens consisted of 1,296 and 1,398 bp, respectively. Phylogenetic analyses revealed that the deduced Ass and Asl of P. annectens had close relationship with that of the cartilaginous fish Callorhinchus milii. Besides being strongly expressed in the liver, ass and asl expression were detectable in many tissues/organs. In the liver, mRNA expression levels of ass and asl increased significantly during the induction phase of aestivation, probably to increase arginine production to support increased urea synthesis. The increases in ass and asl mRNA expression levels during the prolonged maintenance phase and early arousal phase of aestivation could reflect increased demand on arginine for nitric oxide (NO) production in the liver. In the kidney, there was a significant decrease in ass mRNA expression level after 6 months of aestivation, indicating possible decreases in the synthesis and supply of arginine to other tissues/organs. In the brain, changes in ass and asl mRNA expression levels during the three phases of aestivation could be related to the supply of arginine for NO synthesis in response to conditions that resemble ischaemia and ischaemia-reperfusion during the maintenance and arousal phase of aestivation, respectively. The decrease in ass mRNA expression level, accompanied with decreases in the concentrations of arginine and NO, in the skeletal muscle of aestivating P. annectens might ameliorate the potential of disuse muscle atrophy. [ABSTRACT FROM AUTHOR]

18β-glycyrrhetinic acid (18β-GA) is a bioactive component of licorice. The anti-cancer activity of 18β-GA has been studied in many cancer types, whereas its effects in lung cancer remain largely unknown. We first showed that 18β-GA effectively suppressed cell proliferation and inhibited expression as well as activity of thromboxane synthase (TxAS) in non-small cell lung cancer (NSCLC) cells A549 and NCI-H460. In addition, the administration of 18β-GA did not have any additional inhibitory effect on the decrease of cell proliferation induced by transfection with TxAS small interference RNA (siRNA). Moreover, 18β-GA failed to inhibit cell proliferation in the immortalized human bronchial epithelial cells 16HBE-T and another NSCLC cell line NCI-H23, both of which expressed minimal level of TxAS as compared to A549 and NCI-H460. However, 18β-GA abolished the enhancement of cell proliferation induced by transfection of NCI-H23 with pCMV6-TxAS plasmid. Further study found that the activation of both extracellular signal-regulated kinase (ERK)1/2 and cyclic adenosine monophosphate response element binding protein (CREB) induced by TxAS cDNA transfection could be totally blocked by 18β-GA. Altogether, we have delineated that, through inhibiting TxAS and its initiated ERK/CREB signaling, 18β-GA suppresses NSCLC cell proliferation. Our study has highlighted the significance of 18β-GA with respect to prevention and treatment of NSCLC. [ABSTRACT FROM AUTHOR]

