Deletion of Tmem268 in mice suppresses anti-infectious immune responses by downregulating CD11b signaling.

Transmembrane protein 268 (TMEM268) is a novel, tumor growth-related protein first reported by our laboratory. It interacts with the integrin subunit ß4 (ITGB4) and plays a positive role in the regulation of the ITGB4/PLEC signaling pathway. Here, we investigated the effects and mechanism of TMEM268 in anti-infectious immune response in mice. Tmem268 knockout in mice aggravated cecal ligation and puncture-induced sepsis, as evidenced by higher bacterial burden in various tissues and organs, congestion, and apoptosis. Moreover, Tmem268 deficiency in mice inhibited phagocyte adhesion and migration, thus decreasing phagocyte infiltration at the site of infection and complement-dependent phagocytosis. Further findings indicated that TMEM268 interacts with CD11b and inhibits its degradation via the endosome-lysosome pathway. Our results reveal a positive regulatory role of TMEM268 in ß2 integrin-associated anti-infectious immune responses and signify the potential value of targeting the TMEM268-CD11b signaling axis for the maintenance of immune homeostasis and immunotherapy for sepsis and related immune disorders.

Effects of phase-transited lysozyme on adhesion, migration and odontogenic differentiation of human dental pulp cells: An in vitro study.

AIM: To explore the effects of phase-transited lysozyme (PTL) coated dentine slices on cell adhesion, migration and odontogenic differentiation of human dental pulp cells (HDPCs). METHODOLOGY: Cell growth and cell cycle analysis were conducted to verify the biocompatibility of PTL for HDPCs. Cell adhesion, cell morphology and proliferation were explored by DiI staining, Scanning electron microscopy and MTT assay. Cell migration was investigated by Transwell assay. The effects of PTL on odontogenesis and mineralization of HDPCs were assessed by real-time quantitative polymerase chain reaction and Western blot. The mineralization of HDPCs was evaluated by Alizarin red staining. HDPCs were isolated from extracted third molars. The level of statistically significant difference was accepted at p < .05. RESULTS: PTL showed no negative effect on cell cycle of HDPCs and compared with the blank group, HDPCs labelled with DiI staining showed significantly more adhered cells at 48 h (p < .05), extending cell processes and more finger-like or reticular pseudopodia on PTL-coated dentine slices. The results of MTT and Transwell assay showed that PTL promoted the proliferation (p < .05) and migration (p < .01) of HDPCs, respectively. Compared with the blank group, the gene expression of dentine sialophosphoprotein (DSPP), osteopontin and bone sialoprotein in HDPCs cultured on PTL was significantly upregulated on day 3 and 7 (p < .05), while the protein expression of DSPP showed no significant change on both day 7 and day 14. Alizarin red staining showed that PTL promoted more mineralization nodules formation of HDPCs (p < .05). CONCLUSIONS: PTL promoted the adhesion, proliferation and migration of HDPCs on dentine slices, and positively affected odontogenic differentiation and mineralization of HDPCs.

Integrated proteomic analysis to explore the molecular regulation mechanism of IL-33 mRNA increased by black carbon in the human endothelial cell line EA.hy926.

Black carbon (BC) correlates with the occurrence and progression of atherosclerosis and other cardiovascular diseases. Increasing evidence has demonstrated that BC could impair vascular endothelial cells, but the underlying mechanisms remain obscure. It is known that IL-33 exerts a significant biological role in cardiovascular disease, but little is known about the molecular regulation of IL-33 expression at present. We first found that BC significantly increased IL-33 mRNA in EA.hy926 cells in a concentration and time-dependent manner, and we conducted this study to explore its underlying mechanism. We identified that BC induced mitochondrial damage and suppressed autophagy function in EA.hy926 cells, as evidenced by elevation of the aspartate aminotransferase (GOT2), reactive oxygen species (ROS) and p62, and the reduction of mitochondrial membrane potential (ΔΨm). However, ROS cannot induce IL-33 mRNA-production in BC-exposed EA.hy926 cells. Further, experiments revealed that BC could promote IL-33 mRNA production through the PI3K/Akt/AP-1 and p38/AP-1 signaling pathways. It is concluded that BC could induce oxidative stress and suppress autophagy function in endothelial cells. This study also provided evidence that the pro-cardiovascular-diseases properties of BC may be due to its ability to stimulate the PI3K/AKT/AP-1 and p38/AP-1 pathway, further activate IL-33 and ultimately result in a local vascular inflammation.

