Gender-specific SBNO2 and VPS13B as a potential driver of osteoporosis development in male ankylosing spondylitis.

To identify the critical genes and pathways that related to OP development in male AS patients, bioinformatic gene analysis and qRT-PCR validation were performed. SBNO2 and VPS13B were identified as the potential target for OP development, which may be valuable for the prevention of OP in male AS patients. INTRODUCTION: Osteoporosis (OP) is common in men with ankylosing spondylitis (AS). The specific pathogenesis of OP in AS, however, is still unclear. The present study attempted to identify potential genes associated with the development of OP in males with AS. METHODS: Gene expression profiles were downloaded from the GSE73754 and GSE35959 datasets from the Gene Expression Omnibus (GEO). Data from OsteoporosAtlas were downloaded as a supplement. Differentially expressed genes (DEGs) were determined with the limma package. The overlapping DEGs between male AS-related genes and OP-related genes were determined. The DEGs were validated by qRT-PCR in the blood samples of males with AS. Weighted gene co-expression network analysis (WGCNA) was utilized to establish a co-expression network to identify the hub genes. RESULTS: A total of 17 overlapping DEGs were identified; 6 genes in 17 overlapping DEGs were verified as the essential genes in the pathogenesis of OP in male AS by qRT-PCR analysis. After WGCNA, the modules of MEblue (> 0.6) and MEred (> 0.8) were screened out by the correlation analysis and were determined to function mainly in MAPK signaling pathway and osteoclast differentiation. Analysis of the two modules revealed VPS13B and SBNO2 as key genes due to the high degree of correlation. Both genes play an important role in bone metabolism regulation in male AS. Two hub genes MYD88 in MEblue and NCK1 in MEred with high degree of connectivity were selected. CONCLUSIONS: Gender-specific SBNO2 and VPS13B may be key genes involved in OP in male AS.

Long noncoding RNA-JPX predicts the poor prognosis of ovarian cancer patients and promotes tumor cell proliferation, invasion and migration by the PI3K/Akt/mTOR signaling pathway.

OBJECTIVE: To investigate the clinical functions and the detailed mechanism of long noncoding RNA (lncRNA) JPX in human ovarian cancer cell lines. PATIENTS AND METHODS: The expression of JPX in ovarian cancer tissues and cell lines was detected by Real-time polymerase chain reaction (RT-PCR). The correlation between JPX expression and prognosis was analyzed by follow-up data. The OVCAR-3 cell proliferation, invasion and migration were measured by methyl thiazolyl tetrazolium (MTT) assay, cloning formation assay and scratch assay. The cell apoptosis was detected by Bcl-2, Bax, and Caspase-3 activity. PI3K/mTOR inhibitor treatment and Western blot proved that JPX functions associated with PI3K/Akt/mTOR signaling and test the protein levels of p-PI3K, p-Akt, p-mTOR. RESULTS: RT-PCR results showed that the expression of JPX was upregulated in ovarian cancer tissues and ovarian cancer cell lines (p < 0.05), and it was significantly increased in large tumor tissues and metastatic lymph nodes (p < 0.05). The survival rate of high JPX expression patients was much lower than low JPX expression patients (p < 0.05), indicating that high expression of JPX predicted poor prognosis in patients with ovarian cancer. MTT assay, colony formation and scratch assay showed the repression of JPX and resulted with significantly decreased in cell proliferation, invasion and migration of OVCAR-3 cells compared with the control (p < 0.05). PI3K/mTOR inhibitor treatment showed overexpression of JPX could activate the PI3K/Akt/mTOR signaling pathway. Western blot assay showed that the expressions of p-PI3K, p-Akt, p-mTOR were significantly increased after overexpression of JPX (p < 0.05), and after the inhibition of PI3K/Akt/mTOR signaling pathway and overexpression of JPX, the tumor cell proliferation, invasion and migration were significantly repressed, compared with the control (p < 0.05). CONCLUSIONS: JPX could predict the poor prognosis in patients with ovarian cancer, which could promote the tumor cell proliferation, invasion and migration in human ovarian cancer cell lines and inhibited the cell apoptosis through activating PI3K/Akt/mTOR signaling pathway.

