LASP-1 is an actin-binding protein that regulates cytoskeletal dynamics and cell migration. LASP-1 was previously identified in a cDNA library from metastatic breast cancer samples. This protein has since been detected in multiple human cancers, including liver cancer, gastric cancer and pancreatic cancer. S100P is a small calcium-binding protein in the S100 protein family that regulates cellular, physiological and pathological processes in various cancers. However, the clinical significance of LASP-1 and S100P expression in gallbladder cancer (GBC) is not yet clear. In our study, we focused on the clinical significance, biological function and mechanism of LASP-1 in gallbladder cancer and detected LASP-1 and S100P overexpression in GBC tissues. The expression of LASP-1 was significantly correlated with poor prognosis in GBC patients (P < 0.05). Furthermore, down-regulation of LASP-1 expression resulted in the obvious inhibition of proliferation and migration and caused cell cycle arrest by down-regulating S100P via the PI3K/AKT pathway; in mice, tumor volume was significantly decreased. In conclusion, LASP-1 may act as an oncogene to regulate the expression of S100P to influence cellular functions in GBC. LASP-1 could serve as a genetic treatment target in GBC patients.

Novel analytic tools are needed to elucidate the molecular basis of leukemia-relevant gene mutations in the post-genome era. We generated isogenic leukemia cell clones in which the FLT3 gene was disrupted in a single allele using TALENs. Isogenic clones with mono-allelic disrupted FLT3 were compared to an isogenic wild-type control clone and parental leukemia cells for transcriptional expression, downstream FLT3 signaling and proliferation capacity. The global gene expression profiles of mutant K562 clones and corresponding wild-type controls were compared using RNA-seq. The transcriptional levels and the ligand-dependent autophosphorylation of FLT3 were decreased in the mutant clones. TALENs-mediated FLT3 haplo-insufficiency impaired cell proliferation and colony formation in vitro. These inhibitory effects were maintained in vivo, improving the survival of NOD/SCID mice transplanted with mutant K562 clones. Cluster analysis revealed that the gene expression pattern of isogenic clones was determined by the FLT3 mutant status rather than the deviation among individual isogenic clones. Differentially expressed genes between the mutant and wild-type clones revealed an activation of nonsense-mediated decay pathway in mutant K562 clones as well as an inhibited FLT3 signaling. Our data support that this genome-editing approach is a robust and generally applicable platform to explore the molecular bases of gene mutations.

During the past 40 years, more than 400 Sudden Unexplained Deaths (SUDs) have occurred in Yunnan, southwestern China. Epidemiological and toxicological analyses suggested that a newly discovered mushroom called Trogia venenata was the leading culprit for SUDs. At present, relatively little is known about the genetics and natural history of this mushroom. In this study, we analyzed the sequence variation at four DNA fragments among 232 fruiting bodies of T. venenata collected from seven locations. Our ITS sequence analyses confirmed that all the isolates belonged to the same species. The widespread presence of sequence heterozygosity within many strains at each of three protein-coding genes suggested that the fruiting bodies were diploid, dikaryotic or heterokaryotic. Within individual geographic populations, we found significant deviations of genotype frequencies from Hardy-Weinberg expectations, with the overall observed heterozygosity lower than that expected under random mating, consistent with prevalent inbreeding within local populations. The geographic populations were overall genetically differentiated. Interestingly, while a positive correlation was found between population genetic distance and geographic distance, there was little correlation between genetic distance and barium concentration difference for the geographic populations. Our results suggest frequent inbreeding, geographic structuring, and limited gene flow among geographic populations of T. venenata from southwestern China.

Earlier intervention after acute kidney injury would promote better outcomes. Our previous study found that Wnt proteins are promptly upregulated after ischemic kidney injury. Thus, we assessed whether Wnt4 could be an early and sensitive biomarker of tubular injury. We subjected mice to bilateral ischemia/reperfusion injury (IRI). Kidney and urinary Wnt4 expression showed an early increase at 3 hours and increased further at 24 hours post-IRI and was closely correlated with histopathological alterations. Serum creatinine slightly increased at 6 hours, indicating that it was less sensitive than Wnt4 expression. These data were further confirmed by clinical study. Both kidney and urinary Wnt4 expression were significantly increased in patients diagnosed with biopsy-proven minimal change disease (MCD) with tubular injury, all of whom nevertheless had normal estimated glomerular filtration rate (eGFR) and serum creatinine. The increased Wnt4 expression also strongly correlated with histopathological alterations in these MCD patients. In conclusion, this is the first demonstration that increases in both kidney and urinary Wnt4 expression can be detected more sensitively and earlier than serum creatinine after kidney injury. In particular, urinary Wnt4 could be a potential noninvasive biomarker for the early detection of tubular injury.

