Quercetin: a promising virulence inhibitor of Pseudomonas aeruginosa LasB in vitro.

With the inappropriate use of antibiotics, antibiotic resistance has emerged as a major dilemma for patients infected with Pseudomonas aeruginosa. Elastase B (LasB), a crucial extracellular virulence factor secreted by P. aeruginosa, has been identified as a key target for antivirulence therapy. Quercetin, a natural flavonoid, exhibits promising potential as an antivirulence agent. We aim to evaluate the impact of quercetin on P. aeruginosa LasB and elucidate the underlying mechanism. Molecular docking and molecular dynamics simulation revealed a rather favorable intermolecular interaction between quercetin and LasB. At the sub-MICs of ≤256 µg/ml, quercetin was found to effectively inhibit the production and activity of LasB elastase, as well as downregulate the transcription level of the lasB gene in both PAO1 and clinical strains of P. aeruginosa. Through correlation analysis, significant positive correlations were shown between the virulence gene lasB and the QS system regulatory genes lasI, lasR, rhlI, and rhlR in clinical strains of P. aeruginosa. Then, we found the lasB gene expression and LasB activity were significantly deficient in PAO1 ΔlasI and ΔlasIΔrhlI mutants. In addition, quercetin significantly downregulated the expression levels of regulated genes lasI, lasR, rhlI, rhlR, pqsA, and pqsR as well as effectively attenuated the synthesis of signaling molecules 3-oxo-C12-HSL and C4-HSL in the QS system of PAO1. Quercetin was also able to compete with the natural ligands OdDHL, BHL, and PQS for binding to the receptor proteins LasR, RhlR, and PqsR, respectively, resulting in the formation of more stabilized complexes. Taken together, quercetin exhibits enormous potential in combating LasB production and activity by disrupting the QS system of P. aeruginosa in vitro, thereby offering an alternative approach for the antivirulence therapy of P. aeruginosa infections. KEY POINTS: • Quercetin diminished the content and activity of LasB elastase of P. aeruginosa. • Quercetin inhibited the QS system activity of P. aeruginosa. • Quercetin acted on LasB based on the QS system.

Effect of piperine on the inhibitory potential of MexAB-OprM efflux pump and imipenem resistance in carbapenem-resistant Pseudomonas aeruginosa.

The escalating prevalence of carbapenem-resistant Pseudomonas aeruginosa (CRPA) poses a significant threat to global public health through the spread of its 'high-risk' clones. Immediate and decisive research into antimicrobial agents against CRPA is crucial for the development of effective measures and interventions. Overexpression of the MexAB-OprM efflux pump is one of the major mechanisms of CRPA. Since the active efflux of antibacterial agents plays a significant role in mediating drug resistance in CRPA, the inhibition of efflux pumps has become a promising strategy to restore antibacterial potency. Piperine (PIP) has been proven to be a promising efflux pump inhibitor in some bacteria. However, there are no studies on whether PIP can act as a potential efflux pump inhibitor in CRPA. The present study aimed to identify the antibacterial activity of PIP against CRPA and to evaluate the effect on the MexAB-OprM efflux pump. Molecular docking was used to analyze the possible interaction of PIP with the proteins of the MexAB-OprM efflux pump in CRPA. The effect of PIP on the expression of the MexAB-OprM efflux pump was investigated by real-time quantitative PCR (qPCR) and ethidium bromide accumulation efflux assay. The effect of PIP on CRPA imipenem (IPM) resistance was investigated by the checkerboard dilution method. The results demonstrated that PIP exhibited the lowest binding affinity of -9.1 kcal towards efflux pump proteins. A synergistic effect between PIP and IPM on CRPA was observed. More importantly, PIP effectively hindered the efflux of ethidium bromide and IPM by up-regulating MexR gene expression while down-regulating MexA, MexB, and OprM gene expressions. In conclusion, PIP could enhance the antibacterial activity of IPM by inhibiting the MexAB-OprM efflux pump. Our work proved that PIP had the potential to be an efflux pump inhibitor of CRPA.

