Trans-activation response element (TAR) DNA binding protein of 43kDa (TDP-43) is causally related to the neurodegenerative diseases frontotemporal dementia and amyotrophic lateral sclerosis being the hallmark protein in the disease-characteristic neuropathological lesions and via genetic linkage. Histone deacetylase 6 (HDAC6) is an established target of the RNA-binding protein TDP-43. HDAC6 is an unusual cytosolic deacetylase enzyme, central for a variety of pivotal cellular functions including aggregating protein turnover, microtubular dynamics and filopodia formation. All these functions are important in the context of neurodegenerative proteinopathies involving TDP-43. We have previously shown in a human embryonic kidney cell line that TDP-43 knockdown significantly impairs the removal of a toxic, aggregating polyQ ataxin-3 fusion protein in an HDAC6-dependent manner. Here we investigated the influence of TDP-43 and its target HDAC6 on neurite outgrowth.in vitro differentiated SH-SY5Y cells.Human neuroblastoma SH-SY5Y cells with stably silenced TDP-43 showed a significant reduction of neurite outgrowth induced by retinoic acid and brain-derived neurotrophic factor. Re-transfection with TDP-43 as well as HDAC6 rescued retinoic acid-induced neurite outgrowth. In addition, we show that silencing of HDAC6 alone is sufficient to reduce neurite outgrowth of TDP-43 deficiency leads to impairment of neurite growth in an HDAC6-dependent manner, thereby contributing to neurodegenerative events in TDP-43 diseases. Trans-activation response element (TAR) DNA binding protein of 43kDa (TDP-43) is the neuropathological hallmark protein of a new class of neurodegenerative dementias and movement disorders comprising certain types of frontotemporal lobar atrophy (FTLD-TDP) and amyotrophic lateral sclerosis (ALS) . There iHDAC6 was consistently identified by systematic sequencing of RNA isolated by crosslinking immunoprecipitation using TDP-43 antibodies [HDAC6 mRNA and regulates its expression [TDP-43 was originally identified as a protein binding to TAR DNA sequences within human immunodeficiency virus type 1 and acting as a strong transcriptional repressor , apolipotibodies ,14. TDP-pression ,15.HDAC6 is an unusual, cytosolic deacetylase with manifold cellular functions. For example, HDAC6 is centrally involved in misfolded protein and organelle degradation processes . HDAC6 rTDP cells stably expressing TDP-43 directed shRNA compared to control parental SH-SY5Y cells  beyond HDAC6, which remain to be identified.To accommodate the shorter time frames for transient re-transfections, cells were differentiated only with RA for 4d. Under these conditions, we observed no significant difference in the number of neurites per cell Figure  and 3C th Figure , indicatHDAC6) by treating parental cells with different amounts of lentiviral shRNA against HDAC6. This resulted in dose-dependent decrease of HDAC6 protein  that maHdac6-/- mouse embryonic fibroblasts showed reduced activity of the Rho-like GTPase Rac1, which was correlated to the HSP90 deacetylase activity of HDAC6 [Although the overall phenotype of HDAC6 knockout mice is very mild despite abnormal acetylation levels of tubulin and HSP90, at least in testis and spleen , a more of HDAC6 . Finallyof HDAC6 . Our newTDP cells restores neurite outgrowth. Silencing HDAC6 directly causes severe cytoskeletal rearrangements and loss of neurite outgrowth in human neuroblastoma SH-SY5Y cells. Thus, TDP-43 and HDAC6 are in a linear cascade mediating neurite outgrowth. Disturbing this pathway in human TDP proteinopathies may contribute to neurodegeneration.TDP-43 deficiency causes impaired neurite outgrowth. TDP-43 silencing downregulates HDAC6 levels, and transfection of HDAC6 into sh2. Stably silenced shTDP SH-SY5Y cells were described previously [HDAC6 cells were generated by treating parental SH-SY5Y cells with an HDAC6-specific shRNA lentiviral clone , which targets the HDAC6 mRNA in the 3'UTR and therefore allows efficient re-transfection with cDNA. 12,500 cells were treated with 1.5 or 0.22 × 105 transforming units (TU) for 48 h. Selection was performed by adding puromycin (Invivogen) to the culture medium (1 μg/ml).Human neuroblastoma SH-SY5Y cells (ATCC) were grown in Dulbecco's modifies eagle medium: F12 (Biochrom AG) supplemented with 10% fetal bovine serum (PAA Laboratories) under humidified conditions at 37°C and 5%COeviously . Stably Cells were collected and lysed in lysis buffer (50 mM Tris (pH 7.4), 50 mM NaCl, 1% NP-40, 0.1% deoxycholate, and 0.1% SDS, 1× Complete proteinase inhibitor (Roche)). Protein concentration was determined by use of bicinchoninic acid (Pierce Biotechnology). Protein was subjected to SDS-PAGE using 4-12% Bis-Tris NuPAGE gradient gels (Invitrogen) and transferred onto nitrocellulose. Membranes were incubated with rabbit anti-TDP-43 , rabbit anti-HDAC6  or a mouse monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH)  overnight followed by horseradish peroxidase-conjugated secondary antibodies . Bands were visualized with ImmobilonWestern Chemiluminescent HRP Substrate (Millipore) on Hyperfilm ECL high performance chemiluminescence .In vitro differentiation of parental SH-SY5Y cell clones was performed on basis of a previous report with modifications [ications . In brieFor immunostaining, cells were fixed with 4% paraformaldehyde for 20 min at room temperature followed by permeabilization with 1% Triton X-100 in phosphate-buffered saline (PBS) for 30 min. Cells were blocked in 10% normal goat serum and incubated with anti-Flag , anti-Myc  or anti-HDAC6  and/or with Alexa568-coupled phalloidin (Molecular Probes) for 1 h at room temperature followed by incubation with secondary antibody anti-mouse AlexaFluor488 or anti-mouse AlexaFluor647 (both Molecular Probes) for 1 h at room temperature in 1% bovine serum albumin (BSA) in PBS. Cells were washed in PBS and nuclei counterstained with Hoechst 33342  before mounting the cover slips onto slides using fluorescence mounting medium (Dako). Confocal fluorescent images were taken with an AxioImager microscope equipped with an ApoTome Imaging System (Zeiss).Neurites were quantified using Neurolucida software . After manual tracing quantified neurite parameters include total length of neurites (μm), number of neurites and nodes per cell. Mean neurite length was calculated as ratio of total neurite length and number of neurites. Quantified were at least 50 cells per experiment in at least three independent experiments. Statistical analysis was performed with two-sided, paired student's t-test.Cells were transiently transfected 24 h after RA addition with Myc-HDAC6 or Flag-TDP-43 wt using Lipofectamine 2000 (Invitrogen). Constructs pCMV Myc-HDAC6 and pcDNA3.1(-) Flag-TDP-43 have been described previously . Cells wALS: amyotrophic lateral sclerosis; BDNF: brain-derived neurotrophic factor; FTLD: frontotemporal lobar degeneration; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HDAC: histone deacetylase; PBS: phosphate-buffered saline; RA: retinoic acid; shRNA: small hairpin RNA; TDP-43: trans-activation response element (TAR) DNA binding protein of 43kDa; TU: transforming unitsThe authors declare that they have no competing interests.FCF, CS and SSW performed and analyzed the experiments. FCF and PJK designed the study and wrote the paper. PJK is the principal investigator. All authors read and approved the final manuscript.TDP-43 SH-SY5Y cellsRescue of stably silenced sh. Parental SH-SY5Y cells (Ctrl) or stably transduced cells with shRNA against TDP-43 (shTDP) were transfected with either Flag-TDP-43 wt, Myc-HDAC6 or control vector. Cells were lysed, electrophoresed and Western blots sequentially probed with antibodies against TDP-43 (top panel) and HDAC6 (middle panel). Anti-GAPDH probing (bottom panel) was used as a loading control. A, shown is a representative Western blot. Densitometric analysis of TDP-43 levels B, or HDAC6 levels C, of three independent experiments is shown.Click here for file


T1 relaxation-time mapping allows direct myocardial signal quantification and therefore enables true quantitative characterisation of myocardial tissue heterogeneity. Differences between healthy myocardium and scarred tissues can be reliably distinguished from the R1 values derived from pre-contrast T1 maps. In patients with scarred tissues, ΔR1 value derived from both the pre- and the post-contrast T1 maps provides better distinction between grey zone and scar core than either pre-contrast or post-contrast R1 value alone.Cardiac magnetic resonance imaging with late gadolinium enhancement (LGE-CMR) has been the standard tool for assessing regional fibrosis. Tissue heterogeneity quantification by traditional signal-intensity (SI-) based methods is not without limitations. T1 relaxation-time mapping allows direct myocardial signal quantification and therefore enables true quantitative characterisation of myocardial tissue heterogeneity. We aim to explore tissue heterogeneity assessment using T1 maps generated with the modified Look Locker (MOLLI) sequence in patients with previous myocardial infarct.Seven patients with ischaemic infarct history underwent left ventricular scar assessment with standard inversion-recovery gradient-echo sequence 10 minutes post-contrast  in short axis (voxel size 1.8x1.8x8mm) on a 1.5T scanner . Three separate slices of MOLLI images were taken pre- and 20 minutes post-contrast in the same geometry. SI-based tissue heterogeneity assessment on the LGE-CMR images identified the regions of remote healthy myocardium (n=21), scar core (n=21) and grey zone (n=21); 2-standard-deviation SI from the Remote<Grey<Core defined by the full-width-half-maximum method. The corresponding regions-of-interest were identified on the pre- and post-contrast T1 maps. T1 and R1 values (1x103/T1) for the respective regions were analysed. Statistical analyses were performed using Mann-Whitney U and Wilcoxon tests.The T1 relaxation-times for each region were significantly different on both the pre- and the post-contrast T1 maps; p<0.05. At 20 minutes post-contrast, the T1 values were significantly reduced in all regions with the greatest reduction seen in scar core; p<0.05. The R1 and the ΔR1 (defined as (PostContrastR1-PreContrastR1)/PreContrastR1) for each region are plotted in Figure ROC curve analysis showed that the native pre-contrast R1 provided the best prediction for healthy myocardium whereas the ΔR1 provided better prediction for scar core and grey zone than either the pre-contrast or the post-contrast R1 value alone Figure . A cut-oT1 maps acquired from MOLLI sequence allow quantitative assessment of tissue heterogeneity. Differences between healthy myocardium and scarred tissues can be reliably distinguished from the R1 values derived from pre-contrast T1 maps. Potentially, patients without scarred myocardium do not need post-contrast imaging. In patients with scarred tissues, ΔR1 value derived from both the pre- and the post-contrast T1 maps provides better distinction between grey zone and scar core than either pre-contrast or post-contrast R1 value alone.N/A


A priori, a common receptor induced in tumor microvessels, cancer cells and cancer stem-like cells (CSCs) that is involved in tumor angiogenesis, invasiveness, and CSC anoikis resistance and survival, could underlie contemporaneous coordination of these events rather than assume stochasticity. Here we show that functional analysis of the dual endothelin1/VEGFsignal peptide receptor, DEspR,  supports the putative common receptor paradigm in pancreatic ductal adenocarcinoma (PDAC) and glioblastoma (GBM) selected for their invasiveness, CD133+CSCs, and polar angiogenic features. Unlike normal tissue, DEspR is detected in PDAC and GBM microvessels, tumor cells, and CSCs isolated from PDAC-Panc1 and GBM-U87 cells. DEspR-inhibition decreased angiogenesis, invasiveness, CSC-survival and anoikis resistance in vitro, and decreased Panc1-CSC and U87-CSC xenograft tumor growth, vasculo-angiogenesis and invasiveness in nudenu/nu rats, suggesting that DEspR activation would coordinate these tumor progression events. As an accessible, cell-surface ‘common receptor coordinator’, DEspR-inhibition defines a novel targeted-therapy paradigm for pancreatic cancer and glioblastoma. Dear, GenBank accession EF212178.Cancer recurrence and invasiveness remain without effective therapy despite initial therapeutic response of the primary tumor. This is especially true for pancreatic adenoductal carcinoma (PDAC) and glioblastoma multiforme (GBM), both of which have the lowest median survival among cancers despite extensive research. Emerging data implicate cancer stem-like cells (CSCs), or tumor/cancer-initiating cells, which possess stem-like properties of prolonged self-renewal and potential to generate “heterogeneous lineages of cancer cells that comprise the tumor” +/− knockout mouse phenotype +/− knockout mice is not embryonic lethal +/− knockout mice +/- male mice DEspR roles in cancer are deduced from its embryonic-lethal null mutation phenotype resulting in E10.5–12.5 day embryonic lethality characterized by abnormal vasculogenesis