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To clarify the mechanisms of Nosema ceranae parasitism, we deep-sequenced both honey bee host and parasite mRNAs throughout a complete 6-day infection cycle. By time-series analysis, 1122 parasite genes were significantly differently expressed during the reproduction cycle, clustering into 4 expression patterns. We found reactive mitochondrial oxygen species modulator 1 of the host to be significantly down regulated during the entire infection period. Our data support the hypothesis that apoptosis of honey bee cells was suppressed during infection. We further analyzed genome-wide genetic diversity of this parasite by comparing samples collected from the same site in 2007 and 2013. The number of SNP positions per gene and the proportion of non-synonymous substitutions per gene were significantly reduced over this time period, suggesting purifying selection on the parasite genome and supporting the hypothesis that a subset of N. ceranae strains might be dominating infection. [ABSTRACT FROM AUTHOR]

The LIM-homeodomain (LIM-HD) family member Lmx1a has been successfully used to induce dopaminergic neurons from other cell types, thus showing significant implications in replacement therapies of Parkinson's disease, but the underlying mechanism remains elusive. In this study, we used Drosophila eye as a model system to investigate how forced expression of dLmx1a , the fly homolog of human Lmx1a , alters cell identify. We found that ectopic expression of dLmx1a suppresses the formation of Drosophila eye tissue and identified the LIM and HD as two essential domains. dLmx1a requires and physically binds to Chip, a well-known cofactor of LIM-HD proteins. Chip connects two dLmx1a proteins to form a functional tetrameric complex. In addition, we provide evidence showing that dLmx1a expression results in the suppression of two retina determination gene eyes absent ( eya ) and string ( stg ). Taken together, our findings identified Chip as a novel partner of dLmx1a to alter cell differentiation in Drosophila eye through repressing eya and stg expression, and provide an animal model for further understanding the molecular mechanism whereby Lmx1a determines cell fate. [ABSTRACT FROM AUTHOR]

Perivascular adipose tissue (PVAT) has been recognized as an active contributor to vascular function due to its paracrine effects on cells contained within vascular wall. The present study was designed to investigate the effect of diosgenin on adipokine expression in PVAT with emphasis on the regulation of endothelial function. Palmitic acid (PA) stimulation induced inflammation and dysregulation of adipokine expression in PVAT. Diosgenin treatment inhibited IKKβ phosphorylation and downregulated mRNA expressions of proinflammatory cytokines/proteins including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein (MCP-1), and inducible nitric oxide synthase (iNOS), while reduced gene expressions for adiponectin, PPARγ, and arginase 1 (Arg-1) were reversed by diosgenin treatment. Diosgenin enhanced AMPK phosphorylation under basal and inflammatory conditions in PVAT, whereas knockdown of AMPK by SiRNA diminished its modulatory effect, indicating that diosgenin inhibited inflammation in an AMPK-dependent manner. We prepared conditioned medium from PA-stimulated PVAT to induce endothelial dysfunction and found that pre-treatment of PVAT with diosgenin effectively restored the loss of ACh-induced vasodilation and increased eNOS phosphorylation in rat aorta. High-fat diet feeding in rats induced inflammation in PVAT and the impairment of endothelium-dependent vasodilation, whereas these alterations were prevented by oral administration of diosgenin at doses of 20 and 40 mg/kg. In conclusion, the obtained data showed that diosgenin ameliorated inflammation-associated adipokine dysregulation, and thereby prevented endothelial dysfunction. Our findings would shed a novel insight into the potential mechanism by which diosgenin protected endothelial function against inflammatory insult. [ABSTRACT FROM AUTHOR]

Traditional Chinese Medicine Jianpijiedu decoction (JPJD) could improve the general status of liver cancer patients in clinics, especially the symptoms of decreased food intake and diarrhea. In this study, our results showed that the survival rate of the liver cancer with food restriction and diarrhea (FRD-LC) rats was lower than the liver cancer (LC) rats, and the tumor volume of the FRD-LC rats was higher than the LC rats. It was also shown that the high dose of JPJD significantly improved the survival rate, weight, and organ weight when compared with FRD-LC-induced rats. Moreover, JPJD administration upregulated the mRNA and protein levels of ABCC2 and downregulated the mRNA and protein levels of OATP1B2 in liver tissues. However, opposite results were observed in the cancer tissues. In conclusion, the study indicated that the Chinese Medicine JPJD could contribute to the rats with liver cancer which were pretreated with food restriction and diarrhea by regulating the expression of ABCC2 and OATP1B2 in liver tissues and cancer tissues. [ABSTRACT FROM AUTHOR]

The aberrantly activated monocytes and nuclear factor-kappaB (NF-κB) pathway contribute to the pathogenesis of systemic lupus erythematosus (SLE), and the aberrantly activated NF-κB is associated with defects in the anti-inflammatory A20 in SLE. However, whether SLE monocytes express A20 and whether the A20 expression under sustained proinflammatory stimulation is altered to contribute to the uncontrolled NF-κB inflammatory response are unclear. In this study, we found that the freshly isolated monocytes from SLE patients and healthy controls did not differ in expression levels of IL-1β, IκBα and A20. After TNF-α stimulation for 48 h, the monocytes from both groups expressed higher levels of IL-1β and IκBα than the monocytes without TNF-α treatment. Although the increased levels of NF-κB were observed in the nucleus of both the SLE and control monocytes after 24 h of TNF-α stimulation, the enhancement in SLE monocytes was significantly more robust than in the control monocytes. In addition, while the p-IκBα level in healthy monocytes was increased, the p-IκBα level in SLE monocytes was slightly decreased after TNF-α stimulation. Interestingly, after TNF-α treatment, the A20 expression in SLE monocytes was not markedly altered compared with the untreated SLE monocytes; moreover, the SLE monocytes expressed significantly lower A20 than healthy monocytes with TNF-α treatment at each time point. Results in this study demonstrate that TNF-α activates a significant NF-κB inflammatory response in SLE monocytes, which is at least partially mediated by the aberrantly low expression of A20 upon TNF-α stimulation, contributing to the prolonged inflammatory response in SLE. [ABSTRACT FROM AUTHOR]

Psoriasis is a chronic, inflammatory skin disease involving both environmental and genetic factors. According to genome-wide association studies (GWAS), the TNIP1 gene, which encodes the TNF-α–induced protein 3-interacting protein 1 (TNIP1), is strongly linked to the susceptibility of psoriasis. TNIP1 is a widely expressed ubiquitin sensor that binds to the ubiquitin-editing protein A20 and restricts TNF- and TLR-induced signals. In our study, TNIP1 expression decreased in specimens of epidermis affected by psoriasis. Based on previous studies suggesting a role for TNIP1 in modulating cancer cell growth, we investigated its role in keratinocyte proliferation, which is clearly abnormal in psoriasis. To mimic the downregulation or upregulation of TNIP1 in HaCaT cells and primary human keratinocytes (PHKs), we used a TNIP1 specific small interfering hairpin RNA (TNIP1 shRNA) lentiviral vector or a recombinant TNIP1 (rTNIP1) lentiviral vector, respectively. Blocking TNIP1 expression increased keratinocyte proliferation, while overexpression of TNIP1 decreased keratinocyte proliferation. Furthermore, we showed that TNIP1 signaling might involve extracellular signal-regulated kinase1/2 (Erk1/2) and CCAAT/enhancer-binding protein β (C/EBPβ) activity. Intradermal injection of TNIP1 shRNA in BALB/c mice led to exaggerated psoriatic conditions in imiquimod (IMQ)-induced psoriasis-like dermatitis. These findings indicate that TNIP1 has a protective role in psoriasis and therefore could be a promising therapeutic target. [ABSTRACT FROM AUTHOR]

Toll-like receptors are the most important pattern recognition receptors that can recognize conserved molecular structures shared by large groups of pathogens. Here, the aim was to determine the expression and role of TLR2 in peripheral blood mononuclear cells (PBMCs) from patients with cryptococcal meningitis and healthy controls. TLR2 expression was measured using RT-PCR and western blotting. The role of TLR2 in cytokine production by PBMCs after Cryptococcus neoformans exposure was assessed in healthy controls prior to incubation with anti-TLR2. TLR2 mRNA and protein expression were both weaker in patients with cryptococcal meningitis than in healthy controls. Furthermore, pre-incubation of PBMCs from healthy donors with anti-TLR2 led to reduced expression of IFN-γ and IL-12p70, but not of IL-4 and IL-10, following C. neoformans stimulation. Our results suggest that impaired expression of TLR2 may be involved in defective host defense to C. neoformans through an attenuated Th1 response. [ABSTRACT FROM AUTHOR]

