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Hepatocellular carcinoma (HCC) is an invasive malignant neoplasm with a poor prognosis. The development of chemoresistance severely obstructs the chemotherapeutic efficiency of HCC treatment. Therefore, understanding the mechanisms of chemoresistance is important for improving the outcomes of patients with HCC. Eukaryotic translation initiation factor 5A2 (eIF5A2), which is considered to be an oncogene, has been reported to mediate chemoresistance in various types of cancer; however, its precise role in HCC remains unclear. Accumulating evidence has suggested that autophagy serves a dual role in cancer chemotherapy. The present study aimed to investigate the role of autophagy in eIF5A2-mediated doxorubicin resistance in HCC. High expression levels of eIF5A2 in human HCC tissues were observed by immunohistochemistry using a tissue microarray, which was consistent with the results of reverse transcription-quantitative PCR analysis in paired HCC and adjacent healthy tissues. HCC patient-derived tumor xenograft mouse model was used for the in vivo study, and knockdown of eIF5A2 effectively enhanced the efficacy of doxorubicin chemotherapy compared with that in the control group. Notably, eIF5A2 served as a repressor in regulating autophagy under chemotherapy. Silencing of eIF5A2 induced doxorubicin sensitivity in HCC cells by triggering lethal autophagy. In addition, 5-ethynyl-2′-deoxyuridine, lactate dehydrogenase release assay and calcein-AM/PI staining were used to determine the enhanced autophagic cell death induced by the silencing of eIF5A2 under doxorubicin treatment. Suppression of autophagy attenuated the sensitivity of HCC cells to doxorubicin induced by eIF5A2 silencing. The results also demonstrated that knockdown of the Beclin 1 gene, which is an autophagy regulator, reversed the enhanced autophagic cell death and doxorubicin sensitivity induced by eIF5A2 silencing. Taken together, these results suggested eIF5A2 may mediate the chemoresistance of HCC cells by suppressing autophagic cell death under chemotherapy through a Beclin 1-dependent pathway, and that eIF5A2 may be a novel potential therapeutic target for HCC treatment.

Stroke ranks as the second leading cause of disability and death globally. Trigger receptors expressed on myeloid cells (TREM) −1 are responsible for the activation of the innate immune response and also play a critical role in inflammation. In this study, we reported the contribution of TREM-1 after ischemic damage in a rat middle cerebral artery occlusion (MCAO) model. This study also demonstrated that TREM-1 expression was upregulated following cerebral infarction in rats. TREM-1 inhibition was determined using its selective inhibitor, LP17, which indicated a neuroprotective effect on cerebral infarction damage. The findings revealed that inhibition of TREM-1 by administering LP17 improved cerebral damage and decreased ischemic areas and brain water contents. Moreover, LP17 decreased MCAO-induced microglial activation and neurodegeneration, evidenced by a reduction in the expression of microglial Iba-1 and FJ-B positive cells, and reversed neuronal loss. Besides, the contribution of LP17 to ischemic neuronal damage may be associated with a decrease in the production of pro-inflammatory cytokines, and enhanced production of anti-inflammatory cytokine IL-10. Both in vivo and in vitro studies showed that inhibiting TREM-1 attenuated ROS accumulation, lipid per-oxidation (LPO) contents such as malondialdehyde (MDA) and enhanced the superoxide dismutase (SOD) activity after ischemia. Inhibiting TREM-1 alleviated inflammation and pyroptosis found in MCAO rats. This was achieved through the inhibition of the levels of NLRP3, caspase-1, ASC (an apoptosis-associated speck-like protein containing a CARD) and gasdermin D. These results confirmed that inhibiting TREM-1 protects against ischemia-induced neuronal damage and alleviates microglial mediated neuro-inflammation by reducing oxidative stress and pyroptosis. Therefore, blocking TREM-1 expression provides an effective intervention for improving ischemic stroke.

