Most neurodegenerative diseases associated with protein aggregation are hallmarked by activation of astrocytes. However, how astrocytes are activated or which signaling pathways in astrocytes contribute to pathogenesis is not clear. One long-standing question is whether the responses in astrocytes are due to stress or damage in astrocytes themselves, or because of astrocytic responses to cellular stress or damage in neurons. Here, we examine responses in astrocytes induced by expression of disease-associated, aggregation-prone proteins in other cells. We also examine the consequences of these responses in astrocytes in a model for neurodegeneration.Drosophila model for Spinocerebellar ataxia type 3 , a disease caused by expansion of the polyglutamine (polyQ) stretch in the ATXN3 gene. In this Drosophila SCA3 model, eye-specific expression of a biologically relevant portion of the ATXN3 gene, containing expanded polyQ repeats (SCA3polyQ78) was expressed. In a candidate RNAi screen in the Drosophila SCA3 model, we analyzed whether downregulation of expression of specific genes in astrocytes affected degeneration induced by SCA3polyQ78 expression in Drosophila eyes. We next examined the role of astrocytes in response to proteotoxic stress in neurons induced by SCA3polyQ78 expression or amyloid beta peptides, associated with Alzheimer’s disease.We first examined a role for intracellular astrocytic responses in a polyQ78 resulted in the presence of astrocytes in the eye, suggesting putative involvement of astrocytes in SCA3. In a candidate RNAi screen, we identified genes in astrocytes that can enhance or suppress SCA3polyQ78-induced eye degeneration. Relish, a conserved NF-κB transcription factor, was identified as an enhancer of degeneration. Activity of Relish was upregulated in our SCA3 model. Relish can exert its effect via Relish-specific AMPs, since downregulation of these AMPs attenuated degeneration. We next examined Relish signaling in astrocytes on neurodegeneration. Selective inhibition of Relish expression specifically in astrocytes extended lifespan of flies that expressed SCA3polyQ78 exclusively in neurons. Inhibition of Relish signaling in astrocytes also extended lifespan in a Drosophila model for Alzheimer’s disease.Eye-specific expression of SCA3Our data demonstrate that astrocytes respond to proteotoxic stress in neurons, and that these astrocytic responses are important contributors to neurodegeneration. Furthermore, our data demonstrate that activation of NF-κB transcription factor Relish in astrocytes, induced by proteotoxic stress in neurons, enhances neurodegeneration, and that specific Relish inhibition in astrocytes extends lifespan. Our data provide direct evidence for cell-non-autonomous contributions of astrocytes to neurodegeneration, with possible implications for therapeutic interventions in multiple neurodegenerative diseases.The online version of this article (10.1186/s12974-018-1278-2) contains supplementary material, which is available to authorized users. A hallmark of many age-related neurodegenerative diseases is the presence of protein aggregates, which precedes the onset of clinical symptoms. Genetic profiling of transcriptional changes in neurodegenerative diseases resulted in the identification of glial genes whose altered expression could potentially contribute to pathogenesis , 2. BesiThe presence of aggregates in astrocytes can result in their activation and subsequent signaling to neurons ; revieweAstrocytes are involved in essential CNS functions, including metabolic functions, regulating levels of neurotransmitters, maintaining the blood-brain barrier, as well as immune defense, thus contributing to neuronal homeostasis . AstrocyDrosophila melanogaster model of the polyQ disease SCA3. In SCA3, the ATXN3 gene contains an expanded CAG repeat (coding for glutamine) that leads to the expression of a misfolded, aggregation-prone ATXN3 polyglutamine protein. These misfolded polyQ-containing proteins accumulate intracellularly, resulting in neuronal damage and activation of astrocytes [ATXN3 gene containing an expanded polyQ stretch (SCA3polyQ78) exclusively in Drosophila eyes and simultaneously downregulated expression (by RNAi) of candidate genes exclusively in astrocytes. This setup would provide insight into the putative cell-non-autonomous contributions of astrocytes to neurodegeneration induced by proteotoxicity in neurons. In a candidate RNAi screen, we identified genes in astrocytes that cell-non-autonomously influenced degeneration. This demonstrates the relevance of cell-non-autonomous signaling in astrocytes in neurodegeneration. We further explored the contribution of NF-κB transcription factor Relish, which was identified as an enhancer in our screen, and its activity was upregulated in our SCA3 model. We investigated the role of Relish in astrocytes in SCA3, as well as in a model for Alzheimer’s disease.We investigated if and how astrocytes contribute to neurodegenerative diseases in a cell-non-autonomous manner, within the context of an intact animal. Examining astrocytes in an in vivo model is key, given that their morphology and activity change when taken outside their physiological context (reviewed in ). For thtrocytes . In the trocytes –16. We eUAS-MJD.tr-Q27 (8149), UAS-MJD.tr-Q78 (8150), QUAS-mCD8-GFP (30003), UAS myr-RFP (7118), two tub-QS lines (52112 and 30024), alrm-Gal4 (67031); GMR-Gal4 (1104); daughterless-Gal4 (8641); UAS-Relish RNAi #2 (33661); Relish E20 mutant (9457). The following stocks were from Vienna Drosophila Research Center (VDRC): UAS-Relish RNAi #1 (49414-GD), UAS-Attacin A RNAi (50320-GD), UAS-Cecropin A (42859-GD). The stocks used in the screen are all from VDRC. UAS-GFP-Relish has been described [GMR-QF2 (BDSC #59283) nSyb-QF2 (BDSC 51956), the CyO-tub-QS balancer as well as the pQUAST vector were a gift from C. Potter . UAS-necrotic-Abeta42 flies were a gift from D. Crowther  and have been described previously [QUAST-SCA3polyQ27 and QUAST-SCA3polyQ78 transgenic flies by amplifying the corresponding cDNA from gDNA of stock #8149 (UAS-SCA3polyQ27) or #8150 (UAS-SCA3polyQ78), using the following UAS-specific primers: 5′- ATAGGGAATTGGGAATTCGTT-3′ and 5′- CAATTATGTCACACCACAGAA-3′ and cloned into pUAST using EcoRI and XbaI. We generated pQUAST-necrotic-Aβ42 by amplifying from gDNA from UAS-necrotic-Abeta42 flies using 5′-cgaattcaacATGgcgagcaaagtctcgatc-3′ and 5’-ctctagaTTACGCAATCACCACGCCGC-3′ and cloned into pQUAST using EcoRI and XbaI. Constructs were verified by sequencing. Transgenic fly lines were generated in the w1118 background, using BestGene .All fly lines were maintained at 25 °C on standard fly food unless indicated otherwise. The following stocks were obtained from the Bloomington Drosophila stock center : escribed . GMR-QF2yr-RFP 71, two tubalrm-Gal4). We suppressed QF-dependent expression by expressing QS under control of the tubulin promoter. A schematic drawing of the genetic systems involved is shown in Additional files SCA3polyQ78-induced eye degeneration, we used the following fly line: GMR-QF2/(Y); QUAS-SCA3polyQ78:: alrm-Gal4/CyO-tub-QS. In this line, expression of SCA3polyQ78 is suppressed by QS, which inhibits QF2. To analyze genes in astrocytes on the degenerative SCA3 eye phenotype, we crossed this line to different UAS constructs. To quantify eye degeneration by analyzing levels of mCD8-GFP, we used the line GMR-QF2/(Y); QUAS-SCA3polyQ78/CyO-tub-QS: alrm-Gal4: QUAS-mCD8-GFP, and crossed them to UAS lines or w1118 flies. The data depicted are all from female flies, although male flies yielded similar results. To analyze the effect of SCA3polyQ78 in neurons and a possible effect of astrocytes on the SCA3polyQ78-induced pathogenesis, we used the following line: tub-QS/(y); QUAS-SCA3polyQ78:: alrm-Gal4; nSyb-QF2: tub-QS. We used two copies of tub-QS, since one copy was not able to suppress the expression of SCA3polyQ78. We supplemented adult flies with quinic acid to suppress QS and allow expression of SCA3polyQ78 only in adulthood. Data of females are depicted, although data in males yielded similar results.To independently and simultaneously manipulate gene expression in either the eyes or neurons, or astrocytes, we used the QUAS-QF system to express constructs in neurons or in the eyes and UAS-Gal4 to manipulate gene expression in astrocytes (using polyQ78-induced degenerative phenotype (necrotic spots) as shown previously [p < 0.05; **p < 0.01.For each line we analyzed, we calculated the fraction of the eyes that showed a SCA3eviously  of at leribosomal protein 49 (RP49). Analysis of knockdown of gene expression was determined in adult flies.We collected 120 fly heads per point to isolate total RNA using the RNeasy Mini Kit (Qiagen). To collect the heads, flies were frozen in liquid nitrogen and decapitated by vortexing. Fly heads were collected using a mesh. M-MLV reverse transcriptase  was used to transcribe RNA into cDNA. Relative quantification of the gene expression level was determined in CFX ConnectTM (Bio-Rad) by using iQTM SYBR® Green Supermix . For all the samples, gene expression levels were normalized to a housekeeping gene, RP49: 5′-CCGCTTCAAGGGACAGTATC-3′/5′-GACAATCTCCTTGCGCTTCT-3′IM1: 5′-TGCCCAGTGCACTCAGTATC-3′/5′-GATCACATTTCCTGGATCGG-3′IM2: 5′-AAATACTGCAATGTGCACGG-3′/5′-ATGGTGCTTTGGATTTGAGG-3′AttA: 5′-ACAAGCATCCTAATCGTGGC-3′/5′-GGTCAGATCCAAACGAGCAT-3′AttC: 5′-CCAATGGCTTCAAGTTCGAT-3′/5′-AGGGTCCACTTGTCCACTTG-3′DptA: 5′-ACCGCAGTACCCACTCAATC-3′/5′-ACTTTCCAGCTCGGTTCTGA-3′CecA: 5′-GAACTTCTACAACATCTTCGT-3′/5′-TCCCAGTCCCTGGATTGT-3′Relish: 5′-AGCAGTGGCGCACTAAAGTT-3′/5′-GATGGCTGACCATTCGTTTT-3′Primer pairs used for QPCR:p < 0.05; **p < 0.01; ***p < 0.001.Results are average of at least three independent experiments −/+SEM. *polyQ27 or SCA3polyQ78 levels and aggregation and their effect on eye viability (using CD8-GFP), 2-day-old flies were used. At least 30 fly heads per genotype were collected, lysed in Laemli buffer by sonification, and boiled at 95 °C for 5 min. Samples were separated on 12.5% SDS-PAGE gels and transferred to PVDF membranes . After blocking in 5% (w/v) non-fat dried milk in PBST for 1 h, the membrane was incubated with primary antibody overnight at 4 °C. The following are antibodies were used: rat anti-HA-Peroxidase , rabbit anti-GFP  mouse anti-RFP (6G6 ChromoTek) 6E10 Ab was purchased from Covenance and mouse anti-alpha tubulin from Sigma (T5138). After incubation with secondary antibody, ECL (Amersham) signal was detected in a ChemiDocTM Touch (Bio-Rad). The intensity of the bands was analyzed by using ImageJ . Quantification of western blots was of at least three independent experiments unless indicated otherwise, *p < 0.05; **p < 0.01.For examining HA-tagged SCA3tub-QS/(y); QUAS-SCA3polyQ78:: alrm-Gal4/CyO; nSyb-QF2:: tub-QS/TM6B (SCA3polyQ78 line), (2) tub-QS/(y); alrm-Gal4/CyO; nSyb-QF2: tub-QS/TM6B (control line for SCA3polyQ78), (3) QUAS-necrotic-Abeta42:: alrm-Gal4/CyO-QS; nSyb-QF2 (Abeta42 line), and (4) alrm-Gal4/CyO-QS; nSyb-QF2 (control line for Abeta42). These lines were used to test the effect of astrocyte-specific genes on flies that express SCA3polyQ78 in neurons in adulthood or Abeta42 peptides. Expression of SCA3polyQ78 was induced in neurons of adult flies by inhibiting QS through supplementing the food with quinic acid. Quinic acid was used in a concentration of 250 mg/ml with fixed pH of 7. 