with incomplete dorsal aorta formation, and by absent angiogenesis, and failed endocardial-to-mesenchymal transition/migration resulting in thin-walled hearts Given these observations, we tested the hypothesis that human DEspR is a common receptor mechanism in human cancer cells, CSCs, and microvessels, which underlies a putative coordinating paradigm for tumor progression events. To test this hypothesis, we used combinatorial in vitro and in vivo experimental systems testing DEspR roles in two human cancers with the lowest 5-year survival rates and polar vascular phenotypes, pancreatic adenoductal carcinoma (PDAC) and glioblastoma multiforme (GBM).Dear) with Fbxw7 exon-5 on opposite strands  rat xenograft models. In contrast to zero-tumorigenesis of 2-million non-CSC Panc1 tumor cells in orthotopic (n = 20) and heterotopic (n = 20) locations in nude rats (data not shown), 2-million Panc1- and U87-CSCs produced heterotopic tumors in ∼50–100% of nude rats respectively, without pharmacological immune suppression.To analyze DEspR-expression in CSCs representative of multiple CSC-subsets To further explore ligand-specific binding to DEspR, we isolated anchorage independent tumor cells from the Panc1 cell line, Panc1-CSCs. FACS analysis detected successful competition of DEspR ligands, ET1 and VEGFsp thereby blocking, hence reducing, 7c5b2-binding to DEspR+ Panc1-CSCs at 4°C , while cTo define a translational framework, we then studied DEspR expression in pancreatic adenoductal carcinoma and glioblastoma, selected for their high unmet need, similar invasiveness but polar vascular phenotypes. Using 7c5b2-AF568 labeled mAb, immunostaining of human tumor tissue sections detected increased DEspR expression in Grade-IV pancreatic adenoductal carcinoma tumor cells and microvessels, even in areas of stromal desmoplasia , in markHaving validated the 7c5b2 mAb and the experimental cell system for DEspR expression and signal transduction, we studied DEspR roles as a common receptor coordinating mechanism for tumor vessels, nonCSC-tumor cells, and CSCs by analyzing the effects of anti-DEspR 7c5b2 mAb-inhibition in established surrogate in vitro assays for pro-metastatic paradigms: angiogenesis in HUVECs, invasiveness in pancreatic cancer Panc1-cells, and CSC-anoikis resistance, stemness, self-renewal, survival, and tumorigenicity in functionally validated anchorage-independent CSCs derived from Panc1 and glioblastoma-U87 cell lines. These anchorage-independent Panc1 and U87 CSCs represent multiple CSC-subsets in order to better model human tumor heterogeneity To define DEspR functional roles in tumor angiogenesis, we demonstrate that DEspR is increased in tube-forming angiogenic HUVECs but not in quiescent HUVECs , and thaTo first define DEspR roles in invasiveness using adherent nonCSC-Panc1 tumor cells, we demonstrate DEspR-expression in Panc1 cells used in the transwell invasiveness assays since CSCs cultured in suspension preclude the use of established transwell invasiveness assays. Consistent with confocal microscopy analysis , double Using FACs analysis of freshly isolated, non-permeabilized anchorage-independent CSCs to ascertain DEspR-expression on the cell membrane of Panc1-CSCs and U87-CSCs respectively , we deteTo confirm DEspR expression in Panc1-CSCs and U87-CSCs, we first corroborated detection of spliced DEspR-specific RNA, to distinguish it from FBXW7 in cancer cells via Amplification Refractory Mutation System (ARMS) assay To test for increased CSC survival abilities of Panc1- and U87-derived CSCs, a known feature of CSCs To test DEspR roles in anoikis resistance, a requirement for metastasis and local invasiveness pertinent to all cancers especially pancreatic cancer and glioblastoma Having shown DEspR-inhibition effects on individual cancer paradigms, we next tested the “common-receptor coordinating paradigm” by determining the in vivo integrated effects of coordinated inhibition of angiogenesis, tumor cell invasiveness, and CSC anoikis resistance or survival via anti-DEspR mAb therapy. In order to test larger tumor sizes than that attainable in orthotopic nude mouse or nude rat tumors, we analyzed heterotopic PDAC Panc1-CSC and glioblastoma U87-CSC subcutaneous tumors in nude rats.As shown in After validating Panc1-CSC and U87-CSC xenograft heterotopic tumor modeling, we next tested the in vivo effects of DEspR-inhibition. As shown in Similarly, compared to non-treated controls, anti-DEspR mAb treatment decreased U87-CSC glioblastoma xenograft tumor growth P<0.001  and invaHere we show that DEspR is a common receptor expressed in tumor cells, microvessels, and anchorage-independent CSCs, with differential expression in cell- and nuclear-membranes, as well as in the cytoplasm. DEspR is differentially increased in both human pancreatic cancer and glioblastoma in contrast to adjacent normal tissue. This increase is detected at the protein and RNA levels in Panc1- and U87-CSCs.Because of the overlapping transcript organization, DEspR inhibition studies presented here are all done at the protein level, in order to eliminate potential dual DEspR/Fbxw7 knockdown-inhibition at the RNA level. DEspR-inhibition at the protein level decreased in vitro angiogenesis, tumor cell invasiveness, CSC-cell anoikis resistance, survival, and promoted pro-apoptosis balance for both Panc1-CSCs and U87-CSC experimental systems. Concordantly, DEspR-inhibition also decreased in vivo Panc1- and U87-CSC-xenograft tumor volumes, vasculogenesis, invasiveness, and tumor cell survival in the expanding tumor zone. In contrast, the current standard of care for pancreatic cancer, gemcitabine, given intravenously at human equivalent dose had no effect on tumor growth of Panc1-CSC xenograft tumors, thus indicating the potential of DEspR-targeted inhibition for improved clinical therapy for gemcitabine-resistant pancreatic cancer. On the other hand, a standard of care chemotherapy for GBM, temozolomide, prevented early tumor growth of U87 CSC-xenograft tumors (data not shown), but comparative analysis of temozolomide and DEspR-inhibition in tumor progression associated with larger tumors, and in temozolomide-resistant U87-CSCs remain to be studied. Nevertheless, DEspR-inhibition resulting in decreased invasiveness observed in both Panc1-CSC and U87-CSC derived xenograft tumors is of translational importance since both gemcitabine and temozolomide are not effective in invasive PDAC and GBM, respectively. Further testing of DEspR-inhibition on tumor cell invasiveness and tumor vasculo-angiogenesis in orthotopic xenograft tumor models for both PDAC and GBM will be important.The concurrent decrease in invasiveness, tumor vasculo-angiogenesis, and survival in the expanding tumor zones in both Panc1-CSC and U87-CSC xenograft tumors collectively support DEspR as a common receptor mechanism whose activation on different cancer cell players would facilitate contemporaneous tumor progression. Given the complexity of cancers and the expected redundancy of multiple survival, vasculogenic and invasiveness pathways, these data indicate nodal functional significance of DEspR roles in said tumor progression events in both PDAC and GBM.6 per xenograft injection exhibited the expected increased tumorigenicity over non-CSC cancer cell lines, xenograft tumor cell heterogeneity and invasiveness, and reached tumor sizes greater than 2000 mm3 42 days after cell-inoculation. In contrast, in nude mice at ∼1/10 the size of nude rats, injection of 5×106 Panc1 cells were used for tumor formation which reached an average 1700 mm3 only, 71 days post-injection These observations and deductions are strengthened by the fact that septenary tumorigenic CSCs used for study at 2×10Furthermore, these observations are concordant with DEspR-mediated signaling. The combinatorial activation of Src and FAK-phosphoproteins is associated with invasiveness and metastasis Altogether, in vitro and in vivo functional analysis of DEspR supports a “common receptor paradigm” for contemporaneous CSC survival, anoikis resistance, invasiveness and tumor vasculogenesis at the CSC-microvascular niche and invasive tumor edge. We hypothesize that this could comprise a putative trans-cellular mechanism for temporal coordination beyond stochasticity. As an accessible cell-surface, coordinating receptor on nonCSC tumor cells, CSCs and tumor microvessels involved in key tumor-progression events, DEspR inhibition opens a novel targeted therapy approach for pancreatic cancer and glioblastoma.This study was performed 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 Committee on the Ethics of Animal Experiments of Boston University School of Medicine (Permit Number: AN-15160). Euthanasia of study animals was done by removal of vital organs and exsanguination under general anesthesia as stated in AVMA Guidelines 2013.1TMFKGSNE9 at the amino-terminal end of hDEspR as antigen. This antigenic-peptide was previously validated via the development of polyclonal anti-hDEspR ab which detected the expected size product of transcribed, spliced, and translated hDEspR expression construct in permanent Cos1cell transfectants 1TMFKGSNE9 antigenic peptide. Serial dilutions of primary antibodies were incubated at 37°C for 1 hr. The wells were then incubated with horse radish peroxidase (HRP)-labeled anti-IgG (Sigma) at 37°C for 1 hr. Reactions were analyzed at 450 nm after addition of 3,3′5,5′-tetramethylbenzidine substrate at 37°C for 10 minutes.U87 glioblastoma and Panc1 cells were obtained from American Type Culture Collection, ATCC  and maintained according to ATCC-specifications. Human umbilical vein endothelial cells, HUVECs, were obtained from Cascade Biologics, Inc. and maintained according to CBI-specifications. Monoclonal antibody development was custom performed by ProMab Biotechnologies, using a humanDEspR-specific nine amino-acid peptide, MAve−ControlAve]/ControlAve×100.Analysis of ligand-dependent modulation of different signaling pathways by DEspR was custom performed (Kinexus) utilizing the Kinex™ Antibody Microarray System: 506 phosphoprotein-specific antibodies in duplicates or multiple replicates, and 740 pan-specific antibodies of signaling molecules. Effects of ET1- and VEGFsp-DEspR activation on multiplex signaling pathways after 30 minutes of ligand-treatment  was compared with non-activated, non-treated controls, using Cos1-hDEspR permanent cell transfectants developed previously Double immunofluorescence staining was done as described Panc1 and U87-cells in log phase were harvested and subcultured in complete MammoCult® medium  in 5% CO2 humidified incubator at 37°C. After 2–3 weeks in culture, Panc1 and U87 cells were harvested and plated in complete MammoCult® medium containing 0.5% Methylcellulose  in 100 mm ultra-low attachment plates. In vitro experiments were performed using quinary CSCs; xenograft tumors were developed from septenary CSCs. Functional validation of CSC stocks was done through demonstration of maintenance of self-renewal in suspension cultures and anchorage independence through 7 passages, increased tumorigenicity over non-CSC cells, tumor vasculogenesis and tumor cell heterogeneity in xenograft tumors.www.FloJo.com).Panc1-CSCs and U87-CSCs were incubated in ice-cold Hank's balanced salt solution  plus 2% FBS containing: a) 10 µg/ml AF-568 labeled 7c5b2 mAb plus 10 µg/ml AF-488 labeled anti-CD133 ; b) 10 µg/ml AF-568 labeled IgG2b (isotype) plus 10 µg/ml AF-488 labeled IgG1 (isotype). Duplicate samples were incubated for 20 min at 4°C, washed, resuspended in 1% FBS/HBSS, 1% PFA, filtered and analyzed on an LSR-II-FACS instrument. Analysis was done using FloJo Flow Cytometry Analysis Software  containing 2% FBS, 10 µg/ml AF-568 labeled 7c5b2 mAb in the absence of competitors or in the presence of 50-fold molar excess of either ET1 or VEGFsp respectively. Duplicate samples were incubated for 20-minutes at 4°C, processed and analyzed on an LSR-II FACS instrument.5 cells in 0.25 ml of Hank's balanced salt solution containing 2% FBS and increasing concentrations of 7c5b2 mAb (1–100 µg/ml). Incubations were done at 4°C for 20 min and immediately subjected to FACS analysis on a BD™ LSRII Flow cytometer. Each data point was performed in duplicate.7c5b2 mAb was labeled with Alexa Flour 568 using the Alexa Fluor 568 Monoclonal Antibody Labeling Kit (Invitrogen). Specific binding was determined by FACS analysis using 10Angiogenesis assays were done using human umbilical vein cells, HUVECs  using standard angiogenesis conditions with 20,000 cells per well in a 96 -plate Angiogenesis System: Endothelial Cell Tube Formation Matrigel™ Matrix . Different anti-angiogenic conditions were assayed in quadruplicate: basal media (BM) alone ; increasing concentrations of 7c5b2 mAb (0.05–500 nM); BM with 500 nM IgG2b isotype control; BM with anti-hDEspR polyclonal antibody (1∶200); BM with pre-immune serum . After 16 hours, resulting tube formations were analyzed using ImageJ (NIH).Panc1-cell invasion assays were performed as described 88 bp: 5′-TGGACCAGAGAAATTGCTTG-3′, 5′-GAAGTCACCACCCTGACA-3′). Amplification was performed with the QantiTect SYBR Green PCR Kit  