Effects of intra-operative magnesium sulfate infusion on orthognathic surgery: A prospective and randomized controlled trial.

Purpose: To comprehensively understand the effects of intra-operative infusion of magnesium sulfate on patients who underwent orthognathic surgery, including remifentanil consumption, postoperative pain, postoperative nausea and vomiting (PONV), inflammatory response, and serum magnesium levels. Methods: Seventy-five adult patients undergoing orthognathic surgery under general balanced anesthesia were randomly divided into two groups. One group (Group M) received 50 mg/kg of magnesium sulfate in 20 mL 0.9 % saline after intubation, followed by a continuous infusion at a rate of 15 mg/kg/h until 30 min before the anticipated end of surgery. The other group (Group C) received an equal volume of isotonic saline as a placebo. (Clinical trial registration number: chiCTR2100045981). Results: The primary outcome was remifentanil consumption. The secondary outcomes included the pain score assessed using the verbal numerical rating scale (VNRS) and PONV assessed using a Likert scale. Remifentanil comsumption in Group M was lower than Group C (mean ± SD: 0.146 ± 0.04 µg/kg/min vs. 0.173 ± 0.04 µg/kg/min, P = 0.003). At 2 h after surgery, patients in Group C suffered more severe PONV than those in Group M (median [interquartile range, IQR]: 1 [3] vs. 1 [0], mean rank: 31.45 vs. 42.71, P = 0.040). At post-anesthesia care unit (PACU), postoperative pain in Group C was severe than Group M (3 [1] vs. 3 [0], mean rank: 31.45 vs. 42.71, P = 0.013). Changes in haemodynamics and surgical field scores did not differ between the groups (all P > 0.05). The levels of cytokines (IL-4, IL-6, IL-8, IL-10, TNF-a, and MIP-1ß) were not significantly different between the groups after surgery (all P > 0.05). Postoperative serum magnesium levels in Group C were lower than those in Group M (0.74 ± 0.07 mmol/L vs. 0.91 ± 0.08 mmol/L, P = 0.000) and the preoperative level (0.74 ± 0.07 mmol/L vs. 0.83 ± 0.06 mmol/L, P = 0.219). Conclusions: In orthognathic surgery, magnesium sulfate administration can reduce remifentanil requirement and relieve PONV and postoperative pain in the early postoperative phase.

Should we perform sigmoidoscopy for colorectal cancer screening in people under 45 years?

BACKGROUND: The strategy for preventing colorectal cancer is screening by colonoscopy, which offers a direct way for detection and removal of adenomatous polyps (APs). American College of Gastroenterology guidelines recommend that people aged ≥ 45 years should undergo colonoscopy; however, how to deal with people aged ≤ 45 years is still unknown. AIM: To compare the prevalence of APs and high-grade neoplasia between the left and right colon in patients ≤ 45 years. METHODS: A retrospective observational study was conducted at a single tertiary III hospital in China. This study included patients aged 18-45 years with undergoing initial colonoscopy dissection and pathological diagnosis AP or high-grade neoplasia between February 2014 and January 2021. The number of APs in the entire colon while screening and post-polypectomy surveillance in following 1-3 years were evaluated. RESULTS: A total of 3053 cases were included. The prevalence of APs in the left and right colon was 55.0% and 41.6%, respectively (OR 1.7, 95%CI 1.6-2.4; P < 0.05). For APs with high-grade neoplasia, the prevalence was 2.7% and 0.9%, respectively (OR 3.0, 95%CI 2.0-4.6; P < 0.05). Therefore, the prevalence of APs and high-grade neoplasia in the left colon was significantly higher than in the right colon in patients aged ≤ 45 years. There were 327 patients who voluntarily participated in post-polypectomy surveillance in following 1-3 years, and APs were found in 216 cases (66.1%); 170 cases had 1-3 polyps (52.0%) and 46 cases had > 3 polyps (14.1%; OR 0.3, 95%CI 0.1-0.6; P < 0.05). CONCLUSION: This study suggests that flexible sigmoidoscopy would be an optimal approach for initial screening in people aged ≤ 45 years and would be a more cost-effective and safe strategy.

