Transcriptome Based System Biology Exploration Reveals Homogeneous Tumorigenicity of Alimentary Tract Malignancy.

Malignancies of alimentary tract include esophageal carcinoma (ESCA), stomach adenocarcinoma (STAD), colon adenocarcinoma (COAD), and rectum adenocarcinoma (READ). Despite of their similarities in cancer development and progression, there are numerous researches concentrating on single tumor but relatively little on their common mechanisms. Our study explored the transcriptomic data of digestive tract cancers from The Cancer Genome Atlas database, yielding their common differentially expressed genes including 1,700 mRNAs, 29 miRNAs, and 362 long non-coding RNAs (lncRNAs). There were 12 mRNAs, 5 miRNAs, and 16 lncRNAs in the core competitive endogenous RNAs network by RNA-RNA interactions, highlighting the prognostic nodes of SERPINE1, hsa-mir-145, and SNHG1. In addition, the weighted gene co-expression network analysis (WGCNA) illustrated 20 gene modules associated with clinical traits. By taking intersections of modules related to the same trait, we got 67 common genes shared by ESCA and READ and screened 5 hub genes, including ADCY6, CXCL3, NPBWR1, TAS2R38, and PTGDR2. In conclusion, the present study found that SERPINE1/has-mir-145/SNHG1 axis acted as promising targets and the hub genes reasoned the similarity between ESCA and READ, which revealed the homogeneous tumorigenicity of digestive tract cancers at the transcriptome level and led to further comprehension and therapeutics for digestive tract cancers.

Hantavirus Gc induces long-term immune protection via LAMP-targeting DNA vaccine strategy.

Hemorrhagic fever with renal syndrome (HFRS) occurs widely throughout Eurasia. Unfortunately, there is no effective treatment, and prophylaxis remains the best option against the major pathogenic agent, hantaan virus (HTNV), which is an Old World hantavirus. However, the absence of cellular immune responses and immunological memory hampers acceptance of the current inactivated HFRS vaccine. Previous studies revealed that a lysosome-associated membrane protein 1 (LAMP1)-targeting strategy involving a DNA vaccine based on the HTNV glycoprotein Gn successfully conferred long-term immunity, and indicated that further research on Gc, another HTNV antigen, was warranted. Plasmids encoding Gc and lysosome-targeted Gc, designated pVAX-Gc and pVAX-LAMP/Gc, respectively, were constructed. Proteins of interest were identified by fluorescence microscopy following cell line transfection. Five groups of 20 female BALB/c mice were subjected to the following inoculations: inactivated HTNV vaccine, pVAX-LAMP/Gc, pVAX-Gc, and, as the negative controls, pVAX-LAMP or the blank vector pVAX1. Humoral and cellular immunity were assessed by enzyme-linked immunosorbent assays (ELISAs) and 15-mer peptide enzyme-linked immunospot (ELISpot) epitope mapping assays. Repeated immunization with pVAX-LAMP/Gc enhanced adaptive immune responses, as demonstrated by the specific and neutralizing antibody titers and increased IFN-γ production. The inactivated vaccine induced a comparable humoral reaction, but the negative controls only elicited insignificant responses. Using a mouse model of HTNV challenge, the in vivo protection conferred by the inactivated vaccine and Gc-based constructs (with/without LAMP recombination) was confirmed. Evidence of pan-epitope reactions highlighted the long-term cellular response to the LAMP-targeting strategy, and histological observations indicated the safety of the LAMP-targeting vaccines. The long-term protective immune responses induced by pVAX-LAMP/Gc may be due to the advantage afforded by lysosomal targeting after exogenous antigen processing initiation and major histocompatibility complex (MHC) class II antigen presentation trafficking. MHC II-restricted antigen recognition effectively primes HTNV-specific CD4+ T-cells, leading to the promotion of significant immune responses and immunological memory. An epitope-spreading phenomenon was observed, which mirrors the previous result from the Gn study, in which the dominant IFN-γ-responsive hot-spot epitopes were shared between HLA-II and H2d. Importantly, the pan-epitope reaction to Gc indicated that Gc should be with potential for use in further hantavirus DNA vaccine investigations.

Insecticidal activities of constituents of Litsea cubeba fruit extracts effective against the maize weevil (Coleoptera: Curculionidae).

In this study, we investigated the insecticidal activities, including contact toxicity, fumigant toxicity, and repellent activity, of Litsea cubeba fruit extracts against Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae). The extracts, obtained by liquid-liquid extraction in n-hexane, ethyl acetate, chloroform, and water were analyzed by gas chromatography-mass spectrometry. Among the different extract types, chloroform extracts exhibited the strongest repellent, contact, and fumigant activities against S. zeamais. The main components of the chloroform extracts were identified as laurine (21.15%) and 2,6-diisopropyl aniline (16.14%), followed by chlorobutanol (10.54%), 3-O-methyl-N-acetyl-d-glucosamine (10.03%), and 6-methyl-5-hepten-2-one (8.33%). Among the identified components of the chloroform extracts, chlorobutanol showed the strongest fumigant toxicity (LD50 = 21.91 mg/liter), contact toxicity (LD50 = 54.25 µg/adult), and repellent activity against S. zeamais. These results indicate that L. cubeba fruit extracts possess natural insecticide-like activities against S. zeamais.