Methylene blue attenuates renal ischemia-reperfusion injury by negative regulation of NLRP3 signaling pathway.

OBJECTIVE: To investigate the effect of methylene blue (MB) on renal ischemia-reperfusion (IR) injury in mice and its possible relevant mechanisms. MATERIALS AND METHODS: A total of 30 male C57/BL6 mice aged 4 months old were randomly divided into the following three groups: Sham group (n=10), IR group (n=10), and MB group (n=10). Mice in MB group were treated with gavage continuously using methylene blue solution (dosage: 25 mg·kg-1·d-1) until they were 7 months old. Mice in the other two groups were administrated with the same amount of normal saline for gavage. After that, the abdomen of mice in Sham group was opened and closed, and bilateral renal pedicles of mice in IR group and MB group were occluded using a micro-artery clamp for 45 min for modeling. After modeling, the renal tissues and blood samples of mice were taken for detection. The levels of serum creatinine (Scr), blood urea nitrogen (BUN), and malondialdehyde (MDA), and activity of superoxide dismutase (SOD) in mice in each experimental group were detected and statistically analyzed, respectively. The degree of renal tubular necrosis in renal tissues of mice was observed under an optical microscope. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors [interleukin-1ß (IL-1ß), IL-18, IL-10 and transforming growth factor-ß1 (TGF-ß1)] in renal tissues of mice in each experimental group, followed by relevant statistical analyses. The expressions of relevant proteins [NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), nuclear factor-κB (NF-κB), caspase-1, pro-IL-1ß, and IL-1ß] in renal tissues of mice in each experimental group were detected via Western blotting, and the gray value of each band was detected for statistical analysis. RESULTS: It was found in this experimental study that Scr and BUN levels in MB group were significantly lower than those in IR group, and the differences were statistically significant (p<0.05). Compared with that in IR group, the degree of renal tubular necrosis in MB group was significantly alleviated, and the difference was statistically significant (p<0.05). Compared with those in IR group, the levels of inflammatory factors (IL-1ß and IL-18) in MB group were significantly decreased, but the levels of IL-10 and TGF-ß1 in MB group were significantly increased, and the differences were statistically significant (p<0.05). Compared with those in IR group, the activity of SOD in MB group was increased significantly, but the level of MDA was decreased, and the differences were statistically significant (p<0.05). The protein expressions of NLRP3, NF-κB, caspase-1, and pro-IL-1ß in MB group were decreased compared with those in IR group, but the expression of IL-1ß in MB group was increased, and the differences were statistically significant (p<0.05). CONCLUSIONS: We showed that methylene blue can alleviate the apoptosis and inflammatory response induced by renal IR injury in mice, and its relevant mechanism may be related to the fact that methylene blue can negatively regulate NLRP3 signaling pathway.

[Inventory building of phages against extensively drug-resistant Acinetobacter baumannii isolated from wounds of patients with severe burn and related characteristic analysis].