Characterization and genomic analysis of a broad-spectrum lytic phage HZ2201 and its antibiofilm efficacy against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a clinically common conditionally pathogenic bacterium, and the abuse of antibiotics has exacerbated its drug resistance in recent years. This has resulted in extensive reports about the usage of Pseudomonas aeruginosa phage as a novel antibacterial drug. In this study, we isolated a novel phage HZ2201 with a broad lytic spectrum. The lytic rate of this phage against Pseudomonas aeruginosa reached 78.38% (29/37), including 25 multi-drug- and carbapenem-resistant Pseudomonas aeruginosa strains. Transmission electron microscopy revealed that phage HZ2201 belongs to the class Caudoviricetes. Biological characterization showed that phage HZ2201 had an latent period of 40 min, a lytic period of 20 min, and a burst size of 440 PFU/cell, with improved tolerance to temperature and pH. Considering genomic analysis, the HZ2201 genome was a circular double-stranded DNA with a size of 45,431 bp and a guanine-cytosine (G + C) content of 52.16%, and contained 3 tRNAs. 27 of the 74 open reading frames (ORFs) annotated by the Rapid Annotation using Subsystem Technology (RAST) tool could be matched to the genomes of known functions, and no genes related to virulence and antibiotic resistance were found. The phylogenetic tree suggests that phage HZ2201 is highly related to the phage ZCPS1 and PaP3, and ORF57 and ORF17 are predicted to encode a holin and an endolysin, respectively. Cell lysis by HZ2201 proceeds through the holin-endolysin system, suggesting that it is a novel phage. Additionally, we demonstrated that phage HZ2201 has a high inhibitory capacity against Pseudomonas aeruginosa biofilms. The results of our study suggest that phage HZ2201 is a novel potential antimicrobial agent for treating drug-resistant Pseudomonas aeruginosa infection.

An immune-related lncRNA pairs signature to identify the prognosis and predict the immune landscape of laryngeal squamous cell carcinoma.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is the most common squamous cell carcinoma. Though significant effort has been focused on molecular pathogenesis, development, and recurrence of LSCC, little is known about its relationship with the immune-related long non-coding RNA (lncRNA) pairs. METHODS: After obtaining the transcriptome profiling data sets and the corresponding clinical characteristics of LSCC patients and normal samples from The Cancer Genome Atlas (TCGA) database, a series of bioinformatic analysis was conducted to select the differently expressed immune-related lncRNAs and build a signature of immune-related lncRNA pairs. Then, the effectiveness of the signature was validated. RESULTS: A total of 111 LSCC patients and 12 normal samples' transcriptome profiling data sets were retrieved from TCGA. 301 differently expressed immune-related lncRNAs were identified and 35,225 lncRNA pairs were established. After univariate Cox analysis, LASSO regression and multivariate Cox analysis, 7 lncRNA pairs were eventually selected to construct a signature. The riskscore was computed using the following formula: Riskscore = 0.95 × (AL133330.1|AC132872.3) + (-1.23) × (LINC01094|LINC02154) + 0.65 × (LINC02575|AC122685.1) + (-1.15) × (MIR9-3HG|LINC01748) + 1.45 × (AC092687.3|SNHG12) + (-0.87) × (AC090204.1|AL158166.1) + 0.64 × (LINC01063|Z82243.1). Patients were classified into the high-risk group (> 1.366) and the low-risk group (< 1.366) according to the cutoff value (1.366), which is based on the 5-year riskscore ROC curve. The survival analysis showed that the low-risk group had a better prognosis (P < 0.001). The riskscore was better than other clinical characteristics in prognostic prediction and the area under the curves (AUCs) for the 1-, 3-, and 5-year survivals were 0.796, 0.946, and 0.895, respectively. Combining age, gender, grade, stage, and riskscore, a nomograph was developed to predict survival probability in LSCC patients. Then, the riskscore was confirmed to be related with the content of tumor-infiltration immune cells and the model could serve as a potential predictor for chemosensitivity. CONCLUSION: We successfully established a more stable signature of 7 immune-related lncRNA pairs, which has demonstrated a better prognostic ability for LSCC patients and may assist clinicians to precisely prescribe chemo drugs.

An Insight into Antihyperlipidemic Effects of Polysaccharides from Natural Resources.