Background/Aims: The effects of glycyrrhizin treatment in lung cancer remain undetermined, despite extensive studies of the anti-tumor activities of glycyrrhizin. Methods: Lung adenocarcinoma A549 and NCI-H23 cell lines were used in this study. Cell growth was examined by MTS assays, while apoptosis and cell cycle were determined by flow cytometric analysis. Both real-time PCR and western blotting were used to examine the expression levels of thromboxane synthase (TxAS), and TxAS activity was measured using EIA detection of the biosynthesis of TxA2. TxAS was overexpressed in NCI-H23 cells by transfection with TxAS cDNA, while TxAS was inhibited by transfection with TxAS siRNA in A549 cells. For the mouse model of lung adenocarcinoma, the effects of glycyrrhizin on tumor growth were analyzed by western blot evaluation of TxAS, PTEN and survivin. TxAS activity was determined by EIA assay. Results: Glycyrrhizin suppressed cell growth in A549 cells, but not in NCI-H23 cells, by induction of apoptosis. TxAS was overexpressed in A549 cells, but the TxAS levels in NCI-H23 cells were minimal. Moreover, TxAS expression and activity were suppressed by glycyrrhizin. Glycyrrhizin had no additive effects with TxAS siRNA knockdown in suppressing A549 cell growth, whereas it completely suppressed cell growth of NCI-H23 cells transfected with TxAS cDNA. These results were further confirmed by the in vivo study. Conclusion: Our study suggests that the anti-tumor effect of glycyrrhizin in lung adenocarcinoma is, at least in part, TxAS-dependent. Therefore, glycyrrhizin is a promising anti-cancer agent for the treatment of lung adenocarcinoma. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The swamp eel, Monopterus albus, can survive in high concentrations of ammonia (>75 mmol l−1) and accumulate ammonia to high concentrations in its brain (∼4.5 µmol g−1). Na+/K+-ATPase (Nka) is an essential transporter in brain cells, and since NH4+ can substitute for K+ to activate Nka, we hypothesized that the brain of M. albus expressed multiple forms of Nka α-subunits, some of which might have high K+ specificity. Thus, this study aimed to clone and sequence the nka α-subunits from the brain of M. albus, and to determine the effects of ammonia exposure on their mRNA expression and overall protein abundance. The effectiveness of NH4+ to activate brain Nka from M. albus and Mus musculus was also examined by comparing their Na+/K+-ATPase and Na+/NH4+-ATPase activities over a range of K+/NH4+ concentrations. The full length cDNA coding sequences of three nkaα (nkaα1, nkaα3a and nkaα3b) were identified in the brain of M. albus, but nkaα2 expression was undetectable. Exposure to 50 mmol l−1 NH4Cl for 1 day or 6 days resulted in significant decreases in the mRNA expression of nkaα1, nkaα3a and nkaα3b. The overall Nka protein abundance also decreased significantly after 6 days of ammonia exposure. For M. albus, brain Na+/NH4+-ATPase activities were significantly lower than the Na+/K+-ATPase activities assayed at various NH4+/K+ concentrations. Furthermore, the effectiveness of NH4+ to activate Nka from the brain of M. albus was significantly lower than that from the brain of M. musculus, which is ammonia-sensitive. Hence, the (1) lack of nkaα2 expression, (2) high K+ specificity of K+ binding sites of Nkaα1, Nkaα3a and Nkaα3b, and (3) down-regulation of mRNA expression of all three nkaα isoforms and the overall Nka protein abundance in response to ammonia exposure might be some of the contributing factors to the high brain ammonia tolerance in M. albus. [ABSTRACT FROM AUTHOR]

Objective: In this article, the effects of CD33 expression from peripheral blood granulocytes and monocytes on inflammatory response in chronic obstructive pulmonary disease (COPD) were examined. Methods: CD33 and CD64 expression on peripheral blood granulocytes and monocytes from patients with acute exacerbation of chronic obstructive pulmonary disease and healthy control were detected by flow cytometry. To evaluate the effect of CD33 on inflammation immunity in COPD, the correlation between CD33 expression and CD64 expression, as well as inflammatory indices were investigated. Results: We found that the expression of CD64 on granulocytes and monocytes, as well as the levels of C-reacting protein (CRP) and myoglobin (MYO) or the proportion of neutrophils (N%) significantly increased in COPD patients compared to normal control group ( p < 0.01). The expression of CD33 on granulocytes was significantly and negatively correlated with the level of CRP ( r = −0.311, p = 0.012), as well as to the level of MYO ( r = −0.295. p = 0.018). Conclusions: The granulocytes and monocytes in peripheral blood were activated in patients of acute exacerbation in COPD. CD33 on granulocytes had the potential to inhibit inflammation and prevent tissue damage. [ABSTRACT FROM AUTHOR]

We obtained a full cDNA coding sequence of aquaporin 1aa (aqp1aa) from the gills of the freshwater climbing perch, Anabas testudineus, which had the highest expression in the gills and skin, suggesting an important role of Aqp1aa in these organs. Since seawater acclimation had no significant effects on the branchial and intestinal aqp1aa mRNA expression, and since the mRNA expression of aqp1aa in the gut was extremely low, it can be deduced that Aqp1aa, despite being a water channel, did not play a significant osmoregulatory role in A. testudineus. However, terrestrial exposure led to significant increases in the mRNA expression of aqp1aa in the gills and skin of A. testudineus. Since terrestrial exposure would lead to evaporative water loss, these results further support the proposition that Aqp1aa did not function predominantly for the permeation of water through the gills and skin. Rather, increased aqp1aa mRNA expression might be necessary to facilitate increased ammonia excretion during emersion, because A. testudineus is known to utilize amino acids as energy sources for locomotor activity with increased ammonia production on land. Furthermore, ammonia exposure resulted in significant decreases in mRNA expression of aqp1aa in the gills and skin of A. testudineus, presumably to reduce ammonia influx during ammonia loading. This corroborates previous reports on AQP1 being able to facilitate ammonia permeation. However, a molecular characterization of Aqp1aa from A. testudineus revealed that its intrinsic aquapore might not facilitate NH3 transport. Hence, ammonia probably permeated the central fifth pore of the Aqp1aa tetramer as suggested previously. Taken together, our results indicate that Aqp1aa might have a greater physiological role in ammonia excretion than in osmoregulation in A. testudineus. [ABSTRACT FROM AUTHOR]