OBJECTIVE: To build inventory of phages against extensively drug-resistant Acinetobacter Baumannii isolated from wounds of inpatients of burn ICU and analyze related characteristics. METHODS: In 2014 and 2015, 131 strains of extensively drug-resistant Acinetobacter Baumannii were isolated from wounds of inpatients of burn ICU from one hospital in Chongqing. In 2015, 98 strains of extensively drug-resistant Acinetobacter Baumannii were isolated from wounds of inpatients of burn ICU from 6 hospitals in Guangdong province. Above-mentioned 229 strains were collected for conducting experiments as follows: (1) Multilocus sequence typing (MLST) of strains isolated from Chongqing and Guangdong province was analyzed. (2) Sewage co-culture method was applied for isolation of phages with above-mentioned strains and sewage from Chongqing and Guangdong province. Numbers of isolated phages and times of successful isolation and unsuccessful isolation were recorded. (3) The most prevalent subtypes of strains from Chongqing and Guangdong province in 2015 were collected, and their phages respectively underwent cross infection with all strains from Chongqing and those from Guangdong province. The lysis ability of phage was observed when phage underwent cross infection with the same subtype of strain or not the same, and the lytic ratio was calculated. (4) Fluid of phage in one type was randomly selected and equally divided into 3 parts, and its titer was determined by double dilution method. Then each part of phage fluid was subdivided into 3 small parts, which were cultured with LB fluid medium and respectively stored under the condition of -20 ℃, 4 ℃, and room temperature. After being stored for 1 month and 2 months, the titer of phage was determined for evaluating stability of phage. Data were processed with Fisher's exact test, chi-square test, and one-way analysis of variance. RESULTS: (1) The major type of strains from Chongqing in 2014 was ST368 (45%, 31/69), and major types of strains from Chongqing in 2015 were ST75 (26%, 16/62) and ST195 (24%, 15/62), while that from Guangdong province in 2015 was ST977 (46%, 45/98). (2) For strains from Chongqing, isolation effect of phage with sewage of Chongqing (8 times of successful isolation with 9 strains of phages and 1 time of unsuccessful isolation) was better than that with sewage of Guangdong province (1 time of successful isolation with 1 strain of phage and 7 times of unsuccessful isolation). For strains from Guangdong province, isolation effect of phage with sewage of Guangdong province (8 times of successful isolation with 6 strains of phages) was better than that with sewage of Chongqing (7 times of unsuccessful isolation with no phage). These differences were statistically significant (P<0.05 or P<0.01). There was no obvious difference in isolation effect of phage between strains from Chongqing with sewage of Chongqing and strains from Guangdong province with sewage of Guangdong province (P>0.05). (3) The ratios of phages of ST75 and ST977 extensively drug-resistant Acinetobacter Baumannii strains lysing the strains with the same type were respectively 13/16 and 8/9, which were obviously higher than those lysing the strains with different type (respectively 11/115 and 3/53, with χ(2) values respectively 48.23 and 68.46, P values below 0.001). (4) Compared with that before storage, titer of phage under storage condition of -20 ℃, 4 ℃, and room temperature for 1 month decreased by approximately 1 order of magnitude, and that for 2 months decreased by approximately 2 orders of magnitude. After being stored for 1 month and 2 months, there were no statistically significant differences in titer of phage among 3 storage conditions (with F values respectively 1.29 and 1.07, P values above 0.05). CONCLUSIONS: This study has successfully built an inventory covering 229 strains of phages of extensively drug-resistant Acinetobacter Baumannii. MLST of extensively drug-resistant Acinetobacter baumannii varies in different area and different time. Phage can be well isolated using sewage with the same source as that of strain. The lysis ability of phage is closely related to the MLST of strains. Inventory of phages should be built according to regional division. Moreover, phage cultured with LB fluid medium shows good stability without special requirements for storage conditions.

Knockdown of the inflammatory factor pentraxin-3 suppresses growth and invasion of lung adenocarcinoma through the AKT and NF-kappa B pathways.

Pentraxin-3 (PTX3), a modulator of tumor-associated inflammation, is known to be positively correlated with tumor grade and severity of malignancies, but the function and molecular underlying mechanisms of PTX3 remain unclear. In the present study, the expression of PTX3 in human lung adenocarcinoma (LAC) was examined by immunohistochemical assay using a tissue microarray procedure. A loss-of-function experiment was performed to explore the effects of lentiviral vector-mediated PTX3 shRNA (Lv-shPTX3) on cell growth and invasive potential in LAC cell lines (A549 and LETPα-2), assessed by MTT and Transwell assays, respectively. We found that the expression of PTX3 protein was significantly increased in LAC tissues compared with that in adjacent non-cancerous tissues (ANCT) (60.42% vs. 29.17%, P=0.004), and positively correlated with lymphatic invasion of the tumor (P=0.006). Furthermore, knockdown of PTX3 suppressed tumor proliferation and invasion of LAC cells, followed by decreased expression of p-AKT, p-NF-kappa B, PCNA, and MMP-9. Taken together, our findings demonstrate that upregulation of PTX3 expression is correlated with tumor metastasis of LAC patients, and knockdown of PTX3 blocks the development of LAC through inhibition of the AKT and NF-kappa B pathways, suggesting that PTX3 may serve as a potential therapeutic target for the treatment of cancer.