Ad5-EMC6 mediates antitumor activity in gastric cancer cells through the mitochondrial apoptosis pathway.

Endoplasmic reticulum membrane protein complex subunit 6 (EMC6), also known as transmembrane protein 93 (transmembrane protein 93, TMEM93), is an autophagy-related protein. EMC6 overexpression inhibits cancer cell growth and induces apoptosis, but the interaction partners of EMC6 and its cellular responsibilities remain incompletely understood. In this study, we report that adenovirus-mediated ectopic overexpression of EMC6 (Ad5-EMC6) in BGC823 and SGC7901 gastric cancer cells decreases the activity of ERK1/2, down-regulates the levels of BCL-2 protein and phosphorylated BCL-2, increases the expression of tBID and BAX, and decreases mitochondrial membrane potential and subsequently leading to cell apoptosis. In a xenograft tumor model, we found that Ad5-EMC6 impairs the tumorigenesis of SGC7901 gastric cancer cells in nude mice. Additionally, Ad5-EMC6 enhances the sensitivity of gastric cancer cells to the chemotherapeutic drug etoposide. Collectively, these results demonstrate that EMC6-induced apoptosis of gastric cancer cells occurs at least partially through the mitochondrial-mediated apoptosis pathway. Our study suggests a rational basis for the potential clinical application of Ad5-EMC6 in gastric cancer.

Development and validation of an LC-MS/MS method for the determination of a novel thienoquinolin urea transporter inhibitor PU-48 in rat plasma and its application to a pharmacokinetic study.

A specific, sensitive and stable high-performance liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitative determination of methyl 3-amino-6-methoxythieno [2,3-b]quinoline-2-carboxylate (PU-48), a novel diuretic thienoquinolin urea transporter inhibitor in rat plasma. In this method, the chromatographic separation of PU-48 was achieved with a reversed-phase C18 column (100 × 2.1 mm, 3 µm) at 35°C. The mobile phase consisted of acetonitrile and water with 0.05% formic acid added with a gradient elution at flow rate of 0.3 mL/min. Samples were detected with the triple-quadrupole tandem mass spectrometer with multiple reaction monitoring mode via electrospray ionization source in positive mode. The retention time were 6.2 min for PU-48 and 7.2 min for megestrol acetate (internal standard, IS). The monitored ion transitions were mass-to-charge ratio (m/z) 289.1 → 229.2 for PU-48 and m/z 385.3 → 267.1 for the internal standard. The calibration curve for PU-48 was linear over the concentration range of 0.1-1000 ng/mL (r2 > 0.99), and the lower limit of quantitation was 0.1 ng/mL. The precision, accuracy and stability of the method were validated adequately. The developed and validated method was successfully applied to the pharmacokinetic study of PU-48 in rats.

Recombination and repeat-induced point mutation landscapes reveal trade-offs between the sexual and asexual cycles of Magnaporthe oryzae.

The fungal disease caused by Magnaporthe oryzae is one of the most devastating diseases that endanger many crops worldwide. Evidence shows that sexual reproduction can be advantageous for fungal diseases as hybridization facilitates host-jumping. However, the pervasive clonal lineages of M. oryzae observed in natural fields contradict this expectation. A better understanding of the roles of recombination and the fungi-specific repeat-induced point mutation (RIP) in shaping its evolutionary trajectory is essential to bridge this knowledge gap. Here we systematically investigate the RIP and recombination landscapes in M. oryzae using a whole genome sequencing data from 252 population samples and 92 cross progenies. Our data reveal that the RIP can robustly capture the population history of M. oryzae, and we provide accurate estimations of the recombination and RIP rates across different M. oryzae clades. Significantly, our results highlight a parent-of-origin bias in both recombination and RIP rates, tightly associating with their sexual potential and variations of effector proteins. This bias suggests a critical trade-off between generating novel allelic combinations in the sexual cycle to facilitate host-jumping and stimulating transposon-associated diversification of effectors in the asexual cycle to facilitate host coevolution. These findings provide unique insights into understanding the evolution of blast fungus.

