Publications by authors named "Yulong Fu"

32 Publications

Anaerobic condition induces a viable but nonculturable state of the PCB-degrading Bacteria Rhodococcus biphenylivorans TG9.

Sci Total Environ 2021 Apr 9;764:142849. Epub 2020 Oct 9.

Department of Environmental Engineering, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China; Zhejiang Provincial Key Laboratory for Water Pollution Control and Environmental Safety, Hangzhou 310058, China. Electronic address:

Significant microbial removal of highly chlorinated polychlorinated biphenyls (PCBs) requires the cooperation of anaerobic and aerobic bacteria. During the sequencing process of anaerobic dechlorination and aerobic degradation of PCBs, aerobic degrading bacteria have to undergo anaerobic stress. However, the survival strategy of aerobic degrading bacteria under anaerobic condition is not well-understood. In this study, the culturable cells of Rhodococcus biphenylivorans TG9 decreased from 10 CFU/mL to values below the detection limit after 60 days of anaerobic stress while the viable cells remained 10-10 cells/mL, indicating that anaerobic condition induced TG9 entering into the viable but nonculturable (VBNC) state. Cell resuscitation was observed when oxygen was supplied further confirming the VBNC state of TG9. The results of single-cell Raman spectroscopy combined with heavy water indicated the significant decrease of metabolic activity after TG9 entering into the VBNC state. Additionally, the degradation ability of TG9 in the VBNC state was also significantly reduced, while it recovered after resuscitation. Our research proved that entering into the VBNC state is a survival strategy of TG9 under anaerobic conditions, and the limited culturability and degrading capacity could be overcome by resuscitation. The present study provides new insights for improving the remediation efficiency of PCBs contamination.
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http://dx.doi.org/10.1016/j.scitotenv.2020.142849DOI Listing
April 2021

Advanced NSCLC Patients With EGFR T790M Harboring TP53 R273C or KRAS G12V Cannot Benefit From Osimertinib Based on a Clinical Multicentre Study by Tissue and Liquid Biopsy.

Front Oncol 2021 24;11:621992. Epub 2021 Feb 24.

Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Soochow University, Suzhou, China.

Background: Non-small cell lung cancer (NSCLC) patients treated with first-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) almost always acquire resistance, and the development of novel techniques analyzing circulating tumor DNA (ctDNA) have made it possible for liquid biopsy to detect genetic alterations from limited amount of DNA with less invasiveness. While a large amount of patients with EGFR exon 21 p.Thr790 Met (T790M) benefited from osimertinib treatment, acquired resistance to osimertinb has subsequently become a growing challenge.

Methods: We performed tissue and liquid rebiopsy on 50 patients with EGFR-mutant NSCLC who acquired resistance to first-generation EGFR-TKIs. Plasma samples underwent droplet digital PCR (ddPCR) and next-generation sequencing (NGS) examinations. Corresponding tissue samples underwent NGS and Cobas EGFR Mutation Test v2 (Cobas) examinations.

Results: Of the 50 patients evaluated, the mutation detection rates of liquid biopsy group and tissue biopsy group demonstrated no significant differences (41/48, 85.4% vs. 44/48, 91.7%; OR=0.53, 95% CI=0.15 to 1.95). Overall concordance, defined as the proportion of patients for whom at least one identical genomic alteration was identified in both tissue and plasma, was 78.3% (36/46, 95% CI=0.39 to 2.69). Moreover, our results showed that almost half of the patients (46%, 23/50) resistant to first-generation EGFR-TKI harbored p.Thr790 Met (T790M) mutation. 82.6% (19/23) of the T790M positive patients were analyzed by liquid biopsy and 60.9% (14/23) by tumor tissue sequencing. Meanwhile, a wide range of uncommon mutations was detected, and novel mechanisms of osimertinib resistance were discovered. In addition, 16.7% (2/12) of the T790M positive patients with either TP53 R237C or KRAS G12V failed to benefit from the subsequent osimertinib treatment.

Conclusion: Our results emphasized that liquid biopsy is applicable to analyze the drug resistance mechanisms of NSCLC patients treated with EGFR-TKIs. Moreover, we discovered two uncommon mutations, TP53 R273C and KRAS G12V, which attenuates the effectiveness of osimertinib.
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http://dx.doi.org/10.3389/fonc.2021.621992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7943858PMC
February 2021

Autophagy inhibition rescues structural and functional defects caused by the loss of mitochondrial chaperone in .

Autophagy 2021 Jan 25:1-15. Epub 2021 Jan 25.

Research Group Synaptic Plasticity, Hertie Institute for Clinical Brain Research, University of Tübingen , Tübingen, Germany.

We investigated in larval and adult models whether loss of the mitochondrial chaperone is sufficient to cause pathological alterations commonly observed in Parkinson disease. At affected larval neuromuscular junctions, no effects on terminal size, bouton size or number, synapse size, or number were observed, suggesting that we studied an early stage of pathogenesis. At this stage, we noted a loss of synaptic vesicle proteins and active zone components, delayed synapse maturation, reduced evoked and spontaneous excitatory junctional potentials, increased synaptic fatigue, and cytoskeleton rearrangements. The adult model displayed ATP depletion, altered body posture, and susceptibility to heat-induced paralysis. Adult phenotypes could be suppressed by knockdown of , and . The knockdown of components of the macroautophagy/autophagy machinery or overexpression of human broadly rescued larval and adult phenotypes, while disease-associated variants did not. Overexpression of or promotion of autophagy exacerbated defects. AEL: after egg laying; AZ: active zone; brp: bruchpilot; Csp: cysteine string protein; dlg: discs large; eEJPs: evoked excitatory junctional potentials; GluR: glutamate receptor; HO: hydrogen peroxide; mEJP: miniature excitatory junctional potentials; MT: microtubule; NMJ: neuromuscular junction; PD: Parkinson disease; : PTEN-induced putative kinase 1; PSD: postsynaptic density; SSR: subsynaptic reticulum; SV: synaptic vesicle; VGlut: vesicular glutamate transporter.
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http://dx.doi.org/10.1080/15548627.2020.1871211DOI Listing
January 2021

Abnormally activated OPN/integrin αVβ3/FAK signalling is responsible for EGFR-TKI resistance in EGFR mutant non-small-cell lung cancer.