Neuroprotective effects of daphnetin against NMDA receptor-mediated excitotoxicity.

The accumulation of glutamate can excessively activate the N-methyl-d-aspartate (NMDA) receptors and cause excitotoxicity. Daphnetin (Dap), a coumarin derivative, is a protein kinase inhibitor that exhibits antioxidant and neuroprotective properties. However, little is known about the neuroprotective effects of Dap on glutamate-induced excitotoxicity. We evaluated the neuroprotective activities in the primary cultured cortical neurons against NMDA-induced excitotoxicity. Pretreatment with Dap significantly prevented NMDA-induced neuronal cell loss. Dap significantly inhibited the neuronal apoptosis by regulating balance of Bcl-2 and Bax expression. Furthermore, pretreatment of Dap reversed the up-regulation of NR2B-containing NMDA receptors and inhibited the intracellular Ca2+ overload induced by NMDA exposure. In addition, Dap prevented cerebral ischemic injury in mice induced via a 2 h middle cerebral artery occlusion and a 24 h reperfusion in vivo. The findings suggest that Dap prevents the excitotoxicity through inhibiting the NR2B-containing NMDA receptors and the subsequent calcium overload in cultured cortical neurons.

Neuroprotective effects of formononetin against NMDA-induced apoptosis in cortical neurons.

Formononetin (FMNT) is an isoflavone found in many herbs including Trifolium pratense L., Spatholobus suberectus Dunn., and Astragalus mongholicus Bunge. The purpose of this study is to investigate pharmacological properties of FMNT on neurotoxicity induced by N-methyl-D-asparate (NMDA) in primary-cultured cortical neurons. The cell viability was significantly decreased after exposure to NMDA (200 µM) for 40 min. Pretreatment of FMNT (10 µM) for 12 h significantly attenuated the cell loss induced by NMDA exposure. Flow cytometry analysis revealed that treatment of FMNT attenuated the number of apoptotic cells, especially the early phase apoptotic cells, induced by NMDA exposure. Western blot analysis showed that FMNT regulated the expression of apoptosis-related proteins by increasing the levels of Bcl-2 and pro-caspase-3 and decreasing the levels of Bax and caspase-3. These findings demonstrate that FMNT is capable of protecting neurons from NMDA-evoked excitotoxic injury and has a potential perspective to the clinical treatment for neurodegenerative disorders in central nervous system.

Gentiopicroside attenuates morphine rewarding effect through downregulation of GluN2B receptors in nucleus accumbens.

AIMS: Gentiopicroside (Gent) is one of the secoiridoid compound isolated from Gentiana lutea. This compound exhibits analgesic activities and inhibits the expression of GluN2B-containing N-methyl-D-aspartate (NMDA) receptors in the anterior cingulate cortex in mice. Nucleus accumbens (NAc) is a forebrain structure known for its role in drug addiction. However, little is known about the role of Gent on morphine dependence and synaptic transmission changes in the NAc. METHODS: Conditioned place preference (CPP) test and behavioral sensitization of locomotor activity were used to investigate drug-seeking related behaviors. Brain slices containing NAc were prepared, and whole-cell patch-clamp recordings were performed to record the excitatory postsynaptic currents (EPSCs). Expression of proteins was detected by Western blot analysis. RESULTS: Systemic administration of Gent attenuated the CPP effect induced by morphine, but had no effect on morphine-induced behavioral sensitization. Gent significantly reversed overexpression of GluN2B-containing NMDA receptors and dopamine D2 receptors in NAc during the first week of morphine withdrawal. However, the compound did not affect the overexpression of GluN2A-containing NMDA receptors, GluA1, and dopamine D1 receptors. Lastly, Gent significantly reduced NMDA receptors-mediated EPSCs in the NAc. CONCLUSION: Our study provides strong evidence that Gent inhibits morphine dependence through downregulation of GluN2B-containing NMDA receptors in the NAc.

Application of locking plate in long-bone atrophic nonunion following external fixation.

The treatment of atrophic fracture nonunion continues to represent a therapeutic challenge. Large segmental osteopenia is often seen in patients who received uniplanar or hybrid external fixators as the definitive method of fixation for high-energy fractures, and this adds more difficulties to the treatment of fracture nonunion. This retrospective study was designed to assess the outcome of locking compression plating with autologous bone grafting in patients with long-bone atrophic nonunion following external fixation.From January 2004 to December 2009, a series of consecutive patients with atrophic nonunion of the long bone following external fixation were treated with this method in our institution. The clinical outcomes and complications of these patients were retrospectively analyzed. Twenty-seven patients with 28 fracture nonunions were involved in this study. Mean follow-up was 14.2±3.4 months. Bony union was achieved in all 27 patients within a mean 18.6±4.8 weeks after revision surgery. Two patients developed superficial wound infections. No deep infections were found, and no implant failure was seen. Three patients reported minor pain in the donor site of the bone graft, and no other donor site complications were found.Revision osteosynthesis of long-bone atrophic nonunion following external fixation by locking compression plating with autologous iliac crest bone grafting represents a safe and efficacious modality for the treatment of these challenging conditions.