Previous studies have shown that 5,2′,4′-trihydroxy-6,7,5′-trimethoxyflavone (TTF1) is the primary anticancer constituent of the traditional Chinese medicinal plant Sorbaria sorbifolia (SS), which has been applied to treat cancer in China. In this study, we investigated the in vitro and in vivo antitumor effects and biological mechanisms of small-molecule TTF1 nanoparticles (TTF1-NPs). The effects of TTF1-NPs on cell growth and apoptosis were investigated using human hepatoma cells. The molecular changes associated with the effects of TTF1-NPs were analyzed by immunocytochemistry and Western blot analysis. The in vivo effect of TTF1-NPs was investigated using the HepG2 tumor xenograft model. We found that TTF1-NPs exhibited antitumor effects in vitro accompanied by induction of apoptosis in human hepatoma cells. Mechanistically, our data showed that TTF1-NPs induced apoptosis via endoplasmic reticulum stress (ERS) pathway in hepatoma cells. Moreover, inhibition of ERS activation blocked TTF1-NP-induced apoptosis in HepG2 cells. Finally, TTF1-NPs inhibited the growth of HepG2 xenograft tumors. Taken together, our results demonstrated that TTF1-NP-induced apoptosis was mediated at least in part by the ERS pathway and thus inhibited hepatoma tumor growth.

// Rong-Rong Liu 1, * , Ya-Su Lv 1, * , Yue-Xiao Tang 1 , Yan-Fang Wang 1 , Xiao-Ling Chen 2 , Xiao-Xiao Zheng 1 , Shang-Zhi Xie 1 , Ying Cai 1 , Jun Yu 3 , Xian-Ning Zhang 1 1 Department of Cell Biology and Medical Genetics, Research Center for Molecular Medicine, National Education Base for Basic Medical Sciences, Institute of Cell Biology, Zhejiang University School of Medicine, Hangzhou, 310058, China 2 Department of Biological Chemistry, Zhejiang Chinese Medical University, Hangzhou, 310053, China 3 Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Key Laboratory of Multi-Organ Transplantation of Ministry of Public Health, Hangzhou, 310003, China * These authors contributed equally to this work Correspondence to: Xian-Ning Zhang, e-mail: zhangxianing@zju.edu.cn Jun Yu, e-mail: Dr.yujun@gmail.com Keywords: eukaryotic translation initiation factor 5A2, reactive oxygen species, epithelial-mesenchymal transition, migration, hepatocellular carcinoma Received: September 24, 2015 Accepted: February 28, 2016 Published: March 23, 2016 ABSTRACT Eukaryotic translation initiation factor 5A2 ( eIF5A2 ) has been identified as a critical gene in tumor metastasis. Research has suggested that reactive oxygen species (ROS) serve as signaling molecules in cancer cell proliferation and migration. However, the mechanisms linking eIF5A2 and ROS are not fully understood. Here, we investigated the effects of ROS on the eIF5A2-induced epithelial-mesenchymal transition (EMT) and migration in six hepatocellular carcinoma (HCC) cell lines. Western hybridization, siRNA transfection, transwell migration assays, wound-healing assays, and immunofluorescence analysis were used. The protein levels of eIF5A2 in tumor and adjacent tissue samples from 90 HCC patients with detailed clinical, pathological, and clinical follow-up data were evaluated. Overexpression of eIF5A2 was found in cancerous tissues compared with adjacent tissues. We found that eIF5A2 overexpression in HCC was associated with reduced overall survival. Knockdown of eIF5A2 and intracellular reduction of ROS significantly suppressed the invasion and metastasis of HCC cells. Interestingly, N1-guanyl-1, 7-diaminoheptane (GC7) suppressed the intracellular ROS levels. After blocking the EMT, administration of GC7 or N-acetyl-L-cysteine did not reduce cell migration further. Based on the experimental data, we concluded that inhibition of eIF5A2 alters progression of the EMT to decrease the invasion and metastasis of HCC cells via ROS-related pathways.

