Caenorhabditis elegans. It is also important to note that several phytochemicals with higher hydrophobicity or electrophilicity, including phenethyl isothiocyanate and curcumin, markedly induced heat shock proteins, whereas most of the tested nutrients did not. These results suggest that non-specific protein modifications by xenobiotic phytochemicals cause mild proteostress, thereby inducing heat shock response and leading to potentiation of protein quality control systems. We considered these bioactivities to be xenohormesis, an adaptation mechanism against xenobiotic chemical stresses. Heat shock response by phytochemicals may be a fundamental mechanism underlying their various bioactivities.Accumulated evidence shows that some phytochemicals provide beneficial effects for human health. Recently, a number of mechanistic studies have revealed that direct interactions between phytochemicals and functional proteins play significant roles in exhibiting their bioactivities. However, their binding selectivities to biological molecules are considered to be lower due to their small and simple structures. In this study, we found that zerumbone, a bioactive sesquiterpene, binds to numerous proteins with little selectivity. Similar to heat-denatured proteins, zerumbone-modified proteins were recognized by heat shock protein 90, a constitutive molecular chaperone, leading to heat shock factor 1-dependent heat shock protein induction in hepa1c1c7 mouse hepatoma cells. Furthermore, oral administration of this phytochemical up-regulated heat shock protein expressions in the livers of Sprague-Dawley rats. Interestingly, pretreatment with zerumbone conferred a thermoresistant phenotype to hepa1c1c7 cells as well as to the nematode  Interestingly, SFN is a potent inducer of phase II detoxification enzymes, which possess anti-oxidative propertieset al.All living organisms have substantial biological abilities to increase their resistance to various environmental stresses by inducing adaptation mechanisms. For example, plants biosynthesize polyphenols as antioxidants in response to UV light to mitigate oxidative damage, while mammals possess innate and adaptive immune systems for protection from xenobiotic stresses and microbial infections. In these phenomena, moderate stresses serve as driving forces to increase stress resistance, which is termed ‘hormesis,’ for beneficial effects provided by mild biological stress through adaptation mechanismset al. identified a 67-kDa laminin receptor as a receptor for (-)-epigallocatechin-3-gallate (EGCG), a major green tea polyphenol with chemopreventive potentiale.g., heat shock) are known to induce molecular chaperones including heat shock proteins (HSPs) as the major machinery of the protein quality control systemMeanwhile, recent studies have revealed that phytochemicals specifically bind to proteins. In 2004, Tachibana Zingiber zerumbet Smith (Zingiberaceae) (via Michael additionWe previously reported that zerumbone (ZER), a sesquiterpene present in eraceae) , exhibitCaenorhabditis. elegans (C.elegans). In addition, some phytochemicals, but not plant nutrients, were found to be notable HSP inducers. Based on our findings, we propose a hypothesis that non-specific bindings of phytochemicals induce activation of protein quality control systems, resulting in stress-resistant phenotypes.In the present study, we found that ZER binds to numerous proteins in hepa1c1c7 mouse hepatoma cells with low selectivity. ZER-modified proteins were recognized by HSP90 for inducing the expressions of multiple HSPs. Oral administration of ZER led to modifications of biomolecules and HSP70 induction in the livers of Sprague-Dawley (SD) rats, while ZER pretreatment conferred thermoresistance to hepa1c1c7 and ad libitum, both of which were freshly exchanged twice a week.Female BALB/c mice, 6 weeks of age, were purchased from Japan SLC . All mice were quarantined for more than 1 week before starting the experiments, then kept under specific-pathogen free conditions. Mice were maintained in a controlled room at 24±2°C with a relative humidity of 60±5% and a 12-hour light/dark cycle. They were given commercial rodent pellets  and fresh tap water Male SD rats were purchased from Japan SLC at 8 weeks of age and housed in a temperature-controlled (23±2°C) room for at least 2 weeks before beginning the present experiments. Rats were allowed free access to MF pellets and water. All rat experiments were done with male rats. The care and handling of the rats was in accordance with the Guidelines for Animal Care and Use of Otsuka Pharmaceutical Co., Ltd. The experimental protocol using rats have been approved by the animal experiment ethics committee of Otsuka Pharmaceutical Co., Ltd. (#2011-04).C. elegans culturing methodEscherichia coli OP50 at 25°C. All animal experiments using mice and nematodes were performed according to the Guidelines for the Regulation of Animals, as provided by the Experimentation Committee of Kyoto University, which approved the protocol of the present animal experiments (#21-44).Wild-type N2 nematodes were cultured using a standard 2.Hepa1c1c7, the hepatoma cells of mouse originN-hydroxysuccinimide (NHS) were obtained from Pierce . DMEM and FBS were from Gibco BRL . Lipopolysaccharide  was purchased from Difco Labs . Antibodies (Abs) were obtained from the following sources: mouse anti-HSP90, mouse anti-HSP70, and rabbit anti-HSP40 Abs were from Enzo Biochem Inc. ; mouse anti-α-tubulin Ab was from EMD Bioscience ; goat anti-β-actin Ab was from Santa Cruz Biotechnology ; rabbit anti-HSF1 and anti-rabbit Ab HRP-linked IgG Abs were from Cell Signaling Technology ; and HRP-conjugated anti-mouse, HRP-conjugated anti-goat, and FITC-conjugated anti-mouse Abs were from DAKO . HRP-conjugated Streptavidin was purchased from Thermo Scientific . Bio-Adembeads Protein G was obtained from Ademtech SA . Non-specific control small interfering RNA (siRNA) was purchased from Santa Cruz Biotechnology, and HSF1 siRNA and LipofectamineTM 2000 were from Invitrogen . All other chemicals were purchased from Wako Pure Chemical Industries , unless otherwise specified.Zerumbone (ZER) was purified as previously reported (purity >95%)N-bromosuccinimide (1.1 eq) in 50% aqueous acetonitrile system at room temperature for 1 minute. 7-Bromo-2,9,9-trimethyl-6-methylenecycloundeca-2,10-dienone (BrZER) was purified by filtration. BrZER (50 mg) was reacted with glycolic acid (1.5 eq) and silver (I) oxide (2 eq) in diethyl ether at room temperature for 9 hours. The hydroxyl derivative of ZER was purified using thin-layer chromatography  with TLC silica gel and a 60 F254 aluminium sheet . Subsequently, it was reacted with succinic anhydride (2 eq) in pyridine at room temperature for 40 hours to yield the succinylated ZER derivative (SucZER), which was purified on TLC (ethyl acetate: n-hexane = 2:1). Alternatively, ZER (250 mg) was reacted with N-acetyl-L-cysteine (2 eq) in 1N HCl and 50% aqueous acetonitrile at room temperature overnight. NacZER was purified on TLC (chloroform: methanol = 9:1) and subjected to a reverse phase high-performance liquid chromatography (HPLC) device equipped with a YMC-pack ODS-AQ . HPLC was performed with 50% aqueous methanol containing 0.5% acetic acid as an eluent at a flow rate of 3 mL/minute with the UV detector set at 254 nm. Identification of the derivatives was performed by 1H-nuclear magnetic resonance (NMR) analysis using