As expected, both ROD1 mRNA and protein were down-modulated upon hypoxia in a miR-210 dependent manner. ROD1 targeting by miR-210 was biologically significant: the rescue of ROD1 inhibition significantly increased hypoxia-induced cell death. These data highlight the importance of ROD1 regulation by miR-210 for cell homeostasis.Most metazoan microRNA (miRNA) target sites have perfect pairing to the “seed” sequence, a highly conserved region centering on miRNA nucleotides 2–7. Thus, complementarity to this region is a necessary requirement for target prediction algorithms. However, also non-canonical miRNA binding can confer target regulation. Here, we identified a seedless target of miR-210, a master miRNA of the hypoxic response. We analyzed 20 genes that were inversely correlated to miR-210 expression and did not display any complementarity with miR-210 seed sequence. We validated ROD1  as a miR-210 seedless transcript enriched in miR-210-containing RNA-induced silencing complexes. ROD1 was not indirectly targeted by a miR-210-induced miRNA. Conversely, we identified a “centered” miR-210 binding site in ROD1 involving 10 consecutive bases in the central portion of miR-210. Reporter assays showed that miR-210 inhibited ROD1 by the direct binding to this sequence, demonstrating that ROD1 is a  We identified ROD1  as a 2 - 95% N2 was injected for 20 min, yielding about 1% O2HEK-293 cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS. Commercially available human umbilical vein endothelial cells  were grown in EGM-2 (Lonza) containing 2% FBS. Hypoxic conditions were maintained in defined atmosphere chambers (Billups-Rothenberg Inc.); cells were placed in the chamber and then a gas mixture of 5% COg in a micro-centrifuge. Next, lysates were incubated at 4°C with 2.5 µg/plate of anti-c-myc antibody  for 3 hrs and then 50 µl/plate of A/G-agarose beads were added to each sample. After 1 hr, immuno-complexes were washed two times with lysis buffer and resuspended in 200 µl of TRIzol (Invitrogen). RNA was purified and specific mRNAs were measured by quantitative real-time PCR (qPCR). Average values of 3 genes , that previous experiments showed to be RISC-associated but not miR-210 targets Transfected cells were harvested in 1 ml/p15 dish of cold lysis buffer  supplemented with 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, protease inhibitors mixture tablets (Roche) and 100 u/ml of RNasin Plus (Promega). After 30 min at 4°C, samples were pre-cleared for 10 min with 75 µl/plate of A/G-agarose beads (Santa Cruz) and spun at 4°C for 30 minutes at 20.000 3 cells/cm2) at the final concentration of 100 nM. After 16 hrs, cells were re-fed with fresh medium and experiments were performed 24 hrs later. miR-210 was also inhibited using a lentiviral vector that express a “sponge” reporter gene (see supplementary information). Transient miR-210 over-expression was obtained by transfection of pSUPER-pre-miR-210 Locked Nucleic Acid (LNA) oligonucleotides against miR-210 or a control scramble sequence (Exiqon) were transfected by siRNA Transfection Reagent (Santa Cruz), following manufacturer instructions, in 40% confluent HEK-293 (4×10Cells were lysed in 2x Laemmli buffer and boiled for 5 minutes. Proteins were separated in SDS-polyacrylamide gels and transferred to nitrocellulose by standard procedures The RNAhybrid tool (Bielefeld University Bioinformatics Server) was used to calculate the minimum free energy hybridization for short RNA duplexes.Variables were analyzed by both Student’s t test and one way ANOVA. Unless differently specified, a p≤0.05 was deemed statistically significant. n = number of independent experiments. Values are expressed as ±standard error.See In a previous report, we identified a set of 62 miR-210 potential targets, selected for their reciprocal modulation with miR-210 Next, we asked whether ROD1 bound to the RISC also in the presence of physiological miR-210 levels. To this aim, we set-up the reciprocal assay, hypothesizing that miR-210 blockade will lead to a decrease of miR-210 targets bound to RISC. To further validate our results, protein and mRNA levels of ROD1 were evaluated after miR-210 modulation in HEK-293 cells. As shown in cds and 3′UTR) of each transcript were analyzed. We asked if any minor (less represented) isoform contained a putative miR-210-seed binding site. Out of 490 published Expressed Sequence Tags (NCBI), only 4 displayed a miR-210-seed complementary sequence . However, for all of them, the EST portion containing the miR-210-seed complementarity did not align to any region of the genome, likely representing cloning or sequencing artifacts. In conclusion, while we cannot formally exclude that a minor ROD1 isoform may be a canonical miR-210 target, this is an unlikely and rare event.Six ROD1 transcript variants have been annotated (NCBI) and none of these ROD1 isoforms displayed a miR-210-seed complementarity sequence, even though all the parts  and searches for miRNA binding sites in both the 3′UTR and the dulation . Next, w of ROD1 . This co of ROD1 . The the of ROD1 . The minROD1 is a paralog gene of the polypyrimidine tract binding proteins PTBP1 and PTBP2 We and others demonstrated that the anti-apoptotic activity of miR-210 is a fundamental adaptive response to hypoxia In this study, we identified ROD1 as a seedless target of the hypoxia-induced miR-210. ROD1 emerged as the only direct target among 20 seedless genes displaying an inverse correlation with miR-210 expression We considered the possibility that ROD1 was an indirect miR-210 target. Indeed, one can envision a model where a miR-210-induced miRNA targets ROD1 and recruits it to the RISC. For instance, it was recently demonstrated that ROD1 is a target of miR-499 cds. Luciferase reporter assays formally demonstrate that miR-210 inhibits ROD1 by the direct binding to this sequence. The identified miR-210/ROD1 pairing was similar to the “centered pairing” recently described by Bartel and collaborators 2+ concentrations 2+ concentrations When we tried to identify a miR-210 binding site in ROD1, a region with partial pairing to miR-210 was discovered in the Other atypical miRNA binding patterns were recently described Finally, the biological significance of ROD1 targeting was evaluated. The results highlighted the importance of a tight control of ROD1 levels for cell survival in hypoxia. In fact, we found that the rescue of miR-210-dependent down-modulation of ROD1 observed in hypoxic cells increased apoptosis. These data indicate that miR-210 is part of the molecular mechanisms fine tuning ROD1 expression. In keeping with this interpretation, ROD1 levels, that are induced in a mouse model of hindlimb ischemia, are further increased upon miR-210 blockade .in vitro and after infarction in vivoCorrelative evidences also indicate that miR-210 plays a pivotal role in ROD1 regulation. A negative activity of ROD1 in the differentiation control of mammalian cells is emerging. Specifically, ROD1 over-expression blocks the erythroid differentiation of a human leukemia cell line Figure S1miR-210-seedless genes modulated by miR-210. Heat map representing mRNAs down-modulated by miR-210 over-expression (miR-210 column) that displayed an inverse modulation following miR-210 inhibition (anti-210 column) in HUVEC. The only exception is constituted by CTSB, NDUFV2, TCEB2 and VIM that were measured at protein level in miR-210 over-expressing HUVEC. Average values are expressed as fold change. Green and red colors indicate down- or up-regulation, respectively, while gray indicates missing data .(TIF)Click here for additional data file.Figure S2Quantification of miR-210 inhibition. A) HEK-293 were transfected with anti-miR-210 or anti-scramble LNA-oligonucleotides. miR-210 levels were assayed 40 hrs later, confirming an efficient miR-210 knock-down . B) HEK-293 were infected with lentiviruses expressing anti-scramble or anti-miR-210 sponges. miR-210 levels were assayed 72 hrs later, confirming an efficient miR-210 knock-down .(TIF)Click here for additional data file.Figure S3Prediction of the putative binding sites between miR-210 and ROD1 according to the Rna22 algorithm. ROD1 transcript variant 6 (NM_001244898) is the longest ROD1 isoform and it was used for base numeration.(TIF)Click here for additional data file.Figure S4PTBP1 and PTBP2 paralogs are not miR-210 targets. A) HEK-293 were co-transfected with expression vectors for either miR-210 or a scramble sequence and c-myc-Ago2 (mAgo2). Then, c-myc antibody was used to immuno-precipitate the miR-210/mAgo2-containing complexes. Background controls were represented by c-myc-immuno-precipitates derived from cells transfected with miR-210 but not mAgo2. Whereas ROD1 was enriched in the immune-precipitates of the mir-210-loaded RISC, PTBP1 and PTBP2 did not show any significant modulation . B-C) HEK-293 were transfected with plasmids encoding either miR-210 (miR-210) or a scramble sequence (scramble). Then, 24 and 48 hrs later, cell extracts were derived and the and mRNA levels of PTBP1 (B) and PTBP2 (C) were assayed by qPCR (n = 3).(TIF)Click here for additional data file.Figure S5Override of ROD1 down-modulation by hypoxia. HEK-293 were transfected with plasmids encoding ROD1 (pCMV-ROD1) or with vector alone (pCMV) and the next day were exposed to 1% hypoxia for the indicated time. miR-210 and ROD1 levels were assayed by qPCR. Average values are expressed using a log2 scale. Green and red colors indicate down- or up-regulation, respectively .(TIF)Click here for additional data file.Figure S6miR-210 inhibition in ROD1 over-expressing cells further decreases cell survival. HEK-293 (4×103/cm2) were co-transfected with anti-miR-210 or anti-scramble LNA-oligonucleotides and pCMV-ROD1 (ROD1) or vector alone (pCMV). A) HEK-293 growth curve. Data are expressed as % of T0 normoxic control. Significant differences between anti-miR-210 and anti-scramble transfected cells in the same experimental condition are indicated . B) Cell death assessed by Trypan blue exclusion assay. Data are expressed as % of dead cells for each experimental point. Significant differences between anti-miR-210 and anti-scramble transfected cells in the same experimental condition are indicated . C) After 48 hrs of transfection, apoptosis was measured assessing the apoptotic fragmentation of cytoplasmic DNA .(TIF)Click here for additional data file.Table S1RISC-IP of miR-210-seedless genes.(DOC)Click here for additional data file.Table S2miRNAs array upon miR-210 over-expression.(XLSX)Click here for additional data file.Table S3Primers for SYBR-GREEN Real Time PCR.(DOC)Click here for additional data file.Methods S1Supplementary methods.(DOC)Click here for additional data file.


