LncRNA CCAT1 Upregulates ATG5 to Enhance Autophagy and Promote Gastric Cancer Development by Absorbing miR-140-3p.

BACKGROUND: Long noncoding RNA colon cancer-associated transcript 1 (LncRNA CCAT1) is highly expressed in gastric cancer tissues and plays a role in autophagy. However, the underlying mechanism still needs to be further clarified. OBJECTIVE: To study the role of LncRNA CCAT1 in regulating autophagy of gastric cancer cells, analyze its downstream targets, and elucidate the mechanism. METHODS: qPCR detected the expression of LncRNA CCAT1 in gastric cancer cells. The proliferation, migration, and invasion ability of LncRNA CCAT1 and the expression level of autophagy-related proteins in gastric cancer cells were detected. Bioinformatics method predicted the downstream targets of LncRNA CCAT1, and they were verified by dual-luciferase assay. The relationship between LncRNA CCAT1, miR-140, and ATG5 was verified by co-transfection, and the expression levels of ATG5 and ATG5-ATG12 complex proteins were detected. Finally, the role of LncRNA CCAT1 in vivo was confirmed by gastric cancer transplantation model. RESULTS: LncRNA CCAT1 was highly expressed in gastric cancer cells. LncRNA CCAT1 can promote the proliferation, migration, invasion, and autophagy activity of gastric cancer cells. LncRNA CCAT1 can bind to miR-140-3p and regulate its expression, while miR-140-3p further regulates the expression of ATG5. Overexpression of LncRNA CCAT1 can promote tumor growth in nude mice. After LncRNA CCAT1 silencing, the positive expression rate of ATG5 in nude mice was low. CONCLUSION: LncRNA CCAT1 may inhibit the expression of miR-140-3p by sponge adsorption, thus weakening its inhibitory effect on ATG5. Eventually, gastric cancer cells were more prone to autophagy under the pressure of stress.

Clinical and oncologic findings of extraprostatic extension on needle biopsy in de novo metastatic prostate cancer.

This study aimed to explore the clinical and oncologic findings in patients with de novo metastatic prostate cancer (mPCa) and extraprostatic extension (EPE) on biopsy. We retrospectively evaluated data on 630 patients with de novo mPCa between January 2009 and December 2017 in the West China Hospital (Chengdu, China), including evaluating the relationships between EPE and other variables and the association of EPE with survival outcomes by the Chi-square test, Kaplan-Meier curves, and the Cox proportional-hazards model. EPE was found in 70/630 patients, making a prevalence of 11.1%. The presence of EPE on biopsy was associated with higher Gleason scores and higher incidence of neuroendocrine differentiation (NED), intraductal carcinoma of the prostate (IDC-P), and perineural invasion (PNI). Compared with those without EPE, patients with EPE had shorter castration-resistant prostate cancer-free survival (CFS; median: 14.1 vs 17.1 months, P = 0.015) and overall survival (OS; median: 43.7 vs 68.3 months, P = 0.032). According to multivariate analysis, EPE was not an independent predictor for survival. Subgroup analyses demonstrated that patients with favorable characteristics, including negative NED or IDC-P status, Eastern Cooperative Oncology Group (ECOG) score <2, and prostate-specific antigen (PSA) <50 ng ml-1, had worse prognoses if EPE was detected. In patients with PSA <50 ng ml-1, EPE was a negative independent predictor for OS (hazard ratio [HR]: 4.239, 95% confidence interval [CI]: 1.218-14.756, P = 0.023). EPE was strongly associated with other aggressive clinicopathological features and poorer CFS and OS. These data suggest that EPE may be an indicator of poor prognosis, particularly in patients, otherwise considered likely to have favorable survival outcomes.

Optimization of Timing and Times for Administration of Atorvastatin-Pretreated Mesenchymal Stem Cells in a Preclinical Model of Acute Myocardial Infarction.

