Drosophila testis, asymmetric division of germline stem cells (GSCs) is specified by an oriented spindle and cortically localized adenomatous coli tumor suppressor homolog 2 (Apc2). However, the molecular mechanism underlying these events remains unclear. Here we identified Megator (Mtor), a nuclear matrix protein, which regulates GSC maintenance and asymmetric division through the spindle assembly checkpoint (SAC) complex. Loss of Mtor function results in Apc2 mis-localization, incorrect centrosome orientation, defective mitotic spindle formation, and abnormal chromosome segregation that lead to the eventual GSC loss. Expression of mitotic arrest-deficient-2 (Mad2) and monopolar spindle 1 (Mps1) of the SAC complex effectively rescued the GSC loss phenotype associated with loss of Mtor function. Collectively our results define a new role of the nuclear matrix-SAC axis in regulating stem cell maintenance and asymmetric division.In adult  Drosophila male GSC often divides asymmetrically to produce one new stem cell and one gonialblast. The asymmetric division of GSC is specified by perpendicular orientation of the mitotic spindle to the hub-GSC interface and localization of Apc2. Here we show that Tpr/Mtor regulates GSC self-renewal and asymmetric division through the SAC complex. We found that Mtor cell-autonomously required in both GSCs and CySCs to regulate their self-renewal. Loss of Mtor function affects expression and localization of Apc2 and E-cadherin. We further found that Mtor is required for the correct centrosome orientation, mitotic spindle formation, and chromosome segregation. These defects are rescued by SAC complex components, Mps1 and Mad2. These data together suggest that Mtor regulates GSC asymmetric division and maintenance through the mitotic spindle checkpoint complex. We suggest that disruption of the Tpr-SAC pathway might lead to chromosome instability, chromosome lagging, and aneuploidy, stem cell division defects, and thereby tumor development.Like many stem cells, the adult  Drosophila testis provide one of the best genetic systems to study stem cell regulation. At the tip of the Drosophila testis (apex) is a germinal proliferation center, which contains the germline and somatic stem cells that maintain spermatogenesis (Germline stem cells (GSCs) from the ogenesis  1–5]. E–5. EDrosDrosophila testis, we carried out a screen in which a collection of transgenic RNAi lines  [To identify new regulators of stem cell asymmetric division in the Ai lines –22 were n yeast] –25 that n yeast] –29, and n yeast] . Tpr/Mton yeast] , 29–30 on yeast] .Mtor function affects GSC maintenance and asymmetric division. Knockdown Mtor results in Apc2 mis-localization, defects of mitotic spindle formation and chromosome segregation, and eventual GSC loss. Expression of Mad2 and Mps1 of the SAC complex effectively rescued the GSC loss phenotype associated with loss of Mtor function. Our results suggest that a nuclear matrix-SAC axis regulates GSC maintenance and asymmetric division through the Mtor-Mps1/Mad2 pathway.In this study, we found that loss of Mtor in a genetic screen for new regulators of stem cell fates in the Drosophila testis. Mtor knockdown by transgenic RNAi resulted in a significant decrease of GSCs in the testes compared to the wild-type testes. To further understand the function of Mtor in the germline or in the soma, we knocked down Mtor by using cell-type–specific Gal4s. We used three independently generated UAS-MtorRNAi transgenic fly lines. We found that depleting Mtor in the germ cell lineage using Nanos (Nos)-Gal4 resulted in a significant decrease in the number of GSCs associated with the hub compared to the wild-type control  caused defects in GSC differentiation (c587ts>MtorRNAi flies using Traffic Jam (Tj)  and Eyes absent (Eya) (a transcription factor expressed in early cyst cells) markers -Gal4 (escargot (esg)-Gal4 in combination with a temperature-sensitive Gal80 inhibitor , have n96)-Gal4  (ms1096henotype . Furtherregation . These dActts>MtorRNAi)  , suggestorRNAi) .Mtork03905 ), MtorRNAi-2 (BL32941), MtorRNAi-3 (v24265), and rlRNAi (v35641). The sequences used for VDRC knockdown strains are available for each line at https://stockcenter.vdrc.at and sequences for Bloomington knock-down strains are available for each line at http://flystocks.bio.indiana.edu.RNAi stocks used in this study: Nos-Gal4  [upd-Gal4 and c587-Gal4, provided by Ting Xie. esg-Gal4 was obtained from Shigeo Hayashi. Collagen (Cg)-Gal4 [Dilp2-Gal4 [LE-Gal4 [pumpless (ppl)-Gal4 [brachyenteron (byn)-Gal4 [serpenthemo(srphemo)-Gal4 [Aug21-Gal4 [hemolectin (hml)-Gal4 [MS1096-Gal4 [The following Gal4 alleles were used to drive UAS lines: al4VP16) , obtaineCg)-Gal4 , Dilp2-Glp2-Gal4 , LE-Gal4[LE-Gal4 , pumplespl)-Gal4 , brachyeyn)-Gal4 , serpento)-Gal4 , Aug21-Gg21-Gal4 , hemolecml)-Gal4 , and MS1096-Gal4 .Flies were raised on standard fly food at 25°C and at 65% humidity, unless otherwise indicated.Mtor-mutant GSC clones, FRT42D+ and FRT42DMtork03905/Cyo virgin females were mated with males of genotype FRT42Darm-lacZ/Cyo; MKRS, hs-flp/+, or FRTG13Mtork03905/Cyo virgin females were mated with males of genotype FRTG13arm-lacZ/Cyo; MKRS, hs-flp/+. One- or 2-day-old adult males carrying an arm-lacZ transgene in trans to the mutant-bearing chromosome were heat shocked four times at 37°C for 1 hr, at intervals of 8–12 hr. The males were transferred to fresh food every day at 25°C. The testes were removed 1, 2, or 7 days after the first heat-shock treatment and processed for antibody staining.Clones of mutant GSCs were generated as previously described . To geneFRT42D-piM (as a wild-type control) and FRT42D-Mtork03905, we generated the following flies: FRT42Dtub-Gal80/FRT42DMtork03905 (or piM); MKRS, hs-flp/ tub-Gal4,UAS-mCD8.GFP. Three- or 4-day-old adult male flies were heat-shocked twice at 37°C for 45 min, with an interval of 8–12 hr. The flies were transferred to fresh food daily after the final heat shock. The testes were removed at 1, 2, or 7 days after the first heat-shock treatment and processed for antibody staining.To induce MARCM clones of UAS-RNAi transgene flies were crossed with female virgins of genotype Nos-Gal4, upd-Gal4, c587-Gal4; tub-Gal80ts (c587ts), or esg-Gal4, UAS-GFP; tub-Gal80ts (esgts). The flies were cultured at 18°C . Three- to 5-day-old adult flies with the appropriate genotype were transferred to new vials at 29°C  for 3 or 7 days before dissection.Male Normal immunofluorescence staining was performed as described previously with some modifications . BrieflyFor the γ-tubulin staining, testes were dissected in PBS, transferred to 4% formaldehyde in PBS, and fixed for 20 minutes, followed by incubation with methanol for 10 minutes. Then washed for 10 minutes with PBST, and two 10-minutes washes with 5% goat serum PBST. Then incubated in primary antibody in 5% goat serum in PBST overnight at 4°C. Then washed three times, 15 min each in PBST, followed with 2 hrs incubation with secondary antibody in 5% goat serum in PBST. Then washed for at least an hour with PBST, and mounted as above.Caspase-3 activity was assessed using Live Green Caspase Detection Kits  according to standard protocol.Confocal images were obtained by using a Zeiss LSM510 system, and were processed with Adobe Photoshop 7.0. GSCs were scored as Vasa-positive cells adjacent to the hub (detected using Fas3) and containing dot spectrosome (detected using 1B1). Only image with a clear view of the complete hub were used.The following antisera were used: rabbit polyclonal anti-Vasa antibody , rabbit polyclonal anti-β-Gal antibody , mouse monoclonal anti-β-Gal antibody , mouse monoclonal anti-Hts antibody 1B1 , mouse monoclonal anti-Fas 3 antibody , rat polyclonal anti-Tj . P-values were obtained between two groups using the Student’s t-test or between more than two groups by analysis of variance (ANOVA).S1 Figc587ts>LacZRNAi (A), c587ts>MtorRNAi-2 (B), c587ts>EgfrDN (C), and c587ts>rlRNAi (D) flies were examined by staining with anti-vasa , Fas3 , anti-1B1 (green in dot and branched marks the spectrosomes and fusomes respectively) and DAPI (blue). Hub cells are marked by asterisks. In A, white dotted circle with white arrow marks GSCs and yellow dotted circle with yellow arrow marks GB. In B-D, white dotted circles mark expended GSCs or GSC-like cells and white arrowheads point to 1B1 dots. (E-G) GSCs in testes of c587ts>LacZRNAi  and c587ts>MtorRNAi-2 . GSCs in testes of c587ts>LacZRNAi (E) and c587ts>MtorRNAi-2 (F) flies were examined by staining with anti-vasa , Fas3 , anti-Eya  and DAPI (blue). GSCs in testes of c587ts>LacZRNAi (G) and c587ts>MtorRNAi-2 (H) flies were examined by staining with anti-vasa , Tj , and DAPI (blue). In E anf G, white dotted circle with white arrowhead marks GSCs and yellow dotted circle with yellow arrow marks GB. In F and H, white dotted circles mark expended GSCs or GSC-like cells and red arrows point to Tj-positive cells. Green arrows in E and F point to Eya-positive cells. Scale bars are 10 μm in all panels. All flies were cultured for 7 days at 29°C before dissection.(A and B) GSCs in testes of (TIF)Click here for additional data file.S2 Figupd>LacZRNAi (A) and upd>MtorRNAi-2 (B) flies were examined by staining with anti-vasa , anti-1B1 (green in dot and branched marks the spectrosomes and fusomes respectively), and DAPI (blue). (C and D) ISCs in adult posterior midguts of wild-type control (esgts>lacZRNAi) (C) or esgts>MtorRNAi-2 (D) flies were examined by staining with anti-GFP , anti-delta , anti-Pros (red nuclear staining marks enteroendocrine cells) and DAPI (blue). Scale bars are 10 μm in all panels. All flies were cultured for 7 days at 29°C before dissection. (E) UAS-MtorRNAi-2 was expressed with the nine Gal4s listed. Only the ms1096>MtorRNAi-2 flies had wing phenotypes, and the other eight Gal4>MtorRNAi-2 flies had no abnormal phenotypes. All flies were cultured for 7 days at 29°C before examination.(A and B) GSCs in testes of (TIF)Click here for additional data file.S3 FigNos>lacZRNAi) (A) or Nos>MtorRNAi-2 (B), were examined by staining with the indicated antibodies. CySCs (Zfh1 positive cells) located outside GSCs in wild type testis (A), however some CySCs positioned adjacent to the hub cells after some GSCs were depleted in Mtor-deficient testis (B). (C-D’) Testes of wild-type control (Actts>lacZRNAi) (C-C’) or Actts>MtorRNAi-2 (D-D’) flies were stained with anti-Mtor (green) and DAPI (blue). Testes of wild-type control  flies were stained with anti-Mtor (green), NUP98 (red), and DAPI (blue). The broken line circles with asterisks mark the hubs and other broken line circles mark GSCs. All flies were cultured for 7 days at 29°C before dissection. Scale bars are 10 μm in all panels.(A-B) GSCs in testes of wild-type control ((TIF)Click here for additional data file.S4 FigNos>lacZRNAi) (A), and a testis with FRT42D-Mtork03905 mosaic clones  were examined by staining with Caspase 3  and DAPI (blue).  Expression of p35 in the testis of Nos>MtorRNAi-2 (C) or in the testis with FRT42D-Mtork03905 mosaic clones  did not rescue the GSC loss phenotypes of the Mtor mutants. We assessed the rescue of GSC death in the testis of Nos>MtorRNAi-2 (C) or in the testis with FRT42D-Mtork03905 (D) by counting the number of GSC attached to the hub in C and number of GSC clones in D. The testes were examined by staining with Fas3, LacZ (green), Vasa (red), and DAPI (green). White circle lines indicate GSC clones. Asterisks mark hub cells.  