Polygonum cuspidatum (PC) and its active components, resveratrol and emodin, were found to attenuate influenza viral replication in A549 cells. Furthermore, they preferentially inhibited the replication of influenza A virus, including clinical strains isolated in 2009 and 2011 in Taiwan and the laboratory strain A/WSN/33 (H1N1). In addition to inhibiting the expression of hemagglutinin and neuraminidase, PC, emodin, and resveratrol also increased the expression of interferon beta (IFN-β) through Toll-like receptor 9 (TLR9). Moreover, the anti-viral activity of IFN-β or resveratrol was reduced when the A549 cells were treated with neutralizing anti-IFN-β antibodies or a TLR9 inhibitor, suggesting that IFN-β likely acts synergistically with resveratrol to inhibit H1N1 replication. This potential antiviral mechanism, involving direct inhibition of virus replication and simultaneous activation of the host immune response, has not been previously described for a single antiviral molecule. In conclusion, our data support the use of PC, resveratrol or emodin for inhibiting influenza virus replication directly and via TLR-9–induced IFN-β production.Influenza virus infection is a global public health issue. The effectiveness of antiviral therapies for influenza has been limited by the emergence of drug-resistant viral strains. Therefore, there is an urgent need to identify novel antiviral therapies. Here we tested the effects of 300 traditional Chinese medicines on the replication of various influenza virus strains in a lung cell line, A549, using an influenza-specific luciferase reporter assay. Of the traditional medicines tested,  The influenza virus is one of the most infectious respiratory tract pathogens and is annually responsible for significant levels of morbidity, mortality, and economic loss . In receCurrently, there are two classes of antiviral drugs approved by the US Food and Drug Administration (FDA) to treat or prevent influenza virus infections: M2 ion channel inhibitors (amantadine and rimantadine) and neuraminidase inhibitors (oseltamivir and zanamivir) . The useIllicium verum) [One of the raw materials used for the production of oseltamivir is shikimic acid, which is derived from the traditional Chinese spice, star anise (m verum) . Medicinm verum) ,9, howevPolygonum cuspidatum (PC), commonly called Japanese knotweed, is a member of the buckwheat family (Polygonaceae). Eight active compounds have been identified from PC extract: 2-methoxystypandrone (1); emodin (2); resveratrol (3); polydatin (4); emodin-8-O-beta-D-glucopyranoside (5); (E)-3,5, 12-trihydroxystilbene-3-O-beta-D-glucopyranoside-2'- (6); and catechin-3-O-gallate (7); and rubiadin (8). PC has historically been used as a laxative, and occasionally as a food. Recent studies have reported that PC extract and its active components have antipyretic and analgesic activities [tivities –12.Toll-like receptor 9 (TLR9) was first identified as the receptor for unmethylated cytosine-phosphate-guanine motifs in bacteria, and several TLR9 ligands have been identified in viruses and fungi ,14. TLR9The first pathway is a nuclear factor κB (NF-κB)-dependent pro-inflammatory cytokine pathway, and the second involves the activation of type I interferon (IFN) genes through IFN regulatory factor 7 (IRF7) phosphorylation ,16. IRF7In this study, we screened 300 antipyretic Chinese herbal medicines using a cell-based, high-throughput screening system. The active compounds identified during screening were also investigated for their mechanisms of action.2 humidified incubator. pREP4-FluA-Luc was transfected into 293T cells. Stable clones were selected with 250 μg/mL hygromycin B (Roche Diagnostics) and limiting dilution was performed to isolate clones with the highest luciferase activity when infected with influenza A virus. The influenza A/WSN/33 (H1N1) virus (WSN) was obtained from the American Type Culture Collection .Madin-Darby canine kidney (MDCK) cells, human embryonal kidney (293T) cells and A549 lung cancer cells were obtained from the Bioresource Collection and Research Center  and cultured in Dulbecco’s modified Eagle medium  containing 10% fetal bovine serum (Invitrogen) and incubated in a 5% COThe influenza A reporter (pREP4-FluA-Luc) was constructed according to the method of Lutz et al. . Briefly4 cells/well) and incubated at 37°C and 5% CO2 for 24 h. The cells were then washed twice with phosphate buffered saline (PBS). Influenza A virus was diluted with PBS containing 0.2% bovine serum albumin to 10 multiplicity of infection (MOI)/100 μL. Cells were infected with 10 MOI H1N1 and centrifuged at 2000 rpm at room temperature for 1 h. Cells were washed twice with PBS and the test compounds were added. The 96-well plates were then incubated at 33°C and 5% CO2 for 24 h. The supernatants containing the replicated virus were then added to 293T cells stably transfected pREP4-FluA-Luc and incubated at 37°C and 5% CO2 for 24 h. Luciferase activity was determined by a luciferase assay system .A549 cells were seeded into 96-well plates  at 2 μM for 3 h before infection with the influenza virus. For IFN-β neutralization, anti-IFN-β antibodies  were applied together with diluted influenza virus at a concentration of 10 μg/mL.Cell culture supernatants were harvested 24 h after treatment and used directly for measurement of IFN-β concentration. IFN-β concentrations were determined using human IFN-β ELISA kit .Western blotting was performed on A549 cells infected with H1N1 virus and treated with PC and its active components. Cells were harvested, washed with 10 mL of PBS three times, and lysed with mammalian protein extraction reagent  containing a phosphatase inhibitor and a protease inhibitor. Proteins were separated on a 12.5% sodium dodecyl sulfide-polyacrylamide gel electrophoresis (SDS-PAGE) gel and blotted onto a polyvinylidene difluoride membrane. The antibodies used to detect H1N1 hemagglutinin and neuraminidase were generated by immunization of Balb/C mice with H1N1 proteins . For visualization, membranes were probed with an anti-mouse IgG secondary antibody conjugated to horseradish peroxidase. Binding was detected by a chemiluminescent substrate according to the manufacturer’s instructions (Thermo Fisher Scientific). Blots shown are representative of at least three individual experiments.6 cells/well). After incubation at 37°C and 5% CO2 overnight, cells were washed twice with PBS and infected with H1N1 influenza virus (100 plaque forming units (PFU)/well) and kept on ice for 1 h. The cells were treated with PC or its active components in DMEM containing 0.3% agarose. The plate was incubated at 37°C and 5% CO2 for 72 h. The media were removed and the cells were fixed with 4% paraformaldehyde for 1 h at room temperature and subsequently stained with 0.1% crystal violet for 20 min at room temperature.MDCK cells were seeded into 6-well plates , and 5 μg of RNA were reverse-transcribed using a reverse transcriptase for conversion to cDNA (Invitrogen). Primers and probes used for qPCR were selected from a predesigned probe system . The RNA abundance was normalized to glyceraldehyde 3-phosphate dehydrogenase RNA in each sample.50 value for PC was 312 μg/mL.The antiviral activities of PC were evaluated using a virus-inducible reporter system. H1N1 virus (10 MOI) was used to infect A549 lung cancer cells for 1 h, and the cells were then treated with a water extract of PC. The A549 culture supernatant containing the amplified H1N1 was then applied to 293T cells stably transfected with influenza A reporter plasmid (pREP4-FluA-Luc). PC inhibited H1N1 replication in A549 cells as shown by reduced luciferase activity . The IC550 values for rubiadin, resveratrol, emodin, and polydatin were >50 μM, 24.7 μM, 37.3 μM, and >50 μM, respectively. These results indicate that resveratrol and emodin were the most potent active compounds from PC for inhibition of H1N1 viral replication. We also determined the cytotoxicity of PC, rubiadin, resveratrol, emodin, and polydatin on A549 cells. PC, rubiadin, resveratrol, emodin, and polydatin exhibited low toxicity against A549 cells, with GI50 values of >500 μg/mL for PC  were also tested to determine which compound(s) exhibited inhibitory effects on H1N1 replication in A549 cells. The ICL for PC  and >100L for PC .One clinically isolated strain of H3N2 influenza virus (obtained in 2009) and 10 clinically isolated strains of H1N1 influenza viruses (collected in 2009 and 2011) were also treated with PC  and resvAs shown in To investigate the underlying mechanisms of PC, emodin, and resveratrol in inhibiting influenza virus replication, we performed real-time PCR analysis for several antiviral-related genes. A549 cells were treated with PBS (control), the compounds alone, the virus alone, or the compounds and the virus. We found that TLR9 mRNA expression was increased when the cells were treated with PC, emodin, or resveratrol alone or when they were infected with virus and then treated with PC (294 μg/mL), emodin (25 μM), or resveratrol (25 μM). In contrast, the TLR9 mRNA expression level was unchanged when the cells were infected with the virus alone . TLR9 prThe expression MYD88 mRNA was significantly increased with PC treatment. This is significant since MYD88 is involved in signal transduction resulting from TLR9 activation. It has been shown that TLR9 activates the IFN regulatory factors IRF3, IRF5, and IRF7 and the NF-κB family. IRF5 and IRF7 are strong transcriptional activators that lead to the expression of IFN-α and IFN-β. We found that PC, emodin, and resveratrol treatments significantly increased IRF5 and IRF7 mRNA expression . The IRFWe also investigated the expression of IFNα and IFN-β. As shown in Secreted type I IFN induces the expression of myxovirus-resistance protein 1 (MX1), which is important in inhibiting influenza A replication. MX1 mRNA expression levels increased 1.3-fold when cells were treated with H1N1 + PC or resveratrol compared to cells only infected with H1N1 (p < 0.05). In addition, interferon-induced protein IFI-15k (ISG15) and 2′,5′-oligoadenylate synthetase (OAS2) mRNAs exhibited expression patterns that were similar to MX1 .A specific TLR9 inhibitor, Super-iODN, was used to evaluate the importance of TLR9 in inhibiting influenza virus replication. We performed a reporter assay using resveratrol alone or in combination with the TLR9 inhibitor. A549 cells were infected with 10 MOI H1N1 virus for 1 h and subsequently treated with resveratrol, the TLR9 inhibitor, or resveratrol + the TLR9 inhibitor. Inhibition of H1N1 replication by resveratrol was reduced when the cells were treated with the TLR9 inhibitor . This inTo determine the effect of type I IFN on the inhibition of H1N1 replication, we performed a reporter assay using IFN-β alone or in combination with resveratrol. A549 cells were infected with 10 MOI H1N1 virus for 1 h and treated with resveratrol, IFN-β, or IFN-β + resveratrol. Our results indicate that IFN-β alone slightly inhibited viral replication in A549 cells . IFN-β cin vitro.The expression levels of IFN-β were also determined after A549 cells were treated with resveratrol. A549 cells were infected with 10 MOI H1N1 virus and treated with resveratrol, TLR9 inhibitor or TLR9 inhibitor + resveratrol. The expression level of IFN-β was significantly higher in cells treated with resveratrol compared with cells treated with TLR9 inhibitor or TLR9 inhibitor + resveratrol . These rThe aim of this study was to identify potential new treatments for influenza from traditional Chinese herbal medicines and their active components. We created an influenza A virus reporter cell system to screen potential candidates for inhibition of influenza virus replication. In addition, through investigation of the molecular mechanisms underlying these potential new treatments for influenza, we can assess potential new anti-virus pathways for developing more efficacious treatments.In this study, we infected A549 cells with wild type influenza virus and treated them with various compounds. If a compound was effective in mitigating viral replication, viral RNA polymerase was present in lower amounts, leading to a concentration-dependent reduction in luciferase reporter levels. This indicated a concentration-dependent reduction in influenza virus replication.Importantly, this screening process could be completed within 24 h instead of the 2–3 days necessary for plaque formation assays. These results indicated that our system was an effective method to assess influenza A viral titration. Notably, our reporter cell-based screening assay, as with similar assays, showed some level of toxicity to the cells by the test compounds, which could potentially lead to false positives. To avoid false positives, we performed MTT tests to exclude compounds that were toxic to the carrier cells.In our small-scale screening program, we excluded toxic compounds and found that PC, emodin, and resveratrol exhibited significant inhibitory activity against H1N1 and H3N2 influenza A viruses with no significant toxic effect on the host cells. PC, resveratrol, and emodin exhibited potent antiviral activity against multiple subtypes of the influenza A virus, including the A/WSN/33 (H1N1) laboratory strain and the H3N2 and H1N1 clinical strains. Both of these clinically isolated strains have been shown to cause severe human respiratory disease. These strains were utilized in this study since both H3N2 and H1N1 have emerged during the twentieth century and caused four influenza A pandemics .Hemagglutinin is responsible for the binding of the virus to the target cells to initiate viral infection, whereas neuraminidase cleaves the host cellular receptors and facilitates the release of the progeny virus, thus promoting the spread of the infection to neighboring cells ,23. Our The Type I IFN system (IFN-α/β) is the first line of defense against viral infections ,25. ThesThe viral nonstructural protein 1 (NS1) of the influenza A virus prevents the induction of the IFN-β promoter by inhibiting the activation of transcription factors such as IRF3 . Both IR-/- mice [TLR9 is involved in cellular antiviral mechanisms. TLR9 activates two distinct pathways: the NF-κB-dependent proinflammatory cytokine pathway and the IRF7-dependent type I IFN pathway ,30,31. I-/- mice . Our resIn this study, we detected significantly elevated levels of TLR9 and reduced influenza virus A replication in cells treated with PC, resveratrol, or emodin compared to control cells (P < 0.05). This suggests that enhanced TLR9 production and the activation of virus-induced IRF7 induced IFN-β protein expression. Taken together, these data demonstrate that PC, resveratrol, or emodin, in the presence of the influenza A virus, result in the activation of IFN-β through the TLR9-MYD88–IRF7 pathway .In this study, we identified anti-influenza A virus compounds by screening 300 Chinese herbal medicines in a luciferase reporter assay and found that the Chinese herbal compound, PC, and two of its active components, resveratrol and emodin, induced IFN-β only in the presence of influenza virus infection. Moreover, these three compounds exhibited a superior ability to induce the expression of IFN-β and ISGs. PC, resveratrol, and emodin inhibited the growth of influenza A viruses, including the clinically isolated strains H3N2 and H1N1 and the A/WSN/33 (H1N1) laboratory strain. We found that the antiviral activities of PC, resveratrol, and emodin were dependent on IFN production and signaling. This inhibitory effect was associated with the induction of IFN gene expression through the TLR9-MYD88–IRF7 pathway. Together, the findings of this study suggest that synergistic effects of antiviral compounds and IFN-β can protect against influenza virus infections. These insights may lead to the development of novel antiviral therapies for the treatment of influenza.