using the StepOnePlus PCR System  and the following two primer sets: for DEspR . qRT-PCR and cycling parameters were as follow: cDNA synthesis for 15 min at 42°C, thermal inactivation of reverse transcriptase for 3 min at 95°C and 40 cycles of PCR . Data acquisition and analysis were carried out using StepOne software v2.0 .Panc1 cells, U87 cells, Panc1 CSCs and U87 CSCs were lysed with ice-cold Nuclei EZ lysis buffer  and supernants containing cytoplasmic RNA were extracted twice with Phenol:Chloroform (50∶50) followed by ethanol precipitation. Total cytoplasmic RNA concentration was determined by absorbance at 260 nm. After removal and documentation of no residual DNA contamination, cDNA synthesis was performed with the QuantiTect Reverse Transcription Kit  using 400 ng of total cytoplasmic RNA, a DEspR specific primer  or by chemiluminescence using the ECL Western Detection kit .Western blot analysis was done essentially as described 5′-AGGAGCCACTTTTTATACAGTTCTACCCTGATCAAC-3′ and thermo-stable Maxima Reverse Transcriptase as per manufacturer's instructions . Reactions were incubated at 65°C for 2 hours followed by residual RNA hydrolysis at 37°C for 14 hours in 0.5 N NaOH, subsequent neutralization with 1 N HCl, ethanol precipitation, and resuspension in 20 µL of sterile water. We used 2 µL of this DEspR-specific single-strand cDNA stock for PCR-amplification with the forward primer specific for spliced DEspR: GAGC-3′5′-CATGACAATGTTTAAAGG; and the reverse primer being the identical primer used for DEspR-specific cDNA synthesis. Using this primer pair, ARMS amplicon should be 270 bp specific for spliced DEspR .ARMS was performed as described ed DEspR . DetectiNon-CSC and anchorage-independent CSCs were incubated in respective media in adverse conditions  For Panc1 and U87 nonCSC tumor cells, live cells were counted at day-1, 2 and 3 post-set up using Trypan Blue. For CSCs, live CSCs were counted at day-7, 14 and 21 post-set up using Trypan Blue. To test DEspR roles in survival, one million U87-CSCs were incubated in duplicates in the absence (control) or presence of anti-DEspR 7c5b2 mAb (500 nM added once at day 0) and live U87 CSCs counted at various times using Trypan Blue.CSCs (2000/well) were seeded in ultra-low attachment 96-well plate with 500 nM 7c5b2 (treated) and without (control) with 6 replicates. CSCs were cultured in optimal  non-adherent conditions. Anti-hDEspR (7c5b2) mAb was added at seeding, day-2 and day-4. Live and dead CSCs were counted using Trypan Blue on day-5.5 Panc1-CSCs and 1×105 U87-CSCs were grown in 2 ml of complete MammoCult® growth medium without methylcellulose in the presence (500 nM) or absence of 7c5b2 mAb for 24 hours. RNAs from the treated and non-treated samples (controls) were extracted with Trizol reagent (Invitrogen) as described 2 Profiler™ PCR Array  querying 84 genes related to apoptosis. We performed RT-PCR analysis as per manufacturer's instruction using 100 ng of RNA without the amplification step with each sample ran in four technical replicates.1×106 Panc1-CSCs and U87-CSC in 4–5 weeks-old nudenu/nu female rats . Anti-DEspR mAb therapy (n = 6 each for Panc1-CSC and U87-CSC) or gemcitabine treatments (n = 5) commenced at tumor volumes of 300–400 mm3. To inhibit both human-CSC and host-rat DEspR, we used anti-humanDEspR(7c5b2)-mAb and anti-ratDEspR(10a3h10)-mAb at 125 µg/kg/dose each intravenously 2×/week x 4-weeks, beginning on day-14 for Panc1-CSC and day-7 for U87-CSC tumors. Gemcitabine was administered to Panc1-CSC tumor rats at 26 mg/kg/dose IV x 4-weeks equivalent to human dose 2. Contemporaneous mock controls  were infused with vehicle  respectively. Treatments ended when the controls reached maximum allowable tumor size: 6-week study for Panc1-CSC and 3-week study for U87-CSC xenograft tumor models. Tumor volumes were calculated by using the formula (4/3πr12×r2) where r1 is the larger, and r2 the smaller radius as described Heterotopic subcutaneous xenograft tumor models were developed using 2×10Comparative analysis of xenograft tumors from control non-treated rats exhibiting robust tumor growth (n = 3) and from treated rats with robust response (n = 3) was done for invasiveness and cell loss in the expanding tumor zone. Representative Masson Trichrome stained sections were obtained to distinguish the collagen rich fibrous cap from invading tumor cells migrating from the typically cell-rich expanding tumor zone. Contiguous high power fields (HPF) (N = 10–20) were scored for invasiveness through the fibrous cap and into surrounding host dermis or muscle tissue. Invasiveness score: 0, no invasive tumor cells; 10, few invasive cells into fibrous cap; 20, invasive cells in <50% width of fibrous cap in HPF; 30, invasive cells along full-length of fibrous cap in HPF; 40, full thickness traversed by invasive cells or ‘thin fibrous cap’; 50, invading cells into adjacent host tissues. HPFs scored for invasiveness were also scored for cell loss marked by residual ‘cell-ghosts’ or amorphous substance or fibrotic replacement: 0, no cell loss; 10, some cell loss in HPF beneath fibrous cap; 20, >25% cell loss in HPF; 30: >50%; 40, >75% cell loss; 50, 100% cell loss with fibrosis or amorphous substance in HPF.All data were analyzed for normality and descriptive statistics. The following statistical tests were performed using SigmaPlot 11.0 or PRISM 5: one-way analysis of variance (ANOVA) followed by Tukey multiple comparisons test (MCT) for in vitro angiogenesis and invasion assays, xenograft tumor analysis of invasiveness and cell loss scores, and CSC-growth inhibition experiments; two-way ANOVA and Tukey-MCT for apoptosis gene array data; two-way repeated-measures ANOVA and Tukey-MCT for xenograft tumor growth. A P<0.05 was considered statistically significant.Figure S1Representative phosphoproteomic analysis of ligand-specific DEspR-signaling pathways. (A) Human chromosome 4 map with Dear (DEspR) location notated, along with R188 bp, reverse primer for DEspR-specific 1st strand cDNA synthesis which also serves as reverse primer for 88 bp amplicon, F1, forward primer for 88 bp amplicon. (B) Comparative binding affinity of anti-hDEspR mAb candidates (open symbols) as the basis for selecting 7c5b2 (red diamond). The other high binding candidates  did not grow well. (C) Representative phosphoprotein fluorescent readout of DEspR-signaling proteins activated by VEGFsp and ET1 respectively upon stimulation of DEspR+ Cos1 cell-transfectants at t-30 minutes. Red, VEGFsp-induced or ET1-induced activation of signaling phosphoproteins; blue, non-stimulated DEspR+ Cos1 cell-transfectants serving as reference controls. Phosphoproteins tested in duplicate; GenBank gene names listed; phosphorylated amino acids listed in superscript.(PDF)Click here for additional data file.Figure S2Increased DEspR+ expression in pancreatic ductal adenocarcinoma and glioblastoma tumor biopsy cores. Identical exposure settings were used validating comparison of normal pancreas with pancreatic cancer sections, and glioblastoma with normal brain sections respectively; DAPI nuclear stain (blue). (A) Representative low power field  and high field (400×) power immunofluorescence images of DEspR+ immunostaining (red) comparing normal pancreas and pancreatic cancer tumor biopsy cores. Bar, 50 microns (200×), 20 microns (400×). Increased DEspR+ expression detected in tumor cells. (B) Representative LPF-200× and HPF-400× immunofluorescence images of DEspR+ immunostaining (red) comparing normal brain and glioblastoma tumor biopsy cores. Bar, 50-microns (200×), 20-microns (400×). (C) Bar Graph of auto exposure times at identical photomicroscopy settings  representative of immunofluorescence intensity levels (exposure setting≈1/intensity) detected in normal pancreas and DEspR-negative tumor biopsy section biopsy cores vs DEspR+ pancreatic cancer tumor biopsy sections. ANOVA with Tukey's multiple comparisons test, ***, P<0.0001. Black, LPF-200X; Red, HPF-400X fluorescence analysis. (D) Bar Graph of auto exposure times at identical photomicroscopy settings  representative of immunofluorescence intensity levels (exposure setting≈1/intensity) detected in normal brain and DEspR-negative tumor biopsy section biopsy cores (hatched) vs DEspR+ glioblastoma tumor biopsy sections. ANOVA with Tukey's multiple comparisons test, ***, P<0.0001, **, P<0.001. Black, LPF-200X; Blue, HPF-400X fluorescence analysis.(PDF)Click here for additional data file.Figure S3Anti-ratDEspR pAb inhibits VEGFsp-induced and ET1-induced angiogenic sprouting in a rat aortic ring assay. (A) Representative image of aortic ring neovessels in serum; **, ***, ****, secondary, tertiary and quaternary branching; arrow indicates a polygon formed by interconnecting neovessels. (B) Representative image of aortic ring neovessels induced by VEGFsp. (C) Representative image of aortic ring neovessels induced by ET1. (D) Number of neovessels sprouting from rat aortic ring is increased by VEGFsp and ET1 compared to serum and reduced by anti-ratDEspR pAb treatment. One way ANOVA P = 0.0002; Bonferroni's specific pairwise comparison:**, P<0.001; *, P<0.01. (E) Rat aortic ring analysis of angiogenesis measured as # branches per aortic ring. *, P<0.05; VEGFsp, signal peptide for VEGF; ET1,.Endothelin-1.(PDF)Click here for additional data file.Figure S4Apoptosis gene-pathways affected by DEspR-inhibition in Panc1 and U87-CSCs (GenBank nomenclature) and ARMS detection of spliced DEspR-RNA transcript. (A) Real-time qPCR analysis of changes in apoptosis pathway genes after 16-hours of DEspR-inhibition of Panc1-CSCs and U87-CSCs. Black arrows, gene changes that promote apoptosis; white arrows, gene changes that decrease apoptosis. Two-way ANOVA with Tukey-MCT: *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<10−4; ****** P<10−6. (B) Diagram  shows relative location of 36 nucleotide (nt-long) reverse primer for cDNA synthesis (RcDNA), which also serves as the reverse primer for PCR amplification, and 22-nt-long forward ARMS primer (FARMS) that spans the exon 1-to-exon 2 junction. Successful ARMS should detect a predicted 270 bp PCR amplicon of spliced DEspR-RNA. No unspliced DEspR-RNA, predicted size 342-bp amplicon, should be detected based on the 4 nucleotide discrepancy shown in C. (C) ARMS forward primer 22 nucleotide sequence is specific for spliced exon 1-to-exon 2 junction producing a 270 bp amplicon. FARMS forward primer is 4 nucleotides discrepant with unspliced DEspR exon 1-intron junction, hence will not produce the predicted size 342 bp PCR amplicon. ARMS assays can detect single nucleotide substitutions; hence 4 nucleotides discrepancy has increased robustness in accuracy and specificity.(PDF)Click here for additional data file.Figure S5Representative histological and immunofluorescence micrographs of Panc1- CSC nude rat xenograft tumors. (A) H&E stained section showing tumor cells with high mitotic count per HPF in Panc1-CSC xenograft tumor; . (B) Representative merged DEspR+ (red) immunostaining with DIC  overlay showing tumor cell heterogeneity with areas of DEspR+ immunostaining close to DEspR+ microvessels and areas of DEspR(-) tumor cells. (C) Human-specific DEspR+ (red) immunostaining of tumor cells at xenograft tumor edge and DEspR+ adjacent microvessels deriving from human Panc1-CSCs. DIC overlay shows refractive red blood cells within the lumen, indicating connectivity of human-specific DEspR+ Panc1 CSC-derived xenograft tumor blood vessels . (D) Rat-specific anti-DEspR mAb does not immunostain intratumoral vasculature or tumor cells of Panc1-CSC xenograft tumors. RBCs within tumor blood vessels indicate connectivity to host rat circulation. White asterisks, micro-vessel lumen with and without RBCs; bar, 20-microns.(PDF)Click here for additional data file.Figure S6Representative histological and immunofluorescence micrographs of U87-CSC nude rat xenograft tumors . (A) H&E stained section showing invasive U87-CSC xenograft tumor cell cluster (black arrows) encroaching onto the subjacent skeletal muscle. Bar, 10-microns. (B) Merged immunofluorescence with DIC overlay showing human-specific 7c5b2 DEspR+ (red) immunostaining of invasive tumor cells at the tumor edge encroaching into the fibrous capsule, and DEspR+ microvessels with red blood cells in the microvessel lumen (white asterisks). Bar, 20-microns. (C) Merged immunofluorescence with control IgG2b isotype-AF468 showing non-specific red fluorescence in red blood cells (RBCs). Bar, 20-microns. (D) H&E stained section showing thin-walled microvessels (yellow arrows) with RBC-filled lumen (red immunofluorescence) surrounded by tumor cells and stromal cells. Bar, 10-microns. (E) Masson-trichrome stained section of anti-DEspR treated xenograft tumor showing tumor cell ‘island’ circumscribed by collagen and close to a microvessel with stabilized wall architecture. Bar, 10 microns.(PDF)Click here for additional data file.Table S1Comparative analysis of signaling pathways activated upon ET1- and VEGFsp-specific stimulation of hDEspR in DEspR+Cos1 cell-transfectants.(DOCX)Click here for additional data file.Text S1References for (DOCX)Click here for additional data file.