an ARX500  in CDCl3. SucZER and NacZER were separately coupled with carrier proteins using a carbodiimide procedureZER (1 g) was reacted with The obtained hapten-KLH conjugates (0.6 mg/mL in PBS) were emulsified with an equal volume of adjuvant. Six-week-old female BALB/c mice were separately immunized intraperitoneally with these emulsions (100 µL) and repeatedly boosted with immunogens (0.2 mg/mL) emulsified with an equal amount of adjuvant every 2 weeks. For the final boost, 100 µL of immunogens (0.5 mg/mL) without the adjuvant was intraperitoneally injected. Three days after the final boost, the mice were euthanized and serum samples were obtained. The antibodies were purified using a Melon™ Gel IgG Spin Purification Kit .2SO4 solution, binding of the antibody to the antigen was evaluated by measuring optical density at 450 nm.Native BSA and each hapten-BSA conjugate (5 µg/mL) in PBS were used to coat in 96-well plates, then incubated at 37°C for 1 hour. After washing 3 times with PBS containing 0.05% Tween 20 (TPBS), the wells were blocked with a 2% aqueous solution of Block Ace  at 37°C for 1 hour. After washing, ZER and thioether ZER Abs  in TPBS were added, and the wells were incubated at 37°C for 2 hours. After washing, HRP-conjugated anti-mouse IgG (1∶5000) was added and incubated at 37°C for 1 hour. The color developing reaction was performed by addition of TMB substrate solution . After stopping the reaction with 2N HB/B0, in which B is the amount of antibody bound to the coating antigen in the presence of a competitor and B0 is that in the absence of a competitor.For competitive ELISA, primary Abs were added to antigen-coated wells in the presence or absence of competitors. The competitive reactions were performed in PBS containing 0.5% DMSO at 37 °C for 90 minutes. Cross-reactivity of the Abs to the competitors was expressed as 4/200 µL) were treated with ZER  for 30 minutes. After washing, the cells were fixed for 20 minutes in PBS containing 4% paraformaldehyde. The membranes were permeabilized by exposing fixed cells to PBS containing 0.1% Triton X-100 for 30 minutes. The cells were then incubated in PBS containing 2% Block Ace for 1 hour and immunostained with ZER or thioether ZER Ab (1∶10) at 4°C overnight. They were then treated with FITC-conjugated anti-mouse Ab (1∶20) at room temperature for 1 hour and mounted with 50% glycerol in PBS. Images of cellular immunofluorescence were acquired using a MicroRadiance Confocal Laser Scanning microscope (Bio-Rad Laboratories). Propidium iodide (PI) (1 µg/mL) and RNase (100 µg/mL) were added to a secondary Ab solution for nuclear staining. Each competitor was added to the primary Ab solution and competitive assays were performed in PBS containing 0.5% DMSO.Hepa1c1c7 cells (2×105/mL) were seeded into 24-well culture plates, and treated with the sample or vehicle  for various times. Next, they were washed with PBS twice and lysed in lysis buffer , and centrifuged at 6000×g for 5 minutes. Denatured proteins were separated using SDS-polyacrylamide gel electrophoresis (PAGE) on 5% or 10% polyacrylamide gels, then transferred to Immobilon-P membranes . After blocking with PBS containing 2% Block Ace for 1 hour, each membrane was treated with an appropriate specific primary Ab (1∶2000 or 200), followed by the corresponding HRP-conjugated secondary Ab (1∶2000). To detect BioZER-modified proteins, the membrane was treated with HRP-conjugated Streptavidin (1∶10000). The blots were developed using ECL Western blot detection reagents .Hepa1c1c7 cells (1×105/mL) were seeded into 24-well plates and pretreated with BioZER  for 30 minutes at 37°C, followed by exposure to LPS (100 ng/mL) for 24 hours. Each supernatant (100 µL) was mixed with 100 µL of Griess reagent [1% sulfanilamide in 5% phosphoric acid and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in distilled water]. Absorbance was measured at 540 nm.NO production was determined based on the amount of nitrite, a stable end-product of NO. Nitrite concentrations in media supernatants were determined by use of a Griess reaction6/mL) were seeded into 60-mm dishes and washed with PBS twice, then lysed in native lysis buffer . Cell lysates were incubated with ZER (500 µM) for 8 hours, then incubated with non-treated lysates for 0, 2, 5, and 30 minutes. To immobilize generated protein complexes, the mixed lysates were treated with 0.05% glutaraldehyde in native lysis buffer and subjected to SDS-PAGE with 5% polyacrylamide gel for western blot analysis. Lysates pretreated with glutaraldehyde were used as a negative control and the lysates exposed to heat shock (incubation in 43°C waterbath) before glutaraldehyde treatment were used as a positive control.Hepa1c1c7 cells (1×106/10 mL) were seeded into 100-mm dishes and washed with PBS twice, then lysed in lysis buffer for immunoprecipitation . Cell lysates were treated with ZER  for 8 hours and incubated with 0.2% anti-HSP90 Ab in the presence of Bio-Adembeads Protein G for 1 hour. The beads were washed twice with washing buffer  and the immunoprecipitates were eluted using elution buffer .Hepa1c1c7 cells (2×106/5 mL) were seeded into 60-mm dishes and treated with ZER  for 1 or 3 hours. After washing with PBS twice, cells were lysed, and lysates were subjected to a pSer326 HSF1 EIA Kit according to the manufacturer's instructions . Cells exposed to heat shock (incubation at 43°C in waterbath) for 15 minutes were used as a positive control.Hepa1c1c7 cells (1×105/mL) were seeded into 24-well culture plates and treated with a sample or the vehicle  for various times. Alternatively, D3 larvae nematodes were treated with ZER  for 62 hours or exposed to mild heat  in liquid NGM medium. Total RNA was isolated from cells and C. elegans using TRIzol reagent , according to the manufacturer's specifications. The amount and purity of RNA were assessed by spectrophotometry using a SmartSpec® 3000 Spectrophotometer (Bio-Rad Laboratories). cDNA was synthesized using 1 µg of total RNA with an RNA PCR Kit (AMV). Thermal cycling was performed with a 7300 real time PCR system using SYBR green PCR mix , according to the manufacturer's protocol. PCR conditions were as follows: 95°C for 3 minutes, 95°C for 10 seconds, and 60°C for 1 minute (mouse); 50°C for 2 minutes, 95°C for 2 minutes, 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds (C. elegans). The primers and sequences used are summarized in Hepa1c1c7 cells (1×104/mL) were seeded into 24-well plates. An siRNA solution  was added to 50 µL of serum-free Opti-MEM I® . In another microtube, 2 µl of Lipofectamine™ 2000 was diluted in 50 µL of serum-free Opti-MEM I®. After adding the siRNA solution to the Lipofectamine™ 2000 solution, the transfection mixture was incubated for 25 minutes. This transfection mixture was then diluted in 500 µL of serum-free Opti-MEM I®, which was added to wells and cells were incubated for 6 hours. After replacing the medium with DMEM containing 10% FBS, the cells were incubated for another 24 hours. After washing twice with PBS, the cells were lysed and the lysates were subjected to western blot analysis, as described above.Transfection of siRNA was performed using Lipofectamine™ 2000, according to the manufacturer's specifications. Briefly, 24 hours before transfection, cells (1.7×105/mL) were seeded into 24-well plates and pretreated with ZER  for 24 hours at 37°C, or exposed to mild heat . Next, they were exposed to heat