The neocortex of mammalian brains shows prominent oscillatory activity in the local field potential (LFP) and EEG signal within a broad range of frequencies from slow 1-8 Hz) delta/theta band to gamma band (40-100 Hz) and higher frequencies -8 Hz del. Several


Autosomal dominant polycystic kidney disease (ADPKD) is clinically heterogenic. Biomarkers are needed to predict prognosis and guide management. We aimed to profile microRNA (miRNA) in ADPKD to gain molecular insight and evaluate biomarker potential.Small-RNA libraries were generated from urine specimens of ADPKD patients (N = 20) and patients with chronic kidney disease of other etiologies . In this report, we describe the miRNA profiles and baseline characteristics. For reference, we also examined the miRNA transcriptome in primary cultures of ADPKD cyst epithelia (N = 10), normal adult tubule (N = 8) and fetal tubule (N = 7) epithelia.In primary cultures of ADPKD kidney cells, miRNA cistrons mir-143(2) (9.2-fold), let-7i(1) (2.3-fold) and mir-3619(1) (12.1-fold) were significantly elevated compared to normal tubule epithelia, whereas mir-1(4) members (19.7-fold), mir-133b(2) (21.1-fold) and mir-205(1) (3.0-fold) were downregulated (P<0.01). Expression of the dysregulated miRNA in fetal tubule epithelia resembled ADPKD better than normal adult cells, except let-7i, which was lower in fetal cells. In patient biofluid specimens, mir-143(2) members were 2.9-fold higher in urine cells from ADPKD compared to other CKD patients, while expression levels of mir-133b(2) (4.9-fold) and mir-1(4) (4.4-fold) were lower in ADPKD. We also noted increased abundance mir-223(1) (5.6-fold), mir-199a(3) (1.4-fold) and mir-199b(1) (1.8-fold) (P<0.01) in ADPKD urine cells. In ADPKD urine microvesicles, miR-1(2) (7.2-fold) and miR-133a(2) (11.8-fold) were less abundant compared to other CKD patients (P<0.01).We found that in ADPKD urine specimens, miRNA previously implicated as kidney tumor suppressors (miR-1 and miR-133), as well as miRNA of presumed inflammatory and fibroblast cell origin (miR-223/miR-199), are dysregulated when compared to other CKD patients. Concordant with findings in the primary tubule epithelial cell model, this suggests roles for dysregulated miRNA in ADPKD pathogenesis and potential use as biomarkers. We intend to assess prognostic potential of miRNA in a followup analysis. Autosomal dominant polycystic kidney disease (ADPKD) is characterized by unpredictable progression rate and incidence of complications. Research of potential therapeutics is hampered by lack of short-term surrogate markers of therapeutic effects. Reduction in glomerular filtration rate is a late occurrence in the course of the disease that manifests only after >60% of normal renal parenchyma has sustained permanent damage.The diagnostic criteria for ADPKD are based on renal ultrasonography and a positive family history MicroRNAs (miRNA) are small regulatory non-coding RNAs expressed in plants and animals. miRNA primary transcripts (pri-miRNA) are transcribed by RNA polymerase II from independent promoters, or processed from intronic regions, and are thus governed by regulatory mechanisms common to protein-coding genes. Accordingly, abundance of miRNAs, like protein coding mRNAs, is tuned both by cell-specific and cell non-specific regulation. Some miRNA are ubiquitous, while others are strictly limited to a cell type or lineage. Of approximately 600 known human miRNAs Our broad aim in this study is to examine miRNAs in ADPKD in an attempt to translate methodological advantages of miRNA profiling to the need for biomarkers in assessment of disease progression in ADPKD. miRNAs were profiled from nanogram amounts of input total RNA in clinical biofluid specimens from ADPKD patients and other chronic kidney disease (CKD) patients using deep sequencing of multiplexed small-RNA cDNA libraries. In the current report, we describe the urine miRNA profiles in relation to study group. In addition, since cystic proliferation of tubular epithelial cells is pivotal in ADPKD, we profiled miRNA in primary cultures of cyst-derived epithelial cells from ADPKD explants and normal kidneys as a cell culture model for the disease. We intend to evaluate prediction of kidney outcome by baseline miRNA abundance in a followup study.This study was approved by the Institutional Review Boards of the Rockefeller University Hospital and the Weill Medical College of Cornell University. Clinical data and specimens were collected after obtaining participants' written informed consent.Figure S1) Primary tubule epithelial cell cultures were derived from microdissected renal tubules and explanted kidney tissues from adults without parenchymal kidney disease prepared for transplant donation but deemed unsuitable due to renal vascular structural abnormalities (N = 8) or early stage ADPKD ; from surgical nephrectomy of end stage kidneys from patients with ADPKD  or from normal fetal kidneys after elective pregnancy termination . Primary epithelial cells were grown to confluence in cell-type-specific supplemented media according to published techniques . ADPKD patients enrolled in this study were also registered into an ongoing data repository . Total RNA was extracted from 50 ml urine sediment cells and from the ensuing cell-free, ultrafiltration-retained supernatant, a ∼250-fold concentrate of urine particles and macromolecules >100 kDa, thus including vesicle-enclosed RNA  . Small-RNA cDNA libraries were constructed and sequenced with modifications for small amounts of input RNA. Details of head-to-head comparison between precipitation-purified and silica column-purified RNA are shown in Figure S3We collected urine specimens from 20 patients with ADPKD and 20 patients with CKD of other etiologies, matched for age, sex, ethnicity/race and CKD stage Table 1.Extracted total RNA was subjected to barcoded adapter ligation, and the reverse-transcribed small-RNA libraries were deep sequenced on Illumina Genome Analyzer II, as previously described We display miRNA according to our recently published definitions www.spss.com) and the edgeR package for RNA sequencing on R platform t-tests. Individual patient/sample miRNA data are displayed in supplementary tables as counts derived from deep sequencing reads. In addition, counts are aggregated according to miRNA sequence families and precursor cluster cistrons and presented in the respective supplementary tables (see above). Analyses of differential expression between ADPKD and non-ADPKD groups of samples were conducted under the assumption that gene (miRNA) counts in RNA deep sequencing data follow negative binomial distribution. The R/Bioconductor statistical package edgeR was developed with the negative binomial distribution assumption at its core. In generalized linear negative binomial models fitted with edgeR, the dependent variables are the genewise miRNA counts  while the independent variable we included were disease group (e.g. ADPKD vs. other CKD urine cell pellets) and patient sex (when relevant). Fold-change values in miRNA abundance between the two groups of samples are calculated as the ratio of mean CPM in ADPKD to the mean CPM in the non-ADPKD group, and transformed to log base 2. P-values for coefficients in the model are computed by genewise likelihood ratio tests (or quasi-likelihood F-tests). Dispersion, which reflects biological variation, is estimated using the Cox-Reid method, and is inherent in the modeling procedure. Thus, within group variability impacts the statistical inference. Nominal P-values<0.05 are considered significant, provided that false detection rate (FDR) computed with the Benjamini-Hochberg method is less than 0.2.PASW 17.0 Statistics . Based on the addition of defined amounts of synthetic calibrator oligoribonucleotides Small-RNA categories were obtained from primary tubule epithelial cell cultures grown from microdissected renal tubules of adults (i) without parenchymal kidney disease (N = 8), (ii) with early stage  ADPKD (N = 6), (iii) end-stage ADPKD kidneys (N = 4), and from normal fetal kidneys (N = 7) . Sequence reads derived from let-7i(1) (2.3-fold), mir-143(2) (9.1-fold) and mir-3619(1) (11.8-fold) were upregulated, and mir-1(4) (20-fold), mir-133b(2) (20.8-fold) and mir-205(1) (3.1-fold) cistron members were downregulated in primary cultures of cyst epithelial cells compared to normal kidney-derived cells  . Resembling ADPKD-derived cells, fetal cells had similarly lower levels of mir-1(4) and mir-133b(2) members and higher levels of mir-143(2) and mir-3619(1) members compared to normal adult tubular epithelial cells  (miRNA profiles diverged in accordance with sample groups (FDR<0.2) Table 3 Tables S6, S7, S8 in File S1 for details and differential expression). None of the top 15 miRNA precursor clusters or top 40 miRNA sequence families were differentially expressed in immortalized compared to primary cells (Table S8 in File S1), suggesting that miRNA expression and regulatory function in immortalized cell lines may recapitulate their respective properties in primary cultures of kidney epithelial cells. Presence of SV40-miR-S1 and downregulation of miR-141/miR-200c in immortalized cells were the most striking differences in miRNA profiles compared to primary cells (Table S7 in File S1).Segmental origin of the cultured cells, determined in all specimens except end-stage ADPKD kidneys, e.g. proximal straight tubule (PST), thick ascending limb (TAL) and collecting duct (CT) had no impact upon miRNA profiles. However, cells originating in cysts of end-stage ADPKD kidneys had higher content of mir-21(1) (1.6-fold), mir-30a(4) (1.5-fold) and mir-101(2) (3.2-fold) and lower content of mir-143(2) (10-fold), compared to PST, TAL or CT-derived cells . For perspective, we provide miRNA profiles of immortalized kidney epithelial cell lines of tubular and podocyte origin , small-RNA annotation categories and calculated total miRNA content  did not differ by disease group or gender. In cell-free ultrafiltration-retained material total RNA yields were 2-fold lower, but miRNA content per µg total RNA were 2.3-fold higher in ADPKD compared to non-ADPKD CKD specimens, and thus molar miRNA concentrations were overall similar; 68–104 fM (Table S1 in File S1).We collected urine specimens from 20 patients with ADPKD and 20 patients with chronic kidney disease (CKD) of other etiologies, matched for age, sex, ethnicity/race and CKD stage Table 1.Tables S9, S10, S11, S12, S23, S14 in File S1. Compared with CKD of other etiologies, in urine cells from ADPKD mir-143(2) cluster members were upregulated, whereas mir-1(4) and related mir-133b(2) were downregulated. Moreover, there was an increased relative abundance of mir-223(1), mir-199a(3) and mir-199b(1) in ADPKD (Abundance of specific miRNA in urine cells and cell-free ultrafiltration-retained specimens is depicted in in ADPKD Table 4.Table S15 in File S1) Table S16 in File S1) followed similar trends for 6 of the 8 miRNA above, and a similarly derived score discriminated ADPKD from other CKD patients' urine cells  that mapped to the human genome but had no matching small-RNA annotation in primary culture libraries  revealed that no single sequence exceeded a level corresponding to 0.02% of total miRNA . A miRNA of this level typically ranks ∼250 in abundance . Thus, the existence of a previously unidentified yet biologically important kidney tubule epithelial miRNA is effectively ruled-out In this study, we obtained expression profiles of miRNA in ADPKD patient-derived primary cell cultures and urine RNA. Sequence profiles were generated by sequencing of multiplexed small-RNA cDNA libraries constructed in-house from as low as 5 ng total RNA per sample. This technical achievement has not been reported with currently available commercial kits We studied primary cultures of epithelial cells lining ADPKD cysts as a basic model of the disease Compared to normal cells, ADPKD and fetal cells had lower levels of mir-1(4) and mir-133b(2) and higher levels of mir-143(2) and mir-3619(1) members. mir-143(2) members (predominantly miR-145) are highly abundant throughout the urinary tract, particularly in the lower segments. In contrast, mir-1(4) and mir-133b(2) are not abundantly expressed in kidneys (see below). Nonetheless, mir-1(4) and mir-133b(2) were reportedly downregulated in renal cell carcinoma (RCC) specimens and cell lines, and transfection experiments suggested that these clusters of miRNAs may be tumor suppressive in the kidney We hypothesized