Compared with HD1A cells, HD1B cells demonstrated many characteristics similar to lal-/- MDSCs. HD1B cells exhibited increased lysosomes around perinuclear areas, dysfunction of mitochondria skewing toward fission structure, damaged membrane potential, and increased ROS production. HD1B cells showed increased glycolytic metabolism during blockage of fatty acid metabolism to fuel the energy need. Similar to lal-/- MDSCs, the mTOR signal pathway in HD1B cells is overly activated. Rapamycin treatment of HD1B cells reduced ROS production and restored the mitochondrial membrane potential. HD1B cells showed much stronger immunosuppression on CD4+ T cell proliferation and function in vitro, and enhanced cancer cells proliferation. Knockdown of mTOR with siRNA reduced the HD1B cell ability to immunosuppress T cells and stimulate cancer cell proliferation. Therefore, the HD1B myeloid cell line is an “MDSC-like” cell line that can be used as an alternative in vitro system to study how LAL controls various myeloid cell functions.Myeloid-derived suppressor cells (MDSCs) in mouse are inflammatory cells that play critical roles in promoting cancer growth and metastasis by directly stimulating cancer cell proliferation and suppressing immune surveillance. In order to facilitate characterization of biochemical and cellular mechanisms of MDSCs, it is urgent to establish an “MDSC-like” cell line. By cross breeding of immortomouse (simian virus 40 large T antigen transgenic mice) with wild type and lysosomal acid lipase (LAL) knock-out ( MDSCs in the tumor microenvironment have been suggested to have a causative role in directly stimulating cancer cell proliferation and promoting tumor-associated immune suppression. Since MDSCs may serve as a target for preventing tumor growth and metastasis, there is a need to establish in vitro “MDSCs-like” cell lines to facilitate MDSCs studies at the cellular and molecular levels.Myeloid-derived suppressor cells (MDSCs) are myeloid progenitors that are blocked to further differentiate into granulocytes, macrophages, and dendritic cells at various pathogenic conditions ,2. In mi+CD16+ and CD14+CD33+ cells have been linked to heterozygote carriers of LAL mutations in humans  ,9 +CD16+ogenesis . Lal-/- feration , and supferation . Myeloidof MDSCs ,14.lal-/- MDSCs in which their fatty acid generation is blocked [lal-/- MDSCs abilities to stimulate cancer cell proliferation and to suppresses T cell proliferation and function [In order to fully understand the functional role of LAL in MDSCs development, the Affymetrix Genechip microarray assay was performed. The gene profile showed upregulation of metabolic enzyme genes in glycolysis and citric acid cycle in association with over-activation of the mTOR signaling pathway in  blocked . The mTO blocked . The mTO blocked . Inhibitfunction ,18.lal-/- MDSCs [Mitochondria fission (fragment or dot shape) and fusion (filamentous) play critical roles in maintaining functional mitochondria when cells are under metabolic or environmental stress . Studies/- MDSCs , it is elal+/+ HD1A and lal-/- HD1B myeloid lineage cell lines were established from wild type and lal-/- mice that were crossbred with Immortomouse expressing a temperature-sensitive version of simian virus 40 large T antigen. The key characters of MDSCs were analyzed in HD1A and HD1B cell lines. HD1B cells showed higher proliferation than that of HD1A cells. This is accomplished by high consumption of glucose oxidation in the mitochondria to compensate the deficiency of FAO. Similar to its primary precursor lal-/- MDSCs, lal-/- HD1B myeloid cells in vitro showed stronger immunosuppression on T cells, and stronger stimulation on cancer cell proliferation compared with its wild type counterpart HD1A cells. At the cellular level, HD1B cells showed characteristics of lal-/- MDSCs, including over-activation of the mTOR signaling pathway, increased production of reactive oxygen species (ROS), arginase activity, and damaged membrane potential. At the subcellular level, the mitochondrial organization of HD1B cells morphologically showed more fission structure in association with down-regulation of pro-fusion protein Opa1 and phosphorylated activation of pro-fission protein Drp1, while the mitochondrial organization of wild type HD1A cells showed more fusion structure. Establishment of these cell lines will not only facilitate elucidation of cellular and molecular mechanisms that are involved in MDSCs malformation and malfunction, but also provide an in vitro system to screen drugs for pharmacological and immune therapy purposes.In this report, immortalized wild type All scientific protocols involving the use of animals have been approved by the Institutional Animal Care and Use Committee of Indiana University School of Medicine and followed guidelines established by the Panel on Euthanasia of the American Veterinary Medical Association. Animals were housed under Institutional Animal Care and Use Committee-approved conditions in a secured animal facility at Indiana University School of Medicine.lal-/- male mice that had been crossbred with Immortomouse (Charles River Laboratories) expressing a temperature-sensitive version of simian virus 40 large T antigen from an IFN-γ inducible promoter [Peritoneal macrophages were collected from wild type and promoter . Cell suHD1A and HD1B cells were grown in 24-well plates to the desired confluence. The medium were replaced with pre-warmed (37°C) LysoTracker Red DND-99 probe -containing medium for 1 hour. Cells were replaced with fresh medium, and fluorescent signals were examined under the Nikon ECLIPSE Ti inverted fluorescence microscope.The concentration of glucose and pyruvate was measured by the glucose assay kit and pyruvate assay kit (Sigma) respectively according the manufacturer’s instruction. Briefly, HD1A or HD1B cells were washed with PBS before being harvested. The cell pellets were added with pre-warmed water and headed in the 70°C water bath for 10 minutes. After spinning down the cell lysates, for glucose measurement the supernatants were incubated with glucose assay reagent for 15 minutes at room temperature and measured absorbance at the 340 nm. For pyruvate measurement, the supernatants were incubated with pyruvate assay buffer, pyruvate probe solution and pyruvate enzyme mixture for 30 minutes at room temperature and measured absorbance at the 570 nm.The aconitase activity was measured by the aconitase activity assay kit (Sigma) according the manufacturer’s instruction. Briefly, HD1A or HD1B cells were washed with PBS before being harvested. Cells were lysed in the ice-cold assay buffer. After centrifugation, the aconitase activation buffer was added into supernatant and incubated on ice for 1 hour, followed by adding Enzyme Mix, assay buffer and the substrate at 25°C for 30 minute. After addition of developer to the incubation mixture at 25°C for 10 minute, the reactions were measured at the 450 nm absorbance. One unit of the aconitase activity is the amount of enzyme that isomerize 1.0 μmole of citrate to isocitrate per minute at pH 7.4 at 25°C.HD1A and HD1B cells were grown in 24-well plates to the desired confluence. The medium were replaced with pre-warmed (37°C) MitoTracker Green FM -containing medium for 1 hour. Cells were replaced with fresh medium, and fluorescent signals were examined under the Nikon ECLIPSE Ti inverted fluorescence microscope.HD1A and HD1B cells were fixed for 15 min in 4% paraformaldehyde, and permeabilized for 10 min in 0.02% Triton X 100. After washing, cells were blocked with 5% normal goat serum in 1 x PBS for 1 hour followed by incubation of primary goat anti-LAMP1 antibody  overnight. Cells were then incubated with secondary donkey anti-goat antibody conjugated with Cy3  for 1 hour, and co-stained with DAPI. Fluorescent signals were examined under the Nikon fluorescence microscope.HD1A and HD1B cells were lysed in the Cell Lytic M mammalian cell lysis/extraction buffer (Sigma-Aldrich) according to the manufacturer’s instruction. Protein samples were fractionated on a Novex 4–20% Tris-Glycine Mini Gel (Invitrogen). After transferring to the polyvinylidene difluoride membrane (Bio-Rad), the membrane was blotted with 5% nonfat dry milk in 1x PBS, and incubated with rabbit anti-p-S6 (Ser235/236), anti-S6, anti-p-Drp1 (Ser616), anti-Drp1, and anti-actin primary antibodies , or rat anti-LAMP1, rabbit anti-Opa1 antibodies (from Santa Cruz). Following incubation with the secondary antibody that conjugated with horse radish peroxidase, proteins were visualized with chemiluminescent substrate (Thermo Scientific) under ChemiDox MP Image System.HD1A and HD1B cells were treated with solvent, rapamycin (40 nM), or PP242 (40 nM) for 60 minutes. Cells were washed and stained with 2 μM of 2',7'-dichlorodihydrofluorescein diacetate stained  which is nonfluorescent until the acetate group is removed by intracellular esterases and oxidation occurs within the cell (Invitrogen). The incubation was at 37°C for 20 minutes, followed by washing with cold PBS. The ROS level was measured by flow cytometry.2 and 50 μl 50 mM Tris-HCl (pH 7.5) were added, and the enzyme was activated by heating at 56°C for 10 min. Arginine hydrolysis was conducted by incubating the lysate (100 μl) with 100 μl 0.5 M L-arginine (pH 9.7) at 37°C for 60–120 min. The reaction was stopped with 400 μl H2SO4 (96%)/H3PO4 (85%)/H2O . The urea concentration was measured at 540 nm after addition of 25 ml 9% α-isonitrosopropiophenone (dissolved in 100% ethanol), followed by heating at 95°C for 45 min and 10 min at room temperature in the dark. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 μmol urea per minute.HD 1A or HD1B cells were lysed for 30 min at room temperature with 50 μl 0.1% Triton X-100 PBS containing 5 μg pepstatin, 5 μg aprotinin, and 5 μg antipain protease inhibitors per ml. Subsequently, 50 μl 10 mM MnCl+ T cells from the spleen were labeled with carboxyfluorescein diacetate succinimidyl diester  (1 μM in PBS) at room temperature for 5 min, and resuspended in complete medium for 20 min. CD4+ T cells were spun down and cultured in 96-well flat-bottom plates coated with anti-CD3 mAb (2 μg/ml) and anti-CD28 mAb (5 μg/ml) for 4 days in the presence or absence of HD1A or HD1B cells at 37°C. The ratio between HD1A or HD1B cells and CD4+ T cells was 1:30. Cells were harvested and stained with APC-labeled anti-CD4 mAb (eBiosciences). Proliferation of CD4+ T cells was evaluated as CFSE dilution by FACS. To measure T cell secreting lymphokines, OptEIA ELISA kits for IL-2, IL-4 and IFNγ were used according to the manufacturer’s instruction (BD BioScience).Freshly isolated wild type CD4HD1A and HD1B cells were harvested and stained with anti-CD11b-PEcy7 and anti-Ly6G-Apccy7 for flow cytometry analysis.