shock at 45°C in a waterbath for 1 hour. After recovery for 12 hours, cell viability was determined using a Cell Counting Kit-8 . Briefly, after washing with PBS twice, cells were incubated in DMEM containing 5% WST for 1 hour and absorbance of the cell culture medium was measured at 450 nm. Values for cell viability of the positive control cells, which were not exposed to heat shock, were standardized as 100%.Hepa1c1c7 cells (1×10D3 larvae nematodes (10 worms/200 µL) were treated with ZER  for 3 days or exposed to mild heat  in liquid NGM medium. Dead and alive nematodes were counted following exposure to heat shock at 37°C in a waterbath for 1 hour. The survival rate of the positive control nematodes, which were not exposed to heat shock, was standardized as 100%.Twenty-four male SD rats (8 weeks old) were divided into control and experimental groups. Rats were orally administered ZER  in corn oil twice a day for 1 week, then euthanized and the livers were collected. Livers were lysed in PBS or lysis buffer, and the lysates were subjected to competitive ELISA with thioether ZER Ab to determine the concentration of ZER-thiol adducts, or western blot analysis.t-test,Each experiment was performed at least 3 times and values are shown as the mean ± SD where applicable. Statistically significant differences between groups in each assay were determined using a Tukey-Kramer test, Dunnett's test, and Student's in vitro assay results (N-acetyl-L-cysteine (NAC) or ZER  or ZER . Next, h) or ZER . Co-trea) or ZER . To veri) or ZER . It is a) or ZER .50 value of BioZER (9.1 βM) was comparable to that of ZER (12 µM), showing that BioZER can serve as a biological ZER mimic. We then attempted to detect BioZER-bound proteins in lysates from hepa1c1c7 cells by western blotting using HRP-conjugated avidin. Exposure of those cells to BioZER (10 µM) resulted in dramatic formation of BioZER-bound proteins  to confirm its non-selective protein modifications . This chproteins , which dproteins . Thus, Bproteins , which iin vitro assays. When a non-treated hepa1c1c7 cell lysate was added to another lysate containing ZER-modified proteins, the amount of non-bound HSP90 protein was dramatically decreased after 10 and 20 minutes, with a more distinct decrease seen with lysates immediately exposed to heat shock at 43°C  and zinc chloride showed potent activities (Finally, we evaluated the HSP70 inducing activities of various natural compounds including plant primary and secondary metabolites to examine whether this activity is limited to that seen with ZER. Hepa1c1c7 cells were exposed to each compound at nonlethal, maximum concentrations for 6 hours, then HSP70 mRNA expression was determined by qRT-PCR. Among the tested nutrients, only all-tivities , implyintivities . It is iIn the present study, we attempted to uncover the biological consequences of non-specific protein modifications by phytochemicals. We anticipated that ZER, an electrophilic small molecule, would have less binding selectivity to cellular nucleophiles, which was shown by findings regarding its non-selective protein modifications , 2C. On in vitro, while it was bound to numerous cellular proteins with less selectivityHSPs participate in the protein quality control system by binding to hydrophobic regions in denatured proteins, which contributes to prevention of their aggregation and repairs their unfolding structures. HSP90, a major constitutive isoform, is known to repress the expression of inducible HSPs by sequestering HSF1 under a homeostatic conditionC. elegans (et al. showed that HSP expression levels were related to this phenotype in H35 rat hepatoma cellset al. also demonstrated that proteolysis activity in the nucleus is lower than that in cytoplasmWe demonstrated that ZER confers a thermoresistant phenotype to hepa1c1c7 cells and  elegans . ImportaThe present is the first study to widely screen plant food ingredients for their ability to induce HSP70. Our screening test results strongly support our hypothesis that xenobiotic phytochemicals have potential for inducing HSR by providing mild proteostress. Plant secondary metabolites with higher hydrophobicity or electrophilicity, such as phenethyl isothiocyanate, α-humulene, curcumin, and ursolic acid, showed potent HSP70 inducing activities, whereas all of the examined polyphenols were virtually inactive. These contrasting results support the notion that both electrophilicity and hydrophobicity are fundamental properties of phytochemicals to induce HSR presumably through non-specific protein interactions. Considering that HSR is induced by HSP90 recognition of hydrophobic surfaces exposed in denatured proteins, hydrophobicity of phytochemicals may be a crucial structural determinant for their HSP70 inducing activities.et al., who showed that it activated HSF1 in FM3A cellsConsistent with our present results, EGCG and quercetin were shown to suppress HSP70 expression in other cell lineset al. reported an interesting finding that sodium azide, a toxic compound used as antiseptic agent, induced HSP expression and conferred thermoresistance to C. elegans at low, but not high, dosesHormesis is a beneficial effect provided by mild biological stress through adaptation mechanismsIn conclusion, our findings show activation of a protein quality control system by non-specific chemical protein modifications. ZER bound to numerous proteins in both cultured hepatocytes and rat livers, and those modified proteins were recognized by HSP90 for HSR induction . Importa


Drosophila visual system as an in vivo model to examine how the toxicity of Tau varies with phosphorylation status. This system relies on overexpression of Tau from transgenes but is susceptible to position effects altering expression and activity of the transgenes. We have refined the system by eliminating position effects through the use of site-specific integration. By standardising Tau expression levels we have been able to compare directly the toxicity of different isoforms of Tau and Tau point mutants that abolish important phosphorylation events. We have also examined the importance of human kinases in modulating Tau toxicity in vivo. We were able to confirm that human GSK3β phosphorylates Tau and increases toxicity but, unexpectedly, we identified that preventing phosphorylation of Ser404 is a protective event. When phosphorylation at this site is prevented, Tau toxicity in the Drosophila visual system is increased in the presence of GSK3β. Our data suggest that not all phosphorylation events on Tau are associated with toxicity.Hyperphosphorylation of the microtubule associated protein, Tau, is the hallmark of a group of neurodegenerative disorders known as the tauopathies which includes Alzheimer's disease. Precisely how and why Tau phosphorylation is increased in disease is not fully understood, nor how individual sites modify Tau function. Several groups have used the  The tauopathies are a group of neurodegenerative diseases characterised by the accumulation of intra-neural aggregates of the microtubule-associated protein, Tau. They include Alzheimer's disease (AD), fronto-temporal lobar degeneration (FTLD-Tau), Pick's disease, progressive supranuclear palsy and corticobasal degeneration. The microtubule-binding properties of Tau were identified more than 30 years ago but, and despite intense research, its true physiological role remains unclear, thus the cellular mechanisms underpinning