Oryza sativa) receptor-like cytoplasmic kinase subfamily-VII protein, contributes to chitin-triggered immune responses. It is valuable for agriculture because its overexpression confers strong disease resistance to fungal and bacterial pathogens. However, it remains unclear how overexpressed BSR1 reinforces plant immunity. Here we analyzed immune responses using rice suspension-cultured cells and sliced leaf blades overexpressing BSR1. BSR1 overexpression enhances MAMP-triggered production of hydrogen peroxide (H2O2) and transcriptional activation of the defense-related gene in cultured cells and leaf strips. Furthermore, the co-cultivation of leaves with conidia of the blast fungus revealed that BSR1 overexpression allowed host plants to produce detectable oxidative bursts against compatible pathogens. BSR1 was also involved in the immune responses triggered by peptidoglycan and lipopolysaccharide. Thus, we concluded that the hyperactivation of MAMP-triggered immune responses confers BSR1-mediated robust resistance to broad-spectrum pathogens.Plants activate their immune system through intracellular signaling pathways after perceiving microbe-associated molecular patterns (MAMPs). Receptor-like cytoplasmic kinases mediate the intracellular signaling downstream of pattern-recognition receptors. BROAD-SPECTRUM RESISTANCE 1 (BSR1), a rice ( Oryza sativa), CHITIN ELICITOR RECEPTOR KINASE 1 (OsCERK1), a lysine motif (LysM)-receptor-like kinase (RLK), is well-characterized as a protein component of PRRs. OsCERK1 interacts with a receptor-like protein CHITIN ELICITOR BINDING PROTEIN (CEBiP) [2O2), an ROS produced in oxidative bursts, acts as a signaling molecule to induce the transcriptional activation of defense-related genes, biosynthesis of phytoalexin, and programmed cell death [Plants combat pathogens by activating their innate immunity. Microbe/pathogen-associated molecular patterns (MAMPs/PAMPs), which have molecular structures that are conserved in fungi or bacteria, alert plants to pathogen attacks. Chitin , peptidoglycan , lipopolysaccharide , flagellin, and elongation factor-Tu (EF-Tu) are well-known MAMPs . MAMPs a (CEBiP)  to recog (CEBiP) ,5,6 and  (CEBiP) . OsCERK1 (CEBiP) . Thus, t (CEBiP) ,10. Oxid (CEBiP) . Host-de (CEBiP) ,13,14,15ll death ,16.Ustilago maydis to overcome PTI [YAP, encoding an oxidative stress-responsive transcription factor, in U. maydis increases the sensitivity to H2O2 and significantly decreases the pathogenicity. Rice blast fungus (Pyricularia oryzae), which causes a serious disease in rice, releases a catalase-peroxidase B (CPXB)-dependent ROS-degrading activity near conidia [P. oryzae are required for virulence as well as resistance to ROS [P. oryzae mutant strains Δdes1 and Δsir2, which lack transcriptional regulators for extracellular peroxidases and superoxide dismutase, cannot form susceptible lesions because they induce defense responses, including the accumulation of host-derived ROS and the upregulation of defense-related genes [Pathogens have adapted to suppress PTI through the secretion of effectors using type III secretion system (TTSS) and structural variations on MAMPs . Molina come PTI . The del conidia ,20. Enzye to ROS ,22. P. oed genes ,24. ThesArabidopsis thaliana and rice have 147 and 379 RLCK-encoding genes, respectively [A. thaliana, BOTRYTIS-INDUCED KINASE 1 (BIK1) is phosphorylated by PRRs composed of BRI1-ASSOCIATED KINASE 1 (BAK1) and FLAGELLIN SENSING 2 (FLS2) or EF-Tu RECEPTOR (EFR) depending on the ligand -binding [Receptor-like cytoplasmic kinases (RLCKs) contribute to cytoplasmic phosphorylation signaling pathways in PTI. RLCKs are characterized as cytoplasmic proteins that contain a RLK-homologous kinase domain but not a transmembrane domain. ectively ,26. RLCKectively . In A. t-binding ,29. Phos-binding ,31. In r-binding . OsRLCK1-binding . OsRLCK1-binding ,35.Arabidopsis lines [A. thaliana plants overexpressing BSR1 were highly resistant to Pseudomonas syringae pv. tomato DC3000 and Colletotrichum higginsianum. Furthermore, overexpression of BSR1 in rice conferred strong resistance against four rice pathogens: rice blast fungus, brown spot fungus (Cochliobolus miyabeanus), rice leaf blight bacteria (Xanthomonas oryzae pv. oryzae), and Burkholderia glumae, which is the causal agent of bacterial seedling rot and bacterial grain rot [BROAD-SPECTRUM RESISTANCE 1 , a rice RLCK-VII protein, has unique disease control effects when overexpressed. It was identified in a screen for disease resistance in rice Full-length cDNA OvereXpressor (FOX) is lines ,37. The rain rot ,38. To oBSR1 caused significant suppression of chitin-induced defense-responses, including oxidative bursts and the transcriptional activation of defense-related genes [BSR1 decreased resistance to not only fungal but also bacterial diseases [The contribution of BSR1 to the innate immunity of wild-type rice has been analyzed. A knockout of ed genes . BSR1 haed genes . These ied genes . The sildiseases , suggestIn this report, we investigated whether BSR1 contributes to defense responses elicited by bacterial MAMPs. The resulting resistance is almost independent of salicylic acid, a plant hormone related to immunity . TherefoBSR1-knockout lines. These lines were generated in our previous study and contain homozygous frameshift mutations in exon 1 of BSR1 [BSR1-knockout lines produced lower H2O2 concentrations than wild-type cells , DP biosynthetic key transcription factor-encoding gene DITERPENOID PHYTOALEXIN FACTOR (DPF) [PROBENAZOLE-INDUCIBLE PROTEIN 1 (PBZ1), and flavonoid phytoalexin and lignin biosynthetic gene PHENYLALANINE AMMONIA-LYASE 1 (PAL1) were determined. After treatment with peptidoglycan, the inductions of KSL4, DPF, and PBZ1 in knockout cells were significantly weaker than in wild-type, although significant changes in PAL1 transcript level were not detected  momilactone biosynthetic gene OR (DPF) , the repdetected a. Knockinditions . Our liqBSR1 suppressed the elicitation of KSL4 and DPF by LPS -tagged BSR1 and GUS  were generated. The GUS-HPB:OX line was used as a control. The integrities of the inserted constructs were confirmed by western analysis with an anti-HA antibody . These phenotypes were common to all the replicated experiments  of conidia. Considering that the H2O2-degrading activity increased with the conidial concentration, comparisons between H2O2 levels were performed only under the same co-cultivation conditions. When co-cultivated with 8 × 103 mL−1 conidia, no elevation in the H2O2 level was detected in GUS-HPB:OX leaves  was used as the wild-type (WT) plant material. The BSR1-knockout lines bsr1-1#13-1 (KO#1), bsr1-2#16-2 (KO#2), and bsr1-8#5-1 (KO#8) generated with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system in our previous study [Pyricularia oryzae isolate Kyu89-246 , which is compatible with Nipponbare rice plants, was used to prepare the elicitor and for inoculations. Xanthomonas oryzae pv. oryzae (isolate T7174) was used to prepare the elicitor fraction. The culturing methods and P. oryzae and X. oryzae pv. oryzae inoculation techniques were as previously described [Rice  and pBI221 (AF502128), respectively. These DNA fragments were ligated into pENTR/D-TOPO  to construct HPB-tagged BSR1 and GUS  in pMDC32-Mubi-HPB using the Gateway LR Clonase II Plus Enzyme (Invitrogen). pMDC32-Mubi-HPB containing BSR1-HPB or GUS-HPB was introduced into Nipponbare using the Rhizobium radiobacter-mediated transformation method [A DNA fragment containing the maize ateA-SG1  with HinateA-SG1  was digen method . BSR1-HP−1 sucrose; 4.1 mg L−1 N6 salt ; 2 mg L−1 glycine; 0.5 mg L−1 nicotinic acid; 0.5 mg L−1 pyridoxine HCl; 1 mg L−1 thiamine HCl; 100 mg L−1 myo-inositol; 1 mg L−1 2,4-dichlorophenoxyacetic acid; 23.4 mg L−1 MnSO4·4H2O; pH 5.8) was used for liquid cultivation. We treated 1 mL media containing 100 mg suspension-cultured cells with 10 µg mL−1 peptidoglycan from Bacillus subtilis , 50 µg mL−1 LPS from Pseudomonas aeruginosa 10 purified by phenol extraction (Sigma-Aldrich), 10 nM N-acetylchitohexaose (chitin elicitor (CE)), or an autoclaved suspension of X. oryzae pv. oryzae (OD600 = 0.3).Rice suspension-cultured cells were prepared using a previously published method ,39. Modi2O2 concentration was determined at the indicated time using a previously described luminol-dependent chemiluminescence assay [t-test for the experiments with leaf strips.To prepare leaf strips, the sixth leaves of GUS-HPB:OX and BSR1-HPB:OX17 plants at the 6–6.5-leaf stage were used. Two fragments of the leaf blades (8-mm length and 6-mm width) that were slit using bundled razor blades at approximately 0.5-mm intervals were placed in a well of a 12-well plate. Leaf strips were floated on sterile water and incubated at 28 °C for 14–15 h with shaking at 90 rpm, followed by a 1-h incubation in new water. Conidia of rice blast fungus were scraped from the gel surface with sterile water and filtered through a Kimwipe. The conidial concentration in the filtrate was calculated using a hemocytometer. A suspension of autoclaved or living conidia was poured into the wells to the indicated final concentration and incubated at 28 °C. The H−1 peptidoglycan, 50 µg mL−1 LPS, 10 nM N-acetylchitohexaose, an autoclaved suspension of X. oryzae pv. oryzae (OD600 = 0.3), or sterile water. Total RNA extraction and the qRT-PCR were performed as previously described [T (2−ΔΔCt) method with rice Ubiquitin1  as an internal control [Cultured rice cells were frozen in liquid nitrogen after a 3-h treatment with 10 µg mLescribed . Transcr control . The staProtein was extracted from 50 mg leaf blades with 300 µL SDS–urea buffer , 2 × complete inhibitor mix, EDTA-free , 40 mM Tris-HCl: pH 6.8) according to a previously described method . An equa


Correction to:Cell Death Discovery (2018)10.1038/s41420-018-0114-xpublished online 22 October 2018The Article contains an error in Fig. The correct Fig. 2a appears below