J Hematol Oncol 2020 12 7;13(1):169. Epub 2020 Dec 7.

Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Soochow University, Suzhou, 215006, People's Republic of China.

Background: Acquired epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance limits the long-term clinical efficacy of tyrosine kinase-targeting drugs. Although most of the mechanisms of acquired EGFR-TKI resistance have been revealed, the mechanism of ~ 15% of cases has not yet been elucidated.

Methods: Cell viability was analysed using the Cell Counting Kit-8 (CCK-8) assay. Proteome profiler array analysis was performed to find proteins contributing to acquired EGFR-TKI resistance. Secreted OPN was detected by ELISA. Immunohistochemical analysis was conducted to detect expression of integrin αV in NSCLC tissue. The effect of VS-6063 on apoptosis and proliferation of PC9 gefitinib-resistant cells was detected by fluorescence-activated cell sorting (FACS) and clonogenic assays. A mouse xenograft model was used to assess the effect of VS-6063 on the sensitivity of PC9 gefitinib-resistant cells to gefitinib.

Results: OPN was overexpressed in acquired EGFR-TKI-resistant NSCLCs. Secreted OPN contributed to acquired EGFR-TKI resistance by activating the integrin αVβ3/FAK pathway. Inhibition of FAK signalling increased sensitivity to EGFR-TKIs in PC9 gefitinib-resistant cells both in vitro and in vivo.

Conclusions: OPN contributes to acquired EGFR-TKI resistance by up-regulating expression of integrin αVβ3, which activates the downstream FAK/AKT and ERK signalling pathways to promote cell proliferation in NSCLC.
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http://dx.doi.org/10.1186/s13045-020-01009-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7720454PMC
December 2020

KPNB1-mediated nuclear translocation of PD-L1 promotes non-small cell lung cancer cell proliferation via the Gas6/MerTK signaling pathway.

Cell Death Differ 2021 Apr 2;28(4):1284-1300. Epub 2020 Nov 2.

Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Soochow University, 215006, Suzhou, China.

In addition to the role of programmed cell death ligand 1 (PD-L1) in facilitating tumour cells escape from immune surveillance, it is considered as a crucial effector in transducing intrinsic signals to promote tumour development. Our previous study has pointed out that PD-L1 promotes non-small cell lung cancer (NSCLC) cell proliferation, but the mechanism remains elusive. Here we first demonstrated that PD-L1 expression levels were positively correlated with p-MerTK levels in patient samples and NSCLC cell lines. In addition, PD-L1 knockdown led to the reduced phosphorylation level of MerTK in vitro. We next showed that PD-L1 regulated NSCLC cell proliferation via Gas6/MerTK signaling pathway in vitro and in vivo. To investigate the underlying mechanism, we unexpectedly found that PD-L1 translocated into the nucleus of cancer cells which was facilitated through the binding of Karyopherin β1 (KPNB1). Nuclear PD-L1 (nPD-L1), coupled with transcription factor Sp1, regulated the synthesis of Gas6 mRNA and promoted Gas6 secretion to activate MerTK signaling pathway. Taken together, our results shed light on the novel role of nPD-L1 in NSCLC cell proliferation and reveal a new molecular mechanism underlying nPD-L1-mediated Gas6/MerTK signaling activation. All above findings provide the possible combinational implications for PD-L1 targeted immunotherapy in the clinic.
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http://dx.doi.org/10.1038/s41418-020-00651-5DOI Listing
April 2021

Alterations in the Cell Wall of Under Norfloxacin Stress.

Front Microbiol 2020 6;11:554957. Epub 2020 Oct 6.

Department of Environmental Engineering, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, China.

Many microorganisms can enter a viable but non-culturable (VBNC) state under various environmental stresses, while they can also resuscitate when the surroundings turn to suitable conditions. Cell walls play a vital role in maintaining cellular integrity and protecting cells from ambient threats. Here, we investigated the alterations in the cell wall of TG9 at VBNC state under norfloxacin stress and then at resuscitated state in fresh lysogeny broth medium. Electron microscopy analyses presented that TG9 in the VBNC state had a thicker and rougher cell wall than that in exponential phase or resuscitated state. Meanwhile, the results from infrared spectroscopy also showed that its VBNC state has different peptidoglycan structures in the cell wall. Moreover, in the VBNC cells the gene expressions related to cell wall synthesis and remodeling maintain a relatively high level. It indicates that the morphological variations of TG9 at the VBNC state might result from kinetic changes in the cell wall synthesis and remodeling. As a consequence, the alterations in the cell wall of VBNC TG9 may somewhat account for its tolerance mechanisms to antibiotic treatment.
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http://dx.doi.org/10.3389/fmicb.2020.554957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7573542PMC
October 2020

Occurrence and quantification of culturable and viable but non-culturable (VBNC) pathogens in biofilm on different pipes from a metropolitan drinking water distribution system.

Sci Total Environ 2021 Apr 9;764:142851. Epub 2020 Oct 9.

Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, China. Electronic address:

Waterborne pathogens have been found in biofilms grown in drinking water distribution system (DWDS). However, there is a lack of quantitative study on the culturability of pathogens in biofilms from metropolitan DWDS. In this study, we quantified culturable and viable but non-culturable (VBNC) Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa and Vibrio cholerae in biofilms collected from five kinds of pipes (galvanized steel pipe, steel pipe, stainless steel clad pipe, ductile cast iron pipe and polyethylene pipe) and associated drinking water at an actual chlorinated DWDS in use from China. The results of these comprehensive analyses revealed that pipe material is a significant factor influencing the culturability of pathogen and microbial communities. Network analysis of the culturable pathogens and 16S rRNA gene inferred potential interactions between microbiome and culturability of pathogens. Although the water quality met the Chinese national standard of drinking water, however, VBNC pathogens were detected in both biofilms and water from the DWDS. This investigation suggests that stainless steel clad pipe (SSCP) was a better choice for pathogen control compared with other metal pipes. To our knowledge, this is the first study on culturable and VBNC pathogens in biofilms of different pipe materials in metropolitan DWDS.
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http://dx.doi.org/10.1016/j.scitotenv.2020.142851DOI Listing
April 2021

An Optimized Method to Assess Viable O157:H7 in Agricultural Soil Using Combined Propidium Monoazide Staining and Quantitative PCR.