that transcriptome-wide urine miRNA profiles would differ between ADPKD and other chronic kidney diseases, and that alterations discovered in the primary culture model may be recapitulated in the patient specimens. Indeed, abundance of specific miRNA in cellular and extracellular (exosome) specimens differed in ADPKD and non-ADPKD patients. Increased levels of mir-143(2) (urine cells) and decrease in mir-1(4) and mir-133b(2) members (urine cells and extracellular RNA) were found in ADPKD patients, extending the findings from primary cultures to clinical specimens. mir-499(1), albeit rare in kidney cells, was also significantly lower in ADPKD cell-free urine RNA compared to non-ADPKD specimens. miR-499 is encoded within an intron of the sarcomeric myosin gene, Myh7b We observed higher levels of the lymphocyte/monocyte associated miR-223 and fibroblast-enriched miR-199a and miR-199b in ADPKD urine specimens. Monocyte infiltration support a functional role for mir-1(4) and mir-133b(2) members in normal kidney epithelial cells, in which they are present at ∼1,900 RPM (0.19%), see above.In addition to analyzing differential expression , we made use of information uniquely attainable by RNA sequencing. In primary culture profiles, we searched for yet undiscovered human miRNA. However, since miRNA action depends on stoichiometric interaction with their mRNA targets, exceedingly rare miRNA, even if processed and handled as prototypical miRNA, are unlikely to convey detectable post-transcriptional regulation. A study employing large-scale miRNA decoy and sensing libraries combined with RNA sequencing showed that only the most abundant miRNAs in a cell mediate target suppression and that ∼60% of detected miRNAs had no discernible activity Genome-encoded and post-transcriptional alterations in miRNA sequence have bearings on miRNA stability and target repression We profiled miRNA in ADPKD and non-ADPKD-derived kidney epithelial cells and in two sets of ADPKD and non-ADPKD patient urine specimens. We propose a role for repression of mir-1(4) and mir-133b(2) members in the pathogenesis of ADPKD, and for their potential use as biomarkers for monitoring disease progression and response to evolving treatments. However, prospective, longitudinal studies of these miRNA members are required to establish their role as biomarkers of ADPKD. Indeed, we intend to examine prediction of ADPKD progression in a followup longitudinal study of the study participants reported herein. We also suggest that enrichment of monocyte- and fibroblast-specific miRNA in urine from ADPKD patients' urine supports their role in disease progression. Finally, we have demonstrated the feasibility of multiplexed small-RNA cDNA library preparation and sequencing from nanogram-scale input total RNA, and its utility in generating research hypotheses and in the process of biomarker discovery.Figure S1Phase contrast images of confluent normal renal tubule and ADPKD cystic epithelial cell monolayers. (A) primary fetal, (B) primary adult, (C) primary early ADPKD and (D) primary end-stage ADPKD.(EPS)Click here for additional data file.Figure S2Electron micrographs of a representative urine ultrafiltration retentate specimen showing vesicular structures of different sizes; uranyl-acetate (‘negative’) stain.(EPS)Click here for additional data file.Figure S3Parallel miRNA profiling from selected split urine ultrafiltration retentate samples. (exosome preparations). (A) Experimental design – urine specimens were homogenized with TRIzol LS (Life Technologies – Invitrogen), RNA was extracted with chloroform, and the aqueous phase split and subjected to either isopropanol precipitation or miRNeasy silica column purification (Qiagen). For precipitation, glycogen (15 µg) was added prior to isopropanol, and precipitation carried out overnight at −20°C. (B) Differentially extracted miRNA (silica column vs. isopropanol precipitation) as determined with edgeR (EPS)Click here for additional data file.Figure S4miRNA profiles in primary cultures of renal tubule epithelia. (a) Hierarchically clustered samples (columns) and miRNA cistrons (rows). High relative read frequency (log2-transformed) is indicated by bright yellow shades; low frequencies in dark blue. Selected miRNA cistrons are color-shaded to denote dysregulation in ADPKD-descended cultures. (b) MA plot generated showing miRNA cistron differential expression between normal adult kidney and ADPKD cyst-derived epithelial cultures . (c) Multidimensional scaling (MDS) plot of primary culture samples according to miRNA cistron expression profiles .(EPS)Click here for additional data file.File S1Table S1. Small RNA annotation categories across types of study samples, given as percentage of all post-filtering reads. Table S2. Tuschl lab miRNA group definitions. Table S3. Merged mature miRNA profiles of primary kidney cell cultures. Table S4. miRNA precursor (cistron) cluster profiles of primary kidney cell cultures. Table S5. miRNA sequence family profiles of primary kidney cell cultures. Table S6. Merged mature miRNA profiles of pooled primary and immortalized kidney cell cultures. Table S7. miRNA precursor (cistron) profiles of pooled primary and immortalized kidney cell cultures. Table S8. miRNA sequence family profiles of pooled primary and immortalized kidney cell cultures. Table S9. Merged mature miRNA profiles of patients' urine sediment cells. Table S10. iRNA precursor (cistron) cluster profiles of patients' urine sediment cells. Table S11. miRNA sequence family profiles of patients' urine sediment cells. Table S12. merged mature miRNA profiles of patients' urine exosome preparations. Table S13. miRNA precursor (cistron) cluster profiles of patients' urine exosome preparations. Table S14. miRNA sequence family profiles of patients' urine exosome preparations. Table S15. miRNA sequence variants in profiles generated from primary kidney cell cultures. Table S16. miRNA sequence variants in profiles generated from patients' urine sediment cells.Contains. (XLS)Click here for additional data file.


In vivo, TEL-Syk expression in pre-B cells blocks B cell differentiation, leading to lymphoid leukemia. Here, we demonstrate that TEL-Syk introduced into fetal liver hematopoietic cells, which are then adoptively transferred into lethally irradiated recipients, leads to an aggressive myelodysplasia with myelofibrosis that is lethal in mice by 60–75 days. Expression of TEL-Syk induces a short-lived myeloexpansion that is rapidly followed by bone marrow failure and extreme splenic/hepatic fibrosis accompanied by extensive apoptosis. The disease is dependent on Syk kinase activity. Analysis of serum from TEL-Syk mice reveals an inflammatory cytokine signature reminiscent of that found in the sera from patients and mouse models of myeloproliferative neoplasms. TEL-Syk expressing cells showed constitutive STAT5 phosphorylation, which was resistant to JAK inhibition, consistent with deregulated cytokine signaling. These data indicate that expression of TEL-Syk in fetal liver hematopoietic cells results in JAK-independent STAT5 phosphorylation ultimately leading to a uniquely aggressive and lethal form of myelofibrosis.The TEL-Syk fusion protein was isolated from a patient with myelodysplasia with megakaryocyte blasts. Expression of TEL-Syk transforms interleukin-3 (IL-3)-dependent Ba/F3 cells  JAK2V617F is found in >50% of PMF patients and leads to progressive anemia, splenomegaly, myelo-expansion, and fibrosis of the bone marrow. This mutation disrupts autoinhibition of JAK2 and drives deregulated signal transduction downstream of multiple cytokine receptors . TE. TEJAK2VSpleen tyrosine kinase, or Syk, is a non-receptor tyrosine kinase that signals downstream of immunoreceptors and integrins in hematopoietic cells . Syk modin vitro . A. ATEL-Syopoiesis . In Ba/Fopoiesis . In pre-opoiesis . TherefoThis study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the UCSF Institutional Animal Care and Use Committee . All surgery was performed under isoflurane anesthesia, and all efforts were made to minimize suffering.2 inhalation in small chambers within the barrier facility followed by cervical dislocation to ensure death. Tissues were removed for study as described below. Induction of myleodysplasia by retroviral transduction was done in groups of 3–5 mice per experimental retrovirus used. Each experiment usually had 4 different groups (including a control retrovirus cohort).BALB/c (CD45.2) mice were purchased from Taconic Laboratories. Mice were housed in the UCSF rodent barrier facility, under the care of UCSF Laboratory for Animal Resources Center (LARC) staff, which is an AAALAC accredited specific pathogen free facility. Animals were monitored for development of myelodysplasia, following transfer of retrovirally transduced progenitors (see below) daily by LARC staff and by laboratory personnel. Any animals showing evidence of illness, as defined by ruffled fur, poor motility, apparent body weight loss, skin or abdominal swelling or who had a body condition score of 2 or less, were considered to have reached the humane endpoint of experiment and were removed from the study for euthanasia. Mice were also monitored every two weeks by serial blood sampling to look for evidence of myelodysplasia. Euthanasia was conducted by COin vitro cytokine stimulation. For JAK inhibition, we used the JAK inhibitor 1 (EMD Millipore). Anti-Syk antibodies , anti-NTAL , p-Tyr (4G10), phospho-STAT5 and total STAT5 , anti-Erk1 and Erk2 (Santa Cruz Biotech), anti-GFP (Novus), and fluorescently labeled goat anti-mouse or rabbit secondary antibodies were used (LI-COR Biosciences) for immunoblot analysis. Immunoblots were imaged using the Odyssey Infrared Imaging system (LI-COR Biosciences). Antibodies used for flow cytometry included mIgG (Sigma-Aldrich), anti-CD16/CD32 , anti-CD11b (M1/70), anti-Ly6G (1A8), anti-TCRβ (H57-597), anti-CD19 (ID3), anti-NKp46 (9E2), anti-Siglec-F (E50-2440), anti-CD71 (C2F2), anti-TER-119 (TER-119) phospho-STAT5-Alexa Fluor 647 (47) from (BD Biosciences); F4/80 , anti-GFP Biotin  and streptavidin Pacific Orange (Invitrogen).Human TEL-Syk was kindly provided by Dr. Hassan Jumaa . Murine 3VO4 and lysed in RIPA buffer  Triton X-100, 1% (wt./vol) sodium deoxycholate, 0.1% (wt./vol) SDS) containing 1 mM Na 3VO4, 50 mM NaF, 2 mM EDTA, 1 mM Pefabloc, 10 µg/ml of leupeptin, 2 µg/ml of aprotinin, 1 mM dithiothreitol, 1 µg/ml of pepstatin and 1 mM di-isopropyl fluorophosphate. Following addition of sample buffer  and boiling for 10 minutes at 95°C, lysates were separated by SDS-PAGE, transferred to Immobilon-F PVDF membrane (Millipore) and probed with antibodies as indicated.For NTAL phosphorylation, HEK293T cells were washed in ice-cold PBS containing 1 mM Na in vitro kinase assay, lysates from transfected HEK 293T cells were immunoprecipitated with anti-Syk antibody and incubated with protein-A/G Plus agarose beads (Santa Cruz Biotech). Beads were washed in kinase buffer  and then incubated in kinase buffer containing 5 µCi γ-ATP (PerkinElmer) for 20 minutes at 25°C. The reaction was stopped by addition of sample buffer and separated by SDS-PAGE. Gels were fixed and stained with Coomasie blue then dried and exposed to BioMax (Kodak) film.For the + infected fetal liver cells were collected and washed three times in cold PBS. Cells were lysed directly in sample buffer at 5 x106 cells/mL and analyzed by SDS-PAGE followed by immunoblotting.For analysis of phospho-STAT5 and total STAT5, GFP5 splenocytes or bone marrow cells were washed twice in PBS and lysed in sample buffer. Cell lysates were analyzed by SDS-PAGE followed by immunoblotting.For analysis of total phospho-tyrosine and Syk in tissues from mice reconstituted with vector, TEL-Syk KD or TEL-Syk transduced fetal liver cells, 5 x10Retroviral supernatants were generated by transient transfection of HEK-293T cells as previously described . HEK 2936 isolated fetal liver cells were spin-infected at 2000 rpm for 1 hour at 25°C in retroviral supernatants supplemented with 15% FBS, 8 µg/mL polybrene (Sigma), and cytokines, and incubated at 37°C overnight. Cells were washed the next day and placed in IMDM containing 15% (vol/vol) FBS and cytokines. Retroviral infection efficiency was determined by flow cytometry using GFP expression as a marker.Fetal liver cells from day 16-19 embryos were isolated and cultured overnight in Iscove’s Modified Dulbecco’s Medium (IMDM) containing 15% (vol/vol) FBS and cytokines . For retroviral infection, 5-7 x 