SLC34A2-ROS1 (solute carrier family 34 member 2 and ROS proto-oncogene 1) plays an important role in non-small cell lung cancer (NSCLC) progression. However, whether SLC34A2-ROS1 gene can produce circRNA remains unknown. Here, we identified two novel circRNAs (F-circSR1 and F-circSR2) generated from SLC34A2-ROS1 fusion gene, while F-circSR1 has higher expression than F-circSR2. Functional studies through gain- and loss-of-function strategies showed that both F-circSRs promote cell migration in lung cancer cells, whereas they have little effect on cell proliferation. Using the minigene GFP reporter assay, we verified that the flanking complementary sequences with canonical splicing sites are essential for F-circSR biogenesis. Therefore, our findings demonstrate the oncogenic role of F-circSR in NSCLC and highlight its therapeutic potential.Cancer-associated chromosomal translocations are reported to generate oncogenic circular RNA (circRNA), contributing to tumorigenesis. The fusion gene The online version of this article (10.1186/s12943-019-1028-9) contains supplementary material, which is available to authorized users. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer worldwide, accounting for approximately 85% of lung cancers . DespiteROS1 fusion genes from aberrant chromosomal translocations of two separated genes. Among them, SLC34A2-ROS1 fusion gene encodes oncogenic fusion protein to activate downstream signaling cascades such as JAK/STAT, PI3K/Akt and RAS/RAF pathways and promote cell proliferation and survival [SLC34A2-ROS1 gene during tumorigenesis remains unclear.A subtype of NSCLC harbors PML-RARα fusion gene produces f-circPR to promote cell growth in acute promyelocytic leukemia, while MLL-AF9 fusion gene generates f-circM9 that contributes to leukemia progression in vitro and in vivo [EML4-ALK fusion gene can produce two circRNAs [SLC34A2-ROS1 gene generates circRNA is unknown. In this study, we identify the novel circRNA F-circSRs generated from SLC34A2-ROS1 gene. Moreover, F-circSRs, independent from SLC34A2-ROS1 fusion protein, have little effect on cell proliferation, but promote cell migration in lung cancer cells, highlighting the oncogenic role of F-circSRs in NSCLC.Emerging evidences demonstrated that, except for encoding fusion protein, fusion gene could generate circular RNA (circRNA), a covalently-bonded RNA molecule from the back-splicing of linear RNA, to participate in tumor initiation and progression. For example,  in vivo . RecentlcircRNAs , one of circRNAs . HoweverSLC34A2-ROS1 fusion genes expressing long and short transcripts  . As shown in Fig. SLC34A2 exon 2 and 3′ tail of ROS1 exon 37 or 42. Moreover, qPCR results showed that F-circSR1 has much higher expression level than F-circSR2 in HCC78 cells  targeting the back-splicing junction of circRNAs were designed to efficiently knock down F-circSR expression  generated from Additional file 1:Information about primers, siRNAs and full sequence of F-circSR. (DOCX 40 kb)Additional file 2:Figure S1. Absolute quantification of F-circSRs in HCC78 cells using qPCR. Figure S2. Characterization of cellular function of F-circSR in lung cancer cells. Figure S3. Validation of cellular function of F-circSR using pLaccase2 circRNA expression system. Figure S4. Predicted miRNA biding sites in F-circSRs. (DOCX 2870 kb)Additional file 3:Bioinformatics analysis of the cis-elements in the flanking introns of F-circSR. (DOCX 1084 kb)Additional file 4:Supplementary Materials and Methods. (DOCX 2750 kb)


Here, FSC has been further derivatized to optimize the complexation properties of FSC-based chelators for 89Zr-labeling by introducing additional carboxylic groups. These were expected to improve the stability of 89Zr-complexes by saturating the 8-coordination sphere of [89Zr] Zr4+, and also to introduce functionalities suitable for conjugation to targeting vectors such as monoclonal antibodies. For proof of concept, succinic acid derivatization at the amine groups of FSC was carried out, resulting in FSC(succ)2 and FSC(succ)3. FSC(succ)2 was further derivatized to FSC(succ)2 AA by reacting with acetic anhydride (AA). The Zr4+ complexation properties of these chelators were studied by reacting with ZrCl4. Partition coefficient, protein binding, serum stability, acid dissociation, and transchelation studies of 89Zr-complexes were carried out in vitro and the results were compared with those for 89Zr-desferrioxamine B ([89Zr]Zr-DFO) and 89Zr-triacetylfusarinine C ([89Zr]Zr-TAFC). The in vivo properties of [89Zr]Zr-FSC(succ)3 were further compared with [89Zr]Zr-TAFC in BALB/c mice using micro-positron emission tomography/computer tomography (microPET/CT) imaging. Fusarinine C (succ)2AA and FSC(succ)3 were synthesized with satisfactory yields. Complexation with ZrCl4 was achieved using a simple strategy resulting in high-purity Zr-FSC(succ)2AA and Zr-FSC(succ)3 with 1:1 stoichiometry. Distribution coefficients of 89Zr-complexes revealed increased hydrophilic character compared to [89Zr]Zr-TAFC. All radioligands showed high stability in phosphate buffered saline (PBS) and human serum and low protein-bound activity over a period of seven days. Acid dissociation and transchelation studies exhibited a range of in vitro stabilities following the order: [89Zr]Zr-FSC(succ)3 > [89Zr]Zr-TAFC > [89Zr]Zr-FSC(succ)2AA >> [89Zr]Zr-DFO. Biodistribution studies of [89Zr]Zr-FSC(succ)3 revealed a slower excretion pattern compared to [89Zr]Zr-TAFC. In conclusion, [89Zr]Zr-FSC(succ)3 showed the best stability and inertness. The promising results obtained with [89Zr]Zr-FSC(succ)2AA highlight the potential of FSC(succ)2 as a monovalent chelator for conjugation to targeted biomolecules, in particular, monoclonal antibodies.Fusarinine C (FSC) has recently been shown to be a promising and novel chelator for  The physical half-life of 78.4 h is a good match with the biological half-life of mAbs, thus increasing the utility of PET in the development of this class of compound, and its medium mean positron energy of 0.395 MeV allows high-resolution PET imaging Zr4+ from the chelating system [89Zr]Zr4+ has been reported to accumulate in bone, particularly at late time points (3–7 days), which results in a decrease in image contrast, as well as an increase in radiation dose, especially to bone marrow Zr4+. Another potential drawback of FSC as a 89Zr-bifunctional chelator lies in it having three functionalities for derivatization, which, despite facilitating the employment of multivalent concepts for small molecules, are not suitable for conjugation to macromolecules, especially antibodies. Therefore, a further aim of this study was to design a FSC-based mono-functional 89Zr-chelator. As proof of concept, modifications via succinic acid derivatization resulting in FSC(succ)2 acetic anhydride (AA) and FSC(succ)3, see 89Zr-complexes were investigated and compared with [89Zr]Zr-DFO and 89Zr-triacetylfusarinine C ([89Zr]Zr-TAFC). The in vivo properties of [89Zr]Zr-FSC(succ)3 were further compared with [89Zr]Zr-TAFC in BALB/c mice.Recently, we reported that fusarinine C (FSC), which has a 36-membered ring structure and three bidentate hydroxamates to coordinate for 89Zr ,24,25. H89Zr]Zr-oxalate was purchased from Perkin Elmer, Inc  with an activity concentration of 900–1000 MBq per milliliter.All commercially available reagents were of reagent grade and used as supplied with no further purification. Desferrioxamine B was obtained from Genaxxon Bioscience GmbH . Triacetylfusarinine C was prepared and purified as described previously . [89Zr]Z18A 300 Å column  with a flow rate of 3.0 mL/min. The gradient was as follows: 0–1.0 min 10% acetonitrile (CH3CN), 1.0–18.0 min 10–40% CH3CN (gradient A).Purification of compounds was performed via a preparative reversed-phase high-performance liquid chromatography system (preparative RP-HPLC) using a Gilson 322 HPLC pump, a Gilson ultraviolet (UV)/VIS-155 detector , and a Eurosil Bioselect Vertex Plus 300 × 8 mm 5 μm C3CN/H2O/0.1% trifluoroacetic acid (TFA) gradient: 0–0.5 min 0% CH3CN/0.1% TFA, 0.5–7.0 min, 0–55% CH3CN/0.1% TFA (gradient B).Analytical RP-HPLC analysis was performed with an UltiMate 3000 RS HPLC pump, an UltiMate 3000 RS column compartment (column oven temperature was set at 25 °C), an UltiMate 3000 UV-Vis variable wavelength detector , and a Raytest radiometric detector . A Vydac 218 TP5215, 150 × 3.0 mm column , flow rate 1.0 mL/min, and UV-Vis detection at 220/410 nm were employed with the following CHAspergillus fumigatus mutant strain ∆sidG was cultured in iron-free minimal medium with 1% glucose for 36 h at 37 °C and 200 rpm. Biomass was removed by filtration. The media-containing siderophore was collected and concentrated. Additional purification was performed via preparative RP-HPLC  followed by concentration and lyophilization. Analytical RP-HPLC (gradient B): tR = 4.7 min.Fusarinine C was produced as described previously . BrieflyN,N-diisopropylethylamine (DIPEA). After shaking for 0.5 h, FSC(succ)3 was directly isolated via preparative RP-HPLC  and confirmed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The collected fractions were concentrated and lyophilized. FSC(succ)3: yield: 63 mg, (61 µmol), 88%; MALDI TOF-MS: [M + H]+ = 1028.2 . Analytical RP-HPLC (gradient B): tR = 5.6 min.To FSC  dissolved in 1 mL dimethylformamide (DMF), 34.5 mg of succinic anhydride  was added and the pH was adjusted to 4.5 using 2 was the same as that of FSC(succ)3 except that a 2 eq molar excess of succinic anhydride was used. FSC(succ)2 was isolated from the byproducts (FSC-succ and FSC(succ)3) via preparative RP-HPLC . FSC(succ)2: yield: 28 mg, (30 µmol), 43%. MALDI TOF-MS: [M + H]+ = 928.2 . Analytical RP-HPLC (gradient B): tR = 5.4 min.The general synthesis procedure of FSC(succ)2  dissolved in 0.5 mL DMF, 1 mL of acetic anhydride (AA) was added. After shaking for 10 min, FSC(succ)2AA was quickly isolated via preparative RP-HPLC  and confirmed by MALDI-TOF-MS. The collected fractions were concentrated and lyophilized. FSC(succ)2AA: yield: 9 mg, (9.3 µmol), 85%. MALDI TOF-MS: [M + H]+ = 970.1 . Analytical RP-HPLC (gradient B): tR = 5.6 min.To FSC(succ)4 (121 mg) was dissolved in 1 mL 0.1 M hydrochloric acid solution resulting in a concentration of 0.52 M. After dissolving FSC(succ)2AA  or FSC(succ)3  in 1 mL water, a 1.2 eq molar excess of ZrCl4  was added. The reaction solutions were shaken gently for 5 min, then loaded onto a pre-activated Sep-Pak C18 cartridge. After washing the cartridge with 10 mL water, Zr-FSC(succ)2AA and Zr-FSC(succ)3 were eluted with 1mL methanol. The eluate fractions were dried by evaporation under nitrogen and weighed. The identities of the products were confirmed by electrospray ionization mass spectrometry (ESI-MS). Zr-FSC(succ)2AA: yield: 3.6 mg, (3.4 µmol), 65%. ESI-MS: [M + H]+ = 1,055.3 + = 1113.3 Zr-oxalate was mixed with 27 μL of sodium carbonate  and incubated for 3 min at room temperature (RT) [2AA (48.5 μg) or FSC(succ)3 (51.4 μg) was added to the reaction vial. The labeling mixture was allowed to react at RT with a pH between 6.8 and 7.2 for 90 min. The reaction progress was monitored via analytical RP-HPLC (gradient B): [89Zr]Zr-DFO: tR = 4.8 min, [89Zr]Zr-TAFC: tR = 5.9 min, [89Zr]Zr-FSC(succ)2AA: tR = 6.1 min, and [89Zr]Zr-FSC(succ)3: tR = 6.0 min.Approximately 30 MBq (30 µL) of Zr-FSC(succ)2AA or [89Zr]Zr-FSC(succ)3) was combined with 500 µL octanol. The mixture was vortexed for 15 min, and centrifuged for 2 min at 2000 rcf. Subsequently, 50 µL aliquots of the aqueous and the octanol layer were collected, measured in a 2480 Wizard2 Automatic Gamma Counter , and distribution coefficient (logD) values were calculated using Excel 2013 (n = 5) .A volume of 500 µL PBS including 0.5 MBq radioligand 2AA and [89Zr]Zr-FSC(succ)3 was carried out by incubating the radioligands in PBS, a 1000-fold molar excess of ethylenediaminetetraacetic acid (EDTA) solution (radioligand vs. EDTA: 25 µM vs. 25 mM) with different pH , as well as in human serum  for seven days at 37 °C. At selected time points, PBS and EDTA samples were analyzed in triplicate directly via analytical RP-HPLC, while serum aliquots were mixed with 500 µL of CH3CN, vortexed, and centrifuged at 20,000 rcf for 2 min. Then, the precipitate was washed three times using CH3CN before analysis.Determination of the stability of [89Zr]Zr-DFO (50 µL) was mixed with a 1000-fold molar excess of either FSC(succ)2AA or FSC(succ)3 and [89Zr]Zr-TAFC (or [89Zr]Zr-FSC(succ)2AA, [89Zr]Zr-FSC(succ)3) (50 µL each) was mixed with a 1000-fold molar excess of DFO and incubated for seven days at RT. At selected time points, aliquots of the solutions were analyzed in triplicate directly via analytical RP-HPLC. The transchelation was determined by the ratio of [89Zr]Zr-DFO and [89Zr]Zr-FSC derivatives.