Astaxanthin is a carotenoid pigment that possesses potent antioxidative, anti-inflammatory, antitumor, and immunomodulatory activities. Previous studies have demonstrated that astaxanthin displays potential neuroprotective properties for the treatment of central nervous system diseases, such as ischemic brain injury and subarachnoid hemorrhage. This study explored whether astaxanthin is neuroprotective and ameliorates neurological deficits following traumatic brain injury (TBI).

Mannose-binding lectin (MBL), a pattern-recognition molecule in serum, recognizes specific hexose sugars rich in mannose and N-acetylglucosamine on bacterium, yeasts, viruses as well as apoptotic cells. It has been well-identified that MBL has antiviral effects via binding to seasonal influenza H1 and H3 subtype viruses. Influenza A (H7N9) virus, a novel reassortant virus to human population, possesses the surface hemagglutinin (HA) and neuraminidase (NA) genes from duck and wild-bird influenza viruses and internal genes from poultry H9N2 viruses. As of Dec 7th, 2014, a total of 467 human infections and 183 fatal cases have been identified. Here, recombinant human (rh) MBL was tested for its binding and effects on hemagglutination inhibition (HI) and NA activity inhibition (NAI) of avian H7N9, H9N2 and human H3N2 viruses. We discovered that rhMBL exhibited a strong binding to H7N9 virus as human H3N2 did at high virus titers. However, it performed a significantly weaker HI activity effect on H7N9 comparing to those of H3N2 and H9N2, even at a much higher concentration (3.67 ± 0.33 vs. 0.026 ± 0.001 and 0.083 ± 0.02 μg/mL, respectively). Similarly, minor NAI effect of rhMBL, even at up to 10 μg/mL, was found on H7N9 virus while it displayed significant effects on both H3N2 and H9N2 at a lowest concentration of 0.0807 ± 0.009 and 0.0625 μg/mL, respectively. The HI and NAI effects of rhMBL were calcium-dependent and mediated by lectin domain. Our findings suggest that MBL, the host innate molecule, has differential interference effects with human and avian influenza virus and limited antiviral effect against H7N9 virus.

The water-extractable (QWP) and the alkali-extractable (QAP) polysaccharides from quinoa (named QWP and QAP, respectively) and their four polysaccharide sub-fractions (QWP-1, QWP-2, QAP-1 and QAP-2), were isolated and purified by anion-exchange and gel filtration chromatography. QWP-1 and QWP-2 were composed of Rha, Ara, Gal and GalA. QAP-1 and QAP-2 were composed of Rha, Ara, Man, Gal and GalA. Antioxidant and immunoregulatory activities of the polysaccharides were evaluated. The results showed that QWP-1, QWP-2, QAP-1 and QAP-2 had significant antioxidant and immunoregulatory activities. The results suggest that QWP-1, QWP-2, QAP-1 and QAP-2 could be used as potential antioxidants and immunomodulators.

While animal models of controlled cortical impact often display short-term motor dysfunction after injury, histological examinations do not show severe cortical damage. Thus, this model requires further improvement. Mice were subjected to injury at three severities using a Pin-Point™-controlled cortical impact device to establish secondary brain injury mouse models. Twenty-four hours after injury, hematoxylin-eosin staining, Fluoro-Jade B histofluorescence, and immunohistochemistry were performed for brain slices. Compared to the uninjured side, we observed differences of histopathological findings, neuronal degeneration, and glial cell number in the CA2 and CA3 regions of the hippocampus on the injured side. The Morris water maze task and beam-walking test verified long-term (14-28 days) spatial learning/memory and motor balance. To conclude, the histopathological responses were positively correlated with the degree of damage, as were the long-term behavioral manifestations after controlled cortical impact. All animal procedures were approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University School of Medicine.

This study is aimed at investigating the effect of amifostine (AMI) on rat bone marrow stromal stem cells (BMSCs) exposed to 2 Gy radiation. The BMSCs were divided into four groups, namely, group A that received 0 Gy radiation, group B that received 0 Gy radiation and AMI, group C that received 2 Gy radiation, and group D that received 2 Gy radiation and AMI. The proliferation, apoptosis, and distribution of BMSCs in the cell cycle, along with their osteogenesis ability, adipogenesis ability, and ROS production, were subsequently examined. The levels of ALP, PPARγ, P53, and TNFα were determined by Western blotting. The results demonstrated that the proliferation of BMSCs and the levels of ALP in group C were much lower than those in group A. The production of ROS and levels of PPARγ, P53, and TNFα in the group that received 2 Gy radiation were much higher than those in group A. Furthermore, the production of ROS and the levels of PPARγ, P53, and TNFα were much lower in group D than in group C. Additionally, the levels of ALP and extent of cell proliferation were much higher in group D than in group C. The results demonstrated the potential of AMI in reducing the side effects of radiation in BMSCs and in treatment of bone diseases caused by radiation.