Front Microbiol 2020 31;11:1809. Epub 2020 Jul 31.

Department of Environmental Engineering, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, China.

Agricultural soil contaminated by manure is becoming an important source for the transmission of foodborne pathogens. There is an urgent need for a rapid and accurate method for viable pathogen detection in agricultural soil samples. Propidium monoazide (PMA) is a DNA-binding dye that can inhibit the amplification of DNA from dead cells through subsequent quantitative polymerase chain reaction (qPCR), thus allowing for viable cells detection and quantification. The objective of this study was to detect viable O157:H7 in the agricultural soils by PMA-qPCR. In this study, cell extraction and gradient density centrifugation were incorporated before PMA-qPCR to reduce the interference of soil particle including turbidity and a high ratio of dead cells. The optimized treatment conditions were determined as follows, the maximum removal of DNA from dead cells was achieved by 1.067 g/mL Percoll of centrifugation and 50 μM PMA treatment. Under these conditions, the turbidity of paddy soil suspensions decreased from 3500 to 28.4 nephelometric turbidity units (NTU), and the ratio of viable cells to dead cells increased from 0.001 to 1.025%. For typical agricultural soils collected in China, as low as 10colony-forming units (CFU)/g of viable cells could be accurately detected in the presence of a large number of dead cells (10 CFU/g) by the optimized PMA-qPCR. Significantly, with comparable accuracy, the optimized PMA-qPCR assay was more sensitive, accessible and rapid than conventional culture methods. In addition, the viable but non-culturable (VBNC) state of O157:H7 cells in paddy soils, which often escaped the detection by conventional culture methods, could be quantitatively characterized by the optimized PMA-qPCR method. Potentially, the optimized PMA-qPCR can be further applied for viable pathogens detection and give insight into the prevalence of VBNC O157:H7 in agricultural soil.
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http://dx.doi.org/10.3389/fmicb.2020.01809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7411311PMC
July 2020

Induction of Escherichia coli O157:H7 into a viable but non-culturable state by high temperature and its resuscitation.

Environ Microbiol Rep 2020 10 6;12(5):568-577. Epub 2020 Sep 6.

Department of Environmental Engineering, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, 310058, China.

Escherichia coli O157:H7, a causative agent of haemolytic uremic syndrome, can enter into a viable but non-culturable (VBNC) state in response to harsh stress. Bacteria in this state can retain membrane integrity, metabolic activity and virulence expression, which may present health risks. However, virulence expression and resuscitation ability of the VBNC state are not well understood. Here, we induced E. coli O157:H7 into a VBNC state by high temperature, which is commonly used to prevent the proliferation of pathogens in process of soil solarization, composting and anaerobic digestion of organic wastes. The virulence genes were highly expressed in the VBNC state and resuscitated daughter cells. The resuscitation of VBNC cells occurred after the removal of heat stress in Luria-Bertani medium. In addition, E. coli O157: H7 cells can leave the VBNC state and resuscitate with the clearance of protein aggregates. Notably, with the accumulation of protein aggregation and increased levels of reactive oxygen species, cells lost their ability to resuscitate. The results of this study not only can facilitate a better understanding of the health risks associated with the VBNC state but also have the potential to provide a theoretical basis for thermal disinfection processing.
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http://dx.doi.org/10.1111/1758-2229.12877DOI Listing
October 2020

Downregulation of KIAA1199 by miR-486-5p suppresses tumorigenesis in lung cancer.

Cancer Med 2020 08 9;9(15):5570-5586. Epub 2020 Jun 9.

Department of Respiratory Medicine, the First Affiliated Hospital of Soochow University, Suzhou, China.

Lung cancer is the primary cause of death among cancer patients in China, among which nonsmall cell lung cancer (NSCLC) makes up the great majority. Hence, it is imperative to identify the biomarkers and mechanisms involved in NSCLC oncogenesis. Our present research found that KIAA1199 expression was significantly increased in NSCLC and closely related to cell proliferation, motility, and poor prognosis. We demonstrated that knockdown of KIAA1199 reduced NSCLC cell growth and motility in vitro whereas overexpression of KIAA1199 had the opposite effect. Inhibition of KIAA1199 significantly suppressed tumor growth in mouse NSCLC xenograft models. Mechanistically, as an epidermal growth factor receptor (EGFR)-binding protein, KIAA1199 promotes EGFR signaling and regulates EGFR-dependent Src, Erk, and Akt phosphorylation, as well as downstream kinases in the EGF-mediated EMT pathway. We demonstrated that KIAA1199 can function as a direct binding target for miR-486-5p and that miR-486-5p overexpression can attenuate proliferation and migration of NSCLC cells via regulating the EGFR signaling pathways. To conclude, our results defined KIAA1199 as an oncogenic protein that promotes cancer cell proliferation and migration by regulating EGF-mediated signaling pathways. This study provided new insight into NSCLC oncogenesis, which could lead to the development of innovative therapeutic plans for NSCLC.
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http://dx.doi.org/10.1002/cam4.3210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402811PMC
August 2020

Single-cell RNA sequencing identifies novel cell types in Drosophila blood.

J Genet Genomics 2020 04 9;47(4):175-186. Epub 2020 Mar 9.

Center for Precision Disease Modeling, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, 21201, USA; Division of Endocrinology, Diabetes and Nutrition, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, 21201, USA. Electronic address:

Drosophila has been extensively used to model the human blood-immune system, as both systems share many developmental and immune response mechanisms. However, while many human blood cell types have been identified, only three were found in flies: plasmatocytes, crystal cells and lamellocytes. To better understand the complexity of fly blood system, we used single-cell RNA sequencing technology to generate comprehensive gene expression profiles for Drosophila circulating blood cells. In addition to the known cell types, we identified two new Drosophila blood cell types: thanacytes and primocytes. Thanacytes, which express many stimulus response genes, are involved in distinct responses to different types of bacteria. Primocytes, which express cell fate commitment and signaling genes, appear to be involved in keeping stem cells in the circulating blood. Furthermore, our data revealed four novel plasmatocyte subtypes (Ppn, CAH7, Lsp and reservoir plasmatocytes), each with unique molecular identities and distinct predicted functions. We also identified cross-species markers from Drosophila hemocytes to human blood cells. Our analysis unveiled a more complex Drosophila blood system and broadened the scope of using Drosophila to model human blood system in development and disease.
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http://dx.doi.org/10.1016/j.jgg.2020.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321924PMC
April 2020

Anlotinib can overcome acquired resistance to EGFR-TKIs via FGFR1 signaling in non-small cell lung cancer without harboring EGFR T790M mutation.