Aim:The receptor for advanced glycation end-products (RAGE) plays an important role in development of atherosclerosis, and C-reactive protein (CRP) has been found to stimulate its expression in endothelial cells. In this study we investigated how CRP regulated the expression of RAGE in human coronary artery endothelial cells (HCAECs).Methods:HCAECs were treated in vitro with CRP (50 μg/mL) in combination with a variety of inhibitors. ROS generation was determined by immunocytochemistry and flow cytometry. The RAGE expression and phosphorylation of relevant signaling proteins were measured using Western blot analyses.Results:CRP stimulated the expression of RAGE in the cells, accompanied by markedly increased ROS generation, phosphorylation of ERK1/2 and NF-κB p65, as well as translocation of NF-κB p65 to the nuclei. CRP also stimulated phosphorylation of JNK and p38 MAPK. Pretreatment of the cells with the ROS scavenger N-acetyl-L-cysteine, ERK inhibitor PD98059 or NF-κB inhibitor PDTC blocked CRP-stimulated RAGE expression, but pretreatment with the NADPH oxidase inhibitor DPI, JNK inhibitor SP600125 or p38 MAPK inhibitor SB203580 did not significantly alter CRP-stimulated RAGE expression.Conclusion:CRP stimulates RAGE expression in HCAECs in vitro via ROS generation and activation of the ERK/NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Purpose. To explore the association between differential Tim-3 and PD-1 expression patterns and HIV disease progression, and to investigate the impact of common γ-chain cytokines on Tim-3 and PD-1 expression patterns on T cells. Methods. Tim-3/PD-1 expression on the T cells of patients with early and chronic HIV infections was detected. The expression levels and functional profiles of T cells with differential Tim-3 and PD-1 expression patterns induced by γ-chain cytokines were studied. Results. The elevation of differential Tim-3 and PD-1 expression patterns on T cells appeared early in HIV infection. Co-expression of Tim-3 and PD-1 (Tim-3+PD-1+) correlates with more severe exhaustion of T cells during HIV infection. In vitro stimulation of common γ-chain cytokines can induce differential expression patterns of Tim-3 and PD-1 on T cells. The enhancement of Tim-3 and PD-1 expression by common γ-chain IL-2 can inhibit the function of T cells re-stimulated by HIV gag and TCR, not by the re-stimulation of IL-2. Conclusions. The elevation of differential Tim-3 and PD-1 expression patterns on T cells represents a state of T cell exhaustion and can be induced by common γ-chain cytokines. These findings provide insights into HIV pathogenesis and help inform immune intervention strategies. [ABSTRACT FROM AUTHOR]

Transcription factor FOXM1 plays a critical role in maintenance of stem cell pluripotency through stimulating the transcription of pluripotency-related genes in mouse pluripotent stem cells. In this study, we have found that the repression of FOXM1 expression is mediated by FOXM1 3′UTR during retinoic acid-induced differentiation of human pluripotent NT2/D1 embryonal carcinoma cells. FOXM1 3′UTR contains a microRNA response element (MRE) for miR-134, which has been shown to attenuate the expression of pluripotency-related genes post-transcriptionally during mouse embryonic stem cell differentiation. We have determined that miR-134 is induced during RA-induced differentiation of NT2/D1 cells and the overexpression of miR-134 represses the expression of FOXM1 protein but not FOXM1 mRNA. Furthermore, the expression of OCT4 is diminished by FOXM1 knockdown and the OCT4 promoter is regulated directly by FOXM1, suggesting that FOXM1 is required for maintaining the expression of OCT4 in NT2/D1 cells. Together, our results suggest that FOXM1 is essential for human pluripotent stem cells and miR-134 attenuates its expression during differentiation. [ABSTRACT FROM AUTHOR]

Background: Primary immune thrombocytopenia (ITP) is an autoimmune bleeding disorder with an unclear etiology. This study aims to investigate the role of IL-23/Th17 pathway in patients with ITP. Method: The gene expressions of IL-17, IL-23 and their receptors in ITP patients and healthy controls were analyzed by quantitative real-time PCR. ELISA was used to test the IL-17 and IL-23 levels in plasma. Flow cytometry was used to detect the frequency of Th17 cells. The correlation between plasma IL-23 and IL-17 levels, Th17 cells, platelets were analyzed. The level of Th17-related cytokines was measured by ELISA following stimulation with IL-23. Subsequently, the IL-23 and IL-17 levels were measured in patients post-treatment. Results: The PBMCs of ITP patients showed increased mRNA expression levels in each of the following: IL-23p19, IL-12p40, IL-23R, IL-12Rβ1, IL-17A, IL-17F, and RORC. In addition, elevated Th17 cells and plasma IL-17, IL-23 levels were also observed in these ITP patients. Furthermore, it was found that IL-23 levels in plasma are positively correlated with IL-17 levels and Th17 cells, yet negatively correlated with platelet count. Following IL-23 stimulation in vitro, IL-17 levels showed significant elevation. Furthermore, both IL-23 and IL-17 levels decreased after effective treatment. Conclusion: The IL-23/Th17 pathway may be involved in the pathogenesis of ITP through enhancement of the Th17 response. Moreover, our results suggest that the IL-23/Th17 pathway is a potential therapeutic target in future attempts of ITP treatment. [ABSTRACT FROM AUTHOR]

Gambogic acid (GA) is the major active ingredient of gamboges, a brownish to orange resin product from Garcinia hanburyi tree in Southeast Asia. This compound exhibits anti-cancer effect on solid tumors. In this study, we investigated the effects of GA on the growth and differentiation of acute myeloid leukemia cells by growth-inhibition detection, morphological changes observation, nitroblue tetrazolium reduction, and the expression of the relative cell-surface differentiation markers. The results showed that GA could inhibit cell growth and promote differentiation in U937 and HL-60 cells. In addition, GA upregulated the expression of p21waf1/cip1 in the two cell lines. Finally, downregulating the p21waf1/cip1 expression with small interfering RNA partially blocked GA-induced cell growth inhibition and differentiation. These results of this study revealed that GA may be used as one of the investigational drugs for acute myeloid leukemia. [ABSTRACT FROM AUTHOR]

Background: In South China (Gejiu City, Yunnan Province), lung cancer incidence and associated mortality rate is the most prevalent and observed forms of cancer. Lung cancer in this area is called Gejiu squamous cell lung carcinoma (GSQCLC). Research has demonstrated that overexpression of miR-21 occurs in many cancers. However, the unique relationship between miR-21 and its target genes in GSQCLC has never been investigated. The molecular mechanism involved in GSQCLC must be compared to other non-small cell lung cancers in order to establish a relation and identify potential therapeutic targets. Methodology/Principal Findings: In the current study, we initially found overexpression of miR-21 occurring in non-small cell lung cancer (NSCLC) cell lines when compared to the immortalized lung epithelial cell line BEAS-2B. We also demonstrated that high expression of miR-21 could increase tumor cell proliferation, invasion, viability, and migration in GSQCLC cell line (YTMLC-90) and NSCLC cell line (NCI-H157). Additionally, our results revealed that miR-21 could suppress YTMLC-90 and NCI-H157 cell apoptosis through arresting cell-cycle at G2/M phase. Furthermore, we demonstrated that PTEN, RECK and Bcl-2 are common target genes of miR-21 in NSCLC. Finally, our studies showed that down-regulation of miR-21 could lead to a significant increase in PTEN and RECK and decrease in Bcl-2 at the mRNA and protein level in YTMLC-90 and NCI-H157 cell lines. However, we have not observed any remarkable difference in the levels of miR-21 and its targets in YTMLC-90 cells when compared with NCI-H157 cells. Conclusions/Significance: miR-21 simultaneously regulates multiple programs that enhance cell proliferation, apoptosis and tumor invasiveness by targeting PTEN, RECK and Bcl-2 in GSQCLC. Our results demonstrated that miR-21 may play a vital role in tumorigenesis and progression of lung squamous cell carcinoma and suppression of miR-21 may be a novel approach for the treatment of lung squamous cell carcinoma. [ABSTRACT FROM AUTHOR]