250 μl of quinic acid solution was put on top of the food to cover the entire surface and used when the solution was completely absorbed into the food [To determine the lifespan of flies, a maximum of 20 flies was kept in a vial. At least 80 flies per condition were analyzed. Every 2 days flies were transferred into new vials containing fresh food. Data are representative of female flies, although similar results were obtained with males. The following fly lines were used to determine the effect of genes in astrocytes on survival: (1) For each cross, 5 vials of flies, containing a maximum of 20 flies per vial, were analyzed. Here, we analyzed males, as the climbing ability in females varies more between the individuals of a group. To determine the mobility of flies, we analyzed their climbing ability. For this, the vial was divided into four compartments, with the lowest compartment numbered with 1 (slow climbers) and the highest with number 4 (fast climbers). Flies were tapped down to the bottom of vials and were allowed 10 s to climb up, after which a picture was taken. The numbers of flies in each compartment were counted for each time point. To get better separation of the differences, flies in compartment 1 got a score of 1; flies in compartment 2 got a score of 3; flies in compartment 3 got a score of 5, and flies in compartment 4 got a score of 7. Subsequently, the average score of each time point was determined by dividing the total score by the total number of flies. The climbing test was repeated three times for each group, and the average of the three times was shown.Drosophila females were collected in ice-cold PBS, fixed in 3.7% formaldehyde solution (0.1% triton x-100 in PBS) for 15 min, and washed five times in PBS. Next, Drosophila brains were dissected in PBS and stained with DAPI solution  at room temperature. After washing with PBS for three times, the brains were mounted in 80% glycerol on slides and imaged by a Leica SP8 confocal microscope.Heads of ATXN3 gene containing an expansion of the glutamine region  in Drosophila eyes resulted in progressive degeneration of photoreceptor cells [SCA3polyQ27, containing a non-pathogenic length of glutamines  as well as enhancers  of the SCA3 eye phenotype . Downregulation of Relish expression by RNAi, but also downregulation of several other genes in the Relish pathway, suppressed the SCA3 phenotype. While NF-κB has been implicated in activation of astrocytes in neurodegeneration  in our SCA3 model, transcriptional NF-κB targets which help fight infection [SCA3polyQ78, activation of Relish was stronger: AMPs specific for Relish (CecA and DptA) were upregulated more than Dif/Dorsal-specific AMPs , a nfection . In inflnfection ). While Relish RNAi constructs  GFP levels were similar, irrespective of Relish expression , a kinase associated with ensuring genomic integrity and neurodegeneration [SCA3polyQ78. For this, we examined levels of Relish-dependent AMPs in fly heads expressing SCA3polyQ78 in the eyes together with astrocytes-specific targeting of Relish RNAi or Relish overexpression constructs, or in SCA3polyQ78-expressing flies heterozygous for Relish. Expression of Relish RNAi constructs in astrocytes in flies expressing eye-specific SCA3polyQ78 resulted in decreased expression of Relish-specific AMPs (DptA and AttC) in the head to levels comparable to flies not expressing SCA3polyQ78  and examined effects of modulation of Relish expression specifically in astrocytes  that induces SCA3polyQ78 expression is coexpressed with QF-suppressor QS [SCA3polyQ78 is suppressed during development  degeneration also translates into a (long term) survival benefit in flies expressing essor QS  , activity of Gal4 is low and Relish RNAi constructs do not decrease Relish expression , resulting in the secretion of Aβ42 peptides. When expressed in neurons . TherefpolyQ78 expression. We therefore focused on Relish signaling for more in-depth analyses. Relish was specifically activated in astrocytes present in eyes expressing SCA3polyQ78, but not in astrocytes elsewhere in the head  , but indSCA3polyQ78-expressing eyes , including iewed in ), accouniewed in , 41, whiWhereas we show that astrocytes can directly modulate neuronal health, this appeared not to be associated with alteration in the burden of protein aggregates in the brain. However, decreasing the levels of misfolded or aggregated proteins has been suggested as a therapeutic strategy . FurtherTargeting NF-κB signaling in astrocytes to alleviate neurodegeneration may be more broadly applicable: the lifespan of flies expressing Aβ peptides, present in patients with AD, was extended upon astrocyte-specific inhibition of NF-κB Fig. . Thus, dIn conclusion, our data demonstrate that astrocytes respond to cellular stress or damage in neurons induced by neuronal expression of disease-associated, aggregation-prone proteins. These astrocytic responses are important modulators of neurodegeneration. We demonstrate that activation of transcription factor NF-κB Relish occurs in astrocytes in response to proteotoxic stress in neurons, and that in this situation astrocyte-specific inhibition of Relish enhances vitality and extends lifespan. While proteotoxic stress in astrocytes can contribute to neurodegeneration, these data highlight the importance of astrocytic responses to cellular stress or damage in neurons, and provide putative therapeutic potential.Additional file 1:a To model SCA3 in Drosophila eyes, we used the Q (QUAS-QF) system, in which a QUAS-SCA3polyQ78 (or QUAS-SCA3polyQ27) construct was induced by QF2 specifically expressed in the eyes, GMR-QF2. To downregulate gene expression in astrocytes, we used the UAS-Gal4 system. UAS-RNAi constructs were specifically expressed in astrocytes by using a Gal4 expressed in astrocytes, alrm-Gal4. Cell-autonomous roles of genes in SCA3 can be analyzed by coexpressing UAS-SCA3polyQ78 with UAS constructs in the eye, using eye-specific GMR-Gal4. b Independent expression of the QUAS-QF2 system (GFP in the eyes) and the UAS-Gal4 system (RFP in astrocytes) in late pupa. Genotype, GMR-QF2/+; alrm-Gal4/UAS-myr-RFP; QUAS-mCD8-GFP/+. (PDF 186 kb)Genetic setup to analyze cell-non-autonomous or cell-autonomous roles of genes to SCA3. Additional file 2:SCA3polyQ78. The eyes of control flies that express myr-RFP in astrocytes in the absence or presence of SCA3polyQ27 or SCA3polyQ78 were analyzed for expression and localization of RFP. Genotypes: control, GMR-QF2/+; alrm-Gal4::UAS-myr-RFP/+. SCA3polyQ27, GMR-QF2/+; alrm-Gal4::UAS-myr-RFP/QUAS-SCA3polyQ27. SCA3polyQ78, GMR-QF2/+; alrm-Gal4::UAS-myr-RFP/QUAS-SCA3polyQ78. (PDF 4177 kb)Analysis of localization of astrocytes induced by eye-specific expression of Additional file 3:polyQ78-induced eye degeneration. Flies expressing SCA3polyQ78 in the eyes together with alrm-Gal4 were crossed to fly lines containing UAS-RNAi constructs to specifically knock down genes in astrocytes. Progeny expressing SCA3polyQ78 in the eyes with individual genes in astrocytes knocked down were analyzed for SCA3polyQ78-induced eye degeneration. The extent of degeneration was quantified and plotted in a table. The percentage of degeneration in SCA3polyQ78 eyes was set at 100% and the effect of RNAi-mediated gene knockdown in astrocytes was determined by the percentage of deviation from the control. The suppressors of degeneration  are shown in orange, the enhancers (40% or more deviation from the control) in purple. Genotype of the crosses SCA3polyQ78, GMR-QF2/+; QUAS-SCA3polyQ78:: alrm-Gal4/+. RNAi: GMR-QF2/+; QUAS-SCA3polyQ78:: alrm-Gal4/+ together with UAS-RNAi. (PDF 149 kb)RNAi screen to determine the contribution of genes in astrocytes to SCA3Additional file 4:a Activation of Relish in SCA3polyQ78-expressing eyes predominantly occurred in astrocytes. (top) Representative picture of a control eye expressing GFP under the control of the Attacin promoter (att-GFP) and astrocytes expressing RFP or an eye coexpressing SCA3polyQ78. Pictures were taken from live eyes. (bottom) Representative picture of a dissected eye expressing SCA3polyQ78, together with att-GFP and RFP-expressing astrocytes. Nuclei are stained with Hoechst (blue). Note that in the bottom panel there is only an overlap between the RFP and GFP signal in the ommatidia. This may cause the imperfect overlap between the RFP and GFP signal in SCA3polyQ78-expressing flies in the top panel: the RFP signal from the underlying astrocytes. b Efficacy of Relish knockdown. Flies expressing daughterless-Gal4  were crossed to control flies (w1118) or fly lines containing RNAi constructs targeting Relish (Relish RNAi #1 and Relish RNAi #2) and expression of Relish in the progeny was determined. c Modulation of expression in astrocytes does not affect morphology of control eyes. Control flies or fly lines containing RNAi constructs targeting Relish (Relish RNAi #1 and Relish RNAi #2) or expressing Relish were crossed to alrm-Gal4 flies, and the morphology of the eyes was determined. d No cell-autonomous effect of Relish on the degenerative SCA3polyQ78 eye phenotype. Constructs targeting Relish were coexpressed in the eyes expressing SCA3polyQ78. e Expression of Relish RNAi in astrocytes does not influence the relocation of the astrocytes to the eye induced by eye-specific expression of SCA3polyQ78. Images of multiple flies to show similar levels of RFP in the eyes between flies expressing eye-specific of SCA3polyQ78 and astrocyte-specific myr-RFP (RFP) in the absence or presence of Relish RNAi targeted to astrocytes. f Modulating Relish expression in astrocytes does not affect levels of mCD8-GFP in the eyes. Lysates of fly heads expressing eye-specific mCD8-GFP were compared to fly heads expressing eye-specific mCD8-GFP together with astrocyte-specific knockdown or overexpression of Relish constructs or flies heterozygous for Relish (Relish −/+). Analysis of the mCD8-GFP/tubulin ratio of two independent experiments is shown below. Genotypes a control, GMR-QF2/+; alrm-Gal4::UAS-myr-RFP; att-GFP/+. SCA3polyQ78, GMR-QF2/+; alrm-Gal4::UAS-myr-RFP::QUAS-SCA3polyQ78; att-GFP/+.b Control, da-Gal4/+. Relish RNAi #1, UAS-Relish RNAi #1/+; da-Gal4/+. Relish RNAi #2, UAS-Relish RNAi #2/+; da-Gal4/+. c Control, alrm-Gal4/+. Relish RNAi #1, alrm-Gal4/UAS-Relish RNAi#1. Relish RNAi #2, alrm-Gal4/+; UAS-Relish RNAi#2/+. GFP-Relish: alrm-Gal4/UAS-GFP-Relish. d -, GMR-Gal4::UAS-SCA3polyQ78/+. Relish RNAi#1, GMR-Gal4:: UAS-SCA3polyQ78/UAS-Relish RNAi#1. Relish RNAi#2, GMR-Gal4::UAS-SCA3polyQ78/UAS-Relish RNAi #2. Relish overexpression, GMR-Gal4::UAS-SCA3polyQ78/UAS-GFP-Relish.e As in Fig. f -, GMR-QF2/+; alrm-Gal4/+ QUAS-mCD8-GFP. Relish RNAi #1, GMR-QF2/+; alrm-Gal4/UAS-Relish RNAi #1; QUAS-mCD8-GFP/+. Relish RNAi #2, GMR-QF2/+; alrm-Gal4/UAS-Relish RNAi #2; QUAS-mCD8-GFP/+. Relish−/+, GMR-QF2/+; alrm-Gal4; Relish E20/QUAS-mCD8-GFP. Relish overexpression, GMR-QF2/+; QUAS- SCA3polyQ78:: alrm-Gal4/UAS-Relish QUAS-mCD8-GFP. (PDF 1700 kb)Additional file 5:Relish levels in astrocytes on Dif/Dl-dependent gene expression in the head. Expression of Dif/Dl target genes (IM1 or IM2) was determined in the heads of control flies (control), flies expressing SCA3polyQ78 in the eyes (-), and the effect of Relish RNAi targeted to astrocytes (Relish RNAi #1 and Relish RNAi #2), Relish overexpression, or SCA3polyQ78-expressing flies heterozygous for Relish (Relish −/+) on Dif/Dl target gene expression was determined by comparing them to flies only expressing SCA3polyQ78. (PDF 125 kb)Effect of modulating Additional file 6:a Efficacy of knockdown of CeCA or AttA gene expression. Flies expressing daughterless-Gal4  were crossed to control flies (w1118) or fly lines containing RNAi constructs targeting AttA or CecA and expression of CeCA or AttA in the adult progeny was determined. b