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1.
Artigo em Inglês | MEDLINE | ID: mdl-31475764

RESUMO

Branched selectivity in asymmetric allylic C-H alkylation has been favorably enabled by using 2-acylimidazoles as nucleophiles in the presence of chiral phosphoramidite-palladium catalysis. A wide range of terminal alkenes, including1,4-dienes and allylarenes, are nicely tolerated to provide chiral 2-acylimidazoles in moderate to high yields and with high levels of regio-, E/Z- and enantioselectivities. Mechanistic studies by density functional theory (DFT) calculations suggest a nucleophile coordination-enabled inner-sphere attack mode for the enantioselective carbon-carbon bond-forming event.

2.
Mol Med Rep ; 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31485651

RESUMO

Hyperglycemia promotes podocyte apoptosis and contributes to the pathogenesis of diabetic nephropathy (DN). However, the mechanisms of hyperglycemia­induced podocyte apoptosis remain unknown. Recent studies have implicated Src­associated substrate during mitosis of 68 kDa (Sam68) in various cellular processes including RNA metabolism, apoptosis, signal transduction. This study sought to examine the effect of Sam68 on high glucose (HG)­induced podocytes apoptosis, and the mechanism underlying this effect. Immortalized mouse podocytes were exposed to medium containing normal glucose, or HG and Sam68 siRNA, respectively. The expression of Sam68 in podocytes was determined by fluorescence quantitative PCR (qPCR), immunofluorescence and immunoblotting. The role of Sam68 in HG­induced podocyte apoptosis was further evaluated by inhibiting Sam68 expression by Sam68 siRNA and performing flow cytometry. The mRNA and protein expression of pro­apoptosis gene Bax and anti­apoptotic gene Bcl­2 were assessed by qRCR and immunoblotting. In the present study, it was first demonstrated that Sam68 was upregulated in a time and dose­dependent manner in in vitro HG­treated podocytes. Pretreatment with Sam68 siRNA markedly decreased nuclear Sam68 expression. Moreover, the effects of HG­induced apoptosis were also abrogated by Sam68 knockdown in cultured podocytes. Furthermore, HG increased Bax and decreased Bcl­2 protein expression in cultured podocytes, and this effect was blocked by Sam68 knockdown. The results of the present study revealed that Sam68 mediated HG­induced podocyte apoptosis, probably through the Bax/Bcl­2 signaling pathway, and thus may be a potential therapeutic target for DN.

3.
MBio ; 10(5)2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31481383

RESUMO

Eukaryotic cells share a basic scheme of internal organization featuring membrane-based organelles. The use of fluorescent proteins (FPs) greatly facilitated live-cell imaging of organelle dynamics and protein trafficking. One major limitation of this approach is that the fusion of an FP to a target protein can and often does compromise the function of the target protein and alter its subcellular localization. The optimization process to obtain a desirable fusion construct can be time-consuming or even unsuccessful. In this work, we set out to provide a validated set of FP-based markers for major organelles in the budding yeast (Saccharomyces cerevisiae). Out of over 160 plasmids constructed, we present a final set of 42 plasmids, the recommendations for which are backed up by meticulous evaluations. The tool set includes three colors (green, red, and blue) and covers the endoplasmic reticulum (ER), nucleus, Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, and lipid droplets. The fidelity of the markers was established by systematic cross-comparison and quantification. Functional assays were performed to examine the impact of marker expression on the secretory pathway, endocytic pathway, and metabolic activities of mitochondria and peroxisomes. Concomitantly, our work constitutes a reassessment of organelle identities in this model organism. Our data support the recognition that "late Golgi" and "early endosomes," two seemingly distinct terms, denote the same compartment in yeast. Conversely, all other organelles can be visually separated from each other at the resolution of conventional light microscopy, and quantification results justify their classification as distinct entities.IMPORTANCE Cells contain elaborate internal structures. For eukaryotic cells, like those in our bodies, the internal space is compartmentalized into membrane-bound organelles, each tasked with specialized functions. Oftentimes, one needs to visualize organelles to understand a complex cellular process. Here, we provide a validated set of fluorescent protein-based markers for major organelles in budding yeast. Yeast is a commonly used model when investigating basic mechanisms shared among eukaryotes. Fluorescent proteins are produced by cells themselves, avoiding the need for expensive chemical dyes. Through extensive cross-comparison, we make sure that each of our markers labels and only labels the intended organelle. We also carefully examined if the presence of our markers has any negative impact on the functionality of the cells and found none. Our work also helps answer a related question: are the structures we see really what we think they are?