the tauopathies are also poorly understood. More recently, additional properties of Tau have been identified, including an ability to bundle actin filaments, and multiple Tau-binding partners have been identified, including tyrosine kinases. Together, these have led to the suggestion that Tau may act as a signalling scaffold  and through a multitude of post-translational modifications. These include multiple phosphorylation and acetylation events, glycosylation, ubiquitylation and sumolyation amongst others . Four of the five lines  were homozygous viable insertions and none of these showed an eye phenotype in the absence of the GMR-GAL4 driver. Line 3 exhibited the most severe degeneration . We concluded that the Tau-mediated degeneration is highly dependent on positional effects and we would need to control for these if we were going to compare the toxicity of different forms of Tau.We identified the genomic insertion site of UAS-Tau for each line . We next generated four UAS-hGSK3β transgenic lines by random insertion and found each line was expressed to varying amounts (Drosophila heads and found no change in the ratio of soluble to insoluble Tau in these fractions when hGSK3β was co-expressed together with Tau in Drosophila (In order to examine whether human GSK3β (hGSK3β) could also enhance Tau-mediated toxicity in  amounts  yet expr amounts . When hG amounts . The deg amounts . hGSK3β  amounts . Sarcosy amounts . We extrosophila .Drosophila expressing the 2N4RTau transgene inserted into the 68A landing site  recognise known GSK3β target sites in Tau , whereas labelling by antibody AT100 requires phosphorylation of both Thr212 and Ser214 by GSK3β in combination with another kinase . Solubleing site . We founxpressed ; PHF1. Rxpressed ; AT270. xpressed ; AT8. Phxpressed ; AT100. in vitro and acts as a priming kinase for GSK3β .Our failure to detect phosphorylation of Thr212/Ser214 by hGSK3β to generate the AT100 epitope may indicate a requirement for priming at adjacent upstream Ser residues by an additional kinase . One canor GSK3β . The DYRor GSK3β . We genein vivo are essential for increased toxicity, we replaced the individual residues, Ser202, Ser205, Thr212 or Ser404 in 2N4R Tau with non-phosphorylatable alanine. In a fifth mutant, all four of these residues were substituted by alanine (termed Tau4xA). We examined the effect of these mutations on the ability of hGSK3β to phosphorylate 2N4R Tau in Drosophila cells. When 2N4R Tau is expressed in cells, multiple Tau bands were detected using a phospho-independent antibody. These likely correspond to different phosphorylated forms mediated by endogenous kinases. Co-expression of hGSK3β resulted in a retardation of the Tau bands in the gel, indicating increased phosphorylation , with or without a point mutation, R406W, which is found in some autosomal dominant forms of FTLD-Tau . Previoutoxicity . We confransgene . Howeverransgene , the R40s of Tau . In an iotubules  and an i oocytes , Tau R40 oocytes .in vitro but it is not yet clear how each phosphorylation event contributes to Tau toxicity . Details of genetic markers and balancer chromosome are described at Flybase (http://flybase.org). GMR-GAL4 was used to drive expression of UAS-Tau and UAS-kinases in the visual system as previously described  or GenetiVision . All transgenic flies produced were confirmed by sequencing and Splinkerette PCR was used to identify genomic DNA flanking regions . The conescribed .Full length open reading frames (ORFs) of cDNAs for Tau and each kinase were amplified with proof-reading Taq polymerase, cloned into pENTR (Invitrogen) and verified by sequencing. Constructs were recombined into the Murphy collection of Destination vectors supplied by the Drosophila Genomic Resource Centre . The kinases were epitope-tagged with Myc or Flag sequences, while Tau was not tagged.The ORF of 2N4R human Tau cloned into pTW was mutated using the QuikChange Multi kit (Stratagene) and confirmed by sequencing.TM Reverse Transcription System (Promega) following the manufacturer's instructions. 500 ng RNA was used per reaction.RNA was extracted from 5–10 adult fly heads using Tri Reagent (Sigma) and used immediately for cDNA synthesis with the ImProm-IIhttp://www.qstandard.co.uk). Transcript levels for the following genes were quantified: Drosophila actin5c (CG4027), Drosophila GAPDH2 (CG8893), Drosophila EIF-4a (CG9075), mouse CD8a (geneID: 12525), human Tau (geneID: 4137).Quantitative PCR was performed by QStandard  to decapitate. Fly heads were separated from thoraces and abdomens by shaking through a fine sieve.A two-stage, neutral-alkaline extraction procedure was used to maximise recovery of Tau protein. Fly heads were homogenised (20 heads/100 µl) in 100 µl ice-cold homogenisation buffer , pH 6.8, and garnet beads in a FastPrep24 homogeniser . Heads were homogenised twice at 6.5 m/s for 20 s and centrifuged at 20,000 g for 10 min at 4°C. The supernatant was removed and stored on ice. A second homogenisation was then performed by adding 100 µl of ice-cold homogenisation buffer at pH 9.2 to the pellet and centrifuging as above. The supernatants were combined, the pH adjusted to 8.0 and then centrifuged at 20,000 g for 30 min at 4°C. The final supernatant was stored at −80°C.Ice-cold Tris buffer  was added to the fly heads (30 heads/60 µl buffer) and garnet beads. Fly heads were homogenised three times at 6.5 m/s for 20 s as above, then centrifuged at 1,000 g for 5 min at 4°C. The supernatant was removed and stored at −80°C and referred to as the sarcosyl-soluble fraction. N-lauroylsarcosinate (Sigma) was added to the pellet to give a final concentration of 1% (w/v) and the suspension was incubated for 1 h at ambient room temperature with gentle shaking. The suspension was centrifuged at 100,000 g for 1 h at 4°C. The insoluble pellet was resuspended in 50 mM Tris-HCl, pH 7.5, stored at −80°C and referred to as the sarcosyl-insoluble fraction.SDS-PAGE, Western blotting, membrane blocking and probing were all performed by standard protocols. The membrane used was supported nitrocellulose (BioRad) which was blocked for 1 h in 5% milk in Tris-buffered saline (TBS) without Tween-20. Antibodies were diluted in 5% milk in TBS, 0.1% Tween-20 (TBST). Blots were washed with TBST, followed by two washes in TBS without Tween prior to imaging.Primary antibodies raised against: total human Tau (phosphoinsensitive), used at 1/10,000 (Dako); PHF1 human Tau pS396/pS404, used at 1/5,000 ; AT8 human Tau pS202/pT205 at 1/2,000 (Innogenetics); Tau1 human Tau 197–208 at 1/1,000 (Chemicon); AT100 human Tau pT212/pS214 at 1/1,000 (Innogenetics); AT270 human Tau pT181 at 1/1,000 (Innogenetics); 9E10 c-Myc at 1/1,000 (Santa Cruz); M2 Flag-tag at 1/1,000 (Sigma); β-actin at 1/5,000 . Secondary antibodies: Alexa-680 mouse anti-IgG at 1/15,000 (Invitrogen); Alexa-800 mouse anti-IgM at 1/15,000 (Rockland); Alexa-800 rabbit anti-IgG at 1/15,000 (Rockland).Membranes were scanned and quantified using an Odyssey infra-red scanner (Licor Biosciences) and Prism 5 software (GraphPad).A constant amount of recombinant Tau was included in an individual lane on each Tau protein gel to act as a normalisation control.Statistical analysis was performed using Prism 5 (GraphPad). Transcript and protein quantification data were analysed for significance by one-way ANOVA with Bonferroni correction. Resulting p values lower than 0.05 were deemed significant.


