Dissecting direct and indirect genetic effects on chronic obstructive pulmonary disease (COPD) susceptibility

Cigarette smoking is the major environmental risk factor for chronic obstructive pulmonary disease (COPD). Genome-wide association studies have provided compelling associations for three loci with COPD. In this study, we aimed to estimate direct, i.e., independent from smoking, and indirect effects of those loci on COPD development using mediation analysis. We included a total of 3,424 COPD cases and 1,872 unaffected controls with data on two smoking-related phenotypes: lifetime average smoking intensity and cumulative exposure to tobacco smoke (pack years). Our analysis revealed that effects of two linked variants (rs1051730 and rs8034191) in the AGPHD1/CHRNA3 cluster on COPD development are significantly, yet not entirely, mediated by the smoking-related phenotypes. Approximately 30 % of the total effect of variants in the AGPHD1/CHRNA3 cluster on COPD development was mediated by pack years. Simultaneous analysis of modestly (r ² = 0.21) linked markers in CHRNA3 and IREB2 revealed that an even larger (~42 %) proportion of the total effect of the CHRNA3 locus on COPD was mediated by pack years after adjustment for an IREB2 single nucleotide polymorphism. This study confirms the existence of direct effects of the AGPHD1/CHRNA3, IREB2, FAM13A and HHIP loci on COPD development. While the association of the AGPHD1/CHRNA3 locus with COPD is significantly mediated by smoking-related phenotypes, IREB2 appears to affect COPD independently of smoking.     

The Expression Pattern of Classical MHC Class I Molecules in the Development of Mouse Central Nervous System

Classical major histocompatibility complex (MHC) class I, first identified in the immune system, is also expressed in the developing and adult central nervous system (CNS). Although the MHC class I molecules have been found to be expressed in the CNS of different species, a necessary step to elucidate the temporal and spatial expression patterns of MHC class I molecules in the brain development has never been taken. Frozen sections were made from the brains of embryonic and postnatal C57BL/6 J mice, and the expression of H-2D^b mRNA was examined by in situ hybridization. Immunofluorescence was also performed to define the cell types that express H2-D^b in P15 mice. At E10.5, the earliest stage we examined, H2-D^b was expressed in neuroepithelium of the brain vesicles. From E12.5 to P0, H2-D^b expression was mainly located at cerebral cortex, neuroepithelium of the lateral ventricle, neuroepithelium of aquaeductus and developing cerebellum. From P4 to adult, H2-D^b mRNA was detected at olfactory bulb, hippocampus, cerebellum and some nerve nuclei. The major cell types expressing H-2D^b in P15 hippocampus, cerebral cortex and olfactory bulb were neuron. H2-K^b signal paralleled that of H2-D^b and the expression levels of the two molecules were comparable throughout the brain. The investigation of the expression pattern of H-2D^b at both embryonic and postnatal stages is important for further understanding the physiological and pathological roles of H2-D^b in the developing CNS.     

Recombinase-mediated cassette exchange (RMCE) — A rapidly-expanding toolbox for targeted genomic modifications