Hyperlipidemia is a chronic metabolic disease caused by the abnormal metabolism of lipoproteins in the human body. Its main hazard is to accelerate systemic atherosclerosis, which causes cerebrovascular diseases such as coronary heart disease and thrombosis. At the same time, although the current hypolipidemic drugs have a certain therapeutic effect, they have side effects such as liver damage and digestive tract discomfort. Many kinds of polysaccharides from natural resources possess therapeutic effects on hyperlipidemia but still lack a comprehensive understanding. In this paper, the research progress of natural polysaccharides on reducing blood lipids in recent years is reviewed. The pharmacological mechanisms and targets of natural polysaccharides are mainly introduced. The relationship between structure and hypolipidemic activity is also discussed in detail. This review will help to understand the value of polysaccharides in lowering blood lipids and provide guidance for the development and clinical application of new hypolipidemic drugs.

[Research progress on chemical constituents and pharmacological activities of Potentilla].

There are 200-500 species of Potentilla(Rosaceae) worldwide, among which 90 species are widely distributed in China and have a long history of ethnic medicinal use. According to our statistics, a total of 367 compounds have been isolated and identified from plants of this genus, including terpenoids, flavonoids, phenolic acids, tannins, and phenylpropanoids. The medicinal materials made from these plants mainly have antioxidative, blood sugar-lowering, anti-inflammatory, anti-tumor, cardiovascular system-protecting, neuroprotective, and hepatoprotective activities. This study systematically reviews the research progress on chemical constituents and pharmacological activities of Potentilla plants to provide a basis for further research and clinical application.

Anti-inflammatory and antioxidant activities of chemical constituents from the flower buds of Buddleja officinalis.

Five new glycosides including mimenghuasu A and B (1-2), isolinarin (3), cyclocitralosides A and B (4-5), along with forty-seven known compounds were isolated from the flower buds of Buddleja officinalis. These structures were elucidated by extensive spectroscopic analysis (UV, IR, 1 D, 2 D NMR, and MS spectra). The anti-inflammatory activities of the isolated compounds were determined by enzyme-linked immunosorbent assay (ELISA) on the expression of TNF-α (LPS-activated RAW264.7 cells) and MTT experiment on LPS-induced HUVECs proliferation effects. Good suppressive effects on the expression of TNF-α were shown by 4 and 5 with IC50 values of 19.35 and 22.10 µM, respectively, compared to positive control indomethacin (IC50 16.40 µM). In addition to this, some isolated compounds exhibited excellent antioxidant activities including compounds 16, 18, 29, 39, and 47 (IC50 µM: 82.59, 72.94, 33.65, 46.67, and 20.81, respectively) with almost the same or stronger potency with reference to vitamin C as positive control (IC50 81.83 µM).

Research progress on chemical constituents and pharmacological activities of Potentilla / 中国中药杂志

There are 200-500 species of Potentilla(Rosaceae) worldwide, among which 90 species are widely distributed in China and have a long history of ethnic medicinal use. According to our statistics, a total of 367 compounds have been isolated and identified from plants of this genus, including terpenoids, flavonoids, phenolic acids, tannins, and phenylpropanoids. The medicinal materials made from these plants mainly have antioxidative, blood sugar-lowering, anti-inflammatory, anti-tumor, cardiovascular system-protecting, neuroprotective, and hepatoprotective activities. This study systematically reviews the research progress on chemical constituents and pharmacological activities of Potentilla plants to provide a basis for further research and clinical application.

Glucocorticoids protect HEI-OC1 cells from tunicamycin-induced cell damage via inhibiting endoplasmic reticulum stress.

BACKGROUND: To analyze mechanisms of action of glucocorticoid treatment for endoplasmic reticulum stress (ERS) in sensorineural hearing loss (SNHL), we aimed to evaluate the expression and activation status of the protein kinase RNA-like ER kinase (PERK)-C/EBP homologous protein (CHOP) pathway, which is the major pathway in the ERS. METHODS: In the present study, we established an in vitro ERS model using tunicamycin-treated hair-cell-like HEI-OC1 cells. The effect of dexamethasone on proliferation inhibition, apoptosis, and ATF4-CHOP pathway in HEI-OC1 cells was examined by CCK-8 assay, flow cytometry, western blotting, and reverse transcription PCR, respectively. RESULTS: In HEI-OC1 cells, dexamethasone was shown to significantly reduce the tunicamycin-induced expression of ATF4 and CHOP in the context of sustained viability and proliferation, a therapeutic effect that was reversible by co-treatment with a glucocorticoid antagonist. CONCLUSION: Dexamethasone can protect hair-cell-like HEI-OC1 cells from ERS damage, which may be one of the mechanisms of action for GCs in SNHL treatment.

