Many species execute ballistic escape reactions to avoid imminent danger. Despite fast reaction times, responses are often highly regulated, reflecting a trade-off between costly motor actions and perceived threat level. However, how sensory cues are integrated within premotor escape circuits remains poorly understood. Here, we show that in zebrafish, less precipitous threats elicit a delayed escape, characterized by flexible trajectories, which are driven by a cluster of 38 prepontine neurons that are completely separate from the fast escape pathway. Whereas neurons that initiate rapid escapes receive direct auditory input and drive motor neurons, input and output pathways for delayed escapes are indirect, facilitating integration of cross-modal sensory information. These results show that rapid decision-making in the escape system is enabled by parallel pathways for ballistic responses and flexible delayed actions and defines a neuronal substrate for hierarchical choice in the vertebrate nervous system. Many species execute ballistic escape reactions to avoid imminent danger, but this is not always the best strategy. This study shows that zebrafish rapidly assess the salience of an auditory threat to select either an immediate ballistic response or a delayed and more flexible escape that uses a distinct and newly discovered neural pathway. Escape behaviors are fast defensive responses to threats that are typically driven by short sensorimotor reflex arcs . HoweverEscape behavior, triggered by abrupt tactile, auditory, or visual stimuli, has been studied extensively in teleost fish. Central to the escape circuit are the Mauthner cells (M-cells), a bilateral pair of giant reticulospinal neurons that trigger explosive C-start evasive maneuvers with a single action potential –9. HowevTo resolve these questions, we conducted an unbiased circuit-breaking screen to identify specific neurons that drive delayed escapes in zebrafish. We discovered a bilateral cluster of approximately 20 neurons per side in the prepontine hindbrain that initiate delayed escapes. Prepontine escape neurons are only active on trials in which larvae initiate a delayed escape but do not project directly to the spinal cord, indicating that they act as premotor neurons. Finally, results from behavioral experiments suggested that delayed escapes facilitate multimodal integration. Our data reveal that rapid behavioral choice is subserved by parallel pathways for ballistic and flexible delayed escapes and provides an identified and experimentally tractable neuronal substrate to study hierarchical decision-making within the vertebrate nervous system.We used high-speed video to analyze escapes triggered by acoustic/vibrational stimuli in free-swimming larvae that were 6 d post-fertilization (dpf) . Auditory252-Gal4, y293-Gal4, and y330-Gal4; y252-Gal4 and y293-Gal4; n = 10 y293-Gal4 larvae; Figs To identify neurons that subserve delayed escapes, we initiated a circuit-breaking screen using a library of Gal4 lines to selectively chemogenetically ablate subsets of neurons using an engineered nitroreductase variant   and therefore likely reflects the spatial expression pattern of this gene . Con. Cony293ce (DMD) . C-startce (DMD) . These ry293 neurons. For tracing, we sparsely labeled neurons by crossing y293-Gal4 to a heat shock–inducible B3 recombinase and an upstream activating sequence (UAS) reporter with B3 recombinase “blown-out” (blo) recognition sites  on sites . Neuriteon sites . Moreove neurons , they doatonal bHLH transcription factor 7 (atoh7) mutants, which lack retinofugal projections, confirming that retinal signaling is responsible for guiding delayed escape trajectories . This is striking because the VN drives startle responses to abrupt vestibular stimuli in mammals [Delayed escape neurons are located in an unannotated area of the prepontine hindbrain between the locus coeruleus and the cerebellum. The  mammals ,22. The  mammals . Among c mammals  and, at  mammals . Future Why are multiple prepontine escape neurons active during LLC responses? A single M-cell spike defines the latency and direction of an SLC; this spike, however, does not encode the entire movement trajectory but acts together with parallel descending motor pathways . AlthougThe M-cell ventral dendrite receives polysynaptic visual information via the optic tectum ,44; howeAlthough we do not yet have a comprehensive picture of how short- and long-latency escape circuits interact, we can predict features of their organization . PrepontThe M-cell system has given us one of the most complete pictures of neural circuit function underlying behavior in vertebrates and remains an important model for understanding how motor decisions are computed at the cellular level ,43,47. HAll in vivo experimental procedures were conducted according to National Institutes of Health guidelines for animal research and were approved by the NICHD Institutional Animal Care and Use Committee (#18–007).Danio rerio) and Gal4 enhancer trap and transgenic lines used in this study were maintained in a Tüpfel long fin (TL) strain background. Experiments were performed on larvae in the first 8 dpf, before sex differentiation. Embryos were raised in E3 medium supplemented with 1.5 mM HEPES (pH 7.3) (E3h) at 28°C on a 14 h:10 h light:dark cycle with medium changes at least every 2 d unless otherwise described.Wild-type zebrafish (Tg(UAS-E1b:Kaede)s1999t (UAS:Kaede) [huc:Cer pattern to select an initial set of 16 lines with good global coverage and then manually selected additional lines to fill in less well represented areas. For chemogenetic ablation experiments, these lines were crossed to nitroreductase lines Tg(UAS-E1b:BGi-epNTR-TagRFPT-oPre)y268Tg or Tg(UAS:epNTR-TagRFPT-utr.zb3)y362Tg [y362Tg line that includes a synthetic 3′ UTR designed to suppress non-neural expression [http://zebrafish.org). UAS:bloswitch and hsp70l:B3 lines were used for y293Et neuron tracing [Atoh7sa16352 mutants were acquired from the ZIRC [Et(gata2a:EGFP)pku2 (vmat2:GFP) [TgBACmpn202  were as published [Tg(mnx1:GFP)ml2 [Tg(UAS-E1b:synaptophysin-TagRFPT)y261 [Images throughout were registered to the ZBB to enable comparison with other markers . Gal4 liS:Kaede) . We appl3)y362Tg ,50. Gal43)y362Tg , or werepression . Gal4 li tracing . Atoh7sathe ZIRC . The locublished , registe:GFP)ml2  and Tg(UFPT)y261 .y293-Gal4; UAS:bloSwitch fish were crossed to hsp70l:B3. Sparse labeling was achieved by a 25–35-min heat shock at 37°C at 3 dpf to induce B3 recombinase expression. Larvae were then raised under standard conditions and imaged at 6 dpf. Neurons were traced in Imaris 8.4.2, exported as TIFs, and converted to NIFTI for alignment with ANTs to y293-Gal4 as a reference [Embryos were raised in E3h media containing 300 μM N-Phenylthiourea (PTU) starting at 8–22 hpf to suppress melanophore formation with PTU changed at least every 48 h. For imaging at 6 dpf, larvae were anesthetized in 0.24 mg/mL tricaine methanesulfonate (MS-222) for 3 min and mounted in 2.5% low-melting-point agarose in 3D-printed plastic inserts (ABS from Stratasys or clear resin from FormLabs) within #1.5 thickness (0.17 ± 0.005 mm) cover glass-bottom cell-culture chambers (Lab-Tek II 155379). An inverted laser-scanning confocal microscope (Leica TCS SP5 II) equipped with an automated stage, and a 25x/0.95 NA apochromatic water immersion lens (Leica # 11506340) was used to acquire confocal stacks. For labeling individual neurons, eference . To photy293-Gal4;UAS:Kaede larvae raised in PTU. Laser excisions were performed on an upright laser-scanning confocal microscope (Leica TCS SP5 II) equipped with a multiphoton laser (SpectraPhysics MaiTai DeepSee), automated stage, and 20x/1.00 NA apochromatic water-dipping lens (Leica # 11507701). Larvae to be ablated as well as controls were mounted in 2.5% low-melting-point agarose on #1.5 thickness cover glass, which was then inverted for the upright microscope. A 488-nm argon laser line was used to visualize target cells and confirm ablation, whereas the multiphoton laser tuned to 800 nm was used for selective laser excision. The laser was pulsed for 5–1,000 ms at approximately 2.4 W until cell integrity was compromised. Following ablation, larvae were raised in E3h until behavioral testing at 6 dpf. Successful ablations were then confirmed by confocal microscopy—only larvae with 3 cells or fewer remaining on either side were analyzed.For genetic ablations, we used an engineered variant (epNTR) of the bacterial nitroreductase gene, which converts a cell-permeable substrate (metronidazole) into a cell-impermeable cytotoxin ,57. Gal42 (arena light) and by an infrared LED array for imaging purposes from below. During light-spot trials, the arena light was replaced for 3.5 s with a light spot of about 8 μW/cm2 and an approximately 6-mm diameter focused from below. Three seconds after appearance of the light spot, larvae were exposed to an acoustic/vibratory stimulus. Aside from the switching of illumination, control trials to quantify baseline responses were performed under the same conditions. Control and light-spot trials were pseudorandomly presented 20–40 times each at 15-s intervals. The change in illumination elicited O-bends only within the first second, and not at 3 s when the acoustic stimulus was provided. For chemogenetic ablation experiments, larvae were tested individually in 9.7 × 9.7-mm wells of a 3 × 3 grid illuminated by an LED array at approximately 500 μW/cm2 from below. Different stimulus intensities were presented 20 times each in a pseudorandom sequence at 15-s intervals to minimize habituation. Genetically ablated larvae and metronidazole-treated controls were tested in alternation.For light-spot experiments, TL larvae were tested in groups of 15–20 within a 33 × 33-mm corral, which kept larvae in view of a high-speed camera. Larvae were illuminated from above at about 80 μW/cmy293-Gal4 larvae, for which we analyzed 200 ms to ensure that LLCs were absent and not merely delayed.Auditory stimuli consisted of sinusoidal waveforms of 21–36 dB, of 2-ms duration, and nominally 250 or 1,000 Hz, though the acoustic/vibrational stimuli as delivered are intrinsically broadband. Stimuli were delivered with an electrodynamic exciter  controlled by a digital–analog data acquisition card . Behavioral responses of larvae were recorded at 1,000 frames/s with a high-speed camera  fitted with a 50-mm macro lens . For light-spot experiments requiring infrared illumination, cameras were additionally fitted with an infrared filter . Recorded trial bouts were 120 ms in length with acoustic/vibratory stimuli delivered at 30 ms, except for laser excisions of Behavioral responses were analyzed with Flote software . As ongoy293-Gal4 embryos were injected at the one-cell stage with tol1 mRNA and a plasmid containing either UAS:BGi-nls-GCaMP6s.zf2-v2a-nls-dsRed.zf1-afp or UAS:BGi-ChEF-v2a-mCherry-afp, respectively [For calcium imaging or optogenetic stimulation, ectively , screeney293-Gal4 were converted into binary ROIs and projected back onto the larval zebrafish brain by a 460-nm LED (Prizmatic) and digital micromirror device  for durations of 10 or 100 ms controlled by Clampex . Optogenetic-stimulation ROIs covered an area of 50 × 25 μm centered over a prepontine neuron cluster in one hemisphere. Spatial specificity of the setup was previously assessed by photoconversion of Kaede in a targeted area (GCaMP6s- or ChEF-positive neurons were imaged on a custom-built multiphoton microscope with a 20x/0.90 NA water-dipping lens (Olympus) and a Ti-Sapphire laser (Coherent Chameleon Vision-S) tuned to 950 nm and controlled in Matlab (Mathworks) by ScanImage . For calhttp://www.R-project.org/), and Gnumeric (http://projects.gnome.org/gnumeric/). All t tests are two-sided, independent samples . No animals were excluded from analysis except for calcium imaging, for which only SLC- and LLC-responsive animals were analyzed or for which laser ablations were incomplete. Graphs and text report means and standard errors. Box plots show median and quartiles; whiskers show 10%–90%. Bar plots show mean and standard error. N reported in figure legends indicates biological replicates .Analysis was performed with IDL (Harris), R Click here for additional data file.S2 DataExcel spreadsheet containing numerical data underlying supporting figures.