HIV protease is known to cause cell death, which is dependent upon cleavage of procaspase 8. HIV protease cleavage of procaspase 8 generates Casp8p41, which directly binds Bak with nanomolar affinity, causing Bak activation and consequent cell death. Casp8p41 can also bind Bcl2 with nanomolar affinity, in which case cell death is averted. Central memory CD4 T cells express high levels of Bcl2, possibly explaining why those cells do not die when they reactivate HIV. Here, we determine that the Casp8p41-Bcl2 complex is polyubiquitinated and degraded by the proteasome. Ixazomib, a proteasome inhibitor in clinical use, blocks this pathway, increasing the abundance of Casp8p41 and causing more cells to die in a Casp8p41-dependent manner.IMPORTANCE The Casp8p41 pathway of cell death is unique to HIV-infected cells yet is blocked by Bcl2. Once bound by Bcl2, Casp8p41 is polyubiquitinated and degraded by the proteasome. Proteasome inhibition blocks degradation of Casp8p41, increasing Casp8p41 levels and causing more HIV-infected cells to die. Reversible covalent modification of substrates with polyubiquitin chains drives their association with the proteasome and subsequent destruction. It has previously been documented that a complex of other proapoptotic proteins bound to their inhibitors is subject to proteasome-mediated degradation  . To test25–+ T cells were transfected with green fluorescent protein (GFP)-tagged Casp8p41, treated with PIs, and analyzed for GFP-Casp8p41 positivity 6 h later; 10 nM bortezomib increased the percentage of cells positive for GFP-Casp8p41 by 2.5-fold relative to control cells (P = 0.009), and 100 nM ixazomib resulted in a 2.4-fold increase (P = 0.045)   has altered the course of protein production diseases, such as multiple myeloma, in which plasma cells produce excessive amounts of monoclonal immunoglobulins. Long-term treatment with PIs over months to years causes semiselective death of myeloma cells  and impr= 0.045)  and C. Tdy (MAb) . Consistdy (MAb) , 17, Casdy (MAb)  and C, bdy (MAb) .in vitro HIV infection. First, activated primary CD4 T cells were acutely infected with luciferase-expressing HIV and then treated with dimethyl sulfoxide (DMSO) control, bortezomib, or ixazomib. Bortezomib or ixazomib significantly increased killing of HIV-infected T cell cultures over time (P < 0.001 each)  (P < 0.0001) than cells treated with control DMSO (P = 0.72) (P < 0.0001) (P < 0.0001)   at doses01 each) . Interesctively) . In our ctively) . GFP-HIVrol DMSO , but via = 0.72) . As not  0.0001) . Convers 0.0001) . However 0.0001) . Thus, i 0.0001)  yet enhaAbove, we observed that ixazomib causes a transient increase in HIV Luc  and HIV Renilla, as previously described (Renilla luciferase), demonstrating a direct effect on HIV LTR-mediated replication in cells containing HIV-Luc  or the HIV LTR missing the NF-κB-binding sites was linked to luciferase (HIVΔκB-Luc) were transfected into Jurkat T cells, and the transfection efficacy was normalized to that of cotransfected escribed . Treatme HIV-Luc . Consist HIV-Luc . Interna HIV-Luc .P = 0.014 for 5 nM bortezomib; P = 0.004 for 10 nM bortezomib; P < 0.0001 for 50 nM and 100 nM ixazomib)   and B. Txazomib)  and 5D. xazomib) , top, clxazomib) , middle,xazomib) , bottom,+) cells expressing GFP (a marker of procaspase 8) was measured. Ixazomib increased active caspase 3 expression in HIV-infected  cells expressing wild-type procaspase 8 (P = 0.005)  , but notWe next evaluated the effects of bortezomib and ixazomib on HIV-infected J-Lat 10.6 cells compared to the parental non-HIV-infected Jurkat cells using a clonotypic assay in which we evaluated cell survival within mixed populations.+ J-Lat cells should remain at ∼50% over time. Bortezomib and ixazomib treatments both resulted in a decrease in the proportion of viable CTO-positive J-Lat 10.6 cells (P < 0.001). Flow cytometric analysis demonstrated that, despite low-level caspase 3 induction in Jurkat cells, there were significantly more active caspase 3-positive HIV-infected J-Lat cells than uninfected Jurkat cells under the same culture conditions (P < 0.01 for 10 nM bortezomib and 100 nM ixazomib)   and B. S.6 cells  (P < 0.0xazomib)  and E.+ T cells from ART-suppressed patients that reactivation from latency induces Casp8p41 expression (RF), treated with ixazomib or control, and assessed for caspase 3 activation over time (RF-infected and -exposed cells (P < 0.0001), but not in mock-infected cells. This is consistent with our findings in laboratory-adapted HIV-1 strains  (P < 0.001)  , top. To= 0.007) , 41. Ixa< 0.001) , bottom.There is increasing appreciation that the biological processes that favor survival of cancer cells also operate in cells containing the transcriptionally silent reservoir for HIV; examples are epigenetic modifications of DNA and enhanced expression of checkpoint inhibitors , 43. FolOur data demonstrate that an oral proteasome inhibitor that is clinically dosed once weekly functions dually to reactivate HIV and to increase Casp8p41 expression, thereby inducing apoptotic death of T cells in which HIV replicates . Killingex vivo. Acknowledging that the full clinical significance of ex vivo HIV DNA content reductions is unknown, and given that a significant fraction of HIV DNA is replication or otherwise defective  to decrease following single-dose treatment efective , additio+ T cell enrichment cocktail (Stemcell Technologies) or an EasySep human CD4+ T cell isolation kit (Stemcell Technologies); we routinely achieve >95% CD4 T cell purity by these techniques. The following cell lines and sources were used: Jurkat T cells , 293T cells , J-Lat 10.6 cells (NIH AIDS Reagent Program), and JB-6 cells  rat monoclonal antibody , mouse anti-caspase 8 antibody , mouse monoclonal anti-ubiquitin (P4D1) antibody , anti-Bcl-2 (C21) rabbit polyclonal IgG , monoclonal anti-HIV p24 , anti-GAPDH  (14C10) rabbit MAb , histone deacetylase (HDAC) antibody sampler kit , anti-phospho-NF-κB-p65 (Ser 536) rabbit MAb , anti-NF-κBp65 (D14E12) rabbit MAb , anti-phospho-IκBα (Ser32) (14D4) rabbit MAb , anti-IκBα (L35A5) mouse MAb , anti-cleaved caspase 3 (Asp175) antibody , and mouse anti-human PARP . The following antibodies were used for flow cytometry: phycoerythrin (PE)-conjugated mouse anti-HIV core antibody (Beckman Coulter) and allophycocyanin (APC)-conjugated rabbit anti-active caspase 3 antibody .IIIB, from Robert Gallo, and HIV-1RF, from Dean Winslow. The envelope-defective proviral GFP-HIV-Nef-Vpr 426-bp plasmid used for −/Env− was obtained from É. Cohen   . Individ Canada) .4 and 105 on the relevant axis by visual cloud gating. For the analysis shown in Jurkat T cells were transfected with GFP-empty vector and GFP-Casp8p41 constructs, as previously described . IntraceJurkat cells were infected at a multiplicity of infection (MOI) of ∼1.0 with VSV-G GFP HIV in the presence of 6 μg/ml Polybrene (Sigma-Aldrich) for 6 h, washed, and treated or not with ixazomib (100 nM). Cell death was measured with a real-time imaging IncuCyte system (Essen BioScience) using the cell permeability dye IncuCyte cytotox red reagent . Analysis of the data was done using IncuCyte Zoom software (2016B).6-TRABID, CEZANNE, YOD1, OUTLIN, and USP2 and incubated at 37°C for 30 min. Casp8p41-HA protein was eluted and resolved by SDS-PAGE and immunoblotted with HA-horseradish peroxidase (HRP) (Roche).Casp8p41 protein was analyzed for ubiquitin linkage specificity with a UbiCrest deubiquitinase (DUB) enzyme set  . 293T ceRenilla reniformis (pNL-LucR) was obtained from C. Ochsenbauer , as previously reported (+ T cells were HIV infected after activation using phytohemagglutinin (2 μg/ml) and interleukin-2 (IL-2) (50 IU/ml) for 48 h; then, ∼100 ng of p24 in 0.5 ml was added to 106 cells for 6 h and then washed and cultured with 10 IU/ml IL-2. When indicated, cells were lysed with 1× Renilla luciferase lysis buffer and analyzed for Renilla luciferase activity. Viability was assessed using a CellTiter-Glo luminescent cell viability assay (Promega).HIV-1 luciferase-expressing HIV-1 coexpressing reported . The pNLx axis and VIC intensity on the y axis. Gates used to count the droplet events with specific fluorescence properties were defined using graphical tools to outline regions from the positive control, and these gates were applied to all the samples. The positive droplet events were counted within each gate for each sample. The droplet counts were normalized to the RPP30 counts. Total HIV DNA was calculated and expressed in 1 million CD4 cells by the estimated number of RPP30 copies . We validated the use of ddPCR for total HIV DNA estimation using 10 independent evaluations of samples containing a fixed ratio of uninfected Jurkat T cells and chronically HIV-infected 8E5 T cells. The coefficient of variance of the ddPCR was 3.8%, and the detection limit of the assay was found to be 45 HIV DNA copies per million cells.DNA for analysis was extracted using a Qiagen DNA blood minikit , linearized using a Covaris M220 focused ultrasonicator with 3.0 kb minitube blue , and quantified using a NanoDrop 2000 spectrophotometer. The fragmentation length was confirmed via gel electrophoresis. Raindance digital PCR was performed using HIV primers and probes specific for HIV Pol (Hxb2 positions 2536 to 2662) and an RPP30 (RNase P) primer-probe set as previously reported . PCRs wegag gene was followed by a nested real-time PCR with primers and a probe specific for the HIV LTR. The numbers of cells assayed per well ranged from 1,813 to 29,000. The number of cells assayed per well was based on total HIV levels to result in the tightest confidence intervals for binomial distribution (we targeted 30 to 80% positive wells at two dilutions) (DNA was isolated using a Gentra Puregene cell kit (Qiagen). Levels of integrated HIV DNA were measured by Alu-Gag quantitative PCR (qPCR), as previously described . Brieflylutions) .t tests, analysis of variance (ANOVA), or the nonparametric equivalent, as appropriate. The mean values of matched samples were compared using paired t tests. Time course experiments were compared using linear regression. All tests were two sided, and P values of less than 0.05 were considered statistically significant. Statistical analysis was performed using GraphPad Prism 6.Results are depicted as means and standard deviations (SD) unless specifically noted. The mean values of the results of independent experiments were compared using Primary human peripheral blood mononuclear cells were obtained following informed consent according to Mayo Clinic Institutional Review Board (IRB)-approved protocols (IRB numbers 1036-03 and 13-005646) and a Hennepin County Medical Center Human Subjects Research Committee-approved protocol (HSR number 15-3934).


Chimeric antigen receptors (CARs) have undoubtedly revolutionized immunotherapy, especially in the B-cell acute lymphoblastic leukemia (ALL) arena where over 80% of complete remissions are observed in refractory/relapsed (R/R) B-cell ALL patients treated with CD19-directed CAR T-cells (CARTs) . For insPROM1 gene, is a specific marker for MLLr leukemia because PROM1 is an MLL target, especially in MLL-AF4 B-cell ALL [In this sense, loss of CD19 antigen is frequently observed after CD19-directed CARTs therapy in B-cell ALL , 4, but cell ALL –10. Theycell ALL , thus suPROM1/CD133 expression in a large cohort of cytogenetically distinct B-cell ALL subgroups (n = 212 patients) as well as in different subpopulations of normal CD34+ HSPCs obtained across hematopoietic ontogeny from 22-weeks old human fetal liver , cord blood , and adult G-CSF-mobilized peripheral blood/bone marrow . An initial analysis of publicly available RNA-seq data [PROM1 is overexpressed in patients with MLLr B-cell ALL, although its expression is not significantly higher than in other cytogenetic subgroups , which would likely exert “on-target off-tumor” myeloablative, life-threatening toxicity , 12. Becups Fig. . We thenups Fig. . ImportaPROM1 gene product) in the cell surface of BM-derived primary blasts and primografts (PDXs) obtained from 11q23/MLLr (n = 7) and non-MLL (n = 5) B-cell ALL patients, and in comparison with healthy prenatal (22 weeks old FL), perinatal (CB) and adult (PB/BM) CD34+ HSPCs  mediating T-cell activation and another CAR engaging a second antigen  mediating T-cell co-stimulation [Our data demonstrates that mulation . Unfortumulation . Furthermulation , 19.


