Enhanced nuclear localization of YAP1-2 contributes to EGF-induced EMT in NSCLC.

YAP1, a key mediator of the Hippo pathway, plays an important role in tumorigenesis. Alternative splicing of human YAP1 mRNA results in two major isoforms: YAP1-1, which contains a single WW domain, and YAP1-2, which contains two WW domains, respectively. We here investigated the functions and the underlying regulatory mechanisms of the two YAP1 isoforms in the context of EGF-induced epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC). Human NSCLC cell lines express both YAP1-1 and YAP1-2 isoforms-although when compared to YAP1-1, YAP1-2 mRNA levels are higher while its protein expression levels are lower. EGF treatment significantly promoted YAP1 expression as well as EMT process in NSCLCs, whereas EGF-induced EMT phenotype was significantly alleviated upon YAP1 knockdown. Under normal culture condition, YAP1-1 stable expression cells exhibited a stronger migration ability than YAP1-2 expressing cells. However, upon EGF treatment, YAP1-2 stable cells showed more robust migration than YAP1-1 expressing cells. The protein stability and nuclear localization of YAP1-2 were preferentially enhanced with EGF treatment. Moreover, EGF-induced EMT and YAP1-2 activity were suppressed by inhibitor of AKT. Our results suggest that YAP1-2 is the main isoform that is functionally relevant in promoting EGF-induced EMT and ultimately NSCLC progression.

Ambroxol improves lysosomal biochemistry in glucocerebrosidase mutation-linked Parkinson disease cells.

Gaucher disease is caused by mutations in the glucocerebrosidase gene, which encodes the lysosomal hydrolase glucosylceramidase. Patients with Gaucher disease and heterozygous glucocerebrosidase mutation carriers are at increased risk of developing Parkinson's disease. Indeed, glucocerebrosidase mutations are the most frequent risk factor for Parkinson's disease in the general population. Therefore there is an urgent need to understand the mechanisms by which glucocerebrosidase mutations predispose to neurodegeneration to facilitate development of novel treatments. To study this we generated fibroblast lines from skin biopsies of five patients with Gaucher disease and six heterozygous glucocerebrosidase mutation carriers with and without Parkinson's disease. Glucosylceramidase protein and enzyme activity levels were assayed. Oxidative stress was assayed by single cell imaging of dihydroethidium. Glucosylceramidase enzyme activity was significantly reduced in fibroblasts from patients with Gaucher disease (median 5% of controls, P = 0.0001) and heterozygous mutation carriers with (median 59% of controls, P = 0.001) and without (56% of controls, P = 0.001) Parkinson's disease compared with controls. Glucosylceramidase protein levels, assessed by western blot, were significantly reduced in fibroblasts from Gaucher disease (median glucosylceramidase levels 42% of control, P < 0.001) and heterozygous mutation carriers with (median 59% of control, P < 0.001) and without (median 68% of control, P < 0.001) Parkinson's disease. Single cell imaging of dihydroethidium demonstrated increased production of cytosolic reactive oxygen species in fibroblasts from patients with Gaucher disease (dihydroethidium oxidation rate increased by a median of 62% compared to controls, P < 0.001) and heterozygous mutation carriers with (dihydroethidium oxidation rate increased by a median of 68% compared with controls, P < 0.001) and without (dihydroethidium oxidation rate increased by a median of 70% compared with controls, P < 0.001) Parkinson's disease. We hypothesized that treatment with the molecular chaperone ambroxol hydrochloride would improve these biochemical abnormalities. Treatment with ambroxol hydrochloride increased glucosylceramidase activity in fibroblasts from healthy controls, Gaucher disease and heterozygous glucocerebrosidase mutation carriers with and without Parkinson's disease. This was associated with a significant reduction in dihydroethidium oxidation rate of ∼50% (P < 0.05) in fibroblasts from controls, Gaucher disease and heterozygous mutation carriers with and without Parkinson's disease. In conclusion, glucocerebrosidase mutations are associated with reductions in glucosylceramidase activity and evidence of oxidative stress. Ambroxol treatment significantly increases glucosylceramidase activity and reduces markers of oxidative stress in cells bearing glucocerebrosidase mutations. We propose that ambroxol hydrochloride should be further investigated as a potential treatment for Parkinson's disease.

Protection against epithelial damage during Candida albicans infection is mediated by PI3K/Akt and mammalian target of rapamycin signaling.

