Multiwalled carbon nanotubes (MWCNTs) have the potential for widespread applications in engineering and materials science. However, because of their needle-like shape and high durability, concerns have been raised that MWCNTs may induce asbestos-like pathogenicity. Although recent studies have demonstrated that MWCNTs induce various types of reactivities, the physicochemical features of MWCNTs that determine their cytotoxicity and carcinogenicity in mesothelial cells remain unclear. Here, we showed that the deleterious effects of nonfunctionalized MWCNTs on human mesothelial cells were associated with their diameterdependent piercing of the cell membrane. Thin MWCNTs (diameter ∼ 50 nm) with high crystallinity showed mesothelial cell membrane piercing and cytotoxicity in vitro and subsequent inflammogenicity and mesotheliomagenicity in vivo. In contrast, thick (diameter ∼ 150 nm) or tangled (diameter ∼ 2-20 nm) MWCNTs were less toxic, inflammogenic, and carcinogenic. Thin and thick MWCNTs similarly affected macrophages. Mesotheliomas induced by MWCNTs shared homozygous deletion of Cdkn2a/2b tumor suppressor genes, similar to mesotheliomas induced by asbestos. Thus, we propose that different degrees of direct mesothelial injury by thin and thick MWCNTs are responsible for the extent of inflammogenicity and carcinogenicity. This work suggests that control of the diameter of MWCNTs could reduce the potential hazard to human health. environmental health | inflammation | nanotoxicology

The U2AF heterodimer has been well studied for its role in defining functional 3′ splice sites in pre-mRNA splicing, but many fundamental questions still remain unaddressed regarding the function of U2AF in mammalian genomes. Through genome-wide analysis of U2AF-RNA interactions, we report that U2AF has the capacity to directly define ~88% of functional 3′ splice sites in the human genome, but numerous U2AF binding events also occur in intronic locations. Mechanistic dissection reveals that upstream intronic binding events interfere with the immediate downstream 3′ splice site associated either with the alternative exon, to cause exon skipping, or with the competing constitutive exon, to induce exon inclusion. We further demonstrate partial functional impairment with leukemia-associated mutations in U2AF35, but not U2AF65, in regulated splicing. These findings reveal the genomic function and regulatory mechanism of U2AF in both normal and disease states.

Human neural precursor cells grown in culture provide a source of tissue for drug screening, developmental studies and cell therapy. However, mechanisms underlying their growth and differentiation are poorly understood. We show that epidermal growth factor (EGF) responsive precursors derived from the developing human cortex undergo senescence after 30-40 population doublings. Leukemia inhibitory factor (LIF) increased overall expansion rates, prevented senescence and allowed the growth of a long-term self renewing neural stem cell (ltNSC ctx ) for up to 110 population doublings. We established basal gene expression in ltNSC ctx using Affymetrix oligonucleotide microarrays that delineated specific members of important growth factor and signaling families consistently expressed across three separate lines. Following LIF withdrawal, 200 genes showed significant decreases. Protein analysis confirmed LIF-regulated expression of glial fibrillary acidic protein, CD44, and major histocompatibility complex I. This study provides the first molecular profile of human ltNSC ctx cultures capable of long-term self renewal, and reveals specific sets of genes that are directly or indirectly regulated by LIF. Keywords: epidermal growth factor, leukemia inhibitory factor, microarray, neural stem cells, neurosphere. Multi-potent precursor cells derived from the developing or adult brain can be isolated in culture and expanded in the presence of mitogens (for review, see McKay 1997;Gage 2000). In some of these techniques, the cells are grown in free-floating aggregates termed 'neurospheres' (Reynolds et al. 1992). Neurospheres have also been generated from human post-mortem fetal tissues using a variety of growth factors and culturing methods (Svendsen et al. 1996;Chalmers-Redman et al. 1997;Svendsen et al. 1998;Carpenter et al. 1999;Vescovi et al. 1999). Neurospheres have great potential for possible cell therapy applications and as a model for mechanisms of human development and disease. For example, cells derived from neurospheres can migrate and integrate after transplantation into the developing or adult brain (Svendsen et al. 1996;Svendsen et al. 1997a;Flax et al. 1998;Fricker et al. 1999;Rosser et al. 2000;Qu et al. 2001;Ostenfeld et al. 2002b;Wu et al. 2002). We have recently shown that neurospheres isolated from Down syndrome fetal tissue have specific genetic deficits and undergo reduced neurogenesis at later passages (Bahn et al. 2002). However, these intriguing findings underscore the fact that very little is known about the temporal pattern of growth in these cultures or the exact cellular composition of neurospheres.Where clonal analysis has been performed, a number of cells within rodent neurospheres have been shown to self Abbreviations used: BDNF, brain-derived neurtrophic factor; BMP, bone morphogenetic protein; BrdU, 5-bromo-2¢-deoxyuridine; CNTF, ciliary neurotrophic factor; DMEM, Dulbecco's modified Eagle's medium; EFN, ephrin; EGF, epidermal growth factor; EST, expressed sequence tag; FGF-2, basic fibr...