Prostate embryonic development, pubertal and adult growth, maintenance, and regeneration are regulated through androgen signaling-mediated mesenchymal-epithelial interactions. Specifically, the essential role of mesenchymal androgen signaling in the development of prostate epithelium has been observed for over 30 years. However, the identity of the mesenchymal cells responsible for this paracrine regulation and related mechanisms are still unknown. Here, we provide the first demonstration of an indispensable role of the androgen receptor (AR) in sonic hedgehog (SHH) responsive Gli1-expressing cells, in regulating prostate development, growth, and regeneration. Selective deletion of AR expression in Gli1-expressing cells during embryogenesis disrupts prostatic budding and impairs prostate development and formation. Tissue recombination assays showed that urogenital mesenchyme (UGM) containing AR-deficient mesenchymal Gli1-expressing cells combined with wildtype urogenital epithelium (UGE) failed to develop normal prostate tissue in the presence of androgens, revealing the decisive role of AR in mesenchymal SHH responsive cells in prostate development. Prepubescent deletion of AR expression in Gli1-expressing cells resulted in severe impairment of androgen-induced prostate growth and regeneration. RNA-sequencing analysis showed significant alterations in signaling pathways related to prostate development, stem cells, and organ morphogenesis in AR-deficient Gli1-expressing cells. Among these altered pathways, the transforming growth factor β1 (TGFβ1) pathway was up-regulated in AR-deficient Gli1-expressing cells. We further demonstrated the activation of TGFβ1 signaling in AR-deleted prostatic Gli1-expressing cells, which inhibits prostate epithelium growth through paracrine regulation. These data demonstrate a novel role of the AR in the Gli1-expressing cellular niche for regulating prostatic cell fate, morphogenesis, and renewal, and elucidate the mechanism by which mesenchymal androgen-signaling through SHH-responsive cells elicits the growth and regeneration of prostate epithelium. Prostate formation, growth, and regeneration, as well as tumorigenesis, depend on androgens and androgen receptor (AR)-mediated signaling pathways. Tissue recombination assays done more than 30 years ago demonstrated a decisive role for stromal androgen signaling in prostatic epithelium development. However, in the intervening time, the identity of the mesenchymal cells in the urogenital sinus mesenchyme that convey androgen signaling and control prostate epithelium development, morphogenesis, and regeneration has not been determined. In this study, using mouse genetic tools, we demonstrate for the first time that selective deletion of AR in mesenchymal Gli1-expressing cells abolishes early development of prostate tissue and normal prostate formation, and diminishes prostate pubertal growth and regeneration. In addition, using tissue recombination assays, we directly determined an essential requirement for AR expression in mesenchymal Gli1-expressing cells during prostate epithelium development. Our results not only resolve a 30-year-old scientific puzzle by identifying the mesenchymal cell properties of androgen-responsive cells that elicit development of the embryonic prostate epithelium, but also explore a new regulatory mechanism for androgen and Shh signaling-mediated cellular niches in regulating prostatic cell fate, growth, and renewal through paracrine regulation. Given the importance of sex hormone and hedgehog signaling pathways in human development and tumorigenesis, this study extends beyond the field of prostate biology, raising new questions underlying sex hormone and SHH signaling in development and tumorigenesis. Prostate formation, growth, and regeneration depend on androgen receptor (AR)-mediated signaling pathways . During Emerging evidence has shown that sonic hedgehog (SHH) signaling plays a critical role in prostate development, homeostasis, and regeneration through mesenchymal–epithelial interactions ,11. The In this study, we demonstrate an indispensable role of androgen signaling in mesenchymal Gli1-expressing cells in regulating prostate early development, morphogenesis, and regeneration. Specifically, tissue recombination assays showed that urogenital mesenchyme (UGM) containing AR-deficient mesenchymal Gli1-expressing cells combined with wild type urogenital epithelium (UGE) failed to develop normal prostate tissue in the presence of androgens, revealing the decisive role of AR in mesenchymal SHH responsive cells in prostate development. Prepubescent deletion of AR expression in Gli1-expressing cells resulted in severe impairment of androgen-induced prostate growth and regeneration. Using RNA-sequencing approaches, we examined the changes of transcriptome profiles in AR-deficient Gli1-expressing cells, and identified significant alterations in signaling pathways related to prostate development, stem cells, and organ morphogenesis. The data presented in this study elucidate an innovative role of the AR in the Gli1-expressing cellular niche for regulating prostatic cell fate, growth, and renewal, and elucidate a new paracrine regulatory mechanism by which mesenchymal androgen-signaling elicits the growth of prostate epithelium.ROSA26RmTmG/+:ArLoxP/Y:Gli1CreERT2/+ (R26mTmG/+:ArL/Y:Gli1CreER/+), and ArLoxP/Y:Gli1CreERT2/+ (ArL/Y:Gli1CreER/+) mice, as well as R26mTmG/+:Gli1CreER/+controls (Ar gene and activation of membrane-bound green fluorescent protein (mGFP) expression occur in Gli1-expressing cells in these mice  analyses using RNA samples isolated from prostatic GFP-positive cells isolated from both R/+ mice . We idencontrols . Using Gcontrols . Specificontrols . Consistcontrols . An inhiTo address this hypothesis, we performed a series of proof-of-principle experiments. Using quantitative reverse transcription-PCR (qRT-PCR), we observed reduced expression of AR transcripts and its downstream target, probasin, in AR-deficient Gli1-expressing cells . While tProstate embryonic development, subsequent pubertal and adult growth, homeostatic maintenance, and regenerative capability require androgen-mediated paracrine interactions between stromal and epithelial cell compartments ,2 6]. T. T6]. ThSHH signaling plays a critical role in prostatic development, homeostasis, and tumorigenesis through reciprocal epithelial-mesenchymal interactions ,26. ProsTo gain more insight into molecular mechanisms underlying AR action in Gli1-expressing cells, we performed RNA-sequencing analyses with samples isolated from either wild type or AR-deficient Gli1-expressing cells in mouse prostate tissues. We identified aberrant alterations in signaling pathways related to prostate development, stem cells, organ morphogenesis in AR-deficient Gli1-expressing cells. Specifically, the transforming growth factor β1 (TGFβ1) pathway was up-regulated in AR-deficient Gli1-expressing cells. Consistent with this, significant enrichment of the TGFβ1 pathway was observed in DEGs between AR-deficient Gli1-expressing cells. We further performed a series of proof-of-principle experiments to demonstrate that elevated TGFβ1-mediated pathways in AR-deficient mesenchymal Gli1-expressing cells inhibit the growth of prostate epithelium through mesenchymal-epithelial paracrine regulation. We observed elevated expression of TGFβ1 downstream targets and effectors in AR deficient Gli1-expressing cells at embryonic stages and postnatal days 28 and 56, respectively . Since pAll experimental procedures and care of animals in this study were carried out according to the Institutional Animal Care and Use Committee (IACUC) at Beckman Research Institute at City of Hope, and approved by the IACUC. Euthanasia was performed by CO2 inhalation followed by cervical dislocation.Gli1CreER and ROSAmTmG mice were obtained from Jackson Laboratories (stocks 18867 and 7676). ArLox/Y mice were obtained from Dr. Guido Verhoeven [erhoeven . To elicerhoeven ,32. To lAdult male mice were castrated as described previously . For and2O2 for 15 min, blocked in 5% normal goat serum (Gibco) for 1 h, and incubated with appropriate antibodies  and processed into paraffin. Five-micron serial sections were cut and processed from Clearify (American MasterTech Scientific) to PBS through a decreasing ethanol gradient. For histological assessment, hematoxylin and eosin staining was performed as described ,32. For dies see  diluted dies see .Hematoxylin and eosin and immunohistochemistry slides were imaged using an Axio Lab A1 microscope with 10x and 40x Zeiss A-Plan objectives. Images were taken with a Canon EOS 1000D camera and analyzed using Axiovision software (Carl Zeiss). Images of immunofluorescent staining and mTmG signals were acquired on an Olympus Motorized Inverted Research Microscope Model IX81 using 20x and 40x Olympus Plan Fluor objectives, a QImaging RETIGA 2000R camera, and Image-Pro 6.3 software (Media Cybernetics).Mouse serum testosterone levels were measured using a Mouse/Rat Testosterone ELISA kit (Alpco Diagnostic). Mouse blood samples were collected and tested following the manufacturer’s protocol. The concentration of each sample, corresponding to mean of absorbance value, was then calculated from the calibration curve.To prepare Gli1-expressing cells for RNA sequencing, male mice from different genotypes were injected with TM (1 mg) on P14 or TM (125 μg/g body weight) to activate deletion of AR in Gli1-positive cells. On day E16.5 or P28 or P56, the mice were euthanized and their UGS or prostate tissues were collected, minced into small pieces, and digested with 1 mL of collagenase  in DMEM with 10% FBS, DHT (10 nM) and Y-27632 (10 μM) (StemCell Technologies) at 37°C for 90 min. Cells were then digested with TrypLE  at 37°C for 15 min and were centrifuged at 300xg for 5 min. Cells were passed through 40-μm nylon mesh (Fisherbrand), washed twice with DMEM 10% FBS and dissolved in 500 μL of PBS-2% BSA-1 mg/mL DAPI for cell sorting. Cells were sorted for mGFP-positive and tdTomato-negative, or CD24 positive for prostatic luminal epithelial cells . After sRNA samples were extracted with TRIZOL . RNA sequencing libraries were prepared by using SMARTer Ultra Low Input RNA Kit for Sequencing v4 (TaKaRa Clontech) and KAPA Hyper Prep Kit (KAPA Biosystems) according to the manufacturer's protocol. The resulting double stranded cDNA was sheared using a Covaris LE220 Plus (Covaris) with a 200 bp peak in the 50μl volume setting. The fragmented cDNA underwent end repair, 3′ end adenylation and ligation with barcoded adapters. The libraries were validated using the Agilent Bioanalzyer DNA High Sensitivity kit (Agilent), and quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). The sequencing library templates were prepared for sequencing using the Illumina HiSeq SR Cluster V4 Kit. Sequencing runs were performed on an Illumina HiSeq 2500 using the single read mode of 51 cycles of read 1 and 7 cycles of index read with the SBS V4 Kit. Real-time analysis (RTA) 2.2.38 software was used to process the image analysis and base calling.qPCR reactions were performed in triplicate using an Applied Biosystems 7900 Fast sequence detector with SYBR Green PCR master mix . Primers were designed using PrimerQuest (IDT) . All reahttp://heatmapper.ca/expression/) [RNA sequences were aligned to mouse genome assembly mm9 using TopHat v2 , and Ensession/) . To calcession/) ,41 and tession/)  gene setStatistical analyses were performed using GraphPad Prism 6. All data are presented as mean ± s.d. Two group comparisons were analyzed with a two-tailed Student’s t-test and a value of P < 0.05 was taken as statistically significant.S1 Fig(A) and squamous cysts (B) with prominent central keratinization from prostate tissues isolated from 8-week old R26mTmG/+:ArL/Y:Gli1CreER/+ mice. Scale bars, A, B, 200 μm; A’, B’, 20 μm.Representative images of squamous metaplasia (PDF)Click here for additional data file.S2 Fig(A-B) Fluorescence micrographs of mT and mG expression from P56 prostate tissues from male mice of the indicated genotypes. Scale bars, A-B, 20 μm; A’-B’, 10 μm. (C1 to J5) Images of triple-immunofluorescent staining of prostatic tissues with the indicated antibodies. White arrows indicate AR and GFP double positive cells (C5). Pink arrows indicate triple positive cells (D5 and E5). Blue arrows indicate AR-negative, GFP-positive, and E-cadherin negative cells (G5). Green arrows indicate AR-negative, GFP-positive cells with either CD34 (H5) or SMA (I5) staining. Scale bars, C1-4, D1-4, E1-4, F1-4, G1-4, H1-4, I1-4, and J1-4, 20 μm; C5, D5, E5, F5, G5, H5, I5, and J5, 10 μm. (K) Quantification of AR and E-cadherin double positive cells per E-cadherin-positive cells (left panel) and AR and mGFP double positive cells per mGFP-positive cells (right panel) in P56 prostate tissues from male mice of the indicated genotypes. Error bars indicate s.d., analyzed using 2-tailed t-test; n = 6 mice per genotype.(PDF)Click here for additional data file.S3 Fig(A-F’) Representative H&E staining of prostatic lobes from P56 prostates isolated from R26mTmG/+:Gli1CreER/+ or R26mTmG/+:ArL/Y:Gli1CreER/+ mice. (G-L’) Representative H&E staining of prostatic lobes from P56 prostates isolated from androgen supplemented R26mTmG/+:Gli1CreER/+ and R26mTmG/+:ArL/Y:Gli1CreER/+ mice. (M-R’) Representative H&E staining of 8-week old implants from R26mTmG/+:Gli1CreER/+ or R26mTmG/+:ArL/Y:Gli1CreER/+ P14 prostatic lobes. Scale bars, A-R 100 μm; A’-R’ 20 μm.(PDF)Click here for additional data file.S4 Fig(A-B”) IHC analyses for AR expression in different prostatic lobes of regenerated prostates from R26mTmG/+:Gli1CreER/+ or R26mTmG/+:ArL/Y:Gli1CreER/+ mice. (C-D”) IHC analyses for Ki67 expression in different prostatic lobes of regenerated prostates from R26mTmG/+:Gli1CreER/+ or R26mTmG/+:ArL/Y:Gli1CreER/+ mice. Scale bars, 20 μm.(PDF)Click here for additional data file.S5 Fig(A) Relative expression of probasin from Gli1-CreER driven GFP expressing cells and epithelial cells isolated from prostates of either R26mTmG/+:Gli1CreER/+ or R26mTmG/+:ArL/Y:Gli1CreER/+ mice. Both Gli1CreER driven GFP expressing cells and prostatic epithelial cells were isolated and sorted by GFP or CD24 antibody, respectively. RNA samples were prepared and used to generate cDNA. The relative expression levels from three individual experiments were shown. (B—C) Fold changes in labeled expression of genes determined by qRT-PCR analysis using FACS-sorted GFP positive cells from either UGM tissues at day E16.5 (B) or prostate tissues at postnatal day 56 (C) isolated from R26mTmG/+:Gli1CreER/+ or R26mTmG/+:ArL/Y:Gli1CreER/+ mice. Error bars indicate s.d.; *P < 0.05, ** P < 0.01; analyzed using 2-tailed students’ t test. (n = 3 replicates per data point).(PDF)Click here for additional data file.S1 TableSupporting data for (PDF)Click here for additional data file.S2 TableSupporting data for (PDF)Click here for additional data file.S3 TableSupporting data for (PDF)Click here for additional data file.S4 TableSupporting data for (PDF)Click here for additional data file.S5 TableR26mTmG/+:ArL/Y:Gli1CreER/+ mice compared to normal Gli1-expressing cells from age- and sex-matched R26mTmG/+:Gli1CreER/+ controls. Supporting data for List of up-regulated and down-regulated genes from AR-deficient Gli1-expressing cells from (PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.