A total of 66 RTT girls were evaluated, half of whom being treated for 12 months with a dietary supplementation of ω-3 PUFAs at high dosage (docosahexaenoic acid ~71.9 ± 13.9 mg/kg b.w./day plus eicosapentaenoic acid ~115.5 ± 22.4 mg/kg b.w./day) versus the remaining half untreated population. Echocardiographic systolic longitudinal parameters of both ventricles, but not biventricular diastolic measures, improved following ω-3 PUFAs supplementation, with a parallel decrease in the OS markers levels. No significant changes in the examined echocardiographic parameters nor in the OS markers were detectable in the untreated RTT population. Our data indicate that ω-3 PUFAs are able to improve the biventricular myocardial systolic function in RTT and that this functional gain is partially mediated through a regulation of the redox balance.Rett syndrome (RTT) is a devastating neurodevelopmental disorder with a 300-fold increased risk rate for sudden cardiac death. A subclinical myocardial biventricular dysfunction has been recently reported in RTT by our group and found to be associated with an enhanced oxidative stress (OS) status. Here, we tested the effects of the naturally occurring antioxidants  Rett syndrome (RTT) is a genetically determined, neurodevelopmental disorder with autistic features , 2. Althde novo mutation in the X-linked gene encoding MeCP2, a protein known to either activate or repress several transcriptional genes . RT. RTMeCP2n = 33 treated, mean age at supplementation time zero: 13.0 ± 8.6 years; n = 33 untreated, mean age at time zero: 12.4 ± 9.3 years) to either oral supplementation with ω-3 PUFAs oil for twelve months or no treatment.The experimental design was single centre, single blind, and randomized. Patients were randomized at admission  at a dose of 5 mL twice daily, corresponding to docosahexaenoic acid (DHA) 71.9 ± 13.9 mg/kg b.w./day and eicosapentaenoic acid (EPA) 115.5 ± 22.4 mg/kg b.w./day, with a total ω-3 PUFAs 242.4 ± 47.1 mg/kg b.w./day. Use of EPA plus DHA in RTT was approved by the AOUS Ethical Committee.Administered All the subjects, included patients, examined in this study were following a standard Mediterranean diet.E wave, A wave, and E/A ratio) were recorded using pulsed wave (PW) Doppler on the mitral valve. The evaluation of left and right ventricular longitudinal systolic function was performed by (a) mitral annular plane systolic excursion (MAPSE) and tricuspid annular plane systolic excursion (TAPSE), using M-mode, and (b) systolic (S′) and early diastolic (E′) peak velocities, using PW tissue Doppler imaging (TDI) of the lateral (lat) and septal (sep) mitral annulus for left ventricle (LV) and of tricuspidal annulus for right ventricle (RV) in four-chamber apical view. The E/E′lat ratios were determined as surrogate of LV filling pressures.The study was performed using a commercially available echocardiography equipment  as previously reported . BrieflyIn order to reduce operator-dependent bias, all measures were performed by two operators, blinded for clinical and therapeutical data of RTT group.ω-3 PUFAs treated group, blood sampling was performed the day before starting the supplementation and the day after the end of the selected 12-month period.Blood sampling was carried out in all subjects at around 8.00 am after the overnight fast. For the 4, 32 mM HEPES, 5 mM glucose, 1 mM CaCl2), pH 7.4 as a 50% (vol/vol) suspension, and then used for the determination of erythrocyte non protein-bound iron (NPBI).Blood was collected in heparinized tubes, and all manipulations were carried out within 2 h after collection. Blood samples were centrifuged at 2,400 g for 15 min at room temperature. The platelet poor plasma was saved, and the buffy coat was removed by aspiration. The erythrocytes were washed twice with physiological solution, resuspended in ringer solution , 4-hydroxynonenal protein adducts (4-HNE PAs), and NPBI determinations. For all isoprostane determinations, butylated hydroxytoluene (BHT) (90 μM) was added to plasma as an antioxidant and stored under nitrogen at −70°C until analysis.Plasma was used for free isoprostanes  analysis after solid-phase extraction and derivatization steps − precursor ions (m/z 569 and m/z 573) produced from 15-F2t-IsoPs and PGFα2-d4, respectively −precursor ions (m/z 593 and m/z 573) produced from oxidized DHA and the PGFα2-d4, respectively ..ω-3 PUFAω-3 PUFAs (EPA plus DHA) supplementation, significant improvements in systolic longitudinal parameters of both ventricles were observed .ω-3 PUFAs-supplemented group, clinical severity decreased to 25.5 and 30.1% at 6 and 12 months, respectively . Conversely, no significant differences in clinical severity were observed in the unsupplemented group of patients . Significant improvements were observed in the areas of attention, breathing abnormalities, muscular tone, ambulation, autonomic dysfunction, and somatic growth.In the ω-3 PUFAs dietary supplementation. Specifically, ω-3 PUFAs appear to reverse all the examined longitudinal systolic parameters  of the left and right ventricles. Moreover, the improvement in the systolic myocardial function was found to be associated with a marked decrease of OS markers as determined in plasma, whereas no significant changes in the diastolic function were detectable in ω-3 PUFAs-treated patients. Taken as a whole, these findings suggest that OS may play a key role in the systolic performance of the RTT myocardium and that it can be at least partially rescued by ω-3 PUFAs dietary supplementation.Our findings indicate, for the first time, that the subclinical myocardial dysfunction observed in typical RTT can be, at least partially, rescued by 1-year high-dose ω-3 PUFAs action there are changes in membrane structures and gene expression, direct interactions with ion channels, and alterations in eicosanoid biosynthesis [To date, among the molecular mechanisms potentially underlying the ynthesis . In partynthesis  which coynthesis .ω-3 PUFAs in RTT is that the increased isoprostanes levels in RTT are not simply the effect of the peroxidation of the PUFAs precursors following the attack by radical oxygen species (ROS), but rather the effect of a potential dysregulation of the molecular targets of ω-3 PUFAs. Contrary to expectations, the assumed fatty acids are not further oxidized, while the actual endogenous IsoPs production is reduced (the “fatty acid paradox”) together with amelioration of the clinical disease severity [Our current working hypothesis on the beneficial effects of severity .