Light chain deposition disease presenting with gastrointestinal disorder as primary manifestation: report of two cases and literature review.

Light chain deposition disease (LCDD) is an under-recognized condition characterized by deposition of abnormal monoclonal light chains in tissues, leading to organ dysfunction. LCDD involving the gastrointestinal tract is very uncommon, and its diagnosis is challenging. We herein report two cases of LCDD that manifested as inflammatory bowel disease-like symptoms and protein-losing gastroenteropathy. Both patients were women in their early 60s. Tissue biopsies from the gastrointestinal mucosa demonstrated extracellular deposits, which were negative by Congo red staining but positive for κ-light chain by immunohistochemistry. The recent literature on LCDD was reviewed. When patients unexpectedly show extracellular deposits in gastrointestinal biopsy specimens, evaluation of immunoglobulin chains is recommended for diagnosis of LCDD after systemic amyloidosis has been excluded.

Regulating electronic structure of Fe single-atom site by S/N dual-coordination for efficient Fenton-like catalysis.

The activity of single-atom catalysts in peroxymonosulfate activation process is bound up with the local electronic state of metal center. However, the large electronegativity of N atoms in Metal-N4 restricts the electron transfer between center metal atom and peroxymonosulfate. Herein, we constructed Fe-SN-C catalyst by incorporating S atom in the first coordination sphere of Fe single-atom site (Fe-S1N3) for Fenton-like catalysis. The Fe-SN-C with a low valent Fe is found to exhibit excellent catalytic activity for bisphenol A degradation, and the corresponding rate constant reaches 0.405 min-1, 11.9-fold higher than the original Fe-N-C. Besides, the Fe-SN-C/PMS system exhibits ideal catalytic stability under the effect of wide pH range and background substrates by the fast generation of high-valent Fe species. Experimental results and theoretical calculations reveal that the dual coordination of S and N atoms notably increases the local electron density of Fe atoms and electron filling in eg orbital, causing a d band center shifting close to the fermi level and thereby optimizes the activation energy for peroxymonosulfate decomposition via Fe 3d-O 2p orbital interaction. This work provides further development of promising SACs for the efficient activation of peroxymonosulfate based on direct regulation of the coordination environment of active center metal atoms.

Microstructure and Nanoindentation Behavior of FeCoNiAlTi High-Entropy Alloy-Reinforced 316L Stainless Steel Composite Fabricated by Selective Laser Melting.

Selective laser melting (SLM) is one of the metal additive manufactured technologies with the highest forming precision, which prepares metal components through melting powders layer by layer with a high-energy laser beam. The 316L stainless steel is widely used due to its excellent formability and corrosion resistance. However, its low hardness limits its further application. Therefore, researchers are committed to improving the hardness of stainless steel by adding reinforcement to stainless steel matrix to fabricate composites. Traditional reinforcement comprises rigid ceramic particles, such as carbides and oxides, while the research on high entropy alloys as reinforcement is limited. In this study, characterisation by appropriate methods, inductively coupled plasma, microscopy and nanointendation assay, showed that we successfully prepared the FeCoNiAlTi high entropy alloy (HEA)-reinforced 316L stainless steel composites using SLM. When the reinforcement ratio is 2 wt.%, the composite samples show higher density. The SLM-fabricated 316L stainless steel displays columnar grains and it varies to equiaxed grains in composites reinforced with 2 wt.% FeCoNiAlTi HEA. The grain size decreases drastically, and the percentage of the low angle grain boundary in the composite is much higher than in the 316L stainless steel matrix. The nanohardness of the composite reinforced with 2 wt.% FeCoNiAlTi HEA is twice as high as the 316L stainless steel matrix. This work demonstrates the feasibility of using a high-entropy alloy as potential reinforcement in stainless steel systems.