Our previous studies showed that the combination of atorvastatin (ATV) and single injection of ATV-pretreated mesenchymal stem cells (MSCs) (ATV -MSCs) at 1 week post-acute myocardial infarction (AMI) promoted MSC recruitment and survival. This study aimed to investigate whether the combinatorial therapy of intensive ATV with multiple injections of ATV -MSCs has greater efficacy at different stages to better define the optimal strategy for MSC therapy in AMI. In order to determine the optimal time window for MSC treatment, we first assessed stromal cell-derived factor-1 (SDF-1) dynamic expression and inflammation. Next, we compared MSC recruitment and differentiation, cardiac function, infarct size, and angiogenesis among animal groups with single, dual, and triple injections of ATV -MSCs at early (Early1, Early2, Early3), mid-term (Mid1, Mid2, Mid3), and late (Late1, Late2, Late3) stages. Compared with AMI control, intensive ATV significantly augmented SDF-1 expression 1.5∼2.6-fold in peri-infarcted region with inhibited inflammation. ATV -MSCs implantation with ATV administration further enhanced MSC recruitment rate by 3.9%∼24.0%, improved left ventricular ejection fraction (LVEF) by 2.0%∼16.2%, and reduced infarct size in all groups 6 weeks post-AMI with most prominent improvement in mid groups and still effective in late groups. Mechanistically, ATV -MSCs remarkably suppressed inflammation and apoptosis while increasing angiogenesis. Furthermore, triple injections of ATV -MSCs were much more effective than single administration during early and mid-term stages of AMI with the best effects in Mid3 group. We conclude that the optimal strategy is multiple injections of ATV -MSCs combined with intensive ATV administration at mid-term stage of AMI. The translational potential of this strategy is clinically promising. Stem Cells Translational Medicine 2019;8:1068-1083.

Prior switching to a second-line nonsteroidal antiandrogen does not impact the therapeutic efficacy of abiraterone acetate in patients with metastatic castration-resistant prostate cancer: a real-world retrospective study.

Even in the era of novel targeted agents, switching to a second-line nonsteroidal antiandrogen (NSAA) is still widely used in treating metastatic castration-resistant prostate cancer (mCRPC), especially in undeveloped countries. However, whether prior treatment with a second-line NSAA would impact the efficacy of abiraterone acetate (Abi) remains uncertain. In the current study, 87 mCRPC patients treated with Abi were analyzed. Among them, 21 were treated with a second-line NSAA (from bicalutamide to flutamide) before receiving abiraterone, while the remaining 66 received Abi directly. Therapeutic efficacy of Abi was compared between those with and without prior second-line NSAA using Kaplan-Meier curves, log-rank test, and Cox regression models. The therapeutic efficacy of Abi was similar between those with or without the prior switching treatment of flutamide, in terms of either prostate-specific antigen progression-free survival (PSA-PFS, 5.5 vs 5.6 months, P = 0.967), radiographic progression-free survival (rPFS, 12.8 vs 13.4 months, P = 0.508), overall survival (OS, not reached vs 30.6 months, P = 0.606), or PSA-response rate (71.4% [15/21] vs 60.6% [40/66], P = 0.370). This is the first time that the impact of prior switching of treatment to a second-line NSAA on the efficacy of Abi in mCRPC patients has been addressed. Our data support that, use of prior sequential bicalutamide and flutamide does not seem to preclude response to abiraterone, although larger cohort studies and, ideally, a randomized controlled trial are needed. These findings will facilitate doctors' decision-making in the treatment of mCRPC patients, especially for those with previous experience of switching NSAA second-line treatments in the clinic.

Chronic aerobic exercise training alleviates myocardial fibrosis in aged rats through restoring bioavailability of hydrogen sulfide.