Apc2 (white) localization was examined in testes of Nos>MtorRNAi-2 flies. White arrowheads point to Apc2 localization to the hub-GSC interface and red arrows point to Apc2 localization over the GSC cortex. The yellow stars indicate the hub. Scale bars are 10 μm in all panels. Testes of wild-type control ((TIF)Click here for additional data file.S5 FigNos>lacZRNAi) (A) and Nos>MtorRNAi-2  testes. The testes were stained with γ-tubulin (green) and pH3 (red) and DAPI (blue). The broken-line circles with asterisks indicate the hubs. GSCs are highlighted with yellow dotted lines. White arrowheads point to the centrosome localization. Yellow arrows in B and C point to abnormally condensed chromosomes. (D) Abnormal chromosome segregation was observed in Nos>MtorRNAi-2 GSCs. The testes were stained with the α-tubulin (green) and pH3 (red) and DAPI (blue). The broken-line circle with asterisk marks the hub. Yellow arrows point to abnormally segregated chromosomes and white arrows point to abnormal mitotic spindles. (E-H) Mitotic spindles and chromosomes were examined in testes of wild-type control (Nos>lacZRNAi) (E) and Nos>MtorRNAi-2 (F-H) flies. The testes were stained with the α-tubulin (green), γ-tubulin (red), pH3 (red) and DAPI (blue). The white broken-line circles with asterisks mark the hubs. White arrowheads in E point to the centrosome localization. Yellow arrow in F points to the lagging chromosome. Yellow arrows in G and H point to abnormally condensed and segregated chromosomes. All flies were cultured for 7 days at 29°C before dissection. Scale bars are 10 μm in all panels.(A-C) Centrosome orientation was examined in wild-type control ((TIF)Click here for additional data file.S6 FigNos>lacZRNAi) (A), Nos>MtorRNAi-3  and Nos>MtorRNAi-2  flies. The testes were stained with the pH3 (red), α-tubulin (green) and DAPI (blue). The white broken-line circles with asterisks mark the hubs. The yellow broken-line circles mark GSCs. White arrow in B points to the spindle bridge of segregated chromosomes. Yellow arrows in C and D point to abnormally condensed and segregated chromosomes. Yellow arrow in E points to the lagging chromosome.  mitotic spindles and chromosomes were examined in adult posterior midgut ISCs of esg>MtorRNAi-2 and stain with pH3 (red), α-tubulin (green) and DAPI (blue). (H-K) GSCs in testes of Nos>Mtor (H), Nos>mad2 (I), Nos>Apc2 (J), and Nos>E-cad (K) flies were examined by staining with the anti-vasa , Fas3 , anti-1B1 (green in dot and branched marks the spectrosomes and fusomes respectively) and DAPI (blue). White dotted circles with white arrow mark GSCs, yellow dotted circles with yellow arrow mark GB, red dotted circles mark CySCs, and asterisks mark hubs. GSCs in testes of (L) c587>MtorRNAi-2+UAS-Mad2 and (M) c587>MtorRNAi-2+UAS-Mps1 flies were examined by staining with the anti-vasa , Fas3 , anti-1B1 (green in dot and branched marks the spectrosomes and fusomes respectively) and DAPI (blue). Dotted lines mark GSC tumor phenotype, and asterisks mark hub cells. Scale bars are 10 μm in all panels. All flies were cultured for 7 days at 29°C before dissection.(A-D) Mitotic spindles and chromosomes were examined in testes of Control ((TIFF)Click here for additional data file.


WWTR1)-calmodulin-binding transcription activator 1 (CAMTA1) translocations, and recently, a novel Yes-associated protein 1 (YAP1)-transcription factor E3 (TFE3) gene fusion was identified in EHEs. In this study, we examined the expression levels of TFE3 and CAMTA1 via immunohistochemical staining and identified chromosomal alterations using fluorescence in situ hybridization (FISH) assays and RT-PCR tests. Although all of the EHEs were CAMTA1-positive in immunohistochemical staining, only five out of 18 EHEs (27.78%) positively expressed nuclear TFE3. The five TFE3-positive EHEs exhibited TFE3 gene break-apart in FISH assays. YAP1-TFE3 gene fusions were confirmed by RT-PCR. Interestingly, we observed CAMTA1 gene break-apart in all of the five TFE3-positive EHEs via FISH assays, and four out of the five TFE3-positive EHEs exhibited WWTR1-CAMTA1 gene fusions via RT-PCR. These results indicate that these two chromosomal alterations are not mutually exclusive but compossible in EHEs. Finally, primary tumor sites in TFE3-positive EHEs consistently contained single masses (P = 0.0359) with larger sizes (P = 0.0550) compared to TFE3-negative EHEs. Similar to previous reports, we observed well-formed vessels more frequently in TFE3-positive EHEs than in TFE3-negative EHEs (P = 0.0441). In addition, TFE3-positive EHEs tended to more frequently demonstrate high-grade nuclear atypia (P = 0.0654) and hypercellularity (P=0.0987) than TFE3-negative EHEs. Thus, we have now established two clinically distinct subgroups of EHEs: TFE3-positive and TFE3-negative EHEs.Epithelioid hemangioendotheliomas (EHEs) are vascular tumors of intermediate malignancy that can undergo high-grade malignant transformations. EHEs have been characterized by tumor-specific WW domain-containing transcription regulator 1( Epithelioid hemangioendotheliomas (EHEs) are malignant angiocentric vascular neoplasms composed of chains and cords of epithelioid endothelial cells distributed in a myxohyaline stroma . EHEs afWWTR1) gene on 3q23-24 with the calmodulin-binding transcription activator 1 (CAMTA1) gene on 1p36 [YAP1) gene and the transcription factor E3 (TFE3) gene, which defined a distinct subset of EHEs characterized by somewhat different histological morphologies, including focally well-formed vascular channels and variably solid architectures, clinically occurred in young adults [Several studies have reported an EHE-specific translocation resulting in the fusion of the WW domain-containing transcription regulator 1  were included in the study, and the age at diagnosis ranged between 23-82 years (mean ± standard deviation: 49.89 ± 15.26) . Additionally, the longest diameter of single tumor size was much larger in TFE3-positive EHEs (8.24 ± 3.14 cm) compared with TFE3-negative EHEs . Although TFE3-negative EHEs demonstrated small tumor sizes, they were more likely to present multiple lesions in the primary tumor site whereas TFE3-positive EHEs exhibited a single lesion (P = 0.0359).Using immunohistochemistry, the clinical features of five TFE3-positive EHEs were compared to those of 13 TFE3-negative EHEs Table . At diagn = 0/5) occurred, but two distant metastases  developed. Meanwhile, in the TFE3-negative group, two local recurrences , and six distant metastases  developed. No statistically significant difference was observed in the rate of local recurrence (P = 1.0) and distant metastatic (P = 1.0) between TFE3-positive and TFE3-negative EHEs (Table P = 0.3598).In the TFE3-positive group, no local recurrence . In addition, TFE3-positive EHEs tended to more frequently demonstrate high-grade nuclear atypia (P = 0.0654) and hypercellularity (P = 0.0987) than TFE3-negative EHEs, although the difference did not reach statistical significance.We compared the histologic features of EHE cases according to their TFE3 expression state but observed varying degrees of myxohyalinized stroma, tumor necrosis, mitotic activity, spindle and epithelioid cytologic features, and foamy and eosinophilic cytoplasm that seemed irrelevant to their TFE3 expression  in their EHEs study population [TFE3 rearrangement to occur in merely two cases  [n = 4/18) of EHEs and 80.0% (n = 4/5) of TFE3-positive EHEs based on FISH and RT-PCR results. EHE remains an extremely rare disease entity of which TFE3 expression has been scarcely examined. Although we add our current observations to the limited literature covering this subject, we acknowledge that subsequent studies which cover more cases are still required to further clarify such discrepancies, especially on TFE3 expression profile and YAP1-TFE3 gene fusion status that exist between our data and the previous report [A previous study examined CAMTA1 expression profiles in EHEs and identified strong nuclear expression in most cases . Consistpulation . Our datDrosophila discs large tumor suppressor (Dlg1), and zonula occludens-1 protein (zo-1) [WWTR1 and YAP1 share conserved amino acid sequences for the WW domain that can interact with PDZ domains. They are named for the shared homology observed in postsynaptic density protein 95 (PSD95), n (zo-1) . This stn (zo-1) , 7, 11, WWTR1-CAMTA1 and YAP1-TFE3 gene translocations, which were not identified in EHEs having only the WWTR1-CAMTA1 translocation, might reflect a higher degree of genetic heterogeneity in these tumors. Traditionally, sarcomas have been classified into two broad categories: tumors with simple genetic alterations and tumors with complex and unbalanced karyotypes [In this study, we observed that TFE3-positive EHEs had a tendency to present as a single larger mass in younger patients compared with TFE3-negative EHEs and demonstrated histologic findings of the presence of high-grade nuclear atypia and hypercellular areas. We believe that these clinicopathologic findings in EHEs with both ryotypes . Tumors ryotypes , 14. AltAccording to a previous study, tumor size was associated with decreased survival of EHE patients and tumors with >3 mitotic figures/50 high power fields and size >3.0 cm had the worst prognosis . InteresThis retrospective study was approved by the Institutional Review Board of Yonsei University Medical Center . Our inclusion criteria defined a study population of 18 EHE patients who had been histologically diagnosed between 1993-2013. Archival tissues stained with hematoxylin and eosin (H&E) were reviewed by three pathologists .® XT, Ventana Medical System, AZ, USA).Tumors were fixed in formalin and embedded in paraffin. Briefly, 5 μm-thick sections were cut using a microtome, transferred onto adhesive slides, and dried at 62°C for 30 min. Immunohistochemistry with antibodies against CD31 , CD34 , TFE3 , and CAMTA1  was performed using an automated immunohistochemical staining instrument  were used according to the manufacturer's instructions for the detection of translocations involving the TFE3 gene at Xp11.23 and the CAMTA1 gene at 1p36.23, respectively. Break-apart on the slides was evaluated using an epifluorescence microscope . During interphase in normal cells without translocations, green/orange fusion signals appear. Xp11.23 or 1p36.23 loci affected by translocations are indicated by separate and distinct green and orange signals. A case was interpreted as positive when at least 10 out of 50 counted tumor cells (20%) showed separated green and orange signals [FISH was performed on tumor sections after examination by H&E microscopy. A ZytoLight SPEC TFE3 Dual Color Break-Apart Probe  and a MacProbe signals .WWTR1-CAMTA1 fusion transcript or the YAP1-TFE3 fusion transcript: WWTR1 exon 4 forward, 5′-CCGTCAGTTCCACACCAGTGCCTC-3′; CAMTA1 exon 9 reverse, 5′-GGGGCTACAGCAGGGGAGGC-3′; YAP1 exon 1 forward, 5′-CCTGGAGGCGCTCTTGAACG-3′; TFE3 exon 6 reverse, 5′-GTTGCTGACAGTGATGGCTGG3′. The RT-PCR products were analyzed by electrophoresis.Isolation of RNA from formalin-fixed, paraffin-embedded (FFPE) samples was performed following the manufacturer's protocol . RT-PCR was performed as previously published , 6, 7 usAll statistical analyses were performed using GraphPad Prism 5 software, version 5.01  and SPSS for Windows, version 20.0 . For the analysis of age at diagnosis and tumor size, a significant difference between means was determined by t-tests. Sex, tumor multiplicity, and metastasis were compared by Chi-square analyses and Fisher's exact tests.