Thorac Cancer 2020 07 20;11(7):1934-1943. Epub 2020 May 20.

Department of Respiratory Medicine, The First Affiliated Hospital of Soochow University, Suzhou, China.

Background: Although many studies have defined mechanisms of resistance to EGFR-TKIs, acquired resistance remains the major limitation of monotherapy with EGFR-TKIs.

Methods: Cell viability was analyzed using a Cell Counting Kit-8 (CCK-8) assay. EGFR T790M mutation was sequenced on a HiSeq 4000 platform. mRNAs from HCC827 and HCC827 gefitinib-resistant (GR) cells were analyzed by genome analyzer-based deep sequencing. The effect of anlotinib on apoptosis and cell cycle arrest of HCC827 GR was detected by fluorescence-activated cell sorting (FACS) analysis. A mouse xenograft model was used to assess the effect of anlotinib on HCC827 GR cells.

Results: The T790M mutation was found in the PC-9 GR cell line but not in the HCC827 GR cell line. Anlotinib could suppress the growth of HCC827 GR cells by inhibiting FGFR1 in vitro and in a mouse xenograft model. Moreover, FGFR1 was overexpressed in HCC827 GR cells, and the knockdown of FGFR1 reversed gefitinib resistance in HCC827 GR cells. Furthermore, anlotinib induced apoptosis and cell cycle arrest in HCC827 GR cells by increasing the activity of Caspase-3.

Conclusions: FGFR1 overexpression could be the mechanism of EGFR-TKI acquired resistance and anlotinib can suppresse the growth of EGFR-TKI-resistant NSCLC cells without T790M mutation.
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http://dx.doi.org/10.1111/1759-7714.13485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327692PMC
July 2020

EVI5 is an oncogene that regulates the proliferation and metastasis of NSCLC cells.

J Exp Clin Cancer Res 2020 May 11;39(1):84. Epub 2020 May 11.

Department of Respiratory Medicine, the First Affiliated Hospital of Soochow University, Suzhou, 215006, China.

Background: The Ecotropic viral integration site 5 (EVI5), an important protein in regulating cell cycle, cytokinesis and cellular membrane traffic, functions as a stabilizing factor maintaining anaphase-promoting complex/cyclosome (APC/C) inhibitor Emi1 in S/G2 phase. However, the mechanism by which EVI5 promotes malignant transformation of non-small cell lung cancer (NSCLC) remains unknown. In the present study, we addressed the role of EVI5 in NSCLC by regulating tumor growth, migration and invasion.

Methods: The expression levels of EVI5 and miR-486-5p in NSCLC tissues and cells were measured by real-time PCR. Meanwhile, EVI5 and its associated protein expression were analyzed by western blot and co-immunoprecipitation assay. Flow cytometry was performed to determine cell proliferation and apoptosis. CCK-8 and clonogenic assays were used to analyze cell viability. Wound healing, transwell migration and matrigel invasion assays were utilized to assess the motility of tumor cells. To investigate the role of EVI5 in vivo, lung carcinoma xenograft mouse model was applied..

Results: EVI5 was upregulated in NSCLC tissues and cell lines when compared with that in normal tissues and cell line. Knockdown of EVI5 in vitro inhibited tumor cell proliferation, migration and invasion in NSCLC cells. Further, inoculation of EVI5-deficient tumor cells into nude mice suppressed tumor proliferation and metastasis compared to control mice inoculated with unmanipulated tumor cells. These data indicated that EVI5 promote the proliferation of NSCLC cells which was consistent with our previous results. Additionally, we showed that EVI5 was directly regulated by miR-486-5p, and miR-486-5p-EVI5 axis affected the NSCLC migration and invasion through TGF-β/Smad signaling pathway by interacting with TGF-β receptor II and TGF-β receptor I.

Conclusions: Based on these results, we demonstrated a new post-transcriptional mechanism of EVI5 regulation via miR-486-5p and the protumoral function of EVI5 in NSCLC by interacting with Emi1 and/or TGF-β receptors, which provides a new insight into the targeted therapy of NSCLC.
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http://dx.doi.org/10.1186/s13046-020-01585-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212589PMC
May 2020

Exocyst Genes Are Essential for Recycling Membrane Proteins and Maintaining Slit Diaphragm in Nephrocytes.

J Am Soc Nephrol 2020 05 1;31(5):1024-1034. Epub 2020 Apr 1.

Center for Precision Disease Modeling, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland

Background: Studies have linked mutations in genes encoding the eight-protein exocyst protein complex to kidney disease, but the underlying mechanism is unclear. Because nephrocytes share molecular and structural features with mammalian podocytes, they provide an efficient model for studying this issue.

Methods: We silenced genes encoding exocyst complex proteins specifically in nephrocytes and studied the effects on protein reabsorption by lacuna channels and filtration by the slit diaphragm. We performed nephrocyte functional assays, carried out super-resolution confocal microscopy of slit diaphragm proteins, and used transmission electron microscopy to analyze ultrastructural changes. We also examined the colocalization of slit diaphragm proteins with exocyst protein Sec15 and with endocytosis and recycling regulators Rab5, Rab7, and Rab11.

Results: Silencing exocyst genes in nephrocytes led to profound changes in structure and function. Abolition of cellular accumulation of hemolymph proteins with dramatically reduced lacuna channel membrane invaginations offered a strong indication of reabsorption defects. Moreover, the slit diaphragm's highly organized surface structure-essential for filtration-was disrupted, and key proteins were mislocalized. Ultrastructural analysis revealed that exocyst gene silencing led to the striking appearance of novel electron-dense structures that we named "exocyst rods," which likely represent accumulated membrane proteins following defective exocytosis or recycling. The slit diaphragm proteins partially colocalized with Sec15, Rab5, and Rab11.