+ T cells (1:30) for T cell proliferation (96 hours) and lymphokine release study (48 hours), or lysed to test mTOR signaling pathway protein expression, or co-cultured with B16 or LLC cancer cells, or labeled for BrdU incorporation study.HD1A and HD1B cells were transfected with mTOR—specific or scrambled control siRNAs  according to the manufacturer’s protocol . After 48 hours incubation, cells were washed and co-cultured in wells with CFSE-labeled wild type CD4HD1A and HD1B cells were grown in 24-well plates to the desired confluence. Cells were treated with solvent , rapamycin (40 nM), or PP242 (40 nM) for 1 hour, or NAC , or Tempol  overnight. Treated cells were replaced with the pre-warmed (37°C) medium containing JC1  for 1 hour. Labeled cells were replaced with fresh medium and examined under the Nikon inverted fluorescence microscope.For cell proliferation analysis, HD1A and HD1B cells were grown in 24-well plate to the desired confluence. BrdU (BD Biosciences) was added at a final concentration of 10 μM in cell culture medium for 1 hour. Cells were harvested and washed twice with PBS. Cells were fixed and permeabilized with BD Cytofix/Cytoperm buffer, then incubated with DNase I and washed again followed by staining with fluorescent anti-BrdU antibody before analysis by flow cytometry.4 cells per well of 24-well plate). After culture for 3 days, the cells were harvested and analyzed on the LSR II to determine cancer cell proliferation by gating CFSE labeled cells.B16 melanoma or LLC cancer cells were harvested and labeled with CFSE (1 μM in PBS) at room temperature for 5 min. Labeled cancer cells were resuspended in complete medium for 20 min, spun down, and co-cultured with HD1A or HD1B cells  were mixed with HD1A or HD1B cells (2 X 105) and injected subcutaneously at left or right flank sites of C57BL/6 or FVB/N mice. The tumor sizes were measured 14 days post-injection with calipers. The tumor volumes were determined using the formula: (length X width2) /2 [B16 melanoma cells (2 X 10dth2) /2 . At the CD36, CPT1a, CPT1b, CPT1c, Foxo3, Glut1-13, IDO1, IDO2, SIRT1 and the housekeeping gene β-Actin was performed on a StepOnePlusReal-Time PCR System (Applied Biosystems) using Power SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s protocol. The (2-˙ΔΔCt) algorithm was used to determine the relative gene expression.Total RNAs from HD1A or HD1B cells were purified using the Qiagen total RNA purification kit (Qiagen). cDNAs were generated by SuperScript III (Invitrogen). Real-Time PCR for lal-/- mice were crossbred with Immortomouse (Charles River laboratory), which express a temperature-sensitive simian virus 40 T antigen under an INF-γ inducible promoter. HD1A and HD1B cell lines were established and passaged as described in Materials and Methods. As LAL is a lysosome localized enzyme, the lysosome numbers and localization were examined in HD1A and HD1B cells. Western blot analysis showed an increased LAMP1 (a marker for lysosome) expression level in HD1B cells compared with that of HD1A . Mitochondria are double-membrane—bound subcellular organelles of eukaryotic cells with various functions, including oxidative-phosphorylation, apoptosis, and ROS production. In order to fully understand pathogenic malformation and malfunction in HD1B cells, it is necessary to characterize and compare mitochondrial structures in HD1A and HD1B cells for comparison. It has been well documented that the mitochondrial fission and fusion processes play critical roles in governing these mitochondrial functions ,23. Usinlal-/- MDSCs [To see if the structural change leads to functional changes, the mitochondrial membrane potential and ROS production, which are coupled with OXPHOS, were measured in HD1A and HD1B cells. The mitochondrial membrane potential was analyzed by JC1 staining. While most HD1A cells were stained with red fluorescence staining , HD1B cells were stained with less red fluorescence and more green fluorescence (representing damaged mitochondria) . A damag/- MDSCs . Because/- MDSCs , expresslal-/- MDSCs [Previously, Affymetrix GeneChip microarray showed over-activation of the mTOR signaling pathway in association with mitochondrial membrane potential damage and ROS over-production in /- MDSCs . To see + T cells in the anti-CD3 and anti-CD28 antibody coated plate. After 4 days, CD4+ T cell proliferation was analyzed by flow cytometry. Compared with HD1A cells, HD1B cells showed a stronger suppressive function on CD4+ T cell proliferation  were co-cultured with CFSE-labeled splenocyte CD4feration . Lymphok1B cells . These r+ T cell proliferation in the co-culture experiment .Lipids have long been recognized not only as the nutrients for cell growth and structural components, but also as the cell signaling molecules that have the capacity to trigger profound physiological responses. Previously, we have done extensive studies to show that LAL is a critical enzyme that controls inflammation, especially MDSCs development and homeostasis. Deletion of this neutral lipid metabolic controlling enzyme induces massive MDSCs expansion in tastasis ,11–15,25 antigen , we havelal-/- HD1B cells, more intensified lysosomal subcellular structures were observed by both LysoTracker and LAMP1 staining studies compared with HD1A cells , the rate-limiting enzyme for long chain fatty acid entry into mitochondria and fatty acid oxidation (FAO)  . Howeveron (FAO) . When hi+-dependent SIRTs coordinate a switch from glucose to fatty acid oxidation during the acute inflammatory response [Co-regulators for histone acetyltransferases (HATs), including sirtuins (SIRTs) regulate the levels of FOXO acetylation during oxidative stress. NADresponse . At leaslal-/- MDSCs is increased [In the mitochondria, oxidative phosphorylation (OXPHOS) is the metabolic pathway to reform ATP by the oxidation of nutrients. There are several catabolic biochemical processes produce energy (in form of ATP), including glycolysis, the citric acid cycle, and β-oxidation of free fatty acids. In fat catabolism, triglycerides are hydrolyzed to break into fatty acids and glycerol by LAL. Fatty acids are further broken down through a process known as β-oxidation and results in acetyl-CoA, which can be used in the citric acid cycle in mitochondria. β-oxidation of fatty acids with an odd number of methylene bridges produces propionyl CoA, which is converted into succinyl-CoA and fed into the citric acid cycle. In the absence of the regular supply of fatty acids during LAL deficiency, it seems that the energy consumption switches more to the metabolic pathway on extracellular glucose consumption to fuel OXPHOS in HD1B cells. This was first observed by gene microarray analysis, in which the glycolytic metabolic gene profile of bone marrow ncreased . Glycolyncreased . This islal-/- bone marrow Ly6G+ cells  revealed over-activation of the mTOR signaling pathway [lal-/- CD11b+Ly6G+ cell development and expansion [lal-/- Ly6G+ cells showed damaged mitochondrial membrane potential and increased ROS production [lal-/- MDSCs, these abnormal mitochondrial activities can be blocked by treatment with mTOR pharmacological inhibitor rapamycin and PP242  Both lal-/- MDSCs and HD1B cells showed increased glycolytic metabolic activity; 2) Both lal-/- MDSCs and HD1B cells showed over-activation of mTOR signaling; 3) Both lal-/- MDSCs and HD1B cells showed the increased mitochondrial membrane potential damage and altered expression of metabolic molecules involved in mitochondrial functions; 3) Both lal-/- MDSCs and HD1B cells showed increased ROS production; 4) Both lal-/- MDSCs and HD1B cells showed immunosuppressive function on T cell proliferation and lymphokine secretion; 5) All above pathogenic cellular activities were corrected by either mTOR pharmacological inhibitors, or by siRNA knockdown in lal-/- MDSCs and HD1B cells; 6) Both lal-/- MDSCs and HD1B cells showed stimulation on cancer cell proliferation and growth. Taken all together, this newly established HD1B cell line shows similar characteristics of MDSCs from lal-/- mice, and support a concept that LAL supports FAO in myeloid cells and that lysosomal lipolysis contributes to normal function of myeloid cells.In summary, several parallel studies showed resemblances between lal-/- MDSCs and HD1B cells is that HD1B cells are negative for CD11b and Ly6G, two hallmarks for MDSCs. Expression of cell surface markers (e.g. CD11b and Ly6G) depends on extracellular stimuli  in the surround environment in vivo. Since HD1A and HD1B are cell lines cultured in vitro, which are separated from the in vivo environment, CD11b and Ly6G negative expression may result from absence of stimulatory molecules for these two markers in the culture medium. This interesting observation indicates that CD11b and Ly6G may not be responsible for MDSCs malfunction. Therefore, to define an MDSC cell line, one has to rely on its functions, not a few cell surface markers. Nevertheless, both HD1A and HD1B cell lines will be useful in the future to explore the LAL functions in numerous cellular processes and functions of myeloid cells, especially MDSCs. The myeloid cell lines will also be useful for pharmacological identification of drugs to suppress MDSCs expansion and function in multiple inflammation-induced diseases which involve MDSCs.In our characterization, one discrepancy between


UGT1A1). In East Asian populations, the compound homozygous UGT1A1 G71R and Y486D variants are frequently observed in cases with bilirubin levels exceeding 200 μmol/L. In this study, we investigated the spectrum of UGT1A1 variations in Chinese CNS-II patients. We sequenced the enhancer, promoter, and coding regions of UGT1A1 in 11 unrelated Chinese CNS-II patients and 80 healthy controls. Nine of these patients carried variations that are here reported for the first time in CNS-II patients, although they have been previously reported for other types of hereditary unconjugated hyperbilirubinemia. These individual variations have less influence on UGT activity than do the compound homozygous variation (combination of homozygous G71R variant and Y486D variant). Therefore, we propose that the spectrum of UGT1A1 variations in CNS-II differs according to the bilirubin levels.Crigler–Najjar Syndrome type II (CNS-II) is an autosomal recessive hereditary condition of unconjugated hyperbilirubinemia without hemolysis, with bilirubin levels ranging from 102.6 μmol/L to 342 μmol/L. CNS-II is caused by a deficiency of UDP-glucuronyl transferase (UGT), which is encoded by the UDP-glucuronyl transferase 1A1 gene ( Inherited unconjugated hyperbilirubinemia is categorized as Gilbert Syndrome , Crigler–Najjar Syndrome type I , or Crigler–Najjar Syndrome type II , depending on the total bilirubin (TB) levels: the TB levels range from 17.1 μmol/L to 102.6 μmol/L, 102.6 μmol/L to 342 μmol/L, and 342 μmol/L to 769.5 μmol/L in GS, CNS-II, and CNS-I, respectively. CNS-I can be fatal due to kernicterus, which does not respond to phenobarbital treatment. However, the bilirubin level in CNS-II can be controlled by phenobarbital treatment to avoid the neurological damage caused by unconjugated bilirubin [UGT1A1), which forms part of a complex gene locus (UGT1A gene complex). UGT1A encodes a