10+ cells were sorted using a FACS Aria (BD Biosciences) and plated at a density of 2 x 104 in methylcellulose (StemCell Technologies), according to manufacturer’s directions, in 35 mm tissue culture dishes (BD Biosciences) with 1 ng/ml IL-3, 1 ng/ml IL-6, and 5 ng/ml SCF, which is indicated as the “1X” concentration in BALB/c fetal liver cells were isolated at embryonic day 16-19 and infected with retrovirus containing vector alone, Syk, TEL-Syk or TEL-Syk KD. A cytokine mix consisting of 10 ng/mL of murine IL-6, 20 ng/mL of murine IL-3, 100n g/mL of murine SCF, 10 ng/mL of IL-11, and 10 ng/mL of Flt3L in IMDM containing 15% FBS, adapted from Schubbert et al. . M. MJAK2V6in vitro data demonstrating that TEL-Syk drives phosphorylation of STAT5 and colony formation in fetal liver cells even in the presence of JAK inhibitors. Mechanistically, it is evident that these fusions  uncouple the JAK2-STAT5 pathway to drive disease progression.Induction of myeloproliferation with myelofibrosis has also been linked to expression of other tyrosine kinases fused to TEL. In particular, expression of a TEL-Lyn fusion protein, originally isolated from a patient with idiopathic myelofibrosis , in mousOverall, these results demonstrate that expression of the TEL-Syk fusion protein in fetal liver hematopoietic cells leads to rapid myeloproliferation and fatal myelofibrosis in a mouse model. The extent and the aggressive nature of this disease is unusual and will serve as a useful model for studying deregulated signaling in the context of myeloproliferative neoplasms with myelofibrosis. Future work with this model may allow identification of the soluble factor(s) derived from the proliferating myeloid cells that contribute to the extensive stromal fibrosis. Though many investigators have implicated myeloid cell-derived factors in myelofibrosis none have been defined.Figure S1TEL-Syk lead to myeloid cell expansion.Analysis of peripheral blood by flow cytometry. Graphs show total cell numbers (A) and numbers of (B) neutrophils (Ly6G+ CD11b+), (C) B cells (CD19+), and (D) T-cells (TCRβ+) at the indicated days following fetal liver cell transfer. Each data point indicates a single animal. Data are shown as mean ± SEM for vector (n=8), Syk (n=7), TEL-Syk (n=3-7)TEL-Syk KD (n=8). Note that this figure shows absolute numbers, while Figure 2H shows percentages, hence the difference in appearance of CD19+ B cells. (E) Detection of eosinophils (Siglec-F+ Ly6G-) in peripheral blood from a vector and TEL-Syk chimeric mouse at 30 days post cell transfer. Statistical significance was determined by one-way ANOVA. *P< 0.05, **P<0.01, ***P< 0.001.(TIF)Click here for additional data file.Figure S2Expression of TEL-Syk expression in bone marrow or fetal liver cells leads to increased induced tyrosine phosphorylation.(A) Immunoblots of 5x105 sorted GFP+ and GFP- bone marrow cells from vector, TEL-Syk and TEL-Syk KD chimeric mice were stained with the indicated antibodies. (B) Detection of TEL-Syk transcripts by RT-PCR from 1x105 sorted GFP+ and GFP- bone marrow cells from vector, TEL-Syk and TEL-Syk KD chimeric mice. The level of GAPDH was used as a control. (C) Immunoblots of 5x105 sorted GFP+ and GFP- from vector, Syk, TEL-Syk and TEL-Syk KD retrovirally transduced fetal liver cells were stained with the indicated antibodies.(TIF)Click here for additional data file.Figure S3TEL-Syk chimeric mice have abnormal red blood cell morphology.Wright-Giemsa stains of peripheral blood from vector and TEL-Syk transduced mice at 60 days post fetal liver cell transfer. Scale bars correspond to 10 µm.(TIF)Click here for additional data file.Figure S4Expression of TEL-Syk disrupted splenic architecture and induced dysplasia.H&E stained sections of spleens from (A) vector or (B D) TEL-Syk expressing mice. Scale bars correspond to 50 µm (A-C), and 10 μm (D).(TIF)Click here for additional data file.Figure S5TEL-Syk expression induced cellular infiltration and fibrosis in the liver.H&E stained sections of livers from (A) vector or (B) TEL-Syk expressing mice. Masson’s Trichrome stained sections of liver from (C) vector or (D) TEL-Syk chimeras to indicate fibrosis. Scale bars correspond to 500 µm (A-D).(TIF)Click here for additional data file.


Cardiac perfusion MRI requires fast data acquisition to achieve an appropriate combination of temporal resolution, spatial resolution and spatial coverage for clinical studies . We haveTo develop a respiratory motion correction method for joint compressed sensing and parallel imaging acceleration of first-pass cardiac perfusion MRI.Free-breathing first-pass cardiac perfusion MRI with 0.1 mmol/kg of Gd-DTPA (Magnevist) was performed using a modified multi-slice TurboFLASH pulse sequence. Healthy volunteers were imaged on a whole-body 3T scanner  using the standard 12-element body matrix coil array. The relevant imaging parameters include: FOV = 320mmx320mm, image resolution = 1.7mmx1.7mm, slice-thickness = 8mm, TE/TR = 1.3/2.5ms, repetitions=40. An acceleration factor of 8 was used to acquire 10 slices per heartbeat with temporal resolution of 60ms/slice. Data undersampling was performed using a pseudo-random ky-t pattern [Rigid respiratory motion correction significantly increased sparsity in the temporal Fourier domain, which is due to better alignment among frames Fig. . Fig. 2 This work demonstrates feasibility of highly-accelerated first-pass cardiac perfusion MRI without strict breath-holding with rigid respiratory motion correction. Future work will explore the use of non-rigid motion correction. The proposed technique may be useful for imaging patients with impaired breath-hold capabilities.


Caenorhabditis elegans. maco-1 mutants showed abnormal behaviors, including defective locomotion, thermotaxis, and chemotaxis. Expression of human macoilin in the C. elegans nervous system weakly rescued the abnormal thermotactic phenotype of the maco-1 mutants, suggesting that macoilin is functionally conserved across species. Abnormal thermotaxis may have been caused by impaired locomotion of maco-1 mutants. However, calcium imaging of AFD thermosensory neurons and AIY postsynaptic interneurons of maco-1 mutants suggest that macoilin is required for appropriate responses of AFD and AIY neurons to thermal stimuli. Studies on localization of MACO-1 showed that C. elegans and human macoilins are localized mainly to the rough endoplasmic reticulum. Our results suggest that macoilin is required for various neural events, such as the regulation of neuronal activity.Neural signals are processed in nervous systems of animals responding to variable environmental stimuli. This study shows that a novel and highly conserved protein, macoilin (MACO-1), plays an essential role in diverse neural functions in  Caenorhabditis elegans can sense diverse environment stimuli using a nervous system that consists of only 302 neurons. C. elegans exhibit thermotaxis that allows them to remember ambient temperatures and use this information as a cue to seek and remain near food sources. We show here that a novel and conserved protein, MACO-1, is important for proper execution of thermotaxis by C. elegans. The maco-1 gene was originally identified in a novel thermotaxis-defective mutant. Abnormal thermotaxis of maco-1 mutants was weakly but apparently rescued by expressing human MACO-1, suggesting that MACO-1 is functionally conserved among species. MACO-1 protein is required for appropriate regulation of neuronal activity, and the activity of neurons required for thermotaxis is impaired in maco-1 mutants. This analysis provides an important basis for the function of the macoilin family.Any animals, including humans, have to be capable of properly sensing and responding to various environmental stimuli for survival and reproduction. Environmental stimuli are evaluated and, based on past experiences, converted to produce appropriate adaptive behaviors. Even the small, free-living soil nematode  Drosophila melanogasterCaenorhabditis elegansC. elegans, more than 40 percent of the predicted protein products show significant matches to those from other animals Genome projects have determined DNA sequences of various organisms and identified locations of predicted genes in genomes. Surprisingly, numbers of genes are relatively similar across evolutionally divergent animal species. For example, estimated gene numbers are 20000–25000 in human C. elegans is composed of only 302 neurons C. elegans can sense an array of environmental stimuli and produce appropriate behavioral outputs. For example, worms remember cultivation temperatures in association with food conditions C. elegans with food at a certain temperature for several hours, they will migrate to this cultivation temperature along a temperature gradient C. elegansncs-1 animals could be rescued by introducing a wild-type NCS-1 into the AIY interneuron, which is a component of the thermotaxis neural circuit, and that NCS-1 overexpression enhanced the frequency of IT behavior Animals use the nervous system to detect stimuli in the surrounding environment. These stimuli are then assessed based on past experiences, and converted into adaptive behaviors. Many neurons are required to act in coordination to execute these processes. However, it is difficult to address individual neuronal function in relation to other neurons in neural circuits because animal brains generally consist of enormous numbers of neurons. The anatomically characterized nervous system of C. elegans, which was achieved by laser-ablating specific neurons, has been examined previously, and results led to the hypothesis of a neural circuit for thermotaxis The thermotaxis phenotype of C. elegans. maco-1/ttx-8 mutants exhibited abnormalities in thermotaxis and chemotaxis to odorants and sodium chloride (NaCl). The maco-1 gene encodes a conserved novel protein that is expressed in many neurons. A previous study reported that the mouse ortholog of MACO-1, macoilin (C61), showed neuron-specific expression maco-1 mutants is required for thermotaxis behavior. We also found that maco-1 mutants showed abnormal locomotion resulting from frequent turns. Although defective locomotion of maco-1 mutants might have caused abnormal thermotaxis, calcium imaging indicated that MACO-1 is required for activation of AFD and AIY neurons. In vivo co-localization studies have shown that both MACO-1 and human macoilin are localized to the rough endoplasmic reticulum (rER). All together, our results suggest that macoilin is required for a variety of intracellular events that influence basic neuronal function across species.In this study, we found that the novel protein MACO-1 is required for diverse neuronal functions in maco-1(nj21) and maco-1(nj34), were isolated as novel mutants. Two other mutant alleles, maco-1(tm2917) and maco-1(ok3165), were isolated by the National Bioresource Project (Japan) and the C. elegans Gene Knockout Consortium, respectively. All four alleles were genetically recessive (data not shown). Despite obvious defects in thermotaxis after cultivation at 20°C (maco-1(nj21) and maco-1(nj34) mutants were slightly defective in chemotaxis to odorants  than 17°C or 23°C TTX assays. Specifically, during 20°C TTX assays, animals must migrate and remain in the 20°C region, which is shown as the light grey zone in the left schematic of To elucidate molecular mechanisms of thermotaxis, we performed a genetic screen and isolated several mutants that were thermotaxis defective  at 20°C , maco-1(odorants  or to Naodorants . These rmaco-1 gene was identified by mapping it with the snip-single nucleotide polymorphisms (SNPs) method maco-1 mutants was rescued by introduction of the cosmid C09C9. However, thermotaxis was not rescued by the cosmid C15E12 or C03F5  did not rescue abnormal thermotaxis behavior of maco-1 mutants. However, another PCR fragment containing D2092.5 gene (PCR2) could rescue this behavior  and a missense mutation (A805T) (tm2917 mutant has a 780-base pair (bp) deletion and 1-bp insertion just before the first coiled coil domain  . The tm2l domain . The ok3l region .almost full-length genomic maco-1::GFP, which can rescue abnormal thermotaxis of maco-1 mutants .To study subcellular localization of MACO-1, we introduced the  mutants , into wi mutants . Because mutants . Similar mutants . Further mutants , and CFP mutants . These rmaco-1, we constructed the maco-1 promoter::GFP reporter that used an approximately 5-kilobase (kb) upstream region from the ATG initiation codon as a maco-1 