NKL homeobox genes encode developmental transcription factors regulating basic processes in cell differentiation. According to their physiological expression pattern in early hematopoiesis and lymphopoiesis, particular members of this homeobox gene subclass constitute an NKL-code. B-cell specific NKL-code genes generate a regulatory network and their deregulation is implicated in B-cell lymphomagenesis. Epstein-Barr virus (EBV) infects B-cells and influences the activity of signalling pathways including JAK/STAT and several genes encoding developmental regulators. Therefore, EBV-infection impacts the pathogenesis and the outcome of B-cell malignancies including Hodgkin lymphoma and diffuse large B-cell lymphoma (DLBCL). Here, we isolated EBV-positive and EBV-negative subclones from the DLBCL derived cell line DOHH-2. These subclones served as models to investigate the role of EBV in deregulation of the B-cell specific NKL-code members HHEX, HLX, MSX1 and NKX6-3. We showed that the EBV-encoded factors LMP1 and LMP2A activated the expression of HLX via STAT3. HLX in turn repressed NKX6-3, SPIB and IL4R which normally mediate plasma cell differentiation. In addition, HLX repressed the pro-apoptotic factor BCL2L11/BIM and hence supported cell survival. Thus, EBV aberrantly activated HLX in DLBCL, thereby disturbing both B-cell differentiation and apoptosis. The results of our study appreciate the pathogenic role of EBV in NKL homeobox gene deregulation and B-cell malignancies. Hematopoietic stem cells reside in the bone marrow and generate precursor cells for the myeloid and lymphoid lineages. The last steps of B-cell development take place in the germinal centers which are located in lymphoid organs. They include the differentiation into plasma cells (CD38+ CD138+ surface IgG-) or memory B-cells (CD38- CD138- surface IgG+) which express particular cell type specific factors. These developmental processes are mainly regulated at the transcriptional level. Accordingly, several transcription factors like BCL6 and PAX5 act as master genes/factors for B-cell development . MoreoveDLBCL is the most common type of B-cell malignancies . This diEBV is a 172 kb long DNA-virus that belongs to the group of human herpesviruses and is accordingly also named HHV4. It encodes more than 80 genes and enters epithelial and lymphoid cells via the complement receptor CR2/CD21 –13. InfeHere, we established a cell line model to analyze the EBV-mediated impact on B-cell differentiation factors in DLBCL. We show the mechanism and the consequences of EBV in deregulation of the NKL-code member HLX.5´-GAGACCUUCUCUGUCCACUTT-3´ and 5´-AGUGGACAGAGAAGGUCUCTT-3´, and for LMP2A 5´-CUCCCAAUAUCCAUCUGCUTT-3´ and 5´-AGCAGAUGGAUAUUGGGAGTT-3´ [6 cells by electroporation using the EPI-2500 impulse generator  at 350 V for 10 ms. Transfected cells were harvested after 20 h cultivation. For functional examinations treated cells were analyzed by the IncuCyte S3 Live-Cell Analysis System . For detection of apoptotic cells we additionally used the IncuCyte Caspase-3/7 Green Apoptosis Assay diluted at 1:2000 (Essen Bioscience).EBV-negative (ACC No. 47) and EBV-positive subclones of the DLBCL cell line DOHH-2 are held by the DSMZ  and were cultivated as described . The tesGAGTT-3´ . Express5 DOHH-2 cells were washed with phosphate-buffered-saline buffer (PBS) and centrifuged onto acid washed slides. The cells were fixed with methanol / acetic acid (3:1) and subsequently dehydrated via an ethanol series. Cy3 labeled EBV cosmid DNA and Cot DNA  were added to the air-dried slides, denatured and hybridized overnight at 37°C. The slides were then washed with 2xSSC, mounted with Vecta-Shield  and evaluated with fluorescence microscope Axiovert 40 CFL (Zeiss) and software VisiView version 1.6.9 .The detection of EBV in DOHH-2 cells was performed by interphase fluorescence in situ hybridization (FISH) and by polymerase chain-reaction (PCR) analysis as described recently . BrieflyFor PCR analysis, genomic DNA of the cell lines was applied for the detection of episomal, linear, or integrated EBV sequences. The DNA was extracted from PBS washed cells using the High Pure PCR Template Preparation Kit (Roche). The integrity and quality of the DNA was verified by PCR analysis of the human ABL1 gene. The EBV specific PCR primers amplify a 265 bp fragment of a conserved region of the EB2/BMLF1 gene. A parallel reaction was performed with an internal control sequence of 445 bp and amplified with the same primers as the EBV wild type DNA fragment serving as internal control.5´-CGATGAGTAGGAGGGTGACTGG-3´, LMPA2-forward 5´-CCTAGAAATGGTGCCAATGGGC-3´, LMPA2-reverse 5´-GTGTTCCCATAAGAGTCAGAAGC-3´, EBNA1-forward 5´-GATGGTGAGCCTGACGTGCC-3´, EBNA1-reverse 5´-TTGCGCCTGCCTCCATCACC-3´, EBNA2-forward 5´-CGGTTCACCTTCAGGGCCTAGG-3´, EBNA2-reverse 5´-CTGGTAGGACTGGGCGACCGG-3´, EBNA3A-forward 5´-ACCCTGGCCGCCGGATGGCC-3´, EBNA3A-reverse 5´-ACCTGTGCACCCAAGTGTCGCC-3´, EBNA3C-forward 5´-GTGGACACCACCCCATGCTGG-3´, EBNA3C-reverse 5´-GATTCTTCGGTACCGCCTCTGCC-3´, YY1-forward 5´-AAGCAGGTGCAGATCAAGAC-3´, YY1-reverse 5´-CCGAGTTATCCCTGAACATC-3´. The oligonucleotides were obtained from Eurofins MWG . The generated PCR products were subsequently analyzed by agarose gel electrophoresis. Documentation was performed using the Azure c200 Gel Imaging System .Reverse transcription (RT)-PCR was performed using taqpol (Qiagen) and the thermocycler TGradient . The oligonucleotides used for analysis of expressed EBV-encoded genes and a cell line encoded control gene (YY1) were as follows: LMP1-forward 5´-GGAGGCCTTGGTCTACTCCTAC-3´, LMP1-reverse Total RNA was extracted from cell line samples using TRIzol reagent . Primary human total RNA was commercially obtained. We used RNA from peripheral blood mononuclear cells (PBC), lymph node (LN), spleen (SP), and bone marrow (BM) obtained from Biochain/BioCat , and RNA from peripheral CD19-positive B-cells and CD3-positive T-cells obtained from Miltenyi Biotec . cDNA was synthesized from 5 μg RNA by random priming using Superscript II (Invitrogen). RQ-PCR analysis was performed with the 7500 Real-time System, using commercial buffer and primer sets . Quantification of MSX1 was performed as described previously . For norWestern blots were generated by the semi-dry method. Protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma). Proteins were transferred onto nitrocellulose membranes  and blocked with 5% dry milk powder dissolved in PBS. The following antibodies were used: alpha-Tubulin (Sigma), BCL2L11/BIM (Thermo Fisher), IL4R , SMAD1 , STAT3 , phospho-STAT3 (Ser727) , XBP1 (Santa Cruz Biotechnology). For loading control blots were reversibly stained with Poinceau (Sigma) and detection of alpha-Tubulin (TUBA) was performed thereafter. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL . Documentation was performed using the digital system ChemoStar Imager . Densitometric quantification of protein bands was performed using software AzureSpot version 14 (Azure Biosystems). The obtained densities from the protein of interest were correlated to the control bands of TUBA and the calculated values indicated below.Immuno-cytology was performed as follows: cells were spun onto slides and subsequently air-dried and fixed with methanol/acetic acid for 90 s. The phospho-STAT3 antibody  was diluted 1:20 in PBS containing 5% BSA and incubated for 30 min. Washing was performed 3 times with PBS. Preparations were incubated with secondary antibody (diluted 1:100) for 20 min. To detect IgG surface immunoglobulins we used directly labelled anti-IgG antibody diluted at 1:100 . After final washing the cells were mounted in Vectashield , containing DAPI for nuclear staining. Documentation of subcellular protein localization was performed using fluorescence microscope Axio-Imager  configured to a dual Spectral Imaging FISH system .www.NCBI.NLM.gov/GEO) via GSE125420. After RMA-background correction and quantile normalization of the spot intensities, the profiling data were expressed as ratios of the sample mean and subsequently log2 transformed. Data processing was performed via R/Bioconductor using limma and affy packages.Expression profiling datasets of EBV-positive/negative DOHH-2 subclones using HG U133 Plus 2.0 gene chip (Affymetrix) were generated by Dr. Robert Geffers . The primary data are available at Gene Expression Omnibus (http://bonsai.hgc.jp/~mdehoon/software/cluster/index.html).For creation of heat maps showing selected values of gene expression profiling data we used the software CLUSTER (version 2.11) and TREEVIEW (version 1.60) originally developed by Michael Eisen (Public expression profiling datasets GSE3996 and GSE17372 were exploited for HLX expression (probe set ID: 214438_at) in HL and DLBCL patients, respectively. For statistical calculations and data presentations in box plots we used R-based tools.In a previously published study about the virus load in cell lines we described DLBCL cell line DOHH-2 to contain EBV in less than 5% of the cells, demonstrating that this cell culture consisted of both EBV-positive and EBV-negative cells ,29. HereEBV particles enter B-cells via the complement receptor CR2/CD21 . QuantifEBV-negative DOHH-2 cells grew individualized in suspension while EBV-positive DOHH-2 cells created aggregates . SimilarEBV influences the activity of basic B-cell specific genes including BCL6, BACH2, IRF4, MIR155HG and PRDM1 ,18,33,34Recently, we identified a hematopoietic NKL-code which describes the physiological activity of NKL homeobox genes in lymphopoiesis. The B-cell associated code members comprise HHEX, HLX, MSX1 and NKX6-3 and generate a gene network by mutual regulation . RQ-PCR To confirm the role of EBV on HLX we performed siRNA-mediated knockdown of two basic EBV-encoded factors, LMP1 and LMP2A . The resNext, we wanted to define the molecular mechanisms of EBV-mediated deregulation of HLX. In addition to the mutual regulation of NKL-code members NKX6-3 and MSX1, we have described several factors which influence the activity of NKX6-3 in B-cells which may thus contribute indirectly to HLX activation . TherefoActivation of the JAK/STAT-pathway is a general feature of EBV in DLBCL . In partTo investigate this hypothesis we first quantified the concomitant expression levels of STAT3 and HLX in EBV-positive and EBV-negative DOHH-2 cells in comparison to HL cell line L-540 . These dSTAT3 has been found to activate PRDM1 in the course of plasma cell differentiation . AccordiNKX6-3 belongs to the B-cell specific NKL-code and is the only code-member expressed in differentiated plasma cells while HHEX is solely expressed in memory B-cells . ConsistFurthermore, HLX represses in L-540 cells the expression of the pro-apoptotic factor BCL2L11/BIM . ConsistA summary of the results of this study in combination with data from the literature is shown in The EBV-encoded factors LMP1 and LMP2A activate STAT3 in B-cells as indicated here and reported previously –39. The DLBCL has been divided into GC- and ABC-subtypes . EBV is Memory B-cells and plasma cells represent final stages in the course of B-cell development . Plasma BCL2L11/BIM acts as a central apoptotic factor in normal and malignant B-cells ,45. The Together, our data reveal a novel player underlying the oncogenic impact of EBV in differentiation and survival of B-cells. We show that NKL homeobox gene HLX occupies a central position in basic EBV-mediated effects in infected malignant B-cells. In addition, the EBV-positive/negative subclones of DOHH-2 show several deregulated developmental genes involved in B-cell differentiation. Therefore, these cell lines may serve as suitable model to investigate the role of EBV in particular aspects of B-cell malignancies.S1 FigSTR profiling data are shown for DOHH-2 reference cell line (above), and for EBV-positive and EBV-negative DOHH-2 subclones (below), demonstrating their identity.(TIF)Click here for additional data file.S2 FigPublic expression profiling data of EBV-positive and EBV-negative (A) HL patients (GSE3996) and (B) DLBCL patients (GSE17372) show elevated HLX expression in EBV-positive patients as compared to EBV-negative controls in both entities. However, the p-values (obtained by using the Mann-Whitney-U Test) indicated absence of statistical significance.(TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.