Serum proteomic profiling of precancerous gastric lesions and early gastric cancer reveals signatures associated with systemic inflammatory response and metaplastic differentiation.

The noninvasive detection technique using serum for large-scale screening is useful for the early diagnosis of gastric cancer (GC). Herein, we employed liquid chromatography mass spectrometry to determine the serum proteome signatures and related pathways in individuals with gastric precancerous (pre-GC) lesions and GC and explore the effect of Helicobacter pylori (H. pylori) infection. Differentially expressed proteins in GC and pre-GC compared with non-atrophic gastritis (NAG) group were identified. APOA4, a protein associated with metaplastic differentiation, and COMP, an extracellular matrix protein, were increased in the serum of patients with pre-GC lesions and GC. In addition, several inflammation-associated proteins, such as component C3, were decreased in the GC and pre-GC groups, which highlight a tendency for the inflammatory response to converge at the gastric lesion site during the GC cascade. Moreover, the abundance of proteins associated with oxidant detoxification was higher in the GC group compared with that in the NAG group, and these proteins were also increased in the serum of the H. pylori-positive GC group compared with that in the H. pylori-negative GC patients, reflecting the importance of oxidative stress pathways in H. pylori infection. Collectively, the findings of this study highlight pathways that play important roles in GC progression, and may provide potential diagnostic biomarkers for the detection of pre-GC lesions.

RNF115/BCA2 deficiency alleviated acute liver injury in mice by promoting autophagy and inhibiting inflammatory response.

The E3 ubiquitin ligase RING finger protein 115 (RNF115), also known as breast cancer-associated gene 2 (BCA2), has been linked with the growth of some cancers and immune regulation, which is negatively correlated with prognosis. Here, it is demonstrated that the RNF115 deletion can protect mice from acute liver injury (ALI) induced by the treatment of lipopolysaccharide (LPS)/D-galactosamine (D-GalN), as evidenced by decreased levels of alanine aminotransaminase, aspartate transaminase, inflammatory cytokines (e.g., tumor necrosis factor α and interleukin-6), chemokines (e.g., MCP1/CCL2) and inflammatory cell (e.g., monocytes and neutrophils) infiltration. Moreover, it was found that the autophagy activity in Rnf115-/- livers was increased, which resulted in the removal of damaged mitochondria and hepatocyte apoptosis. However, the administration of adeno-associated virus Rnf115 or autophagy inhibitor 3-MA impaired autophagy and aggravated liver injury in Rnf115-/- mice with ALI. Further experiments proved that RNF115 interacts with LC3B, downregulates LC3B protein levels and cell autophagy. Additionally, Rnf115 deletion inhibited M1 type macrophage activation via NF-κB and Jnk signaling pathways. Elimination of macrophages narrowed the difference in liver damage between Rnf115+/+ and Rnf115-/- mice, indicating that macrophages were linked in the ALI induced by LPS/D-GalN. Collectively, for the first time, we have proved that Rnf115 inactivation ameliorated LPS/D-GalN-induced ALI in mice by promoting autophagy and attenuating inflammatory responses. This study provides new evidence for the involvement of autophagy mechanisms in the protection against acute liver injury.

TMEM189 negatively regulates the stability of ULK1 protein and cell autophagy.

ULK1 is crucial for initiating autophagosome formation and its activity is tightly regulated by post-translational modifications and protein-protein interactions. In the present study, we demonstrate that TMEM189 (Transmembrane protein 189), also known as plasmanylethanolamine desaturase 1 (PEDS1), negatively regulates the proteostasis of ULK1 and autophagy activity. In TMEM189-overexpressed cells, the formation of autophagesome is impaired, while TMEM189 knockdown increases cell autophagy. Further investigation reveals that TMEM189 interacts with and increases the instability of ULK1, as well as decreases its kinase activities. The TMEM189 N-terminal domain is required for the interaction with ULK1. Additionally, TMEM189 overexpression can disrupt the interaction between ULK1 and TRAF6, profoundly impairs K63-linked polyubiquitination of ULK1 and self-association, leading to the decrease of ULK1 stability. Moreover, in vitro and in vivo experiments suggest that TMEM189 deficiency results in the inhibition of tumorigenicity of gastric cancer. Our findings provide a new insight into the molecular regulation of autophagy and laboratory evidence for investigating the physiological and pathological roles of TMEM189.