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Recurrent pregnancy loss (RPL) is often associated with dysregulated Annexin A5 (ANXA5) expression. Moreover, the variants of Anxa5, a protein that is enriched in the placenta to prevent coagulation, have been reported to affect RPL risks. The haplotypes M1 [including single nucleotide polymorphisms (SNPs) 1A/C and 27T/C] and M2 (including SNPs 19G/A, 1A/C, 27T/C and 76G/A) of ANXA5 were also reported to affect RPL risks. The present study aimed to investigate the association between the haplotype located in the promoter region of ANXA5 and the risk of RPL. Patients with RPL (n=235) or intrauterine fetus death (IUFD; n=154), as well as healthy control subjects (n=375) were enrolled in the current research. Their haplotypes of ANXA5 were determined using genotyping, and the association between ANXA5 haplotypes and the risk of RPL was accordingly analyzed. A luciferase assay was conducted to investigate the haplotype responsible for ANXA5 activity. Reverse transcription-quantitative PCR, western blot analysis, immunohistochemistry and ELISA were performed to assess the expression level and activity of ANXA5 in patients with RPL. Consequently, the majority (n=214) of patients with RPL had a history of early RPL, whereas 31 patients with RPL had a history of both early and late RPL episodes. A significant difference was found between cases and controls in terms of gravidity and parity, whereas no significant differences were found in terms of age. The percentage of patients with RPL carrying the M2 haplotype of ANXA5 was significantly higher compared with that in control subjects, indicating that the M2 haplotype of ANXA5 was an independent risk of RPL as it influenced the transcription efficiency of ANXA5 promoter. In patients with RPL, ANXA5 activity was suppressed and the mRNA and protein expression levels of Anxa5 were decreased. Thus, the ANXA5 M2 haplotype may be an independent risk factor of RPL by affecting Anxa5 activity. [ABSTRACT FROM AUTHOR]

Polyamines (PAs) play an important regulatory role in many basic cellular processes and physiological and biochemical processes. However, there are few studies on the identification of PA biosynthesis and metabolism family members and the role of PAs in the transition of plant embryogenic calli (EC) into globular embryos (GE), especially in perennial woody plants. We identified 20 genes involved in PA biosynthesis and metabolism from the third-generation genome of longan (Dimocarpus longan Lour.). There were no significant differences between longan and other species regarding the number of members, and they had high similarity with Citrus sinensis. Light, plant hormones and a variety of stress cis-acting elements were found in these family members. The biosynthesis and metabolism of PAs in longan were mainly completed by DlADC2, DlSAMDC2, DlSAMDC3, DlSPDS1A, DlSPMS, DlCuAOB, DlCuAO3A, DlPAO2 and DlPAO4B. In addition, 0.01 mmol∙L−1 1-aminocyclopropane-1-carboxylic acid (ACC), putrescine (Put) and spermine (Spm), could promote the transformation of EC into GE, and Spm treatment had the best effect, while 0.01 mmol∙L−1 D-arginine (D-arg) treatment inhibited the process. The period between the 9th and 11th days was key for the transformation of EC into GE in longan. There were higher levels of gibberellin (GA), salicylic acid (SA) and abscisic acid (ABA) and lower levels of indole-3-acetic acid (IAA), ethylene and hydrogen peroxide (H2O2) in this key period. The expression levels in this period of DlADC2, DlODC, DlSPDS1A, DlCuAOB and DlPAO4B were upregulated, while those of DlSAMDC2 and DlSPMS were downregulated. These results showed that the exogenous ACC, D-arg and PAs could regulate the transformation of EC into GE in longan by changing the content of endogenous hormones and the expression levels of PA biosynthesis and metabolism genes. This study provided a foundation for further determining the physicochemical properties and molecular evolution characteristics of the PA biosynthesis and metabolism gene families, and explored the mechanism of PAs and ethylene for regulating the transformation of plant EC into GE. [ABSTRACT FROM AUTHOR]

This study aims to investigate the role of osteopontin (OPN) genetic polymorphisms in the occurrence of left ventricular hypertrophy (LVH) in Chinese patients with essential hypertension (EH). A total of 1,092 patients diagnosed with EH were recruited. Three single nucleotide polymorphisms (SNP) on the promoter region of the OPN gene, including −66T/G, −156G/GG, and −443C/T were genotyped. The serum thrombin-cleaved OPN levels were studied. Patients were divided into LVH+ ( n = 443) and the LVH− ( n = 649) groups. We found that none of the studied SNPs in the OPN gene was associated with the risk and severity of LVH. The SNPs in the OPN gene did not correlate with the serum OPN levels. However, the serum thrombin-cleaved OPN levels were found to be an independent risk factor for LVH in the EH patients. Multivariate logistic regression analysis showed that serum thrombin-cleaved OPN levels were independently associated with the development of LVH (adjusted OR = 2.47, 95 % CI 1.56-4.01, adjusted P < 0.001). In vitro studies showed that the thrombin-cleaved OPN treatment increased the protein content per cell, the cardiomyocyte surface size, and the expression level of atrial natriuretic peptide protein in a dose-dependent manner. The thrombin-cleaved OPN serum level, but not OPN gene polymorphism, is associated with the development of LVH in EH patients. Key messages: [ABSTRACT FROM AUTHOR]

Background CDK4 is a protein kinase in the CDK family important for G1/S phase cell cycle progression. However, the roles and molecular mechanisms of CDK4 triggering nasopharynx carcinogenesis are still unclear. Methods Lentiviral-vector mediated shRNA was used to suppress CDK4 expression and examine its molecular mechanisms. Using immunohistochemistry, we analyzed CDK4 protein expression in clinicopathologically characterized nasopharyngeal carcinoma (NPC) cases and nasopharyngeal tissues (NPs). Survival curves were plotted by the Kaplan-Meier method and compared using the log-rank test. Results In this investigation, we knocked down CDK4 expression and observed that NPC cell growth and cell cycle progression were significantly blocked by suppressing expression of CCND1, CDK6, and E2F1 as well as elevated p21 expression. Further, we found that reduced CDK4 expression elevated the expression of let-7c, a tumor-suppressive miRNA modulated by E2F1. We found that let-7c was markedly downregulated in NPC tissues compared to NPs and suppressed cell growth and cell cycle progression by modulating p15/p16/CDK4/E2F1 pathway. Finally, CDK4 protein was observed to be overexpressed in NPC tissues and could be considered an unfavorable prognosis factor for NPC patients although its independent prognostic value did not reach statistical significance (p = 0.087). Conclusions Our results demonstrated that overexpressed CDK4 is an unfavorable prognostic factor which suppresses the expression of tumor suppressive-factor let-7c through p21/CCND1/CDK6/E2F1 signaling, and inhibits cell proliferation by p15/p16/CDK4/E2F1 feedback signaling in NPC. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the effects and underlying mechanisms of early and late subconjunctival injection of bevacizumab on the inhibition of corneal neovascularization (NV). Methods: Corneal NV was induced by closed eye contact lens wear followed by a silk suture tarsorrhaphy in rabbits. Weekly subconjunctival injections of bevacizumab (5.0 mg) for 1 month were started immediately (early treatment group) or 1 month after induction of corneal NV with continuous induction (late treatment group). The severity of corneal NV was evaluated. Immunostaining was used to evaluate the intracorneal diffusion of bevacizumab, and the existence of pericytes and smooth muscle cells around the NV. The expression of AM-3K, an anti-macrophage antibody, vascular endothelial growth factor (VEGF) with its receptors (VEGFR1 and VEGFR2), and vascular endothelial apoptosis were also evaluated. Western blot analysis was performed to quantify the expression level of VEGF, VEGFR1 and VEGFR2 on corneal epithelium and stroma in different groups. Results: Early treatment with bevacizumab inhibited corneal NV more significantly than late treatment. Intracorneal diffusion of bevacizumab was not different among different groups. Immunostaining showed pericytes and smooth muscle cells around newly formed vessels as early as 2 weeks after induction. Immunostaining and Western blot analysis showed that VEGF, VEGFR1, and VEGFR2 on corneal stroma increased significantly in no treatment groups and late treatment groups, but not in early treatment group. Bevacizumab significantly inhibited macrophage infiltration in the early but not late treatment group. Sporadic vascular endothelial apoptosis was found at 4 weeks in the late but not early treatment group. Conclusions: Early but not late injection of bevacizumab inhibited corneal NV. Late injection of bevacizumab did not alter macrophage infiltration, and can't inhibit the expression of VEGF, VEGFR1, and VEGFR2 on corneal vessels. The inhibition of corneal NV in early treatment group does not occur via vascular endothelial apoptosis. [ABSTRACT FROM AUTHOR]