Elucidation of the Hdac2/Sp1/miR-204-5p/Bcl-2 axis as a modulator of cochlear apoptosis via in vivo/in vitro models of acute hearing loss.

We previously reported that dysregulation of histone deacetylase 2 (Hdac2) was associated with the prognosis of sudden sensorineural hearing loss. However, the underlying molecular mechanisms are poorly understood. In the present study, we developed an acute hearing loss animal model in guinea pigs by infusing lipopolysaccharides (LPS) into the cochlea and measured the expression of Hdac2 in the sensory epithelium. We observed that the level of Hdac2 was significantly decreased in the LPS-infused cochleae. The levels of apoptosis-inhibition genes Bcl-2 and Bcl-xl were also decreased in the cochlea and correlated positively with the levels of Hdac2. Caspase3 or TUNEL-positive spiral ganglion neurons, hair cells, and supporting cells were observed in the LPS-infused cochleae. These in vivo observations were recapitulated in cell culture experiments. Based on bioinformatics analysis, we found miR-204-5p was engaged in the regulation of Hdac2 on Bcl-2. Molecular mechanism experiments displayed that miR-204-5p could be regulated by Hdac2 through interacting with transcription factor Sp1. Taken together, these results indicated that the Hdac2/Sp1/miR-204-5p/Bcl-2 regulatory axis mediated apoptosis in the cochlea, providing potential insights into the progression of acute hearing loss. To our knowledge, the study describes a miRNA-related mechanism for Hdac2-mediated regulation in the cochlea for the first time.

Research progress in pharmacological effects of polyphenols in Areca catechu L / 中国药理学与毒理学杂志

Betel nut is the dry and mature seed of Areca catechu L., which is originated in Malaysia and cultivated in Yunnan, Hainan and Taiwan and other tropical areas of China. It is also known as big belly, binmen, olive seed, green seed and so on. Betel nut is a dual-use resource for medicine and food, which was first contained in LI Dang's Pharma?ceutical Record. Betel nut tastes bitter, pungent, warm in nature, and belongs to the stomach and large intestine meridian. It contains a variety of chemical components such as alkaloids, phenolic compounds, polysaccharides, fatty acids, amino acids, flavonoids, minerals, terpenoids, and steroids. It has the advantages of promoting digestion, lowering blood pres?sure, anti-depression, anti-oxidation, anti-inflammatory, and anti-parasites, antibacterial and other activities. The content of total phenols in fresh fruits of areca nut was 31.1％, mainly including catechin, isorhamnetin, chrysopanthoxanthin, luteolin, tannin and other polyphenols. The commonly used methods for determination of polyphenols in areca are vanil?lin titration potassium permanganate titration and potassium ferricyanide spectrophotometry. The main activities and mechanisms of areca polyphenols include: ① Antidepressant effect: polyphenols bind to monoamine oxidase type A (MAO-A) receptors that inhibit the production of neurotransmitters, thereby increasing the content of amine transmitters in the brain and playing a therapeutic effect on depression. ② Antioxidant effect: polyphenols contain multiple adjacent hydroxyl groups, which are easily oxidized and can effectively remove superoxide anion free radical, hydroxyl free radi?cal, 1,1-diphenyl-2-picrylhydrazyl radical, showing good antioxidant activity.③Bacteriostatic effect:polyphenols can spe?cifically bind to the surface of bacteria, thus achieving bacteriostatic effect. Studies have found that betel nut polyphenols have varying degrees of inhibitory effects on a variety of bacteria. ④ Inducing apoptosis of lymphocytes: polyphenols deplete the mercaptan in lymphocytes and make them unable to survive, thus inducing apoptosis of lymphocytes.⑤Anti-aging effect: polyphenols have the effect of anti-hyaluronidase and anti-elastase, so as to protect elastin fiber and pro?mote collagen synthesis.⑥Anti-allergic effect:studies have found that polyphenols can reduce ovalbumin induced aller?gic reactions.⑦Other functions:betel nut can freshen breath, eliminate bad breath, and resist the activity of cobra venom. At present, domestic and foreign scholars' research on betel nut mainly focuses on arecoline and its carcinogenicity, mutagenicity, effects on reproductive function, addiction and toxicity to the nervous system, and there are few studies on the positive effects of betel nut, especially on it. There is less research on phenolic ingredients. Therefore, this article reviews the polyphenolic chemical constituents of betel nut, and fully excavates its pharmacological activity to provide a reasonable basis for the scientific use of betel nut.