A Self-Reinforcing Nanoplatform for Highly Effective Synergistic Targeted Combinatary Calcium-Overload and Photodynamic Therapy of Cancer.

While calcium-overload-mediated therapy (COMT) is a promising but largely untapped therapeutic strategy, combinatory therapy greatly boosts treatment outcomes with integrated merits of different therapies. Herein, a BPQD@CaO2 -PEG-GPC3Ab nanoplatform is formulated by integrating calcium peroxide (CaO2 ) and black phosphorus quantum dot (BPQD, photosensitizer) with active-targeting glypican-3 antibody (GPC3Ab), for combinatory photodynamic therapy (PDT) and COMT in response to acidic pH and near-infrared (NIR) light, wherein CaO2 serves as the reservoir of calcium ions (Ca2+ ) and hydrogen peroxide (H2 O2 ). Navigated by GPC3Ab to tumor cells at acidic pH, the nanoparticle disassembles to CaO2 and BPQD; CaO2 produces COMT Ca2+ and H2 O2 , while H2 O2 makes oxygen (O2 ) to promote PDT; under NIR irradiation BPQD facilitates not only the conversion of O2 to singlet oxygen (1 O2 ) for PDT, but also moderate hyperthermia to accelerate NP dissociation to CaO2 and BPQD, and conversions of CaO2 to Ca2+ and H2 O2 , and H2 O2 to O2 , to enhance both COMT and PDT. After supplementary ionomycin treatment to induce intracellular Ca2+ bursts, the multimodal therapeutics strikingly induce hepatocellular carcinoma apoptosis, likely through the activation of the calpains and caspases 12, 9, and 3, up-regulation of Bax and down-regulation of Bcl-2 proteins. This nanoplatform enables a mutually-amplifying and self-reinforcing synergistic therapy.

Mallotucin D, a Clerodane Diterpenoid from Croton crassifolius, Suppresses HepG2 Cell Growth via Inducing Autophagic Cell Death and Pyroptosis.

Hepatocellular carcinoma (HCC) is a major subtype of primary liver cancer with a high mortality rate. Pyroptosis and autophagy are crucial processes in the pathophysiology of HCC. Searching for efficient drugs targeting pyroptosis and autophagy with lower toxicity is useful for HCC treatment. Mallotucin D (MLD), a clerodane diterpenoid from Croton crassifolius, has not been previously reported for its anticancer effects in HCC. This study aims to evaluate the inhibitory effects of MLD in HCC and explore the underlying mechanism. We found that the cell proliferation, DNA synthesis, and colony formation of HepG2 cells and the angiogenesis of HUVECs were all greatly inhibited by MLD. MLD caused mitochondrial damage and decreased the TOM20 expression and mitochondrial membrane potential, inducing ROS overproduction. Moreover, MLD promoted the cytochrome C from mitochondria into cytoplasm, leading to cleavage of caspase-9 and caspase-3 inducing GSDMD-related pyroptosis. In addition, we revealed that MLD activated mitophagy by inhibiting the PI3K/AKT/mTOR pathway. Using the ROS-scavenging reagent NAC, the activation effects of MLD on pyroptosis- and autophagy-related pathways were all inhibited. In the HepG2 xenograft model, MLD effectively inhibited tumor growth without detectable toxicities in normal tissue. In conclusion, MLD could be developed as a candidate drug for HCC treatment by inducing mitophagy and pyroptosis via promoting mitochondrial-related ROS production.

Acute Nitrite Exposure Induces Dysfunction and Oxidative Damage in Grass Carp Isolated Hemocytes.