number of UGTs by alternative splicing, each with various substrate preferences [Liposoluble unconjugated bilirubin is glucuronidated to form hydrosoluble conjugated bilirubin by UDP-glucuronyl transferases (UGT) . This enferences .UGT1A1. In some cases, CNS-II is caused by a combination of a heterozygous Crigler–Najjar-associated structural variant and a GS-associated variant in the promoter region of the gene  [UGT1A1 variants have been reported in CNS-II patients [CNS-II is a rare autosomal recessive hereditary disease and is typically caused by rare homozygous or compound heterozygous mutations in patients . HoweverUGT1A1 was first identified) and November 2014 and found that the compound homozygous G71R variant and Y486D variant were frequently detected in East Asian CNS-II patients whose bilirubin levels were greater than 200 μmol/L (10/13) [We reviewed Crigler–Najjar case reports in NCBI PubMed, EMBASE, HGMD , OMIM (191740) databases between January 1992 . Written informed consent was obtained from all adult subjects or guardians of enrolled minors in the study.Patients were diagnosed with CNS-II using the following criteria: 1) a medical history of persistent unconjugated hyperbilirubinemia with TB > 102.6 μmol/L, comprising predominantly unconjugated bilirubin; 2) normal alanine transaminase and aspartate aminotransferase levels, to exclude hepatitis-related inflammation; 3) hemoglobin levels > 100 g/L (women) or 110 g/L (men) and reticulocyte counts < 1.5%, to exclude potential hemolysis; and 4) TB levels that were reduced with phenobarbital treatment, to distinguish CNS-II from CNS-I patients. Patients who had been on medication during the previous 3 months or who presented with comorbid diseases were excluded to avoid the influence of drugs.Eleven unrelated patients with CNS-II were enrolled between April 2003 and May 2012 in the Department of Infectious Diseases, Southwest Hospital, Third Military Medical University . The bilirubin levels in nine of these patients were under 200 μmol/L (0.818). Aside from severe and persistent jaundice, the physical examination findings were remarkably negative; liver and spleen were not palpable, and no definite neurological abnormalities were described in any of the cases. The patients, who were all Han Chinese, did not appear to be ill, and all were asymptomatic. Clinical data of the patients are listed in Eighty healthy controls were recruited from coworkers in the Department of Infectious Diseases, Southwest Hospital, Third Military Medical University . The healthy controls included 44 men and 36 women, with an average age ± standard deviation of 36.7 ± 11.4 y. All were Han Chinese. They underwent regular physical examination, including medical examination, blood biochemical examination, routine chest X-ray radiography, and abdominal ultrasonography examination at least every 2 years. Liver function tests, including those for TB and conjugated bilirubin, were performed, and the results were found to be normal during the time that they participated in the study.UGT1A1 (GenBank accession number: NT_005120.16) using Primer 3.0 software  and are detailed in the . PCR primers were designed for d in the . PCRs weSNPs were identified using the DNASTAR software  and inspected by at least two independent investigators. The SNP positions and individual genotypes were confirmed by re-amplifying and re-sequencing the SNP site from the same or opposite strand using the corresponding amplification primers. Statistical analysis was performed using SPSS PASW Statistics (version 18.0).In this study, nine variants were detected . The fraUGT1A1 genotypes detected in patients. Patients 1, 2, 3, 4, 5, 6, and 7 were found to carry homozygous or compound heterozygous variations, and the variations included at least one variant that causes a UGT truncation or one variant that is located in the shared exon of the UGT1A gene complex. These compound variations were not detected in the controls. Patient 1 was homozygous for both the G71R variant and the Y486D variant. Patient 2 was heterozygous for the G71R variant and homozygous for the Y486D variant. Patient 3 was homozygous for the P364L variant and heterozygous for the GS-associated promoter variant (c.-40_-39insTA). Patient 4 was heterozygous for both the Y486D variant and the M418fsX423 variant. Patients 5 and 6 were heterozygous for the Q239X variant and homozygous for the GS-associated promoter variant (c.-40_-39insTA). Patient 7 was heterozygous for both the P364L variant and the G71R variant. Patients 8 to 11 carried only a G71R structural variant. Patient 8 was also heterozygous for the GS-associated promoter variant (c.-40_-39insTA) and the c.-3279T>G variant in the enhancer, and this variation was also found in one control ( control . AdditioUGT1A1 variations in 11 Chinese CNS-II patients and nine variants were identified. Nine of the 11 cases carried variations that are reported here for the first time in CNS-II patients (patients 3 to 11). The two variants predicted to cause truncation of UGT (M418fsX423 variant and Q239X variant) were also previously detected in CNS-I patients [In this study, we investigated the spectrum of patients , 14. Thepatients  and was patients , as doespatients . All patpatients , no prevWe reviewed Crigler–Najjar case reports in NCBI PubMed, EMBASE, HGMD and OMIM and found that the spectra are marked variable in different populations. The compound homozygous G71R variant and Y486D variant were detected in 14 of 27 East Asian CNS-II patients , 20–26, UGT1A1 variation detected: single variant homozygous Y486D variant, combination heterozygous G71R variant and homozygous Y486D variant, and compound homozygous G71R variant and Y486D variant . Therefore, in East Asian populations, the Y486D variant is the main pathogenic cause of CNS-II in patients whose bilirubin levels exceed 200 μmol/L, and the GS-associated G71R variant[In these reported East Asian CNS-II cases, the TB levels of the CNS-II patients increased in the following order with respect to the R variant is a synUGT1A1 variations is distinct from the spectrum of UGT1A1 variations reported here. Our study found many variants that have a less marked influence on UGT activity than does the Y486D variant. This variant lowers the bilirubin UGT activity by more than 90% [The previously reported spectrum of than 90% , while tthan 90% , 18–33% than 90% , 40% [16UGT1A1 variation and bilirubin levels (UGT1A1) [However, the two spectra are comparable in terms of the relationship between n levels . The com(UGT1A1) , while sUGT1A1 variants were all identified in CNS-II patients in this study. However, the spectrum of variation in this study was also distinct from the spectrum previously noted for GS. In a Japanese study, the variants that cause a truncation of UGT were absent in GS patients, and variants that are located in the shared exon of the UGT1A gene complex were only observed in 14.1% of GS patients (9/64) [UGT1A gene complex were absent from GS patients [UGT1A gene complex (0.636). These variants can cause the absence or deficiency of the conserved carboxyl terminal of all UGTs, leading to a significant loss of activity in all of the UGT1A subfamily proteins.The c.-40_-39insTA variant and the G71R variant are associated with GS, and the heterozygous Y486D variant or P364L variant have also been observed in GS patients previously  These UGpatients . In thisUGT1A1 encodes only bilirubin UGT [ubin UGT . The actubin UGT . Howeverubin UGT –17, 28. UGT1A1. Bilirubin levels are influenced by other factors besides genetic variation, such as alcohol, fasting, stress, and medication [UGT1A1. It has been reported that CNS-II cases carrying simultaneously UGT1A1 and other gene variations in recent years [CNS-II is an autosomal recessive hereditary disease, and patients 11 and S24 carried only single heterozygous variants in dication . During dication  It may bnt years –33.UGT1A1 variants.The study cautions clinicians that with CNS-II there is a potential risk of drug toxicity and that different bilirubin levels can indicate a corresponding potential disorder of drug metabolism. The c.-40_-39insTA variant, G71R variant, and Y486D variant can influence the metabolism of irinotecan –37. The S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


The diagnosis of acute myocarditis is described to be challenging. Novel techniques like native and contrast enhanced T1 mapping have proven to be superior to standard sequences for differentiation between healthy individuals and acute myocarditis, but a comparison in the clinical setting is not yet done.We sought to assess the diagnostic performance of native and contrast enhanced T1 mapping compared to T2w-imaging, Relative Enhancement (RE) in T1w as well as Late-Gadolinium-Enhancement (LGE).We investigated 284 patients undergoing CMR examination with suspected myocarditis by performing additional pre and post contrast T1 mapping sequences. Furthermore T2 w dark blood turbospin echo (TSE) sequences, pre and early post Gd T1 w dark blood TSE sequences and inversion recovery spoiled gradient echo sequences for LGE were utilized for myocardial tissue evaluation. Assessment of T1 relaxation time and ECV values was based on 3 short axis views and 1 long axis view. For T2w-imaging evaluation the T2-signal intensity (SI) ratio of myocardium compared to skeletal muscle was used. Additionally T2-SI of potential lesions was compared to remote myocardium and the standard deviation. Relative Enhancement (RE) was calculated from pre and post Gd administration T1w-imaging.T2-SI ratio and myocarditis associated LGE pattern, were assessed. Reference standard was an elevated troponin level, and a more than 10-fold elevation was considered relevant (0.14ng/ml).Native T1 and ECV showed good association with relevant troponin elevations. AUC was 75% (p = 0.0004) for native T1 with an optimal threshold of 993ms  and 83% (p < 0.0001) for ECV with and optimal cutoff of 32.4% .AUC for T2w imaging (T2-SI ratio) was 72% (p = 0.0029) with an optimal cutoff of 2.26 .AUC for T2w imaging (T2-SI compared to remote) was 66 % (p = 0.014) with an optimal cutoff of 1.5 standard deviations above remote .Additionally we found good correlation for native T1 and ECV with the Lake-Louise-Criteria.ECV performed significantly better compared to T2-SI in comparison to remote myocardium (p = 0.037) and showed a trend to be superior to T2-imaging in comparison with skeletal muscle (p=0.85).Although ECV and native T1 mapping detect other myocardial alterations besides acute damage, these techniques perform at least equally well in comparison to established CMR-techniques in detecting acute myocardial damage. ECV is superior to comparing T2-SI in the myocardium of remote and lesion and at least comparable with the other established methods for evaluation of acute myocarditis. Considering the potential of T1 mapping supposed to be relatively robust in acquisition, routine T1 mapping might augment the diagnostic value of CMR.N/A.