Several studies have shown that hepatocyte growth factor (HGF) ameliorates chronic renal failure, but its mechanism of action is unclear. This study was designed to test the delivery of HGF in the PCI-neo vector, using the 5/6 nephrectomized rat as a model for chronic renal failure, and to confirm that this protective function is associated with decreased protein expression of transforming growth factor-beta1 (TGF-β1). Rats were randomly divided into the following groups: Control (untreated), PCI-neo (vector control), 5/6 nephrectomy, and PCI-neo-HGF. Rats were sacrificed at both the fifth and ninth week after 5/6 nephrectomy. Kidney specimens were used for pathological examination (hematoxylin-eosin staining), and detection of TGF-β1 protein (Western blot and immunohistochemistry) expression. Blood urea nitrogen, serum creatinine, and 24-h urinary protein excretion (UPE) were increased, renal interstitium was seriously injured, and TGF-β1 protein expression was elevated in 5/6 nephrectomized rats compared to control rats at either time point. Red blood cell and hemoglobin levels decreased in the ninth week after 5/6 nephrectomy. PCI-neo-HGF expression ameliorated the aforementioned changes and decreased TGF-β1 expression, not only in the fifth week, but also in the ninth week after surgery. The process of renal injury in the 5/6 nephrectomized rat was consistent with that of chronic renal failure. The increase in TGF-β1 expression was maintained after 5/6 nephrectomy. HGF relieved chronic renal failure, this protection was associated with down-regulation of TGF-β1 protein expression, and the protective effects were long-term and stable after 5/6 nephrectomy. [ABSTRACT FROM AUTHOR]

The recessive opaque-2 mutant gene (o2) reduces α-zeins accumulation in maize endosperm, changes the amino acid composition of maize kernels, induces an opaque endosperm, and increases the lysine content of kernels. The quality protein maize (QPM) inbred line CA339 (o2o2) and an elite normal inbred line liao2345 (O2O2) were used to construct o2 near-isogenic lines (NILs) by marker-assisted selection (MAS) using the co-dominant SSR marker phi057. Two specific o2 NILs were constructed, named liao2345/o2-1 and liao2345/o2-2. However, the kernel phenotypes of the two o2 NILs were different from each other. liao2345/o2-1 had the wild-type vitreous endosperm, which is similar to its recurrent parent liao2345, while the endosperm of liao2345/o2-2 was opaque, identical to typical o2 mutant individuals. In comparison to their recurrent parent liao2345, the lysine concentration of liao2345/o2-1 was similar and the lysine concentration in liao2345/o2-2 was doubled. SDS-PAGE analysis indicated that liao2345/o2-1 had the same zeins ratio as liao2345, whereas the zeins concentration of liao2345/o2-2 was markedly lower. Sequence and transcript abundance analyses indicated that the CDS of two o2 NILs are derived from CA339, but they have different promoters. The O2 transcript of liao2345/o2-2 is largely inhibited because of an rbg transposable element inserted between the TATA box and initiator codon of liao2345/o2-2. We concluded that different crossing-over patterns during the process of o2 NIL construction resulted in the different kernel phenotypes of the two o2 NILs. We surmise that the reversion of liao2345/o2-1 to wild type was due to the recombination with the wild type liao2345 promoter during introgression and backcrossing. The o2 mutant gene of donor (CA339) is a null mutant because of low O2 expression. However, its CDS probably encodes a protein with normal function which can maintain the normal accumulation of zeins in maize endosperm. [ABSTRACT FROM AUTHOR]

Formation of yellow-red color cocoons in the silkworm, Bombyx mori, occurs as the result of the selective delivery of carotenoids from the midgut to the silk gland via the hemolymph. This process of pigment transport is thought to be mediated by specific cellular carotenoids carrier proteins. Previous studies indicated that two proteins, Cameo2 and CBP, are associated with the selective transport of lutein from the midgut into the silk gland in Bombyx mori. However, the exact roles of Cameo2 and CBP during the uptake and transport of carotenoids are still unknown. In this study, we investigated the respective contributions of these two proteins to lutein and β-carotene transport in Bombyx mori as well as commercial cell-line. We found that tissues, expressed both Cameo2 and CBP, accumulate lutein. Cells, co-expressed Cameo2 and CBP, absorb 2 fold more lutein (P<0.01) than any other transfected cells, and the rate of cellular uptake of lutein was concentration-dependent and reached saturation. From immunofluorescence staining, confocal microscopy observation and western blot analysis, Cameo2 was localized at the membrane and CBP was expressed in the cytosol. What’s more, bimolecular fluorescence complementation analysis showed that these two proteins directly interacted at cellular level. Therefore, Cameo2 and CBP are necessarily expressed in midguts and silk glands for lutein uptake in Bombyx mori. Cameo2 and CBP, as the membrane protein and the cytosol protein, respectively, have the combined effect to facilitate the cellular uptake of lutein. [ABSTRACT FROM AUTHOR]

Background: Apoptosis plays a central role in the pathogenesis of chronic obstructive pulmonary disease (COPD), and this process can be regulated by mitochondrial transcription factor A (mtTFA). Epigenetics is involved in the regulation and modification of the genes involved in lung cancer and COPD. In this study, we determined the expression of mtTFA and its methylation levels in the COPD patients with lung cancer. Methods: Twenty-one squamous cell lung cancer patients, 11 with COPD and 10 without COPD, undergoing pneumonectomy were enrolled. The apoptotic index (AI) of pulmonary vascular endothelial cells was analyzed by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay. The expression of mtTFA mRNA and protein was measured using PCR, immunohistochemistry and Western-blot. Methylation of the mtTFA promoter was detected using bisulfite sequencing PCR. Results: Compared to the non-COPD group, the AI was higher, and expression of mtTFA mRNA and protein was lower in the COPD group (P<0.001). Expression of the mtTFA protein was positively correlated with FEV1/Pre (r = 0.892, P<0.001), and negatively correlated with AI (r = −0.749, P<0.001) and smoke index (r = −0.763, P<0.001). Percentage of mtTFA promoter methylation in the COPD patients was significantly higher compared to the non-COPD patients (P<0.05). Conclusion: These results suggest that the expression of mtTFA mRNA and protein is down-regulated in the lung tissue from the COPD patients with squamous cell lung cancer, and the level of mtTFA protein is related to apoptosis of pulmonary vascular endothelial cells. Aberrant mtTFA methylation may also play an important role in the pathogenesis of COPD. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus (S. aureus) is an important etiological organism in chronic and subclinical mastitis in lactating cows. Given the fundamental role the primary bovine mammary epithelial cells (pBMECs) play as a major first line of defense against invading pathogens, their interactions with S. aureus was hypothesized to be crucial to the establishment of the latter’s infection process. This hypothesis was tested by investigating the global transcriptional responses of pBMECs to three S. aureus strains (S56,S178 and S36) with different virulent factors, using a tag-based high-throughput transcriptome sequencing technique. Approximately 4.9 million total sequence tags were obtained from each of the three S. aureus-infected libraries and the control library. Referenced to the control, 1720, 219, and 427 differentially expressed unique genes were identified in the pBMECs infected with S56, S178 and S36 S. aureus strains respectively. Gene ontology (GO) and pathway analysis of the S56-infected pBMECs referenced to those of the control revealed that the differentially expressed genes in S56-infected pBMECs were significantly involved in inflammatory response, cell signalling pathways and apoptosis. In the same vein, the clustered GO terms of the differentially expressed genes of the S178-infected pBMECs were found to comprise immune responses, metabolism transformation, and apoptosis, while those of the S36-infected pBMECs were primarily involved in cell cycle progression and immune responses. Furthermore, fundamental differences were observed in the levels of expression of immune-related genes in response to treatments with the three S. aureus strains. These differences were especially noted for the expression of important pro-inflammatory molecules, including IL-1α, TNF, EFNB1, IL-8, and EGR1. The transcriptional changes associated with cellular signaling and the inflammatory response in this study may reflect different immunomodulatory mechanisms that underlie the interaction between pBMECs and S. aureus strains during infection by the latter. [ABSTRACT FROM AUTHOR]

Abstract: Background: MicroRNAs (miRNAs) are widely involved in the process of chronic heart failure (HF), which is characterized by reactivation of the fetal gene program. Here, we examined whether the serum expression levels of some HF-related miRNAs in adult HF patients would tend to revert to fetal levels. Methods and results: Serum was obtained from the peripheral venous blood of 22 HF patients, 18 asymptomatic controls, and the umbilical venous blood of 9 fetuses from 9 independent parturitions. Serum pools of the three groups were initially screened against 40 known HF-associated miRNAs via quantitative reverse transcriptase polymerase chain reaction. Twenty-seven miRNAs were stably expressed in the serum pools. Nine miRNAs showed similar expression levels in the HF and fetus groups compared to the controls, two of which (miR-210, miR-30a) were significantly up-regulated in both groups. These miRNAs showed high diagnostic accuracy and correlations with blood N-terminal prohormone of brain natriuretic peptide, identifying them as potential biomarkers for HF. Putative targets of the miRNAs were predicted with online software programs, and the Kyoto Encyclopedia of Genes and Genomes pathway analysis was employed to identify miRNA-regulated functional modules. In particular, miR-210 seemed to be more closely related than miR-30a to the pathological mechanisms of HF, including the calcium signaling, vascular smooth muscle contraction, transforming growth factor-β signaling, and aldosterone-regulated sodium reabsorption pathways. Conclusion: The serum expression levels of some HF-related miRNAs in HF patients tended towards fetal levels. Among them, miR-210 and miR-30a were elevated in the HF and fetus groups. [Copyright &y& Elsevier]