(XLSX)Click here for additional data file.S1 Figy252-Gal4, y293-Gal4, and y330-Gal4. Significant ANOVAs are indicated at the top of each graph, after Bonferroni correction for the 16 comparisons, and *p < 0.05 for post hoc t test compared to nonablated metronidazole-treated non-epNTR expressing sibling controls. Underlying numerical data are included in S2 Data. epNTR, engineered nitroreductase variant; LLC, long-latency C-start; Mav, maximum angular velocity; SLC, short-latency C-start.(A) Comparison of initial bend angle (C1-angle), counterbend angle (C2-angle), and displacement for SLC and LLC responses (same larvae as in (TIF)Click here for additional data file.S2 Figy293-Gal4-, y264-Gal4-, and chata-Gal4-labeled neurons in R1–4. Prepontine neurons labeled by y293-Gal4 are located in R1, in contrast to the anterior (“a”) and posterior trigeminal motor nuclei labeled by chata-Gal4 located in R2 and R3, respectively, and the Mc in R4 labeled by y264-Gal4. (C) Coronal projection of y293-Gal4 prepontine neurons situated between the LC and the CE. CE, cerebellum; d, dorsal; LC, locus coeruleus; m, medial; Mc, Mauthner cell; R1–4, rhombomeres 1–4; Ra, raphe; ZBB, Zebrafish Brain Browser.(A-B) Dorsal (A) and parasagittal (B) projections from ZBB of (TIF)Click here for additional data file.S3 Fign = 9, green), R6 ablation (n = 16 blue), and unablated sibling controls . Significant effects of R1 ablations on LLC and SLC probability; ANOVA F1,102 = 23.37, p < 0.001 and F1,102 = 21.79, p < 0.001, respectively. (C) SLC kinematic measurements after bilateral R1 laser ablation (n's as above). No significant differences (t test). (D) SLC directionality (%Right: percent of SLC responses initiated to the right) after unilateral (left) R1 laser ablation. n = 14 (ablated) and 24 (control). Underlying numerical data are included in (A-B) LLC (A) and SLC (B) responsiveness after R1 ablation ((TIF)Click here for additional data file.S4 Figelavl3:nls-mCar in purple) and neuroanatomic segmentation on the right . Top: coronal projection of the outlined region showing y293-Gal4 , neurod:GFP  [TH-Gal4  [TH (B) and Fndc5 (B'). Image credit: Allen Institute, modified from Allen Developing Mouse Brain Atlas. AMBA, Allen Mouse Brain Atlas; CE, cerebellum; Fndc5, fibronectin type III domain containing 5; HB, hindbrain; LAV, lateral vestibular nucleus; LC, locus coeruleus; MV, medial vestibular nucleus; OT, optic tectum; P56, postnatal day 56; Pp, prepontine area; SUV, superior vestibular nucleus; TH, tyrosine hydroxylase; ZBB, Zebrafish Brain Browser.(A) Bottom: coronal projection through zebrafish rhombomere 1 (slice 512–517 from ZBB) with nuclear labeling on the left  , and TH-nta, LC) . (B) Bot(TIF)Click here for additional data file.S5 Figelavl3:Cer as a reference (gray). (A’) Overlay of co-registered neurons in (A) showing conserved quadripartite morphology (asterisks). Dotted lines indicate areas expanded in (B) and (C). (B) Enlargements of hindbrain from A’ with arrowheads indicating neuron terminals. (C) Enlargements of lateral rhombomere 1 from A’ with arrowheads marking termini in the cerebellar EG. (D) Horizontal projection of confocal stack including prepontine escape neuron cell bodies  after selective photoconversion of Kaede to red and SAG axon rostral termini . (E) Projection of a reconstructed neuron (green) registered to ZBB, with the y256-Gal4 pattern (magenta) that labels the SAG and its projections. (F-G) Horizontal projection (F) and zoom (G) of confocal stack through the caudal hindbrain and anterior spinal cord of a y293-Gal4, UAS:synaptophysin-TagRFPT, mnx1-GFP larva. Scale bars: 50 μm in (A) and (A'); 20 μm in (D-G). EG, eminentia granularis; SAG, statoacoustic ganglion; ZBB, Zebrafish Brain Browser.(A) Dorsal standard-deviation projections of 5 traced prepontine escape neurons with (TIF)Click here for additional data file.S6 Fign = 29 groups of larvae. (B) Percent of LLCs in a rightward direction in the presence of a light spot for atoh7 mutant larvae and siblings. ***p < 0.001, *p < 0.05, n = 5 plates each atoh7−/− and siblings. Underlying numerical data are included in atoh7, atonal bHLH transcription factor 7; C1, initial C-start bend; C2, counterbend; LLC, long-latency C-start.(A) Kinematic parameters for acoustically evoked LLCs performed under broad-field illumination (black) or in the presence of a light spot (blue). (TIF)Click here for additional data file.S1 Videoy293-Gal4. Light stimulation (460 nm) for 10 or 100 ms is indicated by a square in the top right corner of each subframe with timestamp at the bottom right. DMD, digital mirror device.Representative optogenetic trials from 3 ChEF-positive and a ChEF-negative control larvae (bottom right) showing behavioral results to DMD illumination and optogenetic stimulation of prepontine neurons in (AVI)Click here for additional data file.


In addition to seasonal influenza viruses recently circulating in humans, avian influenza viruses (AIVs) of H5N1, H5N6 and H7N9 subtypes have also emerged and demonstrated human infection abilities with high mortality rates. Although influenza viral infections are usually diagnosed using viral isolation and serological/molecular analyses, the cost, accessibility, and availability of these methods may limit their utility in various settings. The objective of this study was to develop and optimized a multiplex detection system for most influenza viruses currently infecting humans.We developed and optimized a multiplex detection system for most influenza viruses currently infecting humans including two type B (both Victoria lineages and Yamagata lineages), H1N1, H3N2, H5N1, H5N6, and H7N9 using Reverse Transcriptional Loop-mediated Isothermal Amplification (RT-LAMP) technology coupled with a one-pot colorimetric visualization system to facilitate direct determination of results without additional steps. We also evaluated this multiplex RT-LAMP for clinical use using a total of 135 clinical and spiked samples (91 influenza viruses and 44 other human infectious viruses).We achieved rapid detection of seasonal influenza viruses  and avian influenza viruses  within an hour. The assay could detect influenza viruses with high sensitivity , comparable to conventional RT-PCR-based approaches which would typically take several hours and require expensive equipment. This assay was capable of specifically detecting each influenza virus  without cross-reactivity with other subtypes of AIVs or other human infectious viruses. Furthermore, 91 clinical and spiked samples confirmed by qRT-PCR were also detected by this multiplex RT-LAMP with 98.9% agreement. It was more sensitive than one-step RT-PCR approach (92.3%).Results of this study suggest that our multiplex RT-LAMP assay may provide a rapid, sensitive, cost-effective, and reliable diagnostic method for identifying recent influenza viruses infecting humans, especially in locations without access to large platforms or sophisticated equipment.The online version of this article (10.1186/s12879-019-4277-8) contains supplementary material, which is available to authorized users. The World Health Organization constantly monitors global influenza activity. It has noted global circulation of H1N1, H3N2, and two lineages of type B (Victoria- and Yamagata-) viruses that cause 670,000 annual deaths worldwide . RecogniVaccination and treatment with antiviral drugs ) are primary interventions to prevent viral infections and their spread. However, vaccine production usually takes 6–12 months to prepare for newly emerging viruses. NAIs also should be taken within the first 48 h following an infection . Thus, rhttps://www.cdc.gov/flu/professionals/diagnosis/molecular-assays.htm). Although the detection time (similar to RIDT) and sensitivity (better than RIDT) of CLIA-waived molecular tests have been improved, they are mostly limited to seasonal flu detection. They are incapable of discriminating seasonal influenza of avian subtypes that can cause human infection . On the other hand, the conventional one-step RT-PCR and real-time RT-PCR assays demonstrated detection limits ranging from 0.1 ~ 1,000 infectious viral genome copies for the same viral RNA samples  and other human infectious viruses obtained from a total of 44 clinical/spiked samples positive for HEV, AdV, PIV, MPV, HboV, HRV, 229E, NL63, OC43, RSVA, RSVB, and MERS-CoV that might potentially be in co-circulation with target viruses and cause similar symptoms. Based on colorimetric assessments, the RT-LAMP reaction using each primer set for H1, H3, H5, and H7, and type B viruses demonstrated no cross-reactivity with other influenza subtypes or human infectious viruses tested  and 18 spiked  samples initially diagnosed with influenza using qRT-PCR  were clinically evaluated using the multiplex RT-LAMP assay developed in this study. For comparison, conventional RT-PCR was performed in parallel. Results showed that 97~100% of seasonal influenza viruses were specifically detected by the multiplex RT-LAMP which exhibited similar sensitivity to qRT-PCR: 35 out of 35 samples in type B (100%), 12 out of 12 samples in H1N1 (100%), and 34 out of 35 samples in H3N2 (97%) without cross-reaction to each other , technical support, and electricity. Thus, the use of chaotropic salt extraction which does not require centrifugation  would imAlthough RT-LAMP assays designed to detect Victoria lineage Type-B viruses has 10 times less sensitivity compared to one-step RT-PCR and qRT-PCR approaches, RT-LAMP reactions for other subtypes exhibited similar or higher sensitivity without cross-reaction to various human infectious viruses and other subtypes of avian influenza viruses. Furthermore, our multiplex RT-LAMP assay was more sensitive than a conventional RT-PCR approach using clinical or spiked samples. Although the RT-LAMP detection method for H7N9 was optimized and evaluated, single spiked sample for clinical verification would be a limitation of the method. The specificity of multiplex RT-LAMP assay demonstrated in this current study might have limitations for use as a primary detection method based on local epidemiological setting. However, this assay’s rapid detection has a valuable contribution by providing diagnostics specifically in areas with prevalent avian influenza viruses infecting humans.Our multiplex RT-LAMP method not only can minimize the use of expensive lab instruments and devices, but also can detect broad-spectrum human influenza viruses and avian influenza viruses infecting humans as accurately and more rapidly than conventional RT-PCR-based detection methods suitable for use in on-site testing. This method will improve and aid in the diagnosis of influenza infections and potentially increase the speed of clinicians to provide appropriate treatment.Additional file 1Figure S1. Specificity of RT-LAMP. The specificity of the RT-LAMP assay was tested using (A) individual and (B) mixed influenza virus samples listed below. RT-LAMP amplicon was confirmed using 2% agarose gel electrophoresis. The positive sample (yellow color) has a typical ladder-like pattern of RT-LAMP reaction. (a) B-Vic:B/Brisbane/60/2008 (Victoria lineage); (b) B-Yam: B/Phuket/3073/2013 (Yamagata lineage); (c) H1N1:A/California/04/2009; (d) H3N2: A/Perth/16/2009; (e) aH5N1:A/Em/Korea/w149/2006; (f) hH5N6 vac: A/Sichuan/26221/2014; (g) aH5N8:A/Em/Korea/w468/2014; (h) aH5N8 vac:A/gyrfalcon/Washington/41088–6/2014; (i) hH7N9:A/Anhui/1/2013; N.C.: Negative control (D.W.). Figure S2. Sensitivity of the RT-LAMP assay compared with conventional methods. To estimate the sensitivity of the RT-LAMP assay, RNA samples from each influenza viruses were 10-fold serially diluted and used as templates for the RT-LAMP assay (A), conventional RT-PCR (B), and real-time qRT-PCR (C). RT-LAMP results are visualized colorimetrically and by gel-electrophoresis. Results of conventional RT-PCR (B) and real-time qRT-PCR (C) are visualized using gel electrophoresis and cycle threshold (Ct) values, respectively. Please see Table Table S1. Primers for detection of influenza viruses of other subtypes and human 3 respiratory disease viruses. (PDF 565 kb):