Ganoderma lucidum, affects adipocyte differentiation and glucose uptake in 3T3-L1 cells.Obesity is a health hazard which is closely associated with various complications including insulin resistance, hypertension, dyslipidemia, atherosclerosis, type 2 diabetes and cancer. In spite of numerous preclinical and clinical interventions, the prevalence of obesity and its related disorders are on the rise demanding an urgent need for exploring novel therapeutic agents that can regulate adipogenesis. In the present study, we evaluated whether a dietary supplement ReishiMax (RM), containing triterpenes and polysaccharides extracted from medicinal mushroom 3H]-glucose.3T3-L1 pre-adipocytes were differentiated into adipocytes and treated with RM (0-300 μg/ml). Adipocyte differentiation/lipid uptake was evaluated by oil red O staining and triglyceride and glycerol concentrations were determined. Gene expression was evaluated by semi-quantitative RT-PCR and Western blot analysis. Glucose uptake was determined with [RM inhibited adipocyte differentiation through the suppresion of expression of adipogenic transcription factors peroxisome proliferator-activated receptor-γ (PPAR-γ), sterol regulatory element binding element protein-1c (SREBP-1c) and CCAAT/enhancer binding protein-α (C/EBP-α). RM also suppressed expression of enzymes and proteins responsible for lipid synthesis, transport and storage: fatty acid synthase (FAS), acyl-CoA synthetase-1 (ACS1), fatty acid binding protein-4 (FABP4), fatty acid transport protein-1 (FATP1) and perilipin. RM induced AMP-activated protein kinase (AMPK) and increased glucose uptake by adipocytes.Our study suggests that RM can control adipocyte differentiation and glucose uptake. The health benefits of ReishiMax warrant further clinical studies. Obesity is increasing at an alarming rate in the developed as well as developing countries . In the There are several lines of evidence to show that obesity triggers the dysregulation of the endocrine function of the adipose tissue ,9. This Ganoderma lucidum, which is well recognized by the Traditional Chinese Medicine (TCM)  glucose as described previously -glucose and 1 mM of deoxy-glucose . After 10 min, the glucose uptake was terminated by washing the cells thrice with cold PBS. The cells were lysed with 0.7 ml of 1% Triton X-100 at 37°C for 20 minutes, and the radioactive glucose uptake was determined by the scintillation counter .Glucose uptake was analyzed by measuring the uptake of 2-deoxy-D-, containing triterpenes and polysaccharides isolated from medicinal mushroom ACS1: acyl-CoA synthetase-1; AMPK: AMP-activated protein kinase; C/EBP-α: CCAAT/enhancer binding protein-α; FABP4: fatty acid binding protein-4; FAS: fatty acid synthase; FATP1: fatty acid transport protein-1; LPL: lipoprotein lipase; PPAR-γ: peroxisome proliferator-activated receptor-γ; RM: ReishiMax; SREBP-1c: sterol regulatory element binding element protein-1c.The authors declare that they have no competing interests.DS designed research; AT-S and BL conducted research; AT-S, BL and DS analyzed data; AT-S and DS wrote the paper; DS had the primary responsibility for final content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/11/74/prepub


C. elegans and Drosophila neurons secrete a cleavage product of VAPB, the N-terminal major sperm protein domain (vMSP). Secreted vMSPs signal through Roundabout and Lar-like receptors expressed on striated muscle. The muscle signaling pathway localizes mitochondria to myofilaments, alters their fission/fusion balance, and promotes energy production. Here, we show that neuronal loss of the C. elegans VAPB homolog triggers metabolic alterations that appear to compensate for muscle mitochondrial dysfunction. When vMSP levels drop, cytoskeletal or mitochondrial abnormalities in muscle induce elevated DAF-16, the Forkhead Box O (FoxO) homolog, transcription factor activity. DAF-16 promotes muscle triacylglycerol accumulation, increases ATP levels in adults, and extends lifespan, despite reduced muscle mitochondria electron transport chain activity. Finally, Vapb knock-out mice exhibit abnormal muscular triacylglycerol levels and FoxO target gene transcriptional responses to fasting and refeeding. Our data indicate that impaired vMSP signaling to striated muscle alters FoxO activity, which affects energy metabolism. Abnormalities in energy metabolism of ALS patients may thus constitute a compensatory mechanism counterbalancing skeletal muscle mitochondrial dysfunction.Mutations in VAPB/ALS8 are associated with amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), two motor neuron diseases that often include alterations in energy metabolism. We have shown that  Vapb function is thought to cause motor neuron disease in ALS8 patients and may predispose individuals to ALS, in general. We have shown that neurons secrete the N-terminal VAPB vMSP into the extracellular environment. The secreted vMSPs signal through Roundabout and Lar-like receptors on striated muscles. This neuron to muscle signaling pathway localizes mitochondria to myofilament I-bands and promotes mitochondrial function. Here we show that loss of VAPB in C. elegans neurons causes metabolic changes in muscles, including altered fat metabolism and elevated DAF-16 FoxO transcription factor activity. DAF-16 promotes muscle triacylglycerol accumulation, increases ATP levels, and prolongs survival in Vapb mutants. However, it does not influence muscle mitochondrial localization nor does it affect oxygen consumption. We also show that Vapb knockout mice exhibit disrupted muscular triacylglycerol and FoxO target gene transcriptional responses to fasting and refeeding. These data indicate that impaired vMSP signaling to muscle triggers an energy deficiency, which induces a protective metabolic response involving FoxO. Hence, some energy metabolism alterations observed in ALS patients might be a consequence of striated muscle mitochondrial dysfunction.ALS patients often present with systemic alterations in energy metabolism, such as dyslipidemia and hypermetabolism of unknown origin. Reduction of  Vapb (VAMP/synaptobrevin-associated protein B) gene. Mutations in Vapb lead to ALS8 that manifests as ALS or late-onset SMA, a motor neuron disease restricted to lower motor neurons Vapb mutations are rare, reduced VAPB mRNA or protein levels have been reported in sporadic ALS patients, a mSOD1 ALS mouse model, and ALS8 patient motor neurons derived from induced pluripotent stem cells ALS8 patients.ALS is a lethal neurodegenerative disease characterized by the combined degeneration of lower and upper motor neurons C. elegans and Drosophila that neurons secrete vMSPs to regulate mitochondrial localization and function in striated muscle VAPB, and its paralog VAPA, are broadly expressed type II membrane proteins that are evolutionarily conserved. These VAPs have been implicated in regulating lipid transport and homeostasis at intracellular organelle contact sites, endoplasmic reticulum (ER) dynamics, and membrane trafficking ALS8 disease. Out of five ALS8 patients studied, five had increased cholesterol levels, four had reduced HDL, three had elevated triacylglycerol levels, and one was diabetic vMSP signaling to muscle mitochondria might be relevant for the energy balance in C. elegans that loss of the VAP homolog VPR-1 causes triacylglycerol (TAG) accumulation in striated body wall muscle. Mosaic analysis and tissue-specific expression studies provide compelling evidence that VPR-1 acts in neurons, not muscles to regulate fat levels. Multiple lines of evidence support the model that impaired vMSP signaling from neurons to muscle increases TAG levels in muscle. We propose that this fat metabolism alteration is part of a compensatory response mediated by the DAF-16/FoxO transcription factor. FoxO promotes muscle fat accumulation, maintains ATP levels during aging, and extends lifespan without influencing muscle mitochondrial morphology, localization, or function. Finally, we provide evidence that skeletal muscle metabolism is abnormal in Vapb mutant mice. Our results support the model that disrupting vMSP signaling to muscle triggers a compensatory response involving FoxO transcription factors.Here we show in vpr-1(tm1411) null mutant hermaphrodites, we noticed that body wall muscles often contain large lipid-like droplets not observed in wild-type controls. These apparent lipid-like droplets were visible in young adults (1–3 days post L4 stage) by differential interference contrast (DIC) microscopy  in muscle, droplets are observed in the muscle belly surrounded by mitochondria (vpr-1(tm1411) mutant muscle shows an expanded muscle belly filled with mitochondria, as previously reported vpr-1 mutant intestinal and epidermal tissues by TEM. Instead, intestinal and epidermal tissues looked similar to wild-type controls, although it is difficult to assess minor differences . These fluorescent compounds can be used to directly visualize fat stores in live tissue vpr-1(tm1411) null mutants contained numerous large Bodipy-FA-stained droplets (vpr-1 mutants (due to defective oogenesis), it is not up-taken by muscle . ESI-MS/MS analysis of the extracts detected a robust increase in TAGs in vpr-1 mutant extracts, but not in the membrane phospholipids phosphatidylethanolamine and phosphatidylcholine  [n = 12] versus 17.3±3.6 droplets/mm2 for vpr-1(tm1411) xbp-1 RNAi [n = 10]; P = 0.28). These data indicate that increased ER stress does not cause the muscle TAG defect in vpr-1 mutants.VAP homologs have been implicated in ER stress pathways  mutants . Second,R stress . Third, vpr-1 is ubiquitously expressed and its homologs have been implicated in regulating lipid dynamics via a cell autonomous mechanism vpr-1(tm1411) mutant hermaphrodites were generated containing the vpr-1 genomic locus and the lineage marker sur-5::GFP expressed from an extrachromosomal array C. elegans, extrachromosomal arrays are spontaneously lost at low frequency during cell division, thereby generating mosaic worms. When these events occur early in development, mosaic worms can be generated with losses in neurons, body wall muscles, intestinal cells, and the germ line.vpr-1 genomic locus in vpr-1(tm1411) null worms rescued the fat metabolism defect in muscle  hermaphrodites to wild-type males. Supplying sperm to the reproductive tract reduces muscle fat levels in vpr-1(tm1411) mutants, as visualized with Bodipy-FAs (vpr-1 mutants (data not shown). However, preventing spermatogenesis in wild-type hermaphrodites using the fog-3(q443) null mutation causes mild muscle fat accumulation, as well as mild mitochondrial morphology defects  background. To test whether VPR-1 expression is sufficient in neurons, we expressed VPR-1 under the control of tissue-specific promoters in vpr-1 null mutants. Consistent with genetic mosaic analysis, VPR-1 expression using the myo-3 muscle-specific promoter or the ges-1 intestine-specific promoter did not influence muscle fat levels. In contrast, over-expressing the vpr-1 cDNA with the unc-119 pan-neuronal promoter completely rescued the muscle fat levels in approximately 30–40% of transgenic mutant worms . Consistent with these observations, driving neuronal expression of the vpr-1 genomic locus instead of the cDNA rescued several vpr-1 mutant phenotypes with increased efficiency. These results indicate that VPR-1 acts cell nonautonomously in