C. elegans undergoes periods of behavioral quiescence during larval molts (termed lethargus) and as adults. Little is known about the circuit mechanisms that establish these quiescent states. Lethargus and adult locomotion quiescence is dramatically reduced in mutants lacking the neuropeptide receptor NPR-1. Here, we show that the aroused locomotion of npr-1 mutants results from the exaggerated activity in multiple classes of sensory neurons, including nociceptive (ASH), touch sensitive (ALM and PLM), and stretch sensing (DVA) neurons. These sensory neurons accelerate locomotion via both neuropeptide and glutamate release. The relative contribution of these sensory neurons to arousal differs between larval molts and adults. Our results suggest that a broad network of sensory neurons dictates transitions between aroused and quiescent behavioral states. C. elegans exhibits a sleep-like state (termed lethargus) that is characterized by the absence of feeding and profound locomotion quiescence. We previously showed that mutants lacking the neuropeptide receptor NPR-1 exhibit increased arousal during larval molts, which is in part mediated by increased secretion of an arousal peptide (PDF-1). Here, we compare the circuits regulating arousal in larval molts and adults. We show that a broad network of sensory neurons arouses locomotion but that the impact of each neuron differs between lethargus and adults. We propose that this broad sensory network allows C. elegans to adapt its behavior across a broad range of developmental and physiological circumstances.Animals switch between periods of behavioral arousal and quiescence in response to environmental, developmental, and circadian cues. Little is known about the circuit mechanisms that produce these behavioral states. During larval molts,  C. elegans locomotion.Animals undergo periods of behavioral quiescence and arousal in response to changes in their environment and metabolic state. Arousal is defined as a state of heightened responsiveness to external stimuli coupled with increased motor activity whereas quiescence is associated with diminished responsiveness and motor activity . QuiesceC.elegans undergoes a prolonged period of profound behavioral quiescence, termed lethargus behavior, whereby locomotion and feeding behaviors are inactive for approximately 2 hours , in, innpr-1naltered . Faster  mutants . This conpr-1 mutants are caused by enhanced sensitivity to environmental cues. In particular, sensory responses mediated by the RMG circuit are enhanced in npr-1 mutants  (Gift from Cori Bargmann)CX4978 nmr-1(ak4) II; npr-1(ky13) XKP6414 glr-1(n2461) III;npr-1(ky13) XKP6415 dpy-19(n1347) glr-2(ak10) IIIVM1123 dpy-19(n1347) glr-2(ak10) III; npr-1(ky13) XKP6740 KP7362 npr-1(ky13) X; nuIs439[nlp-12p::GFP]; nuIs519nuIs472 KP6693 npr-1(ky13) X; nuIs472KP6743 dpy-19(n1347) glr-2(ak10) III; nuIs472KP7194 dpy-19(n1347) glr-2(ak10) III; npr-1(ky13) X; nuIs472KP7195 bzIs17[mec-4p::YC2.12]AQ906 npr-1(ky13) X; bzIS17KP6681 npr-1(ad609) X;kyEx1966 (Gift from Cori Bargmann)CX9396 npr-1(ad609) X;nuEx1519KP6051 npr-1(ad609) X;nuEx1520KP6053 eat-4(ky5) III; npr-1(ky13) X; nuEx1613-1614KP7149, KP7150 eat-4(ky5) III; npr-1(ky13) X; nuEx1615-1616KP7176, KP7177 KP7198, KP7199 eat-4(ky5) III; npr-1(ky13) X; nuEx1640-1641KP7442 npr-1(ky13) X; nuEx1684KP7633 nuEx1613KP7634 nuEx1640ljEx239[sra-6::YC.360]AQ3304 KP7353 npr-1(ky13) X; ljEx239KP7443 npr-1(ky13) X; ljEx239; nuEX1607KP7495 npr-1(ky13) X; ljEx239; nuEX1683KP7191 dpy-19(n1347) glr-2(ak10) III; npr-1(ky13) X; nuEx1637KP7192 dpy-19(n1347) glr-2(ak10) III; npr-1(ky13) X; nuEx1638KP7354, KP7355, KP7356 dpy-19(n1347) glr-2(ak10) III; npr-1(ky13) X; nuEx1642-1644KP7635 nuEx1637KP7636 nuEx1638eat-4 cDNA was amplified by PCR and ligated into expression vectors (pPD49.26) containing the sra-6 (~3.8kb 5’ regulatory sequence: ASH expression), sra-9 (~3kb 5’ regulatory sequence: ASK expression), or mec-4 (~1.1kb 5’ regulatory sequence: Touch neuron expression) promoters.glr-2 genomic DNA was amplified by PCR and ligated into expression vectors (pPD49.26) containing the nlp-12 (~400 bp 5’ regulatory sequence: DVA expression), gcy-28(d) , or glr-1  expression) promoters.ced-3 genomic DNA and GFP were amplified by overlapping PCR and ligated into expression vectors (pPD49.26) (using NheI and SacI restriction sites) containing the sra-6 (~3.8 kb 5’ regulatory sequence: ASH expression) or nlp-12 (~400 bp 5’ regulatory sequence: DVA expression) promoters.myo-2p::NLS-mCherry (KP#1480) and vha-6p::mcherry (KP#1874)). Injection concentration was 40–50 ng/μl for all the expression constructs and 10 ng/μl for coinjection markers. The empty vector pBluescript was used to bring the final DNA concentration to 100 ng/μl. The flp-21 promoter  mutant worms by transgenes co-expressing CED-3 and a fluorescent protein (GFP or mCherry) under the sra-6 (ASH ablation) or nlp-12 (DVA ablation) promoters. ASH or DVA ablations were confirmed after locomotion analysis by fluorescence microscopy.Neurons were ablated in Sensitivity to aldicarb was determined by analyzing the time course of paralysis following treatment with 1 mM aldicarb (Sigma-Aldrich) as previously described . Briefly3, 1.25 mM NaH2PO4, 20 mM glucose, 1 mM CaCl2, and 4 mM MgCl2, bubbled with 5% CO2, 95% O2 at 20°C. Whole cell recordings were carried out at –60 mV using an internal solution containing 105 mM CsCH3SO3, 10 mM CsCl, 15 mM CsF, 4mM MgCl2, 5mM EGTA, 0.25mM CaCl2, 10mM HEPES, and 4 mM Na2ATP, adjusted to pH 7.2 using CsOH. Under these conditions, we only observed endogenous acetylcholine EPSCs. To record GABAergic postsynaptic currents, the holding potential was 0 mV, at which we only observe mIPSCs. All recording conditions were as described ). To image copper and glycerol responses, single animals were placed in a perfusion chamber  under a constant flow rate (0.4 ml min-1) of buffer using a perfusion pencil (AutoMate). Outflow was regulated using a peristaltic pump . 10mM CuCl2 (copper(II)chloride dihydrate, Sigma) or 500mM glycerol (Fisher) were delivered using the perfusion, pencil and switch between control and stimulus solutions was done using manually controlled valves. Solutions contained either 10mM CuCl2 in M13 buffer or 500mM glycerol in 40mM NaCl, 1 mM MgSO4, 1 mM CaCl2 and 5 mM KPO4. The stimulus was delivered for 10 seconds starting on the 10th second from the beginning of the movie. Optical recordings were performed on a Zeiss Axioskop 2 upright compound microscope equipped with a Dual View beam splitter and a Uniblitz Shutter. Images were recorded at 10 Hz using an iXon EM camera (Andor Technology) and captured using IQ1.9 software (Andor Technology). For ratiometric imaging, ROIY tracked the neuron in the yellow channel, and in the cyan channel, ROIC moved at a fixed offset from ROIY. F was computed as FY/FC following a correction for bleed through. No correction for bleaching was required. Ratio changes were detected and parametrized using scripts for MATLAB (The Mathworks). Briefly, the scripts average the F value for 5 preceding and including the marked start stimulus frame (F0) and the 5 frames centered on the marked peak frame (F1). ΔF was equal to (F1—F0) / F0 x 100. Touch-evoked calcium responses in PLM neurons were analyzed as previously described [Using Dermabond topical skin adhesive, individual worms were glued to 2% agarose pads in extracellular saline  and mIPSCs (C-E) were recorded from body wall muscles of adult worms for the indicated genotypes. Averaged traces of stimulus-evoked EPSCs (A), representative traces of mIPSCs (C), and summary data are shown . The number of animals analyzed is indicated for each genotype. Error bars indicate SEM .(TIF)Click here for additional data file.S2 Fignpr-1 aldicarb hypersensitivity was rescued by transgenes expressing NPR-1 in the RMG circuit  but not by those expressed in GABAergic neurons . (B-C) The npr-1 aldicarb hypersensitivity was blocked by mutations inactivating TAX-4/CNG channels or OCR-2/TRPV channels. (D-E) npr-1 aldicarb hypersensitivity was not abolished by mutations inactivating PDF-1 or PDFR-1, (F) but was suppressed by mutations inactivating EAT-4/VGLUT. (G) Capsaicin treatment (2–3 hours) increased aldicarb sensitivity in transgenic animals expressing TRPV1 in ASH neurons, but not in wild type controls.Time courses (120 min) of paralysis of worms on 1 mM aldicarb were plotted for the indicated genotypes. The number of trials is indicated in the parentheses for each genotype. (A) The (TIF)Click here for additional data file.S3 Figeat-4;npr-1  was analyzed in the indicated genotypes. Average motile fraction (A), and average locomotion velocity (B) are plotted. Transgenes that re-instated lethargus quiescence defects in (TIF)Click here for additional data file.S4 Fignpr-1 mutants. (C-D) This defect during L4/A lethargus was rescued by transgenes expressing NPR-1 in the RMG circuit  or in ASH neurons . Values that differ significantly are indicated .Glycerol-evoked calcium transients in ASH were analyzed in L4, L4/A, and adults of the indicated genotypes using cameleon as a calcium indicator. Averaged responses , and the amplitudes of individual trials  are shown for each genotype. Each trace represents the average percentage change in YFP/CFP fluorescence ratio. The light tan rectangle indicates the duration for which 500 mM glycerol was applied. Dark gray shading of each trace indicates SEM of the mean response. (A-B) Glycerol-evoked calcium transients in ASH neurons were significantly reduced during L4/A lethargus, and this effect was abolished in (TIF)Click here for additional data file.S5 Fignpr-1 adults was not suppressed by mutations inactivating glr-1 or nmr-1 glutamate receptors. The number of animals analyzed is indicated for each genotype. Error bars indicate SEM. Values that differ significantly are indicated .Locomotion behavior of single adult worms was analyzed in the indicated genotypes. Average locomotion velocity (A) is plotted. (A) The locomotion defect in (TIF)Click here for additional data file.S6 Fignpr-1 mutants.Touch-evoked calcium transients in PLM were analyzed using cameleon as a calcium indicator. Responses were analyzed in adult animals. Averaged responses (A) and the amplitudes of individual trials (B) are shown for each genotype. Each red trace represents the average percentage change in YFP/CFP fluorescence ratio. The black triangle indicates the time at which the mechanical stimulus was applied. Gray shading indicates the response SEM. Touch-evoked calcium transients in adult PLM neurons were significantly larger in (TIF)Click here for additional data file.