Highly specific capture and direct MALDI-MS analysis of phosphorylated peptides using novel multifunctional chitosan-GMA-IDA-Fe (III) nanosphere.

In this study, we describe a method for highly specific enrichment of phosphopeptides with multifunctional chitosan-glycidyl methacrylate (GMA)-iminodiacetic acid (IDA)-Fe (III) nanospheres for direct analysis by matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). This is the first time that chitosan has been used to create nanospheres support material for selective enrichment of phosphopeptides by modification with GMA, derivatization with IDA, and loading with Fe (III) ions. Chitosan-GMA-IDA-Fe (III) nanospheres with a diameter of 20 to 100 nm have multifunctional chemical moieties which confer unique properties, good dispersibility in highly acidic binding buffers, as well as good biocompatibility and chemical stability which improves their specific interaction with phosphopeptides using various types of acid binding buffers. The process of enrichment is very simple, quick, efficient, and specific. Its high specificity and efficiency for purification of phosphopeptides is reflected in the very low and substoichiometric amounts of phosphopeptides which can be detected, in quantities as low as 1:3,000 M ratios. Compared with other state-of the-art technologies such as the use of conventional Fe(3+)-IMAC and TiO(2), these chitosan nanosphere techniques show superior specificity and sensitivity. Moreover, the resultant chitosan-GMA-IDA-Fe(3+) nanosphere-absorbed phosphopeptides can be either directly analyzed by MALDI-TOF MS analysis or eluted and further analyzed by nano-LC-MS/MS.

Novel multifunctional chitosan-GMA-IDA-Cu(II) nanospheres for high dynamic range characterization of the human plasma proteome.

In this study, we describe characterization of the human plasma proteome based on analysis with multifunctional chitosan-GMA-IDA-Cu(II) nanospheres. Chitosan-GMA-IDA-Cu(II) nanospheres with diameters of 20 to 100 nm have unique properties due to multifunctional chemical moieties, high surface area, high capacity, good dispersibility in buffer solution as well as good biocompatibility and chemical stability which improves their specific interaction with peptides and proteins of the human plasma using different binding buffers. Combining these chitosan-GMA-IDA-Cu(II) nanospheres with MS spectrometry results in a novel strategy which should make it possible to characterize the plasma proteome in a single test. Peptides and proteins adsorbed on the nanosphere can be directly detected by MALDI-TOF-MS. The eluted lower molecular weight peptides and proteins are identified by nano-LC-ESI-MS/MS. A total of 842 unique LMW peptides and 1,682 human unredundant proteins IDs were identified in two different binding buffers, which included relatively low-level proteins (e.g., pg/mL of IL3 Interleukin-3) co-distributed with high-abundance proteins (e.g., 35-55 mg/mL level serum albumin). As such, this nanosphere technique selectively enabled the identification of proteins over a dynamic range of greater than 8 orders of magnitude. Considering this capacity for selective enrichment of peptides and proteins in human plasma, and the large number of LMW peptides and proteins which can be identified, this method promises to accelerate discovery of biomarkers for clinical application.

Functional expression of TRPA1 channel, TRPV1 channel and TMEM100 in human odontoblasts.

TRPA1 and TRPV1 channels respond to external stimulation as pain mediators and form a complex with a transmembrane protein TMEM100 in some tissues. However, their expression and interaction in dental pulp is unclear. To investigate the functional co-expression of TRPA1 channel, TRPV1 channel and TMEM100 in human odontoblasts (HODs), immunohistochemistry, immunofluorescence staining and Western blot were used to study their co-localization and expression in both native HODs and cultured HOD-like cells. Calcium imaging was used to detect the functional interaction between TRPA1 and TRPV1 channels. Immunohistochemistry and multiple immunofluorescence staining of tooth slices showed positive expression of TRPA1 channel, TRPV1 channel and TMEM100 mainly in the cell bodies of HODs, and TRPA1 channel presented more obvious immunofluorescence in the cell processes than TRPV1 channel and TMEM100. HALO software analysis showed that TRPA1 and TRPV1 channels were positively expressed in most TMEM100+ HODs and these three proteins were strongly correlated in HODs (P < 0.01). The protein expression levels of TRPA1 channel, TRPV1 channel and TMEM100 in HODs showed no significant difference (P > 0.05). Double immunofluorescence staining of cultured HOD-like cells visually demonstrated that TRPA1 and TRPV1 channel were both highly co-localized with TMEM100 with similar expressive intensity. Calcium imaging showed that there was a functional interaction between TRPA1 and TRPV1 channels in HOD-like cells, and TRPA1 channel might play a greater role in this interaction. Overall, we concluded that TRPA1 channel, TRPV1 channel and TMEM100 could be functionally co-expressed in HODs.