neurons to regulate muscle fat levels.Genetic mosaics assess the effect of nt worms . These runc-119 neuronal promoter. P56S VPR-1 overexpression in wild-type worms causes increased muscle lipid droplets in most worms  [n = 13] versus 0.9±1.8 droplets/mm2 for wild type [n = 8]). Impaired vMSP signaling causes uncontrolled CLR-1 Lar receptor activity and ectopic Arp2/3-dependent actin filaments in muscle. A reduction of clr-1 or arx-2, which encodes Arp2, rescues the muscle mitochondrial defects, but not the sterility in vpr-1 mutants vpr-1 mutants. clr-1 or arx-2 RNAi restored mitochondria to I-bands, as previously reported vpr-1(tm1411) mutants when compared to the mutant control (2 for vpr-1(tm1411) [n = 12] versus 0.6±1.3 droplets/mm2 for vpr-1(tm1411) clr-1 RNAi  and 3.1±2.0 droplets/mm2 for vpr-1(tm1411) arx-2 RNAi ). Similar results were observed using TEM arx-2/arp2 specifically in wild-type muscle causes mild mitochondrial morphology and fat accumulation defects  daf-16(mu86) double mutants. Muscles of daf-16(mu86) null mutants contain few Bodipy-FA-stained droplets, similar to muscles of wild-type controls. However, muscle fat levels in the double mutants are also low, and strongly reduced when compared to those in vpr-1(tm1411) mutants alone , muscle mitochondria (see below), or sterility of vpr-1(tm1411) mutants. We conclude that the elevated fat levels in vpr-1 null mutants require DAF-16/FoxO activity.The elevated TAGs in ts alone . daf-16 ng rates ; P>0.05,sod-3 promoter (sod-3p::GFP), a direct DAF-16 target vpr-1(tm1411) transgenic worms showed increased GFP expression relative to control transgenic animals (vpr-1(tm1411) mutants show broad GFP expression throughout the body, including the intestine, neurons, vulva muscles, and body wall muscles. The elevated GFP expression is due to DAF-16 because GFP expression is suppressed in transgenic vpr-1(tm1411) daf-16(mu86) double mutants  mutants. An integrated and rescuing transgenic line was used to express DAF-16::GFP under its endogenous promoter. DAF-16::GFP translocates from cytoplasm to nucleus upon loss of insulin signaling, although other mechanisms exist that regulate nuclear DAF-16 activity independent of translocation vpr-1 mutant and control transgenic strains was distributed throughout the cytoplasm and nucleus with no significant difference between the two strains (vpr-1 mutants appear more sensitive to higher temperatures that require increased metabolic activity (To investigate the mechanism(s) by which VPR-1 controls DAF-16/FoxO, we analyzed DAF-16 subcellular localization in  strains . Howeveractivity . We concvpr-1 mutant muscles induce elevated DAF-16 transcriptional activity. If this idea is correct, then inactivating the Arp2/3 complex in vpr-1 mutants should attenuate DAF-16 activity. To assess DAF-16 transcriptional activity, we used the integrated sod-3p::GFP transgenic reporter. arx-2/arp2 RNAi in vpr-1(tm1411) mutants causes a strong reduction in sod-3p::GFP expression in body wall muscle, the intestine, and other cells  mutants contain disorganized and interconnected mitochondrial networks in the muscle belly (daf-16 in vpr-1(tm1411) mutants did not affect muscle mitochondrial morphology or localization  mutants have reduced ATP levels when compared to controls vpr-1 mutants did not decrease over the next two days, as observed in the wild type (vpr-1(tm1411) mutants had higher ATP levels than wild-type controls at the same age  mutants (daf-16 is required for the high ATP concentration in 3-day old mutant adults (P<0.001). ATP levels in daf-16 mutants are similar to wild-type controls (data not shown), as previously shown vpr-1 mutants maintain ATP levels during aging.As the intestine and epidermis are fat storage sites in ild type . 3-day-osame age . Similar mutants . Howevert adults ; P<0.001vpr-1 mutants. Similar to other worm mutants with mild or tissue-specific reduction in mitochondrial function, vpr-1(tm1411) mutants have slightly extended adult lifespan compared to wild-type worms (vpr-1(tm1411) versus 10.5±2.1 days [n = 159] for wild type, P<0.001). daf-16 loss in vpr-1(tm1411) mutants causes a strong reduction in lifespan relative to vpr-1 mutants and wild-type controls (vpr-1(tm1411) daf-16(mu86) [n = 250]; P<0.001). The lifespan of daf-16 single mutants was similar to wild type (data not shown), as previously shown vpr-1 mutants.Based on the abnormalities in energy metabolism, we tested whether DAF-16 influences lifespan in Vapb −/− mice Vapb −/− mice do not exhibit overt defects in energy metabolism. In particular, body weight and glycemia appear normal with age . However, an energy metabolism defect of Vapb deficient mice might be unmasked by modifying insulin supply through feeding and fasting paradigms. In worms and mice, fasting reduces insulin signaling and increases FoxO activity, resulting in altered metabolic gene expression. We used Vapb −/− mice of 2–6 months of age to avoid any confounding effect of the motor dysfunction observed at 18 months P<0.05). In contrast, TAG levels remained unchanged upon fasting in Vapb −/− GA and tibialis anterior (TA) muscles  in response to fasting, but had no effect on fasting induction of other FoxO1 targets such as glucose 6-phosphatase (G6Pase) and pyruvate dehydrogenase kinase (PDK4)  . FoxO1 ae (PDK4) .Vapb −/− muscles, as feeding did not modify expression of these four genes. Vapb genotype did not affect levels of PDK4 mRNA. FoxO1 and FoxO3 expression was down-regulated upon feeding in control TA muscles, but FoxO3 regulation was lost in −/− muscles , and increased expression of the lipogenic transcription factor SREBP1c . This re muscles . The expC. elegans support the model that VAP MSP domains are secreted neurogenic factors that promote striated muscle oxidative metabolism C. elegans, neurons cleave the vMSP and secrete it into the surrounding environment. Secreted vMSPs signal through SAX-3 Roundabout and CLR-1 Lar-like receptors expressed in muscle, down-regulating Lar signaling to the Arp2/3 complex. This signaling pathway restricts actin filament formation to I-bands of the myofilaments, thereby localizing mitochondria to I-bands and promoting mitochondrial function Results from Drosophila and C. elegans that vpr-1/vap loss triggers a robust increase in striated muscle TAG levels. Unexpectedly, this function does not require VPR-1 in muscle. Genetic mosaic and cell-type specific expression studies demonstrate that VPR-1 acts in neurons, consistent with the signaling function. Indeed, muscle vMSP receptors and the downstream Arp2/3 complex mediate this lipid metabolism response. We also found that sperm presence can modulate striated muscle TAG metabolism. Neurons and sperm are two cell types capable of secreting MSP domains VAPs physically interact with multiple proteins involved in lipid binding and transport, such as oxysterol binding protein and ceramide-transfer protein vpr-1/vap loss triggers elevated DAF-16/FoxO activity, resulting in muscle TAG accumulation. Inactivating the Arp2/3 complex largely suppresses these metabolic alterations, as well as the muscle mitochondrial defects. These data support the model that impaired vMSP signaling to muscle triggers elevated FoxO activity. Consistent with this model, over-expressing Arp2 specifically in wild-type muscle causes TAG accumulation and mitochondrial defects. Although we cannot eliminate the possibility that Arp2/3 acts in other tissues, it appears to be a muscle-specific suppressor of vpr-1 mutants. How might the Arp2/3 complex regulate FoxO? One possibility is that vpr-1 mutants go into energy deficit as they age, as mitochondrial dysfunction is thought to increase FoxO activity vpr-1 mutants under standard conditions. However, subtle changes can be more difficult to detect.We show that vpr-1 mutants. The vMSP/ephrin receptor VAB-1 directly interacts with DAF-18/PTEN (phosphatase and tensin homolog deleted on chromosome ten), which regulates FoxO activity Additional mechanisms could also modulate FoxO in Vapb ablation in mice renders muscle lipid stores resistant to fasting, a situation analogous to lipid accumulation in vpr-1 mutant worm muscles. Dysregulated lipid stores in mutant mice is associated with alterations in muscle gene expression consistent with abnormal FoxO1 and FoxO3 activity Vapb −/− mice in the fed state (i.e. in the presence of insulin that decreases FoxO1 activity). Similar results are observed for FoxO3 target genes LC3 and Atrogin-1. These data suggest that FoxO1/3 are less sensitive to insulin inhibition in Vapb −/− mice.In mammals, FoxO transcription factors are critical regulators of energy metabolism, particularly under fasting conditions. We show that Vapb ablation. For instance, VAPB does not appear to influence PDK4 and ATG12 mRNAs. Additionally, some of the mRNAs studied showed uncoupling from circulating insulin levels, consistent with an insensitivity of FoxO1 to insulin. SREBP1c mRNA, which is negatively regulated by FoxO1 Vapb −/− mice, but other mechanisms are likely at work to avoid the major consequences of chronic muscle FoxO activation, such as muscle atrophy C. elegans and Drosophila, remains to be determined.Not all FoxO target genes studied are sensitive to vpr-1 mutants from 6.9±2.5 to 12.9±4.4 days. This lifespan increase may be due to metabolic alterations that compensate for mitochondrial dysfunction. Consistent with this idea, FoxO extends the lifespan of C. elegans with reduced mitochondrial function vpr-1 mutant muscle may reflect an effort to increase energy production. We show that DAF-16 helps vpr-1 mutants maintain ATP levels in 3-day old adults. Among the numerous DAF-16 metabolic genes are those involved in fat synthesis and transport, β-oxidation, the glyoxylate cycle, and gluconeogenesis vpr-1 mutants are more resistant than the wild type to reactive oxygen species and ER stress. Based on identified DAF-16 targets and vpr-1 mutant phenotypes, DAF-16 might increase energy substrate availability in muscle, stimulate anaerobic metabolism, increase oxidative metabolism in non-muscle cells, or decrease ATP consumption. Further studies are necessary to distinguish among these possibilities, as well as other models.A key finding in worms is that DAF-16/FoxO activity prolongs the adult lifespan of vpr-1 loss in worms, Vapb depletion in zebrafish, or Vapb loss in mice does not cause motor neuron degeneration Vapb loss causes neurodegeneration via increased phosphoinositides vap homolog, but many genes with MSP domains. Vertebrate genomes typically encode VAPA and VAPB, which are approximately 60% identical. Vap mutant flies have the most severe developmental defects and the fewest MSP genes in the genome.Metabolic alterations in ALS patients and mouse models are hypothesized to compensate for mitochondrial dysfunction, particularly in skeletal muscle In summary, our results support the model that striated muscle mitochondrial dysfunction alters FoxO activity, which in turn affects energy metabolism and promotes survival. It is possible that reduced vMSP signaling causes some of the mitochondrial and metabolic alterations in ALS patients. Perhaps vMSPs might protect against ALS via effects on skeletal muscle energy metabolism.C. elegans Bristol N2 is the wild-type strain. Worms were grown on NGM plates with NA22 bacteria as the food source sod-3::GFP)], CF1038 daf-16(mu86) I, vpr-1(tm1411)/hT2[bli-4(e937) let-?