The development of pulmonary metastasis is the major cause of death in osteosarcoma, and its molecular basis is poorly understood. In this study, we show that beta4 integrin is highly expressed in human osteosarcoma cell lines and tumor samples. Furthermore, highly metastatic MNNG-HOS cells have increased levels of beta4 integrin. Suppression of beta4 integrin expression by shRNA and disruption of beta4 integrin function by transfection of dominant-negative beta4 integrin was sufficient to revert this highly metastatic phenotype in the MNNG-HOS model without significantly affecting primary tumor growth. These findings suggest a role for beta4 integrin expression in the metastatic phenotype in human osteosarcoma cells. In addition, we identified a previously uncharacterized interaction between beta4 integrin and ezrin, a membrane-cytoskeletal linker protein that is implicated in the metastatic behavior of osteosarcoma. The beta4 integrin-ezrin interaction appears to be critical for maintenance of beta4 integrin expression. These data begin to integrate ezrin and beta4 integrin expression into a model of action for the mechanism of osteosarcoma metastases.

Retinitis pigmentosa (RP) is an inherited blinding disease characterized by progressive loss of retinal photoreceptors. There are numerous rodent models of retinal degeneration, but most are poor platforms for interventions that will translate into clinical practice. The rabbit possesses a number of desirable qualities for a model of retinal disease including a large eye and an existing and substantial knowledge base in retinal circuitry, anatomy, and ophthalmology. We have analyzed degeneration, remodeling, and reprogramming in a rabbit model of retinal degeneration, expressing a rhodopsin proline 347 to leucine transgene in a TgP347L rabbit as a powerful model to study the pathophysiology and treatment of retinal degeneration. We show that disease progression in the TgP347L rabbit closely tracks human cone-sparing RP, including the cone-associated preservation of bipolar cell signaling and triggering of reprogramming. The relatively fast disease progression makes the TgP347L rabbit an excellent model for gene therapy, cell biological intervention, progenitor cell transplantation, surgical interventions, and bionic prosthetic studies.

The GABA(B) receptor is important for the function of auditory neurons. We used Western blotting and immunohistochemical methods to examine the level and localization of GABA(B)R2, a required subunit of a functional GABA(B) receptor, in the rat's central auditory system. Results revealed that this subunit was expressed throughout the auditory system with the level being high in the layers I-V of the auditory cortex, medial geniculate nucleus, dorsomedial and lateral parts of the inferior colliculus, and the molecular and fusiform cell layers of the dorsal cochlear nucleus. Labeled cell bodies were found in all the areas showing immunoreactivity. Neuropil labeling was strong in areas with high overall levels of immunoreactivity. Regional distributions of the receptor subunit revealed clear boundaries of some auditory subnuclei including the dorsal and ventral cochlear nuclei and the lateral superior olivary nucleus. Differences in immunoreactivity were found between the central nucleus and the dorsal cortex of the inferior colliculus and between the dorsal and ventral parts of the ventral nucleus of the lateral lemniscus, although no clear boundaries were observed. No differences in immunoreactivity were found between the core and the belt areas of the auditory cortex and among the subdivisions of the medial geniculate nucleus. The regional distribution of the receptor subunit in auditory structures is consistent with inputs to these structures and the cellular localization of the receptor in auditory neurons supports the contribution of the GABA(B) receptor to synaptic responses in these neurons.

The MYCN gene has a critical role in determining the clinical behavior of neuroblastoma. Although it is known that genomic amplification occurs in high-risk subsets, it remains unclear how MYCN expression is regulated in the pathogenesis of neuroblastomas. Here, we report that MYCN expression was regulated by the oncoprotein MDM2 at the post-transcriptional level and was associated with neuroblastoma cell growth. Increasing MDM2 by ectopic overexpression in the cytoplasm enhanced both mRNA and protein expression of MYCN. Mechanistic studies found that the C-terminal RING domain of the MDM2 protein bound to the MYCN mRNA's AREs within the 3'UTR and increased MYCN 3'UTR-mediated mRNA stability and translation. Conversely, MDM2 silencing by specific siRNA rendered the MYCN mRNA unstable and reduced the abundance of the MYCN protein in MYCN-amplified neuroblastoma cell lines. Importantly, this MDM2 silencing resulted in a remarkable inhibition of neuroblastoma cell growth and induction of cell death through a p53-independent pathway. Our results indicate that MDM2 has a p53-independent role in the regulation of both MYCN mRNA stabilization and its translation, suggesting that MDM2-mediated MYCN expression is one mechanism associated with growth of MYCN-associated neuroblastoma and disease progression.