4.
Sensors (Basel) ; 19(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370171

RESUMO

Recently, automated software vulnerability detection and exploitation in Internet of Things (IoT) has attracted more and more attention, due to IoT's fast adoption and high social impact. However, the task is challenging and the solutions are non-trivial: the existing methods have limited effectiveness at discovering vulnerabilities capable of compromising IoT systems. To address this, we propose an Automated Vulnerability Discovery and Exploitation framework with a Scheduling strategy, AutoDES that aims to improve the efficiency and effectiveness of vulnerability discovery and exploitation. In the vulnerability discovery stage, we use our Anti-Driller technique to mitigate the "path explosion" problem. This approach first generates a specific input proceeding from symbolic execution based on a Control Flow Graph (CFG). It then leverages a mutation-based fuzzer to find vulnerabilities while avoiding invalid mutations. In the vulnerability exploitation stage, we analyze the characteristics of vulnerabilities and then propose to generate exploits, via the use of several proposed attack techniques that can produce a shell based on the detected vulnerabilities. We also propose a genetic algorithm (GA)-based scheduling strategy (AutoS) that helps with assigning the computing resources dynamically and efficiently. The extensive experimental results on the RHG 2018 challenge dataset and the BCTF-RHG 2019 challenge dataset clearly demonstrate the effectiveness and efficiency of the proposed framework.

5.
Biotechnol Bioeng ; 2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31403179

RESUMO

Trichoderma spp. are main producers of peptide antibiotics known as peptaibols. While peptaibols have been shown to possess a range of biological activities, molecular understanding of the regulation of their production is largely unclear, which hampers the production improvement through genetic engineering. Here, we demonstrated that the orthologue of glucose sensors in the outstanding biocontrol fungus Trichoderma longibrachiatum SMF2, TlSTP1, participates in the regulation of peptaibols production. Deletion of Tlstp1 markedly impaired hyphal growth and conidiation, but significantly increased peptaibols yield by 5-fold for Trichokonins A and 2.6-fold for Trichokonins B. Quantitative real-time polymerase chain reaction analyses showed that the increased peptaibols production occurs at the transcriptional levels of the two nonribosomal peptide synthetase encoding genes, tlx1 and tlx2. Transcriptome analyses of the wild type and the Tlstp1 mutant strains indicated that TlSTP1 exerts a regulatory effect on a set of genes that are involved in a number of metabolic and cellular processes, including synthesis of several other secondary metabolites. These results suggest an important role of TlSTP1 in the regulation of vegetative growth and peptaibols production in T. longibrachiatum SMF2 and provide insights into construction of peptaibol-hyperproducing strains through genetic engineering.

6.
Artigo em Inglês | MEDLINE | ID: mdl-31461347

RESUMO

Tuberous sclerosis complex 2, or Tuberin, is a pivotal regulator of the mechanistic target of rapamycin (mTOR) signaling pathway that controls cell survival, proliferation, growth and migration. Loss of Tsc2 function manifests in organ specific consequences, the mechanisms of which remain incompletely understood. Recent single cell analysis of kidney identified Abcg2 expression in renal proximal tubules of adult mice as well as a novel cell population. The impact in adult kidney of Tsc2 knockdown in the ATP binding cassette G2 (Abcg2) expressing lineage has not been evaluated. We engineered an inducible system in which expression of truncated Tsc2, lacking exons 36-37 with an intact 3 prime region and Pkd1, is driven by Abcg2. Here we demonstrate that selective expression of Tsc2fl36-37in the Abcg2pos lineage drives recombination in proximal tubule epithelial and rare perivascular mesenchymal cells which results in progressive proximal tubule injury, impaired kidney function, the formation of cystic lesions, and fibrosis in adult mice. These data illustrate the critical importance of Tsc2 function in the Abcg2 expressing proximal tubule epithelium and mesenchyme during development of cystic lesions and remodeling of kidney parenchyma.