Objective: To investigate the immunologic mechanism by which low-dose mifepristone serves as an anti-implantation contraceptive drug. Design: In vitro study. Setting: University hospital and research laboratory. Patient(s): Fifteen normally cycling patients at the “window of implantation.” Intervention(s): A human endometrial explant system was used to study the effects of low-dose mifepristone (65 and 200 nmol/L) on uterine natural killer (uNK) cells. Endometrial samples were treated with different concentrations of mifepristone. Main Outcome Measure(s): The cytotoxicity of uNK cells to K562 target cells and the expression of perforin (PFN) by uNK cells were examined using a methyl thiazolyl tetrazolium (MTT) assay and double immunohistochemistry, respectively. Result(s): Both uNK cell cytotoxicity and expression of PFN were increased after treatment with 65 or 200 nmol/L mifepristone, and these effects were dose-dependent. Conclusion(s): Mifepristone may negatively regulate implantation by increasing the cytotoxicity of uNK cells, and this increased cytotoxicity may result from increased PFN expression. These findings provide further evidence to support the potential contraceptive activity of low-dose mifepristone. [Copyright &y& Elsevier]

Objective: To investigate the effect of mifepristone on peripheral blood natural killer cell''s (pbNK) cytotoxicity and the expression of the inhibitory receptor CD94/NKG2A and the activated receptor NKG2D on pbNK cells. Design: In vitro study. Setting: University hospital and research laboratory. Patient(s): Twenty healthy nonpregnant women. Intervention(s): Detected the cytolytic activity of pbNK to K562 target cells; measured the expression of CD94/NKG2A and NKG2D on pbNK. Main Outcome Measure(s): Cytotoxicity of pbNK was detected by Methyl thiazolyl tetrazolium. The expression of CD94/NKG2A and NKG2D receptor on pbNK cells were detected by flow cytometry. Result(s): The NK cell cytotoxicity and the expression of inhibitory receptor CD94/NKG2A during the proliferative phase (81.71 ± 11.5, 86.6 ± 9.0) was significantly higher than the secretory phase (60.16 ± 19.2, 60.15 ± 31.0). The NK cells cytotoxicity, after being treated with mifepristone and the expression of inhibitory receptor CD94/NKG2A on pbNK cells treated with 200 nmol/L mifepristone, were significantly increased. Mifepristone had no effect on the expression of activating receptor NKG2D. Conclusion(s): These data suggest that Mifepristone maybe exert its anti-implantation function by increasing NK cytotoxicity. The increasing NK cytotoxicity of mifepristone is not related to CD94/NKG2A and NKG2D. In the secretory phase down-regulated CD94/NKG2A, NKG2D, and NK cytotoxicity may benefit with embryo implantation. [Copyright &y& Elsevier]

AbstractPurpose:The aim of this study was to evaluate the suppression effect of survivin shRNA on the expression of the survivin gene in the human laryngeal cancer cell line Hep-2. Procedures:60 cases of laryngeal squamous-cell carcinoma (LSCC) and 10 cases of normal laryngeal mucosa were examined using immunohistochemistry to determine whether the expression of survivin correlated with tumorigenesis. Three plasmid vectors of short hairpin RNA (shRNA) specific for survivin were designed and generated. Western blot and real-time PCR analysis of survivin expression in Hep-2 cells was performed 48 h after transfection. The growth curve was used to determine the cell proliferation. Propidium iodide (PI) single staining was applied to detect the cell cycle. The apoptosis of the cells was analyzed by flow cytometry with the FITC-annexin-V/PI double staining and PI single staining. Results:68.33 (41 out of 60) of tumors were positive for survivin expression and significantly associated with lymph node metastasis and advanced stage. In contrast, no expression of survivin in normal mucosa was detected. Transfection of Hep-2 cells with survivin shRNA significantly inhibited survivin expression at both the mRNA and the protein level in Hep-2 cells. Downregulation of survivin resulted in increasing the apoptosis index, but the results showed no obvious influence on cell cycle. Conclusions:This study demonstrates that survivin shRNA effectively inhibits survivin gene expression in Hep-2 cells leading to growth suppression and apoptotic induction in Hep-2 cells.Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The JAK/STAT signal transduction pathway regulates many developmental processes in Drosophila. However, the functional mechanism of this pathway is poorly understood. In this report, we identify the Drosophila cyclin-dependent kinase 4 (Cdk4), which exhibits embryonic mutant phenotypes identical to those in the Hopscotch/JAK kinase and stat92E/STAT mutations. Specific genetic interactions between Cdk4 and hop mutations suggest that Cdk4 functions downstream of the HOP tyrosine kinase. We further show that Cyclin D-Cdk4 (as well as Cyclin E-Cdk2) binds and regulates STAT92E protein stability. STAT92E regulates gene expression for various biological processes, including the endocycle S phase. These data suggest that Cyclin D-Cdk4 and Cyclin E-Cdk2 play more versatile roles in Drosophila development. [Copyright &y& Elsevier]

