AQP1 Deficiency Drives Phthalate-Induced Epithelial Barrier Disruption through Intestinal Inflammation.

Di-2-ethylhexyl phthalate (DEHP) is frequently used as a plasticizer to enhance the plasticity and durability of agricultural products, which pose adverse effects to human health and the environment. Aquaporin 1 (AQP1) is a main water transport channel protein and is involved in the maintenance of intestinal integrity. However, the impact of DEHP exposure on gut health and its potential mechanisms remain elusive. Here, we determined that DEHP exposure induced a compromised duodenum structure, which was concomitant with mitochondrial structural injury of epithelial cells. Importantly, DEHP exposure caused duodenum inflammatory epithelial cell damage and strong inflammatory response accompanied by activating the TLR4/MyD88/NF-κB signaling pathway. Mechanistically, DEHP exposure directly inhibits the expression of AQP1 and thus leads to an inflammatory response, ultimately disrupting duodenum integrity and barrier function. Collectively, our findings uncover the role of AQP1 in phthalate-induced intestinal disorders, and AQP1 could be a promising therapeutic approach for treating patients with intestinal disorders or inflammatory diseases.

Real-world impact of chemotherapy on overall survival in craniomaxillofacial osteosarcoma.

OBJECTIVES: The goal of this study was to identify the survival benefit of chemotherapy in craniomaxillofacial osteosarcoma (CMFO) patients based on a US population. MATERIALS AND METHODS: The Surveillance, Epidemiology, and End Results (SEER) database was used to select patients with CMFO from 1988 to 2016. Age and tumor size were grouped by X-tail. Cox analysis were used to estimate hazards ratios (HR) among patients. All of patients were divided into two cohorts by using Propensity Score Matching (PSM) method to evaluate the effect of chemotherapy. All prognostic factors were included in the nomograms which predict the median survival time. RESULTS: 410 patients were included in our study. The results of survival rate, Kaplan-Meier and Cox regression were showed no significant difference between the group of chemotherapy performed and the group without chemotherapy. PSM analysis also demonstrated the limited survival advantage of chemotherapy. Moreover, all factors were further incorporated to construct the novel nomograms and its concordance indices (C-index) for internal validation of OS prediction were 0.749 (95 %CI:0.731-0.767). CONCLUSIONS: Our study did not show the advantage of chemotherapy on the overall survival outcome of CMFO. Although neoadjuvant chemotherapy was currently recommended in clinical treatment, more rigorous randomized controlled trials are still needed. Nomograms would assist clinicians in making more accurate survival evaluation and choosing the optimal medical treatment.

Phthalate drives splenic inflammatory response via activating HSP60/TLR4/NLRP3 signaling axis-dependent pyroptosis.

As the most produced phthalate, di-(2-ethylhexyl) phthalate (DEHP) is a widely environmental pollutant primarily used as a plasticizer, which cause the harmful effects on human health. However, the impact of DEHP on spleen and its underlying mechanisms are still unclear. Pyroptosis is a novel form of cell death induced by activating NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasomes and implicated in pathogenesis of numerous inflammatory diseases. The current study aimed to explore the impact of DEHP on immune inflammatory response in mouse spleen. In this study, the male ICR mice were treated with DEHP (200 mg/kg) for 28 days. Here, DEHP exposure caused abnormal pathohistological and ultrastructural changes, accompanied by inflammatory cells infiltration in mouse spleen. DEHP exposure arouse heat shock response that involves increase of heat shock proteins 60 (HSP60) expression. DEHP also elevated the expressions of toll-like receptor 4 (TLR4) and myeloid differentiation protein 88 (MyD88) proteins, as well as the activation of NF-κB pathway. Moreover, DEHP promoted NLRP3 inflammasome activation and triggered NLRP3 inflammasome-induced pyroptosis. Mechanistically, DEHP drives splenic inflammatory response via activating HSP60/TLR4/NLRP3 signaling axis-dependent pyroptosis. Our findings reveal that targeting HSP60-mediated TLR4/NLRP3 signaling axis may be a promising strategy for inflammatory diseases treatment.

Promoting effect of Fe3+ on gentamicin resistance in Escherichia coli.