Progress in protective effect and mechanism of 6-gingerol on myocardial ischemia/reperfusion injury / 中国药理学与毒理学杂志

The morbidity and mortality of cardiovascular diseases are very high, which has attracted more and more attention all over the world. Common treatment methods for clinical treatment of acute myocardial infarction include direct percutaneous coronary intervention and coronary artery bypass grafting, which can quickly restore blocked coronary blood flow and reduce the infarct size. However, the inevitable ischemia/reperfusion injury will occur during the recovery of coronary blood flow, its pathological mechanism is complicated, and the Western medicine countermeasures are very limited. Among the current drugs for the treatment of cardiovascular diseases, traditional Chinese medicine has become a research hotspot due to its multiple targets, safety, and low side effects. Ginger is the fresh rhizome of Zingiber offici?nale Rosc., a perennial herbaceous plant in the ginger family. It is a dual-purpose resource of medicine and food. Ginger has the functions of relieving the appearance and dispelling cold, warming up and relieving vomiting, resolving phlegm and relieving cough, and relieving fish and crab poison. The chemical components of ginger mainly include volatile oil, gingerol, diphenylheptane, etc.. Among them, 6-gingerol, as the main active component of gingerols, has obvious phar?macological effects in myocardial protection, anti-oxidation, anti-inflammatory, etc.. Studies have shown that 6-gingerol protects myocardium mainly through anti-oxidative stress, anti-inflammatory, inhibiting cell apoptosis, and preventing cal?cium influx. ① Anti-oxidative stress: oxidative stress is a state where oxidation and anti-oxidation in the body are out of balance, and it is also an important factor leading to myocardial damage. Many studies have confirmed that 6-gingerol has an antioxidant effect, and it is considered a natural antioxidant. 6-gingerol can significantly reduce the degree of oxi?dative stress and the level of reactive oxygen species caused by cardiomyocyte damage, and has a significant cardiopro?tective effect. ② Anti-inflammatory: inflammation can cause substantial cell damage and organ dysfunction, which is another important cause of myocardial damage. 6-gingerol can reduce the levels of inflammatory factors such as inter?leukin-6, interleukin-1β, and tumor necrosis factor-αin cardiomyocytes, and at the same time inhibit the TLR4/NF-κB sig?naling pathway, an important regulatory pathway of inflammation, showing that it may improve myocardial damage through anti-inflammatory effects. ③ Inhibition of apoptosis: apoptosis is a complex and orderly process in the autono?mous biochemical process of cells, and one of the main mechanisms of myocardial injury. This process can be roughly divided into three pathways: mitochondria, endoplasmic reticulum, and death receptors. Among them, the mitochondrial pathway plays an important role, and Bcl-2 and Bax located upstream of this pathway can regulate the entire process of cell apoptosis by regulating the permeability of the mitochondrial membrane. Studies have found that the preventive application of 6-gingerol can reduce cell damage, reduce the number of apoptotic cells, reduce the activity of Bax and caspase-3, and increase the expression of Bcl-2. Therefore, 6-gingerol pretreatment can reduce the damage of cardio?myocytes, and its mechanism may be related to the inhibition of apoptosis.④Prevent calcium influx:calcium overload is involved in the pathogenesis of myocardial ischemic injury, which may be related to excessive contracture, arrhythmia, and mitochondrial Ca2+accumulation that impairs myocardial function. 6-gingerol inhibits the increase of intracellular Ca2+concentration by inhibiting L-type calcium current, thereby reducing extracellular Ca2+ influx, thereby avoiding calcium overload and playing a cardioprotective effect. In summary, 6-gingerol can effectively treat and improve myocardial isch?emia/reperfusion injury, and it has great development potential in the fields of medicine and health products.

Differential Levels of Endoplasmic Reticulum Stress in Peripheral Blood Mononuclear Cells from Patients with Sudden Sensorineural Hearing Loss.