Activation of Transient Receptor Potential Ankyrin 1 and Vanilloid 1 Channels Promotes Odontogenic Differentiation of Human Dental Pulp Cells.

INTRODUCTION: Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) are thermosensitive channels that play an important role in thermal sensation or tooth pain by regulating intracellular Ca2+ concentration that is essential for pulp tissue repair. The aim of this study was to evaluate the role of TRPA1 and TRPV1 channels in the odontogenic differentiation of human dental pulp cells (HDPCs). METHODS: HDPCs were isolated from healthy human intact third molars and cultured in odontogenic differentiation medium. Gene and protein expression levels of TRPA1 and TRPV1 channels during the odontogenic differentiation of HDPCs were evaluated by real-time quantitative polymerase chain reaction and Western blot analysis. HDPCs were then treated with channel agonists or antagonists, and the expression levels of odontogenic markers dentin sialophosphoprotein (DSPP) and osteopontin (OPN) were examined. Alkaline phosphatase activity and alizarin red staining were also conducted to detect mineralization levels. RESULTS: Consistent with the mineralization degree and DSPP and OPN expression, messenger RNA and protein expression of TRPA1 and TRPV1 channels was up-regulated during the odontogenic differentiation of HDPCs. The application of TRPA1 or TRPV1 agonists increased the mineralized nodules of alizarin red staining and alkaline phosphatase activity and up-regulated the messenger RNA and protein expression of DSPP and OPN, respectively, with the highest values reached on the seventh day (P < .05). On the contrary, the mineralization level and DSPP and OPN expression could be suppressed by using the antagonists of these 2 channels. CONCLUSIONS: TRPA1 and TRPV1 channels not only showed up-regulated expression along with the odontogenic differentiation of HDPCs but also could affect the odontogenic differentiation by regulating intracellular Ca2+ concentration.

Potent antitumor activities of recombinant human PDCD5 protein in combination with chemotherapy drugs in K562 cells.

Conventional chemotherapy is still frequently used. Programmed cell death 5 (PDCD5) enhances apoptosis of various tumor cells triggered by certain stimuli and is lowly expressed in leukemic cells from chronic myelogenous leukemia patients. Here, we describe for the first time that recombinant human PDCD5 protein (rhPDCD5) in combination with chemotherapy drugs has potent antitumor effects on chronic myelogenous leukemia K562 cells in vitro and in vivo. The antitumor efficacy of rhPDCD5 protein with chemotherapy drugs, idarubicin (IDR) or cytarabine (Ara-C), was examined in K562 cells in vitro and K562 xenograft tumor models in vivo. rhPDCD5 protein markedly increased the apoptosis rates and decreased the colony-forming capability of K562 cells after the combined treatment with IDR or Ara-C. rhPDCD5 protein by intraperitoneal administration dramatically improved the antitumor effects of IDR treatment in the K562 xenograft model. The tumor sizes and cell proliferation were significantly decreased; and TUNEL positive cells were significantly increased in the combined group with rhPDCD5 protein and IDR treatment compared with single IDR treatment groups. rhPDCD5 protein, in combination with IDR, has potent antitumor effects on chronic myelogenous leukemia K562 cells and may be a novel and promising agent for the treatment of chronic myelogenous leukemia.

[Two types of MWNTs with different surface modifications induce differential expression of proteins in RAW264.7 cells].