Objective:To detect the expressions of miRNA-29c,miRNA-30b and those predictive target genes in human endometrium and decidua tissues, and to explore their effects on decidualization of endometria. Methods : Tissue samples including 14 secretory endometria, 16 early pregnancy deciduas and 18 spontaneous miscarriage deciduas were collected. Expressions of miRNA-29c and miRNA-30b were detected by quantitative real-time polymerase chain reaction (qPCR). Expressions of two predicted target genes of miRNA-29c and miRNA-30b, serum and glucocorticoid-inducible kinase 1 (SGK1) and leptin receptor (LEPR), were also detected by qPCR. Results-The expression of miRNA-29c in the spontaneous miscarriage decidua tissues was higher than that in the secretory endometrial tissues (P<0.05). There were not significant differences in expressions of miRNA-30b among three groups (P>0.05). Moreover, expressions of SGK1 and LEPR mRNAs were at highest level in the early pregnancy deciduas, while they were at lowest level in the spontaneous miscarriage deciduas(P<0.05). Conclusions-Expression of miRNA-29c in endometrium may be associated with decidualization by targeting SGK1. Expressions of SGK1 and LEPR in deciduas tissue may be associated with maintaining early pregnancy. [ABSTRACT FROM AUTHOR]

Escin is a natural mixture of triterpene saponins extracted from the seeds of Aesculus wilsonii Rehd. And has been reported to possess the therapeutic effects against neuropathic pain (NP). However, the underlying mechanisms remain unclear. The present study aimed to investigate the therapeutic effects and explore the underlying mechanisms of escin on rats of NP induced by chronic constriction injury (CCI) of sciatic nerve. Rats were treated with escin (7, 14, and 28 mg/kg, i. g.) daily from the third day after the surgery (day 0) for consecutive 14 days. Regular behavior and thermal threshold were measured on days 0, 3, 5, 7, 10 and 14. Investigations into mechanisms involved measurement of inflammatory factors and biochemical factors in dorsal root ganglion (DRG). Inflammatory pain responses and nerve injuries were induced by the CCI model. Tonic pain model and acute inflammatory model induced by formalin or carrageenan were established to evaluated the pharmacological effects of escin on acute inflammatory pain. Corresponding behaviors were monitored and relevant gene expression such as c-fos, mu opioid receptor (MOR) and KCNK1 were detected by qRT-PCR. Investigate the neuroprotective effects of escin on PC12 cell injury induced by lipopolysaccharide (LPS). Cell morphology was observed under inverted microscope and neuroprotective effect of escin on cell activity was assessed by MTT assay. Escin could widen thermal threshold, downregulate the concentration of inflammatory factors like tumor necrosis factor (TNF)-α and interleukin (IL)-1β, suppress the gene expression of toll-like receptor 4 (TLR4), nuclear factor κB (NF-κB), decrease the level of glial fibrillary acidic protein (GFAP) and nerve growth factor (NGF) remarkably. In addition, escin significantly lowered the duration of licking, numbers of flinches and increase in paw edema, showing great therapeutic effects on inflammatory pain responses. Moreover, the activity of injured PC12 cells was significantly improved after escin administrated. Escin exerted the ameliorative effects on NP induced by CCI which may be related to downregulating the release of pro-inflammatory cytokines, suppressing TLR-4/NF-κB signal pathway, thereafter decreasing the level of GFAP and NGF. Image 1 • Escin could downregulate the concentration of inflammatory factors like tumor necrosis factor-α and interleukin-1β. • Escin could suppress toll-like receptor 4/nuclear factor κB signal pathway, decrease the level of glial fibrillary acidic protein and nerve growth factor. • Escin exerted a good therapeutic effect on acute inflammation pain induced by formalin and carrageenan. • Escin had a neuroprotective effect on PC12 cells. [ABSTRACT FROM AUTHOR]