Starting in 1991, the advance of Tyr-recombinases Flp and Cre enabled superior strategies for the predictable insertion of transgenes into compatible target sites of mammalian cells. Early approaches suffered from the reversibility of integration routes and the fact that co-introduction of prokaryotic vector parts triggered uncontrolled heterochromatization. Shortcomings of this kind were overcome when Flp-Recombinase Mediated Cassette Exchange entered the field in 1994. RMCE enables enhanced tag-and-exchange strategies by precisely replacing a genomic target cassette by a compatible donor construct. After “gene swapping” the donor cassette is safely locked in, but can nevertheless be re-mobilized in case other compatible donor cassettes are provided (“serial RMCE”). These features considerably expand the options for systematic, stepwise genome modifications. The first decade was dominated by the systematic generation of cell lines for biotechnological purposes. Based on the reproducible expression capacity of the resulting strains, a comprehensive toolbox emerged to serve a multitude of purposes, which constitute the first part of this review. The concept per se did not, however, provide access to high-producer strains able to outcompete industrial multiple-copy cell lines. This fact gave rise to systematic improvements, among these certain accumulative site-specific integration pathways. The exceptional value of RMCE emerged after its entry into the stem cell field, where it started to contribute to the generation of induced pluripotent stem (iPS-) cells and their subsequent differentiation yielding a variety of cell types for diagnostic and therapeutic purposes. This topic firmly relies on the strategies developed in the first decade and can be seen as the major ambition of the present article. In this context an unanticipated, potent property of serial Flp-RMCE setups concerns the potential to re-open loci that have served to establish the iPS status before the site underwent the obligatory silencing process. Other relevant options relate to the introduction of composite Flp-recognition target sites (“heterospecific FRT-doublets”), into the LTRs of lentiviral vectors. These “twin sites” enhance the safety of iPS re-programming and -differentiation as they enable the subsequent quantitative excision of a transgene, leaving behind a single “FRT-twin”. Such a strategy combines the established expression potential of the common retro- and lentiviral systems with options to terminate the process at will. The remaining genomic tag serves to identify and characterize the insertion site with the goal to identify genomic “safe harbors” (GOIs) for re-use. This is enabled by the capacity of “FRT-twins” to accommodate any incoming RMCE-donor cassette with a compatible design.     

Precision mapping of the human O‐GalNAc glycoproteome through SimpleCell technology

Glycosylation is the most abundant and diverse posttranslational modification of proteins. While several types of glycosylation can be predicted by the protein sequence context, and substantial knowledge of these glycoproteomes is available, our knowledge of the GalNAc‐type O‐glycosylation is highly limited. This type of glycosylation is unique in being regulated by 20 polypeptide GalNAc‐transferases attaching the initiating GalNAc monosaccharides to Ser and Thr (and likely some Tyr) residues. We have developed a genetic engineering approach using human cell lines to simplify O‐glycosylation (SimpleCells) that enables proteome‐wide discovery of O‐glycan sites using ‘bottom‐up’ ETD‐based mass spectrometric analysis. We implemented this on 12 human cell lines from different organs, and present a first map of the human O‐glycoproteome with almost 3000 glycosites in over 600 O‐glycoproteins as well as an improved NetOGlyc4.0 model for prediction of O‐glycosylation. The finding of unique subsets of O‐glycoproteins in each cell line provides evidence that the O‐glycoproteome is differentially regulated and dynamic. The greatly expanded view of the O‐glycoproteome should facilitate the exploration of how site‐specific O‐glycosylation regulates protein function.     

Loss of P53 facilitates invasion and metastasis of prostate cancer cells

Prostate cancer is a lethal cancer for the invasion and metastasis in its earlier period. P53 is a tumor suppressor gene which plays a critical role on safeguarding the integrity of genome. However, loss of P53 facilitates or inhibits the invasion and metastasis of tumor is still suspended. In this study, we are going to explain whether loss of P53 affect the invasion and metastasis of prostate cancer cells. To explore whether loss of P53 influences the invasion and metastasis ability of prostate cancer cells, we first compared the invasion ability of si-P53 treated cells and control cells by wound healing, transwell assay, and adhesion assay. We next tested the activity of MMP-2, MMP-9, and MMP-14 by western blot and gelatin zymography. Moreover, we employed WB and IF to identify the EMT containing E-cad, N-cad, vimentin, etc. We also examined the expression of cortactin, cytoskeleton, and paxillin by immunofluorescence, and tested the expression of ERK and JNK by WB. Finally, we applied WB to detect the expression of FAK, Src, and the phosphorylation of them to elucidate the mechanism of si-P53 influencing invasion and metastasis. According to the inhibition rate of si-P53, we choose the optimized volume of si-P53. With the volume, we compare the invasion and metastasis ability of Du145 and si-P53 treated cells. We find si-P53 promotes the invasion and metastasis in prostate cancer cells, increases the expression and activity of MMP-2/9 and MMP-14. Also, si-P53 promotes EMT and cytoskeleton rearrangement. Further analyses explain that this effect is associated with FAK-Src signaling pathway. Loss of P53 promotes the invasion and metastasis ability of prostate cancer cells and the mechanism is correlated with FAK-Src signaling pathway. P53 is involved in the context of invasion and metastasis.     