Age-related fibrosis is attenuated by aerobic exercise; however, little is known concerning the underlying molecular mechanism. To address this question, aged rats were given moderate-intensity exercise for 12 weeks. After exercise in aged rats, hydrogen sulfide levels in plasma and heart increased 39.8% and 90.9%, respectively. Exercise upregulated expression of cystathionine γ-lyase and 3-mercaptopyruvate sulfurtransferase in heart of aged rats. Furthermore, aged rats were given moderate-intensity exercise for 12 weeks or treated with NaHS (intraperitoneal injection of 0.1 mL/kg per day of 0.28 mol/L NaHS). After exercise in aged rats, Masson-trichrome staining area decreased 34.8% and myocardial hydroxyproline levels decreased 29.6%. Exercise downregulated expression of collagen-I and α- smooth muscle actin in heart of aged rats. Exercise in aged rats reduced malondialdehyde levels in plasma and heart and 3-nitrotyrosine in heart. Exercise in aged rats reduced mRNA and protein expression of C/EBP homologous protein, glucose regulated protein 78, and X-box protein 1. Exercise also reduced mRNA and protein expression of interleukin 6 and monocyte chemotactic protein 1and suppressed activation of c-Jun N-terminal kinase in aging heart. Similar effects were demonstrated in aged rats treated with NaHS. Collectively, exercise restored bioavailability of hydrogen sulfide in the heart of aged rats, which partly explained the benefits of exercise against myocardial fibrosis of aged population.

Serum miR-300 as a diagnostic and prognostic biomarker in osteosarcoma.

In order to determine whether microRNA (miR)-300 is a diagnostic and prognostic biomarker in osteosarcoma, the miR-300 levels in serum of 114 osteosarcoma patients and 114 healthy controls were compared, followed by serum analysis of the differences between the pre-operative and post-operative sera of these osteosarcoma patients. It was observed that the concentration levels of miR-300 in the serum of osteosarcoma patients was significantly higher than those in the serum of healthy controls (P<0.01). Furthermore, the concentration levels of miR-300 in the post-operative serum were significantly reduced when compared with the pre-operative serum levels (P<0.001). High miR-300 levels in serum correlated significantly with clinical stage, distant metastasis and poor survival of osteosarcoma patients. Notably, serum miR-300 was an independent prognostic marker for osteosarcoma. In conclusion, our results suggested that serum miR-300 may be a potential and useful noninvasive biomarker for the early detection of osteosarcoma.

Comparison of Antibody Immune responses to different HIV-1 Envelope Glycoprotein Mutants.

The identification of immunogens is crucial for human immunodeficiency virus type 1 (HIV-1) vaccine development. In our previous study, we demonstrated that HIV-1 envelope glycoprotein mutants based on the equine infectious anemia virus (EIAV)attenuated vaccine enhance immunogenicity, both for DNA immunization alone and as a combined DNA prime-vaccinia boost immunization. An RV144 clinical trial has demonstrated that an envelope protein boost may provide some degree of protection against HIV-1 infection. In order to explore the antibody immune responses to two HIV-1 envelope glycoprotein mutants based on the EIAV vaccine and wild-type envelope glycoprotein, mice and guinea pigs were immunized using a DNA prime-protein boost immunization strategy. The result showed, compared with wild-type gp140, gp140 2M (which contained 2 sites amino acid mutations) and gp140 5M (which contained 5 sites amino acid mutations) increased env-specific IgG and IgG3 binding antibody titers.Gp140 2M resulted in a slight improvement in the neutralizing antibody response against sensitive HIV-1 isolates compared with gp140. These findings have implications for HIV-1 vaccine development based on the HIV-1 CN54 envelope glycoprotein.

Contribution of mitochondrial function to exercise-induced attenuation of renal dysfunction in spontaneously hypertensive rats.

It is well known that exercise training exhibits renal protective effects in animal models of hypertension and chronic renal failure. However, the mechanisms regulating these effects of exercise training remain unclear. This study aimed to investigate the role of mitochondrial function in exercise-induced attenuation of renal injury in spontaneously hypertensive rats (SHR). The adult male SHR and age-matched normotensive Wistar-Kyoto rats (WKY) were given moderate-intensity exercise for 12 weeks or treated with MitoQ10 for 8 weeks. In this work, exercise training in SHR reduced blood pressure, and effectively attenuated renal dysfunction, marked by reduced creatinine excretion, albuminuria, blood urea nitrogen, and glomerular sclerosis. Exercise training in SHR reduced MDA levels in plasma and kidneys and suppressed formation of 3-nitrotyrosine in kidneys. Exercise training suppressed mitochondrial ROS and [Formula: see text] formation, enhanced ATP formation, reduced mitochondrial swelling, and restored electron transport chain enzyme activity in kidneys of SHR. Furthermore, exercise training upregulated protein expression of uncoupling protein 2 and manganese superoxide dismutase in kidneys of SHR. In addition, treatment with mitochondria-targeted antioxidant MitoQ10 exhibited similar renal protective effects in SHR. In conclusion, chronic aerobic exercise training preserved mitochondrial function and abated oxidative stress in the kidneys of SHR, which may in part explain the protective effect of exercise on renal function and structure in hypertensive individuals.