To evaluate the effects of nitrite on the oxidative damage of blood cells of Grass Carp Ctenopharyngodon idella, the isolated hemocytes were exposed to nitrite (0, 1, 10, or 100 mg/L) for up to 24 h. Hemoglobin (Hb) and methemoglobin (MetHb) concentrations, reactive oxygen species (ROS) and malondialdehyde (MDA) levels, mitochondrial membrane potential (∆Ψm), and antioxidant enzyme activity were assayed to assess hematological parameters and the antioxidant defense mechanism. Results showed a remarkable decrease in Hb concentration with increasing nitrite concentration after a 24-h exposure, while the MetHb concentration increased significantly in nitrite exposure groups. The levels of ROS, ∆Ψm, and MDA increased to varying degrees with increases in nitrite exposure concentration and time. The total antioxidant capacity, catalase (CAT) activity, glutathione peroxidase (GPx) activity, and glutathione content showed a trend of rising initially and then decreasing with prolonged exposure time. Superoxide dismutase (SOD) activity was higher in the 1-mg/L nitrite exposure group and lower in the 100-mg/L group than in the control. The relative messenger RNA expression ratios of cat, sod1, and gpx were up-regulated significantly in the 1- and 10-mg/L groups and then declined in the 100-mg/L group. Therefore, it can be concluded that nitrite exposure activates the antioxidant defense mechanism of Grass Carp hemocytes and that the balance of oxidant-antioxidant homeostasis will be undermined by higher nitrite doses or longer exposure periods.

Multifunctional Mechanical Sensing Electronic Device Based on Triboelectric Anisotropic Crumpled Nanofibrous Mats.

Integrating multiple mechanical sensing capabilities in one device is highly desired to mimic the amazing functions of human skin, and it demonstrates promising applications in the human-machine interface and wearable robotic exoskeletons. Yet, challenges remain in how to couple the multisensations using as few modules as possible to increase the compactness and conformality of the electronics. Herein, we report a self-powered multiple mechanical sensing electronic device capable of sensing multiple motion modes of strain ratios, strain direction, and pressure. The self-powered property derives from the triboelectric working mechanism of the sensor. The multiple mechanical sensing is realized by utilizing an anisotropic crumpled nanofibrous membrane as the triboelectric layer and ionic conductor as the electrode layer. For strain ratios and pressure sensing, the output voltages of the sensor changed with the changes of these external stimulus with a comparable sensitivity. More importantly, contributed by the anisotropic structure of the designed crumples, the directional strain sensing is realized by the anisotropic sensitivity in three stretched directions.

A gastro-colic fistula secondary to high-grade B-cell gastric lymphoma in a patient with AIDS: a case report.

To the best of our knowledge, there are no previous reports of a gastro-colic fistula (GCF) secondary to primary high-grade B-cell gastric lymphoma associated with acquired immunodeficiency syndrome (AIDS). Here, we report a 37-year-old man who presented with paroxysmal abdominal pain for 4 months, diarrhea for 15 days and weight loss of 4 kg. He had a history of human immunodeficiency virus (HIV) infection and was diagnosed with AIDS in 2013. The patient was diagnosed with a GCF secondary to primary high-grade B-cell gastric lymphoma by gastroscopy and histopathological examination. Two weeks after diagnosis, he died in another hospital. This is an uncommon case in which the GCF occurred secondary to malignant gastric lymphoma in a patient with AIDS. Supported by the literature, patients with HIV infection who complain of abdominal pain or a mass, severe diarrhea, and weight loss should be assessed for a GCF secondary to lymphoma because of its worse prognosis.

A new model based on gamma-glutamyl transpeptidase to platelet ratio (GPR) predicts prognostic outcome after curative resection of solitary hepatocellular carcinoma.