Hemorrhagic transformation (HT) is a feared complication of cerebral ischemic infarction, especially following the use of thrombolytic therapy. In this study, we examined whether docosahexaenoic acid (DHA; 22:6n-3), an omega-3 essential fatty acid family member, can protect the brain from injury and whether DHA can decrease the risk of HT enhanced by hyperglycemia after focal ischemic injury. Male Sprague-Dawley rats were injected with 50% dextrose (6ml/kg intraperitoneally) to induce hyperglycemia 10min before 1.5h of filament middle cerebral artery occlusion (MCAO) was performed. Treatment with DHA (10mg/kg) 5min before reperfusion reduced HT and further improved the 7-day neurological outcome. It also reduced infarct volume, which is consistent with the restricted DWI and T2WI hyperintensive area. Reduced Evans Blue extravasation and increased expression of collagen IV indicated the improved integrity of the blood-brain barrier (BBB) in DHA-treated rats. Moreover, DHA reduced the expression of the intercellular adhesion molecule-1 (ICAM-1) in the ischemic injured brain. Therefore, we conclude that DHA attenuated hyperglycemia-enhanced HT and improved neurological function by preserving the integrity of BBB and reducing inflammation.

Chronic hepatitis B virus (HBV) infection is a major risk factor for developing hepatocellular carcinoma (HCC), and HBV X protein (HBx) acts as cofactor in hepatocarcinogenesis. In liver tumors from animals modeling HBx- and HBV-mediated hepatocarcinogenesis, downregulation of chromatin regulating proteins SUZ12 and ZNF198 induces expression of several genes, including epithelial cell adhesion molecule (EpCAM). EpCAM upregulation occurs in HBV-mediated HCCs and hepatic cancer stem cells, by a mechanism not understood. Herein we demonstrate HBx induces EpCAM expression via active DNA demethylation. In hepatocytes, EpCAM is silenced by polycomb repressive complex 2 (PRC2) and ZNF198/LSD1/Co-REST/HDAC1 chromatin-modifying complexes. Cells with stable knockdown of SUZ12, an essential PRC2 subunit, upon HBx expression demethylate a CpG dinucleotide located adjacent to NF-κB/RelA half-site. This NF-κB/RelA site is in a CpG island downstream from EpCAM transcriptional start site (TSS). Chromatin immunoprecipitation (ChIP) assays demonstrate HBx-dependent RelA occupancy of NF-κB half-site, whereas RelA knockdown suppresses CpG demethylation and EpCAM expression. Tumor necrosis factor-α activates RelA, propagating demethylation to nearby CpG sites, shown by sodium bisulfite sequencing. RelA-dependent demethylation occurring upon HBx expression requires methyltrasferase EZH2, TET2 a key factor in cytosine demethylation and inactive DNMT3L, shown by knockdown assays and sodium bisulfite sequencing. Co-immunoprecipitations and sequential ChIP assays demonstrate that RelA in the presence of HBx forms a complex with EZH2, TET2 and DNMT3L, although the role of DNMT3L remains to be understood. Interestingly, the human EpCAM gene also has a CpG island downstream from its TSS, and a NF-κB-binding site flanked by CpGs. HepG2 cells derived from human HCC exhibit demethylation of these NF-κB-flanking CpG sites, and HBV replication propagates demethylation to nearby CpG sites. DLK1, another PRC2 target gene, also upregulated in HBV-mediated HCCs, is demethylated in liver tumors at CpG dinucleotides flanking the NF-κB-binding sequence, supporting that this active DNA demethylation mechanism functions during oncogenic transformation.

To study the impact of adjuvant trastuzumab among patients achieving a pathologic complete response (pCR) after trastuzumab-based neoadjuvant systemic therapy (NST).

Tuberculosis (TB) vaccine development has focused largely on targeting T helper type 1 (Th1) cells. However, despite inducing Th1 cells, the recombinant TB vaccine MVA85A failed to enhance protection against TB disease in humans. In recent years, Th17 cells have emerged as key players in vaccine-induced protection against TB. However, the exact cytokine and immune requirements that enable Th17-induced recall protection remain unclear. In this study, we have investigated the requirements for Th17 cell-induced recall protection against Mycobacterium tuberculosis (Mtb) challenge by utilizing a tractable adoptive transfer model in mice. We demonstrate that adoptive transfer of Mtb-specific Th17 cells into naive hosts, and upon Mtb challenge, results in Th17 recall responses that confer protection at levels similar to vaccination strategies. Importantly, although interleukin (IL)-23 is critical, IL-12 and IL-21 are dispensable for protective Th17 recall responses. Unexpectedly, we demonstrate that interferon-γ (IFN-γ) produced by adoptively transferred Th17 cells impairs long-lasting protective recall immunity against Mtb challenge. In contrast, CXCR5 expression is crucial for localization of Th17 cells near macrophages within well-formed B-cell follicles to mediate Mtb control. Thus, our data identify new immune characteristics that can be harnessed to improve Th17 recall responses for enhancing vaccine design against TB.