OBJECTIVE: To compare the different expression of protein in RAW264.7 macrophage cells induced by two types of MWNTs (multi-walled carbon nanotubes) with different surface modifications (acid-treated MWNTs and tau-MWNTs modified by taurine). METHODS: Treating cells with both types of MWNTs in 20 mg/L and 24 h, with a blank-control group set. Cells are lysed by using urea and by ultrasonicating in ice bath, then total proteins of cells are extracted. Using two-dimensional gel electrophoresis to separate total proteins of cells, searching for the differential expressed protein spots on the images of the gels with silver staining. Identifying the differentially expressed proteins via mass spectrometry, and studying the mechanism of effects on cells imposed by two types of MWNTs at protein level. RESULTS: There are 13 spots of protein with notably differential expression among three treated groups (including blank controls). Their functions involve apoptosis-related, calcium-binding, cell-cycle related, DNA synthesis, folding of proteins, and energy metabolism, etc. The results are consistent with our previous studies about the cytotoxicity of Both types of MWNTs including induction of apoptosis and mitochodira damage. CONCLUSION: Both two types of MWNTs could induce alteration of protein expression in RAW264.7 cells. With different surface modifications, they imposed different effects. High throughout proteomics could be applied in toxicity assessment and mechanism investigation about carbon nanotubes.

RNF115 deletion inhibits autophagosome maturation and growth of gastric cancer.

Autophagy is a highly conserved lysosome-dependent degradation system in eukaryotic cells. This process removes long-lived intracellular proteins, damaged organelles, and recycles biological material to maintain cellular homeostasis. Dysfunction of autophagy triggers a wide spectrum of human diseases, including cancer and neurodegenerative diseases. In the present study, we show that RNF115, an E3 ubiquitin ligase, regulates autophagosome-lysosome fusion and autophagic degradation under both nutrient-enriched and stress conditions. Depletion of the RNF115 gene caused the accumulation of autophagosomes by impairing fusion with lysosomes, which results in an accumulation of autophagic substrates. Further investigation suggests that RNF115 interacts with STX17 and enhances its stability, which is essential for autophagosome maturation. Importantly, we provide in vitro and in vivo evidence that RNF115 inactivation inhibits the tumorigenesis and metastasis of BGC823 gastric cancer cells. We additionally show that high expression levels of RNF115 mRNA correlate with poor prognosis in gastric cancer patients. These findings indicate that RNF115 may play an evolutionarily conserved role in the autophagy pathway, and may act to maintain protein homeostasis under physiological conditions. These data demonstrate the need to further evaluate the potential therapeutic implications of RNF115 in gastric cancer.

Effects of at-home bleaching and resin infiltration treatments on the aesthetic and psychological status of patients with dental fluorosis: A prospective study.

OBJECTIVE: This study aims to evaluate the effect of the combination approach of at-home bleaching (HB) and resin infiltration (RI) techniques on different severity degrees of dental fluorosis (DF) and further analyze the psychological changes caused by HB and RI in patients. METHOD: Twenty-two patients (4 males, 18 females, 27.8 ±â€¯1.6 yrs) with 186 fluorotic teeth were included in this study and classified into mild (N = 56), moderate (N = 100) and severe (N = 30) DF groups according to the Dean's index. The treatment effects on patients with DF were assessed by questionnaires including the changes in patients' subjective evaluation of their teeth and psychological status before and after treatments. Standardized digital photographs were taken at each time point of the treatment process, including baseline (T1), after bleaching (T2), immediately after RI treatment (T3) and more than six months after RI treatment (T4). The color alterations (ΔE) between the fluorotic (F2) and the surrounding relatively sound areas (F1) were analyzed. RESULTS: Bad tooth appearance caused 13.64% of patients often depressed, frustrated, or disappointed, whereas 72.72% occasionally had these feelings. After treatment, the satisfaction of DF patients regarding tooth appearance increased from 0% (satisfied) to 58.82% (satisfied) and 23.53% (very satisfied). Moreover, these treatments improved all patients' confidence in smiling, laughing and showing their teeth. The percentage of fluorotic teeth with ΔE values more than 3.0 and 3.7 units decreased gradually from T1 stage to T3 stage in mild and moderate DF groups (p < 0.05), whereas the ΔE value in T3 stage was significantly lower than that of T2 stage in severe DF group (p < 0.05). In T4 stage, no significant difference was observed in the ΔE values between T4 and T3 stages (p > 0.05). CONCLUSION: This study shows the obvious positive aesthetic effect of HB and RI treatment on different severity degrees of DF and the great improvements in psychological discomforts. CLINICAL SIGNIFICANCE: The combination treatment of RI and low concentration HB gel improves the aesthetics of DF and may have a stable effect after 6-months follow-up, suggesting that this approach is a valuable clinical choice for dentists to treat DF.

