Effects of Dietary Chlorogenic Acid Supplementation on Growth Performance, Meat Quality, and Muscle Flavor Substances in Finishing Pigs.

With the prohibition of antibiotics in feed, certain phytocompounds have been widely studied as feed additives. Chlorogenic acid (CGA), a natural polyphenol found in plants, possesses anti-inflammatory, antioxidant, and metabolic regulatory features. The objective of this study was to investigate the effects of dietary chlorogenic acid supplementation on growth performance and carcass traits, as well as meat quality, nutrient value and flavor substances of Duroc × Landrace × Yorkshire (DLY) pigs. Forty healthy DLY pigs (initial body weight (BW): 26.69 ± 0.37) were allotted to four treatment groups and were fed with the control diet, which was supplemented with 25 mg kg-1, 50 mg kg-1, and 100 mg kg-1 CGA, respectively. The trial lasted 100 days. The results suggested that dietary CGA supplementation had no effect (p < 0.05) on the average daily gain (ADG) and feed conversion ratio (FC). Herein, it was found that 50 mg kg-1 CGA-containing diet not only increased the dressing percentage and perirenal fat, but also reduced the rate of muscular pH decline (p < 0.05). In the longissimus thoracis (LT) muscle, the myofiber-type-related genes such as the MyHC IIa and MyHC IIX mRNA levels were increased by 100 mg kg-1 CGA. The results also indicated that the 100 mg kg-1 CGA-containing diet increased the content of crude fat, glycogen, total amino acids, and flavor amino acids, but decreased the inosine and hypoxanthine concentration in LT (p < 0.05). Meanwhile, the lipogenic gene ACC1 mRNA level was elevated by 50 mg kg-1 CGA. Instead, 100 mg kg-1 CGA downregulated the expression level of NT5C2, an enzyme responsible for inosine-5'-monophosphate (IMP) degradation. Additionally, 100 mg kg-1 CGA decreased the malondialdehyde (MDA) content, but increased the glutathione peroxidase (GSH-Px) content as well as antioxidant gene (HO-1, NQO-1, NRF2) mRNA levels in LT muscle. These findings showed that dietary CGA could partly improve carcass traits and muscle flavor without negatively affecting growth performance, and the underlying mechanism may be due to the antioxidant properties induced by CGA.

Integration of solid-phase microextraction and surface-enhanced Raman spectroscopy for in-vivo screening of polybrominated diphenyl ether.

The monitoring of polybrominated diphenyl ethers (PBDEs) is of great significance owing to their high persistence, bioaccumulation, and toxicity to humans and animals. In this study, a sensitive and reproducible probe that integrates solid-phase microextraction and surface-enhanced Raman spectroscopy (SPME-SERS) was developed for screening PBDEs in multiphase specimens, including live fish, water, and electrical products. A roughed Cu fiber with an Ag layer was fabricated with dual functions. BDE-15 was readily extracted and detected on the SPME-SERS probe consisting of propanethiol-modified Ag nanoplates on a Cu wire. A clear linear relationship (R2 = 0.988) was established between the SERS intensity at 782 cm-1 and the logarithmic concentrations (from 100 ppb to 100 ppm), with a detection limit of 15 ppb. This proposed method enables continuous in vivo monitoring in fish without complicated pretreatments. The results obtained by this SPME-SERS approach were validated by high-performance liquid chromatography and showed good agreement. This "extracting and detecting" SPME-SERS method provides a potential tool to monitor the occurrence, formation, and migration of PBDEs.

USP8 inhibitor-induced DNA damage activates cell cycle arrest, apoptosis, and autophagy in esophageal squamous cell carcinoma.