Isolated Skull Cryptococcosis in an Immunocompetent Patient

A 62-year-old immunocompetent rural woman who represents an isolated cryptococcal skull infection without systematic involvement is described. Diagnosis was based on positive India ink staining, positive histopathologic examination, and positive culture. Species identification was performed by growth on Sabouraud dextrose agar and CHROMagar medium and by sequencing of the intergenic and internal transcribed spacer regions of the rRNA genes. This case describes a rare presentation of Cryptococcus neoformans infection in a human immunodeficiency virus-negative patient. The lesions were significantly improved with treatment of daily oral itraconazole 400 mg. A maintenance therapy with a low-dose itraconazole was prescribed to warrant a clinical and mycological eradication. A two-year follow-up did not show any recurrence of infection.     

ATM pathway is essential for ionizing radiation-induced autophagy

BackgroundATM plays an important role in response to DNA damage, while the roles of ATM in radiation-induced autophagy are still unclear in cervical cancer cells.MethodsHuman cervical cancer cells, Hela, were used, and cell models with ATM^?/? and MAPK14^?/? were established by gene engineering. Western blot was implemented to detect protein expression. MDC staining and GFP-LC3 relocalization were used to detect autophagy. CCK-8 was used to detect cell viability. Radiosensitivity was analyzed by colony formation assays. Co-immunoprecipitation was used to detect the interaction between different proteins, and apoptosis was detected by flow cytometry.ResultsAfter radiation autophagy was induced, illustrated by the increase of MAPLC3-II/MAPLC3-I ratio and decrease of p62, and phosphorylation of ATM simultaneously increased. ATM^?/? cells displayed hypersensitivity but had no influence on IR-induced apoptosis. Then inhibitor of ATM, KU55933, ATM and MAPK14 silencing were used, and autophagy was induced by IR more than 200% in control, and only by 35.72%, 53.18% and 24.76% in KU55933-treated cells, ATM^?/? and MAPK14^?/? cells, respectively. KU55933 inhibited IR-induced autophagy by activating mTOR pathways. ATM silencing decreased the expression of MAPK14 and mTOR signals significantly. Beclin's bond to PI3KIII and their interaction increased after IR, while in ATM^?/? and MAPK14^?/? cells this interaction decreased after IR. Both ATM and MAPK14 interacted with Beclin, while ATM^?/? and MAPK14^?/? cells showed no interaction.ConclusionsATM could promote IR-induced autophagy via the MAPK14 pathway, the mTOR pathway, and Beclin/PI3KIII complexes, which contributed to the effect of ATM on radiosensitivity.     

Design and Synthesis of Fluoroacylshikonin as an Anticancer Agent

A series of shikonin derivatives, selectively acylated by various fluorinated carboxylic acids at the side chain of shikonin, were synthesized and their anticancer activity evaluated, in which eight compounds are reported for the first time. Among all the compounds tested, compound S7 showed the most potent anticancer activity against B16‐F10 (malignant melanoma cells), MG63 (human osteosarcoma cells), and A549 (lung cancer cells) with IC₅₀ 0.39 ± 0.01, 0.72 ± 0.04 and 0.58 ± 0.02 µmol/L. Docking simulation of compound S7 was carried out to position S7 into a tubulin active site to determine the probable binding conformation. All the results suggested that compound S7 may be a potential anticancer agent. Chirality 25:757–762, 2013. © 2013 Wiley Periodicals, Inc.