promoter. This construct was then introduced into wild-type animals. We found that strong expression was observed in many neurons  mutants to thermal stimuli. We found that the ratio change in AFD neurons of maco-1(nj34) mutants was significantly less than that of wild-type animals (tax-4(p678) mutants was hardly detected tax-4(p678) mutant do not respond properly to temperature because of defective cGMP-dependent cation channels composed of TAX-2 and TAX-4. The average of maximum ratio change from baseline ± SEM in maco-1(nj34) mutants was slightly larger (4.3±0.8) relative to tax-4(p678) mutants (3.3±0.6) maco-1(nj34) mutants was not completely lost but partially reduced. Similarly, the ratio change in AIY neurons of maco-1 mutants was also lower than that of wild-type animals  is localized to the ER and required for the maturation  of nicotinic acetylcholine receptors maco-1 mutants. We found slightly increased spacing between adjacent SNB-1::GFP puncta (presynaptic marker) in DD motor neurons of maco-1 mutants compared with wild-type animals  I, IK734 maco-1(nj34) I, IK809 maco-1(tm2917) I, IK811 maco-1(ok3165) I, N2; Is[flp-13p::snb-1::gfp] II maco-1(nj21) and maco-1(nj34) mutants were isolated in genetic screens that bypassed temperature-dependent dauer formation of daf-7(e1372) backgrounds 4), 1 mM calcium chloride (CaCl2) and 25 mM potassium phosphate (pH 6)). Dilutions of odorants with ethanol were as follows: 1∶1000 diacetyl, 1 mg/ml pyrazine, 1∶100 isoamyl alcohol, and 1∶100 benzaldehyde. The chemotaxis index was calculated according to Bargmann et al. Animals were grown at 20°C for all behavioral assays in this study, unless otherwise described. The procedure for the thermotaxis assay using radial temperature gradients was based on previously published research by Mori and Ohshima maco-1 promoter::GFP (pMYA3) used in maco-1 promoter region . This fragment was then inserted into the GFP vector pPD95.77 to generate pMYA3. The almost full-length genomic maco-1::GFP (pMYA52) was constructed by PCR amplification of the 5′ 5-kilobase (kb) region of maco-1 gene and 2-kb fragment downstream of 5′ 5-kb fragment using the cosmid C09C9 and wild-type genomic DNA, respectively. These fragments were inserted between the maco-1 promoter and GFP of pMYA3 to generate the almost full-length genomic maco-1::GFP (pMYA52). The almost full-length genomic maco-1 contains a maco-1 promoter region and a 6621-bp fragment of a 7536-bp full-length genomic maco-1 sequence, which includes predicted transmembrane domains and coiled coil domains. The full-length maco-1 cDNA was constructed by PCR amplifications of the 5′ partial maco-1 cDNA (723-bp region of 2694-bp full-length maco-1 cDNA) and the rest of maco-1 cDNA region using a cDNA library and yk1296a05 clone, respectively. These fragments were ligated into pPD49.26 to generate pMYA17. All cell-specific promoter::maco-1 cDNA plasmids were generated by inserting each cell-specific promoter fragment into pMYA17. Cell-specific promoters are as follows: unc-14p for almost all neurons gcy-8p and gcy-18p for AFD neurons ttx-3p for AIY neurons lin-11p for AIZ neurons (unpublished data); odr-1p for AWC neurons glr-3p for RIA neurons (unpublished data). Detailed expression patterns of these cell-specific promoters are shown in maco-1 cDNA and several organelle markers used in glr-1 promoter of glr-1p::YFP::TRAMgcy-8 promoter to generate gcy-8p::YFP::TRAM (pMYA161). The TRAM of pMYA161 was replaced with maco-1 cDNA to generate gcy-8p::YFP::maco-1 (pMYA174). The YFP of pMYA161 was replaced with CFP to generate gcy-8p::CFP::TRAM (pMYA212). The TRAM of pMYA161 was replaced with FLJ10747 cDNA to generate gcy-8p::YFP::FLJ10747 (pMYA182). The glr-1 promoter of glr-1p::MANS::YFPgcy-8 promoter to generate gcy-8p::MANS::YFP (pMYA145), and then YFP of pMYA145 was replaced with CFP to generate gcy-8p::MANS::CFP (pMYA208). The glr-1 promoter of glr-1p::YFP::GPIgcy-8 promoter to generate gcy-8p::YFP::GPI (pMYA153). The glr-1 promoter of glr-1p::CFP::PISgcy-8 promoter to generate gcy-8p::CFP::PIS (pMYA141). The PIS of pMYA141 was replaced with maco-1 cDNA to generate gcy-8p::CFP::maco-1 (pMYA176). Two types of truncated forms of MACO-1, MACO-1 TM (1–453 amino acid) and MACO-1 CC (446–897 amino acid) (unc-14p::maco-1 cDNA and replaced with full-length maco-1 cDNA of unc-14p::maco-1 to generate unc-14p::MACO-1 TM (pMYA107) and unc-14p::MACO-1 CC (pMYA109), respectively. The unc-14p::FLJ10747 shown in unc-14 promoter into pPD49.26 vector to generate pMYA171. The full-length cDNA of human macoilin, FLJ10747 cDNA, was PCR amplified from a Mammalian Gene Collection (MGC) clone (Invitrogen), which contained full-length FLJ10747 cDNA. This product was inserted into pMYA171 to generate unc-14p::FLJ10747 (pMYA172).The no acid) , were PCalmost full-length genomic maco-1::GFP used in gcy-8p::YFP::maco-1 (pMYA174) (20 ng/µl) and gcy-8p::CFP::TRAM (pMYA212) (20 ng/µl) into wild-type animals. This injection was coupled with 120 ng/µl of pTAN124.5 (ges-1p::DsRed) as a co-injection marker. gcy-8p::YFP::FLJ10747 (pMYA182) (50 ng/µl) and gcy-8p::CFP::TRAM (pMYA212) (4 ng/µl) were co-injected into wild-type animals with ges-1p::DsRed (pTAN124.5) (120 ng/µl) as a co-injection marker. ges-1p::NLS-GFP (pKDK66) maco-1(nj34) at 2 and 50 ng/µl, respectively.Transgenic strains were constructed by injecting DNA into the gonad of adult animals Animal behavior was recorded using a custom made tracking and live imaging system. Well-fed single animal was placed on a TTX plate for tracking experiments. An ordinal analog video camera (Olympus) captured freely moving individual animal for 30 min with adequate sampling rate (30 frames/sec). A computer-controlled microscope stage was automatically moved to center animals in the visual field using a custom image analysis algorithm with microscope software . Midlines of recorded animals were extracted from each image, and curvatures at certain positions were calculated by analyzing points along midline with constant intervals. Spatio-temporal curvature maps were constructed by expressing the quantity of curvatures with a color-map . A smoothing filter (3×3 dimensions) was applied to the curvature map to reduce noise effects. Omega turn states  and AIYp::yc3.60 (pNR86.79), were glued onto a 2% agar pad on glass, immersed in M9 buffer, and covered by cover glass. The agar pad and M9 buffer were kept at the initial imaging temperature. Sample preparation was completed within one minute. The sample was then placed onto a Peltier-based thermocontroller  on the stage of a Nikon E600 or Olympus BX61WI microscope at the initial imaging temperature for two minutes. Fluorescence was introduced into W-View  or Dual-View  optic systems. Cyan fluorescent protein  and yellow fluorescent protein  images were simultaneously captured by a CCD camera HiSCA or EM-CCD camera C9000-13 ImagEM (Hamamatsu Photonics). Images were taken with a 200-ms exposure time with 4×4 or 2×2 binning. Temperatures on the agar pad were monitored by the thermometer system, DCM-20 (Tokai Hit and Hamamatsu Photonics). For each imaging experiment, fluorescence intensities of F535 and F480 were measured using AquaCosmos (Hamamatsu Photonics) or MetaMorph (Molecular Device) imaging analysis systems. Relative increases or decreases in intracellular Ca2+ concentrations were measured as increases or decreases in YFP/CFP fluorescence ratios of cameleon protein (ratio change).Error bars in figures indicate standard errors (SEM). The statistical analysis for behavioral experiments was performed by one-way analysis of variance (ANOVA) for multiple comparisons followed by post hoc Dunnett's multiple comparison. A single asterisk and double asterisk indicate statistical significance at p<0.05 and p<0.01, respectively. The value of p is probability.Figure S1maco-1(nj21) or maco-1(nj34) and index of wild-type at each concentration. One asterisk denotes statistical significance at the p<0.05 level and two asterisks denote statistical significance at the p<0.01 level (ANOVA with a Dunnett's post hoc test).Chemotaxis to odorants. (A) Chemotaxis to diacetyl with varying dilutions. (B) Chemotaxis to pyrazine with varying dilutions. (C) Chemotaxis to isoamyl alcohol with varying dilutions. (D) Chemotaxis to benzaldehyde with varying dilutions. Error bar indicates the standard error of the mean (SEM). Asterisks indicate statistically significant differences between the index of (0.26 MB TIF)Click here for additional data file.Figure S2maco-1(nj34) mutants. (A–C) All animals tested were cultivated at 20°C. The y-axis shows the fraction of 20 animals that migrated to the 20°C region. Error bar indicates the standard error of the mean (SEM). (A) Expression of almost full-length genomic maco-1::GFP (50 ng/µl) in most neurons of maco-1(nj34) mutants rescued the abnormal thermotaxis phenotype of maco-1. Statistical significance could not be described due to insufficient assay numbers (two). However, there was an obvious difference between the fraction of 20 of maco-1(nj34) and that of maco-1(nj34); Ex[almost full-length genomic maco-1::GFP] strain . (B) Rescue experiments with varying concentration of FLJ10747, human maco-1 cDNA . Single and double asterisks indicate fractions of 20 of each transgenic strain that was different from fraction of 20 of maco-1(nj34) mutants at the p<0.05 and p<0.01 level, respectively (ANOVA with a Dunnett's post hoc test). (C) n = 60–120 animals. Double asterisks indicate the fraction of 20 of maco-1(nj34); Ex[unc-14p::YFP::maco-1] strain that were different from that of maco-1(nj34) mutants at the p<0.01 level (ANOVA with a Dunnett's post hoc test).Rescue experiments for thermotaxis defects in (0.34 MB TIF)Click here for additional data file.Figure S3maco-1 promoter::GFP (pMYA3) in wild-type. DIC and GFP images are merged. Anterior is to the left. GFP expression was observed in many neurons. Names of several neurons are shown.  Expression of GFP in AFD and AIY neurons. (B) Wild-type. (C) maco-1 mutants. Left panels show merged DIC and GFP images and right panels are GFP images alone. A yellow arrowhead and white arrowhead indicate the cell body of AFD and AIY neurons, respectively. Yellow arrows show dendrites (left side of the yellow arrowhead) and axons (right side of the yellow arrowhead) of AFD neurons. A white arrow shows an axon of AIY neurons. Scale bars = 5 µm.Expression pattern of MACO-1 and neuronal morphology of AFD thermosensory neurons and AIY ineterneurons. (A) Expression of (1.93 MB TIF)Click here for additional data file.Figure S4maco-1(nj34) animals. This transgenic strain showed a partial-rescued phenotype (maco-1(nj34). This strain did not show the rescued phenotype (maco-1(nj34). This strain showed little to no rescued phenotype ::MACO-1, YFP::MACO-1 TM, and YFP::MACO-1 CC. (A–C) Expression of YFP::MACO-1 in almost all neurons and cyan fluorescent protein (CFP)::TRAM (rER marker) in AFD neurons of henotype . (A) YFPhenotype . (J) Merhenotype . (L) Mer(3.51 MB TIF)Click here for additional data file.Figure S5maco-1 mutants. (A–D) SNB-1::GFP expression in six DD motor neurons of L2 larva. We observed transgenic strains, N2; Is[flp-13p::SNB-1::GFP] maco-1(nj34); Is[flp-13p::SNB-1::GFP]. The expression pattern of flp-13p::GFP was shown in maco-1(nj34) mutant.  Arrowheads show cell bodies of DD2, DD3 and DD4 motor neurons. (E) Quantification of distance between adjacent puncta. We measured the distance between adjacent puncta and categorized it into sixteen classes. The x-axis and y-axis show each class and fraction of distance categorized into one of sixteen classes, respectively. Error bar indicates the standard error of the mean (SEM). The statistical difference was determined by using a two-tailed Student's test. A single asterisk indicates statistically significant difference at p<0.05. n = 27 (wild-type) and 30 (maco-1).Slight mislocalization of presynaptic marker, SNB-1::GFP, in (1.42 MB TIF)Click here for additional data file.Table S1Homology search. Results of a homology search by Blast using amino acid sequences of MACO-1 or human homolog of MACO-1 as queries. Several molecules of vertebrate or invertebrate are shown for each survey. The identity and similarity are calculated using the software program, Mac vector.(0.04 MB DOC)Click here for additional data file.Table S2lin-11p as an AIZ promoter, glr-3p as a RIA promoter and odr-1p as an AWC promoter Click here for additional data file.Text S1Supplemental (0.05 MB DOC)Click here for additional data file.