BACKGROUND Sudden sensorineural hearing loss (SSNHL) is currently treated with a combination of drugs, predominantly with glucocorticoids (GCs). However, the mechanisms of action of GCs in SSNHL are unknown. This study aimed to analyze the role of endoplasmic reticulum stress (ERS) in SSNHL pathogenesis and prognosis. MATERIAL AND METHODS In this study, we evaluated the expression and activation status of the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-C/EBP homologous protein (CHOP) pathway in peripheral blood mononuclear cells (PBMCs) from patients with SSNHL and compared them with those in healthy controls. We also compared differences in expression of activating transcription factor 4 (ATF4) and CHOP before and after glucocorticoid treatment in patients with improved and unimproved SSNHL. RESULTS Treatment with GCs significantly improved hearing in 55% of patients with SSNHL. Levels of phosphorylated PERK (p-PERK) and phosphorylated eukaryotic initiation factor 2alpha were increased in PBMCs from patients with SSNHL compared with healthy controls. ATF4 and CHOP expression were also significantly elevated. After treatment, the amount of ATF4 and CHOP proteins in PBMCs in the patients whose SSNHL improved was significantly reduced compared with the levels measured before treatment in all patients with SSNHL. The expression of the ATF4 and CHOP proteins in PBMCs in the unimproved group, however, was not significantly changed relative to pretreatment levels. CONCLUSIONS ERS may play a significant role in the pathogenesis of SSNHL, and the responsiveness of the condition to GC-mediated mitigation of ERS may be one of the key factors that affect patient prognosis.

Panax notoginseng Saponins protect auditory cells against cisplatin­induced ototoxicity by inducing the AKT/Nrf2 signaling­mediated redox pathway.

Cisplatin­induced cytotoxicity, such as nephrotoxicity, neurotoxicity and ototoxicity, restricts the clinical application of this compound. Panax notoginseng Saponins (PNS) exhibit potent free radical scavenging and antioxidant activity. PNS have been demonstrated to reduce cisplatin­induced nephrotoxicity and neurotoxicity. The present study investigated the ability of PNS to protect the auditory HEI­OC1 cell line against ototoxicity induced by cisplatin. PNS induced activation of the AKT/nuclear factor erythroid 2­related factor 2 (Nrf2) signaling pathway. Following pretreatment with PNS, HEI­OC1 cells were treated with cisplatin and cultured for 24 h. The viability of HEI­OC1 cells was examined using a Cell Counting Kit­8 assay. Double staining analysis was used to measure cell apoptosis. The ability of PNS to reduce reactive oxygen species (ROS) levels was assessed by flow cytometry. The levels of phosphorylated (p)­AKT, heme oxygenase 1 (HO­1), NAD(P)H quinone dehydrogenase 1 (NQO1), glutamate­cysteine ligase catalytic (GCLC) and Nrf2 were measured by western blotting. HEI­OC1 cells that were pretreated with PNS exhibited significantly increased cell viability compared with that noted in cells treated only with cisplatin. In addition, PNS suppressed the induction of apoptosis and ROS production following cisplatin treatment. The upregulation of NQO1, HO­1 and GCLC expression in PNS­pretreated cells was associated with p­AKT levels and the activation of Nrf2. These findings suggested that PNS protected auditory cells against ototoxicity induced by cisplatin by activating AKT/Nrf2 signaling. PNS may serve as a potential candidate in regulating cisplatin­induced cytotoxicity.

MicroRNA (miR)-597-5p Inhibits Colon Cancer Cell Migration and Invasion by Targeting FOS-Like Antigen 2 (FOSL2).

Our previous work had shown that FOS-like antigen 2 (FOSL2) is regulated by miR-143-5p in colorectal cancer (CRC). Given that it has been shown by others that FOSL2 is also a target of miR-597-5p in breast adenocarcinoma, the objective of the current work was to determine whether FOSL2 is regulated by miR-597-5p in CRC and the role of miR-597-5p in CRC. MiR-597-5p expression was determined in RNA obtained from 30 paired samples of colon cancer and tumor adjacent normal tissue, as well as in the LoVo (CRC cell line) and FHC (normal colonic epithelial cells) by quantitative real time polymerase chain reaction (qRT-PCR). MiR-597-5p expression was significantly downregulated in both CRC tissue and LoVo cells. Reporter assays using wild-type and miR-597-5p seed mutant FOSL2 confirmed that FOSL2 is a bona fide target of miR-597-5p. Modulating miR-597-5p expression levels in FHC and LoVo cells using antagomir and mimic, respectively, impacted expression of epithelial and mesenchymal cell markers as well as in vitro migration and invasion, without any effect on cell proliferation, showing that miR-597-5p functions as a suppressor of epithelial to mesenchymal transition. Restoration of FOSL2 expression rescued pro-metastatic functional properties of LoVo cells conforming that effect of miR-597-5p was being mediated by targeting FOSL2. Xenograft assays in athymic nude mice showed that miR-597-5p mimic did not reduce tumor incidence or growth in LoVo cells. However, using a hepatic metastasis model showed that miR-597-5p mimic can significantly prevent hepatic metastatic nodule formation as well as FOSL2 expression in these metastatic nodules. Importantly, FOSL2 mRNA and miR-597-5p expression was found to be inversely correlated in an independent cohort of 21 CRC patients Cumulatively our results show that miR-597-5p functions as a suppressor of metastatic progression in CRC by targeting FOSL2. Replenishment of miR-597-5p can be a potential therapeutic target where its expression along with FOSL2 can serve as potential diagnostic markers in CRC.