[89Zr]Zr-FSC(succ)2AA or [89Zr]Zr-FSC(succ)3 for seven days at 37 °C in fresh human serum. Subsequently, 30 µL of the solution was passed through a size exclusion spin column  via centrifugation at 2000 rcf for 2 min. Protein binding of the complexes in triplicate was determined by measuring the activity on the column (non-protein bound) and the activity in the eluate (protein bound) in the gamma counter.The protein binding property was evaluated by incubating Zr-FSC(succ)3  or [89Zr]Zr-TAFC  into the tail vein and sacrificed by cervical dislocation at 6 h post injection (p.i.). Organs , blood, muscle tissue, and bone were dissected, weighed and then measured in the gamma counter to calculate the percentage of injected dose per gram of tissue (% ID/g). Statistical analysis was performed using an independent two-population t-test (significance level p = 0.05) and Origin 6.1 software .For the evaluation of biodistribution, four female BALB/c mice  were intravenously injected with [89Zr]Zr-FSC(succ)3  or [89Zr]Zr-TAFC . MicroPET/CT images were acquired under general anesthesia (isoflurane/O2) for 20 min. Imaging data were recorded via static scans at 80 min and 24 h p.i. The microPET/CT scans were reconstructed and merged with OSEM3D-SPMAP  and Feldkamp . For evaluation of bone uptake, image-based quantitation was carried out by selecting volumes of interest around each knee region and % ID/cm3 were calculated for both time points  and expressed as mean values of left and right knees to compare both compounds.Micro-Positron Emission Tomography/computer tomography (MicroPET/CT) imaging studies were carried out using an Inveon microPET/CT scanner . Healthy female BALB/c mice were injected intravenously with 5 MBq of either Zr-DFO and [89Zr]Zr-TAFC reported previously in which [89Zr]Zr-DFO and [89Zr]Zr-TAFC remain at the origin and free [89Zr]Zr4+ migrates with the solvent front). This change was due to the undesirable spread of [89Zr]Zr-FSC(succ)2AA and [89Zr]Zr-FSC(succ)3 along the ITLC strips.The escribed . Quantit2AA and FSC(succ)3 showed high solubility in water, which is comparable to TAFC but different from recently reported novel octadentate chelators which seem to have poor solubility [89Zr]Zr-FSC(succ)2AA and [89Zr]Zr-FSC(succ)3  indicate higher hydrophilic properties than [89Zr]Zr-DFO and [89Zr]Zr-TAFC . All radioligands showed high stability in PBS and human serum over a period of seven days and no demetallation was observed. The low protein-bound activity (<10%) over the whole monitoring period further confirmed the stability of radioligands in human serum. The logD, protein binding and stability in human serum data for [89Zr]Zr-FSC(succ)2AA and [89Zr]Zr-FSC(succ)3 are summarized in 89Zr]Zr-DFO and [89Zr]Zr-TAFC [Fusarinine C(succ)lubility . The low]Zr-TAFC .89Zr]Zr-DFO, [89Zr]Zr-TAFC, [89Zr]Zr-FSC(succ)2AA and [89Zr]Zr-FSC(succ)3. Data of dissoctiation experiments are summarized in 89Zr]Zr-TAFC, [89Zr]Zr-FSC(succ)2AA and [89Zr]Zr-FSC(succ)3 revealed excellent resistance to transchelation over seven days, in contrast to that for [89Zr]Zr-DFO where almost 60% transchelation occurred, see 89Zr]Zr-FSC(succ)3 remained intact (97.8%), being more stable than [89Zr]Zr-TAFC (94.4%) and significantly higher than [89Zr]Zr-FSC(succ)2AA (76%), see 89Zr]Zr-FSC(succ)3 and [89Zr]Zr-TAFC was more pronounced , see Acid dissociation experiments and transchelation studies were performed to compare the relative kinetic inertness of [89Zr]Zr-DFO in a 1000-fold molar excess of FSC(succ)2AA or FSC(succ)3, or (B) [89Zr]Zr-TAFC, [89Zr]Zr-FSC(succ)2AA and [89Zr]Zr-FSC(succ)3 incubated in a 1000-fold molar excess of DFO. The transchelation ratio of (A) 89Zr from [89Zr]Zr-DFO to FSC(succ)2AA and FSC(succ)3 or (B) from [89Zr]Zr-TAFC, [89Zr]Zr-FSC(succ)2AA and [89Zr]Zr-FSC(succ)3 to DFO was easily calculated due to the significant difference of the retention time of radioligands. Almost quantitative transchelation of 89Zr from [89Zr]Zr-DFO to FSC(succ)2AA or FSC(succ)3 was observed within 1 h. In contrast, [89Zr]Zr-TAFC, [89Zr]Zr-FSC(succ)2AA and [89Zr]Zr-FSC(succ)3 showed a stronger resistance to transchelation to DFO. After 7 days, [89Zr]Zr-FSC(succ)3 showed the highest kinetic inertness (62.1% of intact complex) compared with [89Zr]Zr-TAFC and [89Zr]Zr-FSC(succ)2AA .Transchelation experiments are shown in 89Zr]Zr-FSC(succ)3 and [89Zr]Zr-TAFC were further compared in vivo. Biodistribution data of [89Zr]Zr-FSC(succ)3 and [89Zr]TAFC are presented in 89Zr]Zr-FSC(succ)3 than for [89Zr]Zr-TAFC was observed (0.11 ± 0.08 vs. 0.05 ± 0.01% ID/g). Correspondingly, activity in heart (0.28 ± 0.08% ID/g), lung (0.21 ± 0.04% ID/g), muscle (0.25 ± 0.07% ID/g), and bone (0.24 ± 0.04% ID/g) were also higher. The microPET/CT images of the BALB/c mouse at 80 min p.i. and 24 h p.i. injected with either [89Zr]Zr-FSC(succ)3 or [89Zr]Zr-TAFC further confirmed similar rapid pharmacokinetics, see 89Zr]Zr-FSC(succ)3 showed some gall bladder activity and some minor activity in the small bowel which disappeared in the late images. More importantly, no bone uptake was observed, confirming the high in vivo stability of both compounds. An image-based quantitative comparison of bone uptake in the knee of mice revealed an even lower uptake of [89Zr]Zr-FSC(succ)3 with 0.083 and 0.026% ID/cm3 and of [89Zr]Zr-TAFC with 0.040 and 0.016% ID/cm3 at 80 min and 24 h, respectively, underlining the high in vivo stability of both chelates.Based on the above-mentioned results, Zr4+ and, on the other hand, reduce the number of conjugation groups making them suitable for conjugation to mAbs.The high potential of FSC as a novel multivalent recently . However2, FSC(succ)2AA and FSC(succ)3 were synthesized with satisfactory yields. In our previous report, we discovered that iron protection is required for coupling FSC with targeting vectors ZrCl4 may be a better choice for labeling FSC derivatives in acetate buffer at appropriate pH. The advantage of [89Zr]ZrCl4 over 89Zr-oxalate lies not only in the shorter labeling time but also in avoiding the use of highly concentrated oxalate solution which potentially can result in the production of solid calcium oxalate causing kidney damage. In contrast to HEPES buffer, which requires additional quality control before clinical application, acetate buffer is entirely pharmaceutically compatible. For better understanding the exact coordination structures as well as their influence on stability, the preparation of single crystals of Zr-TAFC, Zr-FSC(succ)2AA and Zr-FSC(succ)3 for X-ray Diffraction was explored using different strategies; however, this failed. Multivalent hydroxamates seem not to crystallize easily, and this may be the reason that Zr(Me-AHA)4 is the only successfully crystallized Zr-hydroxamate compound Zr-DFO, [89Zr]Zr-TAFC, [89Zr]Zr-FSC(succ)2AA and [89Zr]Zr-FSC(succ)3, acid dissociation experiments were performed under acidic conditions. At pH 4, [89Zr]Zr-FSC(succ)3 was the most kinetically inert compound to acid dissociation for seven days, with [89Zr]Zr-TAFC showing moderate stability, followed by [89Zr]Zr-FSC(succ)2AA, and [89Zr]Zr-DFO totally decomposing on day 3. Transchelation studies showed the same but more pronounced trend. [89Zr]Zr-DFO showed total transchelation within 1 h whereas other 89Zr-complexes only showed limited transchelation after seven days. The improved stability and kinetic inertness of [89Zr]Zr-FSC(succ)3, [89Zr]Zr-TAFC, and [89Zr]Zr-FSC(succ)2AA over [89Zr]Zr-DFO were attributed to the macrocyclic structure. The superior stability of [89Zr]Zr-FSC(succ)3 over [89Zr]Zr-TAFC and [89Zr]Zr-FSC(succ)2AA may be as a result of the higher coordination number and better coordination configuration. The lower stability of [89Zr]-Zr-FSC(succ)2AA compared to [89Zr]Zr-TAFC may be related to the loss of the symmetrical structure. However, the superior stability of [89Zr]Zr-FSC(succ)2AA over [89Zr]Zr-DFO highlights the potential of FSC(succ)2 as a monomeric chelator in the development of novel 89Zr-tracers.To assess the relative kinetic inertness of [89Zr]Zr-FSC(succ)3 than [89Zr]Zr-TAFC, resulting in a higher uptake in all major organs. Notably, the bone activity of [89Zr]Zr-FSC(succ)3 was significantly higher than that of [89Zr]Zr-TAFC, possibly mainly reflecting the slower clearance rather than being actual bone uptake. Even though different time points were chosen for an imaging study, this was confirmed by images at 24 h p.i. showing both radioligands without visible bone uptake. [89Zr]Zr-FSC(succ)3 also showed some hepatobiliary excretion in microPET/CT images at the early time point of 80 min, which was not depicted at 24 h p.i. and in the 6 h p.i. biodistribution study. The overall difference in biodistribution may be attributed to the difference in the chelate-radiometal net charge and polarity. [89Zr]Zr-TAFC possesses a positive net charge while [89Zr]Zr-FSC(succ)3 shows an overall negative charge character, which may potentially influence the excretion patterns of radiotracers. However, the difference in clearance rates between [89Zr]Zr-FSC(succ)3 and [89Zr]Zr-TAFC should not be a concern in its final application. Upon conjugation to a targeting biomolecule, the pharmacokinetics of the [89Zr]Zr-FSC(succ)3 complex will be completely superseded by those of biomacromolecules. Higher kidney and lower liver uptake compared to [89Zr]Zr-TAFC indicates a renal excretion pathway, which is consistent with the higher hydrophilicity of [89Zr]Zr-FSC(succ)3.A direct biodistribution comparison revealed a much slower blood clearance of [89Zr]Zr-FSC(succ)2AA showed superiority over [89Zr]Zr-DFO in vitro. Furthermore, [89Zr]Zr-FSC(succ)3 revealed the best stability and inertness in comparison to [89Zr]Zr-DFO, [89Zr]Zr-TAFC and [89Zr]Zr-FSC(succ)2AA. The superiority of FSC(succ)3 was attributed to the higher coordination number and better coordination configuration. The successful synthesis of FSC(succ)2 and FSC(succ)2AA highlights the potential of FSC(succ)2 as a monovalent 89Zr-chelator for the conjugation to mAbs. Further studies of FSC(succ)2-conjugated mAbs, especially in vivo, in comparison with their DFO counterparts and potentially other novel Zr-chelators, are needed to show the true potential of this approach for Immuno-PET.In our study, three FSC derivatives were synthesized and labeled with zirconium-89. [


Scientific Reports 10.1038/s41598-019-49749-y, published online 13 September 2019Correction to: The original version of this Article contained an error in the Abstract.“This retrospective, observational study included 18,165 patients who received anti-vascular endothelial growth factor injections .”now reads:“This retrospective, observational study included 3,380 patients who received anti-vascular endothelial growth factor injections .”This error has now been corrected in the PDF and HTML versions of the Article.