BACKGROUND: This study intends to explore the potential clinical value of gamma-glutamyl transpeptidase to platelet ratio (GPR) and the new multi-factor scoring model for recurrence and prognosis prediction in solitary HCC patients who received radical resection. METHODS: This study retrospectively analyzed 295 HCC patients after curative resection. According to the Receiver Operating Characteristic (ROC) curve, the optimal cut-off value of GPR for predicting prognosis of HCC after resection was determined. The Kaplan Meier method and Cox regression analysis were performed to assess the important potential factors in the prognosis of HCC and determine the independent risk factors. Assign a value to each independent risk factor and establish a new scoring model. Then, using GPR and the new scoring model to evaluate overall survival (OS) and postoperative recurrence probability. RESULTS: When GPR's cut-off value was selected as 0.30, its predictive efficiency for postoperative prognosis was more favorable than those of other cut-off values (0.76, 0.84 and 0.94). GPR, tumor size, microvascular invasion and neutrophil to lymphocyte ratio (NLR) were identified as independent prognostic predictors. Using these variables, a novel prognostic scoring model was devised and established to identify different levels of risk: high, intermediate and low risk groups. We found that patients with high GPR level and of high risk group would have a poorer OS and a higher recurrence rate after radical resection. CONCLUSIONS: GPR may serve as a promising predictor for postoperative prognosis and recurrence probability of HCC, and the new prognostic scoring model may be available for postoperative management among HCC patients.

Growth arrest-specific gene 2 suppresses hepatocarcinogenesis by intervention of cell cycle and p53-dependent apoptosis.

BACKGROUND: Growth arrest-specific gene 2 (GAS2) plays a role in modulating in reversible growth arrest cell cycle, apoptosis, and cell survival. GAS2 protein is universally expressed in most normal tissues, particularly in the liver, but is depleted in some tumor tissues. However, the functional mechanisms of GAS2 in hepatocellular carcinoma (HCC) are not fully defined. AIM: To investigate the function and mechanism of GAS2 in HCC. METHODS: GAS2 expression in clinic liver and HCC specimens was analyzed by real-time PCR and western blotting. Cell proliferation was analyzed by counting, MTS, and colony formation assays. Cell cycle analysis was performed by flow cytometry. Cell apoptosis was investigated by Annexin V apoptosis assay and western blotting. RESULTS: GAS2 protein expression was lower in HCC than in normal tissues. Overexpression of GAS2 inhibited the proliferation of HCC cells with wide-type p53, while knockdown of GAS2 promoted the proliferation of hepatocytes (P < 0.05). Furthermore, GAS2 overexpression impeded the G1-to-S cell cycle transition and arrested more G1 cells, particularly the elevation of sub G1 (P < 0.01). Apoptosis induced by GAS2 was dependent on p53, which was increased by etoposide addition. The expression of p53 and apoptosis markers was further enhanced when GAS2 was upregulated, but became diminished upon downregulation of GAS2. In the clinic specimen, GAS2 was downregulated in more than 60% of HCCs. The average fold changes of GAS2 expression in tumor tissues were significantly lower than those in paired non-tumor tissues (P < 0.05). CONCLUSION: GAS2 plays a vital role in HCC cell proliferation and apoptosis, possibly by regulating the cell cycle and p53-dependent apoptosis pathway.

Symptom distress and interference among cancer patients with osteoradionecrosis of jaw: A cross-sectional study.

OBJECTIVE: Osteoradionecrosis of the jaws (ORNJ) is among the most serious oral complications of head and neck cancer treatment with radiation therapy. This study aimed to examine the level of symptom distress and interference of ORNJ in head and neck cancer patients in China. METHODS: A cross-sectional study was conducted to profile patient reported symptom severity. Ninety-five hospitalized ORNJ patients were recruited. Participants completed the MD Anderson Symptom Inventory-Head and Neck Module-Chinese version. RESULTS: The percentage of participants who reported that they experienced at least one type of symptom was 97.9%, and 85.2% patients reported interference. The 10 most severe symptoms were as follows: limited mouth opening, problem with teeth/gums, difficulty swallowing/chewing, dry mouth, oral malodor, difficulty with voice/speech, dental ulcer, tinnitus/ear obstruction, skin pain/burning/rash, and difficulty hearing. The problem of limited mouth opening was more severe in patients with longer time to onset of ORNJ after radiotherapy. The interference of patients positively correlated with core symptoms (r = 0.612), head and neck symptoms (r = 0.709), and ORNJ symptoms (r = 0.440) (P  < 0.01). The longer time to the onset of ORNJ after radiotherapy was positively and significantly correlated with symptom distress (r = 0.479, P  < 0.001), and mouth opening correlated negatively with symptom distress (r = -0.298, P = 0.003). CONCLUSIONS: ORNJ patients suffered mainly from limited mouth opening and other maxillofacial symptoms. The problem of limited mouth opening was more severe in patients with longer time to onset of ORNJ after radiotherapy. ORNJ patients commonly had symptom distress, which influenced their quality of life.