7.
Mol Biol Rep ; 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31440876

RESUMO

Many Litchi chinensis cv. Baitangying orchards are suffering from a serious fruit cracking problem, but few studies have improved our understanding of the mechanism or the molecular basis of cracking susceptibility in 'Baitangying'. We conducted metabolome and transcriptome analyses of three types of litchi pericarps. To prevent passive progression after fruit cracking from affecting the results, we mainly focused on 11 metabolites and 101 genes that showed the same regulatory status and overlap in pairwise comparisons of cracking 'Baitangying' versus noncracking 'Baitangying' and noncracking 'Baitangying' versus noncracking 'Feizixiao'. Compared with the cracking-resistant cultivar 'Feizixiao', the 'Baitangying' pericarp has higher abscisic acid contents, and the presence of relevant metabolites and genes suggests increased biosynthesis of ethylene and jasmonic acid and decreased auxin and brassinosteroid biosynthesis. The fruit cracking-susceptible trait in 'Baitangying' might be associated with differences in the balance of these five types of hormones between the pericarp of this cultivar and that of 'Feizixiao'. Additionally, combined analyses showed a correspondence between the metabolite profiles and transcript patterns. qRT-PCR validation indicated the reliability of our high-throughput results. The acquired information might help in further studying the mechanisms that mediate fruit cracking susceptibility in 'Baitangying' and other litchi cultivars.

8.
Arch Pharm (Weinheim) ; : e1900127, 2019 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-31441108

RESUMO

Chemotherapy remains a pillar in the treatment and management of various cancers. However, multidrug resistance (MDR) becomes a severe problem after long-term administration of chemotherapy drugs. Overexpression of P-glycoprotein (P-gp) is a significant cause for tumor MDR. Therefore, P-gp inhibition is considered as an effective strategy to reverse MDR. A third-generation P-gp inhibitor tariquidar was selected as a lead compound, and a new series of triazol-N-ethyl tetrahydroisoquinoline based compounds were designed as novel P-gp inhibitors and synthesized through click chemistry. These compounds presented higher reversal activities than the positive-control verapamil (VRP). Among 18 compounds, compound 11 without cytotoxicity reversed MDR in a dose-dependent manner, with a persistent longer chemosensitizing effect and reversibility compared to others. Mechanism studies discovered that compound 11 could escalate the intracellular accumulation of rhodamine-123 and doxorubicin in K562/A02 cells as well as inhibit their efflux from cells. The results obtained suggest that compound 11 is more potent than VRP administered under the same conditions; it may be a potent and safe candidate for P-gp modulation for further development.

9.
Analyst ; 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31432811

RESUMO

Over the past few years, artificial enzymes have attracted enormous attention due to their high stabilities and cost-effective productions. In this work, metal-organic framework-derived SiW12@Co3O4 was synthesized in large quantities by stirring the mixture at ambient temperature and calcination. The obtained SiW12@Co3O4 exhibited a highly inherent peroxidase-like activity and excellent stability. Kinetic studies demonstrated that the synthesized SiW12@Co3O4 had a strong binding affinity to 3,3',5,5'-tetramethylbenzidine (TMB), stronger than HRP had. Specifically, the peroxidase-like activity of SiW12@Co3O4 in an aqueous solution was well maintained after incubation at an elevated temperature, at an extreme pH and for a long time. A SiW12@Co3O4-based method was further developed for H2O2 and one-pot glucose detection with good sensitivity and reliability. The facile synthesis approach is expected to facilitate the practical use of metal-organic frameworks and their derivatives as enzyme mimics in the future.