Increasing evidence suggests that targeting ubiquitin-specific peptidase 8 (USP8) serves as an attractive anti-cancer strategy. However, the role of USP8 inhibitor, DUB-IN-1, in esophageal squamous cell carcinoma (ESCC) cells still needs to be explored. Here, immunohistochemistry was employed to examine the expression of USP8 in ESCC tissues. Cell Counting Kit-8 (CCK-8) was used to evaluate cell proliferation ability, and propidium iodide (PI) was selected to test the effect of DUB-IN-1 on cell cycle. AnnexinV-FITC/PI staining and the activity of caspase 3 were detedcted to evaluate apoptosis. Transmission electron microscope, microtubule-associated protein 1 light-chain 3 (LC3) expression, and acridine orange (AO) staining were selected to check if there was autophagy. Comet assay and γ-H2AX immunofluorescence was used to monitor DNA damage. Rescue experiment was used to determine the key role of of p53 in cell cycle, apoptosis, and autophagy. Results revealed that the leve of USP8 was higher in ESCC tissues than that in tissues adjacent to carcinoma. DUB-IN-1, an USP8 inhibitor, caused DNA damage, led to G2/M phase block by p53-p21 axis, and triggered apoptosis by regulating the p53 target proteins including Bax, Noxa, and Puma. Besides, DUB-IN-1 could stimulate autophagy through p53-dependent adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) activation. Taken together, this study revealed the cytotoxic effects and the mechanism of DUB-IN-1, which indicated that DUB-IN-1 may be a novel inhibitor targeting USP8 that can kill ESCC cells. USP8 inhibitor, DUB-IN-1, treatment could inhibit esophageal squamous cell carcinoma cell growth and induce G2/M cell cycle arrest, apoptosis, and autophagy by DNA damage-induced p53 activation. DUB-IN-1 treatment led to G2/M cell cycle arrest by upregulating the protein level of p21 and triggered apoptosis by modulating the p53 target proteins including Bax, Noxa, and Puma. Meanwhile, DUB-IN-1 treatment stimulated protective autophagy through p53-dependent AMPK activation. Collectively, these findings suggested that DNA damage-triggered p53 activation, p53-Puma/Noxa/Bax, p53-p21, and p53-AMPK pathways were all involved in the effect of DUB-IN-1.

Inhibition of USP1 activates ER stress through Ubi-protein aggregation to induce autophagy and apoptosis in HCC.

The deubiquitinating enzyme USP1 (ubiquitin-specific protease 1) plays a role in the progression of various tumors, emerging as a potential therapeutic target. This study aimed to determine the role of USP1 as a therapeutic target in hepatocellular carcinoma (HCC). We detected USP1 expression in the tumor and adjacent tissues of patients with HCC using immunohistochemical staining. We evaluated the effect of the USP1 inhibitor ML-323 on HCC cell proliferation and cell cycle using a CCK-8 cell-counting kit and plate cloning assays, and propidium iodide, respectively. Apoptosis was detected by annexin V-FITC/Propidium Iodide (PI) staining and caspase 3 (casp3) activity. Transmission electron microscopy and LC3B immunofluorescence were used to detect autophagy. Western blotting was used to detect the accumulation of ubiquitinated proteins, the expression of endoplasmic reticulum (ER) stress-related proteins, and the AMPK-ULK1/ATG13 signaling pathway. We demonstrated that ML-323 inhibits the growth of HCC cells and induces G1 phase cell cycle arrest by regulating cyclin expression. ML-323 treatment resulted in the accumulation of ubiquitinated proteins, induced ER stress, and triggered Noxa-dependent apoptosis, which was regulated by the Activating Transcription Factor 4(ATF4). Moreover, active ER stress induces protective autophagy by increasing AMPK phosphorylation; therefore, we inhibited ER stress using 4-Phenylbutyric acid (4-PBA), which resulted in ER stress reduction, apoptosis, and autophagy in ML-323-treated HCC cells. In addition, blocking autophagy using the AMPK inhibitor compound C (CC), chloroquine (CQ), or bafilomycin A1 (BafA1) enhanced the cytotoxic effect of ML-323. Our findings revealed that targeting USP1 may be a potential strategy for the treatment of HCC.

ML323, a USP1 inhibitor triggers cell cycle arrest, apoptosis and autophagy in esophageal squamous cell carcinoma cells.