Mutations in TNFRSF1A can result in the autosomal dominant TNF receptor-associated periodic syndrome(TRAPS): a complex and heterogeneous systemic autoinflammatory disorder. Misfolding, intracellular aggregation and ligand-independent signalling by mutant TNFR1 play central roles in disease pathophysiology.This work was conducted to study the intracellular signalling pathway activation elicited by mutant TNFR1.To understand the complexity of intracellular signalling pathway perturbation in TRAPS, a prototypic mutant TNFR1 (C33Y), or wild-type TNFR1 (WT), were expressed at near physiological levels in an SK-Hep-1 cell model system. TNFR1-associated signalling pathway intermediates were examined under a range of conditions, employing reverse-phase protein microarray. Peripheral blood mononuclear cells (PBMC) from C33Y TRAPS patients and matched healthy controls were similarly examined.k B, p38, MEK/ERK and JNK MAP kinase pathways, Phosphoinositide 3 kinase, STAT3, JAK2/c-Src, Gsk-3β and transcription factors . Increased activated Jak2/STAT3 may contribute to an “IL6 amplifier” positive feedback loop that promotes and sustains a proinflammatory state.In comparison to cells expressing WT TNFR1 alone, expression of C33Y-TNFR1 in SK-Hep–1 cells and TRAPS patients’ PBMCs revealed a subtle up-regulation of a wide spectrum of signalling intermediates and their phosphorylated forms. These were associated with a proinflammatory/ anti-apoptotic phenotype, including NF-The study thus reveals the pleiotropic effect of a TRAPS-associated mutant form of TNFR1 on multiple inflammatory signalling pathways.None declared.


Each independent CuII cation is penta-coordinated within a distorted square-pyramidal environment defined by a two μ2-O atoms (derived from two 2-carboxy-5-nitrobenzoato anions), two N atoms (bipyridine ligand) and one Cl. Binuclear species are assembled into a two-dimensional supramolecular architecture parallel to (01The asymmetric unit of the title complex, [Cu DOI: 10.1107/S1600536811046782/im2329Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


Dendritic cells (DC) manipulate tissue homeostasis by recognizing dying cells and controlling immune functions. However, the precise mechanisms by which DC recognize different types of dying cells and devise distinct immunologic consequences remain largely obscure. Herein, we demonstrate that Milk-fat globule-EGF VIII (MFG-E8) is a critical mediator controlling DC immunogenicity in inflammatory microenvironments. MFG-E8 restrains DC-mediated uptake and recognition of necrotic cells. The MFG-E8-mediated suppression of necrotic cell uptake by DC resulted in the decreased proinflammatory cytokines production and activated signal components such as STAT3 and A20, which are critical to maintain tolerogenic properties of DC. Furthermore, the DC-derived MFG-E8 negatively regulates the cross-priming and effector functions of antigen-specific T cells upon recognition of necrotic cells. MFG-E8 deficiency enhances an ability of necrotic cell-primed DC to stimulate antitumor immune responses against established tumors. Our findings define what we believe to a novel mechanism whereby MFG-E8 regulates the immunogenicity of DC by modulating the modes of recognition of dying cells. Manipulating MFG-E8 levels in DC may serve as a useful strategy for controlling inflammatory microenvironments caused by various pathological conditions including cancer and autoimmunity. The major functions of MFG-E8 are to regulate immune homeostasis through the phagocytosis of apoptotic cells Milk-fat globule EGF-8 (MFG–E8) is a phosphatidylserine-binding protein secreted by subsets of myeloid cells that signals through engaging αhigh immature DC (iDC) were generated from the bone marrow cells of wild-type or MFG-E8-deficient mice using GM-CSF. The iDC treated with an inflammatory element such as CD40 ligand served as mature DC (mDC), in which MFG-E8 levels were low To investigate whether MFG-E8 has impacts on the activities and immunogenicity of DC, MFG-E8 + regulatory T cell differentiation and suppress antigen-specific adaptive immunity + PI− cells induced by the chemotherapeutic agent cisplatin (CDDP) or γ-irradiation (IR). Necrotic cells were induced by CDDP or γ-irradiation plus the caspase inhibitor zVAD-fms, and were confirmed as Annexin-V− PI+ populations by flow cytometry  -positive OT-I cells  . Moreove-I cells .Programmed necrosis mediates the activation of RIP1/3 kinase and deubiquitinating enzyme CYLD, which is counteracted by caspase-8 or FADD (Fas-associated protein with death domain) These results validate that efficient cross-priming of antigen-specific T cells by DC is correlated with the extent of necrotic cell recognition, which is further enhanced by deficiency or pharmacological inhibition of MFG-E8.Although DC are potent adjuvants for triggering antitumor immunity, the clinical effects of dendritic cell-based vaccination remain unsatisfactory First, wild-type Balb/c mice were immunized with irradiated CT26 colon cancer cells, by which CT26-derived antigens were efficiently recognized by immune cells in the tumor antigen-irrelevant naïve mice As a second model, immature BMDCs generated from wild-type or MFG-E8 KO mice were loaded with necrotic B16-OVA (MO-4) or B16 cells, and then used as a vaccine adjuvant against established B16-OVA tumors. The treatment with wild-type DC primed with necrotic B16 tumor cells was ineffective against established tumors. In contrast, the vaccination of MFG-E8-KO DC loaded with necrotic B16-OVA cells triggered substantial antitumor effects at greater levels than those of wild-type DC loaded with necrotic B16-OVA cells . The vacin vivo settings.These results indicate that pre-immunization or presence of tumor-specific antigens might be required for breaking tolerance, but MFG-E8 inhibition or deficiency enables DC vaccines to further promote tumor eradication in the + T cells in tumor-infiltrating lymphocytes isolated from mice than did necrotic cell-primed WT DC  and Prof. Shigeo Koyasu (Keio University), respectively. All experiments were conducted under a protocol approved by the animal care committees of Hokkaido University.The tumor cells  were obtained from the American Tissue Culture Collection (ATCC). B16 melanoma cells expressing OVA genes (B16-OVA: clone MO4) were kindly provided by Professor Hideo Yagita (Juntendo University) and used as described previously The design, preparation, and transduction of siRNA vectors were performed as described previously 5′-CCGAGACCAACTACTACAACCTGGA-3′ (#1); 5′-CCAACTACTACAACCTGGATGGAGA-3′ (#2); 5′-CCTCCAGCAGCTACAAGACATGGAA-3″(#3); 5′-CCCACTTGGGAAGGCTGGATAATCA-3′ (#4); 5′-CACATCCAGTATGTGGCGTCCTACA-3′ (#5).The phenotype of BMDC was analyzed by flow cytometry using anti-CD11c, anti-CD83, anti-CD86 and anti-MHC-II (BD Bioscience). For intracellular staining, BMDC were treated with brefeldin-A (Sigma-Aldrich), stained with anti-CD11c, fixed, permeabilized with Cytofix/Cytoperm buffer, and stained again with PE-conjugated Abs for phospho-Stat3 (BD Bioscience). In some cases, IFN-γ, IL-10 and IL-12 were quantified by ELISA (BD Bioscience) using supernatant obtained from cultured BMDC.B16 melanoma cells were treated with CDDP or exposed to γ-irradiation (30 Gy) to trigger apoptosis (more than 80%). Other cells were treated with CDDP or γ-irradiation in the presence of caspase inhibitor zVAD-fms or subjected to multiple cycles of freeze-thaw to trigger necrosis. The apoptotic or necrotic cells were labeled with PKH26 red fluorescent dye (Sigma-Aldrich). BMDCs were cocultured with the labeled apoptotic or necrotic cells for 4 h and phagocytosis efficiency evaluated by flow cytometry.BMDC transfected with control or MFG-E8 siRNA were subjected to Western blotting using antibodies against phospho-Stat3, Stat3, A20 .Beta-actin was used as a loading control to check the integrity of each sample. The densitometries of Stat-3, phospho-Stat-3 or A20 were quantified using image J software.The transcriptional activities of DC were assayed as described previously + cells were isolated by magnetic cell sorting (Miltenyi Biotec), and cocultured with naïve CD8+ T cells from the spleens of OT-I mice for 72 h. Intracellular IFNγ or Granzyme-B expression in T cells was then determined by flow cytometry. In some experiments, the tumor cells were pretreated with Necrostatin-1 (100 μM) before the co-culture to evaluate the contribution of necrotic cell uptake to the cross-priming of OVA-specific CTLs.BMDCs were co-cultured in 96-well round-bottom plates for 4 h with apoptotic or necrotic B16-OVA or antigen-irrelevant B16 or EL-4 cells (1∶10 ratio) that had been labeled with PKH26. CD11c6/mouse) once a week for three times. The immunized mice were injected subcutaneously with a mixture of viable CT26 cells and BMDCs transfected with control or MFG-E8 siRNA-encoded lentiviral vectors and pulsed with necrotic CT26 cells or not (1×106/mouse) on day 7 and 14 after tumor inoculation. In additional experiments, C57BL/6 mice were challenged subcutaneously in the flank with B16 melanoma cells on day 0. For the therapy model, intratumoral administration of wild-type or MFG-E8-KO DC (1×106/mouse) pulsed with necrotic B16 or B16-OVA cells were performed once a week for two times. Tumor growth was measured every three days.For in vivo tumor experiments, Balb/c mice were immunized with irradiated CT26 colon cancer cells .Tumor infiltrating lymphocytes were obtained from B16-OVA challenge sites using a Nocoprep (Axis-Shield) cell gradient separation. The frequencies of CD8t test or the two-sample t test with Welch's correction. The differences among three or more groups were determined with a one-way ANOVA. The P values less than 0.05 are considered statistically significant. * p<0.05, ** p<0.01, ns: not significant.The differences between two groups were determined with the Student's Figure S1Necrostatin-1 suppresses necrotic cell death mediated by CDDP and zVAD-fms. EL4 thymoma cells were treated with chemotherapeutic agent cisplatin (CDDP), γ-irradiation (IR) with or without pan-caspase inhibitor zVAD-fms to induce necrosis and apoptosis, respectively. In some instances, RIP-1 kinase inhibitor Necrostatin-1 was added before CDDP and zVAD-fms treatment. The necrotic or apoptotic cell death was shown as annexin-V−/PI+ or annexin-V+/PI+ or – populations, respectively, by flow cytometry.(TIF)Click here for additional data file.Figure S2In vivo vaccination with MFG-E8-KO DC enhanced granzyme-B expression in intratumor CTL. WT or MFG-E8-KO BMDC was loaded with apoptotic or necrotic cells derived from B16-OVA, and inoculated into B16-OVA tumors raised from C57/BL6 mice. The cells were isolated from tumor tissues 5 days after DC treatment, and analyzed for Granzyme-B expression on CD8+T cells by intracellular flow cytometry. Representative data (above) and statistical analysis of total experiments (n = 2) (bottom) is shown.(TIF)Click here for additional data file.