The underlying mechanism of miR-9 upregulation in GC is still unknown. Through searching multiple long non-coding RNA (LncRNA) databases, we here discovered that the long non-coding RNALINC00682 (long intergenic non-protein coding RNA 682) putatively targets miR-9. We show that ectopic overexpression of LINC00682 induced miR-9 downregulation but LMX1A upregulation, inhibiting AGS cell survival, proliferation, migration and invasion. Significant apoptosis activation was detected in LINC00682-overexpressed AGS cells. Contrarily, LINC00682 knockdown induced miR-9 upregulation but LMX1A downregulation, promoting AGS cell survival, proliferation, migration and invasion. In the primary human GC cells, forced LINC00682 overexpression similarly induced miR-9 downregulation and LMX1A upregulation, causing proliferation inhibition and apoptosis activation. Significantly, restoring miR-9 expression by a lentiviral construct reversed LINC00682-induced actions in GC cells. Furthermore, LINC00682 was ineffective in LMX1A KO AGS cells. Importantly, LINC00682 expression levels are significantly downregulated in human GC tissues. We conclude that LINC00682 inhibits GC cell progression via targeting miR-9-LMX1A signaling axis. Gastric cancer (GC) accounts for over 10% of new cancer cases each year . It is oLMX1A gene promoter hyper-methylation in human cancers could be one important cause of its downregulation [miRNA, microRNA-9 (miR-9) [miR-9 upregulation might account for LMX1A downregulation in human GC tissues [miR-9 inhibition upregulated its target LMX1A, thereby inhibiting GC cell survival and proliferation [miR-9 upregulation in human GC is still largely unknown.LIM homeobox transcription factor 1, alpha (LMX1A) is a widely-studied member of LIM-homeodomain (LIM-HD) family protein . As an egulation , 8, 10. Long non-coding RNAs (LncRNAs) are a family of evolutionarily conserved non-coding RNAs (ncRNAs) with over 200 nucleotide long [LncRNAs is commonly detected in GC [LncRNAs regulate almost all important cellular functions, from genomic imprinting, cell proliferation and growth, cell cycle progression to cell differentiation, survival and apoptosis [LncRNA acts as competing endogenous RNA (ceRNA) to sponge target miRNAs [LINC00682 (long intergenic non-protein coding RNA 682) targets miR-9-LMX1A signaling axis to inhibit human GC cell survival and proliferation.ide long –16. Dysred in GC , 18, whied in GC , 17–20. poptosis –16. LncRt miRNAs . The resmiR-9 upregulation in GC tissues  was searched to find possible miR-9-targeting LncRNAs. The LncRNAs were further verified by searching other LncRNA/miRNA databases (StarBase and miRbase). The bioinformatic analyses identified that one LncRNA, LINC00682, putatively targets miR-9, with its percentage over 99%.We hypothesized that LINC00682 on miR-9-LMX1A axis, the lentivirus encoding LINC00682-expressing construct (“LV-LINC00682”) was added to AGS cells. Following selection by the puromycin-containing complete medium, two stable cell lines, “sLi-1” and “sLi-2”, were established. qPCR results confirmed that LINC00682 levels increased over ten folds (versus control cells) in the LV-LINC00682-expressing stable cells  polymerase (PARP)  , as welle (PARP)  and nucle (PARP) . Additiostaining . The conpression –1F nor Apression –1O. ThesLINC00682 overexpression inhibited AGS cell progression in vitro  of LINC00682 were transfected individually to AGS cells. Results from the qPCR confirmed that each siRNA resulted in over 90% reduction of LINC00682 expression in AGS cells  had no detectable effect on LINC00682-miR-9-LMX1A/B expression  in stable primary cancer cells with LV-LINC00682. Forced overexpression of LINC00682 induced miR-9 downregulation  [LINC00682 expression  primary GC patients [LINC00682 expression was examined by qPCR. Results show that LINC00682 levels are significantly downregulated in cancer tissues (“Can”) (LINC00682 downregulation correlates with miR-9 upregulation and LMX1A downregulation in GC tissues (see the results from same set of tissue samples [Expression of patients . LINC006 (“Can”) , when co (“Can”) . TherefomiR-9 depletion led to GC cell apoptosis [LMX1A is hyper-methylated and downregulated in ovarian cancer and cervical cancer , 13. RecLINC00682. One genome wide methylation study between primary and recurrent hepatocellular carcinomas (HCC) has indicated that low levels of LINC00682 methylation were strongly correlated with HCC recurrence and patient disease/recurrent-free survival (DFS) [LINC00682 could function as the ceRNA of miR-9 to promote LMX1A expression, thereby inhibiting GC cell progression.Very few studies have explored the potential biological function of al (DFS) . In the LINC00682 induced miR-9 downregulation but LMX1A upregulation, inhibiting GC cell survival, proliferation migration and invasion. Furthermore, significant apoptosis activation was detected in LINC00682-overexpressed GC cells. On the contrary, LINC00682 knockdown by targeted siRNAs induced miR-9 upregulation but LMX1A downregulation, promoting AGS cell proliferation, migration and invasion. Significantly, LINC00682 expression levels are downregulated in human GC tissues, correlated with miR-9 upregulation and LMX1A downregulation. Thus, LINC00682 downregulation could be at least one reason of miR-9-LMX1A dysregulation in human GC . The antibodies of the present study were obtained from Abcam . The reagents for RNA assays, Lipofectamine 2000 and other transfection reagents were provided by Thermo-Fisher . All sequences, constructs, viruses and plasmids were provided by Shanghai Genechem Co. .Using a previously described protocol AGS cells were cultured . The priLINC00682 was tested by quantitative reverse transcriptase PCR (“qPCR”).As described early , from twΔΔCt method was utilized for the quantification of target mRNA, with GAPDH as the internal control. LINC00682 and miR-9 expression was normalized to U6 RNA. Primers for miR-9, LMX1A, GAPDH and U6 were described previously [LINC00682 were provided by Shanghai Genechem .The detailed protocol for qPCR was described early . The ΔΔCLINC00682 was synthesized by Shanghai Genechem, sub-cloned to a GV248 (hU6-MCS-Ubiquitin-EGFP-IRES-puromycin) vector (Shanghai Genechem). The construct was transfected to HEK-293 cells together with lentiviral packaging plasmids [LINC00682-expressing lentivirus (“LV-LINC00682”). After filtration and enrichment, the lentivirus was added to cultured GC cells (in polybrene medium). Thereafter, puromycin (5.0 μg/mL) was added to select stable cells for 4-5 passages. Control cells were infected with lentivirus with empty vector (“LV-c”).The full-length LINC00682 were individually transfected by Lipofectamine 2000 for 24h (the siRNA concentration at 500 nM). The transfection was repeated another round . Knockdown efficiency was verified by qPCR testing LINC00682 expression. Control cells were transfected with the scramble non-sense control siRNA (“si-C”).Two siRNAs (provided and verified again by Shanghai Genechem) targeting non-overlapping sequences (“Seq1/2”) of LMX1A 3’-UTR construct [LINC00682 siRNA or LV-LINC00682, LMX1A3’-UTR luciferase activity was tested as reported [As reported early , AGS celonstruct , the Renreported .GC cells were seeded into the 96-well plates at 5000 cells per well [As described , GC cellFollowing the genetic treatments, GC cells were seeded onto 12-well plates at 30, 000 cells per well. The EdU (5-ethynyl-20-deoxyuridine) Apollo-567 Kit  was employed to quantify cell proliferation. EdU and DAPI dyes were added to GC cells for 6h. Under a fluorescent microscope cell nuclei were visualized. For each condition 800 nuclei in five random views were included to calculate EdU ratio (EdU/DAPI×100%).Testing cell apoptosis, by the caspase-3 activity assay, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), and Propidium iodide (PI)-Annexin V FACS assay was described in other studies , 25.AGS cells were seeded into six-well plates at 100, 000 cells per well. The two lentiCRISPR-GFP-puro LMX1A knockout constructs  were inFollowing the genetic treatments, the lysis buffer  was added to cultured GC cells. Quantified total cellular lysates were separated by a SDS-PAGE (10%) gel, transferred to a polyvinylidene fluoride blot. The latter was blocked in PBST with 10% milk, and incubated with indicated primary and secondary antibodies. The immunocomplexes were visualized using an ECL substrate kit . Quantification of the target protein bands was through the ImageJ software measuring the total gray. The value was normalized to the loading control.P < 0.05.For statistical analyses the SPSS software (version 18.0) was employed. All values were expressed as the mean ± standard deviation (SD). All differences were considered significant at


Drosophila mushroom body (MB) sequentially gives rise to the MB neuron subtypes. The temporal fate determination pattern of MB neurons can be influenced by extrinsic cues, conveyed by the steroid hormone ecdysone. Here, we show that the activation of Transforming Growth Factor-β (TGF-β) signalling via glial-derived Myoglianin regulates the fate transition between the early-born α’β’ and the pioneer αβ MB neurons by promoting the expression of the ecdysone receptor B1 isoform (EcR-B1). While TGF-β signalling is required in MB neuronal progenitors to promote the expression of EcR-B1, ecdysone signalling acts postmitotically to consolidate theα’β’ MB fate. Indeed, we propose that if these signalling cascades are impaired α’β’ neurons lose their fate and convert to pioneer αβ. Conversely, an intrinsic signal conducted by the zinc finger transcription factor Krüppel-homolog 1 (Kr-h1) antagonises TGF-β signalling and acts as negative regulator of the response mediated by ecdysone in promoting α’β’ MB neuron fate consolidation. Taken together, the consolidation of α’β’ MB neuron fate requires the response of progenitors to local signalling to enable postmitotic neurons to sense a systemic signal.Neuronal diversity is at the core of the complex processing operated by the nervous system supporting fundamental functions such as sensory perception, motor control or memory formation. A small number of progenitors guarantee the production of this neuronal diversity, with each progenitor giving origin to different neuronal types over time. How a progenitor sequentially produces neurons of different fates and the impact of extrinsic signals conveying information about developmental progress or environmental conditions on this process represent key, but elusive questions. Each of the four progenitors of the  Throughout the development of the central nervous system (CNS), a vast number of neuronal types are produced with striking precision. The unique identity of each neuronal cell type and the great cellular complexity in the CNS are established by intricate gene regulatory networks. Disruption of these identity programs leads to neurodevelopmental disorders and defects in cognition. Here, we report an important regulatory mechanism involved in consolidating neuronal fate. We show that during brain development local signalling, derived from interactions between glial cells and neuronal progenitors, is required to promote the expression of a hormone receptor in immature neurons. The perception of a systemic hormonal cue in those postmitotic neurons is fundamental for the consolidation of their neuronal fate. In this context, we additionally uncover an intrinsic regulatory mechanism that