Previous studies have reported that memantine presents evidence of therapeutic benefits in several animal models of ischemic stroke and neurodegenerative diseases. However, the effect of memantine on secondary damage in the ipsilateral thalamus after focal cortical infarction remains undefined. Present study investigated whether memantine has a protective effect on secondary damage in the ipsilateral thalamus after focal cerebral infarction in rats. At 24 h after distal middle cerebral artery occlusion (MCAO), rats in the memantine and vehicle groups were intraperitoneal injected with memantine and isopycnic vehicle, respectively, was once daily administered for consecutive 7 days. Infarct size was evaluated through Nissl staining and sensory decline determined using adhesive removal test. Secondary thalamic damage was assessed using Nissl staining and immunofluorescence 8 days after MCAO. Immunoboltting was used to identify tau and apoptosis-associated proteins in the ipsilateral thalamus after MCAO. Results revealed that memantine ameliorated sensory decline compared to the vehicle controls. Subsequently, tau phosphorylated at threonine 231 (p-tau-231), glycogen synthase kinase3βpY216 (GSK3βpY216) and protein phosphatase 2A (PP2ApY307) were reduced by memantine, causing greater reduction in neuronal loss and inhibition of reactive astrogliosis in the ipsilateral ventroposterior thalamic nucleus (VPN) compared with the vehicle groups. In addition, increase in secondary damage-induced TUNEL-positive cells was blunted by memantine, as demonstrated by the significant reduction in expression of apoptosis-associated proteins. Our results suggest that memantine has a neuro-protective effect on secondary damage in the ipsilateral thalamus following MCAO by inhibiting the activity of GSK3βpY216/PP2ApY307 and down regulating the levels of p-tau-231 protein.

Although excessive plasma adenosine is detrimental in sickle cell disease (SCD), the molecular mechanism underlying elevated circulating adenosine remains unclear. Here we report that the activity of soluble CD73, an ectonucleotidase producing extracellular adenosine, was significantly elevated in a murine model of SCD and correlated with increased plasma adenosine. Mouse genetic studies demonstrated that CD73 activity contributes to excessive induction of plasma adenosine and thereby promotes sickling, hemolysis, multiorgan damage, and disease progression. Mechanistically, we showed that erythrocyte adenosine 5'-monophosphate-activated protein kinase (AMPK) was activated both in SCD patients and in the murine model of SCD. AMPK functions downstream of adenosine receptor ADORA2B signaling and contributes to sickling by regulating the production of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), a negative allosteric regulator of hemoglobin-O2 binding affinity. Preclinically, we reported that treatment of α,β-methylene adenosine 5'-diphosphate, a potent CD73 specific inhibitor, significantly decreased sickling, hemolysis, multiorgan damage, and disease progression in the murine model of SCD. Taken together, both human and mouse studies reveal a novel molecular mechanism contributing to the pathophysiology of SCD and identify potential therapeutic strategies to treat SCD.

Epidermolytic palmoplantar keratoderma (EPPK) is the most frequent form of such keratodermas. It is inherited in an autosomal dominant pattern and is clinically characterized by diffuse yellowish thickening of the skin on the palms and soles with erythematous borders during the first weeks or months after birth. EPPK is generally caused by mutations of the KRT9 gene. More than 26 KRT9 gene mutations responsible for EPPK have been described (Human Intermediate Filament Database, www.interfil.org), and many of these variants are located within the highly-conserved coil 1A region of the α-helical rod domain of keratin 9. Unfortunately, there is no satisfactory treatment for EPPK. Thus, prenatal molecular diagnosis or pre-pregnancy diagnosis is crucial and benefits those affected who seek healthy descendants. In the present study, we performed amniotic fluid-DNA-based prenatal testing for three at-risk pregnant EPPK women from three unrelated southern Chinese families who carried the KRT9 missense mutations p.Arg163Trp and p.Arg163Gln, and successfully helped two families to bear normal daughters. We suggest that before the successful application of preimplantation genetic diagnosis (PGD), and noninvasive prenatal diagnosis of EPPK that analyzes fetal cells or cell-free DNA in maternal blood, prenatal genetic diagnosis by amniocentesis or chorionic villus sampling (CVS) offers a quite acceptable option for EPPK couples-at-risk to avoid the birth of affected offspring, especially in low- and middle-income countries.