Contribution of receptor for advanced glycation end products to vasculature-protecting effects of exercise training in aged rats.

The aim of present work was to investigate the underlying mechanism of vasculature-protecting effects of exercise training in aged rats. Experiment 1: aged rats were given moderate-intensity exercise for 12 weeks. Exercise training suppressed advanced glycation evidenced by reduced activity of aldose reductase, increased activity of glyoxalase 1, reduced levels of methylglyoxal and N(ε)-(carboxymethyl) lysine, and decreased expression of receptor for advanced glycation end products (RAGE) in aged aortas. Experiment 2: aged rats were given moderate-intensity exercise for 12 weeks or treated with FPS-ZM1, an inhibitor of RAGE. Exercise training attenuated aortic stiffening with age marked by reduced collagen levels, increased elastin levels and reduced pulse wave velocity (PWV), and prevented aging-related endothelial dysfunction marked by restored endothelium-mediated vascular relaxation of aortas in response to acetylcholine. Exercise training in aging aortas reduced formation of malondialdehyde, 3-nitrotyrosin and reactive oxygen species, increased GSH/GSSG ratio, suppressed activation of NFκB, and reduced levels of IL-6 and chemokine (C-C motif) ligand 2. Similar effects were demonstrated in aged rats treated with FPS-ZM1. Collectively, exercise suppressed advanced glycation in the aortas of aged rats, which, at least in part, explained the vasculature-protecting effects of exercise training in aged population.

Contribution of renin-angiotensin system to exercise-induced attenuation of aortic remodeling and improvement of endothelial function in spontaneously hypertensive rats.

INTRODUCTION: It is well known that exercise alleviates aortic remodeling and preserves endothelial function in spontaneously hypertensive rats (SHRs). However, the underlying molecular mechanism remains unclear. This study aimed to investigate the role of renin-angiotensin system (RAS) components in exercise-induced attenuation of aortic remodeling and improvement of endothelial function in an animal model of human essential hypertension. METHODS: The 10-week-old male SHR and age-matched normotensive Wistar-Kyoto rats were given moderate-intensity exercise for 12weeks (four groups, n=80-86 in each group). RESULTS: In this work, exercise training reduced blood pressure and effectively attenuated aortic remodeling, marked by a reduction in aortic weight/length, wall thickness, and aortic levels of elastin and hydroxyproline, and improved endothelium-mediated vascular relaxations of aortas in response to acetylcholine. Exercise training in SHR reduced angiotensin II (AngII) levels and enhanced Ang-(1-7) levels in aortas. Exercise training in SHR suppressed aortic angiotensin-converting enzyme (ACE) and AngII type 1 receptor (AT1R) messenger RNA (mRNA) levels and protein expression and up-regulated ACE2, AngII type 2 receptor, and Mas mRNA levels and protein expression. In addition, exercise training in SHR increased levels of microRNA-27a (targeting ACE) and microRNA-155 (targeting AT1R) and decreased levels of microRNA-143 (targeting ACE2) in the aortas. CONCLUSION: Chronic aerobic exercise training improved RAS balance in the aortas, which may in part explain the protective effect of exercise on aortic function and structure. SUMMARY: Chronic aerobic exercise training improved RAS balance in the aortas, which may explain the protective effect of exercise on aortic function and structure, at least in part.

Chronic aerobic exercise training attenuates aortic stiffening and endothelial dysfunction through preserving aortic mitochondrial function in aged rats.