Background: Keratoconus (KC) is a common, degenerative disorder of the cornea, and genetic factors play a key role in its development. However, the genetic etiology of KC is still unclear. This study used the family of twins as material, using, for the first time, multi-omics analysis, to systematically display the changes in KC candidate factors in patients at the DNA, RNA, and protein levels. These can evaluate candidate pathogenic factors in depth and lock onto pathogenic targets.Results: The twins in this study presented classic phenotypes, clear diagnoses, complete case data, and clinical samples, which are excellent materials for genetically studying KC. Whole-exome sequencing was conducted on both the twins and their parents. Transcriptome sequencing was conducted on proband's and health individual's primary human corneal fibroblast cells. Quantitative Real-time PCR and western blot were used to validate the differential gene expressions between the proband and controls. By integrating genomics, transcriptome, and protein level data, multiple consecutive events of KC were systematically analyzed to help better understand the molecular mechanism and genetic basis of KC. The results showed that the accumulation of rare, micro-effect risk variants was the pathogenic factor in this Chinese KC family. Consistent changes in extracellular matrices (ECMs) at the DNA and RNA levels suggested that ECM related changes play a key role in KC pathogenesis. The major gene variants (WNT16, CD248, COL6A2, COL4A3 and ADAMTS3) may affect the expression of related collagens or ECM proteins, thus reducing the amount of ECM in corneas and resulting in KC.Conclusions: This study, the first to explore the genetic etiology of KC via multi-omics analysis under the polygenetic model, has provided new insights into the genetic mechanisms underlying KC and an effective strategy for studying KC pathogenesis in the future. [ABSTRACT FROM AUTHOR]

• This study analyzed IFI30 expression in breast cancer using multiple public database sources and methods. • IFI30 expression was elevated in breast cancer and correlated with tumor stage and prognosis. • IFI30 expression was associated with various immune-related pathways and T cell subsets in breast cancer. • IFI30 overexpression modulated PD-L1 expression and inhibitory efficacy in macrophages. • Targeting the IFI30-PD-L1 axis may be a novel strategy for breast cancer immunotherapy. IFI30 is a lysosomal thiol reductase involved in antigen presentation and immune regulation in various cancers, including breast cancer. Despite its known involvement, the precise mechanism, function, and relationship with the PD-L1 axis and immune response remain unclear. We conducted an extensive investigation into IFI30 mRNA expression in breast cancer utilizing data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Furthermore, we characterized IFI30 mRNA expression across various cell types using publicly available single-cell RNA sequencing datasets, and assessed protein expression through immunohistochemistry using an in-house breast cancer tissue microarray. Functional experiments were performed to elucidate the effects of IFI30 overexpression on PD-L1 expression and inhibitory efficacy in both macrophages and breast tumor cells. Our study unveiled a marked upregulation of IFI30 expression in breast cancer tissues compared to their normal counterparts, with notable associations identified with tumor stage and prognosis. Additionally, IFI30 expression demonstrated significant correlations with various immune-related signaling pathways, encompassing peptide antigen binding, cytokine binding, and MHC class II presentation. Notably, breast cancer samples exhibiting high IFI30 expression in tumor cells displayed high PD-L1 expression on corresponding cells, alongside a diminished ratio of CD8 + T cell infiltration within the tumor microenvironment. Furthermore, ectopic knockdown of IFI30 in both tumor cells and macrophages resulted in a reduction of PD-L1 expression, while conversely, overexpression of IFI30 led to an increase in PD-L1 expression. This study offers new insights into the involvement of IFI30 in breast cancer, elucidating its interplay with the PD-L1 axis and immune response dynamics. Our findings suggest that modulation of the IFI30-PD-L1 axis could serve as a promising strategy for regulating T cells infiltration in breast cancer thus treating breast cancer. [ABSTRACT FROM AUTHOR]