10.
J Zhejiang Univ Sci B ; 20(9): 728-739, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31379143

RESUMO

As one of the most important aquatic fish, Micropterus salmoides suffers lethal and epidemic disease caused by rhabdovirus at the juvenile stage. In this study, a new strain of M. salmoides rhabdovirus (MSRV) was isolated from Yuhang, Zhejiang Province, China, and named MSRV-YH01. The virus infected the grass carp ovary (GCO) cell line and displayed virion particles with atypical bullet shape, 300-500 nm in length and 100-200 nm in diameter under transmission electron microscopy. The complete genome sequence of this isolate was determined to include 11 526 nucleotides and to encode five classical structural proteins. The construction of the phylogenetic tree indicated that this new isolate is clustered into the Vesiculovirus genus and most closely related to the Siniperca chuatsi rhabdovirus. To explore the potential for a vaccine against MSRV, a glycoprotein (1-458 amino acid residues) of MSRV-YH01 was successfully amplified and cloned into the plasmid pFastBac1. The high-purity recombinant bacmid-glycoprotein was obtained from DH10Bac through screening and identification. Based on polymerase chain reaction (PCR), western blot, and immunofluorescence assay, recombinant virus, including the MSRV-YH01 glycoprotein gene, was produced by transfection of SF9 cells using the pFastBac1-gE2, and then repeatedly amplified to express the glycoprotein protein. We anticipate that this recombinant bacmid system could be used to challenge the silkworm and develop a corresponding oral vaccine for fish.

11.
Analyst ; 2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31397451

RESUMO

Subcellular mitochondrion has become a target for improving the therapeutic efficiency and reducing side damage to normal cells via a combination of many therapeutic strategies. However, the underlying molecular mechanisms associated with cell death induced by subcellular dysfunction remain unknown or disputed. In this study, we investigated the dynamic molecular changes of living mitochondria upon phototherapy (photothermal therapy plus photodynamic therapy, PTT & PDT) by surface-enhanced Raman scattering spectroscopy (SERS) and intended to disclose the photo-induced cell death route in breast cancer cells (MCF-7) taking into account the mitochondrion. Indocyanine green (ICG), a Food and Drug Administration (FDA)-approved clinic blood-injection near-infrared angiographic contrast agent and a PTT & PDT drug, was used for the evaluation of the phototherapy effect. The results revealed that the content of phenylalanine (Phe) in mitochondria evidently increased during the phototherapy-induced cell death process. Moreover, the phototherapy-induced cell apoptosis was mainly regulated through the DNA structures. We expect that the understanding of mitochondrial molecular stress responses will be helpful for the diagnosis and therapy of cellular processes associated with mitochondria and provide valuable guidance for the further design and development of more effective therapeutic platforms and methods at the sub-cellular level.

12.
Opt Lett ; 44(16): 3976-3979, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31415526

RESUMO

Next-generation passive optical networks require integrated, polarization-insensitive wavelength-division multiplexing solutions, for which the recently emerging low-loss silicon nitride nanophotonic platforms hold great potential. A novel polarization-insensitive arrayed waveguide grating (AWG) built with silicon nitride waveguides is presented in this Letter. Polarization insensitivity is obtained when both the channel spacing and the center wavelength of the two orthogonal polarization states (i.e., the TE and TM waveguide modes) are simultaneously aligned. In our design, the channel spacing alignment between the polarization states is obtained by optimizing the geometry of the arrayed waveguides, whereas the central wavelength polarization insensitivity is obtained by splitting the two polarization states and adjusting their angle of incidence at the input star coupler to compensate for the polarization mode dispersion of the AWG. A 100 GHz 1×8 wavelength-division multiplexer with crosstalk levels below -16 dB is demonstrated experimentally.

13.
Ying Yong Sheng Tai Xue Bao ; 30(8): 2783-2792, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31418204

RESUMO

In the past decade, the number of islands fixed link with mainland has been increasing rapidly. The construction of such projects improves the accessibility of islands, which results in a rapid increase of tourists and economic income. However, the rapid change of land use and increases of tourists will make the islands more vulnerable. It is urgent to formulate ecosystem management strategy for island ecosystems based on the scientifical prediction of the island ecological vulnerability and accurate identification of sensitive areas. Island ecological vulnerability assessment model was used to evaluate the ecological vulnerability of Zhujiajian Island. Taking 2015 as the starting date, we simulated the changes of tourists and land use, as well as the changes of island ecological vulnerability in the next 20 years. Then, the management strategy was formulated based on the vulnerability assessment and sensitive analysis. The results showed that the ecosystem of Zhujiajian Island was in good condition now, with limited area at moderate and severe vulnerable status. With the rapid increases of tourists and island development intensity, the ecological vulnerability of the island tended to more vulnerability with the increases of severe vulnerability and shrink of low vulnerability. According to the vulnerability assessment and sensitivity analysis, the Zhujiajian Island could be divided into prohibited development zones, restricted development zones and conditional development zones with different ecosystem management strategies.