Downregulating in astrocytes does not affect eye morphology. Control flies or fly lines containing RNAi constructs targeting CeCA or AttA were crossed to alrm-Gal4 flies and the morphology of the eyes was determined. c No effect of modulating of Relish-dependent AMP expression in astrocytes on mCD8-GFP levels in the eyes. Flies expressing eye-specific mCD8-GFP were compared to flies coexpressing RNAi constructs targeting AttA or CecA in astrocytes. Genotypes (a): control, da-Gal4/+. AttA, UAS− AttA/+ da-Gal4/+; CecA, UAS- CecA/+; da-Gal4/+. (b) Control, alrm-Gal 4/+. AttA RNAi, alrm-Gal 4/UAS-AttA RNAi. CecA RNAi, alrm-Gal 4/+; UAS-CecA RNAi/+. (c) As in (b), but with coexpression of QUAS-mCD8-GFP. (c) Control: GMR-QF2/+; alrm-Gal4/+; QUAS-mCD8GFP/+. AttA RNAi, GMR-QF2/+; alrm-Gal4/UAS-AttA RNA; QUAS-mCD8GFP/+. CecA RNAi, GMR-QF2/+; alrm-Gal4/+; UAS-CecA RNAi/QUAS-mCD8GFP. (PDF 289 kb)Additional file 7:a The UAS-Gal4 and the QUAS-QF system function independently. QUAS-QF-driven GFP expression specifically in neurons (GFP); UAS-Gal4-driven expression of myr-RFP specifically in astrocytes (RFP). Nuclei are stained with Hoechst (blue). b Genetic setup to inducibly express SCA3polyQ78 in neurons and simultaneously modulate gene expression in astrocytes. QUAS-QF was used to express SCA3polyQ78; UAS-Gal4 was used to modulate expression in astrocytes. Neuronally expressed QF2  is suppressed by QS (expressed under control of the tubulin (tub) promoter). This suppression is alleviated by quinic acid, resulting in expression of SCA3polyQ78. For details on the fly lines used, see experimental procedures. c Quinic acid does not affect lifespan. Control flies were cultured on standard fly food with or without quinic acid, and the fraction of dead flies was determined over time. d Raising flies at 18 °C does not induce expression of Relish RNAi constructs. Progeny of flies ubiquitously expressing RNAi constructs targeting Relish (Relish RNAi #1 and Relish RNAi #2) raised at 18 °C were analyzed for expression of Relish as in Additional file e Effect of modulating expression of Relish in astrocytes on lifespan. The lifespan of control flies or flies expressing Relish RNAi or overexpression constructs specifically in astrocytes was analyzed. f Effect of modulating Relish expression in astrocytes on impairment of mobility induced by neuronal SCA3polyQ78 expression. Control flies or flies expressing inducible SCA3polyQ78 together with Relish RNAi or Relish overexpression constructs targeted to astrocytes were analyzed for mobility over time. Scoring was done as described in the experimental procedures. g Effect of modulating Relish expression in astrocytes on SCA3polyQ78 levels or aggregation. Head lysates of 15-day-old flies as in Fig. polyQ78 on western blot. Tubulin was used as a control for equal loading. h Expression levels of Abeta42. Two independent lines, QUAS-Abeta42 #5 and QUAS-Abeta42 #7 were analyzed for expression of Abeta42 using 6E10 antibody. Tubulin was used as a control for equal loading. Genotypes: aalrm-Gal4::UAS-myr-RFP; nSyb-QF2/QUAS-mCD8-GFP.ctub-QS/+; alrm-Gal4/+; nSyb-QF2::tub-QS/+. d Genotypes as in Additional file e) -, tub-QS/+; alrm-Gal4/+; nSyb-QF2: tub-QS/+. Relish RNAi #1, Relish RNAi #2, or Relish overexpression, as in ‘-‘, but with UAS-Relish RNAi #1 or #2 or UAS-GFP-Relish. f Genotypes as in Fig. g As in e. h Control, nSyb-QF2/+. Abeta42 #5 or #7, QUAS-Abeta42/+; nSyb-QF2/+. (PDF 609 kb)


Bovine viral diarrhea virus (BVDV) subtypes 1f and 1d were isolated for the first time in Slovenia in 1999 and detected later in a majority of BVDV-infected cattle herds. Here, we report the first nearly complete genome sequences of noncytopathogenic BVDV-1f strain SLO/1170/2000 and cytopathogenic BVDV-1d strain SLO/2416/2002, isolated in Slovenia. Bovine viral diarrhea virus (BVDV) subtypes 1f and 1d were isolated for the first time in Slovenia in 1999 and detected later in a majority of BVDV-infected cattle herds. Here, we report the first nearly complete genome sequences of noncytopathogenic BVDV-1f strain SLO/1170/2000 and cytopathogenic BVDV-1d strain SLO/2416/2002, isolated in Slovenia. Pestivirus A (BVDV-1) and Pestivirus B (BVDV-2), within the Pestivirus genus of the family Flaviviridae (pro-C-Erns-E1-E2-p7-NS2-3(NS2-NS3)-NS4A-NS4B-NS5A-NS5B-3′  is a widespread pathogen of ruminants that generates significant economic losses during acute and persistent infections , 2. Straiviridae . BVDV-1 iviridae . The gen-NS5B-3′ . Genetic-NS5B-3′ .BVDV strain SLO/1170/2000 was isolated in 2000 from a serum sample collected from clinically healthy persistently infected cattle that produced no cytopathic effect on bovine turbinate (BT) cells, while BVDV strain SLO/2416/2002 was obtained from lymph nodes of a 22-month-old dead cow that suffered with mucosal disease and produced cytopathic effect on BT cells.de novo assembly using Genome Sequencer software v.2.9 (Roche) with default parameters. To eliminate assembly errors, all sequenced reads were mapped against the assembled genomes with the Geneious reference mapper  with default parameters. Open reading frames (ORFs) were predicted with the Geneious ORF finder.Total RNA extraction from supernatants of isolates was performed using the QIAamp viral RNA kit . For sequencing of the BVDV strain SLO/1170/2000 genome, a previously described protocol was used . For seqKX577637) from Slovenia, belonging to the subgenotype BVDV-1e, whereas SLO/2416/2002 showed 90.40% nucleotide similarity to BVDV-1d strain BJ1308 from China  and 246,120 reads with a mean read length of 206 nt (for SLO/2416/2002) were obtained, and the assembled genomes had an average read coverage of 225× and 63×, respectively. The nearly complete genomes of SLO/1170/2000 and SLO/2416/2002 were 12,259 and 12,241 nt long with G+C contents of 46.8% and 45.1%, respectively. The single ORF on each genome was 3,898 aa long, and the two ORFs showed 87.7% aa similarity to each other. Compared to the complete sequences of BVDV available in GenBank (using NCBI blastn), strain SLO/1170/2000 displayed only 80.13% nucleotide similarity to strain SLO/2407/2006 (GenBank accession number KX987157 and KY849592, respectively. The raw sequence data were deposited under BioProject number PRJNA576600.The complete genome sequences of these two BVDV isolates, SLO/1170/2000 and SLO/2416/2002, have been deposited in GenBank under the accession numbers


Snx8-/- mice infected with the DNA virus HSV-1 exhibited lower serum cytokine levels and higher viral titers in the brains, resulting in higher lethality. Mechanistically, SNX8 recruited the class III phosphatylinositol 3-kinase VPS34 to MITA, which is required for trafficking of MITA from the ER to perinuclear microsomes. Our findings suggest that SNX8 is a critical component in innate immune response to cytosolic DNA and DNA virus.MITA  is a central adaptor protein in innate immune response to cytosolic DNA. Cellular trafficking of MITA from the ER to perinuclear microsomes after DNA virus infection is critical for MITA activation and onset of innate antiviral response. Here we found that SNX8 is a component of DNA-triggered induction of downstream effector genes and innate immune response.  Infection by virus, such as the DNA virus herpes simplex virus 1, induces the host cells to express proteins that mediate antiviral immune response. The protein called MITA plays an essential role in the process of antiviral immune response. After viral infection, MITA traffics from the endoplasmic reticulum (ER) to perinuclear punctuate structures, a process required for its activation and induction of downstream antiviral genes. How the trafficking of MITA is regulated remains enigmatic. In this study, we found that a protein called SNX8 plays a critical role in regulation of MITA trafficking from the ER to perinuclear punctuate structures. SNX8 does this by recruiting the VPS34-containing translocon machinery to MITA. Deficiency of SNX8 impaired antiviral immune response and rendered the mice more sensitive to DNA virus-induced death. Our results suggest that SNX8 is a critical component in antiviral immune response to DNA virus. Innate immune response is pivotal for host defense against microbial pathogens. Cytosolic DNA derived from invading pathogens triggers signaling events that lead to induction of downstream anti-microbial effector genes –3. It haThe trafficking of MITA is critical for its activity in response to cytosolic DNA . It has Sorting nexin 8 (SNX8) belongs to the sorting nexin protein family, which is involved in endocytosis and endosomal sorting ,17. ReceIFNB1, CXCL10 and IL6 genes induced by HSV-1 and transfected synthetic DNAs ISD (IFN-stimulating DNA) and HSV120 (DNA of 120 basepairs representing the genomes of HSV-1)  . These rIFNB1, ISG56, CXCL10 and IL6 genes induced by HSV-1 and transfected ISD and HSV120 was dramatically inhibited in SNX8-deficient HFFs in comparison to wild-type cells  by the CRISPR-Cas9 method. We found that transcription of pe cells . In control HFFs . In simind ISG56 . Consistnd ISG56 . These rSnx8+/+ and Snx8-/- mice. We found that induction of Ifnb1, Isg56, Cxcl10 and Il6 genes were significantly inhibited in Snx8-/- bone marrow-derived macrophages (BMDMs) and mouse lung fibroblasts (MLFs) in comparison with their wild type counterparts after infection with three different types of DNA viruses, including HSV-1, vaccinia virus (VV), and ectromelia virus (ECTV) , HSV60 and HSV120 (dsDNA 60- or 120-mers representing the genomes of HSV-1) was impaired in Snx8-/- BMDMs and MLFs (Snx8-/- BMDMs (We next investigated innate immune response to DNA virus in primary cells from s (ECTV) . In simi/+ BMDMs . In addinfection . Consistand MLFs . In addiand MLFs . Consist/- BMDMs . Collectin vivo, we infected Snx8+/+ and Snx8-/- mice with HSV-1 by intraperitoneal (i.p.) injection and monitored their survival. The results indicated that Snx8-/- mice were more susceptibility to HSV-1-induced death than their wild-type littermates (Snx8-/- in comparison to their wild-type littermates (Snx8-/- mice infected with HSV-1 (a neurotropic DNA virus) for 6 days showed higher HSV-1 viral loads and genomic DNA copies in comparison to those of infected wild-type littermates , Golgi and endosome , which i-/- MLFs . Further-/- MLFs . These dIt has been demonstrated that during its trafficking, MITA is phosphorylated, activated and recruits IRF3 . We founIFNB1, CXCL10, ISG56 and IL6 .3′3′-cGAMP, Lipofectamine 2000 (InvivoGen); poly(dA:dT), DNA90, ISD, HSV60, HSV120 (Sangon Biotech); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); digitonin and VPS34-IN1 (Sigma); puromycin (Thermo); recombinant IFN-β (R&D Systems); ELISA kits for murine IFN-α and IFN-β (PBL) and IL-6 (Biolegend).Mouse monoclonal antibodies against FLAG and β-actin (Sigma) and HA (Covance); rabbit monoclonal antibodies against phospho-TBK1, TBK1 and SNX8 (Abcam); MITA, VPS34, IRF3, phospho-IRF3, phospho-IκBα and phosphor-MITA (Ser366) , rabbit polyclonal antibody against ERGIC-53/p58 (Sigma); Alexa Fluor 555 Mouse anti GM130 (BD Biosciences) were purchased from the indicated companies. Rabbit and mouse anti-SNX8 sera were raised against a recombinant human SNX8 protein.HEK293 cells were provided by Dr. Gary Johnson . THP-1 cells and HeLa cells were purchased from ATCC. HFFs were provided by Dr. Minhua Luo (Wuhan Institute of Virology). HSV-1 (KOS strain) , vaccinia virus (Tian-Tan Strain)  and ECTV  viruses have been obtained from the indicated resources.Mammalian expression plasmids for HA- or FLAG-tagged VPS34 were constructed