Abstract: Background: Peripheral blood-based gene expression patterns have been investigated as biomarkers to monitor the immune system and rule out rejection after heart transplantation. Recent advances in the high-throughput deep sequencing (HTS) technologies provide new leads in transcriptome analysis. Methods: By performing Solexa/Illumina's digital gene expression (DGE) profiling, we analyzed gene expression profiles of PBMCs from 6 quiescent (grade 0) and 6 rejection (grade 2R&3R) heart transplant recipients at more than 6months after transplantation. Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR) was carried out in an independent validation cohort of 47 individuals from three rejection groups (ISHLT, grade 0,1R, 2R&3R). Results: Through DGE sequencing and qPCR validation, 10 genes were identified as informative genes for detection of cardiac transplant rejection. A further clustering analysis showed that the 10 genes were not only effective for distinguishing patients with acute cardiac allograft rejection, but also informative for discriminating patients with renal allograft rejection based on both blood and biopsy samples. Moreover, PPI network analysis revealed that the 10 genes were connected to each other within a short interaction distance. Conclusions: We proposed a 10-gene signature for heart transplant patients at high-risk of developing severe rejection, which was found to be effective as well in other organ transplant. Moreover, we supposed that these genes function systematically as biomarkers in long-time allograft rejection. Further validation in broad transplant population would be required before the non-invasive biomarkers can be generally utilized to predict the risk of transplant rejection. [Copyright &y& Elsevier]

The IK6 isoform plays an important role in Philadelphia chromosome-positive adult acute lymphoblastic leukemia (Ph + ALL). This study was designed to monitor the expression of the IK6 isoform with associated cytogenetic and molecular abnormalities. The IK6 isoform, cytogenetic and molecular abnormalities were detected in 100 Chinese patients with de novo Ph+ adult ALL. Expression levels of the IK6 isoform and BCR-ABL1 transcripts were monitored during treatment. BCR-ABL1 mutation was identified in 45 paired samples. Strong correlations were found between the expression status of the IK6 isoform and blast cells, additional cytogenetic abnormalities, BCR-ABL1 transcripts, increased risk of relapse, shorter relapse-free survival and overall survival at diagnosis. Higher frequencies of single IK6 expression and ABL mutation, including the types and shifts thereof, were confirmed in relapsed patients. Furthermore, expression of the IK6 isoform was dynamically consistent with BCR-ABL1 transcript levels during treatment in the single expression group, whereas no such correlation was observed in the co-expression group. The expression pattern of the IK6 isoform was altered in three patients from the co-expression group. The findings of this study in Chinese patients with Ph+ adult ALL exhibit some discrepancies with data reported in other countries, thereby enhancing current knowledge on the therapeutic response and prognosis of this disease. [ABSTRACT FROM AUTHOR]

Abstract: Over-expression of cleaved cyclin E in breast tumors is closely associated with tumor progression and resistance to antiestrogens. 17β-Estradiol (E2) has been recently shown to induce cyclin E processing in breast cancer cells. Tamoxifen has been used in patients with estrogen-sensitive breast cancer, yet resistance to antiestrogens and recurrence will appear in some of the patients after its continued use. We therefore addressed possible effects of tamoxifen on the generation of cleaved cyclin E and its signal mechanism(s) in estrogen-responsive MCF-7 breast cancer cells that express both G protein-coupled protein (GPR) 30 and estrogen receptor α (ERα). 4-Hydroxytamoxifen (OHT, tamoxifen's active form) failed to prevent E2-induced proteolysis of cyclin E and migration, but rather triggered cyclin E cleavage coincident with augmented migration. OHT-induced cyclin E truncation also occurred in SK-BR-3 cells that express GPR30 and lack ERα, but not in MDA-MB-231 cells that express neither GPR30 nor ERα. G1, a specific GPR 30 agonist, caused dramatic proteolysis of cyclin E and enhanced migration. Furthermore, OHT-stimulated cleavage of cyclin E and migration were tremendously attenuated by G15, a GPR30 antagonist, or siRNA against GPR30. In addition, inhibitors for EGFR or ERK1/2 remarkably suppressed OHT-induced truncation of cyclin E, suggesting involvement of EGFR signaling. Collectively, our data indicate that OHT contributes to the production of proteolyzed cyclin E via GPR30 with augmented migration in MCF-7 cells. [Copyright &y& Elsevier]

Abstract: In adults with precursor-B lymphoblastic leukemia (BCP-ALL) there remain a majority of patients who fall in an intermediate cytogenetics risk category with a heterogenous outcome. We analyzed immunophenotypic and cytogenetic factors retrospectively in 126 consecutive adults with BCR-ABL negative BCP-ALL who were treated with a pediatric-based protocol at a single institution over a 10 year period. In addition to age, WBC and cytogenetic findings, CD13 positivity was an independent poor prognostic indicator for overall survival (OS, p =0.049), event-free survival (EFS, p =0.013), and relapse-free survival (RFS, p <0.001). The prognostic value of CD13 was primarily seen in patients with normal or intermediate risk cytogenetics. A risk model that includes age>60 years, WBC>30×109/L, SWOG high/very high risk cytogenetics and CD13 positivity, performs better than a risk model of cytogenetics alone for stratifying patients by OS (p =0.001), EFS (p =7×10−4) and RFS (p =8×10−4). Incorporating CD13 into a scoring system provides high discrimination for relapse risk and survival. [Copyright &y& Elsevier]

Adipokine adiponectin (APN) has been recently reported to play a role in regulating bone mineral density (BMD). To explore the mechanism by which APN affects BMD, we investigated BMD and biomechanical strength properties of the femur and vertebra in sham-operated (Sham) and ovariectomized (OVX) APN knockout (KO) mice as compared to their operated wild-type (WT) littermates. The results show that APN deficiency has no effect on BMD but induces increased ALP activity and osteoclast cell number. While OVX indeed leads to significant bone loss in both femora and vertebras of WT mice with comparable osteogenic activity and a significant increase in osteoclast cell number when compared to that of sham control. However, no differences in BMD, ALP activity and osteoclast cell number were found between Sham and OVX mice deficient for APN. Further studies using bone marrow derived mesenchymal stem cells (MSCs) demonstrate an enhanced osteogenic differentiation and extracellular matrix calcification in APN KO mice. The possible mechanism for APN deletion induced acceleration of osteogenesis could involve increased proliferation of MSCs and higher expression of Runx2 and Osterix genes. These findings indicate that APN deficiency can protect against OVX-induced osteoporosis in mice, suggesting a potential role of APN in regulating the balance of bone formation and bone resorption, especially in the development of post-menopausal osteoporosis. [ABSTRACT FROM AUTHOR]

Background: The role of CTGF varies in different types of cancer. The purpose of this study is to investigate the involvement of CTGF in tumor progression and prognosis of human nasopharyngeal carcinoma (NPC). Experimental design: CTGF expression levels were examined in NPC tissues and cells, nasopharynx (NP) tissues, and NP69 cells. The effects and molecular mechanisms of CTGF expression on cell proliferation, migration, invasion, and cell cycle were also explored. Results: NPC cells exhibited decreased mRNA expression of CTGF compared to immortalized human nasopharyngeal epithelial cell line NP69. Similarly, CTGF was observed to be downregulated in NPC compared to normal tissues at mRNA and protein levels. Furthermore, reduced CTGF was negatively associated with the progression of NPC. Knocking down CTGF expression enhanced the colony formation, cell migration, invasion, and G1/S cell cycle transition. Mechanistic analysis revealed that CTGF suppression activated FAK/PI3K/AKT and its downstream signals regulating the cell cycle, epithelial-mesenchymal transition (EMT) and MMPs. Finally, DNA methylation microarray revealed a lack of hypermethylation at the CTGF promoter, suggesting other mechanisms are associated with suppression of CTGF in NPC. Conclusion: Our study demonstrates that reduced expression of CTGF promoted cell proliferation, migration, invasion and cell cycle progression through FAK/PI3K/AKT, EMT and MMP pathways in NPC. [ABSTRACT FROM AUTHOR]