coordinates the hormone response to maintain the final neuronal fate. Drosophila embryo have uncovered that temporal fate specification is governed by the sequential expression of transcription factors in the progenitors, providing temporal identity to their daughter cells , GAL80-asense [UAS-dSmad2-CA [babo9 [hs-FLP, tubP-GAL80, FRT19A; UAS-mCD8::GFP (BL#5134), tubP-GAL80, FRTG13 (BL#5140), tubP-GAL80, FRT40A (BL#5192), hs-FLP, UAS-mCD8::GFP (BL#28832), UAS-mCD8::GFP (BL#5130), GAL4-c708 (BL#50743), GAL4-Repo (BL#7415), UAS-Myoglianin RNAi (BL#31200) [UAS-EcR-B1ΔC655.W650A (BL#6872), tub-GAL80TS (BL#7017), UAS-EcR-B1 (BL#6469), UAS-EcR-A (BL#6470), LexA-GMR26E01 , LexAop-mCD8-GFP , UAS-mCD8-GFP (BL#32194), GAL4-OK107 (BL#854), UAS-Luciferase RNAi (BL#31603), UAS-Kr-h1 RNAi (BL#50685), UAS-LacZ (BL#1777), GAL4-worniu (BL#56553), GAL4-c305a (BL#30829), dSmad21 (BL#44384), babo32 (BL#5399), babok16912 (BL#11207), chinmo1 (BL#59969), UAS-babo-CA (BL#64293) and Kr-h110642 (BL#12380). The following stocks were obtained from Vienna Drosophila RNAi Centre (VDRC): babo RNAi (v106092), dSmad2 RNAi (v14609) and EcR RNAi (v37059). A full list of used genotypes is included in the supplemental information. Mosaic analyses using MARCM to generate NB or single cell clones were performed as previously described [Fly stocks used were as follows: 0-asense , UAS-dSmSmad2-CA  and baboA [babo9 . The folL#31200) , 38, 39,:GFP BL#54, tubP-GPrimary antibodies used in this study were: anti-Fasciclin II , anti-EcR-B1 , anti-Trio , anti-GFP , anti-Deadpan , anti-β-Gal , anti-Chinmo ,Brains were dissected in cold PBS, fixed in 4% formaldehyde and incubated overnight at 4°C with primary antibody. Secondary antibody was incubated for four hours at room temperature. Brains were mounted in Vectashield mounting media (Vector Laboratories). Images were collected with a Zeiss LSM 780 Meta confocal microscope using a 40× 1.4 NA oil immersion objective. To count cell numbers, cell bodies were identified based on mGFP signal. In EcR coding sequence was created using the web-based probe designer (https://www.biosearchtech.com/stellaris-designer) . The probes were labelled with Quasar-570 dye and were purchased from LGC BioSearch Technologies.Wandering third instar larva brains were dissected in cold PBS. Fixation, hybridization and immunostaining were performed as previously described . A set ohttps://yapic.github.io/yapic/) . Training data for background and cell lumen regions were collected on two stacks by using ilastik software [Cell lumens of confocal stacks were detected by training an artificial neural network with the pixel classification software YAPiC  Quantification of number of adult MB neuron cell bodies from OK107>luciferase RNAi and OK107>babo RNAi brains. Statistical comparison to the control: N.S. Not Significant (two tailed t test).  Adult MB lobes from OK107>p35 (C) and OK107>dSmad2 RNAi, p35 (D) brains stained with anti-FasII antibody (magenta). Green: GMR26E01-LexA-α’β’-MB-driven mGFP in C,D. (E) Quantification of number of adult α’β’ MB neuron cell bodies from OK107>p35 (C), OK107>dSmad2 RNAi, p35 (D) and OK107> dSmad2 RNAi brains. Statistical comparison to the control: ***, p<0.001 (two tailed t test); N.S. Not Significant.(A-A’) Single confocal section across the cell body cluster of adult MB neurons as representative picture of the counting procedure. Cells are marked in white. Green: GAL4(DOCX)Click here for additional data file.S2 Figbabo9 (B) (n = 14) and dSmad21 (C) (n = 13) MARCM NB clones induced at NHL and labelled with mGFP (green) using the GAL4-OK107 driver and stained with anti-FasII antibody (magenta). (D-I’) WL3 MB lobes of control , OK107>babo RNAi , OK107>dSmad2 RNAi  brains visualized with GAL4-OK107-driven mGFP (green) and labelled with anti-FasII (D-F’) or Trio (G-I’) antibodies (magenta).(A-C) WL3 MB lobes from control (A) (n = 10), (DOCX)Click here for additional data file.S3 Figbabo9  MARCM MB neuroblast clones (white dashed line) induced at NHL and analysed at WL3. MARCM MB clones visualized with mGFP (green) expressed by the GAL4-OK107 driver and labelled with anti-Chinmo (A-B’) or Abrupt (C-D’) antibodies (magenta).(A-D’) Cell bodies of control  and (DOCX)Click here for additional data file.S4 Figchinmo1 (B), chinmo1; UAS-babo-CA (C) and chinmo1; UAS-dSmad2-CA (D) neuroblast MARCM clones generated at mid-3rd instar, labelled with mGFP (green) using the GAL4-OK107 driver and stained with anti-FasII antibody (magenta). (E) Quantification of α’β’ MB fate defects in control, chinmo1, chinmo1; UAS-babo-CA and chinmo1; UAS-dSmad2-CA neuroblast clones. Statistical comparison to chinmo1: N.S. Not Significant (Fisher’s exact test). (F-I’) Cell bodies of control , chinmo1 , chinmo1; UAS-babo-CA  and chinmo1; UAS-dSmad2-CA  MARCM MB neuroblast clones (white dashed line) induced at NHL and analysed at WL3. MARCM MB clones were visualized with mGFP (green) expressed by the GAL4-OK107 driver and labelled with anti-EcR-B1 antibody (magenta).(A-D) Adult MB lobes from control (A), (DOCX)Click here for additional data file.S5 Figworniu>mGFP (green) brains at WL3, co-labelled with anti-EcR-B1 antibody (magenta).MB progenitors cell bodies from (DOCX)Click here for additional data file.S6 Figworniu>EcR-B1 (A) and worniu>babo RNAi, EcR-B1 (B) brains stained with anti-FasII antibody (magenta). Green: GMR26E01-LexA-α’β’-MB-driven mGFP in A,B. (C) Quantification of number of adult α’β’ MB neuron cell bodies from worniu>EcR-B1 and worniu>babo RNAi, EcR-B1 brains. Statistical comparison to the control: ***, p<0.001 (two tailed t test). (D-E) WL3 MB cell bodies from worniu>EcR-B1 (D) and worniu>babo RNAi, EcR-B1 (E) brains stained with anti-EcR-B1 antibody (magenta). Anti-Abrupt (blue) staining was used as landmark in locating MB neurons. Adult MB lobes from (DOCX)Click here for additional data file.


Aire-deficient mice, in which defective negative selection causes spontaneous and progressive development of autoimmunity. In young and middle-aged mice, Aire deficiency appeared to be protective as supported by the reduced β-gal+ epithelial cells and the enhanced thymic output. However, once the autoimmune phenotype was fully developed in aged Aire-deficient mice, their thymuses underwent accelerated involution. In comparison to the age-matched wildtype littermates, old Aire-deficient mice showed lower numbers of total thymocytes and recent thymic emigrants but more β-gal+ thymic epithelial cells. This phenomenon may partly be attributable to the increased number of activated Th1 cells homing to the thymus. This speculation was further supported by the enhanced thymic aging following repeated challenges with complete Freund’s adjuvant immunization. Taken together, the present study highlights a unique mechanism by which autoimmunity facilitates the senescence of thymic epithelial cells through returning Th1 cells.Although autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus, are frequently associated with premature aging of the thymus, a direct link is missing between autoimmunity and thymic atrophy. Here we monitored the progression of thymic involution in  Thymic involution is characterized by reduced tissue mass and cellularity, disorganized morphology, and diminished generation and exportation of naïve T cells . Loss ofSeveral hypotheses have been proposed to explain thymic atrophy, including aging hematopoietic progenitors, dysfunction of thymic stroma and systemic factors such as elevating levels of sexual hormones . During + T helper (Th) cells have the potential to develop into multiple subsets of effector cells, such as Th1, Th2 and Th17 [lpr and NZB/W F1 mice [Ifng ameliorated nephritis in the MRL/lpr model of SLE [Ifng or Ifngr deficiency accelerated onset and increased incidence of CIA and EAE, indicating a protective role of IFN-γ in suppressing the Th17 responses [Naïve CD4and Th17 . In SLE and Th17 . Treatme F1 mice . In cont F1 mice . And dell of SLE . Collagel of SLE . In factesponses . Given tAire-deficient mice are similarly predisposed to autoimmunity. But the murine disorder is generally milder with a delayed onset [Aire-deficient mice provide a good model for studying the impact of autoimmunity on the thymic involution. In the present study, we compared thymic involution in Aire-deficient mice and the wildtype (WT) littermates. Differential impacts were observed on mice of different ages. While early thymic degeneration was displayed in the absence of Aire, this process was accelerated in aged Aire-deficient mice, possibly due to the increased recirculation of activated CD4+ T cells into the thymus.Autoimmune regulator (Aire) is critically involved in the induction of central tolerance by driving the ectopic expression of a wide range of tissue-restricted antigens in medullary thymic epithelial cells (mTECs) -25. Its ed onset . Therefotm1.1DoiAire/J (+/-Aire) and FVB-Tg (Rag2-EGFP)1Mnz/J mice were purchased from Jackson Laboratory . FVB-Tg (Rag2-EGFP) 1Mnz/J mice were backcrossed for 10 generations onto the C57BL/6 background (termed as RAG2p-GFP in this paper). Aire-/- mice were bred with RAG2p-GFP to generate Aire-/- RAG-GFP mice. The animals were kept in a specific pathogen-free facility at Peking University Health Science Center . The experimental procedures on use and care of animals had been approved by the ethics committee of Peking University Health Science Center No.LA2014178.C57BL/6 congenic mice were purchased from Peking University Health Science Center . B6.129S2-Anti-mouse CD4 PE-Cy7 (RM4-5), anti-mouse CD8a PE (53-6.7), anti-mouse CD62L FITC (MEL-14), anti-mouse Ly51 PE (BP-1) were purchased from BD Biosciences . Anti-mouse CD25 PE (PC61.5), anti-mouse CD44 APC (IM7), anti-mouse CD69 PerCP-Cy5.5 (H1.2F3), anti-mouse CD45 PerCP (30-F11), anti-mouse MHC-II PE (M5/114.15.2), anti-mouse IL-2 FITC (JES6-5H4), anti-mouse IFN-γ APC (XMG1.2), anti-mouse IL-4 FITC (BVD6-24G2), anti-mouse IL-17A PE (eBio17B7) and anti-mouse Foxp3 APC (3G3) were obtained from eBioscience . Anti-mouse EpCam PE-Cy7 (G8.8) was purchased from Biolegend .+ (RTEs) and GFP- (non-RTEs) cells in -/-Aire RAG2p-GFP transgenic mice. For detection of surface molecules, T cells were labeled with the appropriate fluorescent mAbs on ice for 30 min. For detection of cytoplasmic molecules Foxp3, T cells were collected, stained with surface molecules, fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer (eBioscience), and stained with APC-Foxp3 antibody on ice for 30 min. Flow cytometry was then conducted on a Beckman FACS Galios (Beckman Coulter) and data analysis was performed using Kaluza software.For recent thymic emigrant (RTE) analysis, the GFP fluorescence intensity was used to determine GFP-CD4+ thymocytes, peripheral GFP+CD4+ T cells and GFP-CD4+ T cells were stimulated with plate-bound anti-CD3 and anti-CD28 for 12 hours. GolgiStop (brefeldin A 10μg/ml) was added to the culture in the last 4 hours. Cells were fixed by IC fixation buffer for 20 min at room temperature and permeabilized by incubating with Permeabilization Buffer for 10 min at room temperature. After washing, cells were collected and stained with fluorochrome-labeled anti-IL-2, anti-IFN-γ, anti-IL-4 and anti-IL-17A antibodies (1:400 dilution) for 30min on ice. Flow cytometry was then conducted on a Beckman FACS Galios (Beckman Coulter) and data analysis was performed using Kaluza software.CytoSpot assay was performed with the Cytofix/ Cytoperm and GolgiStop kit according to the manufacturer’s instructions (BD Biosciences). Sorted GFPFor BrdU administration, mice received two intraperitoneal injections of BrdU . Lymph nodes were harvested 1 h after the second injection. BrdU incorporation was detected using the APC BrdU Flow Kit (BD Biosciences). After surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm buffer for 30 min on ice and then treated with DNase to expose incorporated BrdU. Subsequently, cells were stained with APC-conjugated anti-BrdU antibody.Cryosections of thymus tissues of different age (16 μm thick) were analyzed for β-gal activity using a Senescence β-Galactosidase Staining Kit according to the manufacturer’s protocol . Cryosections of thymus from CFA group were stained with anti-mouse K5 antibody followed by Cy5-conjugated goat anti-rabbit antibody (Abcam). Images were acquired with Olympus IX51. For quantification analysis, 10 randomly chosen sections were counted using Image J.