Temperature-Dependent HfO2/Si Interface Structural Evolution and its Mechanism.

In this work, hafnium oxide (HfO2) thin films are deposited on p-type Si substrates by remote plasma atomic layer deposition on p-type Si at 250 °C, followed by a rapid thermal annealing in nitrogen. Effect of post-annealing temperature on the crystallization of HfO2 films and HfO2/Si interfaces is investigated. The crystallization of the HfO2 films and HfO2/Si interface is studied by field emission transmission electron microscopy, X-ray photoelectron spectroscopy, X-ray diffraction, and atomic force microscopy. The experimental results show that during annealing, the oxygen diffuse from HfO2 to Si interface. For annealing temperature below 400 °C, the HfO2 film and interfacial layer are amorphous, and the latter consists of HfO2 and silicon dioxide (SiO2). At annealing temperature of 450-550 °C, the HfO2 film become multiphase polycrystalline, and a crystalline SiO2 is found at the interface. Finally, at annealing temperature beyond 550 °C, the HfO2 film is dominated by single-phase polycrystalline, and the interfacial layer is completely transformed to crystalline SiO2.

Fos-like antigen 2 (FOSL2) promotes metastasis in colon cancer.

Among different cancers, incidence and mortality of colorectal cancer (CRC) is one of the highest. KRAS mutation is one of the underlying features in the pathogenesis of CRC with CRC tumors harboring mutant KRAS exhibiting a more aggressive behavior compared to CRC tumors with wild type KRAS. We had earlier shown that the microRNA-143 (miR-143) replenishment not only chemosensitizers CRC cell line with mutant KRAS instead of wild-type KRAS gene, to paclitaxel-mediated cytotoxicity, but also inhibits cell migration and invasion ability. Hence, the study aimed to determine how miR-143 replenishment is inhibiting pre-metastatic behavior in CRC cells with mutant KRAS. Top ten mRNA targets of miR-143 as predicted by TargetScan were evaluated by qRT-PCR in LoVo cells which were performed mock transfection or miR-143 mimic transfection. Evaluation of the changes in cognate mRNA target(s) was done in 30 paired CRC tissue and tumor adjacent normal tissue specimens and in LoVo cells by western blot. Effect of the mRNA target on pro-metastatic behavior was assayed by gain- and loss-of-function studies using a combination of western blotting and in vitro cell proliferation and transwell migration/invasion assay in LoVo cells and in the normal colonic epithelium cell line FHC. In vivo effect of the cognate mRNA target on CRC metastasis was assayed by xenograft assay. Of the 10 predicted mRNA targets, FOSL2 (P < 0.05) and IGFBP5 (P > 0.05) was down regulated in LoVo cells transfected with the miR-143 mimic. FOSL2 mRNA levels were significantly downregulated in CRC tissue specimens compared with adjacent normal tissue (P < 0.05). Immunoblot analysis showed that FOSL2, but not IGFBP5, protein expression is down regulated in LoVo cells after the miR-143 mimic transfection. FOSL2 overexpression in the normal colonic epithelial cell line FHC or siRNA-mediated silencing in LoVo cells induced and repressed, respectively, pro-mesenchymal cell features. Whereas manipulation of FOSL2 expression did not have any effect on cell proliferation rates, silencing its expression inhibited cell migration and invasion ability in vitro. In addition, silencing of FOSL2 expression in the LoVo cells can significantly inhibited invasion of hepatic, while no effect was found for tumorigenic potential. Our results suggest that FOSL2 is a critical regulator of CRC metastasis and might be an important marker for prognostic in CRC patients.