ω-6 and ω-3 PUFAs would actually imply an excessive consumption of these PUFAs in the cell membranes, thus paving the way for a new perspective on the nutritional horizons in RTT. As RTT girls appear to chronically suffer from oxidation of PUFAs, either ω-3  or ω-6 , but, at the same time, benefit from ω-3 PUFAs supplementation, it can be inferred that these patients would need ω-3 PUFAs replacement as a consequence of a persistent PUFAs oxidation within the chronic OS context. On the other hand, it is also possible that, in RTT, the endogenous PUFAs are, for their own nature, more susceptible to the OS as compared to the exogenous ones. Therefore, administered PUFAs may be seen as counteracting this intrinsic defect.Conceivably, an excess of peroxidation end products from ω-3 PUFAs supplementation, as fish oil, in mice with nonalcoholic fatty liver disease is able to prevent hepatic lipid accumulation and improve lipid metabolism without causing oxidative stress [Further research is needed to explore this point, although a very interesting recent report indicates that e stress . This reA further critical new piece of research indicates that cholesterol synthesis is impaired in a mutant mouse model in RTT . This laω-3 PUFAs (or their secondary metabolites) could be related to the modulation of an unrecognized subclinical inflammatory status in RTT, a point certainly in need of further exploration, but well fitting with the known anti-inflammatory properties of ω-3 PUFAs [Moreover, a promising recent line of research suggests that the beneficial actions of -3 PUFAs , 42.On the other hand, a possible explanation for the incomplete rescue of the myocardial dysfunction in RTT could reside in the fact that MeCP2 appears to be involved in myocytes differentiation and maturation . Therefoω-3 PUFAs are able to improve the subclinical biventricular myocardial systolic function observed in typical RTT and that this functional gain is partially mediated through a regulation of the redox balance.Our data indicate that


As such, we have uncovered a novel therapeutic pathway that can be manipulated to block the formation of pathogenic tau species in disease.The accumulation of hyperphosphorylated tau in neurofibrillary tangles (NFTs) is a neuropathological hallmark of tauopathies, including Alzheimer's disease (AD) and chronic traumatic encephalopathy, but effective therapies directly targeting the tau protein are currently lacking. Herein, we describe a novel mechanism in which the acetylation of tau on KXGS motifs inhibits phosphorylation on this same motif, and also prevents tau aggregation. Using a site-specific antibody to detect acetylation of KXGS motifs, we demonstrate that these sites are hypoacetylated in patients with AD, as well as a mouse model of tauopathy, suggesting that loss of acetylation on KXGS motifs renders tau vulnerable to pathogenic insults. Furthermore, we identify histone deacetylase 6 (HDAC6) as the enzyme responsible for the deacetylation of these residues, and provide proof of concept that acute treatment with a selective and blood–brain barrier-permeable HDAC6 inhibitor enhances acetylation and decreases phosphorylation on tau's KXGS motifs  Hyperphosphorylated tau (p-tau) is the primary component of neurofibrillary tangles (NFTs), a pathological hallmark of several neurodegenerative diseases including Alzheimer's disease (AD), frontotemporal dementia with parkinsonism associated with chromosome 17, progressive supranuclear palsy, corticobasal degeneration and chronic traumatic encephalopathy, which has recently gained significant attention due to sports- and military-related injuries . The thr1–42 (Drosophila model of tauopathy (Based on our recent discovery that expression of histone deacetylase 6 (HDAC6) positively correlates with tau burden, while a decrease in HDAC6 activity or expression promotes tau clearance , we furt1–42 , as well1–42 , which h1–42 . HDAC6 l1–42 ,11. In a1–42 , and alsauopathy . Collectin vitro. Furthermore, KXGS motifs are hypoacetylated and hyperphosphorylated in patients with AD, as well as a progressive mouse model of tauopathy (rTg4510), verifying that modulation of this site is linked to disease. Given that tau species phosphorylated on KXGS motifs accumulate in NFTs . While heat-inactivated (h.i.) p300 failed to promote tau acetylation , we demonstrate active p300 catalyzes tau acetylation and prevents tau assembly in a dose-dependent manner . In an effort to identify the key differences between our findings and a previous report .We previously demonstrated that overexpression of HDAC6 promotes the accumulation of p-tau, whereas lowering HDAC6 stimulates p-tau clearance . To gain protein . However protein E and eles report , we testin vitro acetylation reaction with either 4R tau or mutant K259/353R 4R tau, containing a mutation in the lysine residue of the first and last KXGS motifs to prevent acetylation at these sites. As shown in Figure To further investigate the consequence(s) of acetylation on tau, we evaluated the amino acid residues reported to be acetylated by p300, and found all four KXGS motifs in tau's microtubule-binding domain can be modified by acetylation . Given tin vitro acetylation reaction with 4R tau, followed by the addition of cell lysates to the reaction where noted . These results, in combination with our finding that MS275 (Class 1 HDAC inhibitor) does not block the deacetylation of tau on the KIGS motifs, confirm that the effect was due to HDAC6 activity . Given that SIRT1 has recently been implicated as a tau deacetylase , indicating that SIRT1 is not the deacetylase for tau's KIGS motifs.Next, to determine the extent to which HDAC6 regulates the acetylation of tau on the KIGS motifs, we performed the ted Fig. J. A moreted Fig. J. Addingcetylase , we alsoin vitro acetylation reaction with p300 , providing additional evidence that the ability of acetylation to decrease tau polymerization is dependent upon acetylation of the KXGS motifs.To further evaluate the impact of acetylation specifically on KXGS motifs, we introduced either lysine (K) to arginine (R) mutations to prevent acetylation but maintain the charge of the residue or lysine (K) to glutamine (Q) mutations to mimic acetylation on these sites. We thus generated K259/290/321/353 (4KR or 4KQ) mutant proteins, thereby removing all four KXGS motifs. Although acetylation decreased fibrillization of WT full-length tau, acetylation had no effect on filament assembly of the 4KR mutant , inhibiting HDAC6 would be predicted to block phosphorylation of KXGS motifs through increased acetylation.To assess whether phosphorylation similarly blocks