Strategies targeting cross-talk between immunosuppressive renal dendritic cells (DCs) and T regulatory cells (Tregs) may be effective in treating cisplatin (CDDP)-induced acute kidney injury (AKI). Galectin 3 , expressed on renal DCs, is known as a crucial regulator of immune response in the kidneys. In this study, we investigated the role of Gal-3 for DCs-mediated expansion of Tregs in the attenuation of CDDP-induced AKI.Methods: AKI was induced in CDDP-treated wild type (WT) C57BL/6 and Gal-3 deficient  mice. Biochemical, histological analysis, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, real-time PCR, magnetic cell sorting, flow cytometry and intracellular staining of renal-infiltrated immune cells were used to determine the differences between CDDP-treated WT and Gal-3-/- mice. Newly synthesized selective inhibitor of Gal-3 (Davanat) was used for pharmacological inhibition of Gal-3. Recombinant Gal-3 was used to demonstrate the effects of exogenously administered soluble Gal-3 on AKI progression. Pam3CSK4 was used for activation of Toll-like receptor (TLR)-2 in DCs. Cyclophosphamide or anti-CD25 antibody were used for the depletion of Tregs. 1-Methyl Tryptophan (1-MT) was used for pharmacological inhibition of Indoleamine 2,3-dioxygenase-1 (IDO1) in TLR-2-primed DCs which were afterwards used in passive transfer experiments.Results: CDDP-induced nephrotoxicity was significantly more aggravated in Gal-3-/- mice. Significantly reduced number of immunosuppressive TLR-2 and IDO1-expressing renal DCs, lower serum levels of KYN, decreased presence of IL-10-producing Tregs and significantly higher number of inflammatory IFN-γ and IL-17-producing neutrophils, Th1 and Th17 cells were observed in the CDDP-injured kidneys of Gal-3-/- mice. Pharmacological inhibitor of Gal-3 aggravated CDDP-induced AKI in WT animals while recombinant Gal-3 attenuated renal injury and inflammation in CDDP-treated Gal-3-/- mice. CDDP-induced apoptosis, driven by Bax and caspase-3, was aggravated in Gal-3-/- animals and in WT mice that received Gal-3 inhibitor (CDDP+Davanat-treated mice). Recombinant Gal-3 managed to completely attenuate CDDP-induced apoptosis in CDDP-injured kidneys of Gal-3-/- mice. Genetic deletion as well as pharmacological inhibition of Gal-3 in renal DCs remarkably reduced TLR-2-dependent activation of IDO1/KYN pathway in these cells diminishing their capacity to prevent transdifferentiation of Tregs in inflammatory Th1 and Th17 cells. Additionally, Tregs generated by Gal-3 deficient DCs were not able to suppress production of IFN-γ and IL-17 in activated neutrophils. TLR-2-primed DCs significantly enhanced capacity of Tregs for attenuation of CDDP-induced AKI and inflammation and expression of Gal-3 on TLR-2-primed DCs was crucially important for their capacity to enhance nephroprotective and immunosuppressive properties of Tregs. Adoptive transfer of TLR-2-primed WTDCs significantly expanded Tregs in the kidneys of CDDP-treated WT and Gal-3-/- recipients resulting in the suppression of IFN-γ and IL-17-driven inflammation and alleviation of AKI. Importantly, this phenomenon was not observed in CDDP-treated WT and Gal-3-/- recipients of TLR-2-primed Gal-3-/-DCs. Gal-3-dependent nephroprotective and immunosuppressive effects of renal DCs was due to the IDO1-induced expansion of renal Tregs since either inhibition of IDO1 activity in TLR-2-primed DCs or depletion of Tregs completely diminished DCs-mediated attenuation of CDDP-induced AKI.Conclusions: Gal-3 protects from CDDP-induced AKI by promoting TLR-2-dependent activation of IDO1/KYN pathway in renal DCs resulting in increased expansion of immunosuppressive Tregs in injured kidneys. Activation of Gal-3:TLR-2:IDO1 pathway in renal DCs should be further explored as new therapeutic approach for DC-based immunosuppression of inflammatory renal diseases. Cisplatin  is one of the most effective chemotherapeutic agents Upon activation, tissue resident tolerogenic DCs express Indoleamine 2,3-dioxygenase 1 (IDO1) which metabolizes tryptophan to Kynurenine (KYN) in vitro and in vivo approaches we demonstrated that genetic deletion as well as pharmacological inhibition of Gal-3 significantly impaired capacity of TLR-2-primed renal DCs to express IDO1 and produce immunosuppressive KYN which resulted in significantly reduced presence of renal-infiltrated Tregs and notably aggravated CDDP-induced AKI. Therefore, we propose that Gal-3 protects from CDDP-caused nephrotoxicity by promoting TLR-2-dependent activation of IDO1/KYN pathway in renal DCs resulting in increased expansion of immunosuppressive Tregs in injured kidneys.Considerable interest has recently arisen in the intriguing immunomodulatory properties of Galectin 3 , a member of β-galactoside-binding lectins, which regulates numerous biological processes in the kidneys including migration, proliferation and activation of resident and renal-infiltrated immune cells Animals. Male, 6-8-week-old wild type (WT) and Gal-3-/- C57BL/6 mice were used for the induction of CDDP-induced AKI. Breeding pairs of Gal-3-/- and WT C57BL/6 mice of the same substrain were initially obtained from Dr. Daniel Hsu  ad libitum..Induction of AKI To generate CDDP-induced AKI, WT and Gal-3-/- mice were injected with a single, intraperitoneal (i.p) dose of CDDP (16 mg/kg body weight). After mouse euthanasia (72 h after CDDP treatment), both kidneys were excised and blood samples were drawn from the inferior vena cava, as previously described Administration of recombinant Gal-3 . In order to evaluate the effects of rGal-3 in attenuation of CDDP-induced AKI, Gal-3-/- mice received single intravenous injection of rGal-3 , 24 h before CDDP administration -/- animals from control group received only saline.Pharmacological inhibition of Gal-3. Gal-3 inhibitor  was intraperitoneally injected in CDDP-treated WT animals (100 μg/day), for three consecutive days before CDDP administration Evaluation of CDDP-induced AKI. CDDP-induced AKI was evaluated by biochemical and histological analysis, as previously described Biochemical analysis. Serum levels of urea and creatinine were determined to assess the renal function. After blood collection, serum levels of these toxicity markers were measured immediately using assay kits and blood chemistry analyzer, according to the manufacturer's instructions Histopathological Analysis. Kidney tissue was fixed in 10% buffered formalin, embedded in paraffin and cut at 5 μm thickness. Sections were stained with Hematoxylin and Eosin (H&E) and examined in a blinded manner. Histological sections were scored using a semi-quantitative scale designed to assess AKI-associated tubular injury  by a pathologist blinded to the experimental groups . Tubule injury scores (ranging between 0 and 4) were based on the percentage of tubules affected as follows: 0 ≤ 10%, 1 = 10-25%, 2 = 26-50%, 3 = 51-75%, and 4 ≥ 75%, as previously described Immunohistochemistry. In order to assess expression of AGEs, Galectin-3 and TLR2 in the kidneys of WT and Gal-3-/- mice, immunohistochemical (IHC) analysis was performed. Formalin-Fixed Paraffin-Embedded (FFPE) kidney tissue sections were incubated with rabbit anti-mouse AGE antibody , rabbit anti-mouse Galectin 3 antibody  and rabbit anti-mouse TLR2 antibody  overnight at room temperature. Immunoreactivity was visualized by rabbit-specific conjugate  according to the manufacturer's instructions. Sections were photomicrographed with a digital camera mounted on light microscope (Olympus BX51), digitized, and analyzed Analysis was performed on 10 fields per section at 40x magnification. Results are presented as mean count of positive stained cells per field Isolation of Renal-Infiltrated Immune Cells. Isolation of immune cells from the kidneys was conducted as previously described Flow cytometry of renal-infiltrated immune cells. For flow cytometry, 1×106 of renal-infiltrated cells were incubated with anti-mouse CD45, Gr-1, F4/80, CD4, CD11c, CD80, CD86, MHC-II, TLR-2, Gal-3 monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), or allophycocyanin (APC)  following the manufacturer's instructions. Immune cells derived from the kidneys were concomitantly stained for the intracellular content of IFN-γ, IL-17, IL-4, IL-10, IL-12, T-bet, RORγt, STAT3, IDO1, IL-23 and forkhead box P3 (FoxP3) by using the fixation/permeabilization kit and anti-mouse monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), and allophycocyanin (APC) (BD Bioscience). For intracellular cytokine staining, cells were stimulated with 50 ng/mL Phorbol 12-myristate-13-acetate (PMA) and 500 ng/mL Ionomycin for 5 h, and GolgiStop (BD Biosciences) was added. Cells were fixed in Cytofix/Cytoperm, permeated with 0.1% saponin, and stained with fluorescent Abs. Flow