Background: The role of tumor suppressor gene RASSF1A in the esophageal and gastric cardia carcinogenesis is still inconclusive. In this study, the polymorphism, promoter methylation and gene expression of RASSF1A were characterized in esophageal squamous cell carcinoma (ESCC) and gastric cardia adenocarcinoma (GCA).Methods: We firstly analyzed the prevalence of RASSF1A A133S in a total of 228 cancer patients with ESCC (n=112) and GCA (n=116) and 235 normal controls by polymerase chain reaction (PCR) and restriction enzyme-digestion assay. Then, the promoter methylation status of the RASSF1A in ESCC (n=143), GCA (n=92) and corresponding adjacent normal tissues were further investigated using methylation-specific PCR (MSP) approach. Finally, the RASSF1A protein expression were determined in ESCC (n=27), GCA (n=24) and the matched adjacent normal tissues by immunohistochemical method.Results: The frequency of 133Ala/Se and Ser/Ser genotype was significantly higher in GCA patients than in normal controls (19.0% vs. 10.2%, P=0.02). Compared with Ala/Ala genotype, Ala/Se and Ser/Ser genotype significantly increased susceptibility to GCA (OR=2.06, 95% CI=1.09-3.97). However, this polymorphism had no association with ESCC (P=0.69). The promoter methylation of RASSF1A gene was significantly increased the risk to both ESCC (OR=5.90, 95% CI=2.78-12.52) and GCA (OR=7.50, 95% CI= 2.78-20.23). Promoter methylation of RASSF1A gene in ESCC was also associated with age and cancer cell differentiation (for age: OR=3.11, 95% CI=1.10-8.73; for differentiation: OR=0.29, 95% CI=0.12-0.69). RASSF1A positive expression was significantly decreased the risk of GCA (OR=0.16, 95% CI=0.03-0.83). In contrast, there was no statistical significance between RASSF1A positive expression and ESCC. The expression of RASSF1A protein trend to be positively related with older GCA patients (OR=16.20, 95% CI=1.57-167.74).Conclusions: The present findings suggest that alterations of RASSF1A may play an important role in gastric cardia carcinogenesis in terms of polymorphism, promoter hypermethylation and protein expression. Whereas, RASSF1A hypermethylation may probably also be involved in esophageal squamous cell carcinogenesis. [ABSTRACT FROM AUTHOR]

The unique anatomical features of turtles have raised unanswered questions about the origin of their unique body plan. We generated and analyzed draft genomes of the soft-shell turtle (Pelodiscus sinensis) and the green sea turtle (Chelonia mydas); our results indicated the close relationship of the turtles to the bird-crocodilian lineage, from which they split ∼267.9-248.3 million years ago (Upper Permian to Triassic). We also found extensive expansion of olfactory receptor genes in these turtles. Embryonic gene expression analysis identified an hourglass-like divergence of turtle and chicken embryogenesis, with maximal conservation around the vertebrate phylotypic period, rather than at later stages that show the amniote-common pattern. Wnt5a expression was found in the growth zone of the dorsal shell, supporting the possible co-option of limb-associated Wnt signaling in the acquisition of this turtle-specific novelty. Our results suggest that turtle evolution was accompanied by an unexpectedly conservative vertebrate phylotypic period, followed by turtle-specific repatterning of development to yield the novel structure of the shell. [ABSTRACT FROM AUTHOR]

Aim: Early diagnosis of prostate cancer (PCa), which is a clinically heterogeneous-multifocal disease, is essential to improve the prognosis of patients. However, published PCa diagnostic markers share little overlap and are poorly validated using independent data. Therefore, we here developed an integrative proteomics and interaction network-based classifier by combining the differential protein expression with topological features of human protein interaction networks to enhance the ability of PCa diagnosis. Methods and Results: By two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with MS using PCa and adjacent benign tissues of prostate, a total of 60 proteins with the differential expression in PCa tissues were identified as the candidate markers. Then, their networks were analyzed by GeneGO Meta-Core software and three hub proteins (PTEN, SFPQ and HDAC1) were chosen. After that, a PCa diagnostic classifier was constructed by support vector machine (SVM) modeling based on the microarray gene expression data of the genes which encode the hub proteins mentioned above. Validations of diagnostic performance showed that this classifier had high predictive accuracy (85.96∼90.18%) and area under ROC curve (approximating 1.0). Furthermore, the clinical significance of PTEN, SFPQ and HDAC1 proteins in PCa was validated by both ELISA and immunohistochemistry analyses. More interestingly, PTEN protein was identified as an independent prognostic marker for biochemical recurrence-free survival in PCa patients according to the multivariate analysis by Cox Regression. Conclusions: Our data indicated that the integrative proteomics and interaction network-based classifier which combines the differential protein expression and topological features of human protein interaction network may be a powerful tool for the diagnosis of PCa. We also identified PTEN protein as a novel prognostic marker for biochemical recurrence-free survival in PCa patients. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miRNAs), a class of highly conserved small non-coding RNA molecules, are known to play essential roles in central nervous system (CNS) by causing post-transcriptional gene silencing. There is much evidence that miRNAs have specific temporal and spatial expression patterns in the mammal brain, but little is known about the role of the region specificity for the gene regulatory networks of the brain. This study represents the first attempt to perform a profiling analysis of the differential expression of miRNAs between hippocampus and the Marginal division (MrD) of the neostriatum in the rat brain. Results: Microarray was used to detect the expression of 357 miRNAs in hippocampus and the MrD from three rats. A short-list of the most dysregulated 30 miRNAs per rat was generated for data analysis, and the miRNAs that were represented in two or three short-lists were then further analyzed. Quantitative real-time reverse transcriptionpolymerase chain reaction (RT-PCR) was employed to validate the aberrantly expressed miRNAs obtained from the miRNA microarray analysis. A family of 11 miRNAs demonstrated differential expression between the MrD and hippocampus in more than one rat. Amongst these, miR-383 was differentially expressed in all three rats and upregulated to the largest degree in rat one, and the ten other miRNAs, let-7d*, miR-181b, miR-187, miR-195, miR-214, miR-382, miR-411, miR-466b, miR-592 and miR-1224 were differentially expressed in at least two rats. Of these ten, besides miR-382 and miR-411 which were up-regulated in one rat and down-regulated in another, the other eight miRNAs retained a uniform direction of regulation (up-regulation or down-regulation) between different specimens. When further examined by RT-PCR, the aberrantly expressed miRNAs, except miR-383 and let-7d*, demonstrated differential expression that significantly correlated with the microarray findings. Conclusion: This study reported that the miRNA expression patterns in MrD was distinct from that of Hip, suggesting the role of miRNAs in the learning and memory function of the MrD probably different from hippocampus. [ABSTRACT FROM AUTHOR]

Abstract: Background: This study aimed to develop a working animal model for exploring the pathogenesis and molecular mechanisms of premature ovarian failure (POF) as induced by cisplatin. Methods: Female Sprague-Dawley rats were intraperitoneally injected with different doses of cisplatin. The bodyweight, estrous cyclicity, concentration of gonadal hormones, ovarian weight, and histopathology were determined and compared with those of the control group. Gene chips were used to compare the genes that were differentially expressed between the POF and control ovaries. Results: A cisplatin dose of 1.5mg/kg body weight significantly reduced the E2 levels and elevated the gonadotropin levels after two weeks of injection. The treated rats were characterized by estrous cycle disorders and the lack of mature follicles. Thus, these animals could be used as a model for chemotherapy-induced POF. Compared with the controls, the Gene Ontology (GO) categories that were significantly increased in the POF ovaries included “monooxygenase activity,” “cholesterol metabolism,” and “acute inflammatory response.” On the other hand, the GO terms “mitotic spindle organization,” “ovulation,” and “cholesterol biosynthetic process” were downregulated. The upregulation of three proteins CYP2E1, SCARB1, C3 and downregulation of CYP51 were verified in the cisplatin ovaries by Western blot analysis. Conclusions: The immune inflammatory response and reactive oxygen species may damage ovarian function in the cisplatin-induced POF. [Copyright &y& Elsevier]