Surface Passivation of Silicon Using HfO2 Thin Films Deposited by Remote Plasma Atomic Layer Deposition System.

Hafnium oxide (HfO2) thin films have attracted much attention owing to their usefulness in equivalent oxide thickness scaling in microelectronics, which arises from their high dielectric constant and thermodynamic stability with silicon. However, the surface passivation properties of such films, particularly on crystalline silicon (c-Si), have rarely been reported upon. In this study, the HfO2 thin films were deposited on c-Si substrates with and without oxygen plasma pretreatments, using a remote plasma atomic layer deposition system. Post-annealing was performed using a rapid thermal processing system at different temperatures in N2 ambient for 10 min. The effects of oxygen plasma pretreatment and post-annealing on the properties of the HfO2 thin films were investigated. They indicate that the in situ remote plasma pretreatment of Si substrate can result in the formation of better SiO2, resulting in a better chemical passivation. The deposited HfO2 thin films with oxygen plasma pretreatment and post-annealing at 500 °C for 10 min were effective in improving the lifetime of c-Si (original lifetime of 1 µs) to up to 67 µs.

Establishment of a fluorescence quantitative PCR assay for murine polyomavirus detection and its use in the detection in naked mole rats / 中国比较医学杂志

Objective To establish a fluorescence quantitative PCR assay for polyomavirus and to apply this technique in the investigation of its infection rate in naked mole rats. Methods To compare the nucleic acid sequence of murine polyomavirus (Genbank:NC 001515) in NCBI and design primers and probes in its conserved region. To establish a fluorescence quantitavive PCR method for polyomavirus and evaluate the sensitivity and specificity of the method. To infect nine one-day old KM strain suckling mice, and to collect samples of the heart, liver, spleen, lung, kidney, brain, thymus, cecal contents and blood at 21 days after infection. The efficacy of the method was validated by detecting the virus in organs. 62 cecal samples from naked mole rats were tested by the established assay. Results There was obvious fluorescence signal when polyomavirus was used as the template and no fluorescence signal when simian virus 40, murine K virus, MVM and H-1 were used as templates. The detection limit of the assay was 100 copies/μL. Polyomavirus DNA was detected in the heart, liver, spleen, lung, kidney and cecal contents of the mice which were inoculated with polyomavirus. The polyomavirus DNA content was highest in the lung tissue. There was no detectable polyomavirus DNA in the brain, thymus and blood of the infected mice. Sixty-two cecal contents of naked mole rats were tested for polyomavirus and the results were negative. Conclusions The fluorescence quantitative PCR assay for polyomavirus established in this study can effectively detect polyomavirus DNA in animal tissues. The results of investigation of the natural infected polyomavirus of naked mole rats provide a reference for the formulation of microbiological criteria for experimental naked mole rats.

Establishment and application of a RT-PCR detection method for bovine viral diarrhea virus / 中国比较医学杂志

Objective To establish a dual RT-PCR detection method for bovine viral diarrhea virus(BVDV)in bovine-derived samples. Methods The primers were designed and synthesized according to the published BVDV1 and BVDV2 genes containing highly conservative sequences in the 5' untranslated regions(5' UTR)to establish the dual RT-PCR method. The specificity,sensitivity,stability of this method were evaluated. Then 41 bovine-derived samples and 64 bovine plasma samples including bovine calf serum, deproteinized calf serum extract and one lienal polypeptide injection were detected with this method. Results There was no cross reaction with bovine parainfluenza virus type 3(BPIV3), classical swine fever virus(CSFV)and Japanese encephalitis virus(JEV)when samples were detected with the established dual RT-PCR method. The lowest concentration of template DNA for detection of BVDV1 and BVDV2 was 8.87 × 102copies and 6.31 × 102copies per microliter,respectively. Electrophoresis bands of about 151 bp and 303 bp were still amplified and detected after the BVDV1 and BVDV2 cDNA was stored at -30℃ for 12 months. The BVDV positive rate of 41 bovine-derived samples and 64 bovine plasma samples detected with this dual RT-PCR method was 14.6% and 29.7%, respectively. Conclusions The established dual RT-PCR method has the advantages of high efficiency, specificity,sensitivity and stability,and can be used for the detection of BVDV in bovine-derived samples.