Background: Premature drug leakage and inefficient cellular uptake are stand out as considerable hurdles for low drug delivery efficiency in tumor chemotherapy. Thus, we established a novel drug delivery and transportation strategy mediated by biocompatible silk fibroin (SF)-coated nanoparticles to overcome these therapeutic hurdles. Methods: we first synthesised a TME-responsive biocompatible nanoplatform constructed of amorphous calcium carbonate (ACC) cores and SF shells for enhanced chemotherapy by concurrently inhibiting premature drug release, achieving lysosome-targeted explosion and locally sprayed DOX, and monitoring via PAI, which was verified both in vitro and in vivo. Results: The natural SF polymer first served as a "gatekeeper" to inhibit a drug from prematurely leaking into the circulation was demonstrated both in vitro and in vivo. Upon encountering TMEs and targeting to the acidic pH environments of lysosomes, the sensitive ACC nanoparticles were gradually degraded, eventually generating a large amount of Ca2+ and CO2, resulting in lysosomal collapse, thus preventing both the efflux of DOX from cancer cells and the protonation of DOX within the lysosome, releasing multiple hydrolytic enzyme to cytoplasm, exhibiting the optimal therapeutic dose and remarkable synergetic therapeutic performance. In particular, CO2 gas generated by the pH response of ACC nanocarriers demonstrated their imaging capability for PAI, providing the potential for quantifying and guiding drug release in targets. Conclusion: In this work, we constructed TME-responsive biocompatible NPs by coating DOX-preloaded ACC-DOX clusters with SF via a bioinspired mineralization method for efficient therapeutics. This functional lysosome-targeted preservation-strategy-based therapeutic system could provid novel insights into cancer chemotherapy.

QSOX1 (quiescin-sulfhydryl oxidase 1) is involved in various processes, including apoptosis and the development of breast diseases. Here, we investigated the effect of QSOX1 on the meat quality of Simmental cattle by analyzing the correlation between QSOX1 single nucleotide polymorphisms (SNPs), I2 204 C>T and I2 378 C>T, and certain meat quality traits. The effects of QSOX1 on triglyceride synthesis and cell apoptosis were further validated by gene silencing or overexpression in bovine fetal fibroblasts and mammary epithelial cells. The results showed that I2 204 C>T and I2 378 C>T had significant correlations with loin thickness, hind hoof weight, fat coverage, liver weight, heart weight, marbling and back fat thickness (P<0.05). QSOX1 overexpression also increased triglyceride production and suppressed apoptosis. In summary, QSOX1 is an important factor for meat quality, lipid metabolism, and cell apoptosis, indicating that QSOX1 could be used as a biomarker to assist in breeding cattle with superior meat.

Accumulating evidence supports the hypothesis that cancer stem cells (CSCs) are essential for cancer initiation, metastasis and drug resistance. However, the functional association of gastric CSC markers with stemness and epithelial-mesenchymal transition (EMT) signature genes is unclear.

Hemopexin (Hpx) binds heme with extraordinary affinity, and after haptoglobin may provide a second line of defense against the toxicity of extracellular hemoglobin (Hb). In this series of experiments, the hypothesis that Hpx protects neurons from Hb neurotoxicity was evaluated in murine primary cultures containing neurons and glial cells. Contrary to hypothesis, Hpx increased neuronal loss due to micromolar concentrations of Hb by 4- to 12-fold, as measured by LDH release assay; conversely, the neurotoxicity of hemin was completely prevented. The endogenous fluorescence of Hpx was quenched by Hb, consistent with transfer of Hb-bound heme to Hpx. This was associated with precipitation of globin chains, as detected by immunostaining and fluorescent Hb labeling. A portion of this precipitate attached firmly to cells and could not be removed by multiple washes. Concomitant treatment with haptoglobin (Hp) prevented globin precipitation and most of the increase in neuronal loss. Hpx weakly attenuated the increase in culture non-heme iron produced by Hb treatment, quantified by ferrozine assay. However, Hb-Hpx toxicity was iron-dependent, and was blocked by deferoxamine and ferrostatin-1. Up-regulation of cell ferritin expression, a primary cell defense against Hb toxicity, was not observed on western blots of culture lysates that had been concomitantly treated with Hpx. These results suggest that Hpx destabilizes Hb in the absence of haptoglobin, leading to globin precipitation and exacerbation of iron-dependent oxidative cell injury. Combined therapy with hemopexin plus haptoglobin may be preferable to hemopexin alone after CNS hemorrhage.