Esophageal squamous cell carcinoma (ESCC) is a common digestive cancer with high mortality rate due to late diagnosis and drug resistance. It is important to identify new molecular target and develop new anticancer strategy. ML323 is a novel USP1 inhibitor and exhibits anticancer activity against several cancers. Herein, we investigated whether ML323 has some cytotoxity effect on ESCC cells and explored the underlying mechanisms. Results revealed that ML323 impeded esophageal cancer cell viability and colony formation. Meanwhile, ML323 blocked cells at G0/G1 phase concomitant with the reduced protein level of c-Myc, cyclin D1, CDK4 and CDK6. ML323 treatment also triggered DNA damage and active p53. Then, ML323 induced apoptosis by p53-Noxa. Additionally, it stimulated protective autophagy. Co-treatment with CQ or BafA1, two classical autophagy inhibitors, enhanced the cytotoxity of ML323. These findings suggested that USP1 inhibitor (ML323) could be used as a viable anti-ESCC approach.

Enantioselective Synthesis of Chiral Carboxylic Acids from Alkynes and Formic Acid by Nickel-Catalyzed Cascade Reactions: Facile Synthesis of Profens.

We report a stereoselective conversion of terminal alkynes to α-chiral carboxylic acids using a nickel-catalyzed domino hydrocarboxylation-transfer hydrogenation reaction. A simple nickel/BenzP* catalyst displayed high activity in both steps of regioselective hydrocarboxylation of alkynes and subsequent asymmetric transfer hydrogenation. The reaction was successfully applied in enantioselective preparation of three nonsteroidal anti-inflammatory profens (>90 % ees) and the chiral fragment of AZD2716.

Design, synthesis and evaluation of novel bis-substituted aromatic amide dithiocarbamate derivatives as colchicine site tubulin polymerization inhibitors with potent anticancer activities.

As the continuation of our work on the development of tubulin inhibitors with potential anticancer activities, novel bis-substituted aromatic amide dithiocarbamate derivatives were designed by contacting bis-substituted aryl scaffolds (potential anti-tubulin fragments) with N-containing heterocycles (potential anti-tubulin fragments) in one hybrid using the anticancer dithioformate unit as the linker. The antiproliferative activity against three digestive tract tumor cells was evaluated and preliminary structure activity relationships were summarized. Among these compounds, compound 20q exhibited most potent antiproliferative activity against MGC-803, HCT-116, Kyse30 and Kyse450 cells with IC50 values of 0.084, 0.227, 0.069 and 0.078 µM, respectively. In further studies, compound 20q was identified as a novel tubulin inhibitor targeting the colchicine binding site. Compound 20q could inhibit the microtubule assembly and disrupt cytoskeleton in Kyse30 and Kyse450 cells. The results of molecular docking suggested that compound 20q could tightly bind into the colchicine binding site of tubulin by hydrogen bonds and hydrophobic interactions. Compound 20q dose-dependently inhibited the cell growth and colony formation, effectively arrested cells at the G2/M phase and induce mitochondrial apoptosis in Kyse30 and Kyse450 cells. In addition, Compound 20q could regulate the expression of G2/M phase and mitochondrial apoptosis related proteins. Collectively, compound 20q was here reported as a novel tubulin inhibitor with potential anticancer activities.

Untargeted metabolomics reveals the mechanism of quercetin enhancing the bioavailability of ticagrelor.

Ticagrelor is a first-line clinical drug for the treatment of acute coronary syndrome, but its oral bioavailability is relatively low. Flavonoids (polyphenol compounds commonly found in plant foods) seriously affect human metabolism and health. This study compared the effects of quercetin, luteolin and catechin on the pharmacokinetic parameters of ticagrelor and found that quercetin can significantly increase the Cmax and area under the curve from time zero to 36 h (AUC0-36 ) of ticagrelor, that is, quercetin can enhance the bioavailability of ticagrelor, but luteolin and catechin cannot. The difference between the ticagrelor group and the combination of quercetin and ticagrelor was analyzed through untargeted metabolomics methods and multivariate data analysis, which identified changes in the levels of seven metabolites (deoxycholic acid, taurocholic acid, glycocholic acid, glycoursodeoxycholic acid, tryptophan, phenylalanine and kynurenine). Based on the changes of these metabolites, we found that the metabolic pathways of phenylalanine, tyrosine and tryptophan and the biosynthetic pathway of bile acids were changed. A metabolomics study revealed that quercetin improves the oral bioavailability of ticagrelor and that this might rely on changing the metabolic pathways of phenylalanine, tyrosine and tryptophan and the biosynthetic pathway of bile acids. The research results at the metabolic level provide us with a strong basis and direction for further exploring the mechanism underlying quercetin's ability to enhance the bioavailability of ticagrelor, and this may be useful for finding new agents that enhance the bioavailability.