The purpose of this study was to investigate functional alterations of the brain in the early stage of spinal cord injury (SCI) and further investigate how these functional alterations relate to SCI patients' sensorimotor functions.

The aim of the meta-analysis was to provide more solid evidence for the reliability of the new classification. A systematic literature search was performed using PubMed, Armed Forces Pest Management Board Literature Retrieval System, and Google Scholar up to August 2012. A pooled odds ratio (OR) was calculated using either a random-effect or a fixed-effect model. A total of 16 papers were identified. Among the 11 factors studied, five symptoms demonstrated an increased risk for SDD, including bleeding [OR: 13.617; 95% confidence interval (CI): 3.281, 56.508], vomiting/nausea (OR: 1.692; 95% CI: 1.256, 2.280), abdominal pain (OR: 2.278; 95% CI: 1.631, 3.182), skin rashes (OR: 2.031; 95% CI: 1.269, 3.250), and hepatomegaly (OR: 4.751; 95% CI: 1.769, 12.570). Among the four bleeding-related symptoms including hematemesis, melena, gum bleeding, and epistaxis, only hematemesis (OR: 6.174; 95% CI: 2.66, 14.334; P < 0.001) and melena (OR: 10.351; 95% CI: 3.065, 34.956; P < 0.001) were significantly associated with SDD. No significant associations with SDD were found for gender, lethargy, retroorbital pain, diarrhea, or tourniquet test, whereas headache appeared protective (OR: 0.555; 95% CI: 0.455, 0.676). The meta-analysis suggests that bleeding (hematemesis/melena), vomiting/nausea, abdominal pain, skin rashes, and hepatomegaly may predict the development of SDD in patients with DF, while headache may predict otherwise.

Apoptosis has essential roles in a variety of cellular and developmental processes. Although the pathway is well studied, how the activities of individual components in the pathway are regulated is less understood. In Drosophila, a key component in apoptosis is Drosophila inhibitor of apoptosis protein 1 (DIAP1), which is required to prevent caspase activation. Here, we demonstrate that Drosophila CG42593 (ubr3), encoding the homolog of mammalian UBR3, has an essential role in regulating the apoptosis pathway. We show that loss of ubr3 activity causes caspase-dependent apoptosis in Drosophila eye and wing discs. Our genetic epistasis analyses show that the apoptosis induced by loss of ubr3 can be suppressed by loss of initiator caspase Drosophila Nedd2-like caspase (Dronc), or by ectopic expression of the apoptosis inhibitor p35, but cannot be rescued by overexpression of DIAP1. Importantly, we show that the activity of Ubr3 in the apoptosis pathway is not dependent on its Ring-domain, which is required for its E3 ligase activity. Furthermore, we find that through the UBR-box domain, Ubr3 physically interacts with the neo-epitope of DIAP1 that is exposed after caspase-mediated cleavage. This interaction promotes the recruitment and ubiquitination of substrate caspases by DIAP1. Together, our data indicate that Ubr3 interacts with DIAP1 and positively regulates DIAP1 activity, possibly by maintaining its active conformation in the apoptosis pathway.

Podocyte apoptosis is a major mechanism that leads to proteinuria in many chronic kidney diseases. However, the concert mechanisms that cause podocyte apoptosis in these kidney diseases are not fully understood. The Rho family of small GTPases has been shown to be required in maintaining podocyte structure and function. Recent studies have indicated that podocyte-specific deletion of Cdc42 in vivo, but not of RhoA or Rac1, leads to congenital nephrotic syndrome and glomerulosclerosis. However, the underlying cellular events in podocyte controlled by Cdc42 remain unclear. Here, we assessed the cellular mechanisms by which Cdc42 regulates podocyte apoptosis. We found that the expression of Cdc42 and its activity were significantly decreased in high glucose-, lipopolysaccharide- or adriamycin-injured podocytes. Reduced Cdc42 expression in vitro and in vivo by small interfering RNA and selective Cdc42 inhibitor ML-141, respectively, caused podocyte apoptosis and proteinuria. Our results further demonstrated that insufficient Cdc42 or Nwasp, its downstream effector, could decrease the mRNA and protein expression of YAP, which had been regarded as an anti-apoptosis protein in podocyte. Moreover, our data indicated that the loss of stress fibers caused by Cdc42/Nwasp deficiency also decreased Yes-associated protein (YAP) mRNA and protein expression, and induced podocyte apoptosis. Podocyte apoptosis induced by Cdc42/Nwasp/stress fiber deficiency was significantly inhibited by overexpressing-active YAP. Thus, the Cdc42/Nwasp/stress fibers/YAP signal pathway may potentially play an important role in regulating podocyte apoptosis. Maintaining necessary Cdc42 would be one potent way to prevent proteinuria kidney diseases.