The development of the female gametophyte (FG) is one of the key processes of life cycle alteration between the haploid gametophyte and the diploid sporophytes in plants and it is required for successful seed development after fertilization. It is well demonstrated that free nuclear mitosis (FNM) of FG is crucial for the development of the ovule. However, studies of the molecular mechanism of ovule and FG development focused mainly on angiosperms, such as Arabidopsis thaliana and further investigation of gymnosperms remains to be completed. Here, Illumina sequencing of six transcriptomic libraries obtained from developing and abortive ovules at different stages during free nuclear mitosis of magagametophyte (FNMM) was used to acquire transcriptome data and gene expression profiles of Pinus tabulaeformis. Six cDNA libraries generated a total of 71.0 million high-quality clean reads that aligned with 63,449 unigenes and the comparison between developing and abortive ovules identified 7174 differentially expressed genes (DEGs). From the functional annotation results, DEGs involved in the cell cycle and phytohormone regulation were highlighted to reveal their biological importance in ovule development. Furthermore, validation of DEGs from the phytohormone signal transduction pathway was performed using quantitative real-time PCR analysis, revealing the dynamics of transcriptional networks and potential key components in the regulation of FG development in P. tabulaeformis were identified. These findings provide new insights into the regulatory mechanisms of ovule development in woody gymnosperms.

Escherichia coli is one of the most common causal pathogens of mastitis in milk-producing mammals. Toll-like receptor 4 (TLR4) is important for host recognition of this bacteria. Increased activation of TLR4 can markedly enhance the internalization of E. coli. In this study, the relationship between TLR4 and mitogen-activated protein kinase (MAPK) signaling pathways in mediating E. coli internalization was evaluated in sheep monocytes. Using a TLR4-overexpressing transgenic (Tg) sheep model, we explored the bacterial internalization mechanism in sheep. We found that monocytes of Tg sheep could phagocytize more bacteria and exhibited higher adhesive capacity. The specific inhibition of p38 MAPK or c-Jun N-terminal kinase (JNK) or extracellular signal-regulated kinases (ERKs) reduced TLR4-dependent internalization of bacteria into sheep monocytes. Furthermore, the inhibition of MAPK signaling down-regulated the adhesive capacity of monocytes and the expression of scavenger receptors and adhesion molecules. Taken together, the overexpression of TLR4 in transgenic sheep enhanced the internalization of E. coli via MAPK signaling.

Identifying prognostic factors for osteosarcoma (OS) aids in the selection of patients who require more aggressive management. CD133 has been found to be a prognostic factor of certain tumor types. However, the association between CD133 expression and the prognosis of OS remains unknown. In this study, we analyzed the association of CD133 expression in OS with clinical factors and overall survival, and further investigated its potential role in metastasis in vitro. We found CD133 expression in 65.7% (46/70) of OS samples using immunohistochemistry, and it was positively correlated with lung metastasis analyzed by Chi-square test (P=0.002) and shorter overall survival time using the Kaplan-Meier method compared by log-rank test (P=0.000). Multivariate analysis showed that CD133 expression was an independent prognostic factor of patients with OS. To test for direct participation of CD133, we separated CD133(+) and CD133(-) cells in the MG63 cell line using magnetic-activated cell sorting and found that CD133(+) cells were more active in migration by scratch wound-healing assay and invasion by Matrigel invasion assay compared with CD133(-) cells. Elevated mRNA expression of the stemness gene octamer-binding transcription factor 4 (Oct-4) and NANOG, and the metastasis-related receptor C-X-C chemokine receptor type 4 (CXCR4) were also found in CD133(+) cells by reverse transcription-polymerase chain reaction. Thus, expression of CD133 was correlated with lung metastasis and poor prognosis in OS patients. CD133(+) cells may be a type of cancer stem cell with high expression of self-renewal capacity and metastasis-related genes.