Aging is associated with gut dysbiosis – a condition linked with altered central nervous system function (i.e the “gut-brain axis”). Age-related health benefits have been ascribed to the renin-angiotensin system (RAS), mediated partially via the angiotensin (1-7) or Ang(1-7) axis. This pre-clinical study explored dosing of a genetically modified probiotic expressing Ang(1-7) – which we previously showed to induce dose-dependent increases in circulating Ang(1-7) – in modulating the gut-brain axis. Twenty-nine male F344BN rats were randomized at 24 months of age to receive oral gavage of Ang(1-7) Lactobacillus paracasei (LP) zero (control), one, three, or seven times/week over 28 days. At day 29, samples of feces, serum and pre-frontal cortex (PFC) were collected. Microbiome taxonomic analysis of fecal samples was performed via 16S-based PCR. Serum samples were analyzed for tryptophan and downstream metabolites via LC-MS. PFC was evaluated for mRNA expression of select inflammatory cytokines. PCoA revealed that groups differed in the overall fecal microbiota community structure as determined by Unweighted UniFrac. Indices of alpha-diversity, including richness and phylogenetic diversity, displayed significant group differences – with the most dramatic effects observed in the 3-times/week group. Compared to control, serum serotonin and 2-Picolinic Acid were significantly increased in the 3-times/week group. The 3-times/week regimen also significantly reduced COX2, IL1β, and TNFα mRNA expression, and 7-times/week reduced COX2 and IL1β expression in PFC. Therefore, we conclude that short-term treatment with Ang(1-7) LP dose-dependently benefits the gut-brain axis in aged rats, with 3-times/week appearing to be the optimal dosing regimen.


Their chiroptical properties and chiral recognition abilities as chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC) were investigated. Poly-2S and poly-(2Sx-co-31−x) (0.06 < x < 0.71) formed a preferred-handed helical conformation with opposite helical senses after thermal annealing despite possessing the same chiral pendant (h-poly-2S and h-poly-(2Sx-co-31−x)). Furthermore, h-poly-2S and h-poly-(2S0.36-co-30.64) emitted circularly polarized luminescence with opposite signs. h-Poly-2S showed higher chiral recognition abilities toward a larger number of racemates than poly-2S without a preferred-handed helicity and the previously reported preferred-handed poly(diphenylacetylene) derivative bearing the same chiral substituent on half of its pendant phenyl rings. h-Poly-(2S0.36-co-30.64) also exhibited good chiral recognition abilities toward several racemates, though the elution order of some enantiomers was reversed compared with h-poly-2S.A series of optically active poly(diphenylacetylene) derivatives bearing a chiral substituent (poly- It is well known that a pair of enantiomers often show significant differences in their physiological activities. The development of efficient techniques for the separation of enantiomers is therefore important in several fields, especially in the pharmaceutical industry. The direct separation of enantiomers by high-performance liquid chromatography (HPLC) using a chiral stationary phase (CSP) is currently one of the most popular and effective methods for both analyzing the composition of enantiomeric mixtures and purifying such mixtures to give pure enantiomers ,3,4,5,6.4S) (S)-1-phenylethylamine (S)-2. The results revealed that this polymer showed good chiral recognition ability towards various racemates when it was used as a CSP for HPLC and that its chiral recognition ability was significantly dependent on its preferred-handed helical conformation. Notably, the preferred-handed helical conformation of the polymer was induced by the thermal annealing process (h-poly-4S), which was applied after the introduction of the optically active pendants via a polymer reaction. The chiral recognition abilities of h-poly-4S with a preferred-handed helicity were greater than those of poly-4S without a preferred-handed helicity. This result indicated that the macromolecular helicity induced in the polymer backbone by thermal annealing as a consequence of the effect of the chiral pendant groups was playing an important role in the high chiral recognition ability of this material. This finding therefore inspired us to synthesize several other optically active poly(diphenylacetylene)s with the aim of developing more practically useful poly(diphenylacetylene)-based CSPs for HPLC. Although poly(diphenylacetylene)s have pendant phenyl rings on all of the carbons in their main chain, only half of the pendent phenyl rings in poly-4S feature an amide functional group, which could act as effective chiral recognition sites. In this study, we designed and synthesized several novel optically active poly(diphenylacetylene)s bearing chiral or achiral substituents, which were connected to the pendant phenyl rings of the polymer through an amide bond (poly-2S and poly-(2Sx-co-31–x)). We also investigated the chiroptical properties and chiral recognition abilities of these materials as CSPs for HPLC.Optically active poly(phenylacetylene)s with a preferred-handed helicity have been prepared  and some4S)  by the r2S and poly-(2Sx-co-31–x) is shown in 1) were protected as the corresponding n-heptyl esters prior to being polymerized in toluene with WCl6–Ph4Sn at 100 °C. Notably, these conditions have been reported to be effective for the polymerization of diphenylacetylenes bearing ester groups /[DMT-MM] = 2) (S)-2 unit contents of the resulting polymers were found to correspond to approximately one-third of an equivalent of the DMT-MM feed in the first step.The synthetic route used to prepare poly-r groups ,39. It wtroscopy . Poly-2S poly-4S , by the analyses . Poly-(2ysis see . Poly-(2MM] = 2) . The (S)2S showed almost no circular dichroism (CD) in the wavelength region longer than 300 nm ascribed to the absorption of the polymer backbone in N,N-dimethylformamide (DMF) immediately after the preparation of the sample (ε400 = ca. −21) after 5 h at 90 °C or 2 h at 120 °C, which was accompanied by a negligible change in the absorption spectra (h-poly-2S). A similar thermal annealing was necessary for poly-4S to form the preferred-handed helical conformation (h-poly-4S) /[DMT-MM] = 2) (see S)-2 unit contents (x) of the poly-(2Sx-co-31−x)s were determined by 1H-NMR analysis.The other poly-(= 2) see . The (S)2, h-poly-2S or h-poly-(2S0.36-co-30.64) (100 mg) in DMF (1 mL) was used to coat some macroporous silica gel (400 mg) according to a previously reported method [2, h-poly-2S and h-poly-(2S0.36-co-30.64) contents on the silica gel were estimated to be 18.3, 17.6 and 19.9 wt %, respectively, by thermogravimetric analysis. After fractionating the packing materials with sieves, the resulting materials were packed into a stainless steel tube (25 × 0.20 cm (i.d.)) using a conventional high-pressure slurry packing technique with an ECONO-PACKER MODEL CPP-085  [2S), 3500 (h-poly-2S) and 3300 (h-poly-(2S0.36-co-30.64)), respectively, for the elution of benzene with hexane/2-propanol  at a flow rate of 0.1 mL/min. The hold-up times (t0) were estimated using 1,3,5-tri-tert-butylbenzene and acetone as the non-retained compounds for the normal and reverse phases, respectively [A solution of poly-d method , and the, Japan) . The plaectively .2S) or both chiral and achiral units (poly-(2Sx-co-31–x)) and were prepared by the reaction of an optically inactive poly(diphenylacetylene) bearing carboxyl groups with chiral or achiral amines. The resulting polymers were evaluated in terms of their chiroptical properties and chiral recognition abilities toward various racemates such as chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC). We found that poly-2S and poly-(2Sx-co-31–x) folded into a preferred-handed helical conformation (h-poly-2S and h-poly-(2Sx-co-31–x)) by thermal annealing. Notably, the helical senses of these materials varied considerably depending on their chiral unit contents. Furthermore, the circular dichroism (CD) and circularly polarized luminescence (CPL) spectra of h-poly-2S and h-poly-(2S0.36-co-30.64) were almost mirror images of each other. We have demonstrated that these polymers can efficiently resolve various racemates when used as CSPs for HPLC and that the enantioselectivities and elution orders of the enantiomers were significantly influenced by the helical structures induced in these polymers. The elution orders of some enantiomers were reversed on the h-poly-2S- and h-poly-(2S0.36-co-30.64)-based CSPs because of the opposite helicities induced in these polymers. Moreover, poly-2S and h-poly-2S showed higher chiral recognition abilities toward many racemates than the previously reported poly(diphenylacetylene) system bearing the same chiral substituent on only half of its pendant phenyl rings. This result was attributed to an increase in the number of the amide pendants acting as effective chiral recognition sites [In conclusion, we have synthesized a series of optically active poly(diphenylacetylene)s bearing amide functional groups on all of their pendant phenyl rings. These systems consist of a chiral unit (poly-poly-2S0.-co-30.64


Hepatitis E virus genotype 1 (HEV-1) is associated with large epidemics. Notably, HEV subtype 1e (HEV-1e) has caused HEV outbreaks in sub-Saharan Africa. Hepatitis E virus genotype 1 (HEV-1) is associated with large epidemics. Notably, HEV subtype 1e (HEV-1e) has caused HEV outbreaks in sub-Saharan Africa. We report here the second full-length genome sequence of an HEV-1e strain (NG/17-0503) from a recent outbreak in Nigeria in 2017. It shares 94.2% identity with an HEV-1e strain from Chad. AY204877) was obtained from an infected person from France during an outbreak in Chad in 1983 and represents the only complete genome sequence of HEV-1e available (Hepatitis E virus (HEV) is the most common causative agent of acute viral hepatitis. Eight HEV genotypes HEV-1 to HEV-8) have been described, of which five are well recognized as human pathogens ( to HEV-8https://blast.ncbi.nlm.nih.gov) indicated that NG/17-0503 had highest nucleotide identity with the T3 strain. Real-time RT-PCR and consensus nested RT-PCR were conducted as previously described , followed by cDNA synthesis using SuperScript first-strand synthesis , according to the manufacturer’s instructions. Real-time reverse transcription-PCR (RT-PCR) targeting the HEV open reading frames (ORF) 2 and 3 showed viremic HEV infection, with a viral load of 2.22 × 10E+6 IU/ml. Sequence analysis of partial ORF1 (307 bp) and ORF2 401 bp) implemented using the BLASTn search engine (escribed . The 5ʹ 01 bp impMH918640). Sequences were aligned using the MAFFT algorithm  tail, with a G+C content of 57.6%. The genome contains three ORFs  encoding the HEV viral proteins. NG/17-0503 shared the highest identity of 94.2% with the T3 strain from Chad, followed by 88.7% with an HEV-1d Moroccan strain (GenBank accession no. lgorithm .In conclusion, we identified and characterized the full-length genome of an HEV-1e strain circulating during an outbreak in Nigeria in 2017. Sequence and phylogenetic analyses showed that NG/17-0503 is the second full-length HEV-1e genome available to date. Since the Chad HEV-1e strain from 1983 was from a neighboring country of Nigeria, the detection of NG/17-0503 is strongly suggestive of local transmission of this endemic virus over decades.MH918640.The complete genome sequence of NG/17-0503 has been deposited in the GenBank database under the accession no.