-/-Aire mice and WT littermates at indicated age were collected and fixed overnight in 10% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). The degree of lymphocytic infiltrates was evaluated in a blinded fashion. Images were acquired with Olympus IX51. The brightness and contrast were adjusted in Adobe Photoshop CS.Organs from Mice were immunized subcutaneously in the flanks of the lower back, with 5μg of chicken ovalbumin  in 100μl saline or 100μl of complete Freund’s adjuvant  for a total 6 times at 6-, 8-, 10-, 17-, 19- and 21-week-old. At 16-, 27- and 39-week-old, blood, thymus, spleen and the peripheral lymph nodes were collected for phenotype analysis. Serum samples were stored at -80 °C.3) and digested with 2 mg/ml collagenase type IV (Gibco) and 0.1 mg/ml DNase I (Sigma) at 37 °C for 30 min with vortexing every 5 min. Cells from the last 3 digestions were collected. After depletion of thymocytes using anti-CD45 immuno-magnetic bead (Miltenyi Biotec), cells were stained with anti-mouse CD45 PerCP and anti-mouse EpCam PE-Cy7 on ice for 30min. After washing, TECs (CD45-EpCam+) were sorted on a FACS AriaⅡ (BD Biosciences). The purity of the sorted population was > 98%.The thymus was removed and cut into small pieces (< 1 mmIl6 (5’-CTGCAAGAGACTTCCATCCAG-3’ and 5’-AGTGGT ATAGACAGGTCTGTTGG-3’); Lif (5’-ATTGTGCC CTTACTGCTGCTG-3’ and 5’-GCCAGTTGATTCTTG ATCTGGT-3’); Osm (5’-CCCGGCACAATATCCTC GG-3’ and 5’-TCTGGTGTTGTAGTGGACCGT-3’); Ifng (5’-TCAAGTGGCATAGATGTGGAAGAA-3’ and 5’-TGGCTCTGCAGGATTTTCATG-3’); Il4 (5’-ACAGGAGAAGGGACGCCAT-3’ and 5’-GAAGCCCTACA GACGAGCTCA-3’); Actb (5’-TATGGAATCCTGTG GCATC-3’ and 5’-GTGTTGGCATAGAGGTCTT-3’). The quantification was based on delta delta CT calculations and was normalized to β-actin as loading controls.RNA was purified from TEC using Trizol reagent (Invitrogen). cDNA was synthesized using reverse transcription kit (Promega). Quantitative PCR was performed with primers as follows: t test. Throughout the text, figures and figure legends, the following terminology is used to denote statistical significance: *p<0.05, **p<0.01, ***p<0.001.Data are presented as mean values ± standard deviation (SD). Statistical significance between two groups was evaluated by two-tailed unpaired Student loCD80lo mTECs, high in mature MHC-IIhiCD80hi mTECs, and low in the terminally differentiated MHC-IIloCD80loinvolucrin+ mTECs [Aire deficiency results in reduced terminally differentiated involucrin+ mTECs [Aire-/- mice on C57BL/6 background at the age of 6 weeks, a time point before the onset of autoimmune disease [+ senescent cells in thymic frozen slices from Aire-deficient mice. These β-gal+ cells morphologically represented epithelial cells  subsets, absent in immature MHC-IIn+ mTECs . Apart fn+ mTECs . To inve disease . Senesce disease . We founal cells .Aire deficiency on the thymic involution, we examined the general aspects of the thymic function during mouse ontogeny. Although the autoimmune symptoms were mild on C57BL/6 background, lymphocyte infiltration was detected in the salivary gland in all 12-month-old Aire-/- mice  compartment in pression . The pro WT mice . The pat altered . Togethe-/-Aire mice [Aire-/- mice, we compared their activation in the two groups. There was a similar frequency of activated/memory (CD62LloCD44hi) T cells, as well as the expression of activation marker CD69 in the CD4+ compartments of the peripheral lymphoid organs .IFN-γ induced by experimental  atrophy . To exam atrophy . C57BL/6+Ly51lo mTECs versus EpCam+Ly51hi cortical thymic epithelial cells (cTECs) and decreased MHC-II+ cells in mTECs  also have Th1-biased cytokine profile [+ T cells in the mediastinal lymph nodes contribute to myocardial aging [Premature aging of the thymus is one of the major immunological phenotypes in young patients with autoimmune diseases such as RA, SLE, and systemic sclerosis , 13, 38, profile , suggest profile . The accal aging . TogetheIl6, leukemia inhibitory factor (Lif), and oncostatin M (Osm) were found in WT and Aire-deficient thymic epithelial cells (Aire-deficient mice.Both IL-17 and IL-6 contribute to arthritis development at the early onset of RA , 43. Thyal cells , suggestAire-deficient mice on C57BL/6 background [Notably, the autoimmune pathology is very mild in 6-month-old ckground , even thckground . The decckground .Taken together, our finding demonstrates an important link between pathogenic mechanism of autoimmune disease and premature thymic involution. It further suggests that early intervention in young autoimmune disease patients with Th1-biased profile may delay the onset of the disease by postponing the thymic involution.www.aginganddisease.org/EN/10.14336/AD.2018.0608The Supplemenatry material for this article can be found online at:


Dear Editor,Per and Cry, which in turn suppress CLOCK/BMAL1 activity , suggesting a pro-gluconeogenic effect of BMAL1. Supporting this scenario, adenovirus-mediated BMAL1 expression (Ad-BMAL1) in primary hepatocytes greatly prompted forskolin (FSK)-induced gluconeogenic gene expression , while BMAL1 knockdown mediated by adenovirus encoding BMAL1 RNAi (Ad-BMAL1i) inhibited gluconeogenic gene expression  by crossing BMAL1 flox/flox mice with Albumin-Cre transgenic mice. BMAL1 LKO mice were indistinguishable from their wild-type littermates in body weight and blood glucose levels under ad lib and fasted conditions . However, fasting serum insulin levels were relatively lower in BMAL1 LKO mice compared with WT littermates . When challenged with sodium pyruvate, BMAL1 LKO mice showed decreased gluconeogenesis as assayed by pyruvate tolerance test (PTT) . Similarly, the expression of endogenous gluconeogenic genes including G6Pase mRNA expression, Ad-BMAL1 expression increased FSK-induced G6Pase-luc activity in primary hepatocytes . In contrast, FSK-induced CRE-luc (composed of 3 tandem copies of CRE but not IRE sites) activity was not influenced by Ad-BMAL1 expression . Furthermore, the effect of Ad-BMAL1 expression on G6Pase-luc activity was largely abolished when IRE sites but not CRE sites were mutated, suggesting that BMAL1 specifically regulates HDAC5 pathway . Consistent with the results of Ad-BMAL1 expression, knockdown of BMAL1 by Ad-BMAL1i resulted in attenuation of G6Pase-luc activity in primary hepatocytes . HDAC5 is phosphorylated by SIK kinases and sequestered in the cytoplasm under basal conditions. Exposure to FSK triggers HDAC5 dephosphorylation and nuclear translocation where they associate with the promoters of gluconeogenic enzymes such as G6Pase  of endogenous HDAC5 in primary hepatocytes Fig. . BMAL1 cG6Pase-luc activity as well as gluconeogenic gene expression  was enhanced by Ad-BMAL1, however, no such effect was observed for Ad-BMAL1 S42A expression . Furthermore, while FSK-induced HDAC5 dephosphorylation was dramatically promoted by Ad-BMAL1 expression, Ad-BMAL1 S42A failed to show any effect . Based on these results, we further evaluated the importance of BMAL1-HDAC5 interaction on the gluconeogenesis in vivo. Mice were injected intravenously with Ad-GFP, Ad-BMAL1 or Ad-BMAL1 S42A. Although blood glucose levels under ad lib and fasted conditions were not dramatically influenced , the hepatic gluconeogenesis as assessed by PTT as well as hepatic gluconeogenic gene expression was increased in Ad-BMAL1 injected mice but not in Ad-BMAL1 S42A injected mice  or BMAL1 S42A (Ad-BMAL1 S42A). FSK induced Given that HDAC5/FOXO1-dependent increase in gluconeogenesis is a common feature of insulin resistance and obesity Below is the link to the electronic supplementary material.


NL-GF knock-in (KI) mice. Moreover, hippocampal DA and 5-HT contents were significantly decreased in APPNL-GF KI mice. Taken together, our data suggest that SAK3 promotes monoamine DA and 5-HT releases by enhancing the T-type calcium channel and nAChR in the mouse hippocampus.T-type calcium channels in the brain mediate the pathophysiology of epilepsy, pain, and sleep. Recently, we developed a novel therapeutic candidate, SAK3 -2'H-spiro pyridine]-2-ene-3-carboxylate), for Alzheimer’s disease (AD). The cognitive improvement by SAK3 is closely associated with enhanced acetylcholine (ACh) release in the hippocampus. Since monoamines such as dopamine (DA), noradrenaline (NA), and serotonin (5-HT) are also involved in hippocampus-dependent learning and psychomotor behaviors in mice, we investigated the effects of SAK3 on these monoamine releases in the mouse brain. Oral administration of SAK3  significantly promoted DA and 5-HT releases in the naive mouse hippocampal CA1 region but not in the medial prefrontal cortex (mPFC), while SAK3 did not affect NA release in either brain region. The T-type calcium channel-specific inhibitor, NNC 55–0396 (1 μM) significantly antagonized SAK3-enhanced DA and 5-HT releases in the hippocampus. Interestingly, the α7 nicotinic ACh receptor (nAChR) antagonist, methyllycaconitine (1 nM) significantly inhibited DA release, and the α4 nAChR antagonist, dihydro-β-erythroidine (100 μM) significantly blocked both DA and 5-HT releases following SAK3  administration in the hippocampus. SAK3 did not alter basal monoamine contents both in the mPFC and hippocampus. SAK3  administration also significantly elevated DA and 5-HT releases in the hippocampal CA1 region of amyloid-precursor protein (APP) In rblockers . While tblockers . Thus, wNL-GF KI mice, suggesting that decrease in dopaminergic and serotonergic pathways in the hippocampus may be associated with the cognitive impairments seen in APPNL-GF KI mice [NL-GF KI mice. These observations suggest that SAK3 may have potential for improvement of cognitive impairments seen in APPNL-GF KI mice. Supported this idea, we reported that chronic SAK3  administration significantly antagonizes cognitive impairments seen in APPNL-F KI mice [Several postmortem studies have reported decreased density of dopamine receptors in the AD brain including the hippocampus –51. In a KI mice . In addi KI mice . TherefoHere, we observed that SAK3-promoted DA and 5-HT releases reach a peak at 12 min in the hippocampal CA1 region. We previously reported that the brain concentration of SAK3 reaches approximately 0.2 nM within 15 min after SAK3  administration . Since p50 of TTA-A2 on Cav3.1 T-type calcium channel (89 nM) is stronger than that of NNC 55–0396 (6.8 μM) by in vitro assay [in vivo microdialysis. Cav3.1 gene deletion also partially inhibited the SAK3 effect, suggesting that Cav3.1 in part mediates the initiation of hippocampal DA and 5-HT release. We have reported that SAK3 enhances Cav3.1 and 3.3 currents, and that SAK3-promoted hippocampal ACh release is partially attenuated in Cav3.1 KO mice [While T-type calcium channel inhibitor NNC 55–0396 (1 μM) significantly inhibited SAK3-promoted hippocampal DA and 5-HT releases, the same dose of TTA-A2 (1 μM) failed to inhibit SAK3 effect completely. Previous reports indicated that ICro assay , 57. Sinro assay , 57, it ro assay . As NNC ro assay , we here KO mice . Thus, CIn summary, the T-type calcium channel enhancer SAK3 promoted DA and 5-HT, but not NA, release in the mouse hippocampus under naive and AD-like conditions. SAK3 may promote DA and 5-HT releases through activation of the nAChR by enhancing ACh release in the hippocampus. Therefore, SAK3 can facilitate both ACh and DA and 5-HT releases in the hippocampus through enhancing T-type calcium channels. This evidence is particularly important for SAK3-induced improvement of memory deficits and BPSD seen in patients with AD.S1 File(XLSX)Click here for additional data file.