Conclusions: Our findings suggest that the slit diaphragm of nephrocytes requires balanced endocytosis and recycling to maintain its structural integrity and that impairment of the exocyst complex leads to disruption of the slit diaphragm and nephrocyte malfunction. This model may help identify therapeutic targets for treating kidney diseases featuring molecular defects in vesicle endocytosis, exocytosis, and recycling.
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http://dx.doi.org/10.1681/ASN.2019060591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217423PMC
May 2020

Zika virus non-structural protein NS4A restricts eye growth in through regulation of JAK/STAT signaling.

Dis Model Mech 2020 04 30;13(4). Epub 2020 Apr 30.

Department of Biological Sciences, The George Washington University, Washington, DC 20052, USA

To gain a comprehensive view of the changes in host gene expression underlying Zika virus (ZIKV) pathogenesis, we performed whole-genome RNA sequencing (RNA-seq) of ZIKV-infected adult flies. RNA-seq analysis revealed that ZIKV infection alters several and diverse biological processes, including stress, locomotion, lipid metabolism, imaginal disc morphogenesis and regulation of JAK/STAT signaling. To explore the interaction between ZIKV infection and JAK/STAT signaling regulation, we generated genetic constructs overexpressing ZIKV-specific non-structural proteins NS2A, NS2B, NS4A and NS4B. We found that ectopic expression of non-structural proteins in the developing eye significantly restricts growth of the larval and adult eye and correlates with considerable repression of the JAK/STAT reporter, At the cellular level, eye growth defects are associated with reduced rate of proliferation without affecting the overall rate of apoptosis. In addition, ZIKV NS4A genetically interacts with the JAK/STAT signaling components; co-expression of along with the dominant-negative form of or results in aggravated reduction in eye size, while co-expression of in (also known as ) mutant background partially rescues the -induced eye overgrowth phenotype. The function of ZIKV NS4A in regulating growth is maintained in the wing, where ZIKV overexpression in the pouch domain results in reduced growth linked with diminished expression of Notch targets, Wingless (Wg) and Cut, and the Notch reporter, Thus, our study provides evidence that ZIKV infection in results in restricted growth of the developing eye and wing, wherein eye phenotype is induced through regulation of JAK/STAT signaling, whereas restricted wing growth is induced through regulation of Notch signaling. The interaction of ZIKV non-structural proteins with the conserved host signaling pathways further advance our understanding of ZIKV-induced pathogenesis.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/dmm.040816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7197722PMC
April 2020

Forward genetic screen in human podocytes identifies diphthamide biosynthesis genes as regulators of adhesion.

Am J Physiol Renal Physiol 2019 12 30;317(6):F1593-F1604. Epub 2019 Sep 30.

Feinberg Cardiovascular and Renal Research Institute, Northwestern University Feinberg School of Medicine, Chicago, Illinois.

Podocyte function is tightly linked to the complex organization of its cytoskeleton and adhesion to the underlying glomerular basement membrane. Adhesion of cultured podocytes to a variety of substrates is reported to correlate with podocyte health. To identify novel genes that are important for podocyte function, we designed an in vitro genetic screen based on podocyte adhesion to plates coated with either fibronectin or soluble Fms-like tyrosine kinase-1 (sFLT1)/Fc. A genome-scale pooled RNA interference screen on immortalized human podocytes identified 77 genes that increased adhesion to fibronectin, 101 genes that increased adhesion to sFLT1/Fc, and 44 genes that increased adhesion to both substrates when knocked down. Multiple shRNAs against diphthamide biosynthesis protein 1-4 (DPH1-DPH4) were top hits for increased adhesion. Immortalized human podocyte cells stably expressing these hairpins displayed increased adhesion to both substrates. We then used CRISPR-Cas9 to generate podocyte knockout cells for , , or , which also displayed increased adhesion to both fibronectin and sFLT1/Fc, as well as a spreading defect. Finally, we showed that nephrocyte-specific knockdown of Dph1, Dph2, and Dph4 resulted in altered nephrocyte function. In summary, we report here a novel high-throughput method to identify genes important for podocyte function. Given the central role of podocyte adhesion as a marker of podocyte health, these data are a rich source of candidate regulators of glomerular disease.
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http://dx.doi.org/10.1152/ajprenal.00195.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6962514PMC
December 2019

Mutations in Are Implicated in Steroid-Resistant Nephrotic Syndrome.

J Am Soc Nephrol 2019 05 25;30(5):840-853. Epub 2019 Mar 25.

Center for Genetic Medicine Research, Children's National Health System, Washington, DC;

Background: Studies have identified mutations in >50 genes that can lead to monogenic steroid-resistant nephrotic syndrome (SRNS). The gene, which encodes one of the protein components of the nuclear pore complex nucleoporin 160 kD (Nup160), is expressed in both human and mouse kidney cells. Knockdown of impairs mouse podocytes in cell culture. Recently, siblings with SRNS and proteinuria in a nonconsanguineous family were found to carry compound-heterozygous mutations in .

Methods: We identified mutations by whole-exome and Sanger sequencing of genomic DNA from a young girl with familial SRNS and FSGS who did not carry mutations in other genes known to be associated with SRNS. We performed functional validation studies on the mutations using a model.

Results: We identified two compound-heterozygous mutations, and . We showed that silencing of specifically in nephrocytes (fly renal cells) led to functional abnormalities, reduced cell size and nuclear volume, and disorganized nuclear membrane structure. These defects were completely rescued by expression of the wild-type human gene in nephrocytes. By contrast, expression of the mutant allele completely failed to rescue nephrocyte phenotypes, and mutant allele rescued only nuclear pore complex and nuclear lamin localization defects.

Conclusions: Mutations in are implicated in SRNS. Our findings indicate that should be included in the SRNS diagnostic gene panel to identify additional patients with SRNS and homozygous or compound-heterozygous mutations and further strengthen the evidence that mutations can cause SRNS.
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http://dx.doi.org/10.1681/ASN.2018080786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6493979PMC
May 2019

APOL1 risk allele RNA contributes to renal toxicity by activating protein kinase R.

Commun Biol 2018 7;1:188. Epub 2018 Nov 7.