Deletion of TMEM268 inhibits growth of gastric cancer cells by downregulating the ITGB4 signaling pathway.

Transmembrane protein 268 (TMEM268) encodes a novel human protein of previously unknown function. This study analyzed the biological activities and molecular mechanisms of TMEM268 in vivo and in vitro. We found that TMEM268 deletion decreases cell viability, proliferation, and cell adhesion as well as causing S-phase cell cycle arrest and disrupts cytoskeleton remolding. Xenograft tumor mouse model studies showed that TMEM268 deletion inhibits the tumorigenesis of BGC823 gastric cancer cells. In addition, TMEM268-deleted BGC823 cells failed to colonize the lungs after intravenous injection and to form metastatic engraftment in the peritoneum. Molecular mechanism studies showed a C-terminal interaction between TMEM268 and integrin subunit ß4 (ITGB4). TMEM268 knockout promotes ITGB4 ubiquitin-mediated degradation, increasing the instability of ITGB4 and filamin A (FLNA). The reduced ITGB4 protein levels result in the disassociation of the ITGB4/PLEC complex and cytoskeleton remodeling. This study for the first time demonstrates that TMEM268 plays a positive role in the regulation of ITGB4 homeostasis. The above results may provide a new perspective that targeting the TMEM268/ITGB4 signaling axis for the treatment of gastric cancer, which deserves further investigation in the future.

Pharmacokinetics, Tissue Distribution and Excretion of a Novel Diuretic (PU-48) in Rats.

Methyl 3-amino-6-methoxythieno [2,3-b] quinoline-2-carboxylate (PU-48) is a novel diuretic urea transporter inhibitor. The aim of this study is to investigate the profile of plasma pharmacokinetics, tissue distribution, and excretion by oral dosing of PU-48 in rats. Concentrations of PU-48 within biological samples are determined using a validated high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. After oral administration of PU-48 (3, 6, and 12 mg/kg, respectively) in self-nanomicroemulsifying drug delivery system (SNEDDS) formulation, the peak plasma concentrations (Cmax), and the area under the curve (AUC0⁻∞) were increased by the dose-dependent and linear manner, but the marked different of plasma half-life (t1/2) were not observed. This suggests that the pharmacokinetic profile of PU-48 prototype was first-order elimination kinetic characteristics within the oral three doses range in rat plasma. Moreover, the prototype of PU-48 was rapidly and extensively distributed into thirteen tissues, especially higher concentrations were detected in stomach, intestine, liver, kidney, and bladder. The total accumulative excretion of PU-48 in the urine, feces, and bile was less than 2%. This research is the first report on disposition via oral administration of PU-48 in rats, and it provides important information for further development of PU-48 as a diuretic drug candidate.

Liver-specific deletion of Eva1a/Tmem166 aggravates acute liver injury by impairing autophagy.

Acute liver failure (ALF) is an inflammation-mediated hepatocellular injury process associated with cellular autophagy. However, the mechanism by which autophagy regulates ALF remains undefined. Herein, we demonstrated that Eva1a (eva-1 homolog A)/Tmem166 (transmembrane protein 166), an autophagy-related gene, can protect mice from ALF induced by D-galactosamine (D-GalN)/lipopolysaccharide (LPS) via autophagy. Our findings indicate that a hepatocyte-specific deletion of Eva1a aggravated hepatic injury in ALF mice, as evidenced by increased levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), myeloperoxidase (MPO), and inflammatory cytokines (e.g., TNFα and IL-6), which was associated with disordered liver architecture exhibited by Eva1a-/- mouse livers with ALF. Moreover, we found that the decreased autophagy in Eva1a-/- mouse liver resulted in the substantial accumulation of swollen mitochondria in ALF, resulting in a lack of ATP generation, and consequently hepatocyte apoptosis or death. The administration of Adeno-Associated Virus Eva1a (AAV-Eva1a) or antophagy-inducer rapamycin increased autophagy and provided protection against liver injury in Eva1a-/- mice with ALF, suggesting that defective autophagy is a significant mechanism of ALF in mice. Collectively, for the first time, we have demonstrated that Eva1a-mediated autophagy ameliorated liver injury in mice with ALF by attenuating inflammatory responses and apoptosis, indicating a potential therapeutic application for ALF.