Aging leads to large vessel arterial stiffening and endothelial dysfunction, which are important determinants of cardiovascular risk. The aim of present work was to assess the effects of chronic aerobic exercise training on aortic stiffening and endothelial dysfunction in aged rats and investigate the underlying mechanism about mitochondrial function. Chronic aerobic exercise training attenuated aortic stiffening with age marked by reduced collagen concentration, increased elastin concentration and reduced pulse wave velocity (PWV), and prevented aging-related endothelial dysfunction marked by improved endothelium-mediated vascular relaxation of aortas in response to acetylcholine. Chronic aerobic exercise training abated oxidative stress and nitrosative stress in aortas of aged rats. More importantly, we found that chronic aerobic exercise training in old rats preserved aortic mitochondrial function marked by reduced reactive oxygen species (ROS) formation and mitochondrial swelling, increased ATP formation and mitochondrial DNA content, and restored activities of complexes I and III and electron-coupling capacity between complexes I and III and between complexes II and III. In addition, it was found that chronic aerobic exercise training in old rats enhanced protein expression of uncoupling protein 2 (UCP-2), peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), manganese superoxide dismutase (Mn-SOD), aldehyde dehydrogenase 2 (ALDH-2), prohibitin (PHB) and AMP-activated kinase (AMPK) phosphorylation in aortas. In conclusion, chronic aerobic exercise training preserved mitochondrial function in aortas, which, at least in part, explained the aorta-protecting effects of exercise training in aging.

TRIMe7-CypA, an alternative splicing isoform of TRIMCyp in rhesus macaque, negatively modulates TRIM5α activity.

The existence of innate, host-specific restriction factors is a major obstacle to the development of nonhuman primate models for AIDS studies, and TRIM5α is one of the most important of these restriction factors. In recent years, a TRIM5 chimeric gene that was retrotransposed by a cyclophilin A (CypA) cDNA was identified in certain macaque species. The TRIM5α-CypA fusion protein, TRIMCyp, which was expressed in these monkeys, had lost its restriction ability toward HIV-1. We previously found that TRIMe7-CypA, an alternative splicing isoform of the TRIMCyp transcripts, was expressed in pig-tailed and rhesus macaques but absent in long-tailed macaques. In this study, the anti-HIV-1 activity of TRIMe7-CypA in the rhesus macaque (RhTRIMe7-CypA) was investigated. The over-expression of RhTRIMe7-CypA in CrFK, HeLa and HEK293T cells did not restrict the infection or replication of an HIV-1-GFP reporter virus in these cells. As a positive control, rhesus (rh)TRIM5α strongly inhibited the reporter virus. Intriguingly, the anti-HIV-1 activity of RhTRIM5α was significantly reduced in a dose-dependent manner by the co-repression of RhTRIMe7-CypA. Our data indicate that although the RhTRIMe7-CypA isoform does not appear to restrict HIV-1, it may act as a negative modulator of TRIM family proteins, presumably by competitive inhibition.

Intercellular adhesion molecule 1/2 and E-selectin in plasma cell mastitis: immunohistochemical study of 35 cases.

Plasma cell mastitis (PCM) is one of the most frequently encountered inflammatory diseases of the nonlactating breast. Histologically, PCM is characterized by infiltration of relatively abundant plasma cells into the mammary ducts. Its pathogenesis has remained unknown. In this study, we immunolocalized intercellular adhesion molecule (ICAM) 1 and 2 and E-selectin, all of which play pivotal roles in the inflammatory process, in 35 cases of PCM. We then compared the results with those of non-PCM and nonpathologic breast tissue. In the ductal epithelium, ICAM-1 immunoreactivity was significantly more pronounced in PCM than in non-PCM (P = .045). Both ICAM-1 (P < .001) and ICAM-2 (P = .001) were significantly more pronounced in PCM than in nonpathologic breast tissue. However, no significant differences in ICAM-2 and E-selectin immunoreactivity were detected between ductal epithelium of PCM and non-PCM. ICAM-1, but not ICAM-2 or E-selectin, demonstrated significantly higher immunoreactivity in endothelial cells of PCM than in nonpathologic breast (P < .001). These results all suggest that ICAM-1 in both ductal epithelium and endothelium plays important roles in the inflammatory process of PCM, possibly through margination, extravasation, and attachment of plasma cells and lymphocytes, which may result in continuous inflammatory cell homing to ductal epithelial cells.