Effective antitumor gene therapy delivered by polyethylenimine-conjugated stearic acid-g-chitosan oligosaccharide micelles.

Non-viral vesicle composing of low-molecular weight polyethylenimine-conjugated stearic acid-g-chitosan oligosaccharide (CSOSA-g-PEI) was synthesized for gene delivery and therapy. The synthesized CSOSA-g-PEI had good ion-buffer capabilities and DNA-binding capacity, which could form positively charged nano-sized particles (100-150 nm) with plasmid DNA; in vitro gene transfection tests demonstrated that CSOSA-g-PEI presented much lower cytotoxicity and corresponding transfection efficiency in comparison with Lipofectamine 2000 in both human cancer cells (Hela and MCF-7). The gene transfection of CSOSA-g-PEI/pDNA could be further enhanced in the presence of serum or by adding arginine during incubation of CSOSA-g-PEI micelles with plasmid DNA. The biodistribution experiments demonstrated CSOSA-g-PEI conjugate highly localized in the tumor tissue and indicated a persistently increased accumulation. In vivo antitumor activity results showed that CSOSA-g-PEI/plasmid pigment epithelium-derived factor formulation could effectively suppress the tumor growth (above 60% tumor inhibition) without systematic toxicity against animal body after intravenous injection.

Effect of PEG2000 on drug delivery characterization from solid lipid nanoparticles.

The purpose of this work is to develop a PEG2000-modified solid lipid nanoparticles (SLN) intended to encapsulate a drug within their matrix and to study their characteristics. In the present report, drug-loaded SLN were prepared by a novel solvent diffusion method in an aqueous system. Monostearin and PEG2000 were used as carrier material and modifying agent, respectively. The model drug salbutamol sulphate was incorporated to study the characterization of entrapment efficiency, size, zeta potential (charge) and drug delivery characterization. In the test solution of pH 7.2 phosphate buffer, drug-release behavior from SLN suspension exhibited a biphasic pattern. With the monoastearin-based SLN, a distinctly prolonged release over a monitored period of 14 days was observed after a burst drug release in the first 8 hours. Over the monitored period of prolonged release, there was delayed release in the first 5 days with nearly 2.51% of the drug released each day, following which a slightly higher release rate (8.14% per day) appeared in the last 9 days. In contrast, the drug release rate from PEG2000-modified SLN was faster. Nevertheless, further work is required in order to optimize the release behavior of various entrapped drugs. These results also demonstrate that modification with PEG2000 can accelerate release of hydrophilic small molecule drugs from SLN.

Novel self-aggregates of chitosan oligosaccharide grafted stearic acid: preparation, characterization and protein association.

A novel hydrophobically modified chitosan oligosaccharide (CSO) containing 5.4 stearic acid (SA) groups per 100 anhydroglucose units was synthesized by an 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated coupling reaction. The purified copolymer was structurally characterized by IR spectroscopy. Characteristics of self-aggregates of the amphiphilic copolymers were studied by fluorescence spectroscopy and dynamic light scattering. The critical aggregation concentration (cac) value of the self-aggregates in aqueous solution was determined by measuring the fluorescence intensity of pyrene as a fluorescent probe. Mean diameter of self-aggregates in pH 7.0 PBS was 25.0 +/- 14.7 nm with a unimodal size distribution. The diameter, as well as the zeta potential of self-aggregates increased when the pH value of dispersion medium decreased. Bovine serum albumin (BSA) was further enveloped in the interface of different single self-aggregate and formed nanoparticles. The size of BSA-loaded stearic acid modified CSO nanoparticles depended on the pH values of the dispersed aqueous vehicle, and the size diminished when the pH values of the dispersed aqueous vehicle decreased, whilst, the BSA encapsulation efficiency enhanced. The nanoparticles were characterized by Transmission Electron Microscopy (TEM). BSA release from stearic acid modified CSO nanoparticles decreased when the pH values of the delivery media decreased, in the range from 7.2 to 5.8.