by standard molecular biology techniques. The other expression and reporter plasmids were previously described ,19–22.Renilla luciferase) reporter plasmid was added to each transfection. Luciferase assays were performed using a dual specific luciferase assay kit. Firefly luciferase activities were normalized on the basis of Renilla luciferase activities. All reporter assays were repeated for at least three times. Data shown were average values ± SD from one representative experiment.HEK 293 cells were seeded in 24-well dishes and transfected the following day by standard calcium phosphate precipitation method. HeLa cells were transfected by FuGENE. HFFs, MLFs and MEFs were transfected by Lipofectamine 2000. To normalize for transfection efficiency, pRL-TK , reverse transcribed, and subjected to real-time PCR analysis to measure mRNA levels of the tested genes. Data shown are the relative abundance of the indicated mRNA normalized to that of GAPDH: GACAAGCTTCCCGTTCTCAG (forward) and GAGTCAACGGATTTGGTGGT (reverse);IFNB1: TGACTATGGTCCAGGCACAG (forward) and TTGTTGAGAACCTCCTGGCT (reverse);IL6: TTCTCCACAAGCGCCTTCGGTC (forward) and TCTGTGTGGGGCGGCTACATCT (reverse);ISG56: TCATCAGGTCAAGGATAGTC (forward) and CCACACTGTATTTGGTGTCTAGG (reverse);RIG-I: ACGCAGCCTGCAAGCCTTCC (forward) and TGTGGCAGCCTCCATTGGGC (reverse);CXCL10: GGTGAGAAGAGATGTCTGAATCC (forward) and GTCCATCCTTGGAAGCACTGCA (reverse);TGF-β: TACCTGAACCCGTGTTGCTCTC (forward) andGTTGCTGAGGTATCGCCAGGAA (reverse);Gapdh: ACGGCCGCATCTTCTTGTGCA (forward) and ACGGCCAAATCCGTTCACACC (reverse);Ifnb1: TCCTGCTGTGCTTCTCCACCACA (forward) and AAGTCCGCCCTGTAGGTGAGGTT (reverse);Cxcl10: ATCATCCCTGCGAGCCTATCCT (forward) and GACCTTTTTTGGCTAAACGCTTTC (reverse);Isg54: CGAACTACCGTCTGGATGACTG (forward) and CTTCAACCAGCGCCATTGCTTG (reverse);Isg56: ACAGCAACCATGGGAGAGAATGCTG (forward) and ACGTAGGCCAGGAGGTTGTGCAT (reverse);Rig-i: AGCCAAGGATGTCTCCGAGGAA (forward) and ACACTGAGCACGCTTTGTGGAC (reverse);Il6: TCTGCAAGAGACTTCCATCCAGTTGC (forward) and AGCCTCCGACTTGTGAAGTGGT (reverse);ICP22: TGTTTGGAGACCAGACGGTA (forward) andHSV-1 CATCGGAGATTTCATCATCG (reverse);ICP27: GGCCTGATCGAAATCCTAGA (forward) andHSV-1 GTCAACTCGCAGACACGACT (reverse).Cells were lysed in 1 ml NP40 lysis buffer . For each immunoprecipitation, 0.4 ml aliquot of lysate was incubated with 0.5–2 μg of the indicated antibody or control IgG and 25 μl of a 1:1 slurry of Protein-G sepharose  for at least 2 hr. The sepharose beads were washed three times with 1 ml of lysis buffer containing 500 mM NaCl. The precipitates were fractionated on SDS-PAGE, and immunoblot analysis were performed as described ,24.Double-stranded oligonucleotides corresponding to the target sequences were cloned into the pSuper-Retro RNAi plasmid (Oligoengine). The following sequences were targeted for human SNX8 cDNA: #1 (5′-CGGCAGATCTTCTCATATT-3′) and #2 (5′-CGGCAGATCTTCTCATATT-3′).Human VPS34 cDNA: #1 (5′-GATCTGAAACCCAATGCTG-3′) and #2 (5′-GGAATGTGAAGATCAAGAT-3′).Mouse Vps34 cDNA: (5′- GCTGTCCTAGAAGATCCCA-3′)HEK293 cells were transfected with two packaging plasmids, pGag-Pol (10 μg) and pVSV-G (3 μg), and control or VPS34-RNAi retroviral plasmid (10 μg) by calcium phosphate precipitation. The cells were washed 12 hr after transfection and new medium without antibiotics was added for additional 24 hr. The recombinant virus-containing medium was filtered and used to infect THP-1 cells in the presence of polybrene (4 μg/ml). The infected THP-1 cells were selected with puromycin (0.5 μg/ml) for 2 weeks before additional experiments were performed.Establishment of SNX8-deficient HeLa cells and HFFs were performed as described . BrieflyBMDMs were infected with HSV-1 for 18 hr. The culture media were collected for measurement of IFN-β and IL-6 cytokines by ELISA.For preparation of BMDMs, the bone marrow cells were cultured in 10% M-CSF-containing conditional medium from L929 cells for 3–5 days. MEFs were prepared from day 12.5 embryos and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. Primary lung fibroblasts were isolated from approximately 4- to 6-week-old mice. Lungs were minced and digested in calcium and magnesium free HBSS containing 10 μg/ml type II collagenase (Worthington) and 20 μg/ml DNase I (Sigma-Aldrich) for 3 hours at 37°C with shaking. Cell suspensions were filtered through progressively smaller cell strainers (100 and 40 μm) and then centrifuged at 1500 rpm for 5 min. The cells were then plated in culture medium (1:1 DMEM/Ham's F-12 containing 10% FBS). After 1 hour, adherent fibroblasts were rinsed with HBSS and cultured in media.Mice were infected with HSV-1 i.p. The viability of the infected mice was monitored for 12 days. The mouse sera were collected at 6 hr after infection to measure cytokine production by ELISA.Confocal microscopy was performed as previously described . In Briet test was used for statistical analysis with Microsoft Excel and GraphPad Prism Software. For the mouse survival study, Kaplan-Meier survival curves were generated and analyzed by Log-Rank test; P < 0.05 was considered significant.Student’s S1 FigSnx8+/+ and Snx8-/- MLFs (4x105) were infected with VV, ECTV or HSV-1 (MOI = 1) for the indicated times before qPCR analysis.(A) Snx8+/+ and Snx8-/- MLFs (4x105) were infected with HSV-1 (MOI = 1) for the indicated times before immunoblot analysis.(B) Snx8+/+ and Snx8-/- MLFs (4x105) were transfected with ISD, DNA90 and HSV120 (3 μg/ml) for 3 h before qPCR analysis.(C) Snx8-/- MLFs were reconstituted with full length of SNX8 and its truncation mutants by lentiviral-mediated gene transfer. The reconstituted MLFs (4x105) were infected with HSV-1 (MOI = 1) for the indicated times before qPCR analysis.(D) (TIF)Click here for additional data file.S2 Fig5) were firstly transfected with VPS34-RNAi (0.4 μg) for 24 h, then re-transfected with ISRE reporter (0.05 μg) and the indicated expression plasmids (0.1 μg each) for 24 h before luciferase assay.(A) HEK293 cells (1x105) were treated with the indicated doses of VPS34 inhibitor (VPS34-IN1) for 10 h and then infected with HSV-1 (MOI = 1) for the indicated times before qPCR analysis.(B) MLFs (4x10(TIF)Click here for additional data file.


Tumor necrosis factor (TNF) receptor associated factor-2 (TRAF2) knockout (KO) cells were generated to investigate the role of TRAF2 in signaling by TNFR1 and the CD95-type death receptors (DRs) TRAILR1/2 and CD95. To prevent negative selection effects arising from the increased cell death sensitivity of TRAF2-deficient cells, cell lines were used for the generation of the TRAF2 KO variants that were protected from DR-induced apoptosis downstream of caspase-8 activation. As already described in the literature, TRAF2 KO cells displayed enhanced constitutive alternative NFκB signaling and reduced TNFR1-induced activation of the classical NFκB pathway. There was furthermore a significant but only partial reduction in CD95-type DR-induced upregulation of the proinflammatory NFκB-regulated cytokine interleukin-8 (IL8), which could be reversed by reexpression of TRAF2. In contrast, expression of the TRAF2-related TRAF1 protein failed to functionally restore TRAF2 deficiency. TRAF2 deficiency resulted furthermore in enhanced procaspase-8 processing by DRs, but this surprisingly came along with a reduction in net caspase-8 activity. In sum, our data argue for (i) a non-obligate promoting function of TRAF2 in proinflammatory DR signaling and (ii) a yet unrecognized stabilizing effect of TRAF2 on caspase-8 activity. The rec pathway , 3. Ther pathway , 5. The DRs of the CD95 and TNFR1 type are not only able to induce cell death but can also trigger proinflammatory signaling pathways leading to the activation of transcriptions factors of the nuclear factor of kappaB (NFκB) family and mitogen-activated protein (MAP) kinases (MAPKs) –9. TRAF2Here, we knocked out TRAF2 in HCT116-PI3Kmut and HT1080-Bcl2-TNFR2 cells, which are resistant against DR-induced apoptosis downstream of procaspase-8 processing due to inhibition of Bax/Bak-mediated mitochondrial amplification of the apoptotic caspase-8 activity , 22. TRAPIK3CA oncogene causing constitutive degradation of Bax  displayed restored IL8 responsiveness  can promote the initial processing step of procaspase-8 processing  that exist in DR-stimulated cells. This issue must be clarified in future studies. DRs do not trigger necroptosis in HCT116 or HT1080 cells; the anti-necroptotic activity of TRAF2 could therefore not be evaluated in the TRAF2-deficient cells used in this study.Twenty-five years ago, TRAF2 and the TRAF2-interacting E3 ligases cIAP1 and cIAP2 have been identified as components of the TNFR2 signaling complex , 39. AftKO cells . HT1080-Bcl2-TNFR2 cells were obtained by retroviral transfection of HT1080-Bcl2(GFP) cells (described in 21) with a mixture of a TNFR2 and a puromycin encoding vector. HCT116-PIK3CA-mut cells stably transfected with FLIPL and FLIPS were generated as previously described for HaCaT cells (Human Killer TRAIL (TRAIL) was purchased from Enzo Life Sciences and TNF was a kind gift from Prof. Daniela Männel (University of Regensburg). Fc-CD95L and TWEAK contain an internal Flag tag and were produced by transient transfection of HEK293 cells using PEI  as essentially described for other Flag-tagged proteins by Lang et al. . SynthesaT cells .http://arep.med.harvard.edu/human_crispr/). Transfected cells were cultivated for 5 days and then seeded at low density to obtain individual clones. The latter was verified for TRAF2 expression by WB to identify clones with successful KO of TRAF2.To generate TRAF2 KO cells, HCT116-PI3Kmut and HT1080-Bcl2-TNFR2 cells were transfected as described below using polyethylenimine  with a mixture of GeneArt CRISPR Nuclease (CD4 Reporter) vector plasmids (ThermoFisher Scientific) encoding two TRAF2-specific guide RNAs and Cas9. The TRAF2 guide sequences (5′ to 3′) used were as follows: CCTGAGCTGCCGGCATTGCGG and GGACCTGGCGATGGCTGACTGG. The guide RNA sequences were chosen from Table 1 of the supplementary website of Mali et al.   were digested with MfeI and inserted into the plasmid pT2-SVNeo EcoRI site , and the proteins of interest were detected with the corresponding primary antibodies  and HRP-linked secondary antibodies . Antibody complexes were visualized with the ECL solution. Between every step, membranes were washed three times with TBS-Tween [0.05% (v/v)]. The following antibodies were used: anti-caspase-3 (8G10), anti-caspase-9 (#9502), anti-DR4 (D9S1R), anti-DR5 (D4E9), anti-FADD (#2782), anti-phospho-IκBα (Ser32) (14D4), anti-IκBα (L35A5), anti-alpha-Fodrin (#2122), anti-CYLD (D1A10), anti-TRAF1 (45D3), anti-TRAF2 (C192), anti-A20 (D13H3), anti-mouse IgG, HRP-linked antibody (#7076), anti-rat IgG, HRP-linked antibody (#7077) and anti-rabbit IgG, HRP-linked antibody (#7074), which were all from Cell Signaling. Furthermore, anti-PARP (BD Biosciences), HRP-conjugated polyclonal rabbit anti-mouse immunoglobulins , anti-caspase-8 , anti-cIAP1 , anti-tubulin , anti-β-actin , anti-TRAF1 , anti-FLIP , and anti-p52  have been used.Cells (2 × 106 cells per well. Cells were stimulated for 6 h with the indicated reagents and were harvested after a 5-min incubation with trypsin according to the manufacturer's protocol [Application note No. 3021. Rev. 1.3 of NucleoCounter® NC-3000™ (Chemometec)]. Cells were stained using the FAM-FLICA® Caspase-8 Assay Kit . Stained cells were analyzed using NucleoCounter® NC-3000™ software and the values were diagrammed by Graph Pad Prism 5 software.Cells were cultivated in 12-well plates until they reached a density of approximately 3 × 10Measurements