The anti-tumor immunity of natural killer (NK) cells can be paralyzed by the CD73-induced generation of immunosuppressive adenosine from precursor ATP within the hypoxic microenvironment of solid tumors. In an effort to redirect purinergic immunosuppression of NK cell anti-tumor function, we showed, for the first time, that immunometabolic combination treatment with NKG2D-engineered CAR-NK cells alongside blockade of CD73 ectonucleotidase activity can result in significant anti-tumor responses in vivo.NK cells were engineered non-virally with NKG2D.CAR-presenting vectors based on the piggyBac transposon system with DAP10 and CD3ζ co-signaling domains. The anti-tumor immunity of NKG2D.CAR.NK cells in combination with CD73 targeting was evaluated against multiple solid tumor targets in vitro and humanized mouse xenografts in immunodeficient tumor-bearing mice in vivo. Intratumoral migration was evaluated via immunohistochemical staining, while degranulation capacity and IFN-γ production of NK cells were measured in response to solid tumor targets.+ solid tumor targets via mechanisms that might imply alleviation from adenosinergic immunometabolic suppression. CD73 blockade improved the intratumoral homing of CD56+ CAR-NK cells in vivo. These engineered NK cells showed synergistic therapeutic efficacy in combination with CD73 targeting against CD73+ human lung cancer xenograft models. Interestingly, CD73 blockade could inhibit tumor growth in vivo independently of adaptive immune cells, innate immunity or NK cell-mediated ADCC.Our results showed that CD73 blockade can mediate effective purinergic reprogramming and enhance anti-tumor cytotoxicity both in vitro and in vivo by enhancing the killing ability of CAR-engineered NK cells against CD73Immunotherapies targeting the adenosinergic signaling cascade, which act by neutralizing CD73 ectoenzymatic activity, had thus far not been evaluated in humanized tumor models, nor had the implication of innate immunity been investigated. Taken together, our pre-clinical efficacy data demonstrate, for the first time, the potential of targeting CD73 to modulate purinergic signaling and enhance adoptive NK cell immunotherapy via mechanisms that could implicate autocrine tumor control as well as by mediating adenosinergic signaling.The online version of this article (10.1186/s40425-018-0441-8) contains supplementary material, which is available to authorized users. ADO d escape , 4. Furtll types , and enhr cells) . Apart fr cells) . Alongsi subsets , and inh subsets . TherefoNK cells . Arabellor sites . It is bNK cells, specialized effectors of the innate immune system, can respond rapidly to cancer cells due to expression of germline-encoded activating receptors capable of directly binding to pathogen-derived or stress-induced self-antigens . AllogenIn the context of the relationship between adenosine immunosuppression and NK cells’ antitumor immunity, we hypothesize that purinergic signaling blockade may be able to boost cancer immunotherapy with CAR-NK cells. As shown in Scheme + solid tumor targets both in vitro and in vivo. NK cells, engineered using a genomically-stable and clinically-safe non-viral vector system to target NKG2D ligands via DAP10 and CD3ζ co-signaling domains based on the piggyBac transposon system, could mediate a powerful anti-tumor response in conjunction with purinergic blockade :*P < 0.05). In addition, exocytosis of lytic granules containing granzymes and perforin is a prerequisite for the killing ability of NK cells, with CD107a molecules appearing temporarily on the surface. Their expression can be detected as a read-out system for NK cell degranulation [P < 0.01; *P < 0.05), NKG2D.CAR-NK-92 cells displayed significantly enhanced surface CD107a expression in response to the target A549 cells).NKG2D is a key receptor for NK cell activation . Its expnulation . As show+ solid tumor targets in vitro was measured using a 7-AAD/CFSE cell-mediated cytotoxicity assay. Effector cells, either NKG2D.CAR-NK-92 or NK-92 cells, were separately co-incubated with GBM43, GBM10, A549 or PC3 cells at an E/T ratio of 10:1 for 4 h. NKG2D.CAR-NK-92 cells induced a significantly higher percentage of cytolysis towards all solid tumor cell lines when compared to NK-92 cells not bearing the NKG2D.CAR .Patient-derived recurrent (GBM10) and primary GBM43) glioblastoma cells, lung carcinoma cells (A549), and prostate cancer cells (PC3) were maintained in culture prior to analysis. Among these, GBM43 is a primary glioblastoma originally derived from a 69-year-old man who underwent resection of a left temporal glioblastoma. It carries mutant p53 and CDKNA deletion. GBM10, on the other hand, was derived from a recurrent wild-type p53 GBM showing high temozolomide (TMZ) and ionizing radiation resistance. All of them have been maintained as a part of a human cancer xenograft panel as previously described. The killing ability of NKG2D.CAR-NK-92 and NK-92 cells against these CD733 gliobla+ solid tumor cells , further confirming the impairment of anti-cancer immunity by ADO. Targeting CD73 via antibody blockade would similarly prevent accumulation of extracellular ADO and retain NK cell functionality. To investigate whether anti-CD73 antibody could increase NKG2D.CAR-NK-92 cell-mediated cytotoxicity, we co-cultured effector NKG2D.CAR-NK-92 cells in the presence of GBM43, GBM10, A549 or PC3 cells at an E/T ratio of 10:1 in presence of anti-CD73 antibody (10 μg/mL) for 4 h, respectively. NKG2D.CAR-NK-92 cells, in the presence of anti-CD73 antibody, showed a significantly higher percentage of lysis of CD73+ targets   into the right flank of the mice (day 0). 11 days later (day 11), the mice from both anti-CD73 antibody and anti-CD73 antibody + NKG2D.CAR-NK-92 groups started to receive anti-CD73 antibody treatment by intraperitoneal injection (IP) for 3 weeks, once a week. One day later (day 12), the mice in the NKG2D.CAR-NK-92 and anti-CD73 antibody + CAR-NK-92 groups started to receive 5 × 106 NKG2D.CAR-NK-92 cell therapy (IP), respectively, once a week, 3 times in total. Starting on the day of the first injection of NKG2D.CAR-NK-92 cells, all mice were IP administered 2000 IU recombinant human IL-2 (rhIL-2) once every two days. Cytokine administration continued throughout the treatment period. All treatments ended on day 30. Treatment with anti-CD73 antibody alone significantly delayed tumor growth during the first 7-10 days of treatment, but retarded its therapeutic effect once tumor burden increased . NK cells alone were also superior in delaying tumor growth compared to anti-CD73 treatment. In addition, synergistic effects obtained by combining anti-CD73 antibody with NKG2D.CAR-NK-92 cells showed a remarkable and significantly higher delay in tumor growth compared to NKG2D.CAR-NK-92 cells alone  . Meanwhile, there was no significant decrease in body weight of the mice in all four groups throughout the entire treatment period . Besides, these NKG2D.CAR-NK-92 cells also showed stronger lytic activity against CD73- liquid tumor cells (K562) .NK cells, innate lymphocytes, play a primordial role in tumor immunosurveillance and can detect and eliminate cancer cells that are deficient in MHC class I molecules . Given tHowever, TME immunosuppression operates across a broad spectrum of mechanisms which collectively inhibit NK cell anti-tumor responses. Among these, the TGF-β pathway has been extensively characterized , while A2A adenosine receptor, causing dysregulation of effector immune cell subsets (including tumor-infiltrating NK cells and CD8+ T cells), dampening their antitumor immune response [2B adenosine receptors, which are expressed on cancer cells [2A antagonists have been shown to be able to enhance anti-tumor immunity [One of ADO’s key signaling mechanisms is via interaction with adenosine receptors, a group of four G-protein-coupled receptors, most notably the Aresponse . This waer cells . Based oimmunity . Moreove+ solid tumor cells (both GBM10 and A549) can be further increased due to TGF-β. Collectively, this creates a solid tumor niche that fuels cancer progression via mechanisms that lead to higher ADO concentrations and impaired NK cell functions. In this context, CD73 has risen to prominence as a biomarker and a meaningful target for cancer therapy.ADO accumulates upstream of its signaling interactions via its receptors, through an enzymatic cascade that concludes with CD73, an ectonucleotidase which converts AMP into ADO. This cascade makes CD73 a key modulator of the high levels of ADO in the TME of solid tumors. Overexpression of CD73 has been found in broad types of cancer cell lines and patients’ biopsies including breast cancer, colorectal cancer, ovarian cancer, gastric cancer, glioblastoma and gallbladder cancer . Moreoveet al. showed that anti-human CD73 antibody could improve NK cell killing against CD73+ ovarian cancer cells in vitro by acting on mechanisms that may be ascribed to both ADCC and reduced adenosine levels [+ lung tumors in combination with CD73 blockade. Purinergic targeting via CD73 might thus be regulating tumor growth also via mechanisms that exclude immune cell recruitment, a finding that has important clinical implications.In response to this, antibody-mediated CD73 blockade has been reported to show anti-cancer effects when used individually or in combination with other agents , 22, 48.e levels . In our e levels , 44—treae levels , suggest+ solid tumors—including patient-derived primary and recurrent targets—with engineered NK cells by alleviating TME immunometabolic suppression induced by adenosinergic signaling, and enhance intratumoral migration of NK cells in vivo. We observed that hypoxia-induced CD73 ectonucleotidase activity is able to mediate both tumor control as well as innate immunity. By engineering NK cells non-virally using piggyBac vectors, we demonstrated the pre-clinical feasibility of a new cell engineering approach that can mediate stable genomic incorporation of CAR transgenes as a directly translatable immunotherapy of solid tumors.Our results show that targeting CD73 activity can redirect purinergic metabolism and enhance immunotherapy of CD73scid IL2rgtm1Wjl/SzJ (NSG) mice were maintained at the Purdue Center for Cancer Research. All the animal experiments described in this study were approved by the Purdue University Animal Care and Use Committee.Male 6- to 8-week-old NOD.Cg-Prkdc2 environment. A549 cells  were grown in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. PC3 cells  were grown in RPMI1640 supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin.NK-92 cells (directly purchased from ATCC) were maintained in RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 400 U/mL rhIL-2, and 0.1 mM 2-mercaptoethanol. GBM43 and GBM10 cells  were grown in DMEM supplemented with 10% FBS and 1% HEPES. All cell lines were incubated at 37 °C in a humidified 5% CO1,4-butanediol diacrylate (90%), 4-amino-1-butanol (98%), diethyl ether (≥ 99.7%), PBS (10×), and EHNA hydrochloride (≥ 97%) were purchased from Millipore Sigma (USA). 