Primary monocytes are refractory to HIV-1 infection and become permissive upon differentiation into monocyte-derived dendritic cells (MDDCs) or macrophages. Multiple mechanisms have been proposed to interpret HIV-1 restriction in monocytes. Human cellular miRNAs can modulate HIV-1 infection by targeting either conserved regions of the HIV-1 genome or host gene transcripts. We have recently reported that the translation of host protein pur-alpha is repressed by abundant cellular miRNAs to inhibit HIV-1 infection in monocytes. Here, we report that the transcript of another cellular factor, VprBP [Vpr (HIV-1)-binding protein], was repressed by cellular miRNA-1236, which contributes to HIV-1 restriction in monocytes. Transfection of miR-1236 inhibitors enhanced translation of VprBP in monocytes and significantly promoted viral infection; exogenous input of synthesized miR-1236 mimics into MDDCs suppressed translation of VprBP, and, accordingly, significantly impaired viral infection. Our data emphasize the role of miRNA in modulating differentiation-dependent susceptibility of the host cell to HIV-1 infection. Understanding the modulation of HIV-1 infection by cellular miRNAs may provide key small RNAs or the identification of new important protein targets regulated by miRNAs for the development of antiviral strategies. The highly expressed cellular miRNAs miR-125b, miR-150, miR-28, miR-223 and miR-382 repress HIV-1 replication by targeting nef 3′-long-terminal repeat (LTR) region and contribute to viral latency in resting CD4+ T lymphocytes nef transcription and reduces viral production and infectivity, enhances HIV-1 mRNA association with RNA-induced silencing complexes, and sequesters viral mRNA in P bodies for further degradation in silico and predicted to bind with the HIV-1 nef 3′-LTR region + T cells and potentiates HIV-1 replication by targeting host factor MeCP2 (Methyl-CpG binding protein 2) Meanwhile, host cellular miRNAs can modulate HIV-1 infection by targeting either the conserved regions of HIV-1 genome or host gene transcripts, and these miRNAs may play pivotal roles in maintaining viral latency and promoting host defense Primary monocytes are refractory to HIV-1 infection and become permissive upon differentiation into macrophages or dendritic cells (DCs) To uncover the restriction of HIV-1 replication by miRNAs in undifferentiated monocytes, we analyzed the miRNA expression profile in monocytes by miRNA-chip array and compared it with that of their differentiated monocyte-derived DC (MDDC) counterparts. We have recently reported that the translation of host protein pur-alpha was repressed by cellular miRNAs to inhibit HIV-1 infection in monocytes +/vesicular stomatitis virus (VSV)-G in a dose-dependent manner  to form an E3 ubiquitin ligase complex, which targets cellular substrates for proteasome-mediated degradation and G2 cell-cycle arrest in MDDCs . The resWe demonstrated above that VprBP was not expressed in monocytes, as detected at the protein level . To elucConsidering the regulatory role of miRNA on translation, we compared the miRNA expression in monocytes and MDDCs by Agilent miRNA-chip array. Target prediction of miRNAs and alignment of miRNAs with target sequences were achieved using TargetScan. We found the 17–24 bases of VprBP 3′-UTR were targeted by hsa-miR-1236. Expression of miR-1236 was verified with Bulge-loop miRNA qRT-PCR Primer Sets. The expression was adjusted based on U6 small nuclear RNA transcription. miR-1236 expression was significantly enhanced in monocytes compared with MDDCs .Renilla luciferases was calculated. Transfection with miRNA mimics significantly inhibited expression of pGL3cM/VprBP 3′-UTR, and the mutation of conserved binding positions abolished the inhibition mediated by miRNA mimics , is a component that forms a CUL4–DDB1–VprBP/DCAF1 E3 ubiquitin protein ligase complex. As the Vpr binding protein, VprBP is tightly conjugated with Vpr for functioning VprBP functions as the substrate recognition module within CUL4–DDB1–VprBP/DCAF1 E3 ubiquitin protein ligase complex and bridges target proteins to DDB1. HIV-1 Vpr or HIV-2/SIVsmm/mac Vpx can interact with VprBP and assembles with DDB1 to form an E3 ubiquitin ligase complex, which targets cellular substrates for proteasome-mediated degradation and G2 cell-cycle arrest In our miRNA-chip analysis, we also found that other cellular miRNAs, such as miR-15a, miR-15b, miR-16, miR-93, miR-106b and miR-20a, can inhibit HIV-1 infection in monocytes, and Pur-alpha, a host-cell-encoded transcription factor required for enhancing Tat-derived viral transactivation, was targeted for repression in monocytes In summary, our results demonstrate that host factor VprBP was targeted by cellular miR-1236 to modulate monocyte/MDDC differentiation-dependent susceptibility to HIV-1 infection. miRNA-based therapies are being developed, and understanding the modulation of HIV-1 infection by cellular miRNAs may provide key small RNAs or the identification of new important protein targets regulated by miRNAs for antiviral therapeutic interventions.+ monocytes were isolated from PBMCs by using CD14 antibody-coated magnetic beads  as previously described Human peripheral blood mononuclear cells (PBMCs) from healthy donor were purchased from Blood Center of Shanghai . CD14-R+(Vpr+) and an expression plasmid of vesicular stomatitis virus G (VSV-G) protein. Replication competent HIV-1-AD8 (R5 tropic) virus or Vpr deletion AD8 virus were obtained by transfecting HIV-1 vectors of pNLAD8 or pNLAD8-ΔVpr, respectively. Plasmids were kindly provided by Dr. Li Wu . Harvested supernatants that contained viral particles were filtered and quantified with p24gag capture ELISA. Cells were infected with HIV-1-Luc/VSVG (1 ng p24gag) or replication competent HIV-1 (1 ng p24gag) for 2 h, and after washing, cells were further cultured for 2 days (cell line) or 5 days (MDDCs and monocytes). Viral infection was detected by measuring the luciferase activity from the cell lysates or detecting the p24gag level from the supernatant. The HIV-1 p24gag specific monoclonal antibodies are kindly gift of Prof. Yong-Tang Zheng, from Kunming Institute of Zoology, Chinese Academy of Sciences.Single-cycle HIV-Luc/VSV-G was produced by calcium-phosphate-mediated co-transfection of HEK293T cells with the luciferase reporter HIV-1 proviral vector NL-Luc-ECells were transfected by Lipofectamine 2000 (Invitrogen) with plasmids of pCMV-myc/pCMV-myc-VprBP, or with specific duplex siRNA of VprBP or off-target control , or with miR-1236 inhibitor or mimics (GenePharma). Then cells were either collected to detect the condition of expression by Western blotting, or for viral infection as described above. The sequences of siRNA duplex, miRNA inhibitors and mimics were as follows: VprBP siRNA 1# 5′-GGCCCAGAUAACCGAAUAUTT-3′, VprBP siRNA 2# 5′-GCGACUCAUUC UCCAAUAUTT-3′. Plasmid pCMV-myc-VprBP encoding full-length myc-tagged VprBP  was described previouslyhttp://www.targetscan.org). The expression of miRNAs was verified with Bulge-loop miRNA qRT-PCR Primer Sets . The expression was adjusted based on U6 small nuclear RNA transcription, and relative miRNA expression of indicated controls was calculated. PCR was performed on the ABI 7900HT Real-Time PCR system .The mRNA and miRNA expression profiles during monocyte to DC differentiation were screened with Affymetrix U133 plus 2.0 array and Agilent miRNA expression array as described previously 5′-CGGAGCCATCACTGCTTGGAAGAGATTCTTGGCAGAGAGAAGAGGGGACAA-3′ (forward), 5′-GATCTTGTCCCCTC TTCTCTCTGCCAAGAATCTCTTCCAAGCAGTGATGGCTCCGAGCT-3′ (reverse). And point mutations of conserved binding sites of VprBP 3′-UTR for miR-1236 were synthesized as follow: 5′-CGGAGCCATCACTGCTTAACGA CAGTTCTTGGCAGAGAGAAGAGGGGACAA-3′ (forward), 5′-GATCTTGTCC CCTCTTCTCTCTGCCAAGAACTGTCGTTAAGCGTGATGGCTCCGAGCT-3′ (reverse). MiRNA binding reporter pGL3CM-VprBP-3′UTR or mutation was constructed with Bgl II and Sac I. Plasmid pGL3CM was kindly gifted by Dr. Ke Lan .VprBP 3′ UTR was amplified from the total RNA extracted from the HEK293 cell line. Binding sites of VprBP 3′ UTR for miR-1236 were synthesized as follows: A Dual-Luciferase Reporter Assay System  was used to examine the effects of miRNAs on their target genes as described previouslyStatistical analysis was performed using a paired t test with SigmaStat 2.0 .