of independent experiments were collected and analyzed using the one-way ANOVA Bonferroni's multiple comparison test function of the GraphPad Prism5 software .The datasets generated for this study are available on request to the corresponding author.JK, MA, and DS performed experiments and prepared figures. JK and HW designed and analyzed the experiments and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pseudomonas aeruginosa is defined as a “critical” priority pathogen for the development of new antibiotics. Here we report the complete genome sequence of an extensively drug-resistant, Verona integron-encoded metallo-β-lactamase-expressing isolate belonging to the high-risk sequence type 233.Carbapenem-resistant  Pseudomonas aeruginosa is defined as a “critical” priority pathogen for the development of new antibiotics. Here we report the complete genome sequence of an extensively drug-resistant, Verona integron-encoded metallo-β-lactamase-expressing isolate belonging to the high-risk sequence type 233.Carbapenem-resistant  Pseudomonas aeruginosa is a critical threat to public health (P. aeruginosa K34-7 belongs to sequence type 233 (ST233) and is an extensively drug-resistant (XDR), carbapenem-resistant clinical isolate expressing the Verona integron-encoded metallo-β-lactamase (VIM-2)  , before manual curation and validation. Iterative read mapping of Illumina sequences using custom settings in Geneious 10.1.3  and sequenced on a PacBio RS II platform. A standard library of 20-kb fragments was prepared using the BluePippin preparative electrophoresis system  with a 9-kb cutoff and sequenced on a single-molecule real-time (SMRT) cell using P6-C4 chemistry with 360-min movie-time chemistry. Additional whole-genome sequencing (WGS) was performed using an Illumina HiSeq sequencer. Genome assembly involved a P. aeruginosa genomic island 5 (PAGI-5)-like hybrid (aac(3), aac(3)-I, aac(6´)-Il, aadA2, and aph(3´)-IIb], β-lactams , chloramphenicol , trimethoprim (dfrB5), fosfomycin (fosA), sulfonamide (sul1), and tetracycline (tetG), as annotated by the PGAP pipeline . A Vibrio cholerae (P. aeruginosa genomes but not in the ST233 P. aeruginosa PA83 genome (P. aeruginosa K34-7 contains a type I-F CRISPR-Cas system (1656563 to 1664702), previously described in P. aeruginosa strain UCBPP-PA14 (A region (1936000 to 2043700) of 12 direct tandem repeats  encodes a zonular occludens toxin, previously characterized in cholerae . SimilarComplete high-quality bacterial genomes facilitate further research into mechanisms of resistance and their dissemination and aid in the development of new therapies for XDR infections.CP029707 and CP029708.This complete genome project has been deposited at GenBank under the accession numbers


Moreover, DHA lowered protein and mRNA expression levels of lipogenic enzymes such as fatty acid synthase (FAS), acetyl-CoA carboxylase and stearoyl-CoA desaturase-1 (SCD-1) in primary hepatocytes incubated with liver X receptor (LXR) agonist T0901317 or high glucose and insulin. DHA also decreased protein expression of nuclear and precursor sterol response-element binding protein (SREBP)-1, a key lipogenesis transcription factor. We further found that exposure of murine primary hepatocytes to DHA for 12 h increased GPR40 and GPR120 mRNA levels. Specific agonists (Compound A for GPR120 and AMG-1638 for GPR40), hepatocytes from GPR120 knock-out mice and GPR40 selective antagonist (GW1100) were used to assess whether DHA’s antilipogenic effects are mediated through GPR120 or GPR40. Compound A did not decrease SREBP-1 and FAS protein expression in hepatocytes exposed to T0901317 or high glucose with insulin. Moreover, DHA downregulated lipogenesis enzyme expression in GPR120-null hepatocytes. In contrast, AMG-1638 lowered SREBP-1 and SCD-1 protein levels. Additionally, GW1100, a GPR40 antagonist, reversed the antilipogenic effects of DHA. Collectively, our data demonstrate that DHA downregulates the expression SREBP-1-mediated lipogenic enzymes via GPR40 in primary hepatocytes.Nonalcoholic fatty liver disease is a frequent liver malady, which can progress to cirrhosis, the end-stage liver disease if proper treatment is not applied. Omega-3 fatty acids, such as docosahexaenoic acid (DHA) and eicosapentaenoic acid, have been clinically proven to lower serum triglyceride levels. Various physiological activities of omega-3 fatty acids are due to their agonistic actions on G-protein-coupled receptor 40 (GPR40) and GPR120. Lipid droplets (LD) accumulation in hepatocytes confirmed that DHA treatment reduced the number of larger ( >10 μm Nonalcoholic fatty liver disease (NAFLD) affects 80–100 million people in the U.S. alone . Among t® and Lovaza®, are widely used as prescription medications for dyslipidemia [Omega-3 fatty acid provides an energy source as nutrient, but also act as a remedy for serum hyperlipidemia. Commercially available fish oil supplements are one of the most popular over-the-counter medications for the management of elevated serum triglyceride (TG) levels. In the U.S. alone, about 18.8 million adults are taking fish oil supplements . Omega-3ipidemia . Omega-3Omega-3 fatty acids, such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), have been recognized as endogenous ligands for G-protein-coupled receptor 40 (GPR40) and G-protein-coupled receptor 120 (GPR120) ,10. In asrebf1, acacb, fasn, and scd1 which are required to control hepatic TG synthesis . Gene ontology analyses revealed that the genes, including those involved with lipid metabolic processes and cholesterol biosynthetic processes, were mainly altered for similar functional annotations in the liver tissues of fish-oil-fed mice . In addiynthesis .The SREBP1 transcription factor is a critical regulator of fatty acid homeostasis in hepatocytes. Activation of SREBP1 controls the expression of a range of lipogenic enzymes, such as fatty acid synthase (FAS), acetyl-coenzyme A carboxylase (ACC), and stearoyl-CoA desaturase-1 (SCD-1) . Hence, ® staining showed a greater increase in LDs in hepatocytes incubated under the high glucose and insulin condition, but not with T090  in hepatocytes, which reflects the dysregulation of TG fat . We explith T090 A,B. Inteith T090 C. Neverte to DHA D,E. One e to DHA C,D, suggLong-chain fatty acids, such as DHA, act as endogenous ligands on GPR120 and GPR40 and regulates metabolic and inflammatory homeostasis in adipocytes and macrophages ,16. To iWe tested the potential role of GPR40 in antilipogenic effects of DHA in hepatocytes. AMG-1638 is a well-characterized GPR40 agonist in murine species and has shown a specific full agonistic activity compared with other candidates . The enhDHA and EPA are omega-3 fatty acids and known as ligands for GPR40 and GPR120. Although they both have antilipogenic properties, DHA is associated with a greater reduction in serum TG levels compared with EPA . In addiGPCRs are usually downregulated to eschew repercussive effects by persistent stimulation with agonists. Nobili et al. reported that patients administered DHA develop increased levels of GPR120 in hepatocytes . This req/11–coupled receptor GPR40 is expressed mainly in pancreatic β-cells, most of previous studies focused on its potential ability to stimulate insulin secretion. Because GPR40 activation stimulates insulin secretion only in the presence of elevated glucose levels [Because the Gαe levels ,30,31, ie levels . Thus, te levels . They fuTaken together, our findings may help to unravel how DHA alleviates fatty acid accumulation in hepatocytes. Furthermore, these findings support the notion of using specific GPCR agonists as an add-on therapy to manage metabolic syndrome and suggest that GPR40 merits further investigation as an adjuvant therapy for NAFLD.Antibody recognizing precursor sterol regulatory element binding protein-1 (SREBP-1) was obtained from Santa Cruz Biotechnology . Anti-fatty acid synthase (FAS) and antinuclear form of SREBP-1 antibodies were supplied by BD Biosciences . Stearoyl-CoA desaturase-1 (SCD1), phospho-AMP-activated protein kinase (p-AMPK), and acetyl-CoA carboxylase (ACC) antibodies were obtained from Cell Signaling Technology . Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody and T0901317 (T090) were supplied by Calbiochem . Anti-β actin antibody, insulin, and glucose were purchased from Sigma-Aldrich . Horseradish peroxidase-conjugated donkey anti-rabbit IgG, and alkaline phosphatase-conjugated donkey anti-mouse IgG were obtained from Jackson Immunoresearch Laboratories . Eicosapentaenoic acid (EPA), docosahexaenoic acids (DHA), and compound A (CpdA) were supplied by Cayman Chemical . GW1100 and AMG-1638 were kindly donated from LG Chem Ltd. .2.Human hepatoma cell line HepG2 was obtained from American Type Culture Collection . HepG2 cells were grown in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum  with 50 U/mL penicillin and 50 μg/mL streptomycin. All cell lines were maintained at 37 °C in a humidified incubator with 5% COGPR120 knock-out (KO) C57BL/6 mice were kindly donated from LG Chem Ltd. The mice were housed in a pathogen-free animal facility under a standard 12 h light/dark cycle. Animal experiments were conducted under the guidelines of the Institutional Animal Use and Care Committee at Seoul National University .® and rompun®, 24G catheter was cannulated to liver portal vein. After perfusion with Hank’s Balanced Salt solution medium  supplemented with 0.5 mM ethylene glycol tetraacetic acid and 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), liver tissue was digested with low-glucose DMEM containing 1% penicillin/streptomycin, 15 mM HEPES and 1 mg/mL collagenase from Clostridium histolyticum . After tearing digested liver tissue, the isolated cells were washed three times with high-glucose DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 15 mM HEPES, 10 nM dexamethasone. Other cells except primary hepatocytes were all removed by centrifugation.After anesthetizing 8-weeks-old C57BL/6 mice  or GPR120 KO mice with zoletil4.5. mRNA Isolation and Real-Time Quantitative Polymerase Chain Reaction (qPCR)After washing with sterile phosphate-buffered saline (PBS), total RNA was isolated using TRIzol reagent . mRNA was reverse transcribed to cDNA using Maxime RT Premix . Amplified cDNA was analyzed by Bio-Rad CFX Manager™ Software  using iTaq Universal SYBR Green Supermix (Bio-Rad) and SYBR Select Master Mix (Life Technologies). Primer sequences used in experiments were:5′-CTGTGCAGGAATGAGTGGAAG-3′ (mouse GPR120-forward)5′-CTGATGGAGGGTACTGGAAATG-3′ (mouse GPR120-reverse)5′-CTGTGCAGGAATGAGTGGAAG-3′ (human GPR120-forward)5′-CTGATGGAGGGTACTGGAAATG-3′ (human GPR120-reverse)5′-TGGCGCGCCAGCCTGG-3′ (mouse GPR120 KO-forward)5′-CCATATGAAAGCCAGCAGTGCC-3′ (mouse GPR120 KO-reverse)5′-CGCCGTCGGCGCCGTG-3′ (mouse GPR120 WT-forward)5′-CCATATGAAAGCCAGCAGTGCC-3′ (mouse GPR120 WT-reverse)5′-TTTCATAAACCCGGACCTAGGA-3′ (mouse GPR40-forward)5′-CCAGTGACCAGTGGGTTGAGT-3′ (mouse GPR40-reverse)5′- GCCCACTTCTTCCCACTCTA-3′ (human GPR40-forward)5′- AGACCCAGGTGACACAGGAC -3′ (human GPR40-reverse)5′-GTAACCCGTTGAACCCCATT-3′ (mouse and human 18s rRNA protein-forward)5′-CCATCCAATCGGTAGTAGCG-3′ (mouse and human 18s rRNA protein-reverse)g for 20 min to remove debris, and the protein samples were loaded on 8–15% SDS-PAGE gel and transferred to nitrocellulose membrane . Membranes were incubated for 1 h with 5% skim milk  and reacted with primary antibodies overnight at 4 °C. The membranes were washed and incubated with a second antibody for 1 h at room temperature. Protein expression was visualized with LAS3000-mini  using enhanced chemiluminescence (ECL) system reagent . Densitometric analysis was performed by using Multiguage software  and Image J 1.46r.After washing the cultured cells with PBS, cells were lysed in lysis buffer containing 20 mM TrisHCl (pH 7.5), 1% Triton X-100, 137 mM sodium chloride, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 25 mM β-glycerophosphate, 2 mM sodium pyrophosphate, 1 mM phenyl methyl sulfonyl fluoride, and 1 μg/mL leupeptin. Cells were then incubated in ice for 1 h. The cell lysates were centrifuged at 10,000 The left lateral lobe of the liver was sliced, and tissue slices were fixed in 10% buffered-neutral formalin. The liver slices were stained with hematoxylin and eosin (H&E).® 493/503 staining , the 488 nm laser was used and signals were collected with a long pass 505 nm filter. Quantification of LD number and size was performed with MetaMorph, version 7.5 .For BODIPY® SPSS® Statistics 23 . The significance level was set at p value < 0.05 for all comparisons. Although the size of sample is small, Shapiro–Wilk test showed normality of the data and one-way analysis of variance (ANOVA) was used for multiple comparisons. Tukey’s or Dunnett’s tests were used as post hoc analysis methods. Statistical analysis for GSE data was performed by using Student’s t-test and Benjamini–Hochberg test.The analyses were performed with IBM