Abnormal placenta trophoblast proliferation and apoptosis is related to the pathogenesis of preeclampsia. Emerging evidence has also indicated that key pregnancy-associated hormones, such as hCG, progesterone, are found in high concentration at the maternal-fetal interface. The purpose of this study was to investigate the expression of CYP11A, a key enzyme in steroid hormone synthesis and metabolism, in normal pregnancy and severe preeclampsia placenta and to explore the underlying mechanism of the relationship between the altered CYP11A expression and onset of preeclampsia. Immunohistochemistry method was used to study the localization of CYP11A-encoded protein P450scc in the placenta; reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to examine CYP11A expression at mRNA and protein levels in patients with severe preeclampsia and normal placental tissue. CYP11A overexpression in trophoblastic cells was used to evaluate the effect on viability. TUNEL staining was used to determine whether overexpression of CYP11A could affect trophoblastic cell apoptosis. The results showed that CYP11A was selectively expressed in the cytoplasm of the placental trophoblastic cells. CYP11A expression were significantly increased in severe preeclampsia compared with normal pregnancy in both mRNA and protein levels. Multiple regression analysis indicated that CYP11A gene expression was positively correlated to ALT level and Plt, while negatively correlated to INR. Overexpression of CYP11A reduced trophoblastic cell proliferation and induced HTR8/SVneo cells apoptosis through activation of activated caspase-3 expression. These results suggest that abnormally high expression of CYP11A inhibits trophoblastic proliferation and increases apoptosis and therefore could be involved in the pathogenesis of preeclampsia. [ABSTRACT FROM AUTHOR]

Expression of monocyte chemoattractant protein-1 (MCP-1) and CC chemokine receptor 2 (CCR2) and its significance has been demonstrated in some cancer cells in recent clinical studies. However, the role of tumor MCP-1 and CCR2 expression in non-small cell lung cancer (NSCLC) remains unknown. The aim of the present study was to investigate the prognostic significance of MCP-1 and CCR2 expression in NSCLC cells. The relationship between MCP-1 and CCR2 expression in NSCLC cancer cells was examined by immunohistochemical staining of surgical specimens from 134 patients. Sixty-five of these patients had follow-up records. Kaplan-Meier analysis and Cox regression model were used to assess overall survival according to the presence or absence of MCP-1 and CCR2 expression in tumor cells. MCP-1 was detected in cancer cells of 107 NSCLC (79.9 %) and CCR2 was detected in cancer cells of 39 NSCLC (29.1 %). MCP-1 expression was correlated with sex, smoking habits, histology, and tumor size. Presence of MCP-1 in tumor cells was associated with better overall survival ( P = 0.018). By multivariate analysis, MCP-1 expression in cancer cells showed an independent prognostic factor for overall survival ( P = 0.002, hazard ratio [HR] = 0.256, 95 % confidence interval [CI] = 0.106-0.616). There was no significant relationship between CCR2 expression in tumor cells and clinical and pathological characteristics. Also, no significant positive correlation between MCP-1 and CCR2 expression was revealed by Spearman correlation analysis. Our data indicate that MCP-1 is overexpressed in NSCLC cells. Its expression in cancer cells is associated with better survival in NSCLC patients. [ABSTRACT FROM AUTHOR]

Aim: The mechanisms underlying development of chronic hepatitis B virus (HBV) infection are related to immune tolerance, but are as yet incompletely understood. Furin has been found to be essential for maintenance of peripheral immune tolerance mediated by regulatory T cells (Treg). Such effect of furin on chronic HBV infection was investigated in this study. Methods: Peripheral blood from 40 individuals with self-limited HBV infection, 40 patients with asymptomatic persistent HBV infection and 40 patients with chronic hepatitis B (CHB) was collected and mRNA expression levels of furin, transforming growth factor (TGF)-β1 and the Treg-function-related forkhead transcription factor FoxP3 were detected using quantitative real-time polymerase chain reaction. CD4+CD25+FoxP3+ Treg were detected using flow cytometry. Results: Furin mRNA expression in peripheral blood was significantly higher in patients with persistent HBV infection than in individuals with self-limited infection ( P < 0.01), and was much higher in CHB patients than in those with asymptomatic persistent infection ( P < 0.01). Furthermore, furin mRNA was relatively higher in patients with positive hepatitis B e antigen and higher levels of serum HBV DNA (>10 000 copies/mL). In patients with CHB, furin mRNA expression was found to correlate with TGF-β1 mRNA and FoxP3 mRNA expression using Spearman's rank correlation coefficient test. It was 5.7-times higher in CD4+CD25+ T cells than in CD4+CD25- T cells and correlated with the frequency of Treg ( P < 0.05). Conclusion: Furin mRNA expression in peripheral blood correlates with chronic HBV infection and liver damage, and seems to participate in immune inhibitory and anti-inflammatory mechanisms in HBV infection, mediated by TGF-β1 and/or Treg. [ABSTRACT FROM AUTHOR]

Abstract: High level expression of axe1, a gene previously cloned from Volvariella volvacea that encodes an acetyl xylan esterase with two potential N-linked glycosylation sites, has been achieved in Pichia pastoris using a codon-optimized axe1 synthesized by the primer extension PCR procedure. The GC content of the codon-optimized axe1 was 48.62% compared with 55.49% in the native gene. Using the codon-optimized construct, AXE1 expression in P. pastoris was increased from an undetectable level to 136.45U/ml six days after induction of yeast cultures grown in BMMY medium. A further increase (to 463U/ml) was achieved when conditions for yeast culture were optimized as follows: 2.8% methanol, 0.63% casamino acids, and pH 8.0. This latter value represented a 3.4-fold and 246-fold increase in the enzyme levels recorded in non-optimized P. pastoris cultures and in rice straw-grown cultures of V. volvacea, respectively. N-linked glycosylation played an essential role in AXE1 secretion but had only a slight effect on the catalytic activity and stability of the recombinant enzyme. [Copyright &y& Elsevier]

Accumulation of α-synuclein (α-Syn) is a common pathology for both familiar and sporadic Parkinson's disease (PD), enhancing its clearance might be a promising strategy for treating PD. To assess the potential of trehalose in this regard, we investigated its effect on the PC12 cells overexpressing wild type (WT) or A53T mutant α-Syn and the implicated pathway it might mediated. We observed that trehalose promoted the clearance of A53T α-Syn but not WT α-Syn in PC12 cells, and confirmed the increased LC3 and Lysotracker RED positive autolysosomes by using lysotracker and LC3 staining, the enhanced expression of LC3-II in Western blot, and more autophagosomes under Transmission Electron Microscope in a dose dependent manner after the trehalose treatment. The activation of autophagy can be alleviated by applying macroautophagy inhibitor 3-methyladenine (3-MA). In addition, degradation of A53T and WT α-Syn was blocked after Ubiquitin Proteasome System (UPS) inhibitor (MG132) was applied in those PC12 cells overexpressing A53T or WT α-Syn, suggesting that A53T α-Syn could be degraded by both UPS and macroautophagy. But the effect of trehalose on A53T α-Syn is mainly mediated through the macroautophagy pathway, which is not a dominant way for WT α-Syn clearance. Further in vivo research will be needed to verify the effectiveness of trehalose in treating PD. [ABSTRACT FROM AUTHOR]

Preterm delivery (PTD) is the leading cause of infant mortality and morbidity. However, the mechanism at the molecular level is still unknown. Placental inflammatory response and oxidative stress are associated with PTD. Thioredoxin-1 (TRX-1) regulates oxidative stress, inflammation, and the activities of transcription factors. Objectives The objective was to detect in placental tissues the expressions of TRX-1 and the TRX-1-related molecules: tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), thioredoxin-1-binding protein-2 (TBP-2), hypoxia inducible transcription factor 1α (HIF-1α), and forkhead box protein O3A (FoxO3A). Methods PTD was defined as gestation of <37 weeks and term delivery (TD) as ≥37 weeks. The expressions of TRX-1 and TRX-1-related molecules were examined in placental tissues by real-time polymerase chain rection and western blot. Results The expressions of TRX-1, TNF-α, COX-2, HIF-1α, and FoxO3A in the placenta of PTD were significantly higher as compared with TD, but no difference was observed in TBP-2 expression. Discussion These results indicate that TRX-1 may be adaptively induced by the effects of inflammation and oxidative stress, suggesting protective roles for TRX-1 against these effects in the placenta of PTD. [ABSTRACT FROM AUTHOR]