In vivo bone formation and histomorphometry after F intake were also evaluated and related to the proteome. Resistant 129P3/J and susceptible A/J mice were assigned to three groups given low-F food and water containing 0, 10 or 50 ppmF for 8 weeks. Plasma was evaluated for alkaline phosphatase activity. Femurs, tibiae and lumbar vertebrae were evaluated using micro-CT analysis and mineral apposition rate (MAR) was measured in cortical bone. For quantitative proteomic analysis, bone proteins were extracted and analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), followed by label-free semi-quantitative differential expression analysis. Alterations in several bone proteins were found among the F treatment groups within each mouse strain and between the strains for each F treatment group . Although F treatment had no significant effects on BMD or bone histomorphometry in either strain, MAR was higher in the 50 ppmF 129P3/J mice than in the 50 ppmF A/J mice treated with 50 ppmF showing that F increased bone formation in a strain-specific manner. Also, F exposure was associated with dose-specific and strain-specific alterations in expression of proteins involved in osteogenesis and osteoclastogenesis. In conclusion, our findings confirm a genetic influence in bone response to F exposure and point to several proteins that may act as targets for the differential F responses in this tissue.Genetic factors influence the effects of fluoride (F) on amelogenesis and bone homeostasis but the underlying molecular mechanisms remain undefined. A label-free proteomics approach was employed to identify and evaluate changes in bone protein expression in two mouse strains having different susceptibilities to develop dental fluorosis and to alter bone quality.  Fluoride (F) is readily absorbed from the stomach and small intestine, followed by its deposition in calcified tissues and urinary excretion Although the dose of F is highly relevant, it is not the only factor influencing the effects of F on bone. Approximately one third of patients receiving F therapy for osteoporosis are non-responders The influence of genetic background in F-induced responses is also supported by studies comparing different mouse strains. Differences in DF susceptibility/resistance among 12 inbred strains of mice have been observed The present study was designed to investigate the effects of F on bone and the molecular mechanisms that could account for the effects. We used label-free quantitative proteomic analysis complemented by static and dynamic histomorphometry. We chose the A/J and 129P3/J strains of mice as the models since, as discussed above, they have been shown to have distinctly different responses to fluoride exposure. We concluded that F exposure at high levels enhanced bone formation in 129P3/J mice, but not in A/J mice, confirming a genetic influence in the response of bone to F. Several proteins could account for the strain-specific skeletal phenotype after F exposure.2 and stored at −80°C until used.The research protocol was approved by the Institutional Animal Care and Use Committee of the University of São Paulo, Bauru Dental School. Weanling (21 days old) male 129P3/J and A/J mice were obtained from the Jackson Laboratory  and randomly distributed into three groups given low-F diet  and water containing 10 or 50 ppmF added as NaF for 8 weeks. They led to plasma F levels corresponding to those found in humans drinking water containing 1  and 10 mg/L F (areas of endemic fluorosis), respectively Femurs, tibiae and 4th lumbar vertebrae were subjected to micro-CT analysis (n = 8/group). Bones were scanned using the Skyscan 1074HR microCT  at the resolution of 20.7 µm/pixel. Standardized scanning and image reconstruction settings fully automated were used. Hydroxyapatite phantoms (250 mg/cc and 750 mg/cc)  were used to determine bone mineral densities within regions and volumes of interest. Morphometric parameters were calculated by CT-analyser  either in 3D based on a volume model, or in 2D from cross-sectional images. Trabecular regions of the proximal tibiae and L4 vertebrae were used to determine bone mineral density (BMD), bone volume fraction (BV/TV), specific bone surface (BS/BV), bone surface density (BS/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular number (Tb.N), and trabecular bone pattern factor (Pb.Pf). One mm cortical regions within the mid-diaphysis of the femurs were used to determine mean total cross-sectional bone area (B.Ar), mean total cross-sectional bone perimeter (B.Pm), mean total cross-sectional tissue area (T.Ar), mean total cross-sectional tissue perimeter (T.Pm), mean polar moment of inertia (MMI), and cortical BMD.Mice (n = 8/group) were injected intraperitoneally with calcein  at 7 days prior to euthanasia and Alizarin Complexone  2 days prior to euthanasia. Bones were dried to touch and embedded in epoxy, Pelco Eponate 12 (25.7 mL resin +9.3 mL DDSA +16.5 mL NMA +1.6 mL BDMA) . After 24 h cure at 60°C, samples were sawed using a Buehler Isomet Low Speed saw  equipped with a diamond wafer saw blade and 200–300 micron thick sections were cemented to glass slides. Mounted sections were ground using 600-C silicon carbide wet paper  to a final thickness of 100 microns. Calcein and alizarin complexone fluorophores were visualized at 100X and 400X using epifluorescence microscopy, Nikon Eclipse 50i equipped with a XF52 dual band filter set  . Images were captured using a Nikon DXM1200 camera and ACT-1 software .Protein extraction was performed according to the method described by Domenicucci et al. 4HCO3, pH 7.8 for 1 h at room temperature (RT). Then, the samples were diluted to 1 M urea with 50 mM NH4HCO3, pH 7.8. Finally, trypsin  was added (1∶20 w/w), and the solution incubated at 37°C for 18 h. The extracted peptides were concentrated and desalted with Perfect Pure C18 Tips  and analyzed by LC-ESI-MS/MS. The Nanoflow HPLC/MS/MS system consists of a Proxeon EasyNano-LC  coupled to LTQ-VELOS-Ion Trap  MS. The nano-LC system includes a 5 mm L.×0.3 mm I.D. reverse phase trap column packed with 5 µm C18 Zorbax 300SB beads , and nanoflow analytical column (150 mm L.×0.075 mm I.D. with 0.015 mm tip opening) packed with the same reversed phase C18 beads as in the Trap column. Peptides were eluted from the column using a linear gradient profile from 6% Solvent B to 65% solvent B in 30 min, followed by 3 min wash with 95% solvent B . The flow rate was set to 300 nL/min with a maximum pressure of 280 bar. Electrospray voltage and the temperature of the ion transfer capillary were 1.8 kV and 250°C respectively. All samples were analyzed in triplicate, resulting in 18 LC-MS/MS runs. The acquired MS/MS spectra was searched against the Mus musculus database from NCBI (Version Dec/2010) using SEQUEST algorithm in Proteome Discoverer 1.3 software  for protein identification. The minimum number of peptides to identify proteins was set at two. Search results were filtered for a False Discovery rate of 1% employing a decoy search strategy utilizing a reverse database. An additional inclusion criterion for positive identification of proteins was the same protein passing the filter score in at least two different MS analyses from the same time-point group in a total of three MS analyses per group. The label-free semi-quantitative differential expression analysis was performed with SIEVE software 1.3 . Changes in relative protein abundance between the groups (A/B) were regarded as significant when the ratio was ≤0.5 for increase in abundance in B compared to A (B>A) or ≥1.5 for decrease in abundance in B compared to A (B<A), and a p-value <0.05. Identified bone proteins were classified according to their biological processes and cellular localization using Web-based Babelomics (http://babelomics.bioinfo.cipf.es/index.html), MGI Mouse Genome Informatics (http://www.informatics.jax.org/), and Uniprot protein data base (http://www.informatics.jax.org/).For each group, 300 µg of protein were denatured and reduced in a buffer containing 100 µL of 4 M urea, 10 mM DTT and 50 mM NH2 and 1 mM p-NPP were added to 96-well plates. After incubation for 30 min in a water bath, 5 µL of each sample (in duplicate) were added. The plate was kept at 37°C for 60 min. Then the reaction was stopped with 1 M NaOH. The final product (p-nitrophenol) was quantified at 405 nm  and the results were expressed as specific activity (nmol p-nitrophenol min−1 mg−1 of protein).The ALP activity was measured in plasma by enzymatic assay using p-nitrophenyl phosphate (pNPP) as substrate. For the ALP activity assay, 100 µL of a solution containing 25 mM glycine buffer (pH 9.4), 2 mM MgClhttp://rsbweb.nih.gov/ij/).Four independent experiments were performed with individual sample of each group, totalizing 4 different samples per group. Mouse femur samples were extracted as described above. For each group, 30 µg of protein were separated on 10% SDS-PAGE gels and transferred onto a PVDF membrane. After blocking with 1% low-fat milk for 1 h at RT, the membranes were incubated at 4°C overnight with rabbit polyclonal anti-mouse collagen type I  and anti-β-tubulin  in 3% BSA and 0.1% TBS-Tween (TBS-T). The membranes were then washed 3 times for 10 min with 0.1% TBS-T, and incubated for 1 hour at RT with horseradish peroxidase-conjugated secondary antibody . After washing, proteins of interest were detected using the enhanced chemiluminescence technique (ECL) Plus Western Blotting Detection Reagent  according to the manufacturer's recommendations, followed by exposure to x-ray film . Quantification was performed by densitometric analysis using Image J software  and Statistica version 7.0 for windows  were used for all statistical analyzes. Since the assumptions of equality of variance (Bartlett test) and normal distribution of errors (Kolmogorov-Smirnov test) were satisfied, two-way ANOVA and Bonferroni's post hoc tests were carried out for statistical comparisons. In all cases, statistical significance was defined as a p<0.01, p<0.001 and p<0.05 for control, 10 and 50 ppmF groups, respectively) while femur BMD was higher only in control group (p<0.05) (p<0.01) (p<0.01) (p<0.01) (p<0.01)  (p<0.01) (p<0.01) .To assess trabecular and cortical bone morphology after treatment with F we have conducted tibia and femur micro-CT, respectively. The lumbar vertebra was also employed for the trabecular study since it has been shown to be a target for the F effect  [14]. Th(p<0.05) A. Tibia(p<0.01) B,C and (p<0.01) G and in(p<0.01) E Figure.ctively) F Figure.