A novel preparation of solid lipid nanoparticles with cyclosporin A for prolonged drug release.

Solid lipid nanoparticles were prepared by a novel solvent diffusion method in an aqueous system. The lipophilic model drug cyclosporin A was incorporated into SLN to study encapsulation efficiency, zeta potential (charge) and drug delivery. Stearylamine and cyclosporin A were dissolved in ethanol and acetone and the resultant organic solution was dropped into water at 60 degrees C. The drug-loaded SLN suspension quickly formed with an azury color. After burst drug release with 18% of the drug over the first 12 hours, a distinctly prolonged release over a monitored period of 16 days was observed, with nearly 4% of the drug being released each day. These results demonstrate the suitability of SLN produced with the proposed method as a prolonged release formulation for lipophilic drugs.

Preparation and characterization of solid lipid nanoparticles containing peptide.

Solid lipid nanoparticles (SLN) are an alternative colloidal carrier system for controlled drug delivery. However, only a few have been studied regarding the incorporation of peptides into SLN, due to the hydrophilic peptide not easy to enter the lipophilic matrix of SLN. In the present report, peptide-loaded solid lipid nanoparticles were prepared by a novel solvent diffusion method in an aqueous system. The model peptide gonadorelin was incorporated to study the entrapment efficiency, size, zeta potential (charge) and drug delivery characterization. Gonadorelin and monostearin were dissolved in acetone and ethanol at 50 degrees C in water bath, the resultant organic solution was poured into an aqueous containing 1% polyvinyl alcohol (PVA) under mechanical agitation. The peptide-loaded solid lipid nanoparticles were quickly produced and separated by centrifugation. The average volume diameter of gonadorelin-loaded SLN is 421.7 nm and the zeta potential of SLN is -21.1 mV dispersed in distilled water. Up to 69.4% of gonadorelin can be incorporated. In vitro release of gonadorelin from SLN is slow. In the test solution of a 0.1N hydrochloric acid for 2h and then transferred in a pH 6.8 phosphate buffer (simulative gastrointestinal fluid), the drug-release behavior from SLN suspension exhibited a biphasic pattern. After burst drug-release at the first 6h at a percentage of 24.4% of loaded gonadorelin, a distinctly prolonged release over a monitored period of 12 days was observed and nearly 3.81% of drug was released in each day. In the test solution of a pH 6.8 phosphate buffer (simulative intestinal fluid), the drug-release rate from SLN was similar to that in the simulative gastrointestinal fluid. Further, a novel preparation method in the present research for peptide-loaded SLN was established. These results also demonstrate the principle suitability of SLN as a prolonged release formulation for hydrophilic peptide drugs.

Preparation of solid lipid nanoparticles with clobetasol propionate by a novel solvent diffusion method in aqueous system and physicochemical characterization.

Solid lipid nanoparticles (SLN) are a colloidal carrier system for controlled drug delivery. Monostearin SLN were prepared by a novel solvent diffusion method in an acidic aqueous system in order to improve the recovery of the method. The lipophilic model drug clobetasol propionate was incorporated to study the recovery of nanoparticles, entrapment efficacy, zeta potential (charge) and drug delivery characterization. The drug and monostearin were dissolved in acetone and ethanol at 50 degrees C in water bath, the resultant organic solution was poured into an acidic aqueous (pH 1.10) containing 1% polyvinyl alcohol (PVA) under mechanical agitation at room temperature. The drug loaded SLN was quickly produced with an aggregation state and easily separated by centrifugation. The recovery of nanoparticles was markedly increased compared to using a usual aqueous (pH 5.73) containing the same concentration of PVA. After burst drug release at the first 3 h, a distinctly prolonged release over a monitored period of 4 days was observed and nearly 6% drug was released in each day. Further, a novel preparation method and the optimized separation parameters in the present research for SLN were established. These results also demonstrate the principle suitability of SLN as a prolonged release formulation for lipophilic drugs.