cytometry was conducted on a BD Biosciences FACSCalibur and data were analyzed by Flowing Software analysis program.Isolation and TLR-2-priming of DCs. DCs were isolated from the kidneys of healthy WT and Gal-3-/- mice by magnetic cell sorting. Single-cell suspensions of renal-infiltrated mononuclear cells were labelled with CD11c MicroBeads (Miltenyi Biotec). The labelled cells were subsequently positively selected using MACS Column (Miltenyi Biotec) and MACS Separator (Miltenyi Biotec), as previously described 5/ mL) were primed with TLR-2 agonist Pam3CSK4 (300 ng/mL) for 24 h. As a naive control, renal DCs were cultured in the absence of TLR-2 ligand for 24h Pharmacological inhibition of Gal-3 in DCs. For pharmacological inhibition of Gal-3, unprimed and TLR-2-primed renal DCs were cultured in the presence of selective Gal-3 inhibitor  for 24 h, according to previously published protocol Pharmacological inhibition of IDO1 activity in DCs. IDO1 was inhibited in TLR-primed renal DCs (WTDCsPam3CSK4+1-MT) and Gal-3-/-DCs  by using 1-methyl tryptophan , as previously described Transfer of TLR-2 primed DCs in CDDP-treated animals. For transfer-based experiments, TLR-2-primed DCs, isolated from the kidneys of untreated WT and Gal‐3-/- mice , were intravenously injected (5×105cells/mouse) in CDDP-treated WT recipients  or Gal‐3-/- recipients  two days prior CDDP administration Measurement of cytokines. The commercial enzyme-linked immunosorbent assay (ELISA) sets  were used to determine the concentration of IL-12, IL-1β, IL-6, IL-23, IL-17, IFN-γ and IL-10 in serum samples of saline and CDDP-treated animals, according to the manufacturer's instructions Determination of IDO1 activity. IDO1 activity in serums samples of saline and CDDP-treated mice as well as in supernatants of unprimed and TLR-2-primed WTDCs, Gal-3-/-DCs and WTDCsDavanat was determined by spectrophotometric measuring of KYN Isolation of neutrophils. Neutrophils were isolated from healthy WT animals by magnetic cell sorting , according to manufacturer's instructions. Isolated neutrophils were stimulated by lipopolysaccharide  for 18 h and used in the co-culture experiments with Tregs Isolation of Tregs. Tregs were isolated from the population of renal-infiltrated mononuclear cells by magnetic cell sorting , according to manufacturer's instructions Contact-independent co-culture of TLR-2-primed DCs and Tregs. Tregs were co-cultured in the presence of WT DCsPam3CSK4, WTDCPam3CSK4+Davanat or Gal-3-/-DCsPam3CSK4, as previously described Pam3CSK4, WTDCPam3CSK4+Davanat or Gal-3-/-DCsPam3CSK which were seeded in the transwell inserts, and ratio between DCs and Tregs was 1:10.Pam3CSK4, WTDCPam3CSK4+Davanat or Gal-3-/-DCsPam3CSK were collected and used for co-culture experiments with neutrophils, ELISA assay, flow cytometry analysis and intracellular staining of FoxP3, IFN-γ, IL-17 and IL-10 or frozen at -80 °C until gene expression were measured by real-time PCR. Non-primed Tregs or Tregs primed with WTDCsPam3CSK4, WTDCPam3CSK4+Davanat or Gal-3-/-DCsPam3CSK (2x106 cells/ well) were collected, seeded at 24 well plate, cultured for additional 24h in fresh DMEM medium. Supernatants were collected and IFN-γ, IL-17 and IL-10 were determined by ELISA sets , according to the manufacturer's instructions After 48h of culture, non-primed Tregs or Tregs primed with WTDCsCo-culture of Tregs and neutrophils. Non-primed Tregs as well as Tregs, previously primed by WTDCsPam3CSK4, WTDCPam3CSK4+Davanat or Gal-3-/-DCsPam3CSK4, were co-cultured with LPS-activated neutrophils, as previously described Pam3CSK4, WTDCPam3CSK4+Davanat or Gal-3-/-DCsPam3CSK which were seeded in the transwell inserts, and ratio between neutrophils and Tregs was 10:1. After 48 h, the expression of IFN-γ, IL-17 and IL-10 in neutrophils was evaluated by real time PCR analysis. Neutrophils were collected, seeded at 24 well plate (1x106 cells/ well), cultured for additional 24 h in fresh DMEM medium. Supernatants were collected and IFN-γ, IL-17 and IL-10 were determined by ELISA sets , according to the manufacturer's instructions RNA isolation and real‐time PCR analysis. Total RNA from kidneys, Tregs or neutrophils was extracted using TRIzol reagent  according to the manufacturer's instructions. Total RNA (2 μg) was reversely transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kit . qRT-PCR was performed using Power SYBR MasterMix (Applied Biosystems) and miRNA-specific primers for IFN-γ, IL-17, IL-10, FoxP3, T-bet, RORγT, Bax, Bcl-2, caspase-3 and β-actin as a housekeeping gene. qPCR reactions were initiated with a 10 min incubation time at 95 °C followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s in a Mastercycler ep realplex . Relative expression of genes was calculated according to the formula 2-(Ct-Ctactin), where Ct is the cycle threshold of the gene of interest and Ctactin is the cycle threshold value of the housekeeping gene (β-actin) Transfer of Tregs. Tregs were fluorescence-labeled using pre-incubation with carboxyfluorescein diacetate succin-imidyl ester  according to the manufacturer's instructions. For transfer experiments non-primed Tregs, Tregs primed with WTDCPam3CSK4, WTDCPam3CSK4+Davanat or Gal-3-/- DCPam3CSK4 (1x106 Tregs/ mouse) were intravenously injected in CDDP-treated animals, 18 h before induction of AKI.Depletion of Treg cells. For the depletion of Tregs, CDDP-treated WTWTDCPam3CSK4 and Gal-3-/-WTDCPam3CSK4 mice received either cyclophosphamide  3 days before CDDP administration, at a dose of 10 mg/kg which was reported to selectively deplete Tregs but not DCs Isolation and transfer of macrophages in CDDP-treated mice. Macrophages, isolated from healthy WT and Gal-3-/- mice, were stimulated with Pam3CSK4  and injected via the tail vein (1x106/ mouse) 2 h before induction of AKI, as previously suggested .Statistics Data were expressed as the Mean ± standard error of the mean (SEM) for each group. Results were analyzed by Student's t test and Pearson's or Spearman's correlation coefficient. Statistical analyses were performed using SPSS 23.0 for Windows software . The difference was considered significant when p<0.05.-/- mice as reflected by a marked elevation of serum urea and creatinine  and recombinant Gal-3  on the development of CDDP-induced acute renal failure in WT and Gal-3-/- mice  is associated with a massive influx of neutrophils in CDDP-injured kidneys e Figure A-E. CDDPr Figure A. Howevee Figure D, p<0.01e Figure E, p<0.05-/- mice compared to similarly treated WT animals . Intracellular staining revealed that notably higher number of inflammatory T-bet expressing and IFN-γ producing Th1 cells  and WTDCsPam3CSK4 that were cultured in the presence of selective Gal-3 inhibitor-Davanat (WTDCsPam3CSK4+Davanat)  Figure G. Activa) Figure G. Import) Figure G. Signif4 Figure G, p<0.05Pam3CSK4 diminished their capacity to maintain immunosuppressive phenotype of Tregs and to prevent trans-differentiation of Tregs in Th1 or Th17 cells , as evidenced by notably lower serum levels of urea , as evidenced by significantly increased serum levels of urea  by promoting IFN-γ and IL-17-driven inflammation , confirming that expression of Gal-3 on DCs was crucially responsible for DCPam3CSK4-mediated amelioration of CDDP-induced nephrotoxicity  completely abrogated their capacity to attenuate CDDP-induced AKI in CDDP-treated WT  and analyzed their nephroprotective and immunosuppressive effects in CDDP-treated WT  and to promote production of immunosuppressive IL-10 in renal-infiltrated neutrophils, as evidenced by significantly lower number of T-bet-expressing, IFN-γ-, IL-17- and TNF-α-producing neutrophils  in CD4+T cells enabling generation of immunosuppressive CD4+FoxP3+Tregs in inflamed and injured tissues Pam3CSK4  in neutrophils (Figure -/-DCsPam3CSK4or WT DCsPam3CSK4+Davanat were not able to generate immunosuppressive phenotype in activated neutrophils (Figure Pam3CSK4, adoptive transfer of Gal-3-/-DCsPam3CSK4 did not result in expansion of renal-infiltrating Tregs and IL-10 producing neutrophils in the kidneys of CDDP-treated WT and Gal-3-/- recipients (Figure It is well known that Tregs may induce production of immunosuppressive cytokines in neutrophils e Figure E. Additis Figure -9, indicIn conclusion, we propose that expression of Gal-3 in renal DCs is crucially important for their protective effects in CDDP-induced AKI. The main mechanism by which Gal-3 regulates immunosuppressive capacity of renal DCs is relied on the TLR-2-dependent activation of IDO1/KYN pathway and consequent expansion of IL-10-producing renal-infiltrated Tregs which suppress neutrophil-, Th1- and Th17 cell-driven inflammation in CDDP-injured kidneys Figure . ActivatSupplementary figures.Click here for additional data file.