Oridonin induces ferroptosis by inhibiting gamma-glutamyl cycle in TE1 cells.

Oridonin (Ori) is a natural tetracyclic diterpenoid active compound with excellent antitumor activity, but the mechanism of Ori on esophageal cancer cell, TE1, remains unclear. In this study, we examined the levels of intracellular iron, malondialdehyde, and reactive oxygen species after Ori treatment, while interfering with the effects of Ori with ferroptosis inhibitor, demonstrating that Ori's inhibition of TE1 cell proliferation is associated with ferroptosis. To understand the molecular mechanism of Ori, we performed UPLC-MS/MS metabolomics profiling on TE1 cells, which show that gamma-glutamyl amino acids (gamma-glutamylleucine, gamma-glutamylvaline), 5-oxoproline, glutamate, GSH, and GSSG are changed significantly after Ori treatment. Meanwhile, the activity of gamma-glutamyl transpeptidase 1 (GGT1) decreased. This revealed that Ori inhibited the gamma-glutamyl cycle in TE1 cells. Furthermore, we found that Ori can covalently bind to cysteine to form the conjugate oridonin-cysteine (Ori-Cys), resulting in the inhibition of glutathione synthesis, which is consistent with the decrease in the enzymatic activity of glutamate cysteine ligase catalytic subunit (GCLC). Eventually, the value of intracellular GSH/GSSG was reduced, and the enzymatic activity of the glutathione peroxidase 4 (GPX4) was significantly decreased. In conclusion, our experiments indicated that Ori can inhibit the gamma-glutamyl cycle, thereby inducing ferroptosis to exert anti-cancer activity.

Protective effect of metformin on BPA-induced liver toxicity in rats through upregulation of cystathionine ß synthase and cystathionine γ lyase expression.

Human exposure to bisphenol A (BPA) is unavoidable in daily life. Recently, research has showen that BPA could induce oxidative imbalance, thereby causing reproductive toxicity and liver dysfunction. Accumulated evidence has demonstrated that metformin possesses strong anti-oxidative properties. This study aimed to study the mechanism underlying the hepatic-protective effect of metformin on liver injury induced by BPA in rats via the UPLC-MS/MS metabolomics approach. Forty-two male rats were randomly divided into six groups (n = 7), namely the saline group (control), the corn oil group (vehicle), the metformin group (Met), the bisphenol A group (BPA), the bisphenol A and metformin group (BPA + Met), and the bisphenol A and diammonium glycyrrhizinate (positive control) group (BPA + DG). Serum was collected for biochemical analysis and metabolomics, and liver tissue was collected for histopathology and metabolomics in each group. We found that metformin could significantly reduce the levels of liver function enzymes (ALT, AST and GGT) and ameliorate inflammatory cell infiltration and hepatocyte necrosis induced by BPA. On the other hand, metformin could significantly enhance the total antioxidant capacity in BPA rats. Notably, metabolomics data indicated that the principal altered metabolic pathways based on the 26 differential metabolites in liver tissue, and 21 in serum among vehicle, BPA and BPA + Met groups, respectively, including cysteine and methionine metabolism, glutathione metabolism, and arginine biosynthesis and purine metabolism. Additionally, metformin significantly increased cystathionine ß synthase (CBS) and cystathionine γ lyase (CSE), thus reducing serum levels of homocysteine and increasing hepatic levels of cysteine and glutathione in BPA-treated rats. Overall, this study's results provided new insights into the role and mechanism of metformin in BPA-induced liver injury in rats.

Inhibition of deubiquitination by PR-619 induces apoptosis and autophagy via ubi-protein aggregation-activated ER stress in oesophageal squamous cell carcinoma.