Semen is a major vehicle for HIV transmission. Prostatic acid phosphatase (PAP) fragments, such as PAP248-286, in human semen can form amyloid fibrils to enhance HIV infection. Other endogenous or exogenous factors present during sexual intercourse have also been reported to promote the formation of seminal amyloid fibrils.Here, we demonstrated that a synthetic 15-residue peptide derived from the HIV-1 gp120 coreceptor-binding region, designated enhancing peptide 2 (EP2), can rapidly self-assemble into nanofibers. These EP2-derivated nanofibers promptly accelerated the formation of semen amyloid fibrils by PAP248-286, as shown by Thioflavin T (ThT) and Congo red assays. The amyloid fibrils presented similar morphology, assessed via transmission electron microscopy (TEM), in the presence or absence of EP2. Circular dichroism (CD) spectroscopy revealed that EP2 accelerates PAP248-286 amyloid fibril formation by promoting the structural transition of PAP248-286 from a random coil into a cross-β-sheet. Newly formed semen amyloid fibrils effectively enhanced HIV-1 infection in TZM-bl cells and U87 cells by promoting the binding of HIV-1 virions to target cells.Nanofibers composed of EP2 promote the formation of PAP248-286 amyloid fibrils and enhance HIV-1 infection. In 2013, an estimated 35 million people were living with human immunodeficiency virus (HIV) worldwide, and approximately 2.1 million people were newly infected with HIV .Semen acts as a vector for HIV transmission through sexual intercourse and plays an important role in the spread of HIV/AIDS . Semen hMN envelope gp120 glycoprotein blocked T-20-mediated anti-HIV activity [MN gp120 coreceptor-binding region , was found to enhance HIV-1 infection. Gp120 is considered an Ig superantigen (Ig-SAg) [We previously demonstrated that three peptides, termed enhancing peptides (EPs), derived from the HIV-1activity . Coincidactivity –20. Moreactivity . However(Ig-SAg) . Gp120 r(Ig-SAg) . Conserv(Ig-SAg) . Therefo-R-Luc plasmid, HIV-1 Env-encoding plasmids and the peGFP-Vpr plasmid were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. GHOST (3) Hi-5 and HEK-293 T cells were purchased from the ATCC . Plasmids encoding CXCR4-tropic HIV-1 NL4-3, CCR5-tropic HIV-1 SF-162, and dual-tropic 81A and NL4-3 infectious clones were kindly provided by Jan Münch of Ulm University . Polyethyleneimine (PEI), hen egg-white lysozyme (HEWL), bovine insulin, thioflavin T and Congo Red Kits were purchased from Sigma-Aldrich . ProteoStat Amyloid Plaque Detection Kits were purchased from Enzo Life Sciences .The peptide PAP248-286 was synthesized and purified by GL Biochem  into a lyophilized powder. The lyophilized PAP248-286 peptide (>95% purity) was dissolved in phosphate-buffered saline (PBS) to a concentration of 10 mg/ml. EP2 was synthesized and purified by Huada Biotech Company . Lyophilized EP2 (>90% purity) was dissolved in dimethylsulfoxide (DMSO) or deionized water to a concentration of 10 mg/ml. Stock solutions of the peptides were divided into aliquots and stored at -20°C. MT-2 cells, TZM-bl cells, U87-CD4-CCR5 cells, the pNL4-3EThe ability of EP2 to self-assemble into nanofibers was evaluated by two methods based on ThT fluorescence assays. First, a concentration-dependent experiment was performed. For this assay, 5 μl samples containing graded concentrations of EP2  were stained using 195 μl ThT (50 μM in PBS) [Fibrils were generated by incubating 3 mg/ml PAP248-286 in the presence or absence of EP2 (100 μg/ml) with agitation at 1,400 rpm at 37°C in an Eppendorf Thermomixer . Fibril morphology was visualized via TEM at different time points following agitation. To accomplish this, PAP248-286 fibers (300 μg/ml) incubated with or without EP2 (10 μg/ml) were collected at different time points following agitation . All samples were tenfold diluted in PBS buffer. The samples were then deposited on glow-discharged, carbon-coated grids for 2 min and negatively stained with 2% phosphotungstic acid for another 2 min. Amyloid fibril morphology was visualized as described above.To select the optimum concentration of EP2 to foster the aggregation of PAP248-286, graded concentrations of EP2  were agitated with PAP248-286 (3 mg/ml) at 1,400 rpm at 37°C. After screening, 3 mg/ml PAP248-286 in the presence or absence of EP2 (100 μg/ml) was agitated at 37°C for 48 h at 1,400 rpm. Individual solutions of PAP248-286 and EP2 alone were agitated under the same conditions to serve as negative controls. Aggregation was examined at different time points  by ThT and Congo red staining as previously described , 25. ForPAP248-286 (3 mg/ml) was agitated in the presence or absence of EP2 (100 μg/ml) at 1400 rpm at 37°C as described above. The secondary β-sheet structures of PAP248-286, EP2 and the mixture of PAP248-286 and EP2 were examined at different time points after agitation  using CD spectroscopy in the far-UV spectral region between 180 and 260 nm. The formed fibers were diluted 15 times in PBS buffer prior to CD analysis. Therefore, the final concentrations of the PAP248-286 and EP2 fibers were 200 μg/ml and 6.67 μg/ml, respectively. The CD spectra were reported at room temperature using a Jasco 715 spectropolarimeter  equipped with a thermostat-controlled cell housing and cells with a 1-mm path length . Each spEGFP-labeled HIV-1 virions were produced by PEI-mediated cotransfection of 293T cells with proviral DNA expression plasmids and peGFP-Vpr plasmids as previously described [4/ml GHOST (3) Hi-5 cells were incubated with the pretreated HIV-1 virions at 37°C for another 30 min. The cells were then fixed in paraformaldehyde and stained using a ProteoStat Amyloid Plaque Detection Kit and Hoechst 33342 stain (Enzo Life Sciences) for 30 min at room temperature. Finally, the cells were washed three times with PBS buffer and imaged with a laser scanning confocal microscope .The binding of eGFP-labeled HIV-1 virions to PAP248-286 amyloid fibrils was examined by fluorescence microscopy. Briefly, 200 μg/ml PAP248-286, 6.67 μg/ml EP2 and mixtures of PAP248-286 and EP2 collected at different time points following agitation  were stained with Proteostat dye from a ProteoStat Amyloid Plaque Detection Kit at room temperature as previously described . The sam-R-Luc and the HIV-1 Env-encoding plasmid JR-FL via PEI transfection as previously described [4 cells/well and incubated at 37°C overnight. Agitated peptide solutions  were incubated with HIV-1 Env-pseudotyped virus for 5 min. Following this, U87-CD4-CCR5 cells were incubated with the peptide-virus mixtures at 37°C for 3 h. To minimize the toxic effects caused by amyloid fibrils, the culture supernatants were replaced with fresh medium after 3 h. The cells were collected 72 h post-infection, and luciferase activity was detected using a luciferase assay kit . Furthermore, CXCR4-tropic NL4-3, CCR5-tropic SF-162, and dual-tropic 81A and NL4-3 infectious HIV-1 clones were produced by transfection using proviral DNA expression plasmids and PEI transfection reagent as previously described [4 cells/well were incubated for 3 h with different infectious HIV-1 clones in the absence or presence of agitated peptide solutions at different time points . The culture supernatants were then replaced with fresh medium. At 72 h post-infection, the cells were collected, washed and lysed with a lysing reagent, and luciferase activity was detected using a luciferase assay kit . Data were analyzed using SPSS version 19.0 via one-way ANOVA and Dunnett’s post hoc multiple comparisons test .HIV-1 Env-pseudotyped viruses were produced by cotransfecting cells with pNL4-3Eescribed –33. To described , 11. To in vitro, we diluted a DMSO-containing stock solution of EP2 peptides into PBS. ThT, which is the most commonly used dye for detecting amyloid fibril formation, was used to assess the formation of EP2 nanofibers. The results showed that EP2 could form fibrils almost immediately following dilution into PBS buffer in a concentration-dependent manner at room temperature  could not sponentanously form amyloid fibrils at room temperature; this condition was tested for over 72 hours. Thus, unlike EP2, PAP248-286 cannot form fibers under physiological conditions. However, after agitating PAP248-286 at a concentration of 3 mg/ml in PBS at 37°C for 12 h at 1,400 rpm, small fibers were observed under TEM. At 24 h after agitation, branching, needle-like, long amyloid fibrils were revealed, suggesting that PAP248-286 slowly forms amyloid fibrils under agitation .To determine how EP2 (100 μg/ml) affects PAP248-286 amyloid fibril formation, the presence of amyloid fibrils at different time points after agitation  in the presence of EP2 was evaulated by TEM. As shown in We next measured the kinetics of amyloid formation and PAP248-286 aggregation in the presence or absence of EP2. As shown in Beta-sheet aggregation is a distinguishing feature of amyloid fibril formation. To determine the effects of EP2 on PAP248-286 β-sheet aggregation, we evaluated the conformational changes associated with PAP248-286 aggregation in the presence or absence of EP2 by characterizing the CD spectra associated with each condition. As shown in Fluoresence confocal microscopy revealed that HIV-1 virions can bind to PAP248-286 amyloid fibrils . AmyloidBased on the above findings, we utilized an infection assay to investigate whether PAP248-286 amyloid fibrils formed in the presence of EP2 could enhance HIV-1 infection. In the absence of EP2, only PAP248-286 amyloid fibrils collected after 36 to 48 h of shaking at 37°C could increase the infectivity of R5-tropic HIV-1 Env-pseudotyped viruses on U87-CD4-CCR5 cells. No enhancement was observed if the amyloid fibrils were collected prior to 24 h of agitation . ConversSeminal plasma proteins, such as PAP fragments , or bacterial curli proteins may promote the formation of seminal amyloid fibrils, which in turn dramatically enhance HIV-1 infection. These amyloid fibrils in semen are considered an endogenous enhancer of viral infection and are important for sexual transmission of HIV-1 , 30, 34.Numerous envelope glycoprotein complexes disassemble when HIV-1 particles reach target cell surfaces; this disassembly results in the release of gp120 . LysosomMN gp120 coreceptor-binding region (QCKIKQIINMWQEVG), contains the short peptide fragment INMWQG, which is a degradation product of gp120. Furthermore, the sequence of EP2 also overlaps with residues 421–433 (KQIINMWQEVGK), which can be hydrolyzed by Ig. In the present study, we investigated the effects of EP2 on seminal amyloid fibril-mediated enhancement of HIV-1 infection.Many different types of soluble proteins and peptides can form amyloid fibers. These fibers are insoluble, highly ordered, extremely strong and resistant to degradation . In geneAmyloid fibrils are polypeptide-based polymers or highly organized protein filaments that vary in size (commonly at the low micrometer level) and in formation kinetics. Recently, it has been discovered that a subset of short peptides can undergo spontaneous assembly into ordered nanofibrils , 43. NanTheoretically, PAP fragments, such as PAP248-286, do not enhance HIV-1 infection when in monomeric form. Therefore, to act as SEVI amyloid fibrils, PAP fragments must form amyloid fibrils. This formation is controlled by the rate of amyloidogenesis. However, the creation of SEVI amyloid fibrils is challenging due to the stochastic nature of the amyloid assembly process. We further investigated whether the presence of EP2 nanofibers can accelerate the formation of PAP248-286 (a fragment of PAP that is present in human semen) amyloid fibrils. As shown in de novo. As shown in We next determined whether PAP248-286 amyloid fibrils formed in the presence of EP2 enhanced HIV-1 infectivity in a similar manner to semen amyloid fibrils generated The PAP248-286 monomer lacks significant ordered secondary structure. The cross-β-sheet structure common to all amyloid fibrils is created by intermolecular associations of β-sheets that are stabilized by hydrogen bonds , 16. SimPrevious studies have shown that the natural positive charge of PAP248-286 amyloid fibrils (pI = 10.21) facilitates the attachment of HIV-1 virions to target cells. Thus, these amyloid fibrils enhance viral infection by neutralizing the charge repulsion that otherwise exists between HIV-1 virions and host cells . Next, wIn summary, EP2, a 15-residue peptide derived from the HIV-1 gp120 coreceptor-binding region, can self-assemble into nanofibers and accelerate the formation of amyloid fibrils by PAP fragments, such as PAP248-286, in semen. These newly formed amyloid fibrils enhance HIV-1 infection by promoting the binding of HIV-1 virions to target cells. The mechanism underlying how EP2 seeds drive PAP248-286 amyloid formation and thereby enhance HIV-1 infection is detailed in