(q782) qIs48] I;III, SJ4005 zcIs4[hsp-4::GFP], TJ356 zIs356 IV, fog-3(q443) I/hT2[bli-4(e937) let-?(q782) qIs48] I;III, CX3198 sax-3(ky123) X, and XM1004 vpr-1(tm1411) daf-16(mu86)/hT2[bli-4(e937) let-?(q782) qIs48] I;III. Transgenics expressing vit-2p::vit-2::gfp were generated by crossing into the pwIs23 integrated line. RNAi was performed using the feeding method starting at the L1 stage, as previously described arx-2, clr-1, and xbp-1 RNAi clones are from the genome-wide library C. elegans, the marker plasmids pRF4 [rol-6] (60 ng/µl) or myo-3p::mito::GFP (30–60 ng/µl) were mixed with myo-3p::vpr-1 (60 ng/µl), ges-1p::vpr-1(60 ng/µl), unc-119p::vpr-1(60 ng/µl), unc-119p::vpr-1 P56S (60 ng/µl), or myo-3p::arx-2::mCherry (60 ng/µl) and microinjected into the gonads of young adult hermaphrodites. Injected worms were incubated for 24 hours, transferred to new NGM plates, and screened for transgenic progeny. Transgenic lines were selected based on the roller phenotype or GFP expression. Multiple independent transgenic lines were generated for all strains. To conduct genetic mosaic analysis, 10 ng/µl WRM06B28 fosmid DNA containing the vpr-1 genomic locus was mixed with 10 ng/µl pTG96 (sur-5p::GFP) plasmid and microinjected into the gonads of vpr-1(tm1411)/hT2 hermaphrodites. Transgenic lines were selected based on GFP expression. Transgenic lines were maintained as vpr-1(tm1411) homozygotes, as the fosmid rescued the sterility, mitochondria, fat metabolism, slow growth, and embryonic defects. For lineage scoring, approximately 15,000 worms were screened. Transgene loss in the AB lineage was scored by GFP loss in head and tail neurons, the nerve cords, and the excretory gland. Transgene loss in the P1 lineage was scored by GFP loss in the intestine, muscle, somatic gonad, and hyp11. The P2 lineage was scored by GFP loss in numerous body wall muscle cells and hyp11, the P3 lineage was scored by GFP loss in body wall muscle, and the P4 lineage was inferred by a sterile phenotype without GFP loss. Transgene loss in the EMS lineage was scored by GFP loss in the intestine and somatic gonad, while loss in the E lineage was score by exclusive GFP loss in the intestine.To generate transgenic TEM was performed as previously described For the Bodipy-FA experiments, a 5 mM Bodipy-FA  stock solution was prepared in DMSO and kept at −20°C. A 200 µM working solution diluted in distilled water was dropped onto seeded plates and allowed to dry. L4 stage worms were placed on the plates and incubated in the dark for 24 hours at 20°C. Bodipy-FAs can get trapped in intestinal gut granules that are not present in muscle.vpr-1(tm1411) mutants were processed in the same tube and identified based on gonad morphology.Sudan Black B staining was conducted as described in previous studies vpr-1(tm1411) mutant adults were extracted by chloroform-methanol following a modified Bligh/Dyer extraction For the lipid analysis by ESI-MS/MS, lipids from equal masses of wild type and vpr-1(tm1411) mutant hermaphrodites were cultured on the same plates.To assess mitochondrial transmembrane potential, worms were stained using the MitoTracker CMXRos dye , as previously described ATP concentration was measured as described previously, with slight modification Oxygen consumption rates were measured as previously described using the oxygraph system  with minor modifications vpr-1 mutants die during L1–L4 stages and vpr-1 mutants develop slowly. Worms were monitored every day and transferred to flesh NGM plates. Death was scored by failure to respond to touching with a platinum wire. Wild-type worms fed NA22 bacteria have slightly shorter lifespan than worms fed OP50 bacteria.To measure feeding rates, worms were cultured at 20°C and 1-day-old adult worms were placed on new NGM plates. Feeding behavior was recorded using a Zeiss Lumar stereomicroscope with AxioCam MRM digital camera. Measurements were conducted during a 30 second period at room temperature (22°C). The rhythmic contractions of the pharyngeal bulb were counted. For each strain, over 20 worms were counted. To determine lifespan of worms, L4 larval stage worms were placed on new NGM plates seeded with NA22 bacteria and cultured at 20°C. The L4 stage was used because a small percentage of To analyze ER homeostasis, worms were cultured on plates with tunicamycin  from the embryonic stage to adulthood. NGM plates with 0.1% DMSO and 0 or 5.0 µg/ml tunicamycin were prepared. About 30 adult worms were placed on each tunicamycin plate and allowed to lay embryos for 30 minutes. Adult worms were then removed. Twelve hours later the number of hatched embryos was counted and compared with the number of worms that reached the adult stage within 96 hours. We performed at least three independent measurements for each strain.Vapb −/− mice were used and genotyped as described Mouse experiments were performed using the Institutional European Guidelines, under the supervision of an authorized investigator (LD), and approved by the local ethical committee for animal experiments . For RT-qPCR, frozen liver and muscle tissues were placed into tubes containing 5 mm stainless steel beads  and 1 ml of Trizol reagent  and homogenized using a TissueLyser (Qiagen). RNA was prepared from tissue homogenates following Trizol manufacturer's instructions. RNA reverse transcription and SYBR Green real-time PCR assays were performed using the Bio-Rad  iCycler kits and protocols. PCR conditions were 3 min at 94°C, followed by 40 cycles of 45 s at 94°C and 10 s at 60°C. Primers are shown in Triglyceride Colorimetric Assay Kit, Randox Laboratories Limited, UK) as described by the manufacturer. Lipid values were normalized to protein concentration.For TAG analysis, tissue powder was homogenized in lysis buffer  containing protease inhibitors (Sigma P8340) and phosphatase inhibitors (Sigma 8345), centrifuged at 12000×rpm for 15 minutes at room temperature. TAG concentration was determined in duplicate for each sample in 5 µl of supernatant, using the enzymatic method of analysis  Close-up image of a live transgenic vpr-1(tm1441) mutant hermaphrodite expressing mitoGFP in body wall muscle. mitoGFP labels muscle mitochondrial tubules. (B) Image of a single body wall muscle in a transgenic vpr-1(tm1441) mutant hermaphrodite expressing mitoGFP. (C) Close-up image of the boxed region from panel B showing horizontal and vertical cross-sections. Arrowheads indicate lipid-like droplets. Asterisks indicate nucleus. Bar in A, 1 µm; Bars in B and C, 10 µm.Lipid-like droplets and mitochondria in (TIF)Click here for additional data file.Figure S2Transmission electron micrographs of wild-type and mutant adults. Micrographs are transverse sections showing the cuticle (C) and body wall muscle sarcomeres (S) with hypodermis sandwiched in between. A lipid droplet within the hypodermis (HL) is seen in the wild-type panel. The intestine is filled with electron dense and opaque lipid droplets (IL). Yolk lipoprotein complexes (Y) are found between muscle and intestinal tissues also see . Notice (TIF)Click here for additional data file.Figure S3vpr-1 mutants. 1-day-old adult wild-type and vpr-1(tm1411) hermaphrodites were stained using Sudan Black B. Arrowheads indicate fat droplets in body wall muscle. Anterior is to the left in all panels. Boxed regions are magnified 5× below. Low magnification bars, 50 µm; high magnification bars, 10 µm.Sudan Black B staining in wild type and (TIF)Click here for additional data file.Figure S4vpr-1 mutants. (A) Yolk distribution visualized with the vit-2p::vit-2::gfp transgene. vpr-1 mutants accumulate yolk in the pseudocoelom due to failure of oocyte differentiation. GFP uptake is not observed in peripheral tissues. (B) Close-up image showing muscle fat droplets (arrows) in vpr-1 mutants. Bar, 5 µm.Yolk lipoprotein complex distribution in wild-type and (TIF)Click here for additional data file.Figure S5vpr-1 mutant worms. (A) Integrated transgenic lines expressing GFP under the hsp-4 promoter (hsp-4p::GFP) with and without tunicamycin treatment, which induces ER stress. Anterior is to the left in all panels. Bar, 5 mm. (B) Tunicamycin sensitivity in wild-type and vpr-1(tm1411) mutants hermaphrodites. Y-axis indicates the percentage of worms that developed to the adult stage in the presence of 5 µg/ml tunicamycin. Error bars represent SD. Three independent measurements were performed.ER stress assays in wild-type and (TIF)Click here for additional data file.Figure S6vpr-1 mutant hermaphrodite worms fed Bodipy-FAs. Mating with wild type (WT) males provides sperm into the uterus. Sperm presence did not affect the sterility or muscle mitochondrial morphology of vpr-1 mutants (data not shown). Anterior is to the left in all panels. Arrowheads indicate lipid-like droplets. Bar, 5 µm. (B) DIC and fluorescent images of muscle in live transgenic fog-3(q443)/hT2 hermaphrodites containing sperm and unmated fog-3(q443) mutants without sperm. Muscle mitochondrial tubules were visualized using mitoGFP. Arrowheads indicate lipid-like droplets. Asterisks indicate nucleus. Bar, 5 µm.Effect of sperm presence on muscle fat droplets and mitochondria. (A) DIC and fluorescent images of muscle in live 3-day-old (TIF)Click here for additional data file.Figure S7arx-2 under control of the muscle specific myo-3 promoter. Muscle mitochondrial tubules were visualized using mitoGFP. Notice that muscle mitochondrial morphology closely resembles the morphology seen in vpr-1 mutants Effect of ARX-2/Arp2 overexpression on muscle fat droplets and mitochondria. DIC and fluorescent images of muscle in live transgenic wild-type hermaphrodites expressing (TIF)Click here for additional data file.Table S1Primers used for RT-qPCR in mice.(TIF)Click here for additional data file.


HTLV-1 and HTLV-2 trans-activating proteins  differ with respect to their ability to activate genes regulating viral replication, latency, and host cellular immune responses. Although HTLV-2 infections are usually asymptomatic, immunologic alterations are observed. We previously showed that HTLV-2 tax/rex viral load was upregulated in patients with HIV-1/HTLV-2 co-infection. This correlated with higher CD4+ T cell counts and improved health outcomes, possibly due to induction of CC-chemokines.In this study, recombinant Tax1 and Tax2 proteins were expressed in E. coli. PBMCs were incubated with different concentrations of Tax proteins (10-100 pM). Supernatant fluids and cells were harvested at multiple time points for quantitative determinations of MIP-1alpha/CCL3, MIP-1beta/CCL4, RANTES/CCL5, and CCR5 receptor expression.In preliminary experiments it was shown that PMBCs from HTLV-2 infected donors had significantly lower levels of CCR5 expression and higher levels of RANTES/CCL5 compared to HTLV-2 seronegative donors (p<0.05). Tax1 and Tax2-treated PBMCs showed increased viability over a seven day period compared to controls (p<0.01). Both Tax1 and Tax2 induced equally high levels of all three CC-chemokines compared to mock-treated controls (p<0.05). Tax2-treated PBMCs showed a significantly lower percentage of CCR5-PE positive cells compared to mock-treated PBMCs within the gated lymphocyte population (p<0.05).Recombinant Tax2 is a potent modulator of CC-chemokines and CCR5 in vitro. Further investigations are needed to determine the underlying mechanism(s), and whether Tax2 recapitulates the observed effects in vivo.