Kinds of antibiotics are used to prevent and control bacteria infections, unfortunately, the overuse and misuse of antibiotic have promoted the emergence and spread of antibiotic-resistant bacteria. Therefore, understanding the mechanism of antibiotic resistance is very important. This study explores the combined effection of metal ions and antibiotic to the drug resistance of Escherichia coli. Our results found that the minimum inhibitory concentration (MIC) increased as the ammonium ferric citrate concentration increased, especially for gentamicin antibiotic. When the Fe3+ concentration reached 300 µM, the survival of E. coli was stably restored with the increased gentamicin concentration. Exogenous Fe3+ could decrease intracellular gentamicin concentration. On the other hand, Fe3+ treatment together with gentamicin could reduce reactive oxygen species (ROS) production, characterized by decreased levels of NADH and ATP. Furthermore, ROS-scavenging enzymes of superoxide dismutase (SOD) and catalase (CAT) were up-regulated and H2O2 plus gentamicin-mediated killing was restored by Fe3+. These results may have significant implications in understanding bacterial antibiotic-resistant mechanisms based on the external Fe3+ concentration.

LncRNA SNHG4 promotes osteosarcoma proliferation and migration by sponging miR-377-3p.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been identified as crucial regulatory factors in the occurrence and progression of osteosarcoma. METHODS: Quantitative real-time polymerase chain reaction was used for detecting small nucleolar RNA host gene 4 (SNHG4) and miR-377-3p in osteosarcoma cells and tissues. Kaplan-Meier method was applied for evaluating the association between SNHG4 expression and the overall survival of osteosarcoma patients. CCK8, EdU, flow cytometry, and transwell assay were performed to examine the cell proliferation, apoptosis, cycle, and migration of osteosarcoma cells. RESULTS: In our study, we found that lncRNA SNHG4 was highly expressed in osteosarcoma tissues and cell lines. Additionally, the SNHG4 expression was related to distant metastasis, TNM stage, and survival of osteosarcoma patients. Through SNHG4 knockdown, the proliferation of osteosarcoma cells was considerably restrained and the cell apoptosis was induced in vivo and in vitro. Moreover, downregulated SNHG4 inhibited the cell migration and epithelial-mesenchymal transition in HOS and MG63 cells. In mechanism, we found that SNHG4 acts as a competing endogenous RNA to sponge miR-377-3p, which is downregulated in osteosarcoma. Our results showed that there is a negative correlation between SNHG4 and miR-377-3p expression in osteosarcoma patients. CONCLUSION: Taken together, SNHG4 promotes cell proliferation and migration by sponging miR-377-3p in osteosarcoma.

Integrated culture platform based on a human platelet lysate supplement for the isolation and scalable manufacturing of umbilical cord matrix-derived mesenchymal stem/stromal cells.

Umbilical cord matrix (UCM)-derived mesenchymal stem/stromal cells (MSCs) are promising therapeutic candidates for regenerative medicine settings. UCM MSCs have advantages over adult cells as these can be obtained through a non-invasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large-scale manufacturing of UCM MSCs mandatory. A commercially available human platelet lysate-based culture supplement (UltraGROTM , AventaCell BioMedical) (5%(v/v)) was tested to effectively isolate UCM MSCs and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC-like cells were isolated from UCM explant cultures after 11 ± 2 days. After five passages in static culture, UCM MSCs retained their immunophenotype and multilineage differentiation potential. The UCM MSCs cultured under static conditions using UltraGROTM -supplemented medium expanded more rapidly compared with UCM MSCs expanded using a previously established protocol. Importantly, UCM MSCs were successfully expanded under dynamic conditions on plastic microcarriers using UltraGROTM -supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSCs expanded by >13-fold after 5-6 days, maintaining their immunophenotype and multilineage differentiation ability. The present paper reports the establishment of an easily scalable integrated culture platform based on a human platelet lysate supplement for the effective isolation and expansion of UCM MSCs in a xenogeneic-free microcarrier-based system. This platform represents an important advance in obtaining safer and clinically meaningful MSC numbers for clinical translation. Copyright © 2016 John Wiley & Sons, Ltd.

[The measurement of thermal expansion coefficient of Co-Cr alloy fabricated by selective laser melting].