1-(3-aminopropyl)-4-methylpiperazine (98%) was purchased from Alfa Aesar (USA). Dimethyl sulfoxide-D6 (DMSO-D6) (99.9%) was purchased from Cambridge Isotope Laboratories (USA). Tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), and tris-borate-EDTA (TBE) (10×) were purchased from ThermoFisher Scientific (USA). Recombinant human IL-2 (rhIL-2) was gifted from Akron Biotech (USA). RPMI 1640, DMEM, penicillin/streptomycin solution 100× (PS), 2-mercaptoethanol , HEPES (1 M), and trypsin-EDTA were from Gibco . Fetal bovine serum (FBS) was purchased from Corning (USA).pPB-EF1A > hCD247:hKLRC4-KLRK1:P2A:FLAG/hHCST:WPREIn this construct, a human NKG2D-specific CAR (NKG2D-DAP10-CD3ζ) was expressed under the control of the EF1alpha promoter. The NKG2D sequence was derived from previous work.pRP-mCherry-CAG > hyPBaseThis plasmid encodes the hyperactive version of piggyBac transposase under the control of the CMV promoter.All of the plasmids and CAR constructs used in this project were custom-cloned by vectorbuilder.com. The following piggyBac transposon gene expression vectors were used:This polymer was synthesized using a two-step reaction method according to previous reports . BrieflyAll components were diluted in sodium acetate buffer  to the following concentrations: PBAE 447 ; N (0.1 μg/μL); and T (0.1 μg/μL). A series of PBAE/DNA complexes were prepared by adding the PBAE 447 solution into DNA solution. Then the mixture was vortexed gently for 10 s then incubated at RT for 15 min. The complexes were subjected to electrophoresis to examine the association between the polymer and DNA. Herein, electrophoresis was performed on 1% agarose gel and run in TBE buffer at 90 V for 1 h. To characterize the morphology of the corresponding composites, transmission electron microscope (TEM) was performed on FEI Tecnai G2 20, 200 kV. Dynamic light scattering system  was used to measure particle size and zeta potential.5 cells). PBAE/DNA complexes  were then added and incubated for 4 h. After that, 0.5 mL of RPMI 1640 medium with FBS (20%), rhIL-2, 2-mer, and PS (2%) were added to change the final medium back to the normal completed medium and the NK/gene complexes were cultured for additional 48 h. To measure the levels of NKG2D transfection, the cells were stained with FITC-conjugated NKG2D antibody  and analyzed by flow cytometry using BD LSRFortessa (Becton Dickinson). The cell viability after transfection was measured by CCK-8  assay analysis.NK-92 cells were suspended in RPMI 1640 medium without FBS and PS and seeded into 12-well plates  were labeled with CFSE and seeded into 12-well plates. NK-92 and CAR-transfected NK-92 cells (referred to as CAR.NK-92) were then added at a E:T ratios of 10:1. To measure IFN-γ production, GolgiPlug (BD Biosciences) was added followed with the addition of NK cells. After co-culture for 4 h, the cells were collected and fixed with Cytofix-Cytoperm (BD Biosciences) for 20 min at 4°C. Then, the cells were treated with PerCP-Cy5.5-conjugated IFN-γ antibody  in Perm/Wash buffer (BD Biosciences) for 30 min at 4°C. After that, the cells were collected and analyzed by flow cytometry using BD Accuri C6 Plus (Becton Dickinson).CFSE-labeled target cells, seeded into 12-well plates as before, were incubated with NK-92 or CAR.NK-92) at an E:T ratio of 10:1.To detect NK cell degranulation via CD107a staining, PE-conjugated-CD107a antibody  was added at the beginning of the assay. After 1 h of co-incubation, GolgiStop (BD Biosciences) was added. After incubation for an additional 3 h, the cells were collected and analyzed by flow cytometry using BD Accuri C6 Plus (Becton Dickinson). NK cell-mediated cytotoxicity against tumor cells  was analyzed using a 7-AAD/CFSE assay . Briefly, target cells were labeled with CFSE and seeded into 24-well plates. Then the plates were placed in the incubator for at least 4 h to allow for cell attachment. After that, NK-92 and CAR.NK-92 were added at a E:T ratios of 10:1, and co-cultured with target cells for 4 h. Finally, the cells were collected and stained with 7-AAD and cytotoxicity was measured by flow cytometry using BD Accuri C6 Plus (Becton Dickinson).6/sample) were stained with APC-conjugated mouse anti-human CD73  and APC mouse IgG1, κ isotype  for 30 min at 4 °C, respectively. Then, cells were assessed for expression of CD73 using a BD Accuri C6 Plus cytometer (Becton Dickinson). The lytic activity of CAR-NK-92 cells against GBM43, GBM10, A549 or PC3 cells in the presence of anti-CD73  or an adenosine deaminase inhibitor (EHNA hydrochloride) was analyzed using the 7-AAD/CFSE assay according to the procedure described earlier in Materials and Methods (2.9). The final concentrations of anti-CD73 antibody and EHNA hydrochloride were 10 μg/mL and 30 μM, respectively.Detached GBM43, GBM10, A549 or PC3 cells (10n = 25) were subcutaneously (sc) inoculated with 2 × 105 A549 cells, suspended in 100 μL of DMEM medium without FBS and PS, dorsally on the right side. When tumors reached a volume of about 75 mm3, mice were randomly assigned to 4 treatment groups (n = 5/group): (1) untreated mice, (2) mice subjected to intraperitoneal administration (IP) of anti-CD73 antibody (50 μg/mouse) alone, (3) mice subjected to intraperitoneal administration (IP) of 5 × 106 CAR-NK-92 cells alone, and (4) mice subjected to intraperitoneal administration (IP) of both 5 × 106 CAR-NK-92 cells and 50 μg/mouse of anti-CD73 antibody. CAR-NK-92 cells were administered one time per week, 3 times in total. The anti-CD73 antibody was administered 24 h before each injection of CAR-NK-92 cells. Meanwhile, the mice that received NK cell therapy (groups 3 and 4) also received 2000 U rhIL-2 by IP via a single injection every two days. The length (L), width (W), and height (H) of the tumor were measured using a digital caliper, and the volume of the tumor was calculated using the formula: V = 0.52 × L× W × H. Body weights of the mice were also recorded during the treatment. At the end of the experiment, the mice were sacrificed and tumors were harvested for histologic analyses.6-week-old male NSG mice  at a 1:200 dilution. For the quantification of NK-92 cells in the tumors, the stained cells were counted in 5 randomly selected intratumoral fields of each slide under ×200 magnification.P < 0.05 was considered to be statistically significant.Data were presented as mean ± SEM. Statistical analysis was performed using Excel 2007 software (Microsoft office 2007). The difference between two groups was analyzed by a one-way ANOVA analysis. Additional file 1:Figure S1. (A) Scheme of the synthesis of PBAE 447. (B) 1 H NMR spectra of PBAE 447 polymer in DMSO-d6. Figure S2. Cell viability of NK-92 cells after incubation with PBAE 447 polymer at various concentrations for 52 h. Figure S3. Flow cytometric analyses of NKG2D expression in NK-92 cells. NK-92 cells were stained with FITC-NKG2D and positive staining was analyzed. Figure S4. The mean fluorescence intensity (MFI) of NKG2D expression following 5 days’ continuous culture of NKG2D.CAR-NK-92 cells. Figure S5. (A) Cell viability of NK-92 cells after incubation with TGF-β1 for 24 h. (B) The effect of TGF-β1 treatment on NKG2D expression by NK-92 cells. Figure S6. (A) Percentage of NK cells producing IFN-γ intracellularly measured by flow cytometry. (B) Degree of degranulation of NK cells expressed as % CD107a+ cells. Figure S7. Cell viability of NK-92 cells after incubation with adenosine (ADO) at various concentrations for 24 h. Figure S8. Lytic activity of NK-92 cells against (A) GBM43, (B) GBM10, (C) A549 or (D) PC3 cells, in the presence of anti-CD73 antibody (10 μg/mL) and adenosine deaminase inhibitor (ADAi) EHNA (30 μM), respectively. Figure S9. CD73 expression on (A) A549 and (B) GBM10 cells after treatment with TGF-β1 for 24 h. Figure S10. (A) CD73 expression on K562 cells. (B) Lytic activity of NK-92 and piggyBac-NKG2D.CAR-NK-92 cells against CD73- K562 cells. (DOCX 914 kb)


Drosophila imaginal discs as a model system due to its well-characterized regenerative ability after injury or cell death. We genetically-induced cell death and found that the Apoptosis signal-regulating kinase 1 (Ask1) is essential for regenerative growth. Ask1 senses ROS both in dying and living cells, but its activation is selectively attenuated in living cells by Akt1, the core kinase component of the insulin/insulin-like growth factor pathway. Akt1 phosphorylates Ask1 in a secondary site outside the kinase domain, which attenuates its activity. This modulation of Ask1 activity results in moderate levels of JNK signaling in the living tissue, as well as in activation of p38 signaling, both pathways required to turn on the regenerative response. Our findings demonstrate a non-autonomous activation of a ROS sensing mechanism by Ask1 and Akt1 to replace the missing tissue after damage. Collectively, these results provide the basis for understanding the molecular mechanism of communication between dying and living cells that triggers regeneration.How cells communicate to initiate a regenerative response after damage has captivated scientists during the last few decades. It is known that one of the main signals emanating from injured cells is the Reactive Oxygen Species (ROS), which propagate to the surrounding tissue to trigger the replacement of the missing cells. However, the link between ROS production and the activation of regenerative signaling pathways is not yet fully understood. We describe here the non-autonomous ROS sensing mechanism by which living cells launch their regenerative program. To this aim, we used  Drosophila wing imaginal discs and then studied the mechanism that drives living cells to replace the damage zone until it is completely regenerated. We found that the Drosophila Apoptosis signal-regulating kinase 1 (Ask1), a protein that is sensitive to oxidative stress, is a key player in this scenario. This protein acts as an intracellular sensor that upon damage activates the JNK and p38 regenerative signaling pathways. However, high activity of Ask1 can be toxic for the cell. This is controlled by Akt, a kinase downstream the insulin pathway, which attenuates the activity of Ask1 in the living cells that will participate in the regeneration process. In consequence, Ask1 and Akt act synergistically to respond to the stress generated after tissue damage and drive regeneration. Our results provide a first overview within the framework of how insulin signaling inputs could modulate the capacity to overcome tissue damage.One of the early events that occur after tissue damage consists on the production of Reactive Oxygen Species (ROS), that signal to the surrounding tissue to initiate wound healing and regeneration. Many signaling pathways, such as JNK and p38, respond to oxidative stress and are necessary for regenerative growth. As the link between ROS and regenerative signaling is not well understood, we decided to explore the mechanism that underlies this process. To do that, we genetically induced cell death in specific