Transcriptional Induction of Periostin by a Sulfatase 2-TGFß1-SMAD Signaling Axis Mediates Tumor Angiogenesis in Hepatocellular Carcinoma.

Existing antiangiogenic approaches to treat metastatic hepatocellular carcinoma (HCC) are weakly effectual, prompting further study of tumor angiogenesis in this disease setting. Here, we report a novel role for sulfatase 2 (SULF2) in driving HCC angiogenesis. Sulf2-deficient mice (Sulf2 KO) exhibited resistance to diethylnitrosamine-induced HCC and did not develop metastases like wild-type mice (Sulf2 WT). The smaller and less numerous tumors formed in Sulf2 KO mice exhibited a markedly lower microvascular density. In human HCC cells, SULF2 overexpression increased endothelial proliferation, adhesion, chemotaxis, and tube formation in a paracrine fashion. Mechanistic analyses identified the extracellular matrix protein periostin (POSTN), a ligand of αvß3/5 integrins, as an effector protein in SULF2-induced angiogenesis. POSTN silencing in HCC cells attenuated SULF2-induced angiogenesis and tumor growth in vivo The TGFß1/SMAD pathway was identified as a critical signaling axis between SULF2 and upregulation of POSTN transcription. In clinical HCC specimens, elevated levels of SULF2 correlated with increased microvascular density, POSTN levels, and relatively poorer patient survival. Together, our findings define an important axis controlling angiogenesis in HCC and a mechanistic foundation for rational drug development. Cancer Res; 77(3); 632-45. ©2016 AACR.

Flashlight contamination and effectiveness of two disinfectants in a stomatology hospital.

AIM: This study aims to assess the infection risks of flashlight contamination in a stomatology hospital and compare the disinfection effectiveness of alcohol (75%) and disinfecting wipes. BACKGROUND: The flashlight is a basic non-critical medical device in oral and maxillofacial surgery wards. Wounds are mostly found in oral cavities; therefore, reusable flashlights may be a potential source of nosocomial infections (NIs). However, the microbial flora present in flashlights used in hospitals has not yet been explored. METHODS: This study investigated the microbial contamination of 41 flashlights used in a stomatology hospital in Guangzhou in March 2016. RESULTS: Results indicated that 75.6%(31/41) of the flashlights had microbial contamination. Gram-positive bacteria accounted for 72.7%(24/33)of the microbial groups contaminating the flashlights, and Gram-negative bacteria (21.2%, 7/33), and fungi (6.1%, 2/33) constituted the remaining contaminants. The predominantly isolated species was Staphyloccus (66.7%, 22/33), especially Staphylococcus aureus (24.2%, 8/33). Approximately 77.3% (17/22) of the types of bacteria detected in the hands were same as those in the corresponding flashlights. Both the bacterial overstandard and S. aureus detection rates of doctors' flashlights were higher than those of nurses' flashlights (16/17 vs. 14/23, 7/17 vs. 1/23, respectively) (P < 0.05). Moreover, both disinfectants performed excellently, and their eligibility rates were not significantly different (17/17 vs. 14/14) (P > 0.05). CONCLUSION: Flashlights are potential causes of NIs. Disinfecting flashlights could be an effective and practical infection control method.

Contamination of dental goggles and effectiveness of 3 disinfectants in a stomatology hospital.