Mitochondrial respiratory dysfunction has intimate relationship with redox regulation. The key mechanism about how the mitochondrial respiration-defective cells survive oxidative stress is still elusive. Here, we report that transcription factor zinc-finger protein 143 (ZNF143) expression and glutathione peroxidase (GPX) activity are markedly increased in the mitochondrial respiratory-defective cells induced by dominant-negative DNA polymerase γ (POLGdn). In this work, investigation of the cellular antioxidant glutathione (GSH) and enzyme GPX activity in the mitochondrial dysfunction revealed the presence of an increased synthesis of GSH through the activation of GCLC (glutamate-cysteine ligase catalytic subunit) and GCLM (glutamate-cysteine ligase regulatory subunit) gene expression, and also a positive upregulation of glutathione peroxidase 1 (GPX1) activity by the transcription factor ZNF143. Significant increase in gene expression of SepSecS, the key enzyme responsible for selenocysteine transfer RNA (tRNA) synthesis, further confirmed the activation of the selenocysteine synthesis pathway. By using both GPX1 and ZNF143 knockdown, we provided insight into the involvement of ZNF143 in promoting GPX1 activity and protecting cells from oxidative damage and cisplatin treatment in the mitochondrial dysfunction. Furthermore, we reported the possible regulation of mitochondrial transcription factor A (TFAM) in the mitochondrial dysfunction. Our findings delineate an important antioxidant survival pathway that allows the mitochondrial-defective cells to survive oxidative stress and cisplatin treatment.

CD44, a transmembrane glycoprotein expressed in a variety of cells and tissues, has been implicated in tumour metastasis. But the molecular mechanisms of CD44-mediated tumour cell metastasis remain to be elucidated.

Killing cancer cells through the induction of apoptosis is one of the main mechanisms of chemotherapy. However, numerous cancer cells have primary or acquired apoptosis resistance, resulting in chemoresistance. In this study, using a novel chalcone derivative chalcone-24 (Chal-24), we identified a novel anticancer mechanism through autophagy-mediated necroptosis (RIP1- and RIP3-dependent necrosis). Chal-24 potently killed different cancer cells with induction of necrotic cellular morphology while causing no detectable caspase activation. Blocking the necroptosis pathway with either necrostatin-1 or by knockdown of RIP1 and RIP3 effectively blocked the cytotoxicity of Chal-24, suggesting that Chal-24-induced cell death is associated with necroptosis. Chal-24 robustly activated JNK and ERK and blockage of which effectively suppressed Chal-24-induced cytotoxicity. In addition, Chal-24 strongly induced autophagy that is dependent on JNK-mediated phosphorylation of Bcl-2 and Bcl-xL and dissociation of Bcl-2 or Bcl-xL from Beclin-1. Importantly, suppression of autophagy, with either pharmacological inhibitors or small interfering RNAs targeting the essential autophagy components ATG7 and Beclin-1, effectively attenuated Chal-24-induced cell death. Furthermore, we found that autophagy activation resulted in c-IAP1 and c-IAP2 degradation and formation of the Ripoptosome that contributes to necroptosis. These results thus establish a novel mechanism for killing cancer cells that involves autophagy-mediated necroptosis, which may be employed for overcoming chemoresistance.

Fibroblast growth factor receptor (FGFR) signalling has been implicated in pancreas carcinogenesis. We investigated the effect of FGFR inhibition in pancreatic cancer in complementary cancer models derived from cell lines and patient-derived primary tumour explants.