Kidney Disease Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD, 20892, USA.

risk alleles associate with chronic kidney disease in African Americans, but the mechanisms remain to be fully understood. We show that risk alleles activate protein kinase R (PKR) in cultured cells and transgenic mice. This effect is preserved when a premature stop codon is introduced to risk alleles, suggesting that RNA but not protein is required for the effect. Podocyte expression of risk allele RNA, but not protein, in transgenic mice induces glomerular injury and proteinuria. Structural analysis of the APOL1 RNA shows that the risk variants possess secondary structure serving as a scaffold for tandem PKR binding and activation. These findings provide a mechanism by which APOL1 variants damage podocytes and suggest novel therapeutic strategies.
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http://dx.doi.org/10.1038/s42003-018-0188-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220249PMC
November 2018

Channel correlation and BER performance analysis of coherent optical communication systems with receive diversity over moderate-to-strong non-Kolmogorov turbulence.

Appl Opt 2018 Apr;57(11):2890-2899

In this paper, new expressions of the channel-correlation coefficient and its components (the large- and small-scale channel-correlation coefficients) for a plane wave are derived for a horizontal link in moderate-to-strong non-Kolmogorov turbulence using a generalized effective atmospheric spectrum which includes finite-turbulence inner and outer scales and high-wave-number "bump". The closed-form expression of the average bit error rate (BER) of the coherent free-space optical communication system is derived using the derived channel-correlation coefficients and an α-μ distribution to approximate the sum of the square root of arbitrarily correlated Gamma-Gamma random variables. Analytical results are provided to investigate the channel correlation and evaluate the average BER performance. The validity of the proposed approximation is illustrated by Monte Carlo simulations. This work will help with further investigation of the fading correlation in spatial diversity systems.
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http://dx.doi.org/10.1364/AO.57.002890DOI Listing
April 2018

Validating Candidate Congenital Heart Disease Genes in .

Bio Protoc 2017 Jun;7(12)

Center for Cancer and Immunology Research, Children's National Medical Center, 111 Michigan Ave. NW, Washington, DC, USA.

Genomic sequencing efforts can implicate large numbers of genes and mutations as potential disease risk factors. A high throughput model system to validate candidate gene association with pathology is therefore useful. We present such a system employing to validate candidate congenital heart disease (CHD) genes. The protocols exploit comprehensive libraries of UAS-GeneX-RNAi fly strains that when crossed into a 4×Hand-Gal4 genetic background afford highly efficient cardiac-specific knockdown of endogenous fly orthologs of human genes. A panel of quantitative assays evaluates phenotypic severity across multiple cardiac parameters. These include developmental lethality, larva and adult heart morphology, and adult longevity. These protocols were recently used to evaluate more than 100 candidate CHD genes implicated by patient whole-exome sequencing (Zhu ., 2017).
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http://dx.doi.org/10.21769/BioProtoc.2350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5739056PMC
June 2017

Retinoid-Related Orphan Receptor β and Transcriptional Control of Neuronal Differentiation.

Curr Top Dev Biol 2017 27;125:227-255. Epub 2016 Dec 27.

Laboratory of Endocrinology and Receptor Biology, NIDDK, National Institutes of Health, Bethesda, MD, United States. Electronic address:

The ability to generate neuronal diversity is central to the function of the nervous system. Here we discuss the key neurodevelopmental roles of retinoid-related orphan receptor β (RORβ) encoded by the Rorb (Nr1f2) gene. Recent studies have reported loss of function of the human RORB gene in cases of familial epilepsy and intellectual disability. Principal sites of expression of the Rorb gene in model species include sensory organs, the spinal cord, and brain regions that process sensory and circadian information. Genetic analyses in mice have indicated functions in circadian behavior, vision, and, at the cellular level, the differentiation of specific neuronal cell types. Studies in the retina and sensory areas of the cerebral cortex suggest that this orphan nuclear receptor acts at decisive steps in transcriptional hierarchies that determine neuronal diversity.
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http://dx.doi.org/10.1016/bs.ctdb.2016.11.009DOI Listing
March 2018

A Personalized Model of Nephropathy Rescued by the Wild-Type Allele or Dietary Coenzyme Q Supplementation.

J Am Soc Nephrol 2017 Sep 20;28(9):2607-2617. Epub 2017 Apr 20.

Centers for Cancer and Immunology Research and

Clinical studies have identified patients with nephrotic syndrome caused by mutations in genes involved in the biosynthesis of coenzyme Q (CoQ), a lipid component of the mitochondrial electron transport chain and an important antioxidant. However, the cellular mechanisms through which these mutations induce podocyte injury remain obscure. Here, we exploited the striking similarities between nephrocytes and human podocytes to develop a model of these renal diseases, and performed a systematic analysis assessing the role of CoQ pathway genes in renal function. Nephrocyte-specific silencing of , , and , which are genes involved in the CoQ pathway that have been associated with genetic nephrotic syndrome in humans, induced dramatic adverse changes in these cells. In particular, silencing of led to an abnormal localization of slit diaphragms, collapse of lacunar channels, and more dysmorphic mitochondria. In addition, -deficient nephrocytes showed elevated levels of autophagy and mitophagy, increased levels of reactive oxygen species, and increased sensitivity to oxidative stress. Dietary supplementation with CoQ at least partially rescued these defects. Furthermore, expressing the wild-type human gene specifically in nephrocytes rescued the defective protein uptake, but expressing the mutant allele derived from a patient with nephropathy did not. We conclude that transgenic lines carrying mutations in the CoQ pathway genes are clinically relevant models with which to explore the pathogenesis of podocyte injury and could serve as a new platform to test novel therapeutic approaches.
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http://dx.doi.org/10.1681/ASN.2016060626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576924PMC
September 2017

Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes.

Cell Tissue Res 2017 06 8;368(3):615-627. Epub 2017 Feb 8.

Center for Cancer and Immunology Research, Children's Research Institute, Children's National Medical Center, 111 Michigan Avenue NW, Washington, DC 20010, USA.