Respiratory syncytial virus (RSV) causes severe acute lower respiratory tract disease. Retinoic acid-inducible gene-I (RIG-I) serves as an innate immune sensor and triggers antiviral responses upon recognizing viral infections including RSV. Since tripartite motif-containing protein 25 (TRIM25)-mediated K63-polyubiquitination is crucial for RIG-I activation, several viruses target initial RIG-I activation through ubiquitination. RSV NS1 and NS2 have been shown to interfere with RIG-I-mediated antiviral signaling. In this study, we explored the possibility that NS1 suppresses RIG-I-mediated antiviral signaling by targeting TRIM25. Ubiquitination of ectopically expressed RIG-I-2Cards domain was decreased by RSV infection, indicating that RSV possesses ability to inhibit TRIM25-mediated RIG-I ubiquitination. Similarly, ectopic expression of NS1 sufficiently suppressed TRIM25-mediated RIG-I ubiquitination. Furthermore, interaction between NS1 and TRIM25 was detected by a co-immunoprecipitation assay. Further biochemical assays showed that the SPRY domain of TRIM25, which is responsible for interaction with RIG-I, interacted sufficiently with NS1. Suppression of RIG-I ubiquitination by NS1 resulted in decreased interaction between RIG-I and its downstream molecule, MAVS. The suppressive effect of NS1 on RIG-I signaling could be abrogated by overexpression of TRIM25. Collectively, this study suggests that RSV NS1 interacts with TRIM25 and interferes with RIG-I ubiquitination to suppress type-I interferon signaling. Pneumoviridae and contains a negative-sense single-stranded RNA genome. RSV infection is a leading cause of severe acute lower respiratory tract disease and related hospitalization in children and the elderly [Respiratory syncytial virus (RSV) belongs to the family  elderly ,2. DespiThe cellular innate immune system utilizes various sensors including Retinoic acid inducible gene-I (RIG-I) and Toll-like receptors (TLRs) to detect viral infection and activate antiviral immune signaling pathways ,4. AmongLike other viruses, RSV has evolved several strategies to antagonize antiviral immune responses . The rolHEK293T, HEp-2  and A549  cells were maintained in Dulbecco’s High Glucose Modified Eagle’s Medium  containing 10% fetal bovine serum  and 1% penicillin/streptomycin (Hyclone).RSV A2 strain stocks were prepared as described previously . BrieflypEBG-GST-RIG-IN , pEF-IRES (pIRES)-V5-TRIM25 and MAVS-CARD-proline rich domain (PRD)-FLAG were described in a previous study . pIRES-VConventional calcium phosphate transfection and polyethylenimine (PEI) transfection methods were used for transfection. The total plasmid content for each sample condition was adjusted using the empty vector. Low molecular weight polyI:C ; average size from 0.2kb to 1kb) was transfected with PEI solution to mimic intracellular RNAs.Cells were collected and lysed in Triton X-100 cell lysis buffer  containing protease and phosphatase inhibitor cocktail . Lysates were incubated overnight at 4 °C with the corresponding antibody. After the binding reaction, protein A/G resin  were added to samples and further incubated for 2 h at room temperature. The resin was then washed four to five times with cell lysis buffer. Anti-FLAG antibody conjugated resin (Sigma) and anti-V5 antibody conjugated resin (Sigma) were also used for Co-IP in a similar manner. Precipitated proteins were eluted with SDS sample buffer  and subjected to SDS-PAGE and immunoblotting.To precipitate GST-tagged fusion proteins, the cell lysates were incubated with glutathione-conjugated beads (Sigma) at RT for 1–2 h. After incubation, the beads were washed four to five times with cell lysis buffer, followed by SDS-PAGE and immunoblotting.After polyacrylamide gel electrophoresis, target proteins on the gel were transferred to a PVDF membrane . The membrane was incubated overnight at 4 °C with the indicated antibody in 5% bovine serum albumin  solution for binding. After extensive washing with PBST, the membrane was then incubated with the horseradish peroxidase (HRP) conjugated secondary antibody at RT for 2 h. Luminata Forte (Millipore) was used as the HRP substrate. Anti-V5 rabbit antibody , anti-V5 mouse antibody , anti-HA mouse antibody , anti-FLAG mouse antibody , anti-ubiquitin (P4D1) mouse antibody , anti-GST antibody  and HRP conjugated antibodies  were used.HEK293T cells in 24-well plates were transfected with Interferon-β firefly luciferase (0.25 μg/well) and thymidine kinase (TK) renilla reporter plasmids (0.13 μg/well) were transfected along with other constructs as indicated . After 220(dT) primers. Real-time PCR was conducted using synthesized cDNA (2 μL). The mRNA levels of interferon-β and interferon-stimulated gene 15 (ISG15) were determined using the following primers and normalized to those of β-actin: interferon-β-forward; 5′-AAGAGTTACACTGCCTTTGCCATC-3′, interferon-β-reverse; 5′-CACTGTCTGCTGGTGGAGTTCATC-3′, ISG15-forward; 5′-CCTCTGAGCATCCTGGT-3′, ISG15-reverse; 5′-AGGCCGTACTCCCCCAG-3′, β-actin-forward; 5′-TGGAATCCTGTGGCATCCATGAAAC-3′, β-actin-reverse; 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′.Total RNAs were extracted using TRIzol  according to the instruction. cDNAs were generated from RNAs (1 μg) using Superscript III reverse transcriptase  and oligoHEK293T cells were seeded onto fibronectin coated coverslips in a 24-well plate. After 24 h, cells were transfected with FLAG-tagged RSV NS1 and V5-tagged TRIM25 and incubated for 24 h. Then, cells were fixed with 4% paraformaldehyde followed by blocking and permeabilization in permeabilization buffer  at RT in 5 min. The cells were incubated with anti-FLAG mouse and anti-V5 rabbit antibodies in 0.5% BSA solution at 4 °C overnight. Next day, the cells were extensively washed and incubated with FITC- and PE-labeled secondary antibodies at RT for 2 h. The co-localization between RSV NS1-FLAG and TRIM25-V5 was analyzed by Nanoscope K1-Fluo confocal microscope. t-test. A value of p ≤ 0.05 was considered to be significant.Data were presented as the mean ± SEM. Statistical comparisons between the control and treated groups were performed using the Student’s To confirm the suppression of RIG-I-mediated signaling by NS1, RSV NS1 was transfected along with constitutively active RIG-I-2CARDs (RIG-IN). Ectopically expressed NS1 inhibited interferon-β promoter activity that was induced by RIG-IN as determined by the luciferase assays in HEK293T cells, confirming that NS1 itself is capable of inhibiting RIG-I-mediated antiviral signaling A. ConsisBecause TRIM25-mediated RIG-I CARD ubiquitination is an essential step for the successful induction of RIG-I-mediated antiviral responses, we explored the possibility that RSV suppresses RIG-I activation by inhibiting the TRIM25-mediated RIG-ubiquitination. First, we have tested whether RIG-I ubiquitination is suppressed by RSV infection. As seen in Interaction between RIG-IN and NS1 was examined using a GST-pulldown assay to test whether NS1 interacts with RIG-IN similar to NS2. However, we could not detect any interaction (Data not shown). Therefore, interaction between TRIM25 and NS1 was investigated to determine whether NS1 targets TRIM25 to suppress RIG-I ubiquitination. Obvious interaction between ectopically expressed TRIM25 and NS1 was detected by the co-immunoprecipitation assay, whereas interaction between NS2 and TRIM25 was not detected A. FurtheBecause RIG-I ubiquitination is prerequisite for interaction with MAVS, decreased RIG-I CARD ubiquitination by RSV NS1 should result in disruption of RIG-I interaction with MAVS. Thus, the interaction between RIG-IN and MAVS upon virus infection was analyzed. As shown in To confirm that interaction between NS1 and TRIM25 contributes to the interferon-suppressive effect of NS1, the effect of TRIM25 overexpression on the suppression of RIG-I signaling by NS1 was investigated using interferon-β luciferase promoter assays. As shown in Many viruses possess defensive mechanisms against the host immune system for their effective replication. The NS1 and NS2 proteins of RSV have been shown to effectively inhibit the host immune system. NS proteins target diverse proteins related to type-I interferon induction and signal transduction. For example, RSV NS1 upregulates SOCS1 and SOCS3 and triggers STAT2 degradation . RSV NS1In this study, we explored the possibility that these proteins interfere with RIG-I signaling by directly inhibiting RIG-I activation. Indeed, we could demonstrate that NS1 and NS2 suppressed TRIM25-mediated ubiquitination of RIG-I, which is a crucial step for RIG-I activation. Previously, NS2 has been shown to interact with RIG-I, whereas the interaction between NS1 and RIG-I could not be detected . Thus, sThus, it was tempting to test the hypothesis that NS1 targets TRIM25, an E3-ubiquitin ligase responsible for RIG-I ubiquitination. Moreover, several viruses utilize their proteins to interfere with TRIM25 activation and subsequent RIG-I activation. For instance, the Influenza NS1 binds to TRIM25 and inhibits TRIM25 multimerization . The SARInterestingly, the biochemical domain mapping study revealed that the SPRY domain of TRIM25 is responsible for interaction with NS1. Given that the SPRY domain is responsible for interaction with RIG-I, a possible molecular mechanism is that NS1 binds to TRIM25 and sequesters it to prevent its interaction with RIG-I. However, we could not observe a significant reduction in interaction between RIG-I and TRIM25 by NS1 (Data not shown). Further studies are needed to identify the detailed molecular mechanism of RSV NS1-mediated TRIM25 dysfunction.RSV NS2 is known to bind with RIG-I CARD and disrupts its activation  and we sCollectively, this study demonstrates that RSV NS1 interacts with TRIM25 and suppresses TRIM25-mediated RIG-I activation to evade RIG-I-mediated antiviral responses. Further studies including structural analysis of protein interaction and analysis of the interaction modes between RIG-I, TRIM25, NS1 and NS2 need to be conducted.