Mice lacking neuronal Bin1 and expressing Tau P301S showed increased mortality, without increased neuropathology, when compared to neuronal Bin1 and Tau P301S-expressing mice. The loss of Bin1 isoform 1 resulted in reduced excitability in primary neurons in vitro, reduced neuronal c-fos expression as well as in altered microglia transcriptome in vivo. Taken together, our data suggest that the contribution of genetic variation in BIN1 locus to AD risk could result from a cell-autonomous reduction of neuronal excitability due to Bin1 decrease, exacerbated by the presence of aggregated Tau, coupled with a non-cell autonomous microglia activation.BIN1 is the most important risk locus for Late Onset Alzheimer’s Disease (LOAD), after ApoE. BIN1 AD-associated SNPs correlate with Tau deposition as well as with brain atrophy. Furthermore, the level of neuronal-specific BIN1 isoform 1 protein is decreased in sporadic AD cases in parallel with neuronal loss, despite an overall increase in BIN1 total mRNA. To address the relationship between reduction of BIN1 and neuronal cell loss in the context of Tau pathology, we knocked-down endogenous murine Bin1 via stereotaxic injection of AAV-Bin1 shRNA in the hippocampus of mice expressing Tau P301S (PS19). We observed a statistically significant reduction in the number of neurons in the hippocampus of mice injected with AAV-Bin1 shRNA in comparison with mice injected with AAV control. To investigate whether neuronal loss is due to deletion of Bin1 selectively in neurons in presence Tau P301S, we bred Bin1 AD is characterized by cognitive decline associated with accumulation of Amyloid β (Aβ plaques), hyper-phosphorylated misfolded Tau, and neuronal loss . SiBIN1 is ubiquitously expressed. Re. ReBIN1 ividuals ,22,23. Fividuals  as well ividuals .BIN1 Transcription Start Site (TSS), raising the question whether BIN1 expression levels are affected by AD-associated risk alleles, an, anBIN1 Surprisingly, genetic ablation of Bin1 in forebrain excitatory neurons in the context of human Tau P301S expression didn’t affect neuronal number in the hippocampus or cortex. This apparent discrepancy could be explained by the following considerations. First, AAV-mediated Bin1 knock-down completely ablated Bin1 expression , while Tin vitro and that this leads to a reduced Tau toxicity. . Bin1 c-in vivo. Our study suggests that the contribution of genetic variation in BIN1 locus to AD risk might produce moderate effects that could lead to major outcomes with time, consistent with the small effect of common risk variants and with a late onset of Alzheimer’s disease.In summary, our data shows that post-natal Bin1 deletion in excitatory forebrain neurons results in moderate but significant decrease in cell-autonomous neuronal excitability, likely exacerbated by the presence of aggregated Tau, coupled with non-cell autonomous microglia activation S1 Fig(A) Immunofluorescence co-staining of GFP, NeuN (neurons), GFAP (astrocytes), Iba1 (microglia) and Gad67 (inhibitory neurons) in hippocampi of PS19Hemi mice injected with AAV (1/8)-GFP-U6-shRNA scrbl. One representative image per group. Scale bar: 600μm. Arrows mark colocalized staining and x marks non-colocalized staining. Inlet scale bar: 50μm. (B) Plot representing the mean±SD percentage of GFP+ cells co-stained with NeuN, Gad67, GFAP and Iba1, respectively, quantified on 4 animals. (C) Immunohistochemistry of GFP in hippocampi of PS19Hemi mice injected with AAV (1/8)-GFP-U6-shRNA scrbl and AAV (1/8)-GFP-U6-shRNA mBin1, respectively. One representative image per group. Scale bar: 600μm. (D) Plot representing the percentage of GFP staining  for each animal. Circles represent animals injected with AAV (1/8)-GFP-U6-shRNA scrbl: green filled circles represent ipsilateral hemisphere; blue filled circles represent contralateral hemisphere. Squares represent animals injected with AAV (1/8)-GFP-U6-shRNA mBin1: yellow filled squares represent ipsilateral hemisphere; red filled squares represent contralateral hemisphere. Each circle/square represents one single animal.(TIF)Click here for additional data file.S2 Fig(A) Immunohistochemical staining of Iba1 in hippocampi of PS19Hemi mice injected with AAV (1/8)-GFP-U6-shRNA scrbl and AAV (1/8)-GFP- U6-shRNA mBin1, respectively. One representative image per group. Scale bar: 2mm. Inlets scale bar: 100μm. (B) Plot representing the Total Area of hippocampus expressed as Log of mm2 for each animal. (C) Plot representing the Log of the number of microglia cells (based on Iba1 staining) per mm2 for each animal. (D) Plot representing the Log of the Iba1% staining area normalized over the total area of hippocampus for each animal (+2). (E) Plot representing the Log area of microglia dendritic processes in the hippocampus, calculated as area of Iba1 staining normalized by the area of microglia soma for each animal. Circles represent animals injected with AAV (1/8)-GFP- U6-shRNA scrbl: green filled circles represent ipsilateral hemisphere; blue filled circles represent contralateral hemisphere. Squares represent animals injected with AAV (1/8)-GFP- U6-shRNA mBin1: yellow filled squares represent ipsilateral hemisphere; red filled squares represent contralateral hemisphere. Each circle/square represents one single animal. A linear mixed effect model with a 2-way fixed effect model with interaction, a random intercept, and a compound symmetric within-subject correlation structure was employed. P-values were adjusted via the glht single step method. Post-hoc comparisons were analyzed between ipsilateral and contralateral hemispheres within animals, as well as between ipsilateral hemispheres across groups and contralateral hemispheres across groups. Adjusted p-value: * p< 0.05; ** <0.01; *** p<0.001.(TIF)Click here for additional data file.S3 FigA) Western blotting of brain lysates from Bin1flox/+:: Thy1-Cre- and Bin1flox/flox::Thyr1-Cre+ mice show Bin1 expression. Each lane represents lysate from one animal. Tubulin represents loading control. Arrows indicated Neuronal Bin1 isoform 1 (top) and Ubiquitous Bin1 isoform 2 (bottom). (B) Western blotting of brain lysates from Bin1flox/+:: Thy1-Cre-:: PS19Hemi and Bin1flox/flox::Thyr1-Cre+:: PS19Hemi mice show Bin1 expression. Each lane represents lysate from one animal. Tubulin represents loading control. Arrows indicated Neuronal Bin1 isoform 1 (top) and Ubiquitous Bin1 isoform 2 (bottom). (C) Male Bin1flox/flox:: Thy1-Cre+:: PS19Hemi mice  and Bin1flox/+::Thy1-Cre+::PS19Hemi  show reduced time to reach Clinical Score 2 (CS2) relative to Bin1flox/+::Thy1-Cre-::PS19Hemi  mice. Mean time to CS2 WT: 287 days, HET: 281 days (p = 0.419), KO: 250 days (p<0.0001) , relative to WT). (D) Female Bin1flox/flox:: Thy1-Cre+:: PS19Hemi mice  and Bin1flox/+::Thy1-Cre+::PS19Hemi  show reduced time to reach Clinical Score 2 (CS2) relative to Bin1flox/+::Thy1-Cre-::PS19Hemi  mice. Mean time to CS2 WT: 349 days, HET: 308 days (p = 0.045), KO: 275 days (p<0.0001) , relative to WT). (E) Male Bin1flox/flox:: Thy1-Cre+:: PS19Hemi mice  and Bin1flox/+::Thy1-Cre+::PS19Hemi  show reduced time to reach Clinical Score 3 (CS3) relative to Bin1flox/+::Thy1-Cre-::PS19Hemi  mice. Mean time to CS2 WT: 308 days, HET: 287 days (p = 0.202), KO: 256 days (p = 0.008) , relative to WT). (F) Female Bin1flox/flox:: Thy1-Cre+:: PS19Hemi mice  and Bin1flox/+::Thy1-Cre+::PS19Hemi  show reduced time to reach Clinical Score 3 (CS3) relative to Bin1flox/+::Thy1-Cre-::PS19Hemi  mice. Mean time to CS2 WT: 363 days, HET: 317 days (p = 0.112), KO: 308 days (p = 0.001) , relative to WT). Adjusted p-value: * p< 0.05; ** <0.01; *** p<0.001.((TIF)Click here for additional data file.S4 Fig(A) and Total Area of the hippocampus (B), expressed in mm2, in sections from the hippocampi of male and female Bin1flox/+::Thy1-Cre-::PS19Ncar (black filled circles) or Bin1flox/flox:: Thy1-Cre+:: PS19Ncar mice (blue filled circles) mice. Plots representing the Area NeuN staining (C) and Total Area of the hippocampus (D), expressed in mm2, in sections from the hippocampi of male and female Bin1flox/+::Thy1-Cre-::PS19Hemi (gray filled circles) or Bin1flox/flox:: Thy1-Cre+:: PS19Hemi mice (purple filled circles) mice. Plots representing the Area of NeuN staining (E) and Total Area of the cortex (F), expressed in mm2, in sections from the cortexes from male and female Bin1flox/+::Thy1-Cre-::PS19Ncar (black filled circles) or Bin1flox/flox:: Thy1-Cre+:: PS19Ncar mice (blue filled circles) mice. Plots the Area of NeuN staining (G) and Total Area of the cortex (H), expressed in mm2, in sections from the cortexes from male and female Bin1flox/+::Thy1-Cre-::PS19Hemi (gray filled circles) or Bin1flox/flox:: Thy1-Cre+:: PS19Hemi mice (purple filled circles) mice. Each circle represents the value from one animal. 