The Drosophila nephrocyte is a critical component of the fly renal system and bears structural and functional homology to podocytes and proximal tubule cells of the mammalian kidney. Investigations of nephrocyte cell biological processes are fundamental to understanding the insect renal system. Nephrocytes are highly active in endocytosis and vesicle trafficking. Rab GTPases regulate endocytosis and trafficking but specific functions of nephrocyte Rabs remain undefined. We analyzed Rab GTPase expression and function in Drosophila nephrocytes and found that 11 out of 27 Drosophila Rabs were required for normal activity. Rabs 1, 5, 7, 11 and 35 were most important. Gene silencing of the nephrocyte-specific Rab5 eliminated all intracellular vesicles and the specialized plasma membrane structures essential for nephrocyte function. Rab7 silencing dramatically increased clear vacuoles and reduced lysosomes. Rab11 silencing increased lysosomes and reduced clear vacuoles. Our results suggest that Rab5 mediates endocytosis that is essential for the maintenance of functionally critical nephrocyte plasma membrane structures and that Rabs 7 and 11 mediate alternative downstream vesicle trafficking pathways leading to protein degradation and membrane recycling, respectively. Elucidating molecular pathways underlying nephrocyte function has the potential to yield important insights into human kidney cell physiology and mechanisms of cell injury that lead to disease. The Drosophila nephrocyte is emerging as a useful in vivo model system for molecular target identification and initial testing of therapeutic approaches in humans.
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http://dx.doi.org/10.1007/s00441-017-2575-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5429992PMC
June 2017

A Drosophila model system to assess the function of human monogenic podocyte mutations that cause nephrotic syndrome.

Hum Mol Genet 2017 02;26(4):768-780

Center for Cancer and Immunology Research, Children's National Health Systems, 111 Michigan Ave. NW, Washington, DC, USA.

Many genetic mutations have been identified as monogenic causes of nephrotic syndrome (NS), but important knowledge gaps exist in the roles of these genes in kidney cell biology and renal diseases. More animal models are needed to assess the functions of these genes in vivo, and to determine how they cause NS in a timely manner. Drosophila nephrocytes and human podocytes share striking similarities, but to what degree these known NS genes play conserved roles in nephrocytes remains unknown. Here we systematically studied 40 genes associated with NS, including 7 that have not previously been analysed for renal function in an animal model. We found that 85% of these genes are required for nephrocyte functions, suggesting that a majority of human genes known to be associated with NS play conserved roles in renal function from flies to humans. To investigate functional conservation in more detail, we focused on Cindr, the fly homolog of the human NS gene CD2AP. Silencing Cindr in nephrocytes led to dramatic nephrocyte functional impairment and shortened life span, as well as collapse of nephrocyte lacunar channels and effacement of nephrocyte slit diaphragms. These phenotypes could be rescued by expression of a wild-type human CD2AP gene, but not a mutant allele derived from a patient with CD2AP-associated NS. We conclude that the Drosophila nephrocyte can be used to elucidate clinically relevant molecular mechanisms underlying the pathogenesis of most monogenic forms of NS, and to efficiently generate personalized in vivo models of genetic renal diseases bearing patient-specific mutations.
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http://dx.doi.org/10.1093/hmg/ddw428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6074792PMC
February 2017

High throughput in vivo functional validation of candidate congenital heart disease genes in .

Elife 2017 01 20;6. Epub 2017 Jan 20.

Center for Cancer and Immunology Research, Children's National Medical Center, Washington, United States.

Genomic sequencing has implicated large numbers of genes and de novo mutations as potential disease risk factors. A high throughput in vivo model system is needed to validate gene associations with pathology. We developed a -based functional system to screen candidate disease genes identified from Congenital Heart Disease (CHD) patients. 134 genes were tested in the heart using RNAi-based gene silencing. Quantitative analyses of multiple cardiac phenotypes demonstrated essential structural, functional, and developmental roles for more than 70 genes, including a subgroup encoding histone H3K4 modifying proteins. We also demonstrated the use of to evaluate cardiac phenotypes resulting from specific, patient-derived alleles of candidate disease genes. We describe the first high throughput in vivo validation system to screen candidate disease genes identified from patients. This approach has the potential to facilitate development of precision medicine approaches for CHD and other diseases associated with genetic factors.
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http://dx.doi.org/10.7554/eLife.22617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5300701PMC
January 2017

APOL1-G1 in Nephrocytes Induces Hypertrophy and Accelerates Cell Death.

J Am Soc Nephrol 2017 Apr 18;28(4):1106-1116. Epub 2016 Nov 18.

Center for Cancer and Immunology Research, Washington, DC;

People of African ancestry carrying certain APOL1 mutant alleles are at elevated risk of developing renal diseases. However, the mechanisms underlying -associated renal diseases are unknown. Because the gene is unique to humans and some primates, new animal models are needed to understand the function of APOL1 We generated transgenic fly lines expressing the human wild type allele (G0) or the predominant risk allele (G1) in different tissues. Ubiquitous expression of G0 or G1 in induced lethal phenotypes, and G1 was more toxic than was G0. Selective expression of the G0 or G1 transgene in nephrocytes, fly cells homologous to mammalian podocytes, induced increased endocytic activity and accumulation of hemolymph proteins, dextran particles, and silver nitrate. As transgenic flies with either allele aged, nephrocyte function declined, cell size increased, and nephrocytes died prematurely. Compared with G0-expressing cells, however, G1-expressing cells showed more dramatic phenotypes, resembling those observed in cultured mammalian podocytes overexpressing APOL1-G1. Expressing the G0 or G1 transgene in nephrocytes also impaired the acidification of organelles. We conclude that expression of an transgene initially enhances nephrocyte function, causing hypertrophy and subsequent cell death. This new model uncovers a novel mechanism by which upregulated expression of APOL1-G1 could precipitate renal disease in humans. Furthermore, this model may facilitate the identification of APOL1-interacting molecules that could serve as new drug targets to treat -associated renal diseases.
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http://dx.doi.org/10.1681/ASN.2016050550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373456PMC
April 2017

Wnt4 is required for ostia development in the Drosophila heart.

Dev Biol 2016 05 16;413(2):188-98. Epub 2016 Mar 16.