E. coli (EPEC) and enterohemorrhagic E. coli are important causes of morbidity and mortality worldwide. These enteric pathogens contain a type III secretion system (T3SS) responsible for the attaching and effacing (A/E) lesion phenotype. The T3SS is encoded by the locus of enterocyte effacement (LEE) pathogenicity island. The H-NS-mediated repression of LEE expression is counteracted by Ler, the major activator of virulence gene expression in A/E pathogens. A regulator present in EPEC, H-NST, positively affects expression of H-NS regulon members in E. coli K-12, although the effect of H-NST on LEE expression and virulence of A/E pathogens has yet-to-be determined.Enteropathogenic LEE expression and A/E lesion formation on intestinal epithelial cells. We find that H-NST positively affects the levels of LEE-encoded proteins independently of ler and induces A/E lesion formation. We demonstrate H-NST binding to regulatory regions of LEE1 and LEE3, the first report of DNA-binding by H-NST. We characterize H-NST mutants substituted at conserved residues including Ala16 and residues Arg60 and Arg63, which are part of a potential DNA-binding domain. The single mutants A16V, A16L, R60Q and the double mutant R60Q/R63Q exhibit a decreased effect on LEE expression and A/E lesion formation. DNA mobility shift assays reveal that these residues are important for H-NST to bind regulatory LEE DNA targets. H-NST positively affects Ler binding to LEE DNA in the presence of H-NS, and thereby potentially helps Ler displace H-NS bound to DNA.We examine the effect of H-NST on LEE expression and A/E lesion formation likely by counteracting H-NS-mediated repression. We demonstrate that H-NST binds to DNA and identify arginine residues that are functionally important for DNA-binding. Our study suggests that H-NST provides an additional means for A/E pathogens to alleviate repression of virulence gene expression by H-NS to promote virulence capabilities.H-NST induces  Escherichia coli is the prototype of an important family of regulatory proteins that repress transcription of numerous genes in Gram-negative bacteria E. coli K-12, H-NS is a small, 15.9 kDa protein composed of 137 amino acids. H-NS-mediated modulation of gene expression can involve multiple mechanisms including binding of H-NS to regulatory regions of H-NS regulon genes to block association of RNA polymerase or by preventing open-complex formation after RNAP has already associated with the promoter The histone-like nucleoid structuring protein (H-NS) of E. coli K-12 such as proU and bglShigella, Salmonella, enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) In addition to modulating expression of backbone chromosomal genes in Citrobacter rodentium) LEE1-5LEE genes encode a type III secretion system (T3SS) that resembles a needle-like structure, with the EspA protein forming the filament and EspB/EspD forming the pore inserted into the host cell. Effector proteins are secreted through the needle-like structure into the host cell where they manipulate host signaling pathways to subsequently induce disease hns gene encoding H-NS greatly increases transcription of many LEE genes One group of gastrointestinal pathogens that illustrates this interaction of H-NS and pathogenicity island-encoded regulators is the attaching and effacing (A/E) pathogens LEE1 operon encodes the LEE-encoded regulator (Ler), an H-NS-like protein that shares 36% amino acid sequence identity to the DNA-binding C-terminal domain of H-NS. Ler, composed of 123 amino acids (14.3 kDa), is the master positive regulator of EPEC and EHEC LEE virulence genes such as espA and espB, as well as non-LEE-encoded virulence factors such as the long polar fimbria (lpf1) and a serine protease (stcE) LEE2/LEE3 regulatory region, which was identified as a binding target for the Ler C-terminal DNA-binding domain LEE2/LEE3 target DNA involves the side chain of Arg90 being inserted into a narrow minor groove while Arg93 helps in stabilizing the DNA protein complex The first gene in the 5.5-encoded protein of T7 phage (gp 5.5) and the H-NS truncated protein (H-NST) of EPEC, both of which have been shown to interact with H-NS and hinder its repressive activity E. coli K-12 the gp 5.5 protein has been shown to relieve H-NS-mediated repression of the proU promoter in vivobglG promoter region via interaction with the H-NS oligomerization domain, forming a dominant-negative oligomer E. coli K-12 or EHEC but is encoded in the chromosome of some isolates such as EPEC E2348/69, uropathogenic E. coli (UPEC) strain CFT073 and C. rodentium. H-NST from EPEC is encoded by a pathogenicity island located at the asnW locus. Though H-NST exhibits an overall low amino acid sequence similarity to H-NS of only 29%, the first 43 residues of H-NST share 40% similarity to H-NS et al demonstrated that EPEC H-NST negatively affects H-NS-mediated repression of the E. coli housekeeping genes proU and bgl by forming dominant-negative hetero-oligomers with H-NS that render H-NS inactive when tested in a E. coli K-12 background. These authors further demonstrated that Ala16 of H-NST is important for oligomerization and thereby activity E. coli.Inhibition of H-NS activity is also seen with the gene LEE expression. We show that H-NST positively affects levels of LEE-encoded proteins and A/E lesion formation. We demonstrate that H-NST specifically binds to LEE regulatory DNA regions and further show that Ala16 is required for H-NST-mediated increase in the levels of LEE-encoded proteins and induction of A/E lesion formation. Additionally, we determine H-NST Arg60 and Arg63 residues to be important for the ability of H-NST to bind DNA, resulting in the induction of LEE expression and A/E lesion formation. Further, we demonstrate that H-NST is conserved among many human and plant bacterial pathogens, suggesting a global role of H-NST in regulating the expression of the H-NS regulon.In this study, we assess the effect of H-NST on H-NS-mediated regulation of 2.Standard DNA techniques, liquid media and agar plates were used as described Oligonucleotide sequences used for plasmid constructions are listed in hnsT was PCR amplified from EPEC E2348/69 gDNA using the primer set QEH-NST F/QEH-NSTF R, digested with BamHI and HindIII and cloned into the corresponding sites in pQE80 (QIAGEN). Plasmid pQEH-NST encodes a recombinant C-terminal His-tagged H-NST.pQEH-NST: A 262 bp DNA fragment encoding hns was PCR amplified from EPEC E2348/69 gDNA using primer set QEH-NS F/QEH-NS R, digested with BamHI and HindIII and cloned into the corresponding sites in pQE80 (QIAGEN). Plasmid pQEH-NS encodes a recombinant C-terminal His-tagged H-NS.pQEH-NS: A 434 bp DNA fragment encoding H-NST mutant derivatives were constructed by site-directed mutagenesis of pQEH-NST using the QuickChange XL Site-directed Mutagenesis Kit (Agilent) according to manufacturer's instructions. The plasmids pQEH-NST A16V, pQEH-NST A16L, pQEH-NST R60Q and pQEH-NST R60Q/R63Q encoding H-NST substitution mutants were generated using the primer sets H-NSTA16V F/H-NSTA16V R, H-NSTA16L F/H-NSTA16L R, H-NSTR60Q F/H-NSTR60Q R and H-NSTR60Q/R63Q F/H-NSTR60Q/R63Q R respectively.www.ncbi.nlm.nih.gov). H-NST from EPEC strain E2348/69 (YP 002329609) was used as query sequence. Proteins identified at a threshold e-value of 2×10−26 or less with sequence coverage of at least 77% were considered. In addition, a BLASTn search of a database containing a collection of 114 A/E E. coli isolates and 24 reference strains −15 to identify genes encoding hnsT. The multiple amino acid sequence alignment of H-NST was prepared using ClustalW2 BLAST searches were used to identify H-NST present among non-redundant protein sequences in the NCBI database using the BLASTp program with an expect threshold of 10 and the scoring parameters: Blosum 62 matrix, gap cost was 11 for existence and 1 for extension, and conditional compositional score matrix adjustment (C. rodentium ICC168 (YP003365612), Dickeya zeae (WP019843943), Enterobacter sp. SST3 (EJO48231), E. coli 113303 (ESA61347), E. coli 2362-75 (EFR16544), E. coli 2848050 (EMW14361), E. coli 3003 (EII86141), E. coli 536 (YP669831), E. coli C262-10 (AIAP01000001.1), E. coli C639-08 (AIBH01000001.1), E. coli C844-97 (AIBZ01000001.1), E. coli C93-11 (AICD01000002.1), E. coli CFT073 (NP754305), E. coli CUMT8 (EIL77087), E. coli DEC1A (EHU10545), E. coli DEC1B (EHU13728), E. coli DEC1C (EHU11552), E. coli DEC1D (EHU23639), E. coli DEC1E (EHU27188), E. coli DEC2A (EHU30556), E. coli DEC2B (EHU39793), E. coli DEC2C (EHU44940), E. coli DEC2D (EHU46227), E. coli DEC12A (EHX20918), E. coli DEC12E (EHX46652), E. coli DEC15B (EHX98907), E. coli DEC15C (EHX01733), E. coli DEC15D (EHX09544), E. coli DEC15E (EHX13659), E. coli E2348/69 (YP002329609), E. coli E851/71 (ALNX00000000), E. coli HVH 125 (4-2634716) (EQR42309), E. coli HVH HVH 225 (4-1273116) (EQV31905), E. coli HVH 154 (4-5636698) (EQS65207), E. coli HVH 158 (4-3224287) (EQS57084), E. coli KTE100 (EOW07218), E. coli KTE157 (ELJ10809), E. coli KTE16 (ELC28364), E. coli KTE192 (ELH08668), E. coli KTE227 (ELH91245), E. coli MS 21-1 (EFK17805), E. coli MS 57-2 (EGB75116), E. coli MS 115-5 (EFJ95479), E. coli MS 200-1 (EFJ59489), E. coli 042 (CBG35676), E. coli OK1357 (EFZ69143), E. coli TA124 (EHN89354), E. coli TW07793 (EII95637), E. coli UMEA 3022-1 (EQW12951), E. coli UMEA 3108-1 (EQW66083), E. coli UMEA 4076-1 (ERA50029), Klebsiella oxytoca 10-5245 (EHT00587), K. pneumoniae UCICRE 7 (ESM00810), Pantoea ananatis PA13 (YP005993219), Pectobacterium atrosepticum SCRI1043 (CAG74542), Pectobacterium wasabiae CFBP 3304 (WP005969703), Salmonella enterica subsp. arizonae serovar 62:z4,z23:- strain RSK2980 (YP001569976), S. enterica subsp. diarizonae serovar 60:r:e,n,x,z15 strain 01-0170 (ESJ14503), S. enterica subsp. enterica serovar Anatum str. ATCC BAA-1592 (ESJ09538), S. enterica enterica subsp. enterica serovar Hvittingfoss strain A4-620 (EHC52821), S. enterica subsp. enterica serovar Indiana strain ATCC 51959 , S. enterica subsp. enterica serovar Nchanga strain CFSAN001091 (ESJ38946), S. enterica subsp. enterica serovar Sloterdijk str. ATCC 15791 (ESF40572), and Yersinia rohdei ATCC 43380 (WP004713599).H-NST proteins from the following 65 strains were used to generate the multiple sequence alignment: stx1Δstx2 strain TUV93-0 ler. Wild type H-NST and H-NST mutants were produced from pQEH-NST, pQEH-NST A16V, pQEH-NSTA16L, pQEH-NSTR60Q and pQEH-NSTR60Q/R63Q in TUV93-0. Cultures were grown in LB containing ampicillin at 37°C to a density of OD600∼0.5 followed by induction of H-NST expression with 0.5 mM IPTG for 1h. Total cellular protein was precipitated with 5% (vol/vol) trichloric acid, washed with acetone, resuspended in 1× Next Gel sample loading buffer (Amresco). Proteins were resolved on a 4–20% Tris-HCl Criterion precast protein gels (BioRad) and transferred onto an Immobilon-FL polyvinylidene difluoride membrane (Millipore) using a Trans-Blot SD Semi-Dry Transfer Cell (BioRad). The membrane was blocked in Odyssey blocking buffer (Li-Cor Biosciences), exposed to polyclonal antibodies specific to EspA, EspB and GroEL (Sigma), and subsequently to Alexa Fluor 680-conjugated goat anti-rabbit (Invitrogen). A monoclonal Tetra-His anti-mouse antisera (QIAGEN) and Alexa Fluor 680-conjugated goat anti-mouse (Invitrogen) were used to detect His-tagged H-NST. Detection of GroEL served as a loading control for total protein. Proteins were visualized and quantified using an Odyssey Infrared Imaging System with application software version 3.0 (Li-Cor Biosciences) as recommended. The levels of LEE-encoded proteins were normalized to that of GroEL. The western analyses were carried out on four independent biological samples for each strain.The effect of expressing H-NST from pQEH-NST on the production of T3SS-associated proteins was determined in the EHEC O157:H7 EDL933 ΔhnsT and its mutant derivatives were inoculated from freezer stocks into LB medium containing ampicillin and grown statically for about 18 h at 37°C. Cultures were then diluted 1∶3 in DMEM containing 0.2% mannose, ampicillin and 0.3 mM IPTG to induce expression of hnsT, and grown statically at 37°C in 7% CO2 for 1 h. Levels of wild type and mutant H-NST produced in the preinduced TUV93-0 strains were confirmed by western blot analysis. Semi-confluent HeLa cell monolayers grown on glass coverslips to ∼80% confluence