2-way ANOVA with interaction with single-step p-value correction for all endpoints, except Total Area Hippocampus (B) 2-way un-equal variance ANOVA with interaction with single-step p-value correction. Results presented in the figure don’t show statistically significant differences.Plots representing the Area NeuN staining (TIF)Click here for additional data file.S5 Fig(A) Plot representing the Total Distance traveled (mm) by mice at 5 and 6 months of age. Bin1flox/flox:: Thy1-Cre-::PS19Ncar N = 16 (5 month-old), 24 (6 month-old) (black filled circles) or Bin1flox/flox:: Thy1-Cre+:: PS19Ncar mice N = 18 (5 month-old), 24 (6 month-old) (blue filled circles) mice. Bin1flox/flox:: Thy1-Cre-::PS19Hemi N = 10 (5 month-old), 13 (6 month-old) (gray filled circles) or Bin1flox/flox:: Thy1-Cre+:: PS19Hemi mice N = 12 (5 month-old), 20 (6 month-old) (purple filled circles) mice. Two-way ANOVA with Tukey’s multiple comparison test. Main measures for each time point: genotype, time, interaction: genotype and time, followed by post-hoc comparisons to Bin1flox/flox:: Thy1-Cre-::PS19Ncar mice. Bin1flox/flox:: Thy1-Cre+:: PS19Ncar mice show significantly reduced open field exploration at 6m. p-value: * p< 0.05; ** <0.01; *** p<0.001. (B) Plot representing the Startle Amplitude, expressed as arbitrary units, of mice at 6 and 7 months of age. Bin1flox/flox:: Thy1-Cre-::PS19Ncar N = 24 (6 month-old), 23 (7 month-old) (black filled circles) or Bin1flox/flox:: Thy1-Cre+:: PS19Ncar mice N = 23 (6 month-old), 23 (7 month-old) (blue filled circles) mice. Bin1flox/flox:: Thy1-Cre-::PS19Hemi N = 15 (6 month-old), 15 (7 month-old) (gray filled circles) or Bin1flox/flox:: Thy1-Cre+:: PS19Hemi mice N = 19 (6 month-old), 18 (7 month-old) (purple filled circles) mice. Two-way ANOVA with Tukey’s multiple comparison test, main measures for each time point: genotype, time, interaction: genotype and time, followed by post-hoc comparisons to Bin1flox/flox:: Thy1-Cre-::PS19Ncar mice. Bin1flox/flox:: Thy1-Cre+:: PS19Ncar and Bin1flox/flox:: Thy1-Cre+:: PS19Hemi genotypes show reduced auditory startle. p-value: * p< 0.05; ** <0.01; *** p<0.001. (C) Plot representing the percentage Pre-Pulse Inhibition (PPI) of mice at 6 and 7 months of age. Bin1flox/flox:: Thy1-Cre-::PS19Ncar N = 24 (6 month-old), 23 (7 month-old) (black filled circles) or Bin1flox/flox:: Thy1-Cre+:: PS19Ncar mice N = 23 (6 month-old), 23 (7 month-old) (blue filled circles) mice. Bin1flox/flox:: Thy1-Cre-::PS19Hemi N = 15 (6 month-old), 15 (7 month-old) (gray filled circles) or Bin1flox/flox:: Thy1-Cre+:: PS19Hemi mice N = 19 (6 month-old), 18 (7 month-old) (purple filled circles) mice. Two-way ANOVA with Tukey’s multiple comparison test, main measures for each time point: genotype, time, interaction: genotype and time, followed by post-hoc comparisons to Bin1flox/flox:: Thy1-Cre-::PS19Ncar mice. Bin1flox/flox:: Thy1-Cre+:: PS19Hemi genotype mice show reduced Pre-Pulse Inhibition (PPI). p-value: * p< 0.05; ** <0.01; *** p<0.001. (D) Plots representing the percentage of freezing time for male (left plot) and female (right plot) mice during Contextual Fear Conditioning. Two-way ANOVA with Tukey’s multiple comparison test. (E) Plots representing the percentage of freezing time for male (left plot) and female (right plot) mice during Cued Fear Conditioning. Bin1flox/+:: Thy1-Cre-::PS19Ncar (black filled circles) or Bin1flox/flox:: Thy1-Cre+:: PS19Ncar mice (blue filled circles) mice. Bin1flox/+:: Thy1-Cre-::PS19Hemi (gray filled circles) or Bin1flox/flox:: Thy1-Cre+:: PS19Hemi mice (purple filled circles) mice. Each circle represents the value from one animal. Two-way ANOVA with Tukey’s multiple comparison test. Results presented in D and E don’t show statistically significant differences.(TIF)Click here for additional data file.S6 FigA) Western blotting of brain lysates from Bin1flox/+:: Thy1-Cre-,Bin1flox/+::Thy1-Cre+ and Bin1flox/flox::Thyr1-Cre+ mice showing Bin1 expression. Each lane represents lysate from one animal. Gapdh represents loading control. Arrows indicated Neuronal Bin1 isoform 1 (top) and Ubiquitous Bin1 isoform 2 (bottom). (B) Representative immunofluorescence images of Iba1 labeling in select brain regions from Bin1flox/+:: Thy1-Cre- and Bin1flox/flox:: Thy1-Cre+ mice. Scale bar: 300μm. Inlets scale bar: 50μm. (C) Plot representing the number of Iba1+ cells per image. Bin1flox/+:: Thy1-Cre- (black bars) and Bin1flox/flox:: Thy1-Cre+ (blue bars). Boxes represent range, with line representing mean. N = 3 per group. Two-way ANOVA with Tukey’s multiple comparison test. Results presented in C don’t show statistically significant differences.((TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S9 Fig(TIF)Click here for additional data file.S10 Fig(TIF)Click here for additional data file.S1 TableBin1flox/flox:: Thy1-Cre+, Bin1flox/+:: Thy1-Cre 1-Cre+ and Bin1flox/+:: Thy1-Cre 1-Cre- males and females mice at 3 months of age.RNAseq gene expression analysis of microglia from (XLSX)Click here for additional data file.S2 TableBin1flox/flox:: Thy1-Cre+ and the transcriptome of microglia from CK-p25 mice clusters 3, 6 and 7, respectively.List of differentially expressed genes commonly shared by transcriptome of microglia from (XLSX)Click here for additional data file.


V600E-mut-BRAF A375 and G12D-mut-KRAS/high c-myc C8161 melanoma cells. Moreover, TTM protected both melanoma types from toxicity induced by DSF, NC and co-treatment with sub-lethal levels of DSF and the MEK inhibitor, UO126. Toxicity by co-treatment with DSF+OPT was poorly reversed by TTM in C8161 melanoma cells. In contrast to the greater toxicity of 0.1 μM DSF against mutant p53 CRC cells irrespective of their KRAS mutation, TTM did not protect G12V-mut-KRAS/high c-myc SW620 CRC from DSF+OxPt compared to KRAS-WT/BRAF-WT Caco-2 CRC. Our results show that DSF co-treatment with: a) MEK inhibitors may enhance tumour suppression; b) OxPt in CRC may counteract impaired response to cetuximab by KRAS/BRAF mutations and c) as a single treatment, TTM may be less effective than DSF and decreases the efficacy of the latter.Ammonium tetrathiomolybdate (TTM) and disulfiram (DSF) are copper (Cu) chelators in cancer clinical trials partly because Cu chelation: a) restricts the activity of Cu-binding MEK1/2 enzymes which drive tumourigenesis by KRAS or BRAF oncogenic mutations and b) enhances uptake of oxaliplatin (OxPt), clinically used in advanced KRAS-mutant colorectal carcinomas (CRC). Whereas TTM decreases intracellular Cu trafficking, DSF can reach other Cu-dependent intracellular proteins. Since the use of individual or combined Cu chelation may help or interfere with anti-cancer therapy, this study investigated whether TTM modifies the response to DSF supplemented with: 1) UO126, a known MEK1/2 inhibitor; 2) other Cu chelators like neocuproine (NC) or 1, 10-o-phenanthroline (OPT) in wt p53 melanoma cells differing in BRAF or KRAS mutations; 3) OxPt in mutant p53 CRC cells devoid of KRAS and BRAF mutations or harbouring either KRAS or BRAF mutations. TTM was not toxic against Potentiation of melanoma antitumour toxicity of DSF by MEK inhibitor is reversed by TTM.KRAS/c-MYC dysregulation attenuates TTM reversion of melanoma toxicity by DSF + OPT.KRAS/c-MYC dysregulation increases melanoma NC toxicity reversed by TTM.BRAF mutation and lower c-MYC may attenuate toxicity by DSF ± OxPt in colorectal cancer cells BRAFV600-mutant melanoma, approximately 5% of patients with BRAFV600E colorectal cancer respond. Preclinical studies suggest that the lack of efficacy in BRAFV600E colorectal cancer is due to adaptive feedback reactivation of mitogen-activated protein kinase signalling, often mediated by epidermal growth factor receptor (EGFR) –15BRAFV6r (EGFR) , 15. Howr (EGFR) . In color (EGFR) . In prosr (EGFR) . In other (EGFR) . In the r (EGFR) . Howeverr (EGFR) . Similarr (EGFR) , 20. Howr (EGFR) , unlike r (EGFR) , 22. Ther (EGFR) –16 but rr (EGFR) –21, 23. r (EGFR) , 25 in sr (EGFR)  using wtmutation . Since tmutation  which admutation . We alsomutation  and highmutation  comparedmutation  low c-MYmutation  status amutation .wt p33 C8161 melanoma has a G464E mutation in the BRAF P loop region, accompanied by an enhancing KRAS G12D mutation . c-MYC ewt p53 A375 melanoma [CRL-1619] with a homozygous BRAF (V600E) mutation was obtained from the American Type Culture Collection .SW620 colorectal cancer cells harbour two p53 mutations pR273H; P309S), a KRAS (pG12V) homozygous mutation  and have9S, a KRAHT-29 colorectal cancer cells harbour a homozygous p53 mutation (pR273H), a heterozygous BRAF (V600E) mutation .Caco-2 colorectal cancer cells harbour a p53 mutation (E204X) and are wild-type for KRAS, BRAF, PIK3CA and PTEN . These chttp://www.sigmaaldrich.com/life-science/cell-culture/learning-center/mediaexpert/copper.htm]. Whenever indicated, cultures were seeded overnight at 5 × 103 cells per 96 well plates in octuplicates and treatments were added 20 hours after, for a further 24 hours.These cell lines were kept in complete Dulbecco’s containing medium supplemented with 10% foetal calf serum. Although this medium practically does not have Cu supplementation, when supplemented with 10% serum, it provides sufficient copper for cell growth and survival, approx. 50–100 ng/mL since serum albumin is a physiological Cu transporter  and Phase II Trial of Disulfiram With Copper in Metastatic Breast Cancer ClinicalTrials.gov Identifier NCT03323346; study completion date September 2020]. Since KRAS mutation and c-Myc amplification cooperate with KRAS in tumourigenesis [This study examined whether anti-cancer response was enhanced or interfered with by co-treatment between two structurally different Cu chelators currently used in clinical trials like a) TTM [A Phase II Study of igenesis –37, thisBRAF (V600E)/ p53 mut HT-29 CRC cells. It was also noteworthy that TTM attenuated toxicity by DSF ± OxPt preferentially in KRAS WT/ BRAF WT/ p53 mut Caco-2 cells compared to KRAS (G12V)/ p53 mut/ high c-MYC SW-620 cells (G12D mutation to accelerate tumourigenesis [However, the cooperation between mutant KRAS and extent of c-MYC amplification may be different in the CRC cells harbouring a mutated tumour suppressor p53 gene, used in these studies (However, when using another Cu chelator like 0.25-μM OPT, it preferentially killed C8161 cells compared to A375 cells. However, in C8161 cells with concomitant KRAS/c-MYC dysregulation, TTM reversion of melanoma toxicity by DSF + OPT  was dimi20 cells . Althougigenesis , 36, 38. studies . Taken tIn summary, our studies imply that DSF may be the most clinically promising anti-cancer Cu chelator. DSF re-purposing from its long-term clinical use against alcoholism to an anti-cancer drug is based on its ability to act as an ALDH1A1/ALDH2 inhibitor , a propeAli Calderon-Aparicio carried out the melanoma experiments, cooperated with Alejandro Cornejo in the CRC experiments and helped in the Discussion. Alejandro Cornejo carried out the statistical analyses, designed the Graphic Abstract and helped to improve the Discussion. Andrea Orue participated in the Discussion and final revision of this manuscript. Manuel Rieber designed, supervised, provided funding and wrote the final version of this paper.This research was funded by Fonacit-Misión Ciencia sub-proyecto Sistema Publico Nacional de Salud 4-Cancer grant to Manuel Rieber at Instituto Venezolano de Investigaciones Cientificas. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.