We investigated the contamination levels of dental goggles to assess the infection risk in a stomatology hospital and compared the effectiveness of 3 disinfectants, including Swashes (Swashes Enterprise, Co. Ltd., Shenzhen, China), medical ethanol (75%), and chlorine-containing disinfectant (500 mg/L). The results showed that 87.41% of goggles had bacterial contamination. In total, 54% of cultures included gram-positive cocci, making them the major microbial group contaminating the goggles. All 3 disinfectants showed excellent performance in disinfection, and there were no significant differences among the eligibility rates of the 3 disinfectants (97.2% vs 100.0% vs 100.0%, respectively). The study highlights the fact that dental goggles are potential reservoirs of nosocomial pathogens. Disinfection of goggles could be an effective and practical infection control method for dental practice.

[Physiological responses of tubificidae to heavy metal chromium stress].

Tubificidae is now used in the wastewater treatment systems to successfully minimize the sludge production, which has been proved an effective, economical and sustainable technology. But the excess sludge inevitably contains a variety of heavy metals, especially the sludge from industrial wastewater treatment plant. In order to apply tubificidae to these systems, Chromium was selected as pollutant object and the physiological responses of tubificidae to Chromium were studied in this paper. Acute toxicity was analyzed and Median lethal concentrations (LC50) were determined over 96 h periods for Cr. Results indicated that 24 h LC50 and 96 h LC50 were 7.94 mg x L(-1) and 0.49 mg x L(-1), respectively. The duration f tubificidae in Cr solution decreased with increasing Cr concentration. Under the Cr stress, a highest respiration rate was obtained when the concentration of Cr(VI), temperature, pH and DO was 2.50 mg x L(-1), 26 degrees C, 6.0 and 6.0 mg x L(-1), respectively. The order of these factors was the concerntration of Cr(VI), temperature, DO and pH. The respiration experiments demonstrated that low concentration (< 2.50 mg x L(-1)) of Cr could promote the respiration rate of tubificidaes. On the other hand, when the concentration of Cr was 8.00 mg x L(-1), it could remarkably inhibit the respiratory rates of tubificidae.

Suppression of autophagy by FIP200 deletion leads to osteopenia in mice through the inhibition of osteoblast terminal differentiation.

Autophagy is a conserved lysosomal degradation process that has important roles in both normal human physiology and disease. However, the function of autophagy in bone homeostasis is not well understood. Here, we report that autophagy is activated during osteoblast differentiation. Ablation of focal adhesion kinase family interacting protein of 200 kD (FIP200), an essential component of mammalian autophagy, led to multiple autophagic defects in osteoblasts including aberrantly increased p62 expression, deficient LC3-II conversion, defective autophagy flux, absence of GFP-LC3 puncta in FIP200-null osteoblasts expressing transgenic GFP-LC3, and absence of autophagosome-like structures by electron microscope examination. Osteoblast-specific deletion of FIP200 led to osteopenia in mice. Histomorphometric analysis revealed that the osteopenia was the result of cell-autonomous effects of FIP200 deletion on osteoblasts. FIP200 deletion led to defective osteoblast terminal differentiation in both primary bone marrow and calvarial osteoblasts in vitro. Interestingly, both proliferation and differentiation were not adversely affected by FIP200 deletion in early cultures. However, FIP200 deletion led to defective osteoblast nodule formation after initial proliferation and differentiation. Furthermore, treatment with autophagy inhibitors recapitulated the effects of FIP200 deletion on osteoblast differentiation. Taken together, these data identify FIP200 as an important regulator of bone development and reveal a novel role of autophagy in osteoblast function through its positive role in supporting osteoblast nodule formation and differentiation.

A new membrane re-anchored protein originating from GPC3 against hepatoma cells HepG2.