Clinical value of signal transducers and activators of transcription 3 (STAT3) gene expression in human osteosarcoma.

The dysregulation of signal transducers and activators of transcription 3 (STAT3) has been reported to be associated with tumor progression, angiogenesis and metastasis. The purpose of this study was to analyze the clinical value of STAT3 expression in human osteosarcoma. First, semi-quantitative RT-PCR was performed to detect the expression of STAT3 mRNA in normal bone tissues, chondroma tissues and osteosarcoma tissues. Then, immunohistochemistry was performed to detect the expression of STAT3 protein in 76 osteosarcoma tissues and the relationship of STAT3 protein expression with clinicopathologic factors or prognosis of osteosarcoma patients. RNA interference (RNAi) technology was employed to inhibit STAT3 expression. MTT and flow cytometric assays were performed to analyze the effect of STAT3 inhibition on proliferation and apoptosis of osteosarcoma cells. Finally, the expression of STAT3-related target genes were also determined. Results showed that osteosarcoma tissues showed significantly higher expression levels of STAT3 mRNA than normal bone or chondroma tissues (P<0.05). Immunohistochemistry showed that the staining of STAT3 protein was mainly located in cytoplasm of osteosarcoma cells in osteosarcoma tissue samples. The high level of STAT3 protein was associated with poor tumor differentiation and presentation of metastasis (P=0.039 and 0.022). Moreover, the 5-year overall and relapse-free survival rates for osteosarcoma patients with high STAT3 expression were lower than those for patients with low STAT3 expression. In addition, the status of STAT3 protein expression was an independent prognostic factor for both disease-free survival (P=0.0235) and overall survival (P=0.0032). RNAi-mediated STAT3 inhibition could induce proliferation inhibition and apoptosis enhancement in osteosarcoma cells, which might be associated with inhibition of some anti-apoptosis genes. Overall, STAT3 plays crucial roles in osteosarcoma development and might become a potential molecular target for gene therapy of human osteosarcomas.

Hydroxysafflor Yellow A protects spinal cords from ischemia/reperfusion injury in rabbits.

BACKGROUND: Hydroxysafflor Yellow A (HSYA), which is one of the most important active ingredients of the Chinese herb Carthamus tinctorius L, is widely used in the treatment of cerebrovascular and cardiovascular diseases. However, the potential protective effect of HSYA in spinal cord ischemia/reperfusion (I/R) injury is still unknown. METHODS: Thirty-nine rabbits were randomly divided into three groups: sham group, I/R group and HSYA group. All animals were sacrificed after neurological evaluation with modified Tarlov criteria at the 48th hour after reperfusion, and the spinal cord segments (L4-6) were harvested for histopathological examination, biochemical analysis and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. RESULTS: Neurological outcomes in HSYA group were slightly improved compared with those in I/R group. Histopathological analysis revealed that HSYA treatment attenuated I/R induced necrosis in spinal cords. Similarly, alleviated oxidative stress was indicated by decreased malondialdehyde (MDA) level and increased superoxide dismutase (SOD) activity after HSYA treatment. Moreover, as seen from TUNEL results, HSYA also protected neurons from I/R-induced apoptosis in rabbits. CONCLUSIONS: These findings suggest that HSYA may protect spinal cords from I/R injury by alleviating oxidative stress and reducing neuronal apoptosis in rabbits.

Generation and identification of monoclonal antibodies against FNIII domain D of human tenascin-C.

Tenascin-C (TN-C), a key component of extracellular matrix (ECM), is strongly expressed in fetal and cancer tissues. Large-molecular-weight variants of TN-C, including different combinations of its alternative spliced FNIII repeats, are specifically expressed in tissues under certain pathological conditions. Here we report the production of monoclonal antibodies (MAbs) against FNIII domain D (FNIII D) of human TN-C. Complementary DNA encoding the FNIII D region was generated by RT-PCR from human osteosarcoma (OS) cell line, and the recombinant FNIII D-GST fusion protein was expressed and purified. Two hybridoma cell lines secreting monoclonal antibodies (MAbs) against FNIII D were obtained by routine murine hybridoma technique. The MAbs were identified by indirect enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemistry (IHC). Both of them were applicable in Western blot and IHC. With our MAbs, we found TN-C was positive in OS and most of it was among the tumor stroma. To conclude, these MAbs to human FNIII D domain of TN-C may be useful for exploring OS pathogenesis and potential clinical application.