N-acetylglucosaminyltransferase-1 (GCNT1) is a key enzyme that forms core 2-branched O-glycans. Its expression is associated with the progression of several cancers. We established a mouse IgG monoclonal antibody (mAb) against GCNT1 and examined the relationship of GCNT1 expression to the clinicopathological status of PCa. Paraffin-embedded PCa specimens were analyzed by immunohistochemistry for GCNT1 expression using a newly established mouse anti-GCNT1 mAb by ourselves. GCNT1-positive tumor showed significantly higher Gleason score and larger tumor volume. The number of GCNT1-positive cases was significantly lower in cases of organ-confined disease than in cases of extracapsular extension. GCNT1-negative tumors were associated with significantly better prostate-specific antigen (PSA)-free survival compared with GCNT1-positive tumors. Multivariate analysis revealed that detection of GCNT1 expression was an independent risk factor for PSA recurrence. We established new methods for GCNT1 detection from PCa specimens. Immunoblotting was used to examine post-digital rectal examination (DRE) urine from PCa patients. Over 90% of GCNT1-positive PCa patients with high concentrations of PSA showed extracapsular extension. In conclusion, GCNT1 expression closely associates with the aggressive potential of PCa. Further research aims to develop GCNT1 detection in post-DRE urine as a marker for PCa aggressiveness.To identify appropriate candidates for aggressive treatment such as radical prostatectomy or radiation therapy of localized prostate cancer (PCa), novel predictive biomarkers of PCa aggressiveness are essential. Core2 β-1,6- In the United States and Europe, prostate cancer (PCa) is the most common malignancy in men and the second-leading cause of cancer-related death , 2. The Preoperative serum PSA levels and biopsy Gleason scores are conventional and powerful predictors of biological outcomes after radical prostatectomy for PCa , 11. To N-acetyl-glucosaminyltransferase (MGAT5) and Core 2 β -1, 6-N-acetylglucosaminyltransferase-1 (GCNT1) formed GlcNAc β1,6 branching glycan increased PCa aggressiveness [Cell surface carbohydrate structures are altered during carcinogenesis and play important roles in cancer metastasis , 14. Thesiveness , 19.O-glycans by catalyzing the transfer of N-acetylglucosamine from uridine diphosphate-N-acetylglucosamine with a β1, 6-linkage to α-N-acetylgalactosamine of a core 1 O-glycan  against GCNT1 by immunization of a mouse with GCNT1 specific peptide  to evaluISOGEN II Reagent was purchased from Nippon Gene (Japan). A purified rabbit anti-mouse IgG antibody (γ-chain specific) was purchased from Zymed. A horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) antibody and an HRP-conjugated goat anti-mouse IgG antibody were acquired from Cell Signaling Technology. An HRP-conjugated goat anti-mouse IgG antibody was acquired from Millipore. Purified mouse myeloma protein from MOPC 21 , 2-mercaptoethanol, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. Tween-20 was purchased from Wako Pure Chemicals (Japan), as were DMEM and Ham’s F12 medium. Penicillin G/streptomycin solution was from Hyclone. Precision Plus Protein standards Dual Color were from Bio-Rad, and skim milk was from Yukijirushi (Japan).Chinese hamster ovary (CHO) cells were maintained in the alpha modification of Eagle's minimum essential medium (α-MEM) supplemented with 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 10% fetal bovine serum (FBS).Between 2005 and 2011, 250 PCa patients were treated with radical prostatectomy at the Department of Urology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan. The tumor specimens were formalin-fixed and embedded in paraffin. Deparaffinized specimens were incubated with 5 μg/mL of mouse anti-human GCNT1 mAb (clone HU127), followed by incubation with HRP-conjugated goat anti-mouse IgG antibody .g, 5 min) to separate the supernatant and solids. Fifty microliters of the supernatant were spotted on to a nitrocellulose membrane. The membrane fixed with post-DRE urine protein was incubated with an anti-GCNT1 mAb (HU127), followed by an HRP-conjugated secondary antibody. Signals representing GCNT1 were enzymatically detected using the Novex® ECL Chemiluminescent Substrate Reagent Kit (Life Technologies) and visualized in a ChemiDocXRS+ System (Bio-Rad). Signal mean values were measured by Image Lab software (Bio-Rad). Amount of GCNT1 expression was calculated based on the signal mean values of recombinant human GCNT1 . Auto-chemiliminescent signals were subtracted from total signals. Total protein concentration of post-DRE urine samples were measured by a BCA Protein Assay Kit (Pierce). Informed consent was obtained from all patients. All patients provided their written informed consent to participate in this study. The ethical committee of Hirosaki University approved the protocol of this study . The study was performed in accordance with the ethical standards of the Declaration of Helsinki.Post-DRE urine was filled into 50 mL conical tubes, frozen immediately, and stored at -80°C until analysis. Post-DRE urine specimens were collected from 35 patients who underwent radical prostatectomy from 2010 to 2013 at the Department of Urology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan. Frozen samples were thawed overnight at 4°C and briefly centrifuged . Postoperative PSA levels were considered to be increased (PSA recurrence) if they were ≥ 0.2 ng/mL during two consecutive visits in a 1-month interval. Time zero was defined as the day of surgery. Patients with constantly detectable PSA levels (< 0.001 ng/mL) after surgery were recorded as recurrences at time zero. Follow-up intervals were calculated from the date of surgery to the last recorded follow-up .2 + (1 − specificity)2 [The chi-squared test was used to analyze the association of GCNT1 status with clinical and histopathological parameters. PSA-free survival was evaluated using Kaplan–Meier curves and differences between groups were assessed using the log-rank test. We used the SPSS 21.0 software package  for all statistical analyses. Optimal PSA and GCNT1 expression cut-off values were calculated the using the following formula: cut-off = (1 − sensitivity)ficity)2 , 30.To confirm antibody specificity for GCNT1, a mouse anti-human GCNT1 antibody was purified from hybridoma supernatant. Dose-dependent binding of the mouse anti-human GCNT1 mAb (clone HU127) to immobilized recombinant human GCNT1 (rhGCNT1) was detected using HRP-conjugated goat anti-mouse IgG in an enzyme-linked immunosorbent assay (ELISA) . In the To evaluate the role of GCNT1 in PCa progression, PCa specimens were analyzed by immunohistochemistry using the HU127 anti-human GCNT1 mAb. The results demonstrated that GCNT1 was weakly expressed in healthy prostate glands. In contrast, some percentage of PCa cells expressed significant levels of GCNT1 . When coAs shown in 2 + (1 − specificity)2 [To establish a semi-quantitative high-throughput screen for GCNT1 expression, post-DRE urine specimens, which contain high concentrations of PCa proteins, were analyzed by the dot-blotting method using the anti-human GCNT1 antibody . Predictficity)2 , 30  Binding of the core2 β-1,6-N-acetylglucosaminyltransferase-1 (GCNT1)-specific antibodies. Culture supernatants were prepared from HU127 hybridoma cells. Binding of the anti-human GCNT1 monoclonal antibody  or IgG myeloma MOPC 21  to immobilized recombinant human GCNT1 in a concentration-dependent manner (abscissa) was detected using a horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody. Error bars indicate the standard deviation of triplicate measurements. Concentrations of the antibody in supernatants were determined using a sandwich ELISA. The results are representative of two experiments. (B) The anti-human GCNT1 mAb recognized immobilized recombinant human GCNT1, but not BSA. (C) To confirm anti-human GCNT1 mAb specificity, recombinant human GCNT1 and GCNT3 were analyzed using electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. The anti-human GCNT1 mAb specifically recognized GCNT1, but not GCNT3. (D) Immunoblotting and (E) immunocytochemistry revealed the anti-human GCNT1 mAb also specifically recognized GCNT1 in GCNT1-overexpressed CHO cells. Peptide inhibition assay for (F) IHC and (G) dot-blotting methods revealed the GCNT1 signals were inhibited by GCNT1 antigen peptide pre-treated anti-human GCNT1 mAb.((TIF)Click here for additional data file.S2 FigProstate-specific antigen-free survival periods were compared between organ-confined disease (pT2) and extracapsular extension (pT3 and pT4). Survival was analyzed by Kaplan-Meier curves.(EPS)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease of the upper airways frequently associated with asthma. Bacterial infection is a feature of CRSwNP that can aggravate the disease and the response to glucocorticoid treatment.We examined whether the bacterial product lipopolysaccharide (LPS) reduces glucocorticoid receptor (GR) function in control nasal mucosa (NM) fibroblasts and in nasal polyp (NP) fibroblasts from patients with CRSwNP and asthma.in vitro pre-incubated with LPS (24 hours) prior to the addition of dexamethasone. Cytokine/chemokine secretion was measured by ELISA and Cytometric Bead Array. GRα, GRβ, mitogen-activated protein-kinase phosphatase-1 (MKP-1) and glucocorticoid-induced leucine zipper (GILZ) expression was measured by RT-PCR and immunoblotting, GRα nuclear translocation by immunocytochemistry, and GRβ localization by immunoblotting. The role of MKP-1 and GILZ on dexamethasone-mediated cytokine inhibition was analyzed by small interfering RNA silencing.NP (n = 12) and NM fibroblasts (n = 10) were Pre-incubation of nasal fibroblasts with LPS enhanced the secretion of IL-6, CXCL8, RANTES, and GM-CSF induced by FBS. FBS-induced CXCL8 secretion was higher in NP than in NM fibroblasts. LPS effects on IL-6 and CXCL8 were mediated via activation of p38α/β MAPK and IKK/NF-κB pathways. Additionally, LPS pre-incubation: 1) reduced dexamethasone’s capacity to inhibit FBS-induced IL-6, CXCL8 and RANTES, 2) reduced dexamethasone-induced GRα nuclear translocation (only in NM fibroblasts), 3) did not alter GRα/GRβ expression, 4) decreased GILZ expression, and 5) did not affect dexamethasone’s capacity to induce MKP-1 and GILZ expression. MKP-1 knockdown reduced dexamethasone’s capacity to suppress FBS-induced CXCL8 release.The bacterial product LPS negatively affects GR function in control NM and NP fibroblasts by interfering with the capacity of the activated receptor to inhibit the production of pro-inflammatory mediators. This study contributes to the understanding of how bacterial infection of the upper airways may limit the efficacy of glucocorticoid treatment. Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease of the sinus mucosa frequently associated with asthma and with aspirin-exacerbated respiratory disease . Asthma mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) and the glucocorticoid-induced leucine zipper (GILZ)  than in NM (66.57% inhibition) fibroblasts. LPS pre-incubation abrogated dexamethasone inhibition of CXCL8 secretion in both NM and NP fibroblasts, and attenuated dexamethasone inhibition of RANTES in NM fibroblasts (Dexamethasone concentration-dependently inhibited the induction of IL-6 production by 10% FBS in both medium and LPS-pre-treated NM and NP fibroblasts . LPS preroblasts .GRα and GRβ mRNA and protein levels did not differ between NM and NP fibroblasts (data not shown). Moreover, LPS pre-incubation did not affect GRα and GRβ mRNA and protein levels -fold vs 5.76-fold, P<.001; 6 hours: 2.44-fold vs 3.93, P<.01]. Dexamethasone induction of GILZ mRNA was also lower in NP than in NM fibroblasts . LPS increased dexamethasone induction of MKP-1 mRNA, but reduced dexamethasone induction of GILZ mRNA. However, after dividing the values of MKP-1 mRNA induction by dexamethasone by the respective control (medium or LPS) values at each time point, LPS did not significantly reduce dexamethasone’s capacity to induce MKP-1 and GILZ mRNA in nasal fibroblasts  for GILZ. Knockdown of MKP-1 and GILZ did not reduce dexamethasone’s capacity to inhibit the release of IL-6 , does not alter GRα/GRβ expression, decreases GILZ expression, but does not decrease the capacity of dexamethasone to induce GILZ and MKP-1 expression. Finally, we show that glucocorticoid-induced transactivation of MKP-1 is involved in CXCL8 inhibition by dexamethasone. In conclusion, the bacterial product LPS negatively affects GR function in control NM and NP fibroblasts by interfering with the capacity of the activated receptor to inhibit the production of pro-inflammatory mediators. This study contributes to the understanding of how bacterial infection of the upper airways may limit the efficacy of glucocorticoid treatment.S1 Dataset(XLS)Click here for additional data file.S1 FigP<.05 and ***P<.001 versus medium (no DEX).ELISA quantification of IL-6 production in cell supernatants of NM and NP fibroblasts (n = 8 each) pre-incubated with 10% csFBS-supplemented medium with/without LPS  and then incubated with 10% FBS-supplemented medium with/without dexamethasone (DEX) for 24 hours. IL-6 production normalized to each respective control. *(TIF)Click here for additional data file.S2 FigQuantification of total GRα and nuclear GRβ proteins in NM and NP fibroblasts (n = 5–7) pre-incubated with 10% csFBS-supplemented medium with/without LPS (10 μg/ml) for 24 hours.(TIF)Click here for additional data file.S3 Fig–7 M, 1 hour) in NM and NP (n = 5 each) fibroblasts pre-incubated with 10% csFBS-supplemented medium with/without LPS for 24 hours. Representative GRβ Western blot images are shown. Lamin B was used as loading control and α-tubulin was used to assure purity of the nuclear extracts.Quantification of GRβ nuclear translocation in response to dexamethasone (10(TIF)Click here for additional data file.S4 FigMKP-1 (A) and GILZ (B) mRNAs in NM (n = 9–10) and NP (n = 12) fibroblasts pre-incubated with 10% csFBS-supplemented medium with/without LPS  prior to dexamethasone  addition for the indicated times. Data show the ratio of MKP-1 or GILZ mRNA induction by dexamethasone to the respective control (medium or LPS) at each time point.RT-PCR quantification of (TIF)Click here for additional data file.S5 FigA) and GILZ (B) proteins in NM fibroblasts (n = 4–5) pre-incubated with 10% FBS (A) or 10% csFBS-supplemented medium (B) with/without LPS  prior to dexamethasone  addition for one (A) or 18 hours (B). Data show the ratio of MKP-1 or GILZ protein induction by dexamethasone to the respective control (medium or LPS).Quantification of MKP-1 ((TIF)Click here for additional data file.S6 Fig–6 M) for 24 hours. (A) FBS-induced IL-6 production. **P<.01 versus no LPS. Dexamethasone inhibition of FBS-induced IL-6 (B) and GM-CSF (C) release in LPS-pre-incubated cells. ***P<.001 versus LPS alone.NM fibroblasts (n = 6) were transfected with MKP-1/GILZ/negative control siRNAs, as indicated in Methods. Twenty-four hours later, cells were pre-incubated with/without LPS  prior to incubation with 10% FBS-supplemented medium with/without dexamethasone Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S9 Fig(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


D8Rat42-D8Arb23)/Cub (SHR-Lx) strain sensitive to ATRA-induced increase of triacylglycerols and cholesterol under condition of high-sucrose diet. SHR-Lx differs only by 7 genes of polydactylous rat (PD/Cub) origin from its spontaneously hypertensive rat (SHR) progenitor strain.All-trans retinoic acid  is a vitamin A derivative commonly used in the treatment of diverse conditions ranging from cancer to acne. In a fraction of predisposed individuals, the administration of ATRA is accompanied by variety of adverse metabolic effects, particularly by the induction of hyperlipidemia. We have previously derived a minimal congenic SHR.PD-(Lx strains were fed standard diet (STD) and experimental groups were subsequently treated with ATRA (15 mg/kg) via oral gavage for 16 days, while still on STD. We contrasted the metabolic profiles  and cholesterol (C) in 20 lipoprotein fractions) between SHR and SHR-Lx under conditions of standard diet and standard diet + ATRA. We performed transcriptomic analysis of muscle tissue (m. soleus) in all groups using Affymetrix GeneChip Rat Gene 2.0 ST Arrays followed by Ingenuity Pathway Analysis and real-time PCR validation.Adult male rats of SHR and SHR-Lx reacted with substantially greater rise in TG and C concentrations throughout the lipoprotein spectrum (two-way ANOVA strain * RA interaction significant for C content in chylomicrons (CM), VLDL and LDL as well as total, CM and HDL-TG).In response to ATRA, SHR-Sirt3, Il1b, Cpt1b and Pparg) likely to underlie the observed strain specific response to ATRA.According to our modeling of metabolic and signalization pathways using differentially expressed genes we have identified a network with major nodes (including The online version of this article (doi:10.1186/1476-511X-13-172) contains supplementary material, which is available to authorized users. D8Rat42-D8Arb23)/Cub (SHR-Lx hereafter) particularly sensitive to teratogenic actions of ATRA  as major factors . Sirt3: sirtuin 3; Smcr7: mitochondrial elongation factor 2 (Mief2); Il1b: interleukin 1 beta; Cpt1b: carnitine palmitoyltransferase 1b, muscle; Gria2: glutamate receptor, ionotropic, AMPA 2; Pparg: peroxisome proliferator-activated receptor gamma; Aldh3a1: aldehyde dehydrogenase 3 family, member A1; Rdh11: retinol dehydrogenase 11 . (PDF 69 KB)Additional file 1: Figure S1: Validation of microarray results using qPCR. Fold changes are indicated (in log2) for ATRA effect in SHR strains  and the SHR-Additional file 2: Table S1: Two-way ANOVA for triacylglycerol, cholesterol concentrations in 20 lipid subfractions (F1-F20) and lipoprotein particle size. (PDF 279 KB)


Numerous red blood cells are generated every second from proliferative progenitor cells under a homeostatic state. Increased erythropoietic activity is required after myelo-suppression as a result of chemo-radio therapies. Our previous study revealed that the endothelial cell-selective adhesion molecule (ESAM), an authentic hematopoietic stem cell marker, plays essential roles in stress-induced hematopoiesis. To determine the physiological importance of ESAM in erythroid recovery, ESAM-knockout (KO) mice were treated with the anti-cancer drug, 5-fluorouracil (5-FU). ESAM-KO mice experienced severe and prolonged anemia after 5-FU treatment compared to wild-type (WT) mice. Eight days after the 5-FU injection, compared to WT mice, ESAM-KO mice showed reduced numbers of erythroid progenitors in bone marrow (BM) and spleen, and reticulocytes in peripheral blood. Megakaryocyte-erythrocyte progenitors (MEPs) from the BM of 5-FU-treated ESAM-KO mice showed reduced burst forming unit-erythrocyte (BFU-E) capacities than those from WT mice. BM transplantation revealed that hematopoietic stem/progenitor cells from ESAM-KO donors were more sensitive to 5-FU treatment than that from WT donors in the WT host mice. However, hematopoietic cells from WT donors transplanted into ESAM-KO host mice could normally reconstitute the erythroid lineage after a BM injury. These results suggested that ESAM expression in hematopoietic cells, but not environmental cells, is critical for hematopoietic recovery. We also found that 5-FU treatment induces the up-regulation of ESAM in primitive erythroid progenitors and macrophages that do not express ESAM under homeostatic conditions. The phenotypic change seen in macrophages might be functionally involved in the interaction between erythroid progenitors and their niche components during stress-induced acute erythropoiesis. Microarray analyses of primitive erythroid progenitors from 5-FU-treated WT and ESAM-KO mice revealed that various signaling pathways, including the GATA1 system, were impaired in ESAM-KO mice. Thus, our data demonstrate that ESAM expression in hematopoietic progenitors is essential for erythroid recovery after a BM injury. Numerous hematopoietic cells are perpetually generated from hematopoietic stem/progenitor cells that exist primarily in the bone marrow (BM) after birth. Among the various types of hematopoietic cells, red blood cells are indispensable to maintain our day-to-day activities throughout life. Indeed, 2–3 million erythrocytes are produced each second in the adult human. An increase in erythropoiesis is required in times of stress, particularly after receiving chemo-radio therapy for cancer treatment.+ macrophages promote erythroid maturation under both homeostatic and stress conditions by acting like a “niche” for erythroblasts [It is not hematopoietic stem cells (HSCs) or multi-potent hematopoietic progenitor cells (HPCs), but erythroid-specific highly proliferative progenitors, that are thought to play critical roles in supporting the large daily output of red blood cells. Progenitors at the burst forming unit-erythrocyte (BFU-E) level are likely to constitute immature erythroid-restricted progenitors, which possess considerable proliferation potential . These problasts .Accumulating evidence has indicated that molecular crosstalk between erythroblasts and macrophages is important for late erythrocyte maturation. Cell surface proteins such as integrin families or adhesion molecules have been shown to mediate interactions between erythroblasts and central macrophages , 5. SoniWe previously reported that endothelial cell-selective adhesion molecule (ESAM) expression is a biomarker of HSCs in mice and is useful to trace the activation of HSCs upon BM injury , 8. ESAMWild-type (WT) C57BL/6 mice were obtained from CLEA Japan . ESAM-knockout (KO) mice were developed by Dr. T. Ishida  . Mating The 5-FU was purchased from Kyowa-Hakko Kirin . Purified anti-Ly6G and Ly6C/Gr1 (RB6-8C5) monoclonal antibodies (mAbs), phycoerythrin (PE)-conjugated anti-CD3e (145-2C11), Mac1 (M1/70), Ly6G and Ly6C/Gr1 (RB6-8C5), Ter119, CD45.1 (A20), and CD71 (C2) mAbs, fluorescein isothiocyanate (FITC)-conjugated anti-CD34 (RAM34), CD11b/Mac1 (M1/70), Ly6G and Ly6C/Gr1 (RB6-8C5), CD45R/B220 (RA3-6B2), Ter119, and CD3e (145-2C11) mAbs, allophycocyanin (APC)-conjugated anti-CD117/c-Kit (2B8) mAb, phycoerythrin-Cy7 (PE-Cy7)-conjugated anti-Sca1  mAb, PerCPCy5.5-conjugated anti-CD45.2 (104) mAb, Alexa Fluor 647-conjugated anti-CD19 (1D3) mAb, streptavidin- phycoerythrin (SAv-PE), and streptavidin- phycoerythrin-Texas red (SAv-PE-TR) were purchased from BD Pharmingen . Purified anti-CD3 (17A2), Mac1 (M1/70), and Ter119 mAbs, PE-conjugated anti-CD105/Endoglin (MJ7/18) and CD115 (AFS98) mAbs, PE-Cy7-conjugated anti-Ly6G and Ly6C/Gr1 (RB6-8C5) mAb, biotinylated anti-CD41 (eBioMWReg30) mAb, and PerCPCy5.5-conjugated anti-CD16/CD32 (93) mAb were purchased from eBioscience . PE-conjugated anti-CD150 (TC15-12F12.2), CD45R/B220 (RA3-6B2), and IL7Rα (A7R34) mAbs, FITC-conjugated anti-F4/80 (BM8) mAb, APC-Cy7-conjugated anti- CD16/CD32 (93) mAb, and PerCPCy5.5-conjugated anti- CD117/c-Kit (2B8) mAb were purchased from BioLegend . A rat anti-mouse ESAM (1G8) mAb was purchased from BioLegend. The antibody was biotinylated in our laboratory using Sulfo-NHS-LC-Biotin .Cells were obtained from adult mouse tissues as indicated in each experiment. Splenocytes and BM cells of the femora and tibiae were resuspended in staining buffer . Then, the cells were stained with the indicated antibodies. Lin Abs contained anti-Mac1, Gr1, CD3e, CD45R/B220, and Ter119 Abs. A biotinylated anti-ESAM Ab was developed with SAv-PE or SAv-PE-TR. PE-conjugated, SAv-PE, and SAv-PETR Abs were diluted 1:100, and other Abs were diluted 1:50 in staining buffer. Flow cytometry analyses were performed with FACSAria or FACSCanto . Data analyses were performed using the FlowJo software . Absolute cell numbers in each population from a pair of femora and tibiae, or spleen, was calculated by multiplying total cell count by its percentage.The experiments were performed following a protocol described by Chow et al. . Briefly- FCγR-/Lo CD41Lo c-Kit+ Sca1- endoglin+ CD150+ pre colony forming unit-erythrocyte (CFU-E) cells were sorted with FACSAria (BD Bioscience). The purity of the sorted cells was routinely confirmed by using a portion of each sorted population, and was determined to be higher than 97%.WT and ESAM-KO mice were treated with a single 5-FU injection (120 mg/kg) and sacrificed 8 days after treatment. BM cells obtained from the femora and