Astaxanthin is an important immunoactive adjuvant. The present study was conducted to evaluate the effect of glucocorticoid receptor (GR) in astaxanthin alleviates LPS-induced immune stress in Channa argus lymphocyte. After astaxanthin or PDTC treatment, lymphocytes exposed to 10 μg/mL LPS for 24 h, showed significantly decreased NF-κB p65, TNF-α, IL-1β, IL-6, IL-8, NO, NOS, caspase-3, caspase-8 and caspase-9 levels. Upon LPS stimulation, astaxanthin significantly upregulated GR, HSP70 and HSP90 gene expression, in response to inflammatory responses. Treatment with GR inhibitors (RU486) or astaxanthin and 10 μg/ml LPS exposure, showed that RU486 significantly inhibited GR phosphorylation and blocked astaxanthin alleviated LPS-induced NF-κB pathway activation. Meanwhile, RU486 interfered with the anti-inflammatory and increased the inflammatory and apoptotic factors expression levels under LPS stimulation. Collectively, GR plays major role in astaxanthin alleviates LPS-induced immune stress in Channa argus lymphocyte. • Glucocorticoid receptor (GR) interacts with NF-κB to inhibit the inflammatory response in lymphocytes. • Astaxanthin inhibits NF-κB activation by phosphorylation of glucocorticoid receptor (GR) in lymphocytes. • Under the inhibition of GR activity, anti-inflammatory activity of astaxxanthin was attenuated in lymphocytes. [ABSTRACT FROM AUTHOR]

We obtained a full cDNA coding sequence of aquaporin 1aa (aqp1aa) from the gills of the freshwater climbing perch, Anabas testudineus, which had the highest expression in the gills and skin, suggesting an important role of Aqp1aa in these organs. Since seawater acclimation had no significant effects on the branchial and intestinal aqp1aa mRNA expression, and since the mRNA expression of aqp1aa in the gut was extremely low, it can be deduced that Aqp1aa, despite being a water channel, did not play a significant osmoregulatory role in A. testudineus. However, terrestrial exposure led to significant increases in the mRNA expression of aqp1aa in the gills and skin of A. testudineus. Since terrestrial exposure would lead to evaporative water loss, these results further support the proposition that Aqp1aa did not function predominantly for the permeation of water through the gills and skin. Rather, increased aqp1aa mRNA expression might be necessary to facilitate increased ammonia excretion during emersion, because A. testudineus is known to utilize amino acids as energy sources for locomotor activity with increased ammonia production on land. Furthermore, ammonia exposure resulted in significant decreases in mRNA expression of aqp1aa in the gills and skin of A. testudineus, presumably to reduce ammonia influx during ammonia loading. This corroborates previous reports on AQP1 being able to facilitate ammonia permeation. However, a molecular characterization of Aqp1aa from A. testudineus revealed that its intrinsic aquapore might not facilitate NH3 transport. Hence, ammonia probably permeated the central fifth pore of the Aqp1aa tetramer as suggested previously. Taken together, our results indicate that Aqp1aa might have a greater physiological role in ammonia excretion than in osmoregulation in A. testudineus. [ABSTRACT FROM AUTHOR]

Background and purpose Acquisition of resistance to adriamycin (ADR) is one of the most important clinical obstacles in the treatment of breast cancer, but the molecular mechanisms underlying sensitivity to ADR remain elusive. In our previous study, through miRNA microarray and experiments, we have emphasized that miR-222 could promote the ADR-resistance in breast cancer cells. The aim of this study was to explore the possible mechanism by which miR-222 affects sensitivity to ADR. Methods Through pathway enrichment analyses for miR-222, we found that PTEN/Akt/FOXO1 signaling pathway may be of importance. RT-qPCR analyses and western blot assays confirmed the relationship between miR-222 expression and target genes. Immunofluorescence further visually displayed the location of FOXO1. When blocking PTEN/Akt/FOXO1 signaling pathway, we demonstrated the effects of miR-222-mediated ADR resistance by MTT and apoptosis assays. Results RT-qPCR and Western blot results showed that miR-222 expression was negatively correlated with FOXO1 expression. In addition, the subcellular translocation of FOXO1 due to the altered expression of miR-222 was observed from immunofluorescence. Moreover, upregulation of miR-222 expression in MCF-7/S cells is associated with decreased PTEN expression levels and increased phospho-Akt (p-Akt) expression. Conversely in MCF-7/ADR cells, inhibition of miR-222 resulted in increased PTEN expression and decreased p-Akt expression. For further validation, results of the present study also demonstrated that PTEN/Akt/FOXO1 signaling was responsible for the ADR-resistance of breast cancer cells since LY294002, an inhibitor of Akt signaling, partially increased the sensitivity of MCF-7/S cells to ADR. More importantly, we postulated that high expression of miR-222 is closely related to poor overall survival by TCGA database validation. Conclusions Taken together, these data elucidated that miR-222 mediated ADR-resistance of breast cancer cells partly through regulation of PTEN/Akt/FOXO1 signaling pathway and inhibition of miR-222 may improve the prognosis of breast cancer patients. [ABSTRACT FROM AUTHOR]