SynCav1) increases axonal development by increasing both axon length and volume. Moreover, inhibition of Cav-1(Y14A) phosphorylation blunts Rac1/Cdc42-mediated both axonal growth and differentiation of human NPCs and SynCav1(Y14A)-treated NPCs exhibited blunted axonal growth. These results suggest that: (1) SynCav1-mediated dendritic and axonal growth in human NPCs is dependent upon P-Cav-1, (2) P-Cav-1 is necessary for proper axonal growth during early stages of neuronal differentiation, and (3) Rac1/Cdc42CA-mediated neuronal growth is in part dependent upon P-Cav-1. In conclusion, Cav-1 phosphorylation is essential for human neuronal axonal growth during early stages of neuronal differentiation.Proper axonal growth and guidance is essential for neuron differentiation and development. Abnormal neuronal development due to genetic or epigenetic influences can contribute to neurological and mental disorders such as Down syndrome, Rett syndrome, and autism. Identification of the molecular targets that promote proper neuronal growth and differentiation may restore structural and functional neuroplasticity, thus improving functional performance in neurodevelopmental disorders. Using differentiated human neuronal progenitor cells (NPCs) derived from induced pluripotent stem cells (iPSCs), the present study demonstrates that during early stage differentiation of human NPCs, neuron-targeted overexpression constitutively active Rac1 (Rac1CA) and constitutively active Cdc42 (Cdc42CA) enhance expression of P-Cav-1, T-Cav-1, and P-cofilin and increases axonal growth. Similarly, neuron-targeted over-expression of Cav-1 (termed  SynCav1) enhanced MLR formation, receptor-mediated cAMP production, TrkB receptor expression and signaling, and augmented dendritic arborization in primary neurons in vitro  are key regulators of cytoskeletal dynamics . DysreguSynCav1 (Y14A), that prevents Cav-1 phosphorylation at tyrosine (Y) 14, we demonstrated that Cav-1 phosphorylation was required for both SynCav1-mediated and Rac1/Cdc42-mediated neuronal axonal growth and differentiation.Kawauchi and colleagues recently demonstrated that Cav-1 promoted early neurite maturation in an endocytic-dependent manner . HoweverRRID: CVCL_1N86, GM25430) (2. Medium was changed every 2–3 days and cells were split with Accutase and Accumax (Innovative Cell Technologies) when arrived 100% confluency. bFGF was removed from the medium to induce differentiation. Medium was changed every 2–3 days.The use of human induced pluripotent stem cells (hiPSC) was approved by the UCSD IRB/ESCRO (project #:1616206ZX). Human differentiated neuronal progenitor cells (NPCs) were previously derived from hiPSC CVB line (GM25430) . NPCs weGM25430) . NPC medRas-related C3 botulinum toxin substrate 1/Cell division control protein 42 homolog activator , Antibodies used for immunoblotting and immunofluorescence were as follows: P-Cav-1 (#3251), T-Cav-1 (#3267), P-Src (#2101), T-Src (#2108), and GAPDH (#5274) were all from Cell Signaling Technology . P-cofilin (sc-271921) was from Santa Cruz Biotech , microtubule-associated protein 2 (MAP2) (ab5392) was from Abcam , anti-neurofilament SMI 31 (#801601) was from BioLegend . Primary antibodies were visualized using secondary antibodies conjugated to horseradish peroxidase (HRP)  and lumigen ECL ultra . All displayed bands were compared to molecular weight standards . The amount of protein per sample was determined using a dye-binding protein assay . For immunofluorescence, FITC-488, Texas Red-595 and Cy5-647 secondary antibodies were obtained from Molecular Probes .SynCav1), Rac1Q61L  61 to leucine point mutation, constitutively active Rac1), Cdc42Q61L  61 to leucine point mutation, constitutively active Cdc42), and caveolin-1 phospho-mutant  were generated as previously described  or empty vector on day 3 in culture.Lentivirus containing synapsin-caveolin-1  dissolved in NPC medium)] that directly activates Rac1/Cdc42 with various doses and time points . Untreated cells served as negative control. To measure related protein expression level change, cells were harvested directly after activator treatment and prepared for Western blot.Cells were homogenized with cold RIPA lysis buffer followed by 3 cycles of 20-s bursts of sonication at 4°C. The amount of protein per sample was determined using a dye-binding protein assay (Bio-Rad). Electrophoresis was performed on the samples using 4–12% or 10% acrylamide gels (Invitrogen) and transferred to polyvinylidene difluoride membranes (Millipore) by electroelution. Membranes were blocked in blocking solution [20 mM TBS Tween (1%) (TBST) containing 3% bovine serum albumin (BSA)] and then incubated with primary antibodies overnight at 4°C. After 3 washes with TBST, the membrane was incubated with secondary antibody for 1 h at room temperature (RT). Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (Santa Cruz Biotech) was used as secondary antibody. After another 3 times wash with TBST to remove the rest secondary antibody, membrane was incubated with ECL reagent  and prepared for imaging. Bands were compared to molecular weight standards (Santa Cruz Biotech). GAPDH served as the internal reference by which to standardize the other protein bands. The calculated ratio of the control group was normalized to 100%, and the comparisons of other groups to the control group were represented as percentages.Neurons were fixed with 2% paraformaldehyde for 10 min at room temperature and then permeabilized in 0.1% Triton X-100 and blocked with 1% BSA/PBS/Tween (0.05%) for 60 min. Next, cells were incubated with primary antibodies in 1% BSA/PBS/Tween (0.05%) for 24–48 h at 4°C followed by incubation with FITC or Alexa conjugated secondary antibody (1:600 to 1:800) for 1 h at room temperature (RT). Stained cells were mounted in mounting medium containing DAPI and imaged with Olympus confocal microscope system  that included a Photometrics CCD mounted on a Nikon TE-200 inverted epi-fluorescence microscope. Exposure times were set to the same value for all groups every time. Quantitation of intensity of GTP-Rac1/GTP-Cdc42 was conducted by using Image J (NIH). Quantitation of axonal length and volume was conducted by using Autoneuron, which measures 3D image volume stacks (MBF Bioscience) generated by Neurolucida as previously described ; represet-tests or One-Way ANOVA; post hoc comparisons were performed by Student Neuman Keuls tests. Significance was set at p < 0.05. Statistical analysis and graphs were made using Prism 7 .Data were expressed as means ± SE. All data were analyzed by unpaired in vitro (DIV7)  . We havein vitro , therefol growth  . To confirm constitutively activation of Rac1 and Cdc42, antibodies to GTP-bound Rac1 or Cdc42 were used for immunofluorescence (IF). As shown in SynRac1CA and SynCdc42CA, respectively .On DIV4, human NPCs were treated with constitutively active (CA) SynCav1(Y14A)] which prevents phosphorylation at Y14, resulting in a dominant negative Cav-1. At DIV3, NPCs were treated with SynCav1 and SynCav1(Y14A) for 4 days. Western blot assay showed that both SynCav1(Y14A) and SynCav1 significantly increased T-Cav-1, while SynCav1(Y14A) inhibited P-Cav-1, and P-Src expression  treated NPCs exhibited blunted axonal growth  14.Neuronal progenitor cells were treated with a mutated Cav-1 construct . The poSynCav1(Y14A), we found that loss of P-Cav-1(Y14) blunted P-Src (Y416) Rac1/Cdc42-mediated axonal growth, indicating that Rac1/Cdc42-mediated axonal growth in early stages of differentiation is in part dependent on P-Src and P-Cav-1.Previous research showed that Cdc42 activation enhances P-Src, while silencing Cdc42 prevents P-Src . Other wSynCav1 increased dendritic arborization in hippocampal neurons in vivo  provide subcellular and molecular signaling complexes essential for axonal growth and guidance  resulted in profound impaired axonal growth in contrast to SynCav1, which significantly augmented axonal growth. Recent work demonstrated that P-Cav-1 facilitates microRNA insertion into extracellular vesicles by regulating RNA binding proteins in non-neuronal cells  in response to extracellular pro-growth signaling cues. The present study also demonstrated that SynCav1-promoted axonal growth is dependent upon P-Cav-1(Y14). Moreover, inhibition of endogenous phosphorylation of Cav-1 even in the presence of increased T-Cav-1  results in aberrant and blunted axonal growth. In conclusion, targeting Cav-1, specifically Cav-1 phosphorylation in neurons, may serve as a novel therapeutic target to promote axonal growth during early-stage differentiation in the setting of neurodevelopmental disorder such as Down Syndrome or serve to promote functional neuroplasticity after CNS injury (ischemic or traumatic) and in neurodegenerative diseases .In summary, these results show that Cav-1 phosphorylation is critical for axonal growth. Furthermore, because increased Cav-1 phosphorylation occurred with constitutively active Rac1 and Cdc42, but not with The datasets generated for this study are available on request to the corresponding author.SW wrote the manuscript and carried out the cell culture, immunofluorescence microscopy, Western blot, and data analyses. ZZ carried out the cell culture and Western blot. AA-Q provided the NPCs and assisted in cell culture. JL assisted in cell culture and Western blot. CD generated and provided the Rac1 and Cdc42 constructs. DR, PP, and HP assisted in editing and in experimental design. BH conceived the study, assisted in data analyses, provided the phospho-mutant Cav1 construct, and wrote the manuscript.HP and BH are scientific founders of CavoGene LifeSciences LLC and hold equity interest in the company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Scientific Reports 10.1038/s41598-019-51874-7, published online 31 October 2019Correction to: The Acknowledgements section in this Article was omitted. The Acknowledgements section should read:“This project was supported by funds received from the NIH, awarded to MMS (1R01MH116694-01), and to BAN (T32 GM007055)”


Plasmodium falciparum hexose transporter PfHT has emerged as a promising target for anti-malarial therapy, previously identified small-molecule inhibitors have lacked promising drug-like structural features necessary for development as clinical therapeutics. Taking advantage of emerging insight into structure/function relationships in homologous facilitative hexose transporters and our novel high throughput screening platform, we investigated the ability of compounds satisfying Lipinksi rules for drug likeness to directly interact and inhibit PfHT. The Maybridge HitFinder chemical library was interrogated by searching for compounds that reduce intracellular glucose by >40% at 10 μM. Testing of initial hits via measurement of 2-deoxyglucose (2-DG) uptake in PfHT over-expressing cell lines identified 6 structurally unique glucose transport inhibitors. WU-1 -5-methyl-N-[2-(4-methylbenzenesulfonyl)ethyl]-1,2-oxazole-4-carboxamide) blocked 2-DG uptake (IC50 = 5.8 ± 0.6 μM) with minimal effect on the human orthologue class I (GLUTs 1–4), class II (GLUT8) and class III (GLUT5) facilitative glucose transporters. WU-1 showed comparable potency in blocking 2-DG uptake in freed parasites and inhibiting parasite growth, with an IC50 of 6.1 ± 0.8 μM and EC50 of 5.5 ± 0.6 μM, respectively. WU-1 also directly competed for N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) binding and inhibited the transport of D-glucose with an IC50 of 5.9 ± 0.8 μM in liposomes containing purified PfHT. Kinetic analysis revealed that WU-1 acts as a non-competitive inhibitor of zero-trans D-fructose uptake. Decreased potency for WU-1 and the known endofacial ligand cytochalasin B was observed when PfHT was engineered to contain an N-terminal FLAG tag. This modification resulted in a concomitant increase in affinity for 4,6-O-ethylidene-α-D-glucose, an exofacially directed transport antagonist, but did not alter the Km for 2-DG. Taken together, these data are consistent with a model in which WU-1 binds preferentially to the transporter in an inward open conformation and support the feasibility of developing potent and selective PfHT antagonists as a novel class of anti-malarial drugs.Although the  Plasmodium species, the causative agent of malaria, disease prevalence remains alarmingly high, with over 219 million new cases world-wide in 2017 alone 2-DG (2-DG), [3H]D-fructose (D-Frc), [3H]-D-glucose, and [3H]-L-glucose were purchased from American Radiolabels Inc. . GLUT1 short hairpin RNA (shRNA) was obtained through the RNA interference (RNAi) core at Washington University School of Medicine. HEK293 cells were acquired from the American Type Culture Collection (ATCC). Egg phosphatidylcholine (eggPC), palmitoyloleoyl phosphatidylethanolamine (POPE), and palmitoyloleoyl phosphatidic acid (POPA) were purchased from Avanti Polar Lipids Inc. . Lauryl maltose neopentyl glycol (LMNG) detergent was obtained from Anatrace . PEG-biotin cap-ATB-BMPA was purchased from Toronto Research Chemicals, Inc. . FLAG tag rabbit antibody (2368) was obtained from Cell Signaling Technology . All other reagents were purchased from Sigma or as otherwise indicated.WU-1 -5-methyl-N-[2-(4-methylbenzenesulfonyl)ethyl]-1,2-oxazole-4-carboxamide, cat# 002-890-346), WU-2 -1-(2-chloro-6-methylphenyl)urea, cat# 002-892-269), WU-3 (2-amino-5-phenyl-N'-[(E)-methylidene]thiophene-3-carbohydrazide, cat# 002-903-252 and WU-5 -3,5-bis[methylidene]oxan-4-one, cat# 001-840-877) were purchased from MolPort, Riga, Latvia. ). We prepared a 1:900 dilution of 10 mM stock solution of the compounds for an 11.11 μM final concentration. DMSO (0.11% DMSO) and cytochalasin B  were used as negative and positive controls for inhibition, respectively. To initiate the screening assay, cells were washed twice with 50 μl of HBSS per well using an ELx405 plate washer (Biotek), then incubated for 30 minutes (starvation period) after addition of 45 μl of diluted compound using the BiomekFX Liquid Handler (Beckman Coulter). To initiate the uptake, 5 μl of 50 mM glucose was added to the wells  using a Multidrop384 dispenser (Thermo Fisher Scientific). After incubation at room temperature for 120 min, fluorescence was measured using a 2102 EnVision multilabel plate reader (PerkinElmer) at an excitation wavelength of 436 nm (cyan fluorescent protein [CFP]) and emission wavelengths of 485 nm (CFP) and 535 nm (yellow fluorescent protein [YFP]). % inhibition was determined using the plate controls by subtracting the YFP/CFP ratio (FRET ratio) for CB controls from that of the vehicle (DMSO) controls and then calculating the % decrease in signal for each single point compound tested.Black opaque 384-well plates (Greiner Bio-One) were pretreated with 25 μg/ml of polyethylenimine  solution containing 150 mM NaCl to maintain cell adhesion during washing steps. After 20 min of incubation at room temperature, PEI was aspirated and wells were air dried for 5 min. Using a Multidrop384 dispenser (Thermo Fisher Scientific), PfHT-FLIP cells were plated at 20,000 cells/well. After cell plating, plates were left in the sterile hood at room temperature for 45 min before transfer to the incubator which helped reduce edge effects . CompounP. falciparum strain 3D7 was obtained from the Malaria Research and Reference Reagent Resource Center . Parasites were cultured in a 2% suspension of human erythrocytes and RPMI 1640 (Sigma) medium supplemented with 27 mM sodium bicarbonate, 11 mM glucose, 5 mM HEPES, 1 mM sodium pyruvate, 0.37 mM hypoxanthine, 0.01 mM thymidine, 10 μg/ml gentamicin, and 0.5% Albumax (Gibco) at 37°C, 5% O2/ 5% CO2 / 90% N2 atmosphere as previously described 2-DOG . 2-DG uptake was stopped after 2 min by layering the entire reaction over 300 μl of an oil mixture  in Eppendorf tubes and centrifuged at 15,850 × g for 2 mins. The ratio of the estimated intracellular concentration of radiolabel relative to the extracellular concentration were calculated as described -D-glucose  to PfHT proteoliposomes at room temperature. Transport was stopped at different times with 5 ml of ice-cold quench buffer  and then filtered by vacuum on a 0.2-μm mixed cellulose ester filter . Filters were subsequently washed with an additional 10 ml of quench buffer. Scintillation fluid  was added to the filters and the radioactivity counted. [3H]-L-glucose  was used to determine nonspecific transport. Specific uptake was calculated by subtracting nonspecific [3H]-L-glucose from [3H]-D-glucose uptake. Uptake was normalized to the amount of transporter per reaction, determined by analyzing aliquots of transporter-containing liposomes along with a BSA standard curve on an SDS-polyacrylamide gel stained with Blue Bandit protein stain and quantified using an Odyssey infrared imaging system . Data were fitted by least squares nonlinear regression analysis using GraphPad Prism 6.0.Liposomes containing PfHT were made according to Hresko et al. . BrieflyWU-1 inhibitor was added to PfHT liposomes for 10 min at room temperature. Samples  were then incubated for 10 min at room temperature with biotinylated ATB-BMPA  and then placed on ice prior to UV irradiation. Reactions were transferred to a 24-well low protein retention culture dish  and then irradiated at room temperature 5 cm from a Green Spot UV lamp for 1 min (30 sec of light followed by 30 sec of darkness followed by 30 sec of light). Unbound ATB-BMPA was removed using a 0.5 ml Zeba Spin Column . Samples were analyzed by immunoblot analysis using a fluorescently labeled streptavidin . The amount of PfHT protein in each sample was quantified by SDS-PAGE stained with Blue BANDIT protein stain using a BSA standard curve. The streptavidin signal was then normalized to the amount of PfHT protein.Statistical analysis between groups was performed using paired student’s t-test as indicated in Results and Figure Legends.P. falciparum hexose transporter, PfHT. Using our novel FRET-based high-throughput HEK293PfHT screening assay -D-glucose minus ([3H]-L-glucose) into FTPfHT-containing liposomes. Different concentrations of WU-1 were added to the liposomes 20 min prior to the initiation of the transport reaction. Uptake (quenched after 50 sec) was normalized to the amount of FTPfHT in the liposomes. Data were fit by nonlinear regression analysis using GraphPad Prism 6.0 software to calculate the IC50 for WU-1. Data are expressed as mean ± SEM of three independent experiments.WU-1 inhibits the specific uptake ([(TIF)Click here for additional data file.S1 FileFile contains minimal data set used to reach the conclusions drawn in the manuscript including the values behind the means, standard deviations and other measures reported, the values used to build graphs, and the points extracted from images for analysis.(XLSX)Click here for additional data file.


Phosphorylation of p62/SQSTM1 (p62) on Serine 349 (P-Ser349 p62) as well as proteasome dysfunction have been shown to activate the cell protective Keap1/Nrf2 pathway. We showed previously that BAY 11-7085-induced human synovial fibroblast cell death includes autophagy and p62 downregulation. In this work, we have studied expression of P-Ser349 p62 in human synovial fibroblasts. Results showed that P-Ser349 p62 was not detected in synovial cell extracts unless cells were cultured in the presence of proteasome inhibitor (MG132). MG132 revealed P-Ser349 p62 turnover, that was further increased by concomitant autophagy inhibition and markedly enhanced in serum starved cells. Starvation sensitized synovial fibroblasts to BAY 11-7085 while MG132 protected both non-starved and starved cells from BAY 11-7085-induced cell death. Lentivirus mediated overexpression of phosphorylation-mimetic p62 mutant S349E markedly protected synovial fibroblasts from BAY 11-7085. Inhibitor of Keap1-P-S349 p62 interaction, K67, had synergistic effect with MG132. Starvation increased p62 molecular weight, that was reversed by serum and bovine serum albumin re-feeding. Furthermore, starvation markedly induced RAD23B. Increased endo-β-N-acetylglucosaminidase (ENGase) turnover was detected in starved synovial fibroblasts. PNGase F treatment produced faster migration p62 form in human synovial tissue extracts but starvation-like p62 form of higher molecular weight in synovial cell extracts. Co-transfection of NGLY1, with p62 or p62 mutants S349A and S349E markedly stabilized p62 expressions in HEK293 cells. Tunicamycin upregulated p62 and protected synovial fibroblasts from BAY 11-7085-induced cell death. These results showed that P-Ser349 p62 has pro-survival role in human synovial fibroblasts and that de-glycosylation events are involved in p62 turnover. Ubiquitin-proteasome-system (UPS) and autophagy are two separate but interconnected protein degradation systems . UbiquitWe have shown previously that BAY 11-7085-induced human synovial fibroblast cell death includes autophagy and p62 downregulation . In thisP-Ser349 p62 was not detected in serum cultured Figure , line 1 4Cl, was used instead of MHY1485 (results not shown). However, statistical analysis of six different experiments showed that MHY1485 did not significantly increased P-Ser349 p62 in serum starved and MG132 treated cells , phosphorylation-defective p62-HA mutant (S349A), wild type p62-HA (WT) or control EGFP (GFP) Figure . Overexpi.e. phosphorylated on Serine 40) [p62 phosphorylation on Serine 349 is known to activate Nfr2. We thus looked for Nrf2 and activated Nrf2 (rine 40)  in synovWe searched for serum constituent that is able to reverse starvation-induced higher MW p62 form. Results showed that higher MW p62 form, that appeared within minutes of starvation, was rapidly reversed by bovine serum albumin (BSA) Figure , line 6 Two human de-glycosylation enzymes have been cloned and characterized: NGLY1  and ENGaTo test possible involvement of de-glycosylation on p62 and P-S349 p62 turnover, we have transfected human, HA epitope marked, p62 and its S349A and S349E p62 mutants , having p62 has been reported to be degraded by autophagy: autophagy inhibitors increase p62 protein level  and p62 In this work, we have detected stabilization of RAD23B during serum starvation-induced autophagy. Rad23B is known for its roles in DNA repair and proteasomal degradation . It bindHaving interaction with NGLY1, RAD23B, apart from its function in DNA repair , is invoProteasome inhibitors can rescue cells from oxidative stress  as well in vivo. Indeed, proteasome inhibition in mice liver increase both autophagy and Keap1/Nrf2 pathway [in vivo too.Benefic effects of MG132 treatment has been shown in mice model of adjuvant arthritis  and mice pathway . We showHuman primary synovial fibroblasts from osteoarthritis (OA) patients were isolated and cultured as explained previously . Serum cHuman primary synovial fibroblasts and HEK293 cells were collected on ice in lysis buffer . Human sCell and tissue extracts were denaturated and treated with PNGase F (P0704) or O-glycosidase (P0733) plus neuraminidase (P0720), New England BioLabs, for 1–3 h at 37° C, according to the manufacturer's instructions.Synovial fibroblasts were stimulated with different concentrations of proteasome inhibitor MG132 (CAS 133407-82-6), Calbiochem, for 24 hours and then BAY 11-7085  was added for additional 2 hours. Survival assay, MTS , was then performed for additional hour, according to the manufacturer's instructions. Cell survival was expressed as a percentage of surviving cells compared with control cells.4 cells/ 2 ml of culturing medium). Cells were transfected with 3 mg of DNA by the use of linear polyethyleneimine  .Human p62, cloned in pcDNA3.1  was kindFor lentivirus transduction experiments, we created three gene transfer lentiviral plasmids by re-cloning of p62-HA, its phosphorylation mimetic mutant S349E and phosphorylation defective mutant S349A  as HindILentivirus production was done by our GIGA Viral Vectors platform. Briefly, Lenti-X 293T cells (632180), Clontech, were co-transfected with gene transfer lentiviral plasmids, pSPAX2, Addgene and VSV-G encoding plasmid . Viral sFor infection experiments human synovial fibroblasts were plated into 96-wells plates and day after inoculated with lentiviruses caring p62-HA, p62 (S349E), p62 (S349A), EGFP, shRNA p62 or non-targeting NT shRNA, at a multiplicity of infection (MOI) of 5–50. After 5 days of infection BAY 11-7085 was added for 2 hours and then MTS was performed. In parallel, synovial fibroblasts were plated into 24-wells plates and day after infected with lentiviruses caring p62-HA, p62 (S349E), p62 (S349A), EGFP, p62 shRNA or NT shRNA, at 5–50 MOI. After 5 days of infection cells were recuperated in lysis buffer  and analp values were obtained using the Mann–Whitney test and Student's t-test. Values of p < 0.05 were considered as statistically significant.