This study aimed to examine whether hepatocyte growth factor (HGF) can improve renal function in 5/6 nephrectomized rats and investigate whether this function is associated with a decrease in α-smooth muscle actin ( α-SMA) expression in rat glomerulus mesangial cells and renal interstitium. Rats were randomly divided into the following groups: control, PCI-neo, sham-operation, 5/6 nephrectomy, and low-dose and high-dose PCI-neo-HGF. Rats were killed in the ninth week after 5/6 nephrectomy, and the kidney specimens were subjected to pathological examination by Hematoxylin-Eosin staining and detection of α-SMA expression by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry. The results showed that blood urea nitrogen and serum creatinine levels were increased, renal interstitium was injured, and α-SMA expression was elevated in 5/6 nephrectomized rats compared with that in control. The above changes were ameliorated in the rats injected with PCI-neo-HGF vector. At the molecular level we found that PCI-neo-HGF repressed α-SMA expression in mesangial cells stimulated by lipopolysaccharide. In conclusion, our data suggest that HGF can relieve chronic renal failure, and this protection is associated with the down-regulation of α-SMA expression in mesangial cells and renal interstitium. [ABSTRACT FROM AUTHOR]

The present study determined the effects of electroacupuncture on interstitial cells of Cajal and investigated whether changes in the stem cell factor pathway were involved. Animals were assigned to normal, diabetic, diabetic plus sham stimulation, diabetic plus low-frequency stimulation, and diabetic plus high-frequency stimulation groups. Electroacupuncture was performed daily for 8 weeks. In vitro contractility of colonic muscle strips were studied. Expression of c-kit (the marker of interstitial cells of Cajal) and stem cell factor were measured. The results showed that (1) contraction of colonic muscle strips was significantly elevated in low- and high-frequency stimulation groups and (2) in contrast to the diabetic group, the expressions of c-kit and stem cell factor were markedly increased in the low- and high-frequency stimulation groups. These results indicate that both low- and high-frequency stimulation can promote the contractility of colonic muscle strips partially through increasing the number of interstitial cells of Cajal, and these effects could be mediated by an elevated endogenous stem cell factor. [ABSTRACT FROM PUBLISHER]

Ras plays a pivotal role in many cellular activities, and its subcellular compartmentalization provides spatial and temporal selectivity. Here we report a mode of spatial regulation of Ras signaling in the Golgi apparatus by two highly homologous proteins PAQR10 and PAQR11 of the progestin and AdipoQ receptors family. PAQR10 and PAQR11 are exclusively localized in the Golgi apparatus. Overexpression of PAQR10/PAQR11 stimulates basal and EGF-induced ERK phosphorylation and increases the expression of ERK target genes in a dose-dependent manner. Overexpression of PAQR10/PAQR11 markedly elevates Golgi localization of HRas, NRas and KRas4A, but not KRas4B. PAQR10 and PAQR11 can also interact with HRas, NRas and KRas4A, but not KRas4B. The increased Ras protein at the Golgi apparatus by overexpression of PAQR10/PAQR11 is in an active state. Consistently, knockdown of PAQR10 and PAQR11 reduces EGF-stimulated ERK phosphorylation and Ras activation at the Golgi apparatus. Intriguingly, PAQR10 and PAQR11 are able to interact with RasGRP1, a guanine nucleotide exchange protein of Ras, and increase Golgi localization of RasGRP1. The C1 domain of RasGRP1 is both necessary and sufficient for the interaction of RasGRP1 with PAQR10/PAQR11. The simulation of ERK phosphorylation by overexpressed PAQR10/PAQR11 is abrogated by downregulation of RasGRP1. Furthermore, differentiation of PC12 cells is significantly enhanced by overexpression of PAQR10/PAQR11. Collectively, this study uncovers a new paradigm of spatial regulation of Ras signaling in the Golgi apparatus by PAQR10 and PAQR11. [ABSTRACT FROM AUTHOR]

Abstract: Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37°C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein–protein interaction. [Copyright &y& Elsevier]

Focal cerebral ischemia results in an increased expression of matrix metalloproteinase-9 (MMP-9), which induces vasogenic brain edema via disrupting the blood-brain barrier (BBB) integrity. Recent studies from our laboratory showed that baicalin reduces ischemic brain damage by inhibiting inflammatory reaction and neuronal apoptosis in a rat model of focal cerebral ischemia. In the present study, we first explored the effect of baicalin on the neuronal damage, brain edema and BBB permeability, then further investigated its potential mechanisms. Sprague-Dawley rats underwent permanent middle cerebral artery occlusion (MCAO). Baicalin was administrated by intraperitoneally injected twice at 2 and 12 h after the onset of MCAO. Neuronal damage, brain edema and BBB permeability were measured 24 h following MCAO. Expression of MMP-9 protein and mRNA were determined by western blot and RT-PCR, respectively. Expression of tight junction protein (TJP) occludin was detected by western blot. Neuronal damage, brain edema and BBB permeability were significantly reduced by baicalin administration following focal cerebral ischemia. Elevated expression of MMP-9 protein and mRNA were significantly down-regulated by baicalin administration. In addition, MCAO caused the decreased expression of occludin, which was significantly up-regulated by baicalin administration. Our study suggested that baicalin reduces MCAO-induced neuronal damage, brain edema and BBB permeability, which might be associated with the inhibition of MMP-9 expression and MMP-9-mediated occludin degradation. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To investigate the effect of neuregulin 1β (NRG1β) on the neuronal apoptosis and the expressions of Bcl-2 and Bax proteins in experimental dementia model rats. Methods: Thirty adult healthy male Wistar rats were randomly divided into control group, model group and treated group consisting of 10 rats, respectively. The experimental dementia models were established by injecting beta-amyloid protein 1–40 (Aβ1–40) stereotactically into the left lateral ventricle, and treated by injecting NRG1β into right lateral ventricle. The cognitive capacity of rats was evaluated with Y-electric maze. The neuronal apoptosis was counted by TUNEL assay. The expressions of Bcl-2 and Bax were determined with immunohistochemistry assay and double immunofluorescence labeling. Results: The cognitive ability in model group rats decreased, along with the number of neuronal apoptosis and the expressions of Bcl-2 and Bax increased significantly than those in control group (P <0.05). After treatment with NRG1β, the cognitive ability of rats improved, the number of neuronal apoptosis reduced and the expression of Bcl-2 increased while Bax decreased significantly than those in model group (P <0.05). Conclusion: NRG1β could inhibit neuronal apoptosis by regulating the expressions of Bcl-2/Bax to improve the capacity of learning and memory in experimental dementia rats. [Copyright &y& Elsevier]

Summary: CREB3L1/OASIS is a cellular transcription factor synthesized as a membrane-bound precursor and activated by regulated intramembrane proteolysis in response to stimuli like ER stress. Comparing gene expression between Huh7 subclones that are permissive for hepatitis C virus (HCV) replication versus the nonpermissive parental Huh7 cells, we identified CREB3L1 as a host factor that inhibits proliferation of virus-infected cells. Upon infection with diverse DNA and RNA viruses, including murine γ-herpesvirus 68, HCV, West Nile virus (WNV), and Sendai virus, CREB3L1 was proteolytically cleaved, allowing its NH2 terminus to enter the nucleus and induce multiple genes encoding inhibitors of the cell cycle to block cell proliferation. Consistent with this, we observed a necessity for CREB3L1 expression to be silenced in proliferating cells that harbor replicons of HCV or WNV. Our results indicate that CREB3L1 may play an important role in limiting virus spread by inhibiting proliferation of virus-infected cells. [ABSTRACT FROM AUTHOR]

Background: Chronic rejection can prevent long-term survival of organ transplants. Although the beneficial effects of peroxisome proliferator-activated receptor-gamma (PPAR-γ) in reducing graft rejection have been reported, the details of the underlying mechanisms remain unclear, especially in the context of modulating cellular infiltration and preventing vasculopathy and interstitial fibrosis. Methods: The therapeutic effects of the PPAR-γ agonist, rosiglitazone, combined with anti–interleukin-5 are explored in a mouse model of MHC Class II–histoincompatible cardiac transplantation. Results: Rosiglitazone treatment alone marginally increased long-term survival and reduced CD8 T-cell infiltration and vasculopathy in the grafts. However, there was no reduction in collagen deposition and interleukin (IL)-4, IL-5 and eosinophil infiltration were increased. Anti–IL-5 antibody treatment alone reduced eosinophil infiltration and collagen deposition, but had no effect on CD8 T-cell infiltration or vasculopathy. Combined treatment with anti–IL-5 antibody and rosiglitazone prevented graft rejection. Furthermore, rosiglitazone treatment increased adiponectin receptor II expression in grafts and on dendritic cells and T cells in vitro. Graft survival correlated with increased expression in grafts of the inhibitory molecule PD-L1. Conclusions: The findings obtained increase the knowledge on the mode of action of rosiglitazone in promoting the survival of MHC Class II–mismatched cardiac transplants in which the CD8 T cells and eosinophils play key roles. PPAR-γ signaling combined with IL-5 blockade prevents graft rejection. [ABSTRACT FROM AUTHOR]