OBJECTIVES: Targeting the deubiquitinases (DUBs) has become a promising avenue for anti-cancer drug development. However, the effect and mechanism of pan-DUB inhibitor, PR-619, on oesophageal squamous cell carcinoma (ESCC) cells remain to be investigated. MATERIALS AND METHODS: The effect of PR-619 on ESCC cell growth and cell cycle was evaluated by CCK-8 and PI staining. Annexin V-FITC/PI double staining was performed to detect apoptosis. LC3 immunofluorescence and acridine orange staining were applied to examine autophagy. Intercellular Ca2+ concentration was monitored by Fluo-3AM fluorescence. The accumulation of ubi-proteins and the expression of the endoplasmic reticulum (ER) stress-related protein and CaMKKß-AMPK signalling were determined by immunoblotting. RESULTS: PR-619 could inhibit ESCC cell growth and induce G2/M cell cycle arrest by downregulating cyclin B1 and upregulating p21. Meanwhile, PR-619 led to the accumulation of ubiquitylated proteins, induced ER stress and triggered apoptosis by the ATF4-Noxa axis. Moreover, the ER stress increased cytoplasmic Ca2+ and then stimulated autophagy through Ca2+ -CaMKKß-AMPK signalling pathway. Ubiquitin E1 inhibitor, PYR-41, could reduce the accumulation of ubi-proteins and alleviate ER stress, G2/M cell cycle arrest, apoptosis and autophagy in PR-619-treated ESCC cells. Furthermore, blocking autophagy by chloroquine or bafilomycin A1 enhanced the cell growth inhibition effect and apoptosis induced by PR-619. CONCLUSIONS: Our findings reveal an unrecognized mechanism for the cytotoxic effects of general DUBs inhibitor (PR-619) and imply that targeting DUBs may be a potential anti-ESCC strategy.

Nickel-catalyzed C-alkylation of thioamide, amides and esters by primary alcohols through a hydrogen autotransfer strategy.

A simple catalyst of Ni(OAc)2 and P(t-Bu)3 enables selective C-alkylation of thioacetamides and primary acetamides with alcohols for the first time. Monoalkylation of thioamides, amides and t-butyl esters occurs in excellent yields (>95%). Mechanistic studies reveal that the reaction proceeds via a hydrogen autotransfer pathway.

Nickel-Catalyzed Asymmetric Transfer Hydrogenation and α-Selective Deuteration of N-Sulfonyl Imines with Alcohols: Access to α-Deuterated Chiral Amines.

A nickel-catalyzed enantioselective transfer hydrogenation and deuteration of N-sulfonyl imines was developed. Excellent α-selectivity and high deuterium content were achieved by using inexpensive 2-propanol-d8 as a deuterium source. As a highlight, no deuteration of ß-C-H and the remote C-H of N-sulfonyl amines occurred, which is hard to achieve using other imines or by hydrogen isotope exchange with D2O. Mechanism studies indicated a stepwise pathway through the [Ni-D] intermediate.

Quercetin overcomes colon cancer cells resistance to chemotherapy by inhibiting solute carrier family 1, member 5 transporter.

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) remains a significant impediment to the success of cancer chemotherapy. The natural flavonoid Quercetin (Que) has been reported to be able to inhibit P-gp-mediated MDR in various cancer cells. However, the MDR reversal effect of Que on human colon cancer cells and its mechanism at the metabolic level requires further clarification. This study was designed to provide a better understanding of the MDR reversal effect of Que. Our present results showed that 33 µM of Que significantly improved the cytotoxicity of doxorubicin (Dox) to P-gp-overexpressed SW620/Ad300 cells by proliferation and apoptpsis assay. Further mechanism studies demonstrated that Que inhibited the ATP-driven transport activity of P-gp, which in turn increased the intracellular accumulation of Dox. The metabolomics studies based on UPLC-MS/MS analysis revealed that Que could reverse the MDR by significantly blocking D-glutamine and D-glutamate metabolism, and the underlying mechanism is that Que down-regulated the expression of the glutamine transporter solute sarrier family 1, member 5 (SLC1A5) in SW620/Ad300 cells. This is the first time to report that Que was a SLC1A5 inhibitor, which could be served as a template compound to potentially develop novel P-gp-mediated MDR reversal modulators in cancer chemotherapy.

Prefrontal Nectin3 Reduction Mediates Adolescent Stress-Induced Deficits of Social Memory, Spatial Working Memory, and Dendritic Structure in Mice.