[Spectra analysis of dissolved organic matter in pretreatment process of leachate treated by reverse osmosis].

In order to examine the removal of organic matter in the leachate which results in reverse osmosis (RO) membrane fouling, and to provides a reference to select appropriate pretreatment processes of RO, synchronous-scan fluorescence, three-dimensional excitation emission matrix fluorescence spectroscopic and UV-Vis spectrum of the dissolved organic matter (DOM) in different molecular weight range in effluent from each leachate process of "biochemical (UASB+A/O)and UF" pretreatment in some incineration plant were examined. The results of synchronous fluorescence spectra analysis showed that DOM in the wavelength range of 250-320 nm with all the molecular weight and in the wavelength>320 nm with molecular weight>1 KDa was removed obviously by the pretreatment processes. The results of three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectra showed that the pretreatment processes removed low-excitation wavelength tyrosinelike, low-excitation wavelength tryptophan-like and high-excitation wavelength tryptophan-like with all the molecular weight off, and fulvic-like matter and high-excitation wave length tyrosine-like with molecular weight>1 KDa effectively. The results of UV-Vis spectra analysis showed that the pretreatment processes removed DOM of molecular weight>1 KDa with pi-pi transition and DOM of all molecular weight with conjugated system of the benzene ring structure. It was concluded that the removal of both fulvic-like matter and high-excitation wave length tyrosine-like with the wavelength>320 nm, molecular weight<1 KDa and with pi-pi transition should be strengthened for controlling (RO) membrane fouling, when leachate was treated by RO with the pretreatment processes of "biochemical(UASB+A/O)and UF".

The TRIMCyp genotype in four species of macaques in China.

The tripartite motif protein (TRIM)5α/CypA fusion protein TRIMCyp in Old World monkeys is generally considered unable to restrict HIV-1 replication. Monkeys with TRIMCyp can serve as a unique animal model for studies of HIV-1 infection. The present study investigated the distribution and expression status of TRIMCyp in four species of macaques originating from China and its borderlands: pigtail macaques (Macaca nemestrina), rhesus macaques (Macaca mulatta), long-tailed macaques (Macaca fascicularis), and Tibetan macaques (Macaca thibetana). The results revealed that the frequencies of the TRIMCyp genotype were significantly different among different species and even within different populations of the same species. Interestingly, the TRIMCyp genotype was more prevalent among macaques originating from Yunnan and surrounding regions than those from other regions of China. Importantly, TRIMCyp individuals were first identified in Chinese M. mulatta originating from Yunnan, although multiple earlier studies failed to find CypA retrotransposition in this subspecies. Furthermore, TRIMe7-CypA, one of the splicing isoforms of the TRIMCyp transcript was expressed in M. nemestrina and M. mulatta but not M. fascicularis. The intra- and interspecies polymorphisms in the deduced TRIMCyp amino acid sequences of these macaques were also analyzed. Taken together, the data in this study provide important information about the genomic background of TRIMCyp among major species of Chinese macaques.

Contribution of hydrogen sulfide and nitric oxide to exercise-induced attenuation of aortic remodeling and improvement of endothelial function in spontaneously hypertensive rats.