The aim of this study was to confirm the localization of recombinant pGPC3+afp-EGFP which expressed a new re-anchored protein named GPC3+afp-EGFP on the cytoplasmic membrane and to investigate its functions against hepatocellular carcinoma (HCC). EGFP expression in transfected HepG2 cells was observed using fluorescence and a confocal microscope. pGPC3+afp-EGFP expression was detected in membranous and soluble proteins extracted from transfected human embryonic kidney 293 cells by Western blot analysis using GPC3 mAb. The proliferation of transfected HepG2 cells with pGPC3+afp-EGFP (experimental group) was detected using SRB assay and compared to those of transfected HepG2 cells with pGPC3 (control group) and non-transfected HepG2 cells (blank group). Quantitative analysis of mRNA expression of the Fas gene was conducted by real-time PCR using the ß-actin housekeeping gene as the internal control at variable times. Apoptotic HepG2 cells in the three groups were counted and statistically analyzed by a contingency table Chi-square test using Spss 11.5 software and TUNEL assay. Production of both TNF-α and IFN-γ/IL2 was detected by ELISPOT after co-cultivation of transfected HepG2 cells with peripheral blood lymphocytes at different time-points in the experimental group. Green fluorescence was mainly found around the transfected HepG2 cell periphery through fluorescence and confocal microscopy. GPC3+afp-EGFP could not be detected in soluble protein but only in membranous protein. Proliferation curves showed that the proliferative quantities of transfected HepG2 cells in the experimental group decreased, whereas the mRNA expression of the Fas gene increased significantly compared to those of the other two groups. The numbers of apoptotic cells in the experimental group were significantly higher compared to those in the other two groups, as shown by statistical analysis. Both TNF-α and IFN-γ/IL2 were induced and were much higher in the experimental groups than in the diverse control groups at variable times. A new re-anchored protein GPC3+afp-EGFP expressed by recombinant pGPC3+afp-EGFP was localized on the cytoplasmic membrane, and had multiple functions against HCC, such as inhibition of transfected HepG2 cell proliferation, promotion of transfected HepG2 apoptosis and induction of antitumor cytokine excretion.

GPC3 fused to an alpha epitope of HBsAg acts as an immune target against hepatocellular carcinoma associated with hepatitis B virus.

BACKGROUND: The incidence of hepatocellular carcinoma (HCC) in China is closely related to the population infected with hepatitis B virus (HBV). HCC cells with HBV secrete soluble HBsAg into blood but do not express it on the cell membrane. This study aimed to construct and investigate a new glycosyl-phosphatidylinositol (GPI)-anchored protein (GPC3+alpha+EGFP) as a DNA vaccine against HCC associated with HBV. METHODS: A recombinant plasmid (pcDNA3.1(+)/GPC3+ alpha+EGFP) was constructed and verified by restriction endonuclease digestion and sequencing. pcDNA3.1(+)/GPC3+alpha+EGFP was transfected into HepG2 cells (experimental group) using lipofectamine 2000. pEGFP-N1-transfected HepG2 cells were used as a negative control, and non-transfected HepG2 cells served as a blank control. HepG2 cells that steadily expressed the fusion protein GPC3+alpha+EGFP were screened by G418, propagated, and co-cultured with lymphocytes from healthy donors. Cell proliferation was measured by the classic sulforhodamine B assay. Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and Fas gene transcription was determined by quantitative fluorescent PCR. RESULTS: The pcDNA3.1(+)/GPC3+alpha+EGFP plasmid was successfully constructed. In the experimental group, green fluorescence was observed at the cell periphery and in the cytoplasm, whereas in the negative control group, fluorescence was evenly distributed throughout the cell. Proliferation of the experimental group significantly decreased after 72 hours compared to the negative and blank control groups. Furthermore, the number of apoptotic cells was statistically different among the three groups as determined by a contingency table Chi-square test; the experimental group had the highest incidence of apoptosis. Fas gene transcription in the experimental group was higher than in the two control groups, and an increasing trend with time in the experimental group was observed. CONCLUSION: A chimeric, membrane-anchored protein, GPC3+alpha+EGFP, localized to the membrane of HepG2 cells and inhibited proliferation and accelerated apoptosis through a Fas-FasL pathway after co-cultivation with lymphocytes.