14.
Int Wound J ; 2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31418529

RESUMO

To assess the efficacy and safety of dexmedetomidine (DEX) as an adjuvant to local wound infiltration anaesthesia in abdominal surgery, we conducted this meta-analysis. First, the systematic search strategy was performed on PubMed, Embase, and Cochrane Library and five randomised controlled trials (RCTs) involving 294 patients were included. Then, the outcome data were extracted from the studies and their effect sizes were calculated using Review Manager 5. As a result, the addition of DEX significantly reduced visual analogy scores at 6 hours after surgery (mean difference = -0.53[-0.82, -0.25], P < .001), 12 hours after surgery (mean difference = -0.39 [-0.73, -0.05]; P = .03), and 24 hours after surgery (mean difference = -0.20 [-0.29, -0.11], P < .001) and reduced total analgesic consumption within 24 hours after surgery (mean difference = -4.92 [-9.00, -0.84]; P = .02) compared with placebo groups. However, there was no difference in the incidence of postoperative nausea and vomiting (risk ratio = 0.68 [0.41, 1.14]; P = .14). In summary, DEX as a local anaesthetic adjuvant added for local wound infiltration anaesthesia in abdominal surgery could reduce visual analogy scores and postoperative analgesic consumption without changing incidence of postoperative nausea and vomiting.

15.
Environ Sci Technol ; 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31432660

RESUMO

The pertechnetate anion (99TcO4-) is a long-lived radioactive species that is soluble in aqueous solution, in contrast to sparingly soluble 99TcO2. Results are reported for photocatalytic reduction and removal of perrhenate (ReO4-), a nonradioactive surrogate for 99TcO4-, using a TiO2 (P25) nanoparticle suspension in formic acid under UV-visible irradiation. Re(VII) removal up to 98% was achieved at pH = 3 under air or N2. The proposed mechanism is Re(VII)/Re(IV) reduction mediated by reducing radicals (·CO2-) from oxidation of formic acid, not direct reduction by photogenerated electrons of TiO2. Recycling results indicate that photocatalytic reduction of ReO4- exhibits excellent regeneration and high activity with >95% removal even after five cycles. 99Tc(VII) is more easily reduced than Re(VII) in the presence of NO3- with very slow redissolution of reduced 99Tc. This study presents a novel method for the removal of ReO4-/99TcO4- from aqueous solution, with potential application for deep geological disposal.

16.
Int J Mol Sci ; 20(15)2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31390813

RESUMO

Nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductases (CPRs) function as redox partners of cytochrome P450 monooxygenases (P450s). CPRs and P450s in insects have been found to participate in insecticide resistance. However, the CPR of the moth Spodoptera litura has not been well characterized yet. Based on previously obtained transcriptome information, a full-length CPR cDNA of S. litura (SlCPR) was PCR-cloned. The deduced amino acid sequence contains domains and residues predicted to be essential for CPR function. Phylogenetic analysis with insect CPR amino acid sequences showed that SlCPR is closely related to CPRs of Lepidoptera. Quantitative reverse transcriptase PCR (RT-qPCR) was used to determine expression levels of SlCPR in different developmental stages and tissues of S. litura. SlCPR expression was strongest at the sixth-instar larvae stage and fifth-instar larvae showed highest expression in the midgut. Expression of SlCPR in the midgut and fat body was strongly upregulated when fifth-instar larvae were exposed to phoxim at LC15 (4 µg/mL) and LC50 (20 µg/mL) doses. RNA interference (RNAi) mediated silencing of SlCPR increased larval mortality by 34.6% (LC15 dose) and 53.5% (LC50 dose). Our results provide key information on the SlCPR gene and indicate that SlCPR expression levels in S. litura larvae influence their susceptibility to phoxim and possibly other insecticides.