acetylation on the KIGS motifs, we performed an ion Fig. D. The dein vivo, we used a selective HDAC6 inhibitor, ACY-738 . As expected, ac-KIGS levels are significantly increased, while 12E8-positive tau levels are significantly decreased in mice treated with ACY-738 . Notably, the ratio of ac-KIGS to 12E8-positive tau species is increased in mice injected with ACY-738 , demonstrating that HDAC6 inhibition modulates this ratio to favor acetylation. Collectively, these results validate that HDAC6 inhibition is an effective strategy to prevent the accumulation of p-tau species , inhibition of HDAC6 is a promising approach to restore the ac-KIGS:12E8 ratio in disease and alleviate tau burden.In order to further assess human disease relevance, we next evaluated the extent to which KIGS motifs are acetylated or phosphorylated in cortical tissue from AD patients compared to control subjects. In agreement with previous work demonstrating that 12E8-positive p-tau species accumulate in NFTs , we obseSupplementary Material, Fig. S4), acetylation of KXGS motifs decreases tau polymerization  and HDAC6 expression enhances phosphorylation of tau at S262/356 (12E8 epitope), an effect that is blocked by mutation of K259/353 to mimic constitutive acetylation . As summarized in Figure The results presented in the current report demonstrate that modulation of the acetylation state of tau's KXGS motifs is the foundation of a dynamic relationship between HDAC6 and tau. Specifically, we show that HDAC6 inhibition increases the acetylation of KXGS motifs , this may indicate that the pattern of acetylation on tau is determined by more than one deacetylase. As such, future studies will be required to map the specific acetylation sites regulated by different deacetylases, in order to determine the ultimate consequence(s) of modulators of deacetylase (HDAC6 versus SIRT1) activity on tau function and biology. Regardless, given that lysine residues are unique in their ability to participate in electrostatic and hydrophobic interactions .ACY-738 was synthesized and provided by Acetylon Pharmaceuticals, Inc. . We purchased the recombinant proteins p300 , PKA  and HDAC6 . We also purchased phosphatase inhibitor cocktails II and III, nicotinamide, TSA, DNAase, chremophor, acetyl CoA, dextran sulfate (MW 6500–10 000 Da), thioflavin S, and Coomassie blue from Sigma-Aldrich . In addition, we purchased dithiothreitol (DTT) and DMSO , as well as PIC and isopropyl 1-thio-Anti-ac-KIGS antibody was generated by immunizing a rabbit with two synthetic, acetylated-tau peptides spanning residues 255 and 266 (NVKS[ac-K]IGSTENL and NVK[pS][ac-K]IGSTENL), to ensure that phosphorylation on S258 would not prevent the ac-KIGS antibody from detecting acetylated K259. The antisera collected from the animal was then immunodepleted for the unmodified peptide, and subsequently affinity-purified . PHF1  was provided by P. Davies . 12E8 (anti–pS262/S356) was provided by P. Seubert . Tau 5  was provided by L. Binder . In addition, we purchased anti-acetyl-lysine (1:1000) and anti-MARK2 (1:1000) from Cell Signaling Technology, Inc. , anti-tubulin (1:4000) was purchased from Sigma-Aldrich, anti-HDAC6 (1:1000) was purchased from EMD Millipore and anti-GAPDH (1:10 000) was purchased from Meridian Life Science, Inc. . E1  was generated by our group against amino acid residues 19–33 within exon 1 of human tau ,20,44. SMARK2/Par1-myc was kindly provided by B. Lu , while GFP and 4R-V5 tau constructs for mammalian expression were cloned into pcDNA3.1 in our lab ,44. A hum isopropyl 1-thio-d-galactopyranoside for 4 h, and the cells were collected by centrifugation and washed in 1× TBS before storage at −80°C. The cell pellets were resuspended in lysis buffer  and lysed with three freeze–thaw cycles using liquid nitrogen and a tepid water bath. These lysates were centrifuged to remove cellular debris, and NaCl was then added to the supernatants at 500 mm. These were heated at 80°C for 10 min, and this was followed by cooling on ice for 10 min. The precipitates were removed by centrifugation, and the supernatant was loaded onto a 5-ml HiTrap Mono S column  using the ÄKTA FPLC system . The column was washed with 10 column volumes of wash buffer , and Tau proteins were eluted with a linear gradient of NaCl at 20 mm/min. Fractions of 1 ml were collected and analyzed by SDS– PAGE and Coomassie blue staining. Fractions containing high concentrations of purified tau protein were pooled and trice dialyzed against 200 volumes of 10 mm HEPES buffer at pH 7.4. Protein concentrations were determined using the BCA Protein Assay Kit .Human cDNAs encoding either 4R0N WT or mutant tau were subcloned into pET-30a bacterial expression vectors (EMD Millipore) and transformed in Rosetta 2(DE3) pLysS competent cells (EMD Millipore) for expression. Overnight cultures were grown to saturation, and these were used at 1/100 to inoculate larger cultures in the morning. The cells were then grown in Luria Broth at 37°C in a shaker rotating at 220 rpm until the OD600 reached 0.5 absorbance units. Tau expression was then induced with 0.5 mm was incubated with 0.5 µg of human recombinant p300 in buffer A  with 125 µm acetyl CoA for 4 h at 30°C . To heat-inactivate p300, the enzyme was heated at 95°C for 10 min. For the PKA phosphorylation reaction, 8 µm of recombinant tau was incubated with 0.3 U of active or h.i. (95°C for 10 min) PKA per pmol of Tau at 37°C for 4 h in buffer A with 2 mm ATP.Tau protein at a final concentration of 8 µg for 15 min at 4°C. Protein concentration was determined by BCA protein assay, an equal amount of protein was added to the acetylation reaction, vehicle (DMSO) or HDAC6 inhibitors were added to the reaction where noted, and samples were incubated at 30°C for 16 h.HEK-293T cells were transfected with human HDAC6 or mock plasmid, and 24 h after transfection were lysed in buffer A, sonicated briefly and centrifuged at 16 000 × To assess deacetylation with recombinant human HDAC6, following the acetylation reaction, different concentrations of HDAC6 were added to the reaction in the presence or absence of vehicle (DSMO) or ACY-738 (where noted). Heat inactivation of HDAC6 was performed by heating the enzyme at 95°C for 10 min. Reactions were incubated at 30°C for 16 h, and analyzed by immunoblot, tau filament assembly and pelleting analysis.m HEPES, pH 7.4, 50 mm NaCl, 1.5 mm MgCl2, 0.5 mm DTT, 2.5 mm EGTA, 0.1 mm EDTA) with 0.04 mg/ml of dextran sulfate to induce assembly. In all experiments described, the tau concentration employed was 8 µm (0.33 mg/ml). To study the effect of acetylation