BACKGROUND: The ability of epithelial cells (ECs) to discriminate between commensal and pathogenic microbes is essential for healthy living. Key to these interactions are mucosal epithelial responses to pathogen-induced damage. METHODS: Using reconstituted oral epithelium, we assessed epithelial gene transcriptional responses to Candida albicans infection by microarray. Signal pathway activation was monitored by Western blotting and transcription factor enzyme-linked immunosorbent assay, and the role of these pathways in C. albicans-induced damage protection was determined using chemical inhibitors. RESULTS: Transcript profiling demonstrated early upregulation of epithelial genes involved in immune responses. Many of these genes constituted components of signaling pathways, but only NF-κB, MAPK, and PI3K/Akt pathways were functionally activated. We demonstrate that PI3K/Akt signaling is independent of NF-κB and MAPK signaling and plays a key role in epithelial immune activation and damage protection via mammalian target of rapamycin (mTOR) activation. CONCLUSIONS: PI3K/Akt/mTOR signaling may play a critical role in protecting epithelial cells from damage during mucosal fungal infections independent of NF-κB or MAPK signaling.

Fine-mapping across diverse ancestries drives the discovery of putative causal variants underlying human complex traits and diseases.

Genome-wide association studies (GWAS) of human complex traits or diseases often implicate genetic loci that span hundreds or thousands of genetic variants, many of which have similar statistical significance. While statistical fine-mapping in individuals of European ancestries has made important discoveries, cross-population fine-mapping has the potential to improve power and resolution by capitalizing on the genomic diversity across ancestries. Here we present SuSiEx, an accurate and computationally efficient method for cross-population fine-mapping, which builds on the single-population fine-mapping framework, Sum of Single Effects (SuSiE). SuSiEx integrates data from an arbitrary number of ancestries, explicitly models population-specific allele frequencies and LD patterns, accounts for multiple causal variants in a genomic region, and can be applied to GWAS summary statistics. We comprehensively evaluated SuSiEx using simulations, a range of quantitative traits measured in both UK Biobank and Taiwan Biobank, and schizophrenia GWAS across East Asian and European ancestries. In all evaluations, SuSiEx fine-mapped more association signals, produced smaller credible sets and higher posterior inclusion probability (PIP) for putative causal variants, and captured population-specific causal variants.

Genetic architecture of the inflammatory bowel diseases across East Asian and European ancestries.

Inflammatory bowel diseases (IBDs) are chronic disorders of the gastrointestinal tract with the following two subtypes: Crohn's disease (CD) and ulcerative colitis (UC). To date, most IBD genetic associations were derived from individuals of European (EUR) ancestries. Here we report the largest IBD study of individuals of East Asian (EAS) ancestries, including 14,393 cases and 15,456 controls. We found 80 IBD loci in EAS alone and 320 when meta-analyzed with ~370,000 EUR individuals (~30,000 cases), among which 81 are new. EAS-enriched coding variants implicate many new IBD genes, including ADAP1 and GIT2. Although IBD genetic effects are generally consistent across ancestries, genetics underlying CD appears more ancestry dependent than UC, driven by allele frequency (NOD2) and effect (TNFSF15). We extended the IBD polygenic risk score (PRS) by incorporating both ancestries, greatly improving its accuracy and highlighting the importance of diversity for the equitable deployment of PRS.

Correction: Oral and Vaginal Epithelial Cell Lines Bind and Transfer Cell-Free Infectious HIV-1 to Permissive Cells but Are Not Productively Infected.

[This corrects the article DOI: 10.1371/journal.pone.0098077.].

Fgf10 Signaling-Based Evidence for the Existence of an Embryonic Stage Distinct From the Pseudoglandular Stage During Mouse Lung Development.

The existence during mouse lung development of an embryonic stage temporally and functionally distinct from the subsequent pseudoglandular stage has been proposed but never demonstrated; while studies in human embryonic lung tissue fail to recapitulate the molecular control of branching found in mice. Lung development in mice starts officially at embryonic day (E) 9.5 when on the ventral side of the primary foregut tube, both the trachea and the two primary lung buds emerge and elongate to form a completely separate structure from the foregut by E10. In the subsequent 6 days, the primary lung buds undergo an intense process of branching to form a ramified tree by E16.5. We used transgenic mice allowing to transiently inhibit endogenous fibroblast growth factor 10 (Fgf10) activity in mutant embryos at E9, E9.5, and E11 upon intraperitoneal exposure to doxycycline and examined the resulting lung phenotype at later developmental stages. We also determined using gene arrays the transcriptomic response of flow cytometry-isolated human alveolar epithelial progenitor cells derived from hESC or hiPSC, grown in vitro for 12 or 24 h, in the presence or absence of recombinant FGF10. Following injection at E9, the corresponding mutant lungs at E18.5 appear almost normal in size and shape but close up examination indicate failure of the right lung to undergo lobar septation. At E9.5, the lungs are slightly hypoplastic but display normal differentiation and functionality. However, at E11, the lungs show impaired branching and are no longer functional. Using gene array data, we report only a partial overlap between human and mouse in the genes previously shown to be regulated by Fgf10 at E12.5. This study supports the existence of an embryonic stage of lung development where Fgf10 signaling does not play a function in the branching process but rather in lobar septation. It also posits that functional comparisons between mouse and human organogenesis must account for these distinct stages.