Caenorhabditis elegans adapts to unfavorable environmental conditions by pausing reproductive development and entering a stress-resistant larval stage known as dauer. The transition into dauer is marked by vast morphological changes, including remodeling of epidermis, neurons, and muscle. Although many of these dauer-specific traits have been described, the molecular basis of dauer-specific remodeling is still poorly understood. Here we show that the nidogen domain-containing protein DEX-1 facilitates stage-specific tissue remodeling during dauer morphogenesis. DEX-1 was previously shown to regulate sensory dendrite formation during embryogenesis. We find that DEX-1 is also required for proper remodeling of the stem cell-like epidermal seam cells. dex-1 mutant dauers lack distinct lateral cuticular alae during dauer and have increased sensitivity to sodium dodecyl sulfate. Furthermore, we find that DEX-1 is required for proper dauer mobility. We show that DEX-1 is secreted from the seam cells during dauer, but acts locally in a cell-autonomous manner. We find that dex-1 expression during dauer is regulated through DAF-16/FOXO–mediated transcriptional activation. Finally, we show that dex-1 acts with a family of zona pellucida domain-encoding genes to regulate dauer-specific epidermal remodeling. Taken together, our data indicate that DEX-1 is an extracellular matrix component that plays a central role in C. elegans epidermal remodeling during dauer.Phenotypic plasticity is a critical component of an organism’s ability to thrive in a changing environment. The free-living nematode  Tissue remodeling is an important component of stress-induced phenotypic plasticity. For example, desert locusts are capable of altering their morphology between distinct “gregarious” and “solitarious” phases based on population density , into a reproductive adult. However, under unfavorable environmental conditions, C. elegans larvae arrest their development at the second larval molt and enter the stress-resistant dauer stage  mutants. Furthermore, we show that DEX-1 can function as a secreted protein and localizes in or near dauer alae. Finally, we find that dex-1 is upregulated in seam cells during dauer in a DAF-16–dependent manner. DEX-1 was previously shown to function with a zona pellucida (ZP) domain-containing protein, DYF-7, to mediate dendrite extension  component that plays a role in modulating cell shape of several cell types throughout development.The decision to enter dauer typically results in an all or none phenotype where dauers have every dauer morphological characteristic. However, mutations in some genes necessary for dauer formation result in an intermediate dauer phenotype, called partial dauers, in which the animals will display some, but not all, dauer characteristics . Here, wogenesis . We findxtension . Our datadhesion , and datN2 strain and the following mutant strains were used: DR27daf-16(m27) I, FX01126 cut-1(tm1126) II, CHB27 dex-1(ns42) III, UP2571 dex-1(cs201)III; csEx402[dex-1p::dex-1a + unc-199p::gfp], CB1372daf-7(e1372) III, DR129daf-2(e1370) unc-32(e189) III, RB1574cut-6(ok1919) III, RB1629cut-5(ok2005) X, SP1735dyf-7(m537) X. All mutant strains were backcrossed at least twice. dex-1(cs201) was generated using standard EMS mutagenesis protocols (dex-1(ns42) was a gift from Dr. Maxwell Heiman . cut-1(tm1126) was provided by the Mitani Consortium . The following transgenic strains were used: ST65ncIs13[ajm-1::gfp] was used to observe the apical junctions of the seam cells ] V ::gfp, pMH125 dex-1p(2.1 kb)::gfp  and dex-1p::dex-1(ecto)::sfgfp (pJC15) contain the 2.1 kb dex-1 promoter and dex-1 (isoform a) complementary (cDNA) from pMH7 [see sfgfp was inserted either at an internal endogenous BglII restriction site to generate a full-length fusion tagged upstream of the first nidogen domain, or inserted at the 3′ end of a cDNA truncated before the transmembrane domain. See accompanying article (The following plasmids were a generous gift from Dr. Maxwell Heiman: pMH7 kb)::gfp . The surdex-1(ns42) animals were injected with 20 ng/μl of plasmid and 80 ng/μl of unc-122p::gfp, unc-122p::rfp or sur-5::gfp as the co-injection marker. dex-1(cs201) animals were injected with 30 ng/μl of pJC15 or pJC24 and 50 ng/μl of co-injection marker pHS4 (lin-48p::mrfp).Animals containing extrachromosomal arrays were generated using standard microinjection techniques (Domain schematics were constructed using the wormweb.org Exon-Intron Graphic Maker. Domain locations were determined using the Simple Modular Architecture Research Tool domain prediction software .daf-16(m27) mutants using plates with high pheromone concentrations (EC90) (daf-7(e1372) or daf-2(e1370), dauers were induced using the restrictive temperature of 25°  . For temt-test. For confocal microscopy of the IL2 neurons, dauers were mounted on 10% agarose pads and anesthetized with 0.1 or 0.01 M levamisole. Animals were imaged using a Zeiss LSM 880 confocal microscope.Unless otherwise specified, animals were mounted onto 4% agarose pads and immobilized with 0.1 or 0.01 M levamisole for dauers and nondauer or partial dauers, respectively. In our hands, dauers frequently lay in a dorsal-ventral position following anesthesia. Therefore, to image the lateral side, dauers were immobilized by mounting on 4% agarose pads with Polybead Polystyrene 0.10 μm microspheres  . Seam cedex-1(ns42) and N2 animals, dauer larvae were induced using pheromone plates and processed for high-pressure freezing and freeze substitution modified from previously established methods , 0.1% uranyl acetate (Polysciences) in 2% H20, and 100% acetone. Samples were held at −90° for 110 hr, then warmed to −20° at the rate of 5° per hour (14 hr). Samples were then held at −20° for 16 hr, then warmed to 0° at the rate of 5° per hour (4 hr). Samples were washed three times in prechilled (0°) 100% acetone and incubated at 0° for 1 hr after the final wash. Samples were then warmed to room temperature and washed an additional two times with 100% acetone. Samples were infiltrated with 1:1 Polybed812 (Polysciences) resin:acetone for 6 hr, 2:1 resin:acetone for 14 hr, and 100% resin for 72 hr. All infiltration steps were incubated on an orbital shaker at room temperature. Samples were then embedded in molds in 100% resin plus DMP-30 hardener (Polysciences) and baked at 60° for 48 hr, then 70 nm sections were cut with a diamond knife using a PowerTome PC ultramicrotome and collected onto formvar-coated copper slot grids. Samples were imaged with a Philips CM200 transmission electron microscope.For transmission electron microscopy of  methods . Using ON2) control. Dose-response curves and LD50 values were determined by testing 20 dauers per treatment at each concentration, with three independent experiments. The LD50 and 95% confidence interval of each concentration was calculated using probit analysis in Minitab 18. LD50 values were considered significantly different if the 95% confidence intervals did not overlap. Significant difference was denoted with a single asterisk. Single concentration assays were conducted at 0.1% SDS with 20 dauers for each genotype and three independent experiments. Data were analyzed using a one proportion exact method analysis in Minitab 18 and considered significantly different if the 95% confidence intervals did not overlap. Significant difference was denoted with a single asterisk.Sodium dodecyl sulfate (SDS) dose-response assays were performed using 12-well culture dishes containing M9 buffer and specified concentrations of SDS. Dauers were exposed to SDS for 30 min and scored as alive if movement was observed following stimulation with an eyelash. Each concentration was tested in triplicate with each experiment containing a separate wild-type  and wild-type dauers were transferred to seeded NGM plates and allowed to recover for 10 min. After recovery, each animal was scored individually for 2 min, and then removed from the plate. Data were analyzed by an unpaired t-test, using GraphPad Prism 6 software.Pharyngeal pumping assays were modified from previous established methods . dex-1. The fluorescence intensities of the V2pap, V2ppp, and V3pap seam cells were measured using established methods (dex-1p::gfp and daf-16(m27); dex-1p::gfp was controlled by using the same transgene in both wild-type and daf-16(m27) backgrounds. Multiple independent lines were examined. To control for potential variation in copy number between the dex-1p(IRS∆)::gfp strain and dex-1p::gfp, we examined multiple independent lines. The data were analyzed using one-way ANOVA and Bonferroni’s multiple comparisons test. Ten animals were measured for each genotype.To analyze dex-1(ns42) dauer animals expressing extrachromosomal dex-1p::dex-1 and sur-5::gfp  body diameter data for dauers and L3s. Figure S2 shows the results of fluorescent bead feeding and pharyngeal pumping assays. Figure S3 shows that dauer-specific lag-2p::gfp is present in dex-1(ns42) dauers. Figure S4 shows that starved and pheromone-induced dauers are morphologically similar. Figure S5 describes dyf-7(m537) dauer morphology. Figure S6 contains a dose-response curve of SDS resistance of wild type and single- and double-mutants. Figure S7 contains gene schematics of cuticlin (CUT) mutants used in this study. Supplemental material available at Figshare: https://doi.org/10.25386/genetics.7077146.All strains and plasmids are available upon request. Table S1 contains all plasmid constructs used in this study. Figure S1 shows the C. elegans dauers have a distinctive morphology due to radial shrinkage that leads to a thin appearance compared with nondauers (dex-1(ns42) mutants produce dauers that are defective in radial shrinkage, leading to a “dumpy dauer” phenotype (dex-1(ns42) mutant L3s show no differences in body size compared with wild-type L3s . Radial shrinkage in dauers is correlated with the formation of longitudinal cuticular ridges on the lateral sides of the animal, called the alae (dex-1(ns42) mutant dauers are indistinct compared with wild-type dauers  mutant dauers were able to survive significantly higher concentrations of SDS than wild-type nondauer animals, they were sensitive to 1% SDS  allele results in a premature stop codon in exon 9, encoding the predicted zonadhesin-like functional domain (dex-1(cs201), which introduces a point mutation at the splice donor site of intron 4 (dex-1(cs201) cDNA suggests this results in multiple transcripts with read through into intron 4, with all introducing a new stop codon 10 bp into this intron (dex-1(cs201) mutants have a high rate of early larval lethality . Similar to dex-1(ns42), dex-1(cs201) dauers are defective in radial shrinkage, alae formation, and SDS resistance  to SDS compared with dex-1(ns42) (dex-1(ns42) is a hypomorphic allele; however, due to the early larval lethality of dex-1(cs201), all further experiments were done using the dex-1(ns42) background, unless otherwise noted.The previously isolated l domain    combineddex-1(ns42) mutants. Dauers suppress pharyngeal pumping and have a buccal plug that prevents feeding (dex-1(ns42) mutant dauer intestines; however, we occasionally observed them in the buccal cavity . Additionally, we found no difference in the rate of pharyngeal pumping between dex-1(ns42) and wild-type dauers . These data suggest that while pharyngeal pumping is efficiently suppressed, dex-1(ns42) dauers have low-penetrance defects in buccal plug formation. Finally, we examined dex-1(ns42) dauers for the presence of dauer-specific gene expression of lag-2p::gfp in the IL2 neurons during dauer (dex-1(ns42) dauers showed appropriate dauer-specific expression .We characterized additional dauer-specific phenotypes in  feeding . Nondauedex-1(ns42) mutation would affect the initial decision to enter dauer. The dauer formation decision is based on the ratio of population density to food availability during early larval development  mutant dauers . The C. elegans insulin/IGF-like and TGF-β signaling pathways function in parallel with regulate dauer formation (DAF-2 or the TGF-β homolog DAF-7 results in a daf-c phenotype. The dex-1(ns42) alae formation defects were not suppressed in the daf-7(e1372) or daf-2(e1370) mutant backgrounds. While nonnull alleles were used in these crosses, these data may suggest that DEX-1 acts outside of the dauer