C.elegans germline cells.The extracellular matrix protein Hemicentin (HIM-4) is an extracellular factor that regulates anillin (ANI-1) for germ cell membrane stabilization and contractile ring formation in  Caenorhabditis elegans germline cells.Cell division requires constriction of an actomyosin ring to segregate the genetic material equally into two daughter cells. The spatial and temporal regulation of the contractile ring at the division plane primarily depends on intracellular signals mediated by the centralspindlin complex and astral microtubules. Although much investigative work has elucidated intracellular factors and mechanisms controlling this process, the extracellular regulation of cytokinesis remains unclear. Thus far, the extracellular matrix protein Hemicentin (HIM-4) has been proposed to be required for cleavage furrow stabilization. The underlying molecular mechanism, however, has remained largely unknown. Here, we show that HIM-4 and anillin (ANI-1) genetically act in the same pathway to maintain the rachis bridge stability in the germline. Our FRAP experiments further reveal that HIM-4 restricts the motility of ANI-1. In addition, we demonstrate that HIM-4 is recruited to the cleavage site in dividing germ cells and promotes the proper ingression of the cleavage membrane. Collectively, we propose that HIM-4 is an extracellular factor that regulates ANI-1 for germ cell membrane stabilization and contractile ring formation in  The ECM is a tissue-specific assembly of molecules that reside and function outside of the cell. Specific resident cells secrete these molecules, mainly proteoglycans and large, multidomain, fibrous proteins, which form extracellular fibrils and supramolecular networks . ECM proCaenorhabditiselegans (C. elegans) embryo cell division  zygotes.Apart from the roles in tissue organization, some of the ECM proteins are also involved in cell division. Previous work has shown that chondroitin proteoglycans (CPGs) are required for division . RNA intdivision . In cpg- embryos . HoweverRecently, another extracellular matrix protein, Hemicentin (HIM-4), has been proposed to be required for germline syncytium stabilization. Depletion of HIM-4 resulted in effects on the germ cell, including membrane destabilization, cleavage furrow retraction, and cytokinesis failure, resulting in multinucleated cells in the germline . SimilarC. elegans, which contain a single representative called HIM-4 . Simultaneously depletion of ANI-1 and HIM-4 phenocopies the single depletion of each for germline compartmentation, rachis bridge size, and cleavage furrow constriction rate of the dividing germ cells. This suggests that HIM-4 regulates ANI-1 for membrane stabilization and contractile ring formation. Our work, therefore, demonstrates that the ECM protein HIM-4 mediates two biological events: (1) stabilization of germ cell membrane and (2) promotion of contractile ring constriction.In this study, using C. elegans gonad rachis to stabilize syncytial architecture (him-4(e1266) (him-4(RNAi) and him-4(e1266) germ cells are multinucleated (him-4(RNAi) germ cells started to divide in the mitotic zone, and the cleavage membranes remained stable  during the imaging periods. On the contrary, about 10% of the him-4(RNAi)–treated dividing germ cells showed cleavage membrane retraction at a later stage of furrow ingression, resulting in binucleation   mutant l4(e1266) . In the 4(e1266) , but 15%ucleated . To studcleation  (Dong etcleation . Membrancleation . These rVideo 1him-4(RNAi).Download videoGerm cells expressing GFP::PH; mCherry::HIS in C. elegans germline (him-4(e1266)  and him-4(RNAi)  is significantly larger than that of control germ cells  (The size of the rachis bridge is another parameter that can be assessed for intercellular bridge stability within the germline . To detegermline . Notably n = 74) . To ensu n = 74) . Collectani-2(RNAi) but enlarged in ani-1(RNAi) gonads (ani-1(RNAi) germline cells were enlarged to a very similar extent to that of him-4(RNAi) (ani-1(RNAi) (him-4(RNAi) and him-4(e1266) was observed in ani-1(RNAi);him-4(e1266) (him-4(e1266) line depleted of ANI-1 by RNAi. The maximum diameter of the rachis bridge in him-4(e1266);ani-1(RNAi) was significantly larger than the control but not significantly different from him-4(e1266) (him-4(e1266) mutant and then measured rachis bridge diameter. The enlargement of rachis bridge diameter in HIM-4 depletion would be resolved if it is caused by the reduction of ANI-1 activity. Our results show a statistically significant decrease in the average rachis bridge diameter of him-4(e1266);ani-2(RNAi) compared with that of him-4(e1266) (him-4(e1266) could be slightly rescued by the depletion of ANI-2. Taken together, our results demonstrate that ANI-1 and HIM-4 are required for intercellular bridge stability and probably act in the same pathway.In addition to HIM-4, the anillin actomyosin scaffold protein family members ANI-1 and ANI-2 also localized in the rachis  Fig 1C)Fig 1C), ) gonads  (Amini e) gonads . Interes-4(RNAi) . In addi-1(RNAi) , and sim4(e1266) . Therefo4(e1266) . This su4(e1266) . This dehim-4(RNAi) were compared, but no significant difference was observed (data not shown). We then examined whether HIM-4 regulates the mobility of ANI-1 by analyzing the diffusion behavior of HIM-4 and ANI-1 on the rachis membrane in the mitotic zone by fluorescence recovery after photobleaching (FRAP) (him-4(RNAi)  . HIM-4 sg (FRAP) . On the g (FRAP) . If HIM--4(RNAi) . This re-4(RNAi) . As a co-4(RNAi) , whereas-4(RNAi) . Importa-4(RNAi)  and NMY--4(RNAi)  after phVideo 2Download videoFRAP experiment of GFP::HIM-4 germline.Video 3Download videoFRAP experiment of GFP::ANI-1 germline.Video 4him-4(RNAi) germline.Download videoFRAP experiment of GFP::ANI-1; Video 5Download videoFRAP experiment of GFP::ANI-2 germline.Video 6Download videoFRAP experiment of NMY-2::GFP germline.Video 7him-4(RNAi) germline.Download videoFRAP experiment of GFP::ANI-2; Video 8him-4(RNAi) germline.Download videoFRAP experiment of NMY-2::GFP; C. elegans germlines, ANI-1 and HIM-4 accumulated in the rachis and formed ring-like structures at the rachis bridges (him-4(RNAi)  . Collecti & ii and iii). The intensity of this newly formed patch then gradually increased and moved with the ingressing cleavage furrow  and him-4(e1266) dividing germ cells were delayed to 694 and 799 s, respectively (him-4(RNAi) dividing germ cells (him-4(RNAi) and him-4(e1266) (him-4(RNAi) and him-4(e1266) required 190 s to reach 10% membrane ingression. The constriction rate to 10% membrane ingression was 6.2 × 10−4 μm/s in the control germ cells, whereas it was significantly reduced to 3.57 × 10−4 μm/s and 4.23 × 10−4 μm/s in him-4(RNAi) and him-4(e1266) germ cells, respectively  . Becauseectively .Video 11Download videoCytokinesis of NMY-2::GFP; mCherry::HIS dividing germ cells.Video 12him-4(e1266) dividing germ cells.Download videoCytokinesis of NMY-2::GFP; mCherry::HIS; him-4(RNAi) germ cells (him-4(RNAi) germ cells took longer time to recruit ANI-1 to the same level as the control for similar extend of ingression in the early anaphase  germ cells co-expressing NMY-2::GFP and mCherry::HIS were imaged and the rate of membrane ingression was quantified (him-4(RNAi) and him-4(e1266) dividing germ cells, ani-1(RNAi) dividing germ cells took longer to reach 80% membrane ingression compared with the control germ cells (−4 μm/s) was significantly lesser than the control germ cells (ani-1(RNAi) dividing germ cells had no differences in measured constriction rate characteristics compared with him-4(RNAi) and him-4(e1266) dividing germ cells (him-4(e1266);ani-1(RNAi) dividing germ cells have no significant differences from him-4(RNAi), him-4(e1266), and ani-1(RNAi)  . CollectVideo 13ani-1(RNAi) dividing germ cells.Download videoCytokinesis of NMY-2::GFP; mCherry::HIS; Video S14him-4(e1266);ani-1(RNAi) dividing germ cells.Download videoCytokinesis of NMY-2::GFP; mCherry::HIS; In this article, we have expanded our understanding of the role played by the extracellular matrix protein Hemicentin beyond the established role as a scaffold protein to include a regulatory role in germline cytokinesis. HIM-4 stabilizes the cleavage membrane in the mitotic and meiotic transition zones to prevent the formation of multinucleated germ cells. This stabilization requires ANI-1. We observed that HIM-4 retains ANI-1 at the rachis bridges and that it is required for proper cytokinesis in germ cells. Upon anaphase onset, HIM-4 accumulates at the outer leaflet of the division plane membrane and moves together with the cleavage furrow as its ingresses. Depletion of HIM-4 compromised the proper recruitment of ANI-1 at the division plane, which in turn reduces the rate of contractile ring formation. Based on these results, we propose that HIM-4 regulates ANI-1 and consequently stabilizes germ cell cytokinesis, ensuring a stable syncytial gonad.C. elegans germline, HIM-4 is mainly secreted by gonadal leader cells as a monomer that cross-links to form complexes in the rachis P2-mediated cortical recruitment . ImportaC. elegans strains used in this study are listed in Table S1. All of the strains were cultured at 22°C on NGM plates seeded with Escherichia coli strain OP50.The C. elegans strains used in this study.Table S1 ani-1 was generated using CRISPR/Cas9-triggered homologous recombination as described previously  and ani-1-sgR (CAAGACATCTCGCAATAGG). A homologous repair template construct of ani-1 was obtained via insertion of the two homology arms of ani-1 into pDD282 digested with ClaI and SpeI, and two primer pairs were used for the construction: one pair of primers to amplify the 5′ homology arm of the ani-1 genome insertion site  and one pair of primers to amplify the 3′ homology arm .An N-terminal GFP knock-in transgenic strain of endogenous eviously . The CasE. coli were transformed with a dsRNA expression plasmid derived from L4440 with the sequence encoding the gene of interest; these plasmids were obtained from the RNAi library. To produce RNAi-feeding plates expressing dsRNA, the bacteria were grown in LB with 100 μg/ml ampicillin overnight at 37°C and then seeded onto NGM plates containing 100 