Chronic stress may disrupt the normal neurodevelopmental trajectory of the adolescent brain (especially the prefrontal cortex) and contribute to the pathophysiology of stress-related mental illnesses, but the underlying molecular mechanisms remain unclear. Here, we investigated how synaptic cell adhesion molecules (e.g., nectin3) are involved in the effects of adolescent chronic stress on mouse medial prefrontal cortex (mPFC). Male C57BL/6N mice were subjected to chronic social instability stress from postnatal days 29 to 77. One week later, the mice exposed to chronic stress exhibited impaired social recognition and spatial working memory, simplified dendritic structure, and reduced spine density in the mPFC. Membrane localization of nectin3 was also altered, and was significantly correlated with behavioral performance. Furthermore, knocking down mPFC nectin3 expression by adeno-associated virus in adolescent mice reproduced the stress-induced changes in behavior and mPFC morphology. These results support the hypothesis that nectin3 is a potential mediator of the effects of adolescent chronic stress on prefrontal structural and functional abnormalities.

Synthesis and in vitro biological evaluation of novel derivatives of Flexicaulin A condensation with amino acid trifluoroacetate.

As our research focus on anticancer drugs, two series of novel derivatives of Flexicaulin A (FA), an ent-kaurene diterpene, condensation with amino acid trifluoroacetate were synthesized, and their anti-proliferative activity against four human cancer cell lines (TE-1, MCF-7, A549 and MGC-803) were evaluated. Compared with FA, the anticancer activity and solubility of most derivatives were significantly improved. Among them, compound 6d had the best activity, and its IC50 value against Esophageal cancer cells (TE-1) was up to 0.75 µM. Subsequent cellular mechanism studies showed that compound 6d could inhibit the proliferation of cancer cells, the formation of cell colonies, and increase the level of ROS on TE-1 cells. In addition, 6d could up-regulate the expressions of SAPK/JNK pathway-associated proteins (p-ASK1, p-MKK4 and p-JNK) and pro-apoptotic proteins (Bak, Bad and Noxa), remarkably increase the ratio of Bax to Bcl-2 and activate Cleaved Caspase-3/9/PARP. These results indicate that compound 6d induces apoptosis through the ROS/JNK/Bcl-2 pathway and holds promising potential as an anti-proliferative agent.

GAS1 Deficient Enhances UPR Activity in Saccharomyces cerevisiae.

Beta-1,3-glucanosyltransferase (Gas1p) plays important roles in cell wall biosynthesis and morphogenesis and has been implicated in DNA damage responses and cell cycle regulation in fungi. Yeast Gas1p has also been reported to participate in endoplasmic reticulum (ER) stress responses. However, the precise roles and molecular mechanisms through which Gas1p affects these responses have yet to be elucidated. In this study, we constructed GAS1-deficient (gas1Δ) and GAS1-overexpressing (GAS1 OE) yeast strains and observed that the gas1Δ strain exhibited a decreased proliferation ability and a shorter replicative lifespan (RLS), as well as enhanced activity of the unfolded protein response (UPR) in the absence of stress. However, under the high-tunicamycin-concentration (an ER stress-inducing agent; 1.0 µg/mL) stress, the gas1Δ yeast cells exhibited an increased proliferation ability compared with the wild-type yeast strain. In addition, our findings demonstrated that IRE1 and HAC1 (two upstream modulators of the UPR) are required for the survival of gas1Δ yeast cells under the tunicamycin stress. On the other hand, we provided evidence that the GAS1 overexpression caused an obvious sensitivity to the low-tunicamycin-concentration (0.25 µg/mL). Collectively, our results indicate that Gas1p plays an important role in the ageing and ER stress responses in yeast.

Postnatal nectin-3 knockdown induces structural abnormalities of hippocampal principal neurons and memory deficits in adult mice.