Aberrant expression of microRNAs (miRNAs) has been associated with clinical outcome in patients with chronic lymphocytic leukemia (CLL). To identify a powerful and easily assessable miRNA bio-marker of prognosis and survival, we performed quantitative reverse-transcription polymerase chain reaction (qRT-PCR) profiling in 104 CLL patients with a welldefined chromosome 17p status, and we validated our findings with miRNA microarray data from an independent cohort of 80 patients. We found that miR-15a, miR-21, miR-34a, miR-155, and miR-181b were differentially expressed between CLLs with chromosome 17p deletion and CLLs with normal 17p and normal karyotype, and that miR-181b was downregulated in therapy-refractory cases. miR-21 expression levels were significantly higher in patients with poor prognosis and predicted overall survival (OS), and miR-181b expression levels significantly predicted treatment-free survival. We developed a 21FK score (miR-21 qRT-PCR, fluorescence in situ hybridization, Karyotype) to stratify patients according to OS and found that patients with a low score had a significantly longer OS time. When we evaluated the relative power of the 21FK score with the most used prognostic factors, the score was the most significant in both CLL cohorts. We conclude that the 21FK score represents a useful tool for distinguishing between good-prognosis and poor-prognosis CLL patients. (Blood. 2010;116(6):945-952)

Mutations in the ATP13A2 gene are associated with KuforRakeb syndrome (KRS) and are found also in patients with various other types of parkinsonism. ATP13A2 encodes a predicted lysosomal P5-type ATPase that plays important roles in regulating cation homeostasis. Disturbance of cation homeostasis in brains is indicated in Parkinson disease pathogenesis. In this study, we explored the biological function of ATP13A2 as well as the pathogenic mechanism of KRS pathogenic ATP13A2 mutants. The results revealed that wild-type ATP13A2, but not the KRS pathogenic ATP13A2 mutants, protected cells from Mn 2؉ -induced cell death in mammalian cell lines and primary rat neuronal cultures. In addition, wild-type ATP13A2 reduced intracellular manganese concentrations and prevented cytochrome c release from mitochondria compared with the pathogenic mutants. Furthermore, endogenous ATP13A2 was up-regulated upon Mn 2؉ treatment. Our results suggest that ATP13A2 plays important roles in protecting cells against manganese cytotoxicity via regulating intracellular manganese homeostasis. The study provides a potential mechanism of KRS and parkinsonism pathogenesis.Mutations in ATP13A2 were initially identified in patients with Kufor-Rakeb syndrome (KRS), 2 an atypical form of inherited parkinsonism. KRS is characterized by juvenile-onset autosomal recessive nigro-striatal-pallidal-pyramidal neurodegeneration with clinical features of Parkinson disease (PD) plus spasticity, supranuclear upgaze paresis, and dementia (1). Homozygous and heterozygous mutations in ATP13A2 are also found in patients with various parkinsonism, including juvenile parkinsonism, young-onset PD, early-onset PD, and familial PD (2-9). ATP13A2 encodes a predicted lysosomal P5-type cationtransporting ATPase with multiple transmembrane domains. It is highly expressed in the brain, especially in the substantia nigra, the region with characteristic dopaminergic neuronal loss in PD. Ypk9, a yeast ortholog of ATP13A2, was shown to function as a manganese transporter to protect cells from excess Mn 2ϩ exposure, whereas loss of Ypk9 increases the sensitivity of yeast to Mn 2ϩ toxicity (10, 11). Previous studies have suggested that cation disturbance is involved in pathogenesis of PD neurodegeneration. Increased levels of cations, including iron and aluminum, are found in the substantial nigra of the PD patient brain (12, 13). Chronic occupational exposure to copper and/or manganese is associated with higher incidence of PD in a case-control study (14). Furthermore, excess levels of Mn 2ϩ accumulation in brains associated with occupational exposure, psychostimulant drug abuse, and liver disease result in an atypical form of parkinsonism in human (15).PD and parkinsonism are believed to be consequences of interactions between both genetic and environmental components (16,17). In this study, we aimed to explore the connection between the KRS-associated ATP13A2 genetic defect and manganese-associated toxicity. Our results show that wild-type ATP13A2 (ATP13A2WT), but not KRS-as...