Hepatocellular carcinoma (HCC) can be derived from malignant transformed adult hepatic progenitor cells. However, the regulatory factors and molecular mechanisms underlying the process are not well defined. Our previous microRNA (miRNA) microarray analysis revealed a significant decrease of miR-200a level in F344 rat HCC side population (SP) fraction cells versus their normal counterparts. In the present study, we further investigated the effect of miR-200a on hepatic oval cell (HOC) phenotypes. We first confirmed downregulated miR-200a levels in rat hepatoma cells compared with WB-F344 cells. Next, by lentivirus-mediated loss-of-function studies, we showed that stable knockdown of miR-200a confers a mesenchymal phenotype to WB-F344 cells, including an elongated cell morphology, enhanced cell migration ability and expression of epithelial mesenchymal transition (EMT)-representative markers. Concomitantly, several cancer stem cell (CSC)-like traits appeared in these cells, which exhibit enhanced spheroid-forming capacity, express putative hepatic CSC markers and display superior resistance to chemotherapeutic drugs in vitro. Furthermore, bioinformatics analysis, luciferase assays and western blot analysis identified β-catenin (CTNNB1) as a direct and functional target of miR-200a. Knockdown of miR-200a partially activated Wnt/β-catenin signaling, and silencing of β-catenin functionally attenuated anti-miR-200a effects in vitro in WB-F344 cells. At length, in vivo xenograft assay demonstrated the acquisition of tumorigenicity of WB-F344 cells after miR-200a siliencing. Collectively, our findings indicate that miR-200a may function as an important regulatory factor in neoplastic transition of HOCs by targeting the β-catenin pathway.

Gallbladder cancer is the most common malignant tumor of the biliary tract, and this condition has a rather dismal prognosis, with an extremely low five-year survival rate. To improve the outcome of unresectable and recurrent gallbladder cancer, it is necessary to develop new effective treatments and drugs. The purpose of the present study was to evaluate the effects of cordycepin on human gallbladder cells and uncover the molecular mechanisms responsible for these effects. The Cell Counting Kit-8 (CCK-8) and colony formation assays revealed that cordycepin affected the viability and proliferation of human gallbladder cancer cells in a dose- and time-dependent manner. Flow cytometric analysis showed that cordycepin induced S phase arrest in human gallbladder cancer cell lines(NOZ and GBC-SD cells). Cordycepin-induced apoptosis was observed using an Annexin V/propidium iodide (PI) double-staining assay, and the mitochondrial membrane potential (ΔΨm) decreased in a dose-dependent manner. Additionally, western blot analysis revealed the upregulation of cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP and Bax and the downregulation of Bcl-2, cyclin A and Cdk-2 in cordycepin-treated cells. Moreover, cordycepin inhibited tumor growth in nude mice bearing NOZ tumors. Our results indicate that this drug may represent an effective treatment for gallbladder carcinoma.

Receptor for advanced glycation end products (RAGE) signaling is involved in a series of cell functions after spinal cord injury (SCI). Our study aimed to elucidate the effects of RAGE signaling on the neuronal recovery after SCI. In vivo, rats were subjected to SCI with or without anti-RAGE antibodies micro-injected into the lesion epicenter. We detected Nestin/RAGE, SOX-2/RAGE and Nestin/MAP-2 after SCI by Western blot or immunofluorescence (IF). We found that neural stem cells (NSCs) co-expressed with RAGE were significantly activated after SCI, while stem cell markers Nestin and SOX-2 were reduced by RAGE blockade. We found that RAGE inhibition reduced nestin-positive NSCs expressing MAP-2, a mature neuron marker. RAGE blockade does not improve neurobehavior Basso, Beattie and Bresnahan (BBB) scores; however, it damaged survival of ventral neurons via Nissl staining. Through in vitro study, we found that recombinant HMGB1 administration does not lead to increased cytokines of TNF-α and IL-1β, while anti-RAGE treatment reduced cytokines of TNF-α and IL-1β induced by LPS via ELISA. Meanwhile, HMGB1 increased MAP-2 expression, which was blocked after anti-RAGE treatment. Hence, HMGB1/RAGE does not exacerbate neuronal inflammation but plays a role in promoting NSCs differentiating into mature neurons in the pathological process of SCI.

The success of genome-wide association studies has paralleled the development of efficient genotyping technologies. We describe the development of a next-generation microarray based on the new highly-efficient Affymetrix Axiom genotyping technology that we are using to genotype individuals of European ancestry from the Kaiser Permanente Research Program on Genes, Environment and Health (RPGEH). The array contains 674,517 SNPs, and provides excellent genome-wide as well as gene-based and candidate-SNP coverage. Coverage was calculated using an approach based on imputation and cross validation. Preliminary results for the first 80,301 saliva-derived DNA samples from the RPGEH demonstrate very high quality genotypes, with sample success rates above 94% and over 98% of successful samples having SNP call rates exceeding 98%. At steady state, we have produced 462 million genotypes per week for each Axiom system. The new array provides a valuable addition to the repertoire of tools for large scale genome-wide association studies.