(p<0.01) . Statist(p<0.01) B,D,E,F (p<0.01) C  (p<0.05) (Slight dose-dependent increases in the rate of new bone deposition were observed for both strains but they were significant only in the 50 ppmF-treated 129P3/J group when compared to the control and 10 ppmF groups (ctively) A,B. Com(p<0.05) A,B.p = 0.014)  .A total of nine differential analyses for relative protein abundance was performed with SIEVE software 1.3 , three comparisons among the F treatments for 129P3/J strain, three among the F treatments for A/J strain and three between the strains for each F treatment. For the 129P3/J strain, comparisons between control and experimental groups showed an increase in level of 29 and 126 proteins in 10 ppmF and 50 ppmF groups, respectively, while only 1 protein decreased in level in 50 ppmF group . CompariProteins with differences in abundance in each comparison were classified according to their biological process and cellular location . Severalp = 0.262) but there was a significant increase in the level of collagen type I in 129P3/J compared to A/J strain  for all F treatment groups  chain precursors), the major protein of bone, was more abundant in control 129P3/J mice compared to control A/J mice. These data were further confirmed by western blotting analysis. Since mechanical properties of bone tissue depend both on the mineral and matrix (primarily type I collagen fibrils) constituents, as well as their geometrical arrangement, the greater abundance of collagen type I in the 129P3/J strain could explain the higher mechanical strength Exportin-2 (CSE1L) is a protein transport that mediates importin-alpha re-export from the nucleus to the cytosol after import substrates have been released into the nucleoplasm CHD7 was more abundant in 129P3/J mice and, along with CHD4, it was up-modulated by 50 ppmF. Although no role has been reported for CHD-4 and CHD-7 in bone, another chromatin remodeling protein, namely CReMM/CHD9, is expressed by osteoprogenitors cells in mature bone and regulates gene promoters of osteocalcin Proteins associated with endoplasmatic reticulum (ER) stress, such as eukaryotic translation initiation factor 2 alpha kinase 3 precursor (eIF2αk3) or PKR-like endoplasmic reticulum kinase (PERK), were also affected by F. ER stress during osteoblast differentiation activates the PERK-eIF2α-ATF4 signaling pathway involved in the promotion of gene expression essential for osteogenesis, such as osteocalcin and bone sialoprotein The treatment with high levels of F increased expression of SHIP1/2 in both A/J and 129P3/J mice. SHIP dephosphorylates the 5′-phosphate group from phosphatidylinositol-3,4,5-triphosphate (PIP3), the major product of phosphatidylinositol 3-kinase (PI3-K), inactivating it. This signaling is implicated in macrophage-colony stimulating factor and receptor activator of nuclear factor-κB ligand-mediated osteoclast activation Another identified protein involved in bone resorption and osteoclast regulation with differences in abundance is Nox4. NADPH oxidase is an enzyme system responsible for producing osteoclastic superoxide radicals, promoting bone resorption Detoxification enzymes, such as AFB1-AR and CBR, showed an increase in abundance after 50 ppmF treatment in 129P3/J mice compared to the control group. The CBR was identified in mice lung tissue and may have a role in the detoxification of xenobiotics and of toxic aldehydes derived from lipid peroxidation processes in vivo and in vitro, by still uncertain mechanisms These results suggest that F targets molecular mechanisms in both osteoblasts and osteoclasts. This is consistent with the fact that F acts on osteoblasts and osteoclasts Taken together, 129P3/J and A/J mice have intrinsic molecular differences related to bone metabolism A. They One of the major obstacles of osteoporosis therapy with F is the difficulty in prospectively identifying which patients might benefit from therapy, since not all patients respond to F treatment S1 Figureth lumbar vertebrae from A/J and 129P3/J mice treated with 0, 10 or 50 ppm F. Values are mean ± SD, n = 8/group. A =  Bone mineral density (BMD); B =  Specific bone surface BS/BV; C =  Bone volume fraction (BV/TV); D =  Bone surface density (BS/TV); E =  Trabecular number (Tb.N); F =  Trabecular thickness (Tb.Th); G =  Trabecular separation (Tb.Sp); H =  Trabecular bone pattern factor (Tb.Pf). *p<0.05 and **p<0.01 represent significant differences between strains, for each group.BMD and histomorphometric parameters in trabecular region of 4(DOCX)Click here for additional data file.S2 FigureBiological process distribution of the identified bone proteins with differences in abundance among F treatments in A/J and 129P3/J mice, n = 8/group.(DOCX)Click here for additional data file.S3 FigureCellular distribution of the identified bone proteins with differences in abundance among F treatments in A/J and 129P3/J mice, n = 8/group.(DOCX)Click here for additional data file.S4 FigureBiological process distribution of the identified bone proteins with differences in abundance between the strains (A/J and 129P3/J) for each F treatment, n = 8/group.(DOCX)Click here for additional data file.S5 FigureCellular distribution of the identified bone proteins with differences in abundance between the strains (A/J and 129P3/J) for each F treatment, n = 8/group.(DOCX)Click here for additional data file.S1 TableComplete list of identified proteins with differences in abundance in the comparison between control 129P3/J and 10 ppmF-treated 129P3/J mice.(DOCX)Click here for additional data file.S2 TableComplete list of identified proteins with differences in abundance in the comparison between control 129P3/J and 50 ppmF-treated 129P3/J mice.(DOCX)Click here for additional data file.S3 TableComplete list of identified proteins with differences in abundance in the comparison between 10 ppmF-treated 129P3/J and 50 ppmF-treated 129P3/J mice.(DOCX)Click here for additional data file.S4 TableComplete list of identified proteins with differences in abundance in the comparison between control A/J and 10 ppmF– treated mice.(DOCX)Click here for additional data file.S5 TableComplete list of identified proteins with differences in abundance in the comparison between control A/J and 50 ppmF-treated A/J mice.(DOCX)Click here for additional data file.S6 TableComplete list of identified proteins with differences in abundance in the comparison between 10 ppmF-treated A/J and 50 ppmF-treated A/J mice.(DOCX)Click here for additional data file.S7 TableComplete list of identified proteins with differences in abundance in the comparison between control 129P3/J and control A/J mice.(DOCX)Click here for additional data file.S8 TableComplete list of identified proteins with differences in abundance in the comparison between 10 ppmF-treated 129P3/J and 10 ppmF-treated A/J mice.(DOCX)Click here for additional data file.S9 TableComplete list of identified proteins with differences in abundance in the comparison between 50 ppmF-treated 129P3/J and 50 ppmF-treated A/J mice.(DOCX)Click here for additional data file.


D8Mit343–D8Mit312 for the number of OMCs and D8Mit86–D8Mit89 for their distribution, designated as mast cell in the ovary of MRL/MpJ 1 (mcom1) and mcom2, respectively. Among MC migration-associated genes, ovarian expression of chemokine (C-C motif) ligand 17 at mcom1 locus was significantly higher in MRL/MpJ than in C57BL/6N, and positively correlated with the expression of OMC marker genes. These results indicate that the appearance of neonatal OMCs in MRL/MpJ is controlled by environmental factors and filial genetic factors, and that the abundance and distribution of OMCs are regulated by independent filial genetic elements.In MRL/MpJ mice, ovarian mast cells (OMCs) are more abundant than in other mouse strains, and tend to distribute beneath the ovarian surface epithelium at birth. This study investigated the factors regulating the appearance of neonatal OMCs in progeny of the cross between MRL/MpJ and C57BL/6N strains. F1 neonates had less than half the number of OMCs than MRL/MpJ. Interestingly, MRLB6F1 had more neonatal OMCs than B6MRLF1, although they were distributed over comparable areas. Furthermore, in MRL/MpJ fetuses for which parturition was delayed until embryonic day 21.5, the number of OMCs was significantly higher than in age-matched controls at postnatal day 2. These results suggest that the number of OMCs was influenced by the environmental factors during pregnancy. Quantitative trait locus analysis using N2 backcross progeny revealed two significant loci on chromosome 8:  Mast cells (MCs) reside in most tissues and act as sentinel cells in both innate and adaptive immunity lpr/lpr mice, are models for autoimmune diseases that resemble human systemic lupus erythematosus and rheumatoid arthritis i.e., metaphase-specific apoptosis of meiotic spermatocytes, heat shock resistance of spermatocytes found in experimental cryptorchidism, existence of testicular oocytes in newborn males, and development of ovarian cysts originating from the rete ovarii MRL/MpJ mice originate from C57BL/6J (0.3%), C3H/HeDi (12.1%), AKR/J (12.6%), and LG/J (75.0%) strains. MRL/MpJ mice and their mutant strain, MRL/MpJ-We have observed that neonatal MRL/MpJ mice possess numerous MCs in the ovary compared to other inbred mouse strains including C57BL/6N This study was approved by the Institutional Animal Care and Use Committee convened at the Graduate School of Veterinary Medicine, Hokkaido University . The investigators adhered to the Guide for the Care and Use of Laboratory Animals of Hokkaido University, Graduate School of Veterinary Medicine .MRL/MpJ and C57BL/6N mice purchased from Japan SLC  were used in this study. Timed mating was established by housing females with males overnight. At noon of the following day, females were checked for the presence of a vaginal plug, and the embryos were recorded as embryonic day 0.5 (E0.5). C57BL/6N mice were mated with MRL/MpJ mice to produce F1 and F2 progeny. In addition, 200 female N2 backcross progeny (BMM) were generated for QTL analysis by mating female B6MRLF1  with male MRL/MpJ mice.2) as previously described 2) Collected ovaries were fixed in 4% paraformaldehyde overnight, embedded in paraffin, and cut into 3-µm sections. Deparaffinized sections were stained with 1% toluidine blue in 70% ethanol for 30 min. The number of metachromatic MCs per total ovarian area was measured as the OMC density  suspended in sesame oil (20 mg/ml) every 24 h from E17.5 to E20.5 Heparinized plasma was collected from female fetuses of MRL/MpJ and C57BL/6N mice at E18.5 of normal pregnancy. Plasma estradiol and progesterone were measured by enzyme-linked immunosorbent assay .www.informatics.jax.org) were used for a genome-wide scan, with a mean intermarker distance of 10–20 cM, to genotype the C57BL/6N or MRL/MpJ allele . Linkage analyses were performed using the Map Manager QTX20b program http://phenome.jax.org).For several genes coded as highly significant QTL, single nucleotide polymorphisms (SNPs) specifically associated with non-synonymous coding polymorphisms were determined in MRL/MpJ- and C57BL/6N-type genomes using the Mouse Phenome Database (MPD) from Jackson Laboratory  and treated with DNase . Complementary DNA (cDNA) was synthesized from the RNA using ReverTra Ace  and random primers (Promega). Each cDNA, adjusted to 1.0 µg/µL, was used for quantitative real-time PCR (qPCR) with Brilliant III SYBR Green QPCR Master Mix . Gene-specific primer pairs are listed in U test was used to compare two groups. The Kruskal-Wallis test was used for comparisons of more than two groups, and multiple comparisons were performed using Scheffé’s method. Correlations were evaluated using Pearson’s correlation test.Results are expressed as mean ± SEM and were analysed using nonparametric methods. The Mann-Whitney To determine the pattern of inheritance, the appearance of OMCs was compared between MRL/MpJ and C57BL/6N mice and their F1 and F2 progeny at P0. Metachromatic MCs were abundant in ovaries of MRL/MpJ mice , but rarOMC density was 50-fold higher in MRL/MpJ than in C57BL/6N mice , while vBased on the results of histoplanimetric analyses of F1 progeny, it was supposed that environmental factors during pregnancy affect the abundance of OMCs in perinatal mice. To test this hypothesis, parturition was delayed by injecting pregnant MRL/MpJ and C57BL/6N mice with progesterone, and OMC density was compared between E21.5 fetuses and age-matched P2 pups. In MRL/MpJ mice, MCs were abundant in the ovary at E21.5 , and theTo identify the genetic loci controlling the appearance of OMCs in neonatal MRL/MpJ mice, a genome-wide QTL analysis was performed using 200 BMM mice at P0. Since both OMC abundance and their distribution beneath the ovarian SE were unique phenotypes of MRL/MpJ mice, OMC density, SEMC density, and SEMC ratio