Adriamycin (Adr) is a potent chemotherapeutic agent for chemotherapy of breast cancer patients. Despite impressive initial clinical responses, some developed drug resistance to Adr-based therapy and the mechanisms underlying breast cancer cells resistance to Adr are not well known. In our previous study, in vitro, we verified that miR-222 was upregulated in Adr-resistant breast cancer cells (MCF-7/Adr) compared with the sensitive parental cells (MCF-7/S). Here, miR-222 inhibitors or mimics were transfected into MCF-7 cell lines. RT-qPCR and western blot were used to detect the expression of p27 kip1 . Immunofluorescence showed that miR-222 altered the subcellular location of p27 kip1 in nucleus. MTT was employed to verify the sensitivity of breast cancer cell lines to Adr. Flow cytometry showed the apoptosis and cell cycles of the cells after adding Adr. The results showed that downregulation of miR-222 in MCF-7/Adr increased sensitivity to Adr and Adr-induced apoptosis, and arrested the cells in G1 phase, accompanied by more expressions of p27 kip1 , especially in nucleus. Furthermore, overexpressed miR-222 in MCF-7/S had the inverse results. Taken together, the results found that miR-222 induced Adr-resistance at least in part via suppressing p27 kip1 expression and altering its subcellular localization, and miR-222 inhibitors could reverse Adr-resistance of breast cancer cells. These results disclosed that the future holds much promise for the targeted therapeutic in the treatment of Adr-resistant breast cancer. [ABSTRACT FROM AUTHOR]

• The ethanol extract of Usnea diffracta shows a positive antiatherogenic effect. • The ethanol extract of Usnea diffracta promotes lipid metabolism. • The ethanol extract of Usnea diffracta reduces the plaque area in atherosclerotic model rats. • The mechanism may be related to the inhibition of TLR5/NF-κB signaling pathway. Usnea diff ;racta Vain. (U. diffracta) belonging to the Usnea genus, is widely used as a folk medicine for the treatment of ulcer, abdominal pain, diarrhea, malaria and so on. However, the antiatherogenic effect of U. diffracta has not yet been reported. This study aims to investigate the antiatherogenic effects of the ethanol extract of U. diffracta and its mechanism. A high fat diet and VD3 were used to establish the atherosclerotic rat model, with 0.004 g/kg/d of simvastatin as a positive control, fed with 0.7, 1.4, and 2.8 g/kg/d of Usnea ethanol extract for 21 days. The blood, liver, and aorta samples from each rat were collected after the last administration. Pharmacodynamic effects were evaluated. The inflammation related factors, the gene expressions of Toll-like receptor 5 (TLR5), myeloid differentiating factor 88 (MyD88), and nuclear factor-κB (NF-κB) were detected. Compared with the model group, simvastatin and ethanol extract of U. diffracta can significantly reduce the serum levels of triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), Ca2+, AST, ALT, the liver contents of total cholesterol (TC), TG, AI and liver index, as well as significantly increase the contents of high-density lipoprotein cholesterol (HDL-C) both in serum and liver (p < 0.01 or p < 0.05). The serum level of ox-LDL can be significantly reduced by simvastatin, low and medium U. diffracta ethanol extract doses (p < 0.01). In addition, simvastatin and low dosage of U. diffracta ethanol extract can significantly reduce the liver content of LDL-C (p < 0.01). U. diffracta ethanol extract shows a positive antiatherogenic effect. Furthermore, the mechanism may be related to promoting the expression of serum IL-10 and inhibition of TLR5/NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]