It is well known that exercise training attenuates aortic remodeling and improves endothelial function in spontaneously hypertensive rats (SHR). However, the underlying molecular mechanism remains unclear. Hydrogen sulfide (H(2)S) and nitric oxide (NO), as two established physiologic messenger molecules, have important roles in the development of aortic remodeling and endothelial dysfunction in hypertensive animals and patients. In this work, it was found that exercise training had no significant effect on blood pressure, but effectively attenuated baroreflex dysfunction in SHR. Exercise training in SHR attenuated aortic remodeling and improved endothelium-mediated vascular relaxations of aortas in response to acetylcholine. Interestingly, exercise training in SHR restored plasma H(2)S levels and aortic H(2)S formation and enhanced levels of mRNA for cystathionine γ-lyase in aortas. Furthermore, exercise training in SHR resulted in augmentation of nitrite and nitrate (NOx) contents and reduction of asymmetric dimethylarginine contents of aortas, upregulation of dimethylarginine dimethylaminohydrolase 2, and phosphorylation of nitric oxide synthase 3, but had no significant effect on protein levels of NOS3. In addition, exercise training could effectively reduce malondialdehyde production and suppressed formation of O(2) (-), and OONO(-) in aortas of SHR through enhancing activities of superoxide dismutase and catalase, and suppressing NADPH oxidase activity. In conclusion, exercise training ameliorates aortic hypertrophy and endothelial dysfunction, possibly via restoring bioavailabilities of hydrogen sulfide and nitric oxide in SHR.

Proteome of melamine urinary bladder stones and implication for stone formation.

Melamine can cause urinary stones related to nephropathy of the kidney and hyperplasia or carcinoma of the bladder, but the mechanism of stone formation is not well understood. In this study, male rats were administered melamine for thirteen weeks to establish melamine bladder stone models and the stones were analysed by Fourier transform infrared (FTIR) spectroscopy, powder X-ray diffraction (XRD), energy dispersive X-ray (EDX) spectroscopy, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) and western blot, respectively, for the composition and proteome, and to explore the implication of proteins for stone formation. The results showed bladder stones were composed of predominant melamine and a few amount of proteins. The proteins had a wide range of molecular weights and 1051 proteins were identified. Gene Ontology (GO) classification of the identified proteins showed most proteins were from injured cells, involved in various metabolic processes and had binding functions. Of the identified proteins, there were a few inflammatory proteins and urinary proteins. Physicochemical characteristics of the identified proteins showed that 67.1% proteins' isoelectric points (pI) value was below 7.0, 91.1% proteins' grand average of hydropathicity (GRAVY) scores were below 0 and nearly half of the proteins were stable. Our data indicated proteins might play an important role in melamine bladder stone formation.

[Role of poly (ADP-ribose) polymerase 1 on DNA methylation variation induced by B(a)P in human bronchial epithelial cell].

OBJECTIVE: To investigate DNA methylation variation in human cells induces by B(a)P, and to explore the role of PARP1 during this process. METHODS: The changes of DNA methylation of 16HBE and its PARP1-deficient cells exposed to B(a)P (1.0, 2.0, 5.0, 10.0, 15.0, 30.0 µmol/L) were investigated by immunofluorescence and high performance capillary electrophoresis, and simultaneously, the expression level of PARP 1 and DNMT 1 were monitored dynamically. RESULTS: The percentage of methylated DNA of overall genome (mCpG%) in 16HBE and 16HBE-shPARP1 cells were separately (4.04 ± 0.08)% and (9.69 ± 0.50)%. After being treated by 5-DAC for 72 hours, mCpG% decreased to (3.15 ± 0.14)% and (6.07 ± 0.54)%. After both being exposed to B(a)P for 72 hours, the mCpG% in 16HBE group (ascending rank) were separately (5.10 ± 0.13), (4.25 ± 0.10), (3.91 ± 0.10), (4.23 ± 0.27), (3.70 ± 0.15), (3.08 ± 0.07); while the figures in 16HBE-shPARP1 group (ascending rank) were respectively (10.63 ± 0.60), (13.08 ± 0.68), (9.75 ± 0.55), (7.32 ± 0.67), (6.90 ± 0.49) and (6.27 ± 0.21). The difference of the results was statistically significant (F values were 61.67 and 60.91, P < 0.01). For 16HBE group, expression of PARP 1 and DNMT 1 were 141.0%, 158.0%, 167.0%, 239.0%, 149.0%, 82.9% and 108.0%, 117.0%, 125.0%, 162.0%, 275.0%, 233.0% comparing with the control group, whose difference also has statistical significance (t values were 11.45, 17.32, 32.24, 33.44, 20.21 and 9.87, P < 0.01). For 16HBE-shPARP1 group, expression of PARP 1 and DNMT 1 were 169.0%, 217.0%, 259.0%, 323.0%, 321.0%, 256.0% and 86.0%, 135.0%, 151.0%, 180.0%, 229.0%, 186.0% comparing with the control group, with statistical significance (t values were 9.06, 15.92, 22.68, 26.23, 37.19 and 21.15, P < 0.01). When the dose of B(a)P reached 5.0 µmol/L, the mRNA expression of DNMT 1 in 16HBE group (ascending rank) were 125.0%, 162.0%, 275.0%, 233.0% times of it in control group, with statistical significance (t values were 12.74, 24.92, 55.11, 59.07, P < 0.01); while the dose of B(a)P reached 2.0 µmol/L, the mRNA expression of DNMT 1 in 16HBE-shPARP1 group were 135.0%, 151.0%, 180.0%, 229.0%, 186.0% of the results in control group, and the differences were statistically significant (t values were 23.82, 40.17, 32.69, 74.85, 46.76, P < 0.01). CONCLUSION: The hypomethylation of 16HBE cells induced by B(a)P might be one important molecular phenomenon in its malignant transformation process. It suggests that PARP1 could regulate DNA methylation by inhibiting the enzyme activity of DNMT1, and this effect could be alleviated by PARP1-deficiency.