Myocardial phase velocity mapping studies have generally been acquired using Cartesian k-space coverage and respiratory gating ,2. AcquiThe sequence (TR=30ms) consists of five interleaved spiral k-space paths (23ms). Reference and 3-directional velocity encoded data  are acquired in consecutive cardiac cycles following a single dummy cycle (breath-hold duration 21 cardiac cycles). A black-blood suppression pulse (6ms) is output on alternate phases to reduce blood flow artefacts and retrospective gating allows full coverage of the cardiac cycle. Forty phases are reconstructed  with an acquired spatial resolution of 2.4 x 2.4 x 8mm (reconstructed 1.2x1.2mm).Basal, mid and apical short axis slices were acquired in 6 healthy volunteers on a Siemens Skyra 3Tesla scanner. Radial, circumferential and longitudinal velocities were calculated pixel-wise and as an average over six semi-automatically segmented regions of the left ventricle.Figure We have developed a phase velocity mapping technique which allows 3D myocardial velocities to be acquired in a single breath-hold. The spiral k-space coverage results in reasonable temporal resolution (33ms) and the implementation of retrospective ECG-gating allows analysis of the entire cardiac cycle, including atrial contraction. Future work will include implementing an on-line correction map to reduce off-resonance blurring and parallel imaging to reduce the breath-hold duration.Heart Research UK grant:RG2584.


The relevance of these defenses to the control of HIV-1 in vivo remains to be elucidated. We hypothesized that cellular restriction of HIV-1 replication plays a significant role in the observed suppression of HIV-1 in "elite controllers", individuals who maintain undetectable levels of viremia in the absence of antiretroviral therapy (ART). We comprehensively compared the expression levels of 34 host restriction factors and cellular activation levels in CD4+ T cells and sorted T cell subsets between elite controllers, HIV-1-infected (untreated) non-controllers, ART-suppressed, and uninfected individuals.Several host-encoded antiviral factors suppress HIV-1 replication in a cell-autonomous fashion 2 = 0.597, p < 0.0001), viral load , and expression of ISG15 , a marker of interferon exposure. APOBEC3C, APOBEC3D, CTR9, TRIM26, and TRIM32 were elevated in elite controllers with respect to ART-suppressed individuals, while levels were comparable to uninfected individuals and non-controllers.Expression of schlafen 11, a codon usage-based inhibitor of HIV-1 protein synthesis, was significantly elevated in CD4+ T cells from elite controllers as compared to both non-controllers (p = 0.048) and ART-suppressed individuals (p = 0.024), with this effect most apparent in central memory CD4+ T cells. Schlafen 11 expression levels were comparable between controllers and uninfected individuals. Cumulative restriction factor expression was positively correlated with CD4+ T cell activation (rHost restriction factor expression typically scales with cellular activation levels. However, the elevated mRNA and protein expression of schlafen 11, despite low activation and viral load, violates the global pattern and may be a signature characteristic of HIV-1 elite control. To date, there is no effective cure or prophylactic vaccine for HIV-1 infection. Antiretroviral therapy (ART) has dramatically decreased the morbidity and mortality associated with HIV-1 infection. However, there is a demand for alternative clinical management strategies due to the necessity of lifelong therapy, evolution of antiretroviral resistance, toxicity issues, and substantial costs of current regimens Naïve (CD45RO-CCR7+CD27+CD57-); [2] Central Memory (CD45RO+CCR7+CD27+); [3] Transitional Memory (CD45RO+CCR7-CD27+); and [4] Effector Memory (CD45RO+CCR7-CD27-) , followed by 15 min centrifugation at 12,000 g at 4°C. RNA was extracted from the aqueous layer using the miRNeasy Mini kit (Qiagen) with on-column DNAase treatment (Qiagen RNase-Free DNase Set) and eluted in 30 μl of RNase-free water. DNase-treated, clean RNA was transcribed into cDNA using random primers and the SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen), according to manufacturer’s instructions. Quantitative real-time PCR utilized custom-made TaqMan® Low Density Arrays (TLDA) from Applied Biosystems following the manufacturer’s instructions. TaqMan Low Density Array (TLDA) cards  are 384-well microfluidic cards with eight ports, each containing 48 connected wells. The primers and probes for each assay were preloaded and dried onto the designated duplicate wells. All probes used on our card are conjugated to 6-carboxyfluorescein (FAM). Black hole quencher 1 (BHQ1) was used as a quencher for all probes. The Cumulative Restriction (CuRe) TLDA card is designed to run 4 samples in duplicate on one card. Each port tests against 34 different antiviral genes and a panel of housekeeping genes. All assays and their respective target genes are listed in Table th gene is notated as ei, i = 1,2,…,n, assuming there are n genes. Missing values were imputed using the minimum expression value across samples for each gene, respectively. The median gene expression profile is calculated for the HIV-1-uninfected control group. A reference sample with the maximum number of genes that are closest to the median gene expression profile is the chosen from the uninfected control group. The reference expression value for the ith gene is notated as ri, i = 1,2,…,n. The gene expression profile for each sample was standardized by calculating the ratio against the reference sample. The CuRe score for a sample is the cumulative fold-difference in antiviral gene expression with respect to a control individual, expressed by the following formula:The expression value for the iEight cryopreserved PBMC samples  were chosen for protein quantitation. CD4+ T cells were negatively selected from PBMC as described previously. 15 μl of cellular lysates in NuPAGE LDS Sample Buffer with 2-mercaptoethanol  were resolved by NuPAGE 4-12% gel, then transferred to PVDF membrane. The membrane was blocked by 5% non-fat dry milk/TBST and incubated with anti-SLFN11 mAb : sc-374339) or anti-GAPDH  XP® Rabbit mAb #5174) in 5% BSA/TBST at 4C overnight. The next day, the membrane was washed and incubated with Cell Signal Tech anti-mouse or anti-rabbit HRP antibodies respectively and developed with PerkinElmer Western Lightning ECL Pro. The immunoblotting bands were quantified with ImageJ64 software. The quantified SLFN11 protein expression levels were normalized to corresponding GAPDH protein levels to ensure equal loading.Cryopreserved PBMCs were quickly thawed, washed in FACS buffer  and surface stained for 30 min at 4°C. Activation panel included the following antibodies: CD3-ECD , CD4-PacificBlue , CD8-QDot605 , CD14-APC/Cy7 , CD19-APC/Cy7 , CD38-PE , HLA-DR-FITC , BD Pharmingen), and a live/dead marker (Invitrogen) emitting in the aqua wavelength. After staining, the cells were washed three times, fixed in 2% paraformaldehyde, and events were collected on a Becton Dickinson LSR-II, using FACS DIVA software (BD Biosciences). Data were analyzed using FlowJo software . FMOs were carried out for compensation purposes. T cell activation levels are reported as the percentage of CD4+ T cells co-expressing HLA-DR and CD38 and the mean fluorescence intensity (MFI) of HLA-DR.A battery of statistical tests including unpaired t tests and Pearson’s r correlation coefficient were applied to data using GraphPad Prism v5.0c.APOBEC: Apolipoprotein B mRNA editing enzyme; BST-2: Bone marrow stromal cell antigen 2; SAMHD1: SAM domain and HD domain-containing protein 1; TRIM: Tripartite motif; ISG: Interferon-stimulated gene; CDKN1A: Cyclin-dependent kinase inhibitor 1A; PAF1: RNA polymerase II associated factor; EIF2AK2: Eukaryotic translation initiation factor 2-alpha kinase 2; HERC5: HECT domain and RLD 5; IFITM: Interferon induced transmembrane; MOV10: Moloney leukemia virus 10 homolog; SLFN11: Schlafen family member 11; TLDA: TaqMan® low density array; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; 18S: 18S ribosomal RNA; ACTB: Beta-actin; PPIA: Peptidylprolyl isomerase A; RPLP0: 60S acidic ribosomal protein P0; UBC: Ubiquitin C; HN: HIV negative controls; EC: Elite controllers; AS: ART suppressed; NC: Non-controllers.The authors declare that they have no competing interests.MAM, RASR, TL, ES and SKP performed gene expression and flow cytometry-based experiments, ML and MD performed western blotting, and XD performed statistical analyses. RH and SGD coordinated patient recruitment. MAM, SKP, JKW, DFN, SGD, MSS and HEAG designed the studies. MAM and SKP wrote the paper. All authors read and approved the final manuscript.Table S1. Subject characteristics. Table S2. P values for inter-disease state gene expression comparisons.FACS-based sorting of CD4+ T cell subpopulations. PBMCs were isolated by Ficoll density gradient, stained with aqua amine reactive dye, and then stained with fluorescently-conjugated monoclonal antibodies against CD3, CD4, CD45RO, CD27, CCR7, CD57 and CD14. After gaining on lymphocytes and singlets, and excluding non-viable cells (using amine reactive dye) and CD14+ monocytes, CD3+CD4+ cells were sorted as illustrated: (1) Naïve (CD45RO-CCR7+CD27+CD57-); (2) Central Memory (CD45RO+CCR7+CD27+); (3) Transitional Memory (CD45RO+CCR7-CD27+); and (4) Effector Memory (CD45RO+CCR7-CD27-). Click here for file