Multi-component reactive transport modeling of natural attenuation of an acid groundwater plume at a uranium mill tailings site.

Natural attenuation of an acidic plume in the aquifer underneath a uranium mill tailings pond in Wyoming, USA was simulated using the multi-component reactive transport code PHREEQC. A one-dimensional model was constructed for the site and the model included advective-dispersive transport, aqueous speciation of 11 components, and precipitation-dissolution of six minerals. Transport simulation was performed for a reclamation scenario in which the source of acidic seepage will be terminated after 5 years and the plume will then be flushed by uncontaminated upgradient groundwater. Simulations show that successive pH buffer reactions with calcite, Al(OH)3(a), and Fe(OH)3(a) create distinct geochemical zones and most reactions occur at the boundaries of geochemical zones. The complex interplay of physical transport processes and chemical reactions produce multiple concentration waves. For SO4(2-) transport, the concentration waves are related to advection-dispersion, and gypsum precipitation and dissolution. Wave speeds from numerical simulations compare well to an analytical solution for wave propagation.

Cowpox virus genome encodes a second soluble homologue of cellular TNF receptors, distinct from CrmB, that binds TNF but not LT alpha.

We show the cowpox genome (Brighton Red strain) contains a single copy gene, crmC, expressed at late times during viral infection, encoding a soluble, secreted protein whose sequence marks it as a new member of the TNF receptor family. The cysteine-rich protein contains 186 amino acids, the N-terminal 21 of which constitute a signal peptide, and two potential N-linked glycosylation sites. The approximately 25-kDa recombinant protein binds TNF specifically and completely inhibits TNF-mediated cytolysis. The strongest sequence homologues are the ligand-binding regions of the type II cellular TNF receptor (TNFRII) and CrmB, a distinct pox virus gene also encoding a soluble TNF binding protein. Unlike TNFRII and CrmB, CrmC does not bind lymphotoxin (LT alpha, TNF beta) and lacks the conserved (but nonhomologous) approximately 150-residue C-terminal domain of CrmB proteins. The presumed function of CrmC is viral inhibition of host-elicited TNF.

Cowpox virus contains two copies of an early gene encoding a soluble secreted form of the type II TNF receptor.

The inverted terminal repeats of the DNA of cowpox virus (Brighton Red strain) contain the crmB gene, an additional member of a family of viral genes that modify cytokine responses to infection. The crmB gene is transcribed from an early promoter. The primary product is a 355-amino-acid protein containing a signal peptide sequence and three potential N-linked glycosylation sites. The mature gene product is a secreted soluble protein that has an apparent molecular mass of 48 kDa. TNF alpha and TNF beta bind to this protein in a competitive manner, consistent with the sequence of its N-terminal 176 amino acids, which closely resembles the ligand-binding domains of the type II (75-kDa) human TNF receptor. The sequence of the C-terminal 161 amino acids of the CrmB protein is unlike that of human TNF receptors, but overall, the CrmB protein is similar to the T2 proteins of the leporipoxviruses (48% identity) and the predicted product of the G4R/G2R open reading frame of variola virus (85% identity), suggesting that not only the TNF-binding domains but also the C-terminal regions contribute to the functions of these viral proteins. These results show that orthopoxiviruses such as cowpox virus encode secreted forms of TNF receptors that can contribute to the modification of TNF-mediated antiviral processes.

Transcription of the terminal loop region of vaccinia virus DNA is initiated from the telomere sequences directing DNA resolution.

The telomeres of vaccinia virus DNA are transcribed at late times after infection. Analysis of cDNAs of RNA transcripts of the terminal loop region of the viral DNA shows that both inverted and complementary forms of the terminal loop region are transcribed. These late RNAs, which contain 5' poly(A) sequences, do not appear to encode any proteins. The transcriptional start sites for most of these RNAs are within the sequences that direct the resolution of concatemeric DNA replication intermediates (M. Merchlinsky and B. Moss, 1989, J. Virol. 63, 4354-4361). This suggests that the process of DNA resolution may involve transcription initiated from the telomere sequences required for resolution.