The early postnatal stage is a critical period of hippocampal neurodevelopment and also a period of high vulnerability to adverse life experiences. Recent evidence suggests that nectin-3, a cell adhesion molecule, mediates memory dysfunction and dendritic alterations in the adult hippocampus induced by postnatal stress. But it is unknown whether postnatal nectin-3 reduction alone is sufficient to alter hippocampal structure and function in adulthood. Here, we down regulated hippocampal expression of nectin-3 and its heterophilic adhesion partner nectin-1, respectively, from early postnatal stage by injecting adeno-associated virus (AAV) into the cerebral lateral ventricles of neonatal mice (postnatal day 2). We found that suppression of nectin-3, but not nectin-1, expression from the early postnatal stage impaired hippocampus-dependent novel object recognition and spatial object recognition in adult mice. Moreover, AAV-mediated nectin-3 knockdown significantly reduced dendritic complexity and spine density of pyramidal neurons throughout the hippocampus, whereas nectin-1 knockdown only induced the loss of stubby spines in CA3. Our data provide direct evidence that nectins, especially nectin-3, are necessary for postnatal hippocampal development of memory functions and structural integrity.

Downregulation of esophageal cancer-related gene 4 promotes proliferation and migration of hepatocellular carcinoma.

Esophageal cancer-related gene 4 (ECRG4) is a candidate tumor suppressor gene, which is involved in cell apoptosis, migration, infection and inflammation responsiveness; however, its expression level and clinical significance in hepatocellular carcinoma (HCC) remains unclear. In the present study, the authors aim to evaluate the clinical significance and potential role of ECRG4 in HCC. Level of ECRG4 protein expression in HCC and peripheral tissues was investigated in tissue specimens obtained from 56 consecutive HCC patients by immunohistochemistry. Cell proliferation, cell migration and invasion regulations were examined by MTT curves, flow cytometry, Transwell assays and western blotting. ECRG4 expression was weak positive in normal liver cells but was downregulated in HCC cells in vivo or in vitro. A decreased expression of ECRG4 was associated with the age of the patients, metastasis and Ki-67 proliferation index. However, decreased ECRG4 expression was not associated with differentiation, tumor size, the presence of portal vein tumor thrombosis, satellite lesions, tumor relapse or mortality. Further investigations revealed that ectopic expression of ECRG4 inhibited cell proliferation, migration and invasion and promoted cell apoptosis in SMMC-7721 cells, which was mediated by the regulation of BAX and B cell lymphoma-2, in addition to the upregulation of epithelial-mesenchymal transition markers. In conclusion, the results of the present study indicated that ECRG4 was downregulated in HCC and served important roles in promoting cell proliferation and migration.

Associations of CYP2C9 and CYP2A6 Polymorphisms with the Concentrations of Valproate and its Hepatotoxin Metabolites and Valproate-Induced Hepatotoxicity.

The aim of this study was to compare genetic polymorphisms and concentrations of hepatotoxic metabolites in patients with epilepsy and liver injury and those with normal liver function receiving valproate monotherapy to identify risk factors for VPA-induced hepatotoxicity. A total of 279 Chinese patients with epilepsy were divided into an abnormal liver function (ANLFT) group (n = 79) and a normal liver function (NLFT) group (n = 200). Polymerase chain reaction-restriction fragment length polymorphism PCR-RFLP and nested PCR were applied to identify the frequency of two SNPs in candidate genes. Serum concentrations of VPA and its major metabolites were determined by Ultra-Performance Liquid Chromatography-tandem mass spectrometry UPLC-MS/MS. Significant differences were found in genotype distributions of CYP2A6 and CYP2C9 between the two groups. The values of 4-ene-VPA and 2,4-diene-VPA in the ANLFT group were significantly higher than in the NLFT group. Only CYP2A6 polymorphisms had associations with the concentrations of 4-ene-VPA and 2,4-diene-VPA. CYP2A6*1/*4 and CYP2A6*4/*4 variant carriers had higher CDR4-ene-VPA and CDR2,4-diene-VPA values than CYP2A6*1/*1 carriers. The logistic regression analysis showed that CYP2C9 and CYP2A6 were significant risk factors for hepatotoxicity by increasing the risk by 7.50 and 5.13 times, respectively. These findings provide preliminary evidence that CYP2A6 and CYP2C9 are associated with hepatotoxicity. However, only the CYP2A6 polymorphism was found to be associated with concentrations of 4-ene-VPA and 2,4-diene-VPA. Potential important risk factors include mutated genotypes of CYP2C9 and CYP2A6 and higher concentrations of VPA, 4-ene-VPA and 2,4-diene-VPA.