were measured as QTs. All QTs had gradient distributions, suggesting a polygenic pattern of inheritance . SEMC deD4Mit42  , with one highly significant peak on D8Mit248–D8Mit312   , Table 17.12 cM) , Table 1D2Mit340 , MRL/MpJ-type D13Mit17 , and C57BL/6N-type D14Mit11-D14Mit141  , with one highly significant peak on D8Mit343–D8Mit312  overlapping the highly significant locus for OMC density, and the other peak on D8Mit86–D8Mit89  (mast cell in the ovary of MRL/MpJ 1 (mcom1) and mcom2, respectively.For SEMC density, suggestive loci were mapped to MRL/MpJ-type 4.28 cM) , Table 14.28 cM) . Signifi2.93 cM) , Table 1D4Mit42  and MRL/MpJ-type D9Mit18  , which overlapped with mcom2 (Suggestive QTL for SEMC ratio were mapped to C57BL/6N-type 1.49 cM) , Table 1th mcom2 , Table 1D8Mit312 (47.12 cM) and D6Mit74 (23.70 cM) for both OMC and SEMC densities. Additional QTL interactions were identified between D8Mit248 (44.99 cM) and D19Mit33 (51.76 cM) for SEMC density, and between D8Mit89 (62.93 cM) and D19Mit91 (40.53 cM) for SEMC ratio.Epistatic interactions were examined to determine QTL responsible for the appearance of OMCs. QTL interaction was identified between MRL/MpJ mice homozygous QTL on Chr 8 and heterozygous QTL on Chr 6 showed the highest values for OMC and SEMC densities, while QTL on Chr 6 had no effect on OMC number in mice with heterozygous QTL on Chr 8 . BMM micmcom1 locus associated with MC migration and chemotaxis were evaluated as candidates for the regulation of OMC density, because MCs were the only immune cell type present in the ovary of neonatal MRL/MpJ mice mcom1, including Interleukin 15 (Il15), Matrix metallopeptidase 2 (Mmp2), Metallothionein 1–4 (Mt1–Mt4), Chemokine (C-C motif) ligand 17 (Ccl17), Ccl22, and Chemokine (C-X3-C motif) ligand 1 (Cx3cl1). In contrast, no genes associated with the MC migration or chemotaxis were reported at mcom2.Genes on the Mt4 and Ccl17 expression were significantly higher in MRL/MpJ than in C57BL/6N mice, while Il15 level was significantly lower in MRL/MpJ  in P0 MRL/MpJ mouse ovary Expression levels of these MC chemoattractant genes at P0 were compared between MRL/MpJ and C57BL/6N ovaries by qPCR.  MRL/MpJ . Express MRL/MpJ . ExpressThe appearance of OMCs varies according to mouse strain In MRL/MpJ fetuses with artificially delayed parturition, OMC density at E21.5 was significantly higher than in age-matched mice at P2. Fetuses were exposed to high doses of steroid hormones during late pregnancy, and both of estradiol and progesterone levels have been shown to affect MC migration D8Mit248–D8Mit312 on Chr 8 , whereas SEMC ratio was linked to a second locus, MRL/MpJ-type D8Mit86–D8Mit89 on Chr 8 . These results clearly demonstrate that OMC abundance and distribution are regulated by independent genetic factors derived from mcom1 and mcom2, respectively.Because both the abundance of OMCs and their distribution beneath the ovarian SE were phenotypes unique to MRL/MpJ mice, three parameters were selected for QTL analysis: OMC and SEMC densities, as well as SEMC ratio. A high OMC density was correlated with the MRL/MpJ-type D6Mit74 and D19Mit91–D19Mit33 loci showed epistatic interactions with the mcom1, and mcom2, respectively. The genotypes on these chromosomes affected the appearance of MCs only in mice carrying MRL/MpJ homozygosity at mcom1 and mcom2. These results indicate that these loci on Chrs 6 and 19 are positive modifiers of mcom1 and mcom2. QTL analysis using F2 progeny would be useful for identifying relevant loci on other chromosomes, and evaluating interactions between mcom1 and mcom2.In addition, mcom1 locus could be major determinants of OMC number. Based on differences in gene expression between MRL/MpJ and C57BL/6N mice, two possible candidate genes were highlighted in mcom1: Mt4 at 46.26 cM, and Ccl17 at 46.85 cM. In contrast, no genes associated with the MC migration or chemotaxis were located at mcom2, suggesting that other molecular mechanisms, such as non-coding RNA, could regulate MC distribution.The results of the QTL analysis suggested that genes at the Ccl17 overexpression increases the number of MCs in chronic contact hypersensitivity, and Ccl17-deficient mice have reduced numbers of MCs in the skin Ccl17 expression was upregulated in MRL/MpJ mice compared to C57BL/6N, and was positively correlated with the expression of the MC marker gene Tpsb2, suggesting Ccl17 as a strong candidate gene at the mcom1 locus.Mt1 through Mt4 was detected in the P0 ovary, but only Mt4 was upregulated in MRL/MpJ mice relative to C57BL/6N, although no correlation was observed between the expression of Mt4 and Tpsb2. Therefore, the overexpression of Mt4 could induce MC migration in an all-or-none rather than a concentration-dependent manner.Metallothioneins (MTs), comprising four subfamilies designated MT1 to MT4 in mammals, have ∼85% homology to CCL17 at the amino acid level, and immune cells migrate chemotactically toward MT1 and MT2 mcom1 and mcom2 would regulate the early follicular development of neonatal mice by altering the appearance of MC.Gene expression profiles in oocytes change dramatically during early follicular development, including immune response genes such as interleukins and chemokines mcom1 and mcom2, respectively. The identification of genes responsible for the appearance of OMCs helps to clarify the mechanisms underlying organ- and age-specific recruitment of MCs, and the role of these cells in early follicular development.In summary, the appearance of OMCs was found to be closely associated with both environmental factors and filial genetic backgrounds. The environmental factors influenced the number but not the distribution of OMCs, but both were influenced by the filial genetic background and regulated by Table S1(DOC)Click here for additional data file.Table S2(DOC)Click here for additional data file.


We report two novel reduced-height maize mutants caused by mutations within a putative inositol polyphosphate 5-phosphatase-encoding gene. Auxin-mediated internode elongation probably requires regulation of inositol and/or phospholipid metabolism. Zea mays L.), as in other grass species, stem elongation occurs during growth and most noticeably upon the transition to flowering. Genes that reduce stem elongation have been important to reduce stem breakage, or lodging. Stem elongation has been mediated by dwarf and brachytic/brevis plant mutants that affect giberellic acid and auxin pathways, respectively. Maize brevis plant1 (bv1) mutants, first identified over 80 years ago, strongly resemble brachytic2 mutants that have shortened internodes, short internode cells, and are deficient in auxin transport. Here, we characterized two novel bv1 maize mutants. We found that an inositol polyphosphate 5-phosphatase orthologue of the rice gene dwarf50 was the molecular basis for the bv1 phenotype, implicating auxin-mediated inositol polyphosphate and/or phosphoinositide signalling in stem elongation. We suggest that auxin-mediated internode elongation involves processes that also contribute to stem gravitropism. Genes misregulated in bv1 mutants included genes important for cell wall synthesis, transmembrane transport, and cytoskeletal function. Mutant and wild-type plants were indistinguishable early in development, responded similarly to changes in light quality, had unaltered flowering times, and had normal flower development. These attributes suggest that breeding could utilize bv1 alleles to increase crop grain yields.In maize ( Zea mays, plant height at maturity largely results from stem elongation  in wheat and dwarf-3 in sorghum have increased plant yields by reducing stem lodging (anther ear1 (an1), dwarf plant1 (d1), dwarf plant2 (d2) dwarf plant3 (d3), dwarf plant5 (d5), dwarf plant8 (D8), and dwarf plant11 (D11) are deficient in gibberellic acid (GA) biosynthesis or signalling. A second class, including brachytic1 (br1), brachytic2 (br2), and brevis plant1  are gibberellin insensitive. GA mutants have stunted growth habits very early in development and the mutations affect organ size and sex determination  encodes an ATP-binding cassette phosphoglycoprotein  light ratios. We found that bv1 encodes an inositol polyphosphate 5-phosphatase (5PTase) with WD40 domains. The two mutant genotypes had non-synonymous substitutions within conserved residues of the inositol phosphatase domain, indicating that inositol polyphosphate and/or phosphoinositide signalling is necessary for auxin-mediated cell elongation. Auxin and phosphoinositide/inositol polyphosphate signalling are known components of the stem gravitropic response. Auxin-responsive gene upregulation within the mutant plants suggested the presence but misdistribution of auxin. In addition, genes involved in the production of cell wall and cytoskeleton proteins were misexpressed in the mutant, suggesting perturbation of vesicular transport. Although we did not find evidence of use of bv1 in the past use in plant breeding, we suggest that allelic variation of the gene may contribute to future plant improvement.Here, we have described two novel br1, br2, and bv1 maize mutants, were identified within two M2 or M3 segregating families originating from the seed of a B73 maternal parent crossed with a B73 paternal parent whose pollen underwent ethyl methanesulphonate (EMS) mutagenesis as described by Two maize plants with reduced height and short internodes, similar to 4NO3, 60g of Micronutrient Mix  and 800g of MgSO4.7H2O dissolved in 20 l of water, and administered in a 100:1 water:solution mix. B73 wild-type and 1302 plants were grown in Turface only (high R:FR) or in Turface covered with grass sod (low R:FR) from germination until anthesis and silking  maize mutants was obtained from The Maize Genetics Cooperation Stock Center located at the University of Illinois Urbana/Champaign . We crossed the 1301 and 1302 mutants to inbred lines A619 and Mo17 to generate F2 mapping populations. Mapping populations were grown in Guelph, Ontario, Canada. In the summer of 2009, a small mapping population consisting of 73 F2 plants of a cross between 1302 and A619 was grown. In the summers of 2010 and 2011, 2122 and 1325 plants of the F2 of a cross between 1301 and the inbred Mo17 were grown in the same location. Finally, in 2012, tissue from 491 individuals of the F2 of 1301×Mo17 with the bv1 phenotype were collected. For each population, the heights of individual plants were scored at maturity and classified as tall or short.Seed of homozygous 2 plants, using either a GeneEluteTM Plant Genomic DNA Mini prep kit  or a CTAB-based protocol adapted in-house for 96-well plate extractions. An initial genotyping of 73 individuals was done at the Analytical Genetics Technology Centre of the Princess Margaret Hospital in Toronto, Canada, using the iPLEX Gold Assay  using 68 polymorphic single-nucleotide polymorphisms (SNPs) between B73 and A619 over the 10 maize chromosomes  markers and SNP markers. SSR markers used included umc1060, umc1966, umc1747, umc1155, umc1822, and bnlg609 from MaizeGDB (http://www.maizegdb.org/ssr.php). PCR for genotyping with SSR markers was performed under the following conditions: 1min of initial denaturation at 95 °C, 35 cycles of 30s denaturation at 95 °C, 30s annealing at 55 °C, and 40s extension at 72°C, followed by 10min of final extension at 72 °C. Additional SNPs  were stored in 1.5ml of RNAlater . At this stage of development for inbred B73, the shoot apical meristem has transitioned into the production of male reproductive structures. Elongation initiates sequentially, with lower internodes initiating growth prior to the upper internodes  of 0.05  as medium and the same nutrients solution as described above. At 14 