PURPOSE: To investigate the thermal expansion coefficient of different processing parameters upon the Co-Cr alloy prepared by selective laser melting (SLM) technique, in order to provide technical support for clinical application of SLM technology. METHODS: The heating curve of self-made Co-Cr alloy was protracted from room temperature to 980°C centigrade with DIL402PC thermal analysis instrument, keeping temperature rise rate and cooling rate at 5 K/min, and then the thermal expansion coefficient of 9 groups of Co-Cr alloy was measured from 20°C centigrade to 500°C centigrade and 600°C centigrade. RESULTS: The 9 groups thermal expansion coefficient values of Co-Cr alloy heated from 20°C centigrade to 500°C centigrade were 13.9×10(-6)/K,13.6×10(-6)/K,13.9×10(-6)/K,13.7×10(-6)/K,13.5×10(-6)/K,13.8×10(-6)/K,13.7×10(-6)/K,13.7×10(-6)/K,and 13.9×10(-6)/K, respectively; when heated from 20°C centigrade to 600°C centigrade, they were 14.2×10(-6)/K,13.9×10(-6)/K,13.8×10(-6)/K,14.0×10(-6)/K,14.1×10(-6)/K,14.1×10(-6)/K,13.9×10(-6)/K,14.2×10(-6)/K, and 13.7×10(-6)/K, respectively. CONCLUSIONS: The results showed that the Co-Cr alloy has good matching with the VITA VMK 95 porcelain powder and can meet the requirement of clinic use.

Oct4 and Nanog directly regulate Dnmt1 to maintain self-renewal and undifferentiated state in mesenchymal stem cells.

The roles of Oct4 and Nanog in maintaining self-renewal and undifferentiated status of adult stem cells are unclear. Here, increase in Oct4 and Nanog expression along with increased proliferation and differentiation potential but decreased spontaneous differentiation were observed in early-passage (E), hypoxic culture (H), and p21 knockdown (p21KD) mesenchymal stem cells (MSCs) compared to late-passage (L), normoxic culture (N), and scrambled shRNA-overexpressed (Scr) MSCs. Knockdown of Oct4 and Nanog in E, H, and p21KD MSCs decreased proliferation and differentiation potential and enhanced spontaneous differentiation, whereas overexpression of Oct4 and Nanog in L, N, and Scr MSCs increased proliferation and differentiation potential and suppressed spontaneous differentiation. Oct4 and Nanog upregulate Dnmt1 through direct binding to its promoter, thereby leading to the repressed expression of p16 and p21 and genes associated with development and lineage differentiation. These data demonstrate the important roles of Oct4 and Nanog in maintaining MSC properties.

c-Jun N-terminal kinase 1 negatively regulates osteoblastic differentiation induced by BMP2 via phosphorylation of Runx2 at Ser104.

Runx2 plays a crucial role in osteoblastic differentiation, which can be upregulated by bone morphogenetic proteins 2 (BMP2). Mitogen-activated protein kinase (MAPK) cascades, such as extracellular signal-regulated kinase (ERK) and p38, have been reported to be activated by BMP2 to increase Runx2 activity. The role of cjun-N-terminal kinase (JNK), the other kinase of MAPK, in osteoblastic differentiation has not been well elucidated. In this study, we first showed that JNK1 is activated by BMP2 in multipotent C2C12 and preosteoblastic MC3T3-E1 cell lines. We then showed that early and late osteoblastic differentiation, represented by ALP expression and mineralization, respectively, are significantly enhanced by JNK1 loss-of-function, such as treatment of JNK inhibitor, knockdown of JNK1 and ectopic expression of a dominant negative JNK1 (DN-JNK1). Consistently, BMP2-induced osteoblastic differentiation is reduced by JNK1 gain-of-function, such as enforced expression of a constitutively active JNK1 (CA-JNK1). Most importantly, we showed that Runx2 is required for JNK1-mediated inhibition of osteoblastic differentiation, and identified Ser104 of Runx2 is the site phosphorylated by JNK1 upon BMP2 stimulation. Finally, we found that overexpression of the mutant Runx2 (Ser104Ala) stimulates osteoblastic differentiation of C2C12 and MC3T3-E1 cells to the extent similar to that achieved by overexpression of wild-type (WT) Runx2 plus JNK inhibitor treatment. Taken together, these data indicate that JNK1 negatively regulates BMP2-induced osteoblastic differentiation through phosphorylation of Runx2 at Ser104. In addition, unraveling these mechanisms may help to develop new strategies in enhancing osteoblastic differentiation and bone formation.