We demonstrate through PdO doping that creation of heterojunctions on Co3O4 nanoparticles can quantitatively adjust band-gap and Fermi energy levels to study the impact of metal oxide nanoparticle semiconductor properties on cellular redox homeostasis and hazard potential. Flame spray pyrolysis (FSP) was used to synthesize a nanoparticle library in which the gradual increase in the PdO content (0-8.9%) allowed electron transfer from Co3O4 to PdO to align Fermi energy levels across the heterojunctions. This alignment was accompanied by free hole accumulation at the Co3O4 interface and production of hydroxyl radicals. Interestingly, there was no concomitant superoxide generation, which could reflect the hole dominance of a p-type semiconductor. Although the electron flux across the heterojunctions induced upward band bending, the E(c) levels of the doped particles showed energy overlap with the biological redox potential (BRP). This allows electron capture from the redox couples that maintain the BRP from -4.12 to -4.84 eV, causing disruption of cellular redox homeostasis and induction of oxidative stress. PdO/Co3O4 nanoparticles showed significant increases in cytotoxicity at 25, 50, 100, and 200 μg/mL, which was enhanced incrementally by PdO doping in BEAS-2B and RAW 264.7 cells. Oxidative stress presented as a tiered cellular response involving superoxide generation, glutathione depletion, cytokine production, and cytotoxicity in epithelial and macrophage cell lines. A progressive series of acute pro-inflammatory effects could also be seen in the lungs of animals exposed to incremental PdO-doped particles. All considered, generation of a combinatorial PdO/Co3O4 nanoparticle library with incremental heterojunction density allowed us to demonstrate the integrated role of E(v), E(c), and E(f) levels in the generation of oxidant injury and inflammation by the p-type semiconductor, Co3O4.

It has been recently proposed by a genome-wide association study (GWAS) meta-analysis that the CCDC62 variant rs12817488 is a new risk locus associated with Parkinson's disease (PD). In this study, we aimed to investigate the association between rs12817488 and PD in a Chinese cohort. A total of 341 PD patients and 423 matched controls were recruited in Eastern China. Our results showed that the A allele of rs12817488 was significantly associated with an aggravated risk of PD (p = 0.006) and represented a major allele in contrast to a minor one in Caucasians. Genotype distributions also differed between PD patients and controls (p = 0.011 for AA/AG/GG). Further analysis showed that the association of rs12817488 with PD only existed in females. We also investigated the protein level of CCDC62 in peripheral blood mononuclear cells from 41 AA or GG carriers and found an apparently higher expression in PD patients carrying the AA genotype. A potential interaction was found between two estrogen-related loci, i.e. rs12817488/CCDC62 and rs2697962/PRDM2, particularly in the female stratum. In conclusion, our study demonstrated for the first time a significant association between the rs12817488 polymorphism and PD predisposition in a Chinese population with gender variations and provides new insight regarding the variant's protein expression and estrogen-related genetic interaction.

Epidermolytic palmoplantar keratoderma (EPPK) is a relatively common autosomal-dominant skin disorder caused by mutations in the keratin 9 gene (KRT9), with few therapeutic options for the affected so far. Here, we report a knock-in transgenic mouse model that carried a small insertion-deletion (indel) mutant of Krt9, c.434delAinsGGCT (p.Tyr144delinsTrpLeu), corresponding to the human mutation KRT9/c.500delAinsGGCT (p.Tyr167delinsTrpLeu), which resulted in a human EPPK-like phenotype in the weight-stress areas of the fore- and hind-paws of both Krt9(+/mut) and Krt9(mut/mut) mice. The phenotype confirmed that EPPK is a dominant-negative condition, such that mice heterozygotic for the K9-mutant allele (Krt9(+/mut)) showed a clear EPPK-like phenotype. Then, we developed a mutant-specific short hairpin RNA (shRNA) therapy for EPPK mice. Mutant-specific shRNAs were systematically identified in vitro using a luciferase reporter gene assay and delivered into Krt9(+/mut) mice. shRNA-mediated knockdown of mutant protein resulted in almost normal morphology and functions of the skin, whereas the same shRNA had a negligible effect in wild-type K9 mice. Our results suggest that EPPK can be treated by gene therapy, and this has significant implications for future clinical application.

A high affinity polyclonal antibody-based enzyme linked immunosorbent assay (ELISA) was developed for the quantification of zeranol in bovine urine. On the basis of urine matrix studies, the optimized dilution factors producing insignificant matrix interference were selected as 1:5 in pretreatment. In the improved ELISA, the linear response range was between 0.02 and 1 microg/ml, and the detection limit was 0.02 microg/ml for the assay. The overall recoveries and the coefficients of variation (CVs) were in the range of 82% to approximately 127% and 3.5% to approximately 8.8%, respectively. Thirty-six bovine urine samples spiked with zeranol (ranging from 0.2 to 10 microg/ml) were detected by the ELISA and liquid chromatography (LC) method, and good correlations were obtained between the two methods (R(2)=0.9643). We conclude that this improved ELISA is suitable tool for a mass zeranol screening and can be an alternative for the conventional LC method for zeranol in bovine urine.