leaf tips , 40 d after planting, approximately 5mmternodes . InternoTranslated coding sequence similarity searches were performed against the UniProt database  and UniProt .The Integrative Genomics Viewer  was consistent with the 3:1 segregation ratio expected for a monogenic recessive allele . Hybrids between 1301 and 1302 mutants failed to complement, suggesting the same gene in both genotypes caused the mutant phenotype. The progeny of crosses between both mutants (1301 and 1302) and br2 mutant was wild type. The progeny of 1302 and bv1-1 (formerly br3) had reduced height . In addition, in both wild-type and mutant genotypes, leaf emergence occurred more quickly among the plants not exposed to the low R:FR treatment than in those exposed. Beginning 30 d after planting, at approximately the V4–V5 stage, low R:FR-treated plants had up to one less emerged leaf tip than plants not exposed to low R:FR conditions . Thus, the low R:FR treatment-induced increase of plant height and decrease of leaf emergence rate were not bv1 dependent. Consistent with the similar response of wild-type and mutant plants to changes in the R:FR ratio, spraying bv1-1301 mutant plants and wild-type plants with IAA did not alter the plant heights of either genotype (data not shown).Low ratios of red:far-red light and auxin treatment can induce cell expansion . At V14, when the tassel had emerged from the whorl, the maximum internode lengths of bv1-1301 and bv1-1302 mutants were 5.08 and 4.13cm, respectively . In contrast, the maximum B73 internode length was 14.23cm. Lengths of internodes 1 and 2 did not significantly differ . From internodes 3 to 8, the differences between wild-type internodes and mutant internodes increased. Lengths of internodes 8 –14 differed the most between mutant and wild-type plants. The differences varied from 9.2 to 11.9cm for bv1-1301 mutants and from 9.9 to 12.0cm for bv1-1302 mutants . These short internodes resulted in a severely reduced plant height at maturity  (. Bv1-1302 developed one more leaf (P<0.05) than the other genotypes with no significant difference in days to anthesis . These results showed that stem elongation can be independent of plant developmental timing.Flowering time is often highly positively correlated with plant height and leaf number (e.g. (P>0.05) . Bv1-130anthesis ). The nu2 mutants and 3447 mutant and wild-type F2 plants of a bv1-1301×Mo17 cross to map bv1-1301 between nt 160 660 813 and 160 758 241 (~97kb) on the ZmB73 RefGen_v2 pseudochromosome 5 long arm . This map location coincides with the interval on chromosome 5 to which bv1 and is less than 2Mb away from the region in the IBM2 2008 Neighbors map estimated for bv1 . Eight F2 plants had recombinant genotypes at the SNP markers flanking the ~97kb interval . There were five uncharacterized genes in this region (ZmB73_5b_FGS): GRMZM2G012046, AC211269.3_FGP003, GRMZM2G366698, GRMZM2G078272, and GRMZM2G377600. bv1-1301 mutant and Mo17 SNPs within GRMZM2G012046 and GRMZM2G377600 defined the outer boundaries of the ~97kb interval, and we observed recombinants between a SNP position within the gene and the mutant phenotype for both genes  is the closest rice homologue to GRMZM2G366698  and is located on rice chromosome 2: 16 360 196–16 364 326, a region syntenic to maize chromosome 5. Maize genes GRMZM2G012046 and GRMZM2G078272 flanking GRMZM2G366698 also had homologous genes in the same region of rice chromosome 2, Os02g0475300  and Os02g0478550 , respectively. The putative gene spanned ~13kb and had three annotated splice variants, GRMZM2G366698_T01 , GRMZM2G366698_T02 , and GRMZM2G366698_T03 . The putative GRMZM2G366698_P01 protein encoded a putative 5PTase. It contained an inositol polyphosphate phosphatase catalytic domain SMART: SM00128 (bv1 has a similar phenotype to four d50 rice mutants (Supplementary Table S1 at JXB online). Among the eight plants with recombinants within the ~97kb interval , seven wild-type individuals were heterozygous across the locus 1301/Mo17, and the one brachytic mutant was homozygous 1301/1301 for this region. Thus, the height phenotype and GRMZM2G366698 genotype were completely linked across more than 3900 F2 plants. We did not determine whether any of the other four genes within the interval were also completely linked with the bv1 phenotype.We assayed 491 Fth genes . GRMZM2GP>0.05). The mean coverage of each gene’s transcript across genotypes was 15×, 0.51×, and 13×, respectively. Cufflinks predicted six different isoforms of GRMZM2G366698 within B73, Bv-1301, and the 1302 and 1301 bv1 mutants. These were the three splice variants reported in the reference genome annotation and three additional potentially novel splice variants (see Supplementary Tables S2 and S3 at JXB online). Although the isoform with highest abundance differed across genotypes (Supplementary Table S2), these isoform differences are likely stochastic. Across our sample of 22 446 expressed genes (FPKM >1), the most highly expressed isoform differed in 4223 (18.8%).We performed RNA-Seq analyses of RNA harvested from the sixth internodes of wild-type and mutant plants shortly after the shoot had transitioned to generating reproductive tissues. Within the ~97kb interval, transcript abundances were similar between the three mutant and wild-type genes for which we detecteded transcripts  causing an S-to-L change at position 565 of 766 in GRMZM2G366698_P01  transition in exon 4 that caused a putative G-to-R amino acid change at position 367 of GRMZM2G366698_P01 supported by 34 out of 34 RNA-Seq reads for this region (24 unique reads) . Using Sanger sequencing of amplified genomic DNA, we obtained 99% of all intronic sequences of GRMZM2G366698 from bv1-1301 and bv1-1302 plants and found no nucleotide changes relative to the B73 reference genome. We confirmed the bv1-1301 allele through Sanger sequencing, but were unable to amplify GC-rich exon 10, the location of the bv1-1302 mutation, from any genotype. Among the three expressed genes in the ~97kb interval, we identified no other nucleotide differences between bv1-1301, bv1-1302, B73, and Bv1-1302 reads and the B73 reference genome. We concluded that point mutations in GRMZM2G366698 caused the reduced-height phenotypes of bv1-1301 and bv1-1302 mutants.Reads derived from 6698_P01  and 6A. e reads)  and 6B. d50 gene has the highest homology with A. thaliana FRAGILE FIBER3 , a gene necessary for stem mechanical strength and proper cell wall development, we noted that bv1 and d50 were as similar to the putative A. thaliana inositol polyphosphate 5 phosphatase proteins At5PTase12 (At2g43900), At5PTase13 (At1g05630), and At5PTase14 (At2g31830) as they were to FRA3. In protein BLAST searches, over 97% of the bv1 and d50 protein sequences overlapped these putative Arabidopsis proteins, all with over 51% identity. For example, 99% of GRMZM2G366698_P01 aligned with FRA3 with 53% identity, and 97% of GRMZM2G366698_P01 aligned with At5PTase13 with 54% identity. At5PTase12 (At2g43900) and At5PTase13 (At1g05630) exhibited phosphatase activity towards InsP3, while At5PTase14 (At2g31830) and FRA3 exhibited phosphatase activity towards InsP3 and PtdIns but had the highest affinity for PtdInsP2 P2 . The simbv1-1301 and bv1-1302 internode tissue relative to B73 internode tissue, respectively , 42 genes were differentially expressed in the two mutants relative to B73 and were not differentially expressed between a Bv1/Bv1 bv1-1302 sister-line and B73 (see Supplementary Table S5 at JXB online). We assigned putative functions to 16 of these genes (S-transferase (GST 29); and (vii) GRMZM2G04902, an isochorismatase family protein rutB. Genes with lower transcript levels in mutant than in wild-type internode tissue included: (i) GRMZM2G118786, which encodes a putative actin-related protein 2/3 complex subunit 3; and (ii) GRMZM2G103273, which encodes a β-galactosidase. Functional relationships among genes can be inferred from co-expression. We tested for enrichment of the 27 differentially expressed genes  within groups of genes co-expressed across maize development . Because genes that are differentially expressed between wild-type and mutant plants are co-regulated across development, BV1 probably affects a discrete biological process that operates in diverse tissues. Genes within Zm_mod07 are more highly expressed in root tissues of different ages (P=0.0002) and mature leaf (P=0.003) relative to other tissues  GRelopment . Of the bv1-1301 mutant mapped to a five-gene-containing, ~97kb chromosomal region on chromosome 5 . One missense mutation converted serine to leucine in bv1-1301, and a second missense mutation converted glycine to arginine in bv1-1302. Among genes expressed within internodes and that were in the ~97kb interval, we detected no additional nucleotide differences between wild-type and mutant genes. Mutant and wild-type internodes have similar bv1 transcript abundances.We have presented four independent lines of evidence to support GRMZM2G366698 as Bv1/Br3. First, the mosome 5 . Fine mabr2 gene encodes a P glycoprotein auxin transporter, and the mutant is defective in auxin transport P2 levels are reduced. Although the abundance of auxin is unchanged, the distribution and polar transport of auxin are affected , which is one of seven subunits of the actin-related protein complex , which is responsible for the majority of β-galactose activity against xyloglucan. Misexpressed genes encoding membrane-associated transporters include GRMZM2G153920 and GRMZM2G180659. The sorbitol transporter-like protein GRMZM2G153920 is up-regulated in bv1 relative to wild-type internodes. Sorbitol transporter-like proteins can transport pentoses as well as polyols and different hexoses  identified in mapping studies. In a segregating population derived from crosses between an Illinois High Oil population and an Illinois Low Oil population, 2 of inbred lines K05×W65 that maps close to bv1. Nonetheless, in an association mapping panel, Pfeiffer et al. (2014) found little correlation between bv1 alleles and plant height. Similarly, SNP variation flanking the sorghum gene model homologous to bv1, Sobic.004G145500, was uncorrelated with plant height variation in a set of 377 diverse sorghum accessions . Although maize height and flowering time are consistently genetically correlated  in contrast to other reduced-height maize mutants such as anther ear1 (an1), dwarf plant1 (d1), dwarf plant3 (d3), dwarf plant5 (d5), dwarf plant8 (d8) and dwarf plant11 (d11)  and B73 (wt) plants grown with low R:FR reflected light (surrounding turfgrass) and high R:FR reflected light (no turfgrass) at different times after planting.Fig. S2. Number of visible leaf tips of bv1-1302 (bv1) and B73 (wt) plants grown with low R:FR reflected light (turfgrass) and high R:FR reflected light (no turfgrass).Fig. S3. Internode lengths of field grown bv1-1301, bv1-1302 and B73 plants measured 66 d after planting.Fig. S4. Genotypes of plants with crossovers that mapped the bv1 mutation to a ~97kb region on chromosome 5 (5:160 660 813–160 758 241).Fig. S5. Multiple sequence alignment of maize GRMZM 2G366698_P01 subsequence and 20 homologous proteins across diverse plant species showing amino acid changes (in red rectangle) caused by the mutations in bv1-1301 (A) and bv1-1302 (B).Table S1. Markers designed.Table S2. Transcript abundance of GRMZM2G366698 across sample pools.Table S3. Exon coding regions for the three potentially novel splice variants identified in the RNA-Seq reads.Table S4. Number of differentially expressed genes between mutants bv1-1301 and bv1-1302 and wild-type genotypes Bv1-1302 and B73.Table S5. All 68 genes differentially expressed in bv1-1301 and bv1-1302 relative to B73 .