Natural killer (NK) cells are important in host to eliminate circulating tumour cells (CTCs) in turn preventing the development of tumour cells into metastasis but the mechanisms are very poorly defined. Here we find that the expression level of miR-296-3p is much lower in the non-metastatic human prostate cancer (PCa) cell line P69 than that in the highly metastatic cell line M12, which is derived from P69. We demonstrate that miR-296-3p directly targets and inhibits the expression of intercellular adhesion molecule 1 (ICAM-1) in the malignant M12. The data from clinical tissue microarrays also show that miR-296-3p is frequently upregulated and ICAM-1 is reversely downregulated in PCa. Interestingly, ectopic expression of miR-296-3p in P69 increases the tolerance to NK cells whereas knockdown of miR-296-3p in M12 reduces the resistance to NK cells, which both phenotypes can be rescued by re-expression or silencing of ICAM-1 in P69 and M12, respectively. These results are also manifested in vivo by the decrease in the incidence of pulmonary tumour metastasis exhibited by knockdown of miR-296-3p in M12 when injected into athymic nude mice via tail vein, and consistently down-expression of ICAM-1 reverses this to increase extravasation of CTCs into lungs. Above results suggest that this newly identified miR-296-3p-ICAM-1 axis has a pivotal role in mediating PCa metastasis by possible enhancing survival of NK cell-resistant CTC. Our findings provide novel potential targets for PCa therapy and prognosis.

The Chinese giant salamander belongs to an old lineage of salamanders and endangered species. Many studies of breeding and disease regarding this amphibian had been implemented. However, the studies on the ultrastructure of this amphibian are rare. In this work, we provide a histological and ultrastructural investigation on posterior esophagus of Chinese giant salamander. The sections of amphibian esophagus were stained by hematoxylin & eosin (H&E). Moreover, the esophageal epithelium was observed by transmission electron microscopy (TEM). The results showed that esophageal epithelium was a single layer epithelium, which consisted of mucous cells and columnar cells. The esophageal glands were present in submucosa. The columnar cells were ciliated. According to the diverging ultrastructure of mucous vesicles, three types of mucous cells could be identified in the esophageal mucosa: i) electron-lucent vesicles mucous cell (ELV-MC); ii) electron-dense vesicles mucous cell (EDV-MC); and iii) mixed vesicles mucous cell (MV-MC).

The paper presents studies of Bose-Einstein Correlations (BEC) for pairs of like-sign charged particles measured in the kinematic range [Formula: see text] 100 MeV and [Formula: see text] 2.5 in proton collisions at centre-of-mass energies of 0.9 and 7 TeV with the ATLAS detector at the CERN Large Hadron Collider. The integrated luminosities are approximately 7 [Formula: see text]b[Formula: see text], 190 [Formula: see text]b[Formula: see text] and 12.4 nb[Formula: see text] for 0.9 TeV, 7 TeV minimum-bias and 7 TeV high-multiplicity data samples, respectively. The multiplicity dependence of the BEC parameters characterizing the correlation strength and the correlation source size are investigated for charged-particle multiplicities of up to 240. A saturation effect in the multiplicity dependence of the correlation source size parameter is observed using the high-multiplicity 7 TeV data sample. The dependence of the BEC parameters on the average transverse momentum of the particle pair is also investigated.

The biological role of monocytes and macrophages in B-cell non-Hodgkin lymphoma (NHL) is not fully understood. We have previously reported that monocytes from patients with B-cell NHL have an immunosuppressive CD14(+)HLA-DR(low/-) phenotype that correlates with a poor prognosis. However, the underlying mechanism by which CD14(+)HLA-DR(low/-) monocytes develop in lymphoma is unknown. In the present study, we found that interleukin (IL)-10, which is increased in the serum of patients with B-cell NHL, induced the development of the CD4(+)HLA-DR(low/-) population. Using peripheral blood samples from patients with B-cell NHL, we found that absolute numbers of CD14(+) monocytic cells with an HLA-DR(low/-) phenotype were higher than healthy controls and correlated with a higher International Prognostic Index score. IL-10 serum levels were elevated in lymphoma patients compared with controls and were associated with increased peripheral monocyte counts. Treatment of monocytes with IL-10 in vitro significantly decreased HLA-DR expression and resulted in the expansion of CD14(+)HLA-DR(low/-) population. We found that lymphoma B cells produce IL-10 and supernatants from cultured lymphoma cells increased the CD14(+)HLA-DR(low/-) population. Furthermore, we found that IL-10-induced CD14(+)HLA-DR(low/-) monocytes inhibited the activation and proliferation of T cells. Taken together, these results suggest that elevated IL-10 serum levels contribute to increased numbers of immunosuppressive CD14(+)HLA-DR(low/-) monocytes in B-cell NHL.