areas of  Drosophila imaginal discs, which are well-characterized epithelial sacs capable to regenerate after genetically-induced apoptosis or when parts are physically removed . Compiliewed in –4. Actuaiewed in –7. Althoiewed in .Drosophila . ,47. Ask1sk1RNAi) . Similarsk1RNAi) . To discci>), where the Ask1RNAi transgene was expressed. It is well known that apoptosis in discs induces compensatory proliferation of nearby cells  resulted in an increase of mitosis compared to the neutral  or to the Ask1 knockdown  conditions. However, combining genetic ablation in the salE/Pv zone with Ask1 knockdown in the nearby anterior compartment, the number of mitosis associated to damage did not increase   in the anterior compartment (sk1RNAi) . This obDrosophila Ask1 locus encodes two protein isoforms of different lengths, Ask1-RB and Ask1-RC, with predicted molecular weights of 136.5 kDa and 155.5 kDa, respectively. Both have a protein kinase-like domain that contains a highly conserved core of threonines conferring functionality to the protein , which we hypothesized that could be key in Ask1 regulation (The iewed in ). It hasectively . In humain vitro . This isterminus . Howevergulation , S2 Fig.Ask1 mutant backgrounds, both the general and the mitosis-associated P-Thr levels were reduced but not abolished (Ask1RNAi discs did not show any effect on P-Thr levels in mitosis (Ask1RNAi suppressed the low endogenous P-Ser83 Ask1 levels (Active Ask1 can be traced with antibodies against the phosphorylated threonine residues of the highly conserved kinase domain (henceforth P-Thr)  and the bolished , and Ask mitosis . This di mitosis . Ectopic1 levels .Ask1RNAi expression   . In the Ask1 at serine 83 to alanine and cloned it into a UAS vector (UAS-Ask1S83A). In parallel, we also cloned a wild-type form of Ask1 (UAS-Ask1WT). We ectopically expressed Ask1WT in the posterior compartment of the wing disc , which resulted in an increase of P-Ser83, in addition to low levels of P-Thr , did not show a rise in P-Ser83, but strong activation of P-Thr ((hh>), where the transgene was activated. This observation concurs with the P-Ser83 residue as responsible for the attenuation of P-Thr.In contrast to the high activity of Ask1 in dying cells (high P-Thr), the presence of P-Ser83 in living cells could be indicative of tolerable levels of Ask1 achieved by attenuation of P-Thr activity. To test this hypothesis, we mutated of P-Thr . Interesof P-Thr . The higUAS-Ask1WT or UAS-Ask1S83A transgenes in the anterior compartment without cell death did not cause any defects in wing morphology, size or vein and intervein patterning . In contrast, expression of UAS-Ask1S83A led to a fall to only 29% of individuals being capable of regenerate and the rest showed strong effects on wing morphology, with altered pattern of veins and interveins, as well as the appearance of notches, which together are indicative of disrupted regeneration , were required for Ser83 phosphorylation.Next, we decided to identify the upstream signal responsible for Ser83 phosphorylation. In mammalian cells, attenuating phosphorylation of Ask1 is driven by the serine-threonine Akt kinase, the core kinase of the insulin pathway . We wondin vivo, we specifically inhibited this kinase with a UAS-AktRNAi in a stripe of cells at the center of the disc, the ptc domain (ptc>AktRNAi), and found that P-Ser83 was reduced (UAS-myr-Akt1.S), a constitutively activated membrane-anchored form of Akt1  domain motif for the Akt kinase   pattern, although conservation is limited to birds and mammals (dark versus light shades of violet). On the right, the domain highlighted in cyan is conserved all across the diverse taxa, from human (top) to sponges (bottom), and it contains a serine (red background) that corresponds to the fly Ser83. On both panels, another domain matching a degenerated xxRxx[ST] pattern that is only conserved within vertebrate sequences is shown as a green block. Right alignment also includes at the bottom the consensus sequence of a PFAM domain of unknown function (DUF4071) that starts two residues before the Ser83 conserved block; this block within the domain shows high conservation across the different protein families considered when building the corresponding model.Two blocks of conserved columns pinpointing the aforementioned residues were selected from the whole-length alignment of 50 MAP3K5/Ask1 protein orthologs provided on the (TIF)Click here for additional data file.S3 FigAsk1MB06487 homozygous mutant background and are strongly reduced in the Ask1MB06487 mutant background over the deficiency Def(3R)50636. Quantification of mean pixel intensity for P-Thr in mitotic and for P-Thr in non-mitotic  cells are shown in separate graphics. P-Thr mean Pixel intensity in mitotic cells of the wild-type discs (+/+), 113.92±20.60, S.D. (n = 10); of the Ask1MB06487/Ask1MB06487 discs, 104.43±9.54, S.D. (n = 10); and of the Ask1MB06487/Def(3R)BSC636 88.37±13.03, S.D. (n = 10). Mean pixel intensity of the basal levels in interphase cells of the wild-type, 50.44±6.27, S.D. (n = 20); of the Ask1MB06487/Ask1MB06487, 41.65±4.80, S.D. (n = 10); and of the Ask1MB06487/Def(3R)BSC636, 27.22±5.73, S.D. (n = 18). (C) Inhibition of the endogenous Ask1 P-Ser83 levels after expressing the UAS-Ask1RNAi in the posterior compartment, using the hh-Gal4 driver. Scale bars: 5μm.(A) Mitotic (P-H3 positive) and Ask1 P-Thr. Lower right images: Zoom of mitotic cells from the white square. TP-3: TO-PRO-3 nuclear staining. (B) The basal levels of P-Thr in wild-type (WT) discs (mitotic and interphase cells) decrease in the (TIF)Click here for additional data file.S4 Figptc-Gal4 UAS-rpr (ptc>rpr) and stained with P-Ser83 (n = 5). (B) SalE/Pv>hid discs stained with P-Thr (n = 12) and P-Ser83 (n = 5). YP-1: YO-PRO-1 nuclear staining. Purple: salE/Pv>rpr ablated zone. (C) Staining of Ask1 P-Ser83 after physical injury, (n = 6). Square: magnified image. Scale bars: 50μm.(A) Left: wild-type disc stained with P-Ser83. Right: disc in which apoptosis has been induced with the driver (TIF)Click here for additional data file.S5 FigsalE/Pv>rpr cell death (purple) and blocking Pi3K (ci>dp110DN) in the anterior compartment (black). (B-D) Staining of P-Akt (B), Ask1 P-Ser83 (C) and Ask1 P-Thr (D). White-dotted line indicates anterior-posterior boundary. TP3: TO-PRO-3 nuclear staining. Scale bars: 50μm.(A) Design of the experiments in B, C and D with (TIF)Click here for additional data file.S6 FigPurple area shows where transgenes were activated.(TIF)Click here for additional data file.S7 FigsalE/Pv>rpr, and two examples of salE/Pv>rpr ci>Ask1RNAi. Thermal LUT was used to detect pixel differences. TP-3: TO-PRO-3 nuclear staining. Purple indicates the dead zone. Colorized red indicates the ci>Ask1RNAi zone. (B) Quantification of the P-JNK in raw images. A and P indicate the approximate areas measured for pixel intensity in n = 15 for each genotype. (C-E) Live imaging of a puc-Gal4, UAS-GFP used as a reporter of the JNK pathway. Discs were cut and cultured for 6 hours. White lines indicate the wound site. White arrows point to the endogenous puc expression zone at the tip of the notum. (C) Wild type. (D) Ask1MB06487 heterozygous. The nuclear marker NucRed Live647 was added in the culture medium. Scale bar: 50μm. (E) Quantification of puc-Gal4, UAS-GFP reporter at the wound site (n = 13 discs each genotype). Error bars indicate standard deviation. **P<0.01, ***P<0.001.(A) P-JNK staining in wild type, (TIF)Click here for additional data file.S1 Appendixgenewise alignment of the human M3K5_HUMAN protein sequence against the genomic segment of 5kbp upstream/downstream that is centered on the annotated fly Ask1 gene transcription start site. No alignment was reported for the first 126 residues of the human protein where the Ser83 RxRxx[ST] domain has been described.This file contains the plain text (PDF)Click here for additional data file.S2 Appendixhttp://eggnogdb.embl.de) homologs cluster KOG4279, comprising human MAP3K5 and its counterpart in fly Ask1-PC, we filtered out 49 proteins that were confidently annotated as MAP3K5 orthologs on different taxa plus fly Ask1-PB isoform downloaded from FlyBase, which are listed here : Homo sapiens ENSP00000351908 (M3K5_HUMAN),Pan troglodytes ENSPTRP00000043317 (K7CFQ9_PANTR), Gorilla gorilla ENSGGOP00000022651 (G3S3K1_GORGO), Macaca mulatta ENSMMUP00000021508 (F7G2W1_MACMU), Pongo abelii ENSPPYP00000019077 (H2PKF0_PONAB), Otolemur garnettii ENSOGAP00000000498 (H0WGN9_OTOGA), Callithrix jacchus ENSCJAP00000015659 (F7GZH9_CALJA), Rattus norvegicus ENSRNOP00000051338 (D3ZW27_RAT), Mus musculus ENSMUSP00000093485 (M3K5_MOUSE), Cavia porcellus ENSCPOP00000003829 (H0V2Q4_CAVPO),Bos taurus ENSBTAP00000011949 (F1MXH6_BOVIN), Pteropus vampyrus ENSPVAP00000009127 (ens9127_PTEVA), Mustela putorius furo ENSMPUP00000014726 (G9K9I6_MUSPF), Loxodonta africana ENSLAFP00000000494 (G3UDL3_LOXAF), Echinops telfairi ENSETEP00000012543 (ens12543_ECHTE), Canis lupus familiaris ENSCAFP00000000362 (F1PA02_CANFA), Ailuropoda melanoleuca ENSAMEP00000010579 (D2H0V2_AILME), Tursiops truncatus ENSTTRP00000002388 (ens2388_TURTR), Dasypus novemcinctus ENSDNOP00000014966 (ens14966_DASNO), Sarcophilus harrisii ENSSHAP00000014955 (G3WHQ0_SARHA), Gallus gallus ENSGALP00000022482 (F1NYS9_CHICK), Meleagris gallopavo ENSMGAP00000013731 (G1NK21_MELGA), Taeniopygia guttata ENSTGUP00000011202 (H0ZKT1_TAEGU), Pelodiscus sinensis ENSPSIP00000018784 (K7GES3_PELSI), Xenopus tropicalis ENSXETP00000005271 (F7A415_XENT), Gasterosteus aculeatus ENSGACP00000009628 (G3NWA9_GASAC), Oreochromis niloticus ENSONIP00000007016 (I3JDS6_ORENI), Danio rerio ENSDARP00000046640 (E7F2L3_DANRE), Takifugu rubripes ENSTRUP00000002505 (H2RQT9_TAKRU), Tetraodon nigroviridis ENSTNIP00000022322 (Q4RFA2_TETNG),Branchiostoma floridae JGI219597 (C3XTE7_BRAFL), Drosophila melanogaster FBpp0089256 (Ask1-PB_DROME) and FBpp0289484 (Ask1-PC_DROME), D. ananassae FBpp0120816 (B3LXS9_DROAN),Culex quinquefasciatus CPIJ008487-PA (B0WN02_CULQU), Anopheles gambiae AGAP000747-PB (QEA6_ANOGA), Aedes aegypti AAEL008311-PA (Q16Z55_AEDAE), Bombyx mori BGIBMGA010545-TA (E9JEG8_BOMMO), Danaus plexippus EHJ64716 (G6DP69_DANPL),Apis mellifera GB14238-PA (H9KAX6_APIME), Nasonia vitripennis NV13729-PA (K7IXA9_NASVI), Acyrthosiphon pisum ACYPI009039-PA (J9K801_ACYPI),Tribolium castaneum TC000430-PA (TCOGS2_TRICA), Daphnia pulex DappuP328832 (P32883_DAPPU),Caenorhabditis elegans F59A6.1 (NSY-1_CAEEL), C. japonica CJA14063 (K7I1N3_CAEJA),C. briggsae CBG02299 (A8WUN1_CAEBR), Strongylocentrotus purpuratus SPU_010872tr (H3I9Y9_STRPU), Amphimedon queenslandica PAC_15725247 (15725247_AMPQE), Trichoplax adhaerens TriadP10277 (B3RYN2_TRIAD).Many positions are preserved from sponges to humans, including serine/threonine residues, whilst other show a dual pattern of conservation with specific substitutions between chordate and invertebrate species. From the whole set of 338 sequences on 164 species that define the EggNOG ((PDF)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.