tibiae were stained with FITC- anti-Lin Abs in combination with PE-anti-CD150, PerCPCy5.5-anti-c-Kit, APC-anti-endoglin, PE-Cy7-anti-Sca1, APC-Cy7-anti-FCγR, and biotinylated anti-CD41 Abs. Subsequent to staining with SAv-PE-TR, Lin5 or 1 × 105 BM cells were plated for counting BFU-E or CFU-E, respectively. At 5-FU day 0, 2 × 105 splenocytes were plated to count both BFU-E and CFU-E. 1 × 106 or 2 × 105 5-FU-treated splenocytes (day 8) were plated for counting BFU-E or CFU-E, respectively. Cells were plated in Methocult M3334 for CFU-E assays and in Methocult SF M3436 for BFU-E assays. To evaluate colony numbers in peripheral blood (PB), nucleated cells derived from 100 μL of PB were plated in methylcellulose media (Methocult GF M3434). To evaluate colony numbers in sorted common myeloid progenitors (CMPs) or granulocyte-monocyte progenitors (GMPs), cells were plated in Methocult GF M3434. CFU-E and BFU-E were scored at 3 and 9 days after incubation, respectively. Granulocyte colony-forming units (CFU-G), macrophage colony-forming units (CFU-M), granulocyte-macrophage colony-forming units (CFU-GM), and mixed erythroid-myeloid colony-forming units (CFU-Mix) were scored at 8 to 10 days after incubation. An absolute number of each colony isolated from a pair of femora and tibiae or spleen was calculated by colony count and total cell number.Splenocytes and BM cells were isolated from WT and ESAM-KO mice. Nucleated cells were plated in methylcellulose media . Then, 2 × 10Total RNA samples from sorted cells were isolated using the RNeasy Mini Kit . Then, the RNA samples were amplified and converted into cDNA using the ovation Pico WTA System V2 . Gene expression profiling using the SurePrint G3 mouse 8x60K microarray assay  was performed by Takara Bio . Two criteria were used to normalize and compare the values of WT and ESAM-KO pre CFU-E and define the signal ratios: more than a 2-fold increase or less than a 2-fold decrease. Bioinformatic analyses were conducted with Ingenuity Pathway Analysis software . The raw data have been accepted in Gene Expression Omnibus (GEO), a public repository for microarray data, aimed at storing Minimum Information About Microarray Experiments (MIAME). Access to data concerning this study can be found under GEO experiment accession number (GSE 73496).t-test. Error bars used throughout represent the standard error of the mean (SEM). Statistical significance was taken at values of *P < 0.05, **P < 0.01, and ***P < 0.001.Statistical analyses were conducted using a standard Student’s We have previously reported that ESAM-KO mice exhibit a more life-threatening pancytopenia than WT mice within 10 days after a single 5-FU injection . In this- Sca-1+ c-KitHi (LSK) cells in the BM, which contains HSCs and multipotent HPCs, did not differ between WT and ESAM-KO mice [- Sca-1- c-Kit+ fraction, which contains non-lymphoid multipotent progenitors, and estimated the number of common myeloid progenitors (CMPs), granulocyte-monocyte progenitors (GMPs), and megakaryocyte-erythrocyte progenitors (MEPs) [- Ter119+ CD71Hi or erythroblast subpopulations defined by forward scatter (FSC) in combination with Ter119 and CD71 [ESAM-KO mice showed slight anemia, even under homeostatic conditions. Because the number of Lin-KO mice , 12, we and CD71 , 14. The- Ter119+ CD71Hi, or Ter119Hi CD71Hi FSCLo mature erythroid progenitor levels also significantly decreased  was observed in ESAM-KO mice compared to that in WT mice, but this difference was not significant , LSK cells, CMPs, and GMPs between WT and ESAM-KO mice, a slight decrease in MEPs in ESAM-KO mice’s spleens was repeatedly detected . A signiecreased . To compnificant . Becausenificant . Taken t- Ter119+ CD71Hi mature erythroid progenitors in the BM of ESAM-KO mice was drastically reduced compared to that of WT mice (Med CD71Hi) population in BM cells and CD71Hi FSCHi or the CD71Hi FSCLo erythroblast population in Ter119Hi cells was smaller in ESAM-KO mice compared to that in WT mice  treatment. Slight anemia was observed in ESAM-KO mice after PHZ administration in a trend consistent with that seen in the homeostatic condition . Howeverr spleen . These rEpoR) in ESAM-KO immature erythroid progenitors was higher compared to that in WT mice  . Serum e WT mice . Regardi WT mice . Bone moat day 8 . These r+ T cells, CD19+ B cells, MAC1+ myeloid cells, and LSK cells was not significantly different between 5-FU-treated WT and ESAM-KO mice at day 0 and day 8 S5 Fig). - and ESAM+ subpopulations, and tested their differentiation potential. Methylcellulose colony assays showed that ESAM+ MEPs exhibited higher colony-forming activity than ESAM- MEPs. Interestingly, the ESAM+ MEP fraction contained a substantial number of primitive progenitor CFU-Mix  [BM macrophages can be transplanted by standard BM transplantation methods and are candidates for the erythroid progenitor niche , 20. Thereatment . Intrigut shown) . However- FCγR-/Lo CD41Lo c-Kit+ Sca1- endoglin+ CD150+ cells were sorted from 5-FU-treated WT and ESAM-KO mice, and subjected to microarray analyses. Bioinformatic analyses using the Ingenuity Pathway Analysis (IPA) software revealed that the ESAM deficiency affected various signaling pathways such as “hematological system development”, “cell-to-cell signaling and interaction,” and “cellular movement” (Gata1 were down-regulated in ESAM-KO mice (Ikzf1) expression was up-regulated in ESAM-KO mice. A previous study revealed that IKZF1 is a negative regulator for erythrocyte formation via repression of GATA1 [Eed) is a subunit of the polycomb repressive complex 2. Furthermore, Xie reported that Eed-KO BM HSCs are defective in differentiation [To determine the molecular mechanisms involved in the compromised erythropoiesis of ESAM-KO mice, Linovement” . We focuovement” . Then, oovement” . Interes-KO mice . On the of GATA1 , 25. Thentiation . We founntiation . In summWhile hematopoietic progenitors maintain a continuous cellular turnover of mature blood cells under a homeostatic state, they start to proliferate more actively after marrow ablation. Proliferation of early erythroid progenitors is also observed after acute anemia resulting from bleeding or hemolysis , 17, 27.Adult ESAM-KO mice are slightly anemic, but otherwise healthy under a homeostatic state; however, they experienced severe pancytopenia after 5-FU treatment . We prev+ macrophages in the BM also become ESAM-positive after 5-FU treatment because these macrophages are known to be critical for erythroid recovery from acute anemia or myeloablation [Transplantation analyses suggested that ESAM expression in hematopoietic cells, but not in the BM environment, is essential for erythroid recovery. Furthermore, when sorted MEPs from 5-FU-treated BM were subjected to colony assays, ESAM-KO mice showed reduced BFU-E formation activity compared to WT mice. Therefore, it is important to consider how the up-regulation of ESAM in hematopoietic cells contributes to erythroid recovery . One posablation . AdditioGata1, some genes including hemoglobin alpha chain complex (Hba) and hemoglobin beta chain complex (Hbb), which encode globin proteins, were down-regulated in ESAM-KO mice, suggesting that ESAM deficiency negatively affects Hb synthesis after BM injury. GATA1 is also required for the differentiation of the BFU-E stage to the CFU-E stage [- Ter119+ CD71Hi mature erythroid progenitors was significantly lower in 5-FU-treated ESAM-KO mice compared to that in 5-FU-treated WT mice . Additional research on the human ESAM will lay the foundation for clinical applications. Furthermore, in the field of regenerative medicine, ESAM-positive cells in combination with other specific markers will be useful to purify active erythroid progenitor cells.S1 FigHi cells were analyzed with respect to FSC.  In the left graph, the percentage of Ter119Med CD71Hi proerythroblasts (ProE.) in whole cells is shown. In the right graph, percentages of Ery.A (Ter119Hi CD71Hi FSCHi), Ery.B (Ter119Hi CD71Hi FSCLo), and Ery.C (Ter119Hi CD71Lo FSCLo) erythroblast populations within Ter119Hi cells are shown . Data are shown as mean ± SEM. Statistically significant differences are represented by asterisks .BM cells (A-B) and splenocytes (C-D) were isolated from WT and ESAM-KO mice to perform FACS analyses.  Representative FACS profile of BM cells (A) and splenocytes (C) stained with antibodies against Ter119 and CD71. In the right panel, Ter119(PDF)Click here for additional data file.S2 FigHi cells are shown . Data are shown as means ± SEM. Statistically significant differences are represented by asterisks .WT and ESAM-KO mice were injected with 150 mg/kg 5-FU. Then, 8 days after treatment, BM cells (A-B) and splenocytes (C-D) were isolated to perform FACS analyses.  Representative FACS profile of BM cells (A) and splenocytes (C) are shown.  In the left graph, the percentage of proerythroblasts (ProE.) in whole cells is shown. In the right graph, percentages of Ery.A, Ery.B, and Ery.C erythroblast populations within Ter119(PDF)Click here for additional data file.S3 Fig5 BM cells or splenocytes were plated for counting BFU-E. Each bar represents the number of BFU-E in BM (left graph) or spleen (right graph). (C) The number of c-Kit- Ter119+ CD71Hi cells in the BM (left graph) or spleen (right graph) are shown. Data are shown as mean ± SEM.WT and ESAM-KO mice were treated with 40 mg/kg of PHZ intraperitoneally for 2 consecutive days (days 0 and 1) to then sacrifice the mice and analyze erythroid recovery at day 6 (n = 3 in each). (A) Hemoglobin concentration in PB is shown. (B) 1 × 10(PDF)Click here for additional data file.S4 FigEpoR were evaluated. (D) Femurs were flushed with 0.3 mL of PBS. Cells were lysed by a freeze and thaw cycle. Cell debris was removed by centrifugation. Then, the concentration of SCF in the BM was analyzed using a mouse SCF ELISA kit. (E-F) Expression levels of SCF (E) and Bmp-4 (F) in BM cells and splenocytes are shown.  RNA samples were isolated using a PureLink RNA Mini Kit. Reverse transcription reactions were performed using a High Capacity RNA-to-cDNA Kit. Relative expression of each gene relative to GAPDH were evaluated according to the Taqman Gene Expression Assay Protocol.  Data are shown as mean ± SEM. Statistically significant differences are represented by an asterisk (*P < 0.05).(A-F) Comparison analyses between WT and ESAM-KO mice treated with 150 mg/kg of 5-FU were performed. Serum total bilirubin (T-bil) (A) and serum erythropoietin (EPO) (B) were evaluated (n = 3 in each). T-bil was analyzed using Vetscan VS2 and serum EPO was analyzed by BML, INC. (C) Pre CFU-E progenitor cells were sorted from pooled BM cells (two mice in each), and gene expression levels of (PDF)Click here for additional data file.S5 Fig+ and ESAMHi cells, respectively.ESAM expression levels on BM LSK fractions from C57BL/6 WT mice after 5.5Gy total body irradiation (TBI) were examined using a FACS analysis. Each panel shows a representative histogram of ESAM expression level on LSK at day 0 (control), 4, and 8 after TBI. Dashed lines show background levels with an isotype control Ab. Tinted lines show ESAM expression levels of LSK after TBI. The solid line, which represents ESAM expression levels at day 0 is added to each panel. Upper and lower numbers in each histogram indicate the percentages of ESAM(PDF)Click here for additional data file.S6 Fig- or ESAM+ MEPs were sorted from C57BL/6 WT mice under steady state or 8 days after 150 mg/kg of 5-FU injection. Then 1,500 cells were plated in methylcellulose media, Methocult GF M 3434. Under the steady state, an ESAM+ MEP population could not be detected. After 10 days, CFU-Mix colonies were enumerated according to shape and color under an inverted microscope (n = 3 in each). N.D. means “not done”. Data are shown as mean ± SEM. Statistically significant differences are represented by an asterisk (** P < 0.01).ESAM(PDF)Click here for additional data file.S7 FigGenes that were differently expressed between WT and ESAM-KO pre CFU-E (Bioset 1) were compared with those in WT and Eed-KO HSCs (Bioset 2) using the NextBio software. The Bioset 2 was extracted from the GEO experiment accession number GSE 51084. (A) Venn diagram shows the number of common and unique genes in both sets. (B) Significance of the overlap between 2 gene subsets. The scale bar indicates–log . The comparison shows a positive correlation between these 2 biosets.(PDF)Click here for additional data file.