Several types of fibrous stone called asbestos have been an unexpected cause of human cancer in the history. This form of mineral is considered precious in that they are heat-, friction-, and acid-resistant, are obtained easily from mines, and can be modified to any form with many industrial merits. However, it became evident that the inspiration of asbestos causes a rare cancer called malignant mesothelioma. Because of the long incubation period, the peak year for malignant mesothelioma is expected to be 2025 in Japan. Thus, it is necessary to elucidate the mechanisms of asbestos-induced mesothelial carcinogenesis. In this review, we summarize the cutting edge results of our 5-year project funded by a MEXT grant, in which local iron deposition and the characteristics of mesothelial cells are the key issues.

Exposure to asbestos is a risk for malignant mesothelioma (MM) in humans. Among the commercially used types of asbestos (chrysotile, crocidolite, and amosite), the carcinogenicity of chrysotile is not fully appreciated. Here, we show that all three asbestos types similarly induced MM in the rat peritoneal cavity and that chrysotile caused the earliest mesothelioma development with a high fraction of sarcomatoid histology. The pathogenesis of chrysotile-induced mesothelial carcinogenesis was closely associated with iron overload: repeated administration of an iron chelator, nitrilotriacetic acid, which promotes the Fenton reaction, significantly reduced the period required for carcinogenesis; massive iron deposition was found in the peritoneal organs with high serum ferritin; and homozygous deletion of the CDKN2A/2B/ARF tumour suppressor genes, the most frequent genomic alteration in human MM and in iron-induced rodent carcinogenesis, was observed in 92.6% of the cases studied with array-based comparative genomic hybridization. The induced rat MM cells revealed high expression of mesoderm-specific transcription factors, Dlx5 and Hand1, and showed an iron regulatory profile of active iron uptake and utilization. These data indicate that chrysotile is a strong carcinogen when exposed to mesothelia, acting through the induction of local iron overload. Therefore, an intervention to remove local excess iron might be a strategy to prevent MM after asbestos exposure.

Tumor cells form immune escape and subsequently obtain unlimited proliferation ability due to the abnormal immune surveillance mediated by immune checkpoints. Among this class of immune checkpoints, PD-1/PD-L1 was recognized as an anticancer drug target for many years, and so far, several monoclonal antibodies have achieved encouraging outcome in cancer treatment by targeting the PD-1/PD-L1 signaling pathway. Due to the inherent limitations of antibodies, the development of small molecule inhibitors based on PD-1/PD-L1 signaling pathway is gradually reviving in decades. In this review, we summarized a number of small molecule inhibitors based on three different therapeutic approaches interfering PD-1/PD-L1 signaling pathway: (1) blocking direct interaction between PD-1 and PD-L1; (2) inhibiting transcription and translation of PD-L1; and (3) promoting degradation of PD-L1 protein. The development of these small molecule inhibitors opens a new avenue for tumor immunotherapy based on PD-1/PD-L1 signaling pathway.