formation decision to generate dauer alae  mutant dauer seam cells are larger and have jagged, rectangular edges, unlike the smooth, elongated seam cells of wild-type dauers  driven by a 5.7 kb 5′ dex-1 upstream promoter. We observed bright fluorescence in the seam cells and glia socket cells of the anterior and posterior deirid neurons starting in the predauer L2 (L2d) stage. Expression of dex-1p::gfp in the seam cells and deirid socket cells persisted throughout dauer  tagged with super-folder GFP (sfGFP) under the control of its endogenous promoter. This dex-1p::sfgfp::dex-1 construct rescued the SDS phenotype in dex-1(ns42) mutant dauers, suggesting it is functional (dex-1(ns42) dauers expressing the dex-1p::sfgfp::dex-1 construct, indicative of dominant negative effects of the transgene.To determine the subcellular localization of nctional . During nctional . Interesnctional . The mosDEX-1 contains a putative transmembrane domain, but was previously suggested to be secreted through cleavage of the large extracellular domain ::sfgfp construct that truncates the C-terminal transmembrane domain. Consistent with a role as a secreted protein, the truncated DEX-1 construct rescued the SDS resistance  radial shrinkage in a mosaic pattern similar to that seen with the full-length sfgfp::dex-1 construct . We did not observe alae rescue in cells that did not express nuclear sur-5::gfp . Although both dex-1(ns42) mutant and wild-type dauers initially respond to mechanical stimulation, the dex-1 mutant dauers have significantly reduced locomotion and display slightly uncoordinated body movements  and wild type  dauer locomotion defects  mutant background. While mutations in daf-16 result in animals incapable of forming dauers, under high-pheromone concentrations, daf-16(m27) mutants can enter into a partial dauer state with some dauer morphological characteristics (daf-16(m27) partial dauers are identifiable by body morphology and the presence of indistinct lateral alae (dex-1p::gfp was significantly reduced in daf-16(m27) partial dauers compared to wild type, suggesting that DAF-16 regulates dex-1 seam cell expression during dauer   . Within es (IRS) . To detel dauers . Taken tDEX-1 acts with the ZP-domain protein DYF-7 to regulate primary dendrite extension during embryogenesis (dyf-7(m537) mutant for defects in dauer morphogenesis. Unlike dex-1 mutants, dyf-7 mutants are unable to enter dauer under typical dauer-inducing environmental conditions .ogenesis . We thercut-1 and cut-5 are expressed in the seam cells, while cut-6 is expressed in the surrounding hypodermis , the seam cells of the CUT mutant dauers were enlarged with jagged edges (dex-1(ns42) mutants, we found that cut-1(tm1126) and cut-5(ok2005) dauers were more sensitive to SDS compared with wild-type dauers  mutant dauers were resistant to the standard 1% SDS treatment  proteins are a family of ZP-domain proteins originally isolated from nematode cuticles  with cut-1(tm1126), cut-5(ok2005), and cut-6(ok1919). It is worth noting that, while the cut mutations are all deletion alleles , these data should be interpreted with caution as dex-1(ns42) is a hypomorphic allele. The dex-1; cut-1 double mutant did not enhance SDS sensitivity beyond the dex-1 single mutant, suggesting that they may act in the same pathway to regulate dauer remodeling ; dyf-7 double mutant (dex-1(cs201) . High levels of SNED1 expression promote invasiveness during breast cancer metastasis , the dex-1(ns42) allele is a non-sense mutation late in the coding region (dex-1(cs201) allele suggests that dex-1(ns42) is hypomorphic. Therefore, our dex-1 genetic interaction experiments should be interpreted with caution. In addition to this complication, our double-mutant experiments do not provide a clear interaction pathway. For example, our results suggest that deletion of cut-6 abrogates loss of cut-1. One possible explanation is a compensatory mechanism in the ECM, where loss of one ECM protein leads to increased expression of other structural components. This was previously shown in cases of osteogenesis imperfecta, where mutations in type I collagen led to increases in levels of thrombospondin and fibronectin (Our data suggests that g region . Our isoDEX-1 is another seam-specific epidermal matrix component that, along with ZP CUTs, facilitates apical constriction of the seam and formation of dauer alae (DEX-1 acts in a cell-autonomous manner directly beneath the edges of the lateral alae. Although expression of dex-1 in the hypodermis was sufficient to partially rescue dauer-specific radial shrinkage, hypodermal expression failed to rescue the lateral alae phenotype. We therefore propose DEX-1 may act as a secreted protein during dauer with restricted localization to the cuticle or ECM immediately above the apical membrane of the seam cells. During embryogenesis, DEX-1 is secreted and localized to the dendritic tips (DEX-1 may serve to couple physical interactions between the remodeled cuticular ECM and seam cell shape. Failure of these tissues to properly compact and thicken due to a loss of DEX-1 could lead to an overall weakening of the cuticle, and thus result in the SDS sensitivity observed in dex-1 mutant dauers. Previous research demonstrated a role for autophagy in dauer-specific seam cell remodeling (It has been proposed that biochemical compaction of the CUTs in the extracellular space between the seam and the hypodermis causes radial constriction, and thus forms the lateral alae via a “CUT tether” (uer alae . Our dattic tips . DEX-1 m


Drosophila vacuolar peduncle (vap), a presumed GAP enzyme, is associated with enhanced basal autophagy rate and physiological alterations resulting in a wasteful cell energy balance, a hallmark of overactive autophagy. By contrast, starvation-induced autophagy was disrupted in vap mutant conditions, leading to a block of maturation into autolysosomes. This phenotype stem for exacerbated biogenesis of PI(3)P-dependent endomembranes, including autophagosome membranes and ectopic fusions of vesicles. These findings shed new light on the neurodegenerative phenotype found associated to mutant vap adult brains in a former study. A partner of Vap, Sprint (Spri), acting as an endocytic GEF for Rab5, had the converse effect of leading to a reduction in PI(3)P-dependent endomembrane formation in mutants. Spri was conditional to normal basal autophagy and instrumental to the starvation-sensitivity phenotype specific of vap. Rab5 activity itself was essential for PI(3)P and for pre-autophagosome structures formation. We propose that Vap/Spri complexes promote a cell surface-derived flow of endocytic Rab5-containing vesicles, the traffic of which is crucial for the implementation of a basal autophagy rate.Basal autophagy is as a compressive catabolic mechanism engaged in the breakdown of damaged macromolecules and organelles leading to the recycling of elementary nutrients. Thought essential to cellular refreshing, little is known about the origin of a constitutional rate of basal autophagy. Here, we found that loss of  Autophagy  is a conserved self-eating process with homeostatic and cytoprotective functions important in many aspects of human health , 2. AutoIn parallel to its induction by stress, a basal rate of autophagy is taking place constitutively in most cells for a renewal of damaged organelles or elimination of misfolded proteins or aggregates –5. This ATG genes (AuTophagy Genes) are coordinating advancement in the process .(A) A structure-function analysis of Vap domain mutants was performed based on the capacity of over-expressed Vap to antagonize fat body-cell PI(3)P as shown in Fig 6E . Mean GFP area signals (left chart) or mean foci number (right chart) of selected cells were calculate for w- control clones  and spri(RI) clones . The difference between the two representations is accounted by the small size of the spri- foci. Error bars are standard errors; Significances are from Student’s t-tests.(B) Quantification of GFP:FYVE signals of perinuclear and cytoplasmic PI(3)P pools from w- and vap1 starved fat tissues . Note the accumulation of autophagosomes and amphisomes structures in the vap1 mutant, consistent with a block of the autophagy flux at late maturation stage (see text).(C) Characteristic vesicular autophagy-structures were compared in equivalent number of TEM sections of control, w1118/ hsFLP12; UAS-GFP:myc:2xFYVE / UAS-Vap:myc16.4; Act>CD2>GAL4/+. Assay: w1118/ hsFLP12; UAS-GFP:myc:2xFYVE/ +; Act>CD2>GAL4 / UAS-Vap:mycR695K. w1118/ hsFLP12; UAS-GFP:myc:2xFYVE / +; Act>CD2>GAL4; Vap:myc22.2. w1118/ hsFLP12; UAS-GFP:myc:2xFYVE / UAS-Vap:mycN15.1; Act>CD2>GAL4/+. w1118/ hsFLP12; UAS-GFP:myc:2xFYVE / UAS-Vap:mycB59.1; Act>CD2>GAL4/+. w1118/ hsFLP12; UAS-GFP:myc:2xFYVE/ +; Act>CD2>GAL4/ UAS-Vap:myc17.3.Genotypes. (A) Control: 1*. Martin G a, Yatani a, Clark R, Conroy L, Polakis P, Brown a M, et al. GAP domains responsible for ras p21-dependent inhibition of muscarinic atrial K+ channel currents. Science 1992; 255(5041): 192–4.2*. Tocque B, Delumeau I, Parker F, Maurier F, Multon MC, Schweighoffer F. Ras-GTPase activating protein (GAP): A putative effector for Ras. Cellular Signalling. 1997. p. 153–8.(TIF)Click here for additional data file.S6 Figw- and dominant-negative Rab5 expressing cell clones were generated using the Act>CD2>Gal4 flipout cassette method together with the GFP:FYVE marker. Fed or 3h-starved fat cells of early mid-3rd larvae were analyzed in fixed tissues focusing either in middle cell or below the cell surface close to cortical actin.(A-B”) Control, w- clones shows normal PI(3) P distribution in fed or starved fat cells (A).Control, Rab5-DN prevents the formation of PI(3)P at pericentral and cell surface locations. Instead, fluorescent GFP:FYVE probe remains throughout the cytosol. In starved cells, residual PI(3)P vesicles are apparent in the cytosol and in the cell surface (B-B’). Rab5-DN clones are missing the pericentral actin network in fed and starved cells (arrows) as analyzed by phalloïdin staining (B”).In fed cells, Rab5-DN clones are associated with expression of GFP:Atg8a marker . No GFP-labeled autophagosomes are detected in the starved Rab5-DN expressing cells and fluorescent probe remains over the cytosol .(C) Rab52-null fat-cell clones were generated by mitotic recombination as in  in fed and 3h-starved animals and analyzed for the presence of mCherry-labeled autophagosomes using p-mChAtg8a made of the natural Atg8a promoter  . Not any red-labeled autophagosomes are present in fed cells and rare mCherry-labeled structures are associated with the Rab5-/- clone in starved cells (arrows) compared to neighboring wild-type cells. Note the large-sized Rab5 mutant cells in A through D.(D) w- cell clones is associated with GFP:FYVE marker expression and analyzed for phalloïdin staining, revealing thin pericentral punctuate structures in all fed cells and larger ones in all 3h-starved cells (E’ inset: higher magnification images). Image stacks helps to visualized the actin structures in fed cells (E”). Scale bars in all panels = 20 μm.(E-E”) Control, w1118/ hsFLP12; UAS-GFP:myc:2xFYVE, Act>CD2>GAL4/+. Assay: w1118/ hsFLP12; UAS-GFP:myc:2xFYVE, Act>CD2>GAL4/ UAS-Rab5DN. (C) w1118/ hsFLP12; Act>CD2>GAL4, UAS-GFP:Atg8a/ UAS-Rab5DN. (D) w1118/ hsFLP12; Rab52, FRT40A/ 2xUAS-EGFP, FRT40A, Fb-GAL4; p-mChAtg8a/+. (E-E”) Control as in A.Genotypes. (A-B’) Control: (TIF)Click here for additional data file.S7 Figw- and mutant, vap1 fat bodies as in Fig 4B. These were compared to starved control, w- cells . Fed mutant and starved control cells shows elevated number of p62 bodies and non-overlapping aggregates of Rab7:GFP, suggesting that autophagy is at least partially stimulated in the fed vap mutant conditions. Scale bar = 10 μm.The state of endogenous p62/SQSTM1 autophagy-flux marker and late endosome Rab7:GFP marker were recorded in well fed cells of control, (TIF)Click here for additional data file.S8 FigScale bars = 20 μm.(TIF)Click here for additional data file.S1 Tablespri(RI) fat cells were always showing incomplete penetrance . This feature is likely in relation to the variegating nature of the spri gene [Note that analyzed autophagy defects of pri gene  or the R(TIF)Click here for additional data file.S1 File(ZIP)Click here for additional data file.S2 File(ZIP)Click here for additional data file.