These results suggest that Rab39a negatively regulates the LPS-induced autophagy in macrophages.Rab39a has pleiotropic functions in phagosome maturation, inflammatory activation and neuritogenesis. Here, we characterized Rab39a function in membrane trafficking of phagocytosis and autophagy induction in macrophages. Rab39a localized to the periphery of LAMP2-positive vesicles and showed the similar kinetics on the phagosome to that of LAMP1. The depletion of Rab39a did not influence the localization of LAMP2 to the phagosome, but it augments the autophagosome formation and LC3 processing by lipopolysaccharide (LPS) stimulation. The augmentation of autophagosome formation in Rab39a-knockdown macrophages was suppressed by Atg5 depletion or an inhibitor for phosphatidylinostol 3-kinase (PI3K). Immunoprecipitation analysis revealed that Rab39a interacts with PI3K and that the amino acid residues from 34 Rab GTPases localize to specific subcellular organelles and regulate various membrane trafficking ,2. The rToll-like receptors (TLRs) are pattern recognition receptors to detect infection by recognizing the conserved pathogen-associated molecular patterns such as lipopolysaccharide (LPS) and trigger innate immune responses to defend invading microbes ,11. HoweIn macrophages, the stimulation by LPS or other TLR ligands induces a unique lysosomal degradation pathway for cytoplasmic materials termed autophagy -19. AutoIn this study, we characterized the functions of Rab39a in membrane trafficking, phagocytosis and autophagy induction in macrophages. We found that Rab39a interacts with class III phosphatidylinositol 3-kinase (PI3K) and regulates autophagy induced by various TLR stimulations. These results imply the possibility that Rab39a is a potent target molecule in the clinical therapy for the sepsis syndrome.Animal experiments in this study were approved by the Hamamatsu University School of Medicine Animal Care Committees at the Center Animal Care facility (permit number: 2012074). Mice were sacrificed by cervical dislocation and all efforts were made to minimize suffering.Raw264.7 and HEK293T cells were obtained from ATCC and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 25 μg/ml penicillin G and 25 μg/ml streptomycin. Bone marrow-derived macrophages (BMM) were differentiated from bone marrow cells of C57BL/6 mice by culturing in DMEM supplemented with 30% L929-conditional medium, 10% FBS and above antibiotics.Dpn I, followed by the transformation. Transfection of Raw264.7 or HEK293T with plasmid was performed using an MP-100 electroporator  or X-tremeGENE 9 (Roche), respectively, according to the manufacturer’s instructions.Construction of the plasmid for EGFP-Rab39a was described previously . PCR forRat anti-mouse LAMP2 monoclonal antibody (SouthernBiotech), rabbit anti-LC3 polyclonal antibody (MBL), mouse anti-tubulin monoclonal antibody (Sigma-Aldrich), rabbit anti-p62 antibody (MBL), mouse anti-ubiquitin antibody (MBL), rabbit anti-Beclin1 antibody (MBL), rabbit anti-GM130 antibody (MBL), mouse anti-EGFP monoclonal antibody (Clonetech), rat anti-EGFP monoclonal antibody  , mouse anti-c-Myc antibody (Wako), rabbit anti-Vps34 antibody (CST), mouse anti-UVRAG antibody (MBL) and rabbit anti-ATG14L antibody (MBL) were used as primary antibodies. Alexa488- or Alexa546-conjugated anti-IgG antibodies (Invitrogen) and horseradish peroxidase-conjugated anti-IgG antibodies (Dako) were used as secondary antibodies.Immunofluorescence and thin-section electron microscopic analyses were performed as described previously ,24. For 5 cells) grown on 35-mm glass dishes were allowed to phagocytose latex beads for 2 h. FRAP analysis was performed using an FV1000-D confocal microscope (Olympus) with a 60×/1.4 numerical aperture oil-immersion objective lens. The area of the phagosome surrounded by EGFP-Rab39a was photobleached using a 410-nm laser at 15–20% power for 300 ms after pre-bleached images were acquired. Following photobleaching, the recovery of fluorescence was monitored at every 2 sec using a 488-nm laser at 1-2 % power for the phagosomes containing latex beads. Fluorescence intensities were quantified using the ImageJ (http://rsb.info.nih.gp/ij/).Fluorescence recovery after photobleaching (FRAP) analysis was performed as described previously . BrieflysiRNA duplexes were synthesized by Sigma-Aldrich as listed in 3VO4, and protease inhibitor cocktail (Roche), respectively. Cell lysates were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then subjected to immunoblot analysis using anti-LC3 antibody (1:500 v/v), anti-tubulin antibody (1:1000 v/v), anti-p62 antibody (1:500 v/v), anti-c-Myc antibody (1:1000 v/v) and mouse anti-EGFP antibody (1:1000 v/v).For immunoblot (IB) analysis, macrophages or HEK293T cells were extracted with 8 M urea or immunoprecipitation (IP) buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride, 1 mM NaIP analysis was performed as described previously . For IP t-test was used to assess the statistical significance of differences between the two groups. To assess the proportions of cells with LC3-dot or Beclin1-dot, three or four independent experiments were conducted, and more than 200 cells were counted for each condition. Fluorescence with more than 1 μm-diameter was judged as LC3-dot or Beclin-1 dot.The paired or unpaired two-sided Student’s E. coli-containing phagosomes  macrophages. LAMP2 localized to the phagosomes in Rab39a-KD macrophages as in control macrophages , we examined the effect of Rab39a depletion on autophagy induction. To investigate the effect of Rab39a depletion on classical autophagy pathway, we treated Rab39a-KD macrophages with rapamycin and found that Rab39a depletion does not influence autophagy induced by rapamycin -κB ,39 that Beclin1-Vps34 complex regulates distinct membrane trafficking pathways by interacting with different regulatory proteins ,41. It iIn summary, our results suggest that TLR stimulation induces autophagy via the activation of two distinct pathways, i.e., increase of p62 expression and activation of class III PI3K. Rab39a could interact with PI3K and negatively regulate its activity in induction of autophagy by TLR stimulation. Therefore, Rab39a depletion resulted in the augmentation this type of autophagy. Our results also imply the possibility that Rab39a is a potent target molecule to control the sepsis syndrome because Rab39a regulates the inflammatory responses by controlling Caspase-1 activity and autophagy induction in response to LPS stimulation.Figure S1Rab39a localizes to lysosomes. (A) Projection of focal planes with y-z and x-z side views of E. coli. Raw264.7 macrophages expressing EGFP-Rab39a were infected with Alexa-405 labeled E. coli for 2 h and immunostained with anti-LAMP2 antibody. An arrowhead indicates a Rab39a-positive, LAMP2-positive E. coli-containing phagosome.(TIF)Click here for additional data file.Figure S2LAMP2 localization to phagosomes in Rab39a-KD macrophages. (A) RT-PCR analysis of Raw264.7 macrophages transfected with siRNA duplexes for Rab39a. (B) Quantitative analysis of Rab39 transcription. Rab39a mRNA level was assessed by RT-qPCR using GAPDH mRNA as a control. (C) Localization of LAMP2 to E. coli-containing phagosomes in Rab39a-KD macrophages. Raw264.7 macrophages transfected with siRNA for Rab39a were incubated with TexasRed-labeled E. coli for 2 h and were immunostained with anti-LAMP2 antibody. (D) The proportion of LAMP2-positive phagosomes in Rab39a-KD macrophages. Data represent the mean and SD of three independent experiments. *p < 0.05 (unpaired Student’s t-test).(TIF)Click here for additional data file.Figure S3Rapamycin-induced autophagy in Rab39a-KD macrophages. Raw264.7 macrophages transfected with control or Rab39a siRNA were treated with rapamycin at 50 μg/ml for 2 h. (A) Cells were fixed and immunostained with anti-LC3 antibody. (B) Cell extracts were subjected to immunoblot analysis for anti-LC3 antibody.(TIF)Click here for additional data file.Figure S4Characterization of LPS-induced autophagosome formation in Rab39a-KD macrophages. (A) Thin-section electron micrograph of Rab39a-KD macrophages. Raw264.7 macrophages transfected with control or Rab39a siRNA were treated with LPS at 10 ng/ml for 24 h and observed by thin-electron microscopy. An arrowhead indicates the electron dense-aggregation. (B) Co-localization of LC3 or p62 and ubiquitin in Rab39a-KD macrophages treated with LPS. Raw264.7 macrophages transfected with Rab39a siRNA were treated with LPS for 24 h and immunostained with anti-LC3 or anti-p62 and anti-ubiquitin antibodies.(TIF)Click here for additional data file.Figure S5Augmentation of autophagy induced by LPS in Rab39a-KD BMM. (A) Immunofluorescence analysis of LC3 distribution. BMM transfected with control or Rab39a siRNA were treated with LPS for 24 h and immunostained with anti-LC3 antibody. (B) Immunoblot analysis of LC3 processing in BMM treated with LPS. Raw264.7 macrophages transfected with control or Rab39a siRNA were treated with LPS for 24 h and subjected to immunoblot analysis using indicated antibodies. *, non-specific band.(TIF)Click here for additional data file.Figure S6A) The quantification of band intensity for LC3-II in ( The ratio of band intensity for LC3-II/tubulin at each condition to that in control macrophages is shown. Data represent the mean and SD of three independent experiments. *p < 0.05 (paired Student’s t-test). (B) The quantification of band intensity for immunoprecipitated proteins with EGFP-Rab39a in p < 0.05 (paired Student’s t-test).(TIF)Click here for additional data file.Figure S7Expression level of p62 in Rab39a-KD macrophages. Raw264.7 macrophages transfected with control or Rab39a siRNA were treated with LPS for 24 h. Cell extracts were subjected to immunoblot analysis for anti-p62 antibody.(TIF)Click here for additional data file.Figure S8Interaction of Rab39a with Beclin1 or Bcl2. (A) HEK293T cells were transfected with plasmid for EGFP-Rab39a or EGFP-Rab39b. Whole cell lysates (WCL) were used for immunoprecipitation (IP) with anti-Beclin1 antibody, followed by immunoblot analysis (IB) with anti-GFP antibody. For detection of input, aliquots of 5 μg of WCL were used. (B) HEK293T cells were transfected with plasmids for Myc-Bcl2 and EGFP-Rab39a or EGFP-Rab39b. WCL were used for IP with anti-GFP antibody, followed by IB with anti-Myc antibody. For detection of input, aliquots of 5 μg of WCL were used.(TIF)Click here for additional data file.Figure S9Subcellular localization of Rab39b and Rab39a_M1 in macrophages. Raw264.7 macrophages were transfected with the expression plasmid for EGFP-Rab39a, EGFP-Rab39b or EGFP-Rab39a_M1 and immunstained with anti-LAMP2 antibody  or anti-GM130 antibody .(TIF)Click here for additional data file.Figure S10Enlarged images of  Quantification for the fluorescence intensities across the arrows in right panels is also shown.(TIF)Click here for additional data file.Figure S11th to 41st of Rab39a is important for interaction of Rab39a with Beclin1.Amino acid residues from 34 (A) ClustalW alignment of the amino acid sequences of Rab39a, Rab39b, Rab39a_M1 and Rab39b_M1 is shown. (B) HEK293T cells were transfected with plasmids for Myc-Beclin1 and EGFP-Rab39a, EGFP-Rab39b, EGFP-Rab39a_M1 or EGFP-Rab39b_M1. WCL were used for IP with anti-GFP antibody followed by IB with anti-Myc antibody. For detection of input, aliquots of 15 μg of WCL were used. (C) Quantification of band intensity for immunoprecipitated Beclin1. Band intensity of Beclin1 for IP/WCL in p < 0.05 (paired Student’s t-test).(TIF)Click here for additional data file.Movie S1FRAP analysis of EGFP-Rab39a on the phagosome. This movie corresponds to (MOV)Click here for additional data file.Table S1Primer list for plasmid constructions.(XLS)Click here for additional data file.Table S2siRNA list for gene silencing.(XLS)Click here for additional data file.Table S3Primer list for real time-quantitative PCR.(XLS)Click here for additional data file.