In this study, we aimed to explore the long noncoding RNA expression pattern in squamous cell lung cancer (SQCC) on a genome-wide scale. Total RNAs were extracted from 16 lung SQCC patients’ normal and matched lung cancer tissues by Trizol reagent. The expression level of genome-wide scale lncRNA and mRNA was determined by microarray. qRT-PCR was used to validate the lncRNA expression level in 47 patients. Data analyses were performed using R and Bioconductor. A total of 2,748 up and 852 down regulated probes were identified to be significantly and differentially expressed in tumor tissues. The annotation result of their co-expressed mRNAs showed that the most significantly related category of GO analysis was development and differentiation, while the most significantly related pathway was cell cycle. Subgroup analysis identified that 46 and 18 probes were specifically differentially expressed in smoking and moderately differentiated tumors, respectively. Our study indicated that clusters of lncRNAs were significantly and differentially expressed in SQCC compared with normal tissues in the same subject. They may exert a significant role in lung cancer development and could be potential targets for future treatment of SQCC.

The published data revealed conflicting results of the polymorphism of MDR1 exon 26 SNP C3435T on the pharmacokinetics of cyclosporine; thus, the aim was to conduct a meta-analysis of significant magnitude to investigate the influence of SNP C3435T on the pharmacokinetics of cyclosporine. A literature search was conducted to locate the relevant papers by using the PubMed electronic source from 1997 and onwards. The pharmacokinetic parameters, including AUC(0-4), AUC(0-12), AUC(0-inf), C(max), CL/F and trough concentration (C(0)), were extracted and a meta-analysis was performed by using Stata version 9.1. A total of 14 papers concerning 1036 individuals were included in the meta-analysis. The overall results showed no major influence of SNP C3435T on the pharmacokinetic parameters, including AUC(0-4), AUC(0-inf), CL/F, C(max) and C(0), although AUC(0-12) was lower in subjects with CC genotype. A subanalysis by ethnic population showed that C(0) was lower in Caucasian individuals harbouring CC genotype. In conclusion, our meta-analysis of available studies has thus far failed to demonstrate a definitive correlation between the SNP C3435T in MDR1 gene and alterations in P-glycoprotein function that can result in altered pharmacokinetics of cyclosporine, although it was indicated in this meta-analysis that the carrier of CC genotype of the SNP C3435T of MDR1 had lower cyclosporine exposure presented as AUC(0-12) than those with at least one T allele. There seems to be ethnic differences in the relationship between the SNP C3435T of MDR1 and cyclosporine pharmacokinetics.

Objective To evaluate the effect of different protocols of intratympanic dexamethasone injection in patients with unilateral sudden sensorineural hearing loss(SSNHL) who have failed to respond to typical medical treatment (including systemic steroid treatment). Methods From January 2005 to January 2008, 71 patients who were diagnosed with unilateral SSNHL and failed typical medical treatment received intratympanic dexamethasone injection. Four injection protocols were employed: injection of 0.3 ml dexamethasone(5 mg/ml) three times a week for 3 weeks for a total dose of 13.5 mg (Group I, n=16); injection of 0.6 ml (5 mg/ml) dexamethasone three times a week for 3 weeks for a total dose of 27 mg (Group II, n=18); injection at 0.3 ml(5mg/ml) week for 6 weeks for a total dose of 9 mg (Group III, n=18); injection at 0.3 ml (5 mg/ml)/2 days for a total dose of 4.5 mg (Group IV n=19). Hearing recovery was assessed by pure tone audiogram. Results The total effective rate was 37.5%, 38.89%, 33.33% and 36.84% for each treatment protocol, respectively, with no statistical difference between them (P > 0.05). The results suggest that a total dose of 4.5 mg (injected once a week for 3 weeks) is the most adequate protocol, and increasing dose or injection frequency yields no additional benefits. Conclusion Intratympanic dexamethasone significantly improves the prognosis of SSNHL. Small dose at low injection frequency is sufficient. Further multicenter studies are needed to determine the standard treatment protocol.