[Fluorescence of early stage leachates DOM using RO membrane as tertiary treatment].

In the present study, fluorescence spectra of the dissolved organic matter (DOM) extracted from early stage leachate samples obtained from biologically pretreated leachate fed to RO tertiary treatment, the permeated, the concentrate, and liquids collected after cleaning the membrane with acid and then with base were determined. The results of synchronous fluorescence spectra analysis showed the RO membrane effectively removed the high content of organic matter corresponding to short wavelengths of 280, 340, and 370 nm of the feed. Liquids collected after cleaning membrane with acid and base showed obvious influence on the organic pollutant matters in the range of 300-420 nm. The results of three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectra showed three protein-like peaks, namely low-excitation wavelength, tyrosine-like high-excitation wavelength, tyrosine-like high-excitation wavelength, tryptophan-like and two fulvic-like peaks, visible and ultra visible fulvic-like were found in the feed. The permeated contains two peaks with higher intensity than the feed, low-excitation wavelength tyrosine-like and high excitation wavelength tyrosine-like, while the other three peaks were shown in the concentrate. The acid and the base cleaning had great influence on the molecule chemical structure of the organic pollutants on the RO membrane and caused obvious location shifts. It can be concluded that the RO mainly separated the fulvic matter in the early stage leachate and the fouling consisted of fulvic-like matter together with the protein-like, and low-excitation wavelength tyrosine-like.

Effect of PARP-1 deficiency on DNA damage and repair in human bronchial epithelial cells exposed to Benzo(a)pyrene.

Benzo[a]pyrene is a ubiquitously distributed environmental pollutant known to cause DNA damage, whereas PARP-1 is a nuclear enzyme that is activated by damaged DNA and plays an important role in base excision repair and genomic stability. Here, 16HBE and its PAPR1-deficient cells were exposed to BaP, and the DNA damage level and repair ability of both cell lines were measured by alkaline comet assay. The results showed that cell viability of both cell lines decreased in a dose-dependent manner when exposed to BaP, but there was no significant difference between two cell lines. Comet assay showed that BaP caused DNA damage in both cell lines at an obvious dose- and time-dependent manner. Compare with 16HBE, the PARP1-deficient cells were more sensitive to the damage caused by BaP. The results of DNA repair experiment showed that both cell lines can recover from the damage in a time-dependent pattern. The relative repair percentage of PARP1-deficient cells were generally lower than that of 16HBE at all exposed concentrations at the early stage of repair, but tended to be closer between two cell lines at the later period. According to results, we came to the conclusion that PARP1-deficient cells were more sensitive to BaP in contrast to normal 16HBE; DNA repair capacity in PARP1-deficient cells decreased significantly at the early stage of repair, but increased to the equivalent level of normal 16HBE in the later period. PARP-1 plays an important role in early repair of DNA damage caused by BaP in 16HBE notwithstanding the main repair work is taken by NER pathway.