[The sealing effects of three desensitizers on tubule:an SEM investigation].

PURPOSE: The purpose of this in vitro study was to evaluate the sealing effects on tubule by three different dentin desensitizers. METHODS: 48 freshly extracted human premolars were selected. Their buccal dentin was exposed and a standardized circular area was isolated. They were randomly divided into three experiment groups and one control group. Dentin desensitizers included Systemp desensitizer, Seal&protect desensitizer and Gluma desensitizer. Twelve teeth were prepared for each test group (36 teeth total), and the other twelve teeth were selected as a control group. After the removal of cement, the dentin surfaces were cleaned, treated. Then the teeth were vertically cleaved into two sections. The surface and section of these teeth were observed by means of a scanning electron microscope. RESULTS: All 3 desensitizers could seal the tubules on dentin surface. Systemp desensitizer's dentin permeability was better than Seal&protect, but Gluma had no dentin permeability. CONCLUSION: 3 desensitizers had sealing effect on tubule, systemo desensitizer was the best one.

[Advances in investigation of calcified root canal therapy].

Calcified root canal occurs often during root canal therapy. It's important to understand various methods to deal with calcified root canal, which may increase the successful rate. From anatomy, radiography, chemical root canal preparation, ultrasonic root canal preparation and endodontic microscopic system etc., this paper reviews the present research about the treatment techniques for calcified root canal.

ROS-mediated ERK activation in delayed protection from anoxic preconditioning in neonatal rat cardiomyocytes.

BACKGROUND: The activation of extracellular signal-regulated kinase1/2 (ERK1/2) has been shown to be important signaling pathway in the ischemic preconditioning (IPC) response. Recently, some studies suggest a key role for the mitochondrial ATP-sensitive potassium channel (mKATP) as both a trigger and an end effector of acute and delayed protection of IPC. Hence, this study was undertaken to elucidate the relationship between mKATP and ERK1/2 in the delayed protection mechanism of anoxic preconditioning (APC). METHODS: An APC model was established using cultured neonatal rat cardiomyocytes. Pharmacological agents [diazoxide, 5-hydroxydecanoate (5-HD), 2-mercaptopropionylglycine (MPG), and PD98059] were used to modulate mKATP and ERK1/2 activation. Cellular injury was evaluated by measuring cellular superoxide dismutase (SOD) activity, cell viability, and lactate dehydrogenase (LDH) release. The generation of cellular reactive oxygen species (ROS) and the activation of ERK1/2 were determined at different time points starting from the beginning of preconditioning with anoxia or diazoxide (an mKATP opener). RESULTS: Cell viability and SOD activity in the APC [(81.9 +/- 11.4)%, (13.6 +/- 3.7) U/L] and diazoxide [(79.2 +/- 12.4)%, (16.5 +/- 4.6) U/L] groups were significantly higher than in the anoxia/reoxygenation (A/R) [(42.2 +/- 7.3)%, (8.8 +/- 2.8) U/L] group (all P < 0.01). LDH activity in the APC group [(101.9 +/- 18.9) U/L] and diazoxide group [(97.5 +/- 17.7) U/L] was significantly lower than in the A/R group [(250.5 +/- 43.6) U/L] (all P < 0.01). Both APC and diazoxide simultaneously facilitated intracellular ROS generation and rapid ERK1/2 activation. But the effects of APC and diazoxide were remarkedly attenuated by 5-HP (an mKATP blocker) and by MPG (a free radical scavenger). In addition, the ERK1/2 inhibitor PD98059 also abolished the cellular protective effects induced by diazoxide. CONCLUSION: mKATP may mediate ERK1/2 activation during anoxia preconditioning by generating ROS, which then triggers the delayed protection of APC in rat cardiomyocytes.

Simple flow cytometric method used to assess lipid accumulation in fat cells.

Adipogenesis of preadipocytes in culture has been frequently used to study the molecular basis and effect of drugs on fat cell conversion. However, after adipogenic induction, cells respond to the inducing agent with various speeds of conversion and fat accumulation, which complicates direct molecular and biochemical analyses. Here we present a simple and sensitive method to detect and quantify fat accumulation inside cells by flow cytometry. Using this method, we detected elevated levels of cytoplasmic granularity that correlated well with an increased level of fat accumulated inside cells after adipogenic conversion. We further demonstrated the ability of this method to monitor and quantify fat cell maturation within a complex population of cells and to identify and collect the fat cells with similar fat storage for further analysis. Flow cytometry offers distinct advantages over existing detection systems for cytoplasmic lipid staining and lipid extraction and could represent a powerful analytical tool to monitor the effect of chemicals and biological molecules on fat cell conversion and maturation. Moreover, in combination with a cell sorting facility, our method offers a simple and efficient means of collecting fat cells of specific status for further analysis.