were co-cultured with an initial number of ∼1×106 bacteria in DMEM supplemented with 2% FBS. After 3 h of infection cells were washed with Hanks buffer (Invitrogen), fresh media was added, and cells were incubated for an additional hour. At 4 h post infection cell monolayers were washed once with Hanks buffer, and fixed in 4% formamide in 1× PBS. Coverslips were washed three times with 1× PBS, cells were permeabilized with 0.1% Triton X-100 in 1× PBS, and F-actin was stained using Alexa Fluor 488 phalloidin (Invitrogen) diluted 1∶50. Coverslips were mounted on slides using Prolong Gold Antifade Reagent (Invitrogen). FAS assays were independently conducted at least three times for each strain. Actin-stained cells were visualized using an AxioSkop microscope equipped with a 40× objective and images were captured with an AxioCam MR3 digital camera using AxioVision v. 4.8 software (Carl Zeiss MicroImaging Inc).The effects of wild type H-NST and H-NST mutants on the ability of EHEC O157:H7 TUV93-0 to induce A/E lesion formation on HeLa cell monolayers was evaluated using the fluorescent actin staining assay (FAS) 600 ∼0.5 prior to the induction with 0.5 mM IPTG. Proteins were produced for 1 h and cells were then harvested by centrifugation at 7,667 g for 20 min at 4°C. Cell pellets were suspended in binding buffer  to a final volume of 40 ml. Cells were lysed by two passages through a Microfluidics LV1 micro-fluidizer, and then the lysed cell suspension was centrifuged at 26,536 g for 70 min at 4°C. The supernatant of the lysate was then filtered through a 0.2 µm pore size filter (Millipore) and applied to a HisTrap FF column  coupled to an ÄKTAprime plus system . After sample application the column was first washed with 30 ml of binding buffer at a flow rate of 2 ml/min and then washed with 50 ml of 6% elution buffer  in binding buffer. Protein was eluted with 100% elution buffer and eluates were buffer exchanged into storage buffer  and concentrated using an Amicon Ultra Centrifugal Filter Device with a 3 kDa MW cut-off value (Millipore). Protein samples were analyzed by SDS-PAGE using a 4–20% Tris-HCl Criterion precast gel (BioRad), and visualization with GelCode Blue Stain Reagent (Thermo Scientific), a technique that provides nanogram-level detection. The purity of the H-NST preparation was about 95% as estimated from the stained gel.Recombinant H-NS, H-NST, and H-NS mutant derivatives were produced from pQEH-NS, pQEH-NST, pQEH-NSTA16V, pQEH-NSTA16L, pQEH-NSTR60Q and pQEH-NSTR60Q/R63Q in BL21-DE3 (pLysS) (Novagen). Cells were grown in 1 L of LB medium at 37°C to an optical density of OD600 ∼0.5 before the expression of ler was induced with 0.2% arabinose for 2 h. Cells were harvested by centrifugation at 7,667 g at 4°C for 20 min. Purification of Ler-Myc-His was as described for H-NST with the exception that 10% glycerol was omitted from all buffers. Purified Ler was buffer exchanged and concentrated using Amicon Ultra Centrifugal Filter Device with a 10 kDa MW cut-off value.Recombinant Ler-Myc-His protein was produced from pVS45 in DH5α LEE regulatory regions were prepared as described 2O, chloroform extracted three times, ethanol precipitated in 125 µM NaCl, and then resuspended in 300 µl dH2O. The oligonucleotides (5 µg) were fluorescently labeled in 100 µl reactions using a ten-fold molar excess of Alexa Fluor® 790 Carboxylic Acid, Succinimidyl Ester, penta (triethylammonium) Salt (Life Technologies) in 100 mM Sodium Tetraborate (pH 8.5). Reactions were allowed to proceed overnight at room temperature in the dark. Labeled oligonucleotides were purified using G-25 spin columns  according to manufacturer's directions.Fluorescently-labeled oligonucleotides used for PCR amplification of DNA fragments containing LEE1 P2, LEE1 P1P2 and LEE3 regulatory regions were PCR amplified from EHEC strain TUV93-0 gDNA using the fluorescently-labeled primer sets LEE1P2 F/LEE1P2 R, LEE1P1P2 F/LEE1P2 R and LEE3 F/LEE3 R respectively. The LEE1 P2 and LEE1 P1P2 DNA fragments contain the LEE1 regulatory region ranging from positions −112 to +33 and −301 to +33 relative to the transcription initiation site for the proximal LEE1 promoter respectively. The LEE3 DNA fragment contains the sequence from +83 to +210 relative to the transcription initiation site for LEE3. The primer set rssB F/rssB R was used to amplify a 99 bp long unlabeled DNA fragment containing part of rssB, which served as nonspecific DNA target. Amplified DNA products were purified using G-50 spin columns  according to manufacturer's directions.DNA fragments encoding LEE1 P2, LEE1 P1P2 or LEE3 regulatory regions and then incubated for 20 min at 30°C in binding buffer . Unlabeled LEE DNA fragments added in 6-fold excess and unlabeled rrsB DNA fragment added in 12-fold excess served as specific and nonspecific competitor DNA, respectively. Binding reactions containing both H-NST and H-NS were preincubated for 10 min at room temperature to allow interaction between proteins before the DNA fragment was added. Further, binding experiments where Ler was added to reactions already containing H-NST and/or H-NS bound to DNA were first incubated for 10 min at room temperature, and then Ler was added followed by 20 min of incubation at 30°C. DNA fragments were separated by polyacrylamide gel electrophoresis (PAGE) using a 4–20% TBE Criterion precast gel run in 1× TBE at 52 amps for 60 min (BioRad). Fluorescently-labeled DNA fragments were visualized using an Odyssey Imaging System at 800 nm with application software version 3.0 (Li-Cor Biosciences). The EMSA analyses were carried out at least three times using proteins from at least two different protein purification preparations.Purified H-NS, Ler, H-NST and mutant H-NST derivatives (concentrations as indicated in the figure legends) were incubated for one minute prior to addition of 24 ng of fluorescently-labeled DNA fragment containing the E. coli K-12 LEE and the virulence-associated A/E lesion phenotype remains to be elucidated. To evaluate the effect of H-NST on LEE-encoded protein levels, we cloned hnsT from EPEC under the control of an IPTG-inducible tac promoter in pQE80 and produced H-NST from this construct in EPEC strain E2348/69. Production of H-NST from pQEH-NST in EPEC grown to the exponential phase in DMEM did not affect levels of LEE-encoded proteins (unpublished data), which might be due to the possibility that an effect of H-NST is masked by a relatively high basal level of LEE-encoded proteins present in EPEC. Since the abundance of LEE-encoded proteins in EHEC is minimal in the exponential phase stx-deleted derivative of EDL933 that exhibits Ler and H-NS-mediated regulation of the LEE similar to EPEC. Interestingly, production of H-NST from pQEH-NST in EHEC grown in LB to exponential phase (OD600∼0.4) induced the expression of the LEE as reflected by increased levels of the T3SS-secreted proteins EspA and EspB by 5.4-fold and 3.1-fold, respectively, as opposed to the vector control , suggesting that the H-NST proteins from both these A/E pathogens affect LEE expression. These results strongly suggest that production of H-NST has a dominant-negative effect on the H-NS-mediated repression of LEE expression in the exponential growth phase. Since our data indicated that H-NST positively affects levels of LEE-encoded proteins, we further investigated whether the LEE-encoded global virulence gene regulator Ler was required for regulation by H-NST. To assess this we provided H-NST from pQEH-NST in a ler-deleted derivative of EHEC TUV93-0, and found that production of H-NST increased EspA and EspB levels by 1.9-fold and 1.2-fold respectively , the latter which has been associated with Crohn's disease E. coli including some reference strains from the Human Microbiome Project (NIH). Interestingly, H-NST also is conserved in other human pathogens such as Klebsiella pneumonia and K. oxytoca as well as in Salmonella enterica, where H-NST is present in three subspecies and eight serovars that are associated with food poisoning outbreaks worldwide Dickeya zeae, P. atrosepticum, Pectobacterium wasabiae and Pantoea ananatis, the latter of which is an opportunistic human pathogen causing bacteremia Salmonella and in plant pathogens that utilize H-NS-mediated regulation of virulence factors. This widespread distribution suggests a global regulatory effect of H-NST, prompting us to further investigate the molecular basis for H-NST function on the regulation of LEE expression.Since H-NST from EPEC E2348/69 positively affects the LEE we carried out BLAST searches to determine the presence of H-NST among A/E pathogens and other  the LEE . Those sLEE DNA complex, and was suggested to play a role in Ler regulation of LEE expression The current literature suggests that H-NST, like Ler, functions by negatively affecting H-NS-mediated repression of gene expression probably by displacing H-NS bound to regulatory DNA sites proU expression and that H-NST from UPEC containing valine at position 16 is nonfunctional LEE expression and the A/E lesion phenotype, we constructed the H-NST A16V mutant using site-directed mutagenesis of pQEH-NST. Evaluation of the sequence around Ala16 revealed a potential heptadic repeat within the coiled-coil element of H-NST of which Ala16 is the first residue . Actin filaments are stained by FITC-phalloidin in this assay to visualize condensed actin indicative of A/E lesions. FAS assays for EHEC typically involve a 5–6 h incubation time LEE-encoded proteins and induce pedestal formation, further supporting the functional importance of the Ala16 residue. The decreased activity of H-NST mutant at the Arg60 and Arg63 residues suggest functional importance of these residues, which potentially could be involved in DNA-binding.To determine whether the decreased ability of the H-NST mutants to induce accumulation of LEE operon expression through binding to DNA. The LEE1-encoded ler is expressed from two promoters named the distal (P1)- and proximal (P2) promoters with the distal promoter being the major promoter for LEE1 expression LEE1 and LEE2/LEE3 operons has been well studied LEE DNA LEE1 and LEE3 DNA targets contained in EHEC using electrophoretic mobility shift assays (EMSA) .H-NS and Ler both modulate s (EMSA) . We puriLEE1 containing both P1 and P2 promoters, we evaluated the binding of H-NST to the LEE1 P1P2 DNA target. H-NST bound specifically to LEE1 P1P2 DNA as demonstrated by a downshift of the bound labeled fragment in the presence of specific unlabeled LEE1 P1P2 DNA as opposed to the presence of unlabeled nonspecific rrsB DNA , which migrated at the same position regardless of whether one or both proteins were present , suggesting that H-NST is dispensable for LEE expression under the growth in DMEM. However, H-NST when expressed from an inducible promoter positively affected LEE-encoded protein levels and subsequently A/E lesion formation of EHEC (The expression of virulence factors including those of A/E pathogens encoded in the  of EHEC  and 4A–BLEE target DNA in vitro. We demonstrated that H-NST binds to DNA fragments containing the regulatory regions of LEE1 and LEE3 (LEE-encoded protein levels and A/E lesion formation (LEE contained in A/E pathogens.Arginine residues are commonly involved in DNA-binding by proteins and LEE3 , which iormation  and 4E–Formation , suggestproU and bgl in E. coli K-12 LEE and A/E lesion formation (proU expression, which was suggested to be due to the inability of the A16V mutant to form functional oligomers LEE-encoded protein levels and A/E lesion formation (We demonstrated that the Ala16 residue, which is known to be functionally important for H-NST to positively affect the expression of the H-NS-repressed genes ormation  and 4C-Dormation , 4C–D, sormation , suggestLEE3 regulatory region pre-bound by H-NS (LEE3 regulatory region. It is possible that H-NST when bound to DNA can change DNA topology to a conformation that is more suitable for DNA-binding by Ler than H-NS. The finding that H-NST affects LEE expression independently of ler further supports a model in which H-NST positively regulates expression through H-NS. Whether H-NST does so by modulating the DNA curvature by binding to DNA and/or prevents the binding of H-NS to DNA by forming H-NST/H-NS oligomers as previously suggested Interestingly, our data indicated that H-NST positively affects the binding of Ler to the  by H-NS , suggest