17.
Int J Biochem Cell Biol ; 115: 105591, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31442607

RESUMO

GATA-6 is a transcription factor that participates in cell lineage differentiation and organogenesis in many tissue types. The abnormal expression of GATA-6 is associated with the development of diverse cancers. GATA-6 acts as an oncogene or tumor suppressor based on tumor origin. Here, we investigated the effects of GATA-6 on lung squamous cell carcinoma (LSCC). We found that GATA-6 was significantly reduced in LSCC tissues compared with the paired normal tissues. The overexpression of GATA-6 inhibited LSCC cell proliferation and migration. Importantly, a luciferase reporter assay and chromatin immunoprecipitation assay demonstrated that GATA-6 negatively regulated the expression of sonic hedgehog (Shh) by directly binding to its promoter region. Furthermore, N-Shh stimulation rescued the inhibition of LSCC cell proliferation and migration upon GATA-6 overexpression. Thus, GATA-6 inhibited the proliferation and migration of LSCC cells by transcriptionally inhibiting the expression of Shh, indicating that targeting GATA-6 may be a potential approach for LSCC therapy.

18.
Phys Med Biol ; 2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31422959

RESUMO

In this study, we propose to automatically remove Moiré image artifact induced by system instabilities in grating-based x-ray interferometry imaging using convolutional neural network (CNN) technique. This method reduces Moiré image artifact in image-domain via a learned image post-processing procedure, rather than developing signal retrieval optimization algorithms to minimize the inconsistencies between acquired phase stepping data and assumed signal model. To achieve this aim, we train the CNN network using numerical dataset synthesized from both natural images and experimentally acquired Moiré artifact-only images. In particular, a novel approach is developed to generate a large number of different high quality Moiré artifact-only images from finite groups of experimental phase stepping data. Both numerical and experimental results demonstrate that the developed CNN method is able to effectively remove the undesired Moiré image artifact. As a result, the image quality of a practical grating-based x-ray interferometry system can be greatly improved.

19.
Chem Commun (Camb) ; 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31455967

RESUMO

Single-chain magnets (SCMs) are one-dimensional (1D) coordination polymers that exhibit magnetic bistability and have potential application in high-density information processing. Usually SCMs are magnetically isolated chains stacked by hydrogen bonds or ππ stacking; however, with the rapid development of metal-organic frameworks (MOFs), coordination chains assembled in MOFs have also shown SCM behaviour when the 1D magnetic correlation is strong enough in comparison to the other two dimensionalities. The high-dimensional framework of MOFs could also help in the formation of coordination chains that cannot form in isolated one-dimensional coordination systems. This feature article highlights the progress in this field by summarizing cobalt(ii)-based MOFs exhibiting SCM behaviour and classifying them into categories according to the bridging ligands within the CoII chains, to provide fundamentals for further studies on the magnetic properties of coordination chains assembled in MOFs.

20.
Hum Mol Genet ; 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31272105

RESUMO

Lower lobe predominant pulmonary cysts occur in up to 90% of patients with Birt-Hogg-Dubé (BHD) syndrome, but the key pathologic cell type and signaling events driving this distinct phenotype remain elusive. Through examination of the LungMAP database, we found that folliculin (FLCN) is highly expressed in neonatal lung mesenchymal cells. Using RNA-Seq, we found that inactivation of Flcn in mouse embryonic fibroblasts (MEFs) leads to changes in multiple Wnt ligands, including a 2.8-fold decrease in Wnt2. This was associated with decreased TCF/LEF activity, a readout of canonical WNT activity, after treatment with a GSK3-α/ß inhibitor. Similarly, FLCN deficiency in HEK293T cells decreased WNT pathway activity by 76% post GSK3-α/ß inhibition. Inactivation of FLCN in human fetal lung fibroblasts (MRC-5) led to ~100-fold decrease in Wnt2 expression and a 33-fold decrease in Wnt7b expression- two ligands known to be necessary for lung development- furthermore, canonical WNT activity was decreased by 60%. Classic WNT targets such as AXIN2 and BMP4, and WNT enhanceosome members including TCF4, LEF1, and BCL9, were also decreased after GSK3-α/ß inhibition. FLCN-deficient MRC-5 cells failed to upregulate LEF1 in response to GSK3-α/ß inhibition. Finally, we found that a constitutively active ß-catenin could only partially rescue the decreased WNT activity phenotype seen in FLCN-deficient cells, whereas silencing the transcription factor TFE3 completely reversed this phenotype. In summary, our data establish FLCN as a critical regulator of the WNT pathway via TFE3 and suggest that FLCN-dependent defects in WNT pathway developmental cues may contribute to lung cyst pathogenesis in BHD.

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