μg/ml ampicillin and 1 mM IPTG, followed by incubation at room temperature for 8 h. To deplete the two genes, the corresponding bacterial strains were mixed at a 1:1 ratio based on the optical densities of the cultures. Worms were then raised on these RNAi-feeding plates for 24–72 h before microscopy imaging.RNAi was performed according to the feeding methods described by Timmons et al . HT115 with a MicroPoint system . Photobleaching experiments were performed using the MicroPoint targeted illumination acquisition routine in MetaMorph (Molecular Devices). For photobleaching, a 60×/1.45 NA oil immersion objective was used, and the number of pulses and the MicroPoint laser power were adjusted in different samples. Time-lapse single-plane images were acquired under a 488-nm excitation laser: two frames were imaged before photobleaching, and the regions of interest (ROIs) were then photobleached. Images were acquired after photobleaching at 20-s intervals according to the parameter settings.FRAP experiments were conducted within worms expressing GFP::ANI-1, GFP::ANI-2, NMY-2::GFP, and GFP::HIM-4 under control or ROI1) and ROI2 (I(t)ROI2) was first normalized by subtracting the intensity of the background region ROI3 (I(t)ROI2) using the following formula: For data analysis, the shuffling of the time-lapse images was first adjusted with the stacks-shuffling plugin in ImageJ. Then, the intensity of the bleached region (ROI1), the non-bleached rachis region (ROI2), and the background region (ROI3) was measured with ROI manager in ImageJ. Thereafter, the obtained raw fluorescence data were normalized with easyFRAP software. In detail, the intensity of ROI1 (I(t)C. elegans hermaphrodites. The worms were anesthetized with 0.1% tetramisole and mounted on a 3–5% agarose pad with a coverslip, which was sealed with petroleum jelly. To analyze the germ cell cytokinesis process and cytological phenotypes under gene depletion, time-lapse with Z-stack images were acquired with an inverted spinning disk confocal microscope (Yokogawa CSU-X1) under a 60×/1.4 NA objective, or using a Nikon A1R confocal microscope under a 100×/1.4 NA objective, or using a ZEISS LSM 880 microscope with an AiryScan module or with normal confocal module under a 63×/1.4 NA objective.All of the microscopy imaging experiments were performed with young adult To measure the diameter of rachis bridges, a Z-stack  from the surface to the bottom layer of the germline was acquired using a Nikon A1R confocal microscope with a 100×/1.4 NA objective. For each germ cell, the clearest middle layer of the rachis bridge in the meiotic pachytene zone was used to perform measurements. The diameter of the rachis bridges was measured with ImageJ.t test  using GraphPad Prism software.To measure furrow ingression, cleavage furrow fluorescence accumulation, and chromosome segregation kinetics, single central-plane time-lapsed images of NMY-2::GFP; HIM-4::GFP; GFP::ANI-1 and PH::GFP with mCherry::HIS or PH::mCherry were acquired at 10- or 20-s intervals upon anaphase onset, using an inverted spinning disk confocal microscope with a 60×/1.4 NA objective. The position of the furrow tip was manually pointed to in each frame showing the PH::GFP, PH::mCherry, NMY-2::GFP, or ANI-1::GFP signal. Furrow ingression was calculated based on the normalized cortical distance . Chromosome segregation was calculated based on the normalized chromosome segregation distance . Statistical significance between different treatments or samples was determined by applying


Recognition of viral RNA by the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), including RIG-I and MDA5, initiates innate antiviral responses. Although regulation of RLR-mediated signal transduction has been extensively investigated, how the recognition of viral RNA by RLRs is regulated remains enigmatic. In this study, we identified heterogeneous nuclear ribonucleoprotein M (hnRNPM) as a negative regulator of RLR-mediated signaling. Overexpression of hnRNPM markedly inhibited RNA virus-triggered innate immune responses. Conversely, hnRNPM-deficiency increased viral RNA-triggered innate immune responses and inhibited replication of RNA viruses. Viral infection caused translocation of hnRNPM from the nucleus to the cytoplasm. hnRNPM interacted with RIG-I and MDA5, and impaired the binding of the RLRs to viral RNA, leading to inhibition of innate antiviral response. Our findings suggest that hnRNPM acts as an important decoy for excessive innate antiviral immune response. Infection by virus, such as the RNA virus Sendai virus, induces the host cells to express proteins that mediate antiviral immune responses. Upon infections, the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) detects the intracellular viral RNA and initiates innate immune responses. Although the regulation of RLR-mediated signal transduction has been extensively investigated, how the recognition of viral RNA by RLRs is regulated remains enigmatic. In this study, we found that a protein called hnRNPM plays an important role in the process of antiviral immune response. hnRNPM does this by impairing the binding of the RLRs to viral RNA. Our results suggest that hnRNPM is an inhibitor of RNA virus-induced signaling which provides a critical control mechanism of viral RNA sensing for the host to avoid excessive and harmful immune response. Innate immune response provides the first line of host defense against invading microbial pathogens . Upon inViral nucleic acids are major PAMPs that are sensed by the host cells after viral infection. Extracellular viral RNA is recognized by transmembrane and endosomal Toll-like receptor 3 (TLR3), which is expressed mostly in immune cells . GeneticBoth RIG-I and MDA5 contain two tandem caspase-recruitment domains (CARDs) at their N terminus, which mediate downstream signaling; a central DExD/H helicase domain with an ATP-binding motif; and a C-terminal RNA-binding domain –14. VariRLR-mediated innate antiviral responses are regulated by distinct mechanisms. For examples, TRIM25, TRIM4, Riplet , TRIM13, USP4, USP3, USP15, USP21, CKII, PP1α/γ, and TRIM38 have been reported to regulate the post-translational modifications of RLRs –27. MEX3The heterogeneous nuclear ribonucleoprotein M (hnRNPM) contains three RNA recognition motif (RRM) domains. It has been shown that hnRNPM is involved in pre-mRNA splicing and diverse aspects of RNA metabolism, including translational control, telomere biogenesis, mRNA stability, and trafficking, 32. HerIFNB1, ISG56 and CXCL10 genes  poly(I:C) was markedly increased in hnRNPM-deficient cells  domains . Domain Since hnRNPM is a heterogeneous nuclear ribonucleoprotein that contains three RRMs , we deteFinally, we investigated whether hnRNPM regulates sensing of viral RNA by RIG-I and MDA5. The ‘‘footprint” experiments showed that overexpression of hnRNPM inhibited the binding of RIG-I to SeV RNA . ConversRecognition of viral RNA by RLRs is essential for the initiation of innate antiviral response initiation. Although the regulation of RLR-mediated downstream signaling have been extensively investigated, little is known about the regulatory mechanisms of the recognition of viral RNA by RLRs. Recently, we identified ZCCHC3 as a co-receptor for RIG-I and MDA5 by facilitating the sensing of viral RNA . In addiOverexpression of hnRNPM inhibited SeV-triggered activation of ISRE, NF-κB, and the IFN-β promoter, while deficiency of hnRNPM had the opposite effects. In addition, the replication of RNA virus was decreased in hnRNPM-deficient cells compared with control cells. These data established a critical role for hnRNPM in innate immune response to viral RNA.We found that hnRNPM underwent re-distribution between nucleus and cytoplasm during RNA virus infection. hnRNPM was mostly localized in the nucleus in rest cells. Following infection, hnRNPM was translocated from the nucleus to cytoplasm, which was impaired in RIG-I knockdown cells. Furthermore, hnRNPM was also translocated from the nucleus to cytoplasm in IFN-β-treated cells. These results suggest that hnRNPM translocation is dependent on RLRs.Several evidences suggest that hnRNPM inhibits RNA virus-triggered innate immunity by antagonizing RNA sensing of RLRs. Firstly, viral infection caused export of hnRNPM from the nucleus to cytoplasm, indicating a cytoplasmic role of hnRNPM after viral infection. Second, hnRNPM was associated with RIG-I and MDA5 and their interactions were important for the functions of hnRNPM. Third, overexpression of hnRNPM inhibited the binding of RIG-I to SeV RNA. Conversely, deficiency of hnRNPM enhanced the binding of RIG-I to SeV RNA. Recombinant hnRNPM caused approximately 10- and 3-fold decrease of the affinities of RIG-I and MDA5 to dsRNA respectively. These results collectively suggest that hnRNPM is an inhibitor for RLR-mediated innate immune response by impairing viral RNA sensing by RIG-I and MDA5.Our results revealed that the classical cellular mRNA binding regions (RRM) of hnRNPM differs from its viral RNA binding regions. The mutants containing partial RRM regions such as 1–149, 1–281 and 71–281 are lack of viral RNA binding activity while the mutant 281–653 being lack of all three RRM domains had a stronger affinity to viral RNA.Although hnRNPM binds to dsRNA, the binding is insufficient for regulating RLR-mediated signaling. Several truncations of hnRNPM, aa281-653 and aa204-729, that could bind to RNA but failed to interact with RIG-I and MDA5, had no effects on SeV-triggered activation of the IFNB promoter. hnRNPM appeared to prefer to bind to the 5’- terminus of SeV RNA, but inhibited the binding of RIG-I to diverse regions of SeV RNA. The regions of RIG-I that interact with hnRNPM overlapped with its viral RNA binding regions. These results collectively suggest the functions of hnRNPM on RLR-mediated innate immune response are mostly dependent on its association with RLRs instead of RNA binding.Interestingly, we also found that hnRNPM deficiency enhanced DNA virus -triggered expression of downstream genes. However, hnRNPM deficiency had no significant effects on the induction of downstream genes by transfected DNA mimics such as HSV120, HT-DNA and VACA70. These results suggest that hnRNPM regulates HSV-1-triggered innate immune responses in a viral DNA-independent manner. Previous studies have implicated that RIG-I also detected dsRNA produced by herpesviruses –45. We pIn conclusion, our study suggests that hnRNPM is a decoy of innate antiviral response by impairing the sensing of viral RNA by RLRs, which provides a critical control mechanism of viral RNA sensing for the host to avoid excessive immune response.3-biotin and streptavidin agarose resin (Thermo); protein G sepharose ; anti-Flag affinity gel ; RNAiso plus (Takara); and recombinant IFN-β (R&D systems) were purchased from the indicated companies.Poly(dA:dT), poly(I:C)-HWM, poly(I:C)-LWM, poly(I:C)-fluorescein, 5’ppp-dsRNA, RNase inhibitor, M-MLV and Lipofectamine 2000 (Invivogen); HT-DNA (Sigma); DTT (Fermentas); RNase I (Ambion); polybrene (Millipore); recombinant IFN-β (PeproTech); SYBR (Bio-Rad); dual-specific luciferase assay kit (Promega); puromycin and EZ-link psoralen-PEGAnti-Flag (F3165), anti-β-actin (A2228) and anti-β-tubulin (T8328) were from Sigma-Aldrich. Anti-phospho-IκBα (Ser32/36) (5A5), anti-phospho-IRF3 (Ser396) (4D4G), anti-phospho-STAT1 (Tyr701) (58D6) and anti-phospho-p65 (Ser536) were from Cell Signaling Technology. Anti-LMNB1 (12987-1-AP) was from ProteinTech. Anti-G3BP1(210-323aa) (611126) was from BD Biosciences. Anti-HA (16B12) was from Covance. Anti-TBK1 (ab109735), anti-phospho-TBK1 (Ser172) (ab109272) were from Abcam. Anti-IRF3 (FL-425), anti-p65 (C-20) (sc-372), anti-STAT1 (C-111) were from Santa Cruz Biotechnology. Goat anti-mouse IgG (R37116) or donkey anti–rabbit (R37119) conjugated to Alexa Fluor 594 and goat anti-mouse IgG (A-10684) conjugated to Alexa Fluor 488 were purchased from Thermo Fisher. Mouse antisera against hnRNPM, IκBα, RIG-I and MDA5 were raised against purified recombinant human hnRNPM (1-729aa), IκBα, RIG-I (1-200aa) and MDA5 (1-200aa). Rabbit antisera against SeV was raised against purified SeV.Human embryonic kidney 293 , Human acute monocytic leukemia cell line (THP-1) and Henrietta Lacks  cells were purchased from American Type Culture Collection, and Vero cells were purchased from China Center for Type Culture Collection . HEK293T cells were originally provided by G. Johnson . The strains (BL21 and DH5α) were purchased from ATCC.SeV, VSV (Indiana Strain), NDV and HSV-1 were previously described , 47. EMCThe following oligonucleotides were used to stimulate cells:VACV70: 5’-CCATCAGAAAGAGGTTTAATATTTTTGTGAGACCATGGAAGAGAGAAAGAGATAAAACTTTTTTACGACT-3’;HSV120: 5’-AGACGGTATATTTTTGCGTTATCACTGTCCCGGATTGGACACGGTCTTGTGGGATAGGCATGCCCAGAAGGCATATTGGGTTAACCCCTTTTTATTTGTGGCGGGTTTTTTGGAGGACTT-3’.RIG-I or MDA5 and their mutants, Flag or HA-tagged hnRNPM and their truncation mutants, pGEX6p-1-GST-hnRNPM and pGEX6p-1-GST-MDA5 (280–1025) were constructed by standard molecular biology techniques. The other expression and reporter plasmids were previously described .HEK293 cells were transfected by standard calcium phosphate precipitation method. To normalize for transfection efficiency, 0.01 μg of pRL-TK (Renilla luciferase) reporter plasmid was added to each transfection. Luciferase assays were performed using a dual-specific luciferase assay kit. Firefly luciferase activities were normalized on the basis of Renilla luciferase activities.Double-stranded oligonucleotides corresponding to the target sequences were cloned into the pSuper. Retro-RNAi plasmid (Oligoengine).The following sequences were targeted for hnRNPM mRNA.The hnRNPM-shRNA#1 targeting sequence: 5’-GGCATAGGATTTGGAATAA-3’The hnRNPM-shRNA#2 targeting sequence: 5’-GCAATCGCTTTGAGCCATA-3’.The RIG-I-shRNA targeting sequence: 5’-CCGTGATTCCACTTTCCTG-3’HEK293 or THP-1 cells were transduced with plentiCRISPRv2-hnRNPM-sgRNA viruses for five days. Puromycin-resistant individual clones were selected and analyzed by immunoblotting to determine the efficiency of hnRNPM knockout.The hnRNPM-gRNA#1 targeting sequence: 5’-GGCGACGGAGATCAAAATGG-3’.The hnRNPM-gRNA#2 targeting sequence: 5’-GGCGGCGACGGAGATCAAAA-3’.Total RNA was isolated for qPCR analysis to measure mRNA abundance of the indicated genes. Data shown are the relative abundance of the indicated mRNA derived from human cells normalized to that GAPDH respectively. Gene-specific primer sequences were as described , 47.HNRNPM:Q-PCR primers for Forward Sequence: TCCTGAACGCCCACAGCAACTTReverse Sequence: TGCCTTTGCTCAGATGGTTGGCCells were lysed in NP-40 lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1mM EDTA, 1% NonidetP-40, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mM phenyl-methylsulfonyl fluoride). The lysates were subjected to immunoprecipitation and immunoblotting analysis with the indicated antibodies.Nuclear and cytoplasmic fractions extraction of HEK293 cells were generated according to the instruction of Nuclear and Cytoplasmic Protein Extraction Kit .Poly(I:C) and 5’ppp-dsRNA was conjugated to biotin by UV (365 nm wave-length) cross-linking for 1 hour. HEK293 cells transfected with the indicated plasmids were lysed in Pre-lysis buffer pre-treated with DEPC. Lysates were incubated with biotinylated-5’ppp-dsRNA or biotinylated-poly(I:C) for 2 hours at room temperature, and then incubated with streptavidin beads for another 1 hour at room temperature. The beads were washed four times with lysis buffer and analyzed by immunoblots with the indicated antibodies.The pGEX-6p-1-GST plasmids encoding hnRNPM and MDA5(280–1025) were transformed into BL21 competent cells. Expression of the proteins was induced with 0.1 mM IPTG at 16°C for 24 hours. The proteins were purified with GST resins and eluted with elution buffer . To obtain purified RIG-I, Flag-RIG-I plasmid was transfected into HEK293 cells by standard calcium phosphate precipitation method. The expressed Flag-RIG-I protein was immunoprecipitated with anti-Flag affinity gel and eluted with 3×Flag peptides.Purified recombinant protein hnRNPM and MDA5/RIG-I were incubated in PBS with RNase I at 4°C for 3 hours. Lysates were respectively immunoprecipitated with anti-MDA5 and protein G beads (50 ul) or anti-Flag affinity gel at 4°C for another 2 hours. GST was used as negative control. Bound proteins were analyzed by immunoblots with the indicated antibodies.MST analysis was performed using a NanoTemper Monolith NT.115 instrument (NanoTemper Technologies GmbH). For detecting affinity between dsRNA and GST-hnRNPM, Flag-RIG-I or GST-MDA5, 20 nM Cy5-labeled 25 bp dsRNA  was mixed with different concentrations of proteins in PBS with 100 mM Tris-HCl (pH 8.8). GST was used as negative control. Samples were loaded into Premium Coated Capillaries and MST measurements were performed using 20% MST power and 40% LED power at 25°C. Laser-on and -off times were 30 and 5 s respectively. NanoTemper Analysis 1.2.20 software was used to fit the data and to determine the apparent Kd values.HEK293 cells were transfected with the indicated HA- or Flag-tagged plasmids for 20 hours, then infected with SeV for 1 hour, washed with medium and cultured for 2 more hours. Cell lysates were immunoprecipitated with IgG or anti-HA (2 μg) and protein G beads (50 μl) at 4°C for 3 hours. The immunoprecipitates were treated with diluted RNase I (1:25 in PBS) at 37°C for 5 min. The bead-bound immunoprecipitates were washed for 3 times with lysis buffer containing RNase inhibitors. The protein and RNA complexes were eluted with 200 μL TE buffer containing 10 mM DTT at 37°C for 30 min. The RNA was extracted using Trizol reagent before qPCR analysis for SeV RNA. The SeV genome primer sequences were described in Confocal microscopy was performed as previously described . Briefly5) cultured in 12-well plates were infected with viruses at the respective MOI for 1 hour, then washed with PBS and cultured with 1 ml fresh complete medium. The plates were incubated for 36 hours post-infection at 37°C, 10% CO2. The media were collected and used for plaque assays on monolayers of Vero cells seeded in 24-well plates. The Vero cells were infected by incubation for 1 hour at 37°C with serial dilutions of collected media. After infection for 1 hour, the cells were overlaid with 1.5% methylcellulose and then incubated for about 48 hours. The overlay was removed, and cells were fixed with 4% paraformaldehyde for 15 min and stained with 1% crystal violet for 30 min before plaque counting.Host cells  Effects of hnRNPM-deficiency on HSV-1-induced transcriptions of downstream genes. hnRNPM-KO and control THP-1 cells were left un-infected or infected with HSV-1 for the indicated times before qPCR analysis. (B) Effects of hnRNPM-deficiency on cytosolic dsDNA-induced transcriptions of downstream genes. hnRNPM-KO and control THP-1 cells were transfected with the indicated nucleic acids (2 μg/ml) for 4 h before qPCR analysis.(TIF)Click here for additional data file.S2 Fig(A) hnRNPM had no effect on the interaction of RLR with their adaptor. HEK293 cells were transfected with the indicated plasmids before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. (B&C) Domain mapping of the interactions between hnRNPM and RIG-I or MDA5.HEK293 cells were transfected with the indicated plasmids before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. The results were schematically presented in (TIF)Click here for additional data file.S3 FigSupplementary data for (TIF)Click here for additional data file.S4 FigHEK293 cells were infected with SeV for indicated times. Cell lysates were then immunoprecipitated with control IgG or anti-hnRNPM. The immunoprecipitates were treated with RNase I and bound-RNA was extracted for qPCR analysis. nt, nucleotides.(TIF)Click here for additional data file.S5 Fig(A) Supplementary data for (TIF)Click here for additional data file.S1 TableThe SeV genome primer sequences used in Q-PCR were described in the table.(DOC)Click here for additional data file.


An outbreak of hand, foot and mouth disease among children in Thailand peaked in August 2017. Enterovirus A71 subgenogroup B5 caused most  cases. Severe disease  was observed in 1 patient. Coxsackievirus A6 was detected in 6.0% (29/482) of patients, and coxsackievirus A16 was detected in 2.7% (13/482) of patients. Hand, foot and mouth disease (HFMD) and herpangina, caused primarily by enterovirus A, commonly affect children and result in painful blisters in the buccal cavity and on the soles of the hands and feet. In rare situations, enterovirus infection can lead to severe neurologic complications, notably aseptic meningitis, encephalitis, acute flaccid paralysis, and death in young children (A nationwide outbreak of HFMD caused by coxsackievirus A6 (CV-A6) affected many children in Thailand in 2012 . We tested clinical specimens from 482 children requiring hospital care for HFMD (n = 435) and herpangina (n = 47) that were submitted to King Chulalongkorn Memorial Hospital  during January 1–October 30, 2017, from 12 provinces with reported HFMD outbreaks  Figure 1We subjected specimens to 2 real-time reverse transcription PCRs (RT-PCRs). The first RT-PCR identifies enterovirus A71 (EV-A71), CV-A6, and CV-A16   Figure 1Comparison of full-length VP1 nucleotide sequences of subgenogroup B5 viruses between strains isolated in Thailand during this study and those previously isolated in Thailand showed 93.3%–100.0% identity  Table. SA severely ill 9-month-old boy in Nakhon Sawan Province was given a diagnosis of HFMD and required prolonged hospitalization because of sequelae caused by severe brain damage from encephalitis and myocarditis. Laboratory tests on rectal swab samples identified EV-A71 subgenogroup B5. An 18-month-old girl from Uttaradit Province who suddenly died during development of HFMD was positive for CV-A9.Enteroviruses associated with HFMD in Thailand during 2012–2016 are usually CV-A6 or CV-A16 (Our study had limitations. Because samples were obtained only from provinces with reported HFMD outbreaks, we might have missed HFMD-associated enterovirus in other regions. Limited availability of demographic and clinical data also prevented more in-depth analysis of disease severity and clinical associations. Additional deaths caused by EV-A71 infections might have been missed. Thus, without accurate and complete reporting of severe complications associated with EV-A71 infections, clinical implications from such infections could not be determined.Effective prevention of widespread HFMD is needed. The approved EV-A71 vaccine containing subgenogroup C4 and ongoing development of B2- and B4-based vaccines can elicit cross-neutralizing antibodies against circulating EV-A71 subgenogroups, which might prove to be effective in preventing subgenogroup B5 infection, which is predominant in Thailand (Additional information on enterovirus A71 infection, Thailand, 2017.


Scientific Reports 10.1038/s41598-019-50792-y, published online 01 October 2019Correction to: In Figure 4, panel F (Cadia case study) is erroneously a duplicate of panel D (Xinmo case study). The correct Figure 4 appears below as Figure