Center for Cancer and Immunology Research, Children's Research Institute, Children's National Medical Center, 111 Michigan Ave. NW, Washington, DC 20010, USA; Department of Pediatrics, George Washington University School of Medicine and Health Sciences, Washington, DC 20010, USA. Electronic address:

The Drosophila ostia are valve-like structures in the heart with functional similarity to vertebrate cardiac valves. The Wnt/β-catenin signaling pathway is critical for valve development in zebrafish and mouse, but the key ligand(s) for valve induction remains unclear. We observed high levels of Wnt4 gene expression in Drosophila ostia progenitor cells, immediately prior to morphological differentiation of these cells associated with ostia formation. This differentiation was blocked in Wnt4 mutants and in flies expressing canonical Wnt signaling pathway inhibitors but not inhibitors of the planar cell polarity pathway. High levels of Wnt4 dependent activation of a canonical Wnt signaling reporter was observed specifically in ostia progenitor cells. In vertebrate valve formation Wnt signaling is active in cells undergoing early endothelial-mesenchymal transition (EMT) and the Wnt9 homolog of Drosophila Wnt4 is expressed in valve progenitors. In demonstrating an essential role for Wnt4 in ostia development we have identified similarities between molecular and cellular events associated with early EMT during vertebrate valve development and the differentiation and partial delamination of ostia progenitor cells in the process of ostia formation.
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http://dx.doi.org/10.1016/j.ydbio.2016.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4857614PMC
May 2016

Feedback induction of a photoreceptor-specific isoform of retinoid-related orphan nuclear receptor β by the rod transcription factor NRL.

J Biol Chem 2014 Nov 7;289(47):32469-80. Epub 2014 Oct 7.

From the Laboratory of Endocrinology and Receptor Biology, NIDDK, and

Vision requires the generation of cone and rod photoreceptors that function in daylight and dim light, respectively. The neural retina leucine zipper factor (NRL) transcription factor critically controls photoreceptor fates as it stimulates rod differentiation and suppresses cone differentiation. However, the controls over NRL induction that balance rod and cone fates remain unclear. We have reported previously that the retinoid-related orphan receptor β gene (Rorb) is required for Nrl expression and other retinal functions. We show that Rorb differentially expresses two isoforms: RORβ2 in photoreceptors and RORβ1 in photoreceptors, progenitor cells, and other cell types. Deletion of RORβ2 or RORβ1 increased the cone:rod ratio ∼2-fold, whereas deletion of both isoforms in Rorb(-/-) mice produced almost exclusively cone-like cells at the expense of rods, suggesting that both isoforms induce Nrl. Electroporation of either RORβ isoform into retinal explants from Rorb(-/-) neonates reactivated Nrl and rod genes but, in Nrl(-/-) explants, failed to reactivate rod genes, indicating that NRL is the effector for both RORβ isoforms in rod differentiation. Unexpectedly, RORβ2 expression was lost in Nrl(-/-) mice. Moreover, NRL activated the RORβ2-specific promoter of Rorb, indicating that NRL activates Rorb, its own inducer gene. We suggest that feedback activation between Nrl and Rorb genes reinforces the commitment to rod differentiation.
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http://dx.doi.org/10.1074/jbc.M114.605774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4239602PMC
November 2014

An isoform of retinoid-related orphan receptor β directs differentiation of retinal amacrine and horizontal interneurons.

Nat Commun 2013 ;4:1813

Laboratory of Endocrinology and Receptor Biology, National Institutes of Health, NIDDK, 10 Center Drive, Bethesda, Maryland 20892-1772, USA.

Amacrine and horizontal interneurons integrate visual information as it is relayed through the retina from the photoreceptors to the ganglion cells. The early steps that generate these interneuron networks remain unclear. Here we show that a distinct retinoid-related orphan nuclear receptor β1 (RORβ1) isoform encoded by the retinoid-related orphan nuclear receptor β gene (Rorb) is critical for both amacrine and horizontal cell differentiation in mice. A fluorescent protein cassette targeted into Rorb revealed RORβ1 as a novel marker of immature amacrine and horizontal cells and of undifferentiated, dividing progenitor cells. RORβ1-deficient mice lose expression of pancreas-specific transcription factor 1a (Ptf1a) but retain forkhead box n4 factor (Foxn4), two early-acting factors necessary for amacrine and horizontal cell generation. RORβ1 and Foxn4 synergistically induce Ptf1a expression, suggesting a central role for RORβ1 in a transcriptional hierarchy that directs this interneuron differentiation pathway. Moreover, ectopic RORβ1 expression in neonatal retina promotes amacrine cell differentiation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3671912PMC
http://dx.doi.org/10.1038/ncomms2793DOI Listing
December 2013

Loss of Bicc1 impairs tubulomorphogenesis of cultured IMCD cells by disrupting E-cadherin-based cell-cell adhesion.

Eur J Cell Biol 2010 Jun 9;89(6):428-36. Epub 2010 Mar 9.

Division of Translational Cancer Research and Therapy, State Key Laboratory of Molecular Oncology, Cancer Hospital and Institute, Chinese Academy of Medical Sciences, Beijing 100021, China.

The Bicaudal-C (Bic-C) gene was originally discovered in Drosophila melanogaster. The gene product Bic-C is thought to serve as an RNA-binding molecule targeting diverse proteins at the post-transcriptional level. Recent research has shown this gene to be conserved in many species, from Caenorhabditis elegans to humans. Disruption of this protein can disturb the normal migration direction of the anterior follicle cell of Drosophila oocytes, while mutation of a mouse Bicc1 (a mouse homologue of Bic-C) results in phenotypes mimicking human hereditary polycystic kidney disease (PKD). However, the cellular function of Bicc1 gene products in mammalian systems remains largely unknown. In this study, we established stable IMCD (mouse inner medullary collecting duct) cell lines, in which Bicc1 was silenced by short hairpin RNA inhibition (shRNA). We show that inhibition of Bicc1 disrupted normal tubulomorphogenesis and induced cystogenesis of IMCD cells grown in three dimensional cultures. To determine what factors contributed to the defect, we systematically examined biological changes of Bicc1-silenced IMCD cells. We found that the cells had significant defects in E-cadherin-based cell-cell adhesion, along with abnormalities in actin cytoskeleton organization, cell-extracellular matrix interactions, cell proliferation, and apoptosis. These findings suggest that lack of Bicc1 leads to disruption of normal cell-cell junctions, which in turn impedes establishment of epithelial polarity. These cellular defects may initiate abnormal tubulomorphogenesis and cystogenesis of IMCD cells grown in vitro. The observation of aberrant cellular behaviors in Bicc1-silenced IMCD cells reveal functions for Bicc1 in renal epithelial cells and provides insight into a potential pathogenic mechanism of polycystic kidney disease.
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http://dx.doi.org/10.1016/j.ejcb.2010.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2886128PMC
June 2010