Methanocalculus chunghsingensis sp. nov., isolated from an estuary and a marine fishpond in Taiwan.

Three novel halotolerant, hydrogenotrophic methanogens, designated strains K1F9705bT, K1F9705c and O1F9704a, were isolated from an estuary in Eriln Shi, Taiwan, and from a nearby marine water aquaculture fishpond. These isolates were irregular cocci that stained Gram-negative. Strains K1F9705bT and K1F9705c were non-motile, but strain O1F9704a was weakly motile with flagella. They were able to use formate and H2/CO2 to form methane, but they could not catabolize acetate, methanol, trimethylamine or secondary alcohols. Acetate was required for cell growth. Tungsten greatly stimulated the growth of strains K1F9705bT and K1F9705c, but did not affect the growth of strain O1F9704a. Optimal pH and temperature for growth of these three isolates were respectively 7.2 and 37 degrees C. Optimal NaCl concentration for growth was 0.5% for strain O1F9704a and 1.0% for strains K1F9705c and K1F9705bT. Moreover, all strains grew well at up to 8-12% NaCl. Analysis of the 16S rRNA gene revealed that these isolates are members of the genus Methanocalculus, but are distinct from Methanocalculus taiwanensis, Methanocalculus pumilus and Methanocalculus halotolerans, with sequence similarities of 98.4, 98.3 and 98.2%, respectively. In addition, strain K1F9705bT possessed 85, 80, 37, 29 and 10% DNA-DNA relatedness to strain K1F9705c, strain O1F9704a, M. pumilus, M. halotolerans and M. taiwanensis, respectively. Analysis of protein profiles and the Mr of surface (S)-layer glycoprotein subunits showed that these three new isolates are closely related to, but distinct from, known Methanocalculus species. A novel species, Methanocalculus chunghsingensis sp. nov., is proposed for strains K1F9705bT, K1F9705c and O1F9704a. The type strain is K1F9705bT (=OCM 772T=DSM 14646T).

[Different roles of ERK(1/2) and p38 MAPK(alpha/beta) in cellular signaling during cardiomyocyte anoxia preconditioning].

Preconditioning (PC) exhibits earlier and delayed protection. But the mechanism of cellular signaling in delayed protection of PC remains unclear. We explored the roles of ERK(1/2) and p38 MAPK(alpha/beta) (p38(alpha/beta)) in delayed protection of anoxia preconditioning (APC). The anoxia/reoxygenation (A/R) injury and APC models were established in cultured neonatal rat cardiomyocytes. An ERK(1/2) inhibitor (PD98059) and a p38(alpha/beta) blocker (SB203580) were applied and their effects on A/R and APC models were observed. The cellular contents of MDA, SOD, cell viability and LDH release was measured at the end of the study. ERK(1/2) and p38 MAPK total activity was measured by in-gel myelin basic protein phosphorylation assay at different points during sustained anoxia. The results obtained are as follows: (1) PD98059 (but not SB203580), administered in preconditioning anoxia phase in APC group, abolished completely the delayed protection of APC; (2) SB203580 administered in sustained anoxia phase in A/R group could relieve cell injury induced by anoxia, but not by PD98059; (3) the highest activity of ERK(1/2) and p38 MAPK induced by anoxia appeared at 4 h after the beginning of sustained anoxia. APC inhibited the over activation of both ERK(1/2) and p38 during the following sustained anoxia. These results suggest that ERK(1/2) activation during preconditioning may be an important link of cell signal transduction in the mechanism of APC delayed protection. p38(alpha/beta) activation at the preconditioning stage dose not participate in signaling of APC delayed protection. The excessive activation of p38(alpha/beta) is possibly a key factor in mediating cell injury induced by sustained anoxia. The inhibition of p38(alpha/beta) excessive activation during subsequent sustained anoxia might play a role in delayed protection mechanism of APC.