Oral and vaginal epithelial cell lines bind and transfer cell-free infectious HIV-1 to permissive cells but are not productively infected.

The majority of HIV-1 infections worldwide are acquired via mucosal surfaces. However, unlike the vaginal mucosa, the issue of whether the oral mucosa can act as a portal of entry for HIV-1 infection remains controversial. To address potential differences with regard to the fate of HIV-1 after exposure to oral and vaginal epithelium, we utilized two epithelial cell lines representative of buccal (TR146) and pharyngeal (FaDu) sites of the oral cavity and compared them with a cell line derived from vaginal epithelium (A431) in order to determine (i) HIV-1 receptor gene and protein expression, (ii) whether HIV-1 genome integration into epithelial cells occurs, (iii) whether productive viral infection ensues, and (iv) whether infectious virus can be transferred to permissive cells. Using flow cytometry to measure captured virus by HIV-1 gp120 protein detection and western blot to detect HIV-1 p24 gag protein, we demonstrate that buccal, pharyngeal and vaginal epithelial cells capture CXCR4- and CCR5-utilising virus, probably via non-canonical receptors. Both oral and vaginal epithelial cells are able to transfer infectious virus to permissive cells either directly through cell-cell attachment or via transcytosis of HIV-1 across epithelial cells. However, HIV-1 integration, as measured by real-time PCR and presence of early gene mRNA transcripts and de novo protein production were not detected in either epithelial cell type. Importantly, both oral and vaginal epithelial cells were able to support integration and productive infection if HIV-1 entered via the endocytic pathway driven by VSV-G. Our data demonstrate that under normal conditions productive HIV-1 infection of epithelial cells leading to progeny virion production is unlikely, but that epithelial cells can act as mediators of systemic viral dissemination through attachment and transfer of HIV-1 to permissive cells.

Effects of 1-MCP on chlorophyll degradation pathway-associated genes expression and chloroplast ultrastructure during the peel yellowing of Chinese pear fruits in storage.

The peel yellowing is an important pigment physiological process of green fruit ripening, which mainly results from chlorophyll degradation in the fruit peel. In this work, two typical cultivars with different ripening speed, a slow ripening pear 'Emerald' (Pyrus bretschneideri Rehd. cv. Emerald) and a fast ripening 'Jingbai' (Pyrus ussuriensis Maxim. cv. Jingbai) were used to investigate the molecular mechanism of chlorophyll degradation in pear yellowing/ripening during postharvest storage. The fruits after harvest were treated with 1-methylcyclopropene (1-MCP), an ethylene action inhibitor at 1.0 µLl(-1) to determine its effect on chloroplast ultrastructure and the expression of chlorophyll degradation associated genes in peel tissues. Our results show that the pears treated with 1-MCP had a lower ethylene production rate and higher chlorophyll content compared to those of untreated fruit. The more intact chloroplasts with well-organised grana thylakoids and small plastoglobuli were maintained in the peel of 1-MCP treated fruit for up to 30 and 15 d in 'Emerald' and 'Jingbai', respectively. The expression of chlorophyll degradation associated genes: pheophorbide a oxygenase (PAO), non-yellow colouring (NYC), NYC1-like (NOL), stay-green 1(SGR1), was suppressed, while no significant change was found in chlorophyllase 1 (CHL1) and red chlorophyll catabolite reductase (RCCR) in both cultivar fruits treated with 1-MCP. These results suggest that 1-MCP can delay chlorophyll degradation by inhibiting ethylene production and suppressing the gene expression of PAO, NYC, NOL and SGR1, which are closely associated with chlorophyll catabolic pathway.

A biphasic innate immune MAPK response discriminates between the yeast and hyphal forms of Candida albicans in epithelial cells.

Discriminating between commensal and pathogenic states of opportunistic pathogens is critical for host mucosal defense and homeostasis. The opportunistic human fungal pathogen Candida albicans is also a constituent of the normal oral flora and grows either as yeasts or hyphae. We demonstrate that oral epithelial cells orchestrate an innate response to C. albicans via NF-κB and a biphasic MAPK response. Activation of NF-κB and the first MAPK phase, constituting c-Jun activation, is independent of morphology and due to fungal cell wall recognition. Activation of the second MAPK phase, constituting MKP1 and c-Fos activation, is dependent upon hypha formation and fungal burdens and correlates with proinflammatory responses. Such biphasic response may allow epithelial tissues to remain quiescent under low fungal burdens while responding specifically and strongly to damage-inducing hyphae when burdens increase. MAPK/MKP1/c-Fos activation may represent a "danger response" pathway that is critical for identifying and responding to the pathogenic switch of commensal microbes.