on tau polymerization, dextran sulfate was added to the acetylation reaction, and incubated at 37°C for 4 h before analysis.Tau polymerization was performed at 37°C in buffer A  for 30 min. thioflavin S binding intensity was measured by integrating the curve between the ranges of 460–600 nm using the Cary Eclipse Scan software.Tau filament assembly was monitored by fluorescence of thioflavin S binding using a Cary Varian Eclipse Spectrofluorometer  with an excitation wavelength of 440 nm and a slit width 10 nm, and emission spectrum collected from 460 to 600 nm and a 10 nm slit width. Measurements were performed at room temperature after incubating 30 μl of tau reaction with 90 μl of 10 µg for 75 min at 4°C to separate free and aggregated tau. The pellets containing aggregated and polymerized tau were resuspended in 30 µl 1× sample buffer. Then the pellets were visualized by SDS–PAGE on 10% Tris–glycine gels by Coomassie blue staining.Samples from assembly reactions were centrifuged at 100 000 × g magnification, and the average filament number, average filament length and total filament length per field was measured manually using Image J freeware. The length bar was also measured so these lengths could be converted into nanometers.Reaction samples were diluted 4× in Buffer A, and 10 μl was absorbed onto carbon/formvar-coated 400 mesh copper grids  for 45 s. These were then stained with 2% uranyl acetate (Electron Microscopy Sciences) for 45 s, and the grids were examined with a Philips 208S electron microscope . For quantification, four images were collected randomly at three predetermined coordinate locations that remained fixed throughout these studies. These 12 images were collected at 5000 × HeLa and HEK-293T cells were maintained in Opti-Mem (Life Technologies) supplemented with 10% h.i. FBS (Life Technologies) and 1% PenStrep (Life Technologies) passaged every 3–4 days based on 90% confluency. For plasmid transfections, 2 μg plasmid DNA  was combined with Lipofectamine 2000 reagent (Life Technologies) for 20 min in 500 μl Opti-Mem (serum-free media), and this mixture was added to cells for 4 h. The transfection mixture was then replaced with fresh complete media.m Tris–HCl (pH 7.4), 274 mm NaCl, 5 mm KCl, 5 mm EDTA, 1% Triton X-100, 1 mm PMSF, protease inhibitor cocktail and phosphatase inhibitor cocktails II and III, followed by sonication. Samples were centrifuged at 16 000 × g for 15 min, and a standard BCA Protein Assay Kit (Pierce Biotechnology) performed on the supernatant. Thirty micrograms of protein from each sample were diluted in dH2O, 2× Tris–glycine SDS sample buffer (Life Technologies) and 5% beta-mercaptoethanol (Sigma-Aldrich), and heat-denatured for 5 min at 95°C. Samples were run on 10% or 4–20% SDS–PAGE Tris–glycine gels (Life Technologies), and transferred to PVDF membrane (Millipore). Membranes were blocked in 5% non-fat dry milk in TBS/0.1% Triton X-100, and incubated overnight in primary antibody diluted in 5% milk in TBS/0.1% Triton X-100 rocking at 4°C. Membranes were incubated in HRP-conjugated secondary antibodies  for 1 h at room temperature, and detected by ECL (PerkinElmer). Bands were quantified using Scion Image by analyzing pixel density, and protein levels were normalized to the protein loading control.Cells were harvested in lysis buffer containing 50 mm Tris base, pH 8, 274 mm NaCl, 5 mm KCl, 1% Triton X-100, PIC, 1 mm PMSF, phosphatase inhibitor cocktails II and III, 5 mm nicotinamide and 1 μm trichostatin A), sonicated and centrifuged for 15 min at 16 000 × g at 4°C to remove cellular debris. Supernatants were collected, protein concentration was determined by BCA assay and samples were processed as described above for immunoblotting.Human and mice brains were weighed and homogenized in 10× volume of homogenate buffer (50 mKm), as determined by a titration curve. The enzymatic reaction was monitored over 30 min for the trypsin catalyzed release of 7-amino-4-methoxy-coumarin after deacetylation of the lysine side chain in the peptide substrate and the linear rate of the reaction was calculated.Potency of each compound was determined using recombinant enzymes and a kinetic method described previously . BrieflyAll animal procedures were approved by the Mayo Institutional Animal Care and Use Committee and are in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals .For pharmacokinetic studies with ACY-738, mice were dosed with 10 mg/kg in 0.5% hydroxypropylmethyl cellulose/saline by intraperitoneal injection. Mice were sacrificed at the given times after dosing, and plasma and brain were collected. Compound was extracted and measured by LC/MS/MS using a matrix-matched standard curve to quantitate levels.For the efficacy study, male FVB (Charles River Laboratories) at 2 months of age were injected (s.c.) with vehicle  or 0.5 mg/kg ACY-738 for 3 days, and euthanized by cervical dislocation 1 h after the last injection. Brains were quickly removed, hemisected and frozen on dry ice. The right forebrain was used for biochemical analysis, and the cerebellum was used to measure drug levels . For the aging study, male rTg4510 mice were aged to the indicated time points, euthanized by cervical dislocation, their brains were quickly removed, hemisected and frozen on dry ice. The right forebrain was used for biochemical analysis.Human postmortem brain tissue was provided by the brain bank at Mayo Clinic Jacksonville. For these studies, frontal cortex from six control patients  and six AD patients  was used. The average age of control patients was 86 years, while the average age of AD patients was 70 years. All AD patients were Braak Stage 6.Levels of total tau, ac-KIGS and 12E8 in brain homogenates was measured by a sandwich immunoassay using the Meso Scale Discovery System. Briefly, a 96-well MSD plate was coated with the capture antibody , and incubated overnight. The next day, blocking buffer was added to prevent non-specific binding, the plate washed, and standards (recombinant acetylated/phosphorylated tau) or samples were added to the appropriate wells. Following incubation, the plate was washed prior to adding the SULFO-TAG-labeled detection antibody . Tau levels were detected by adding MSD Read Buffer and reading the light emission at 620 nm after electrochemical stimulation using the MSD Sector Imager 2400.t-tests. Differences among groups with more than two conditions were analyzed using one-way ANOVA, with post hoc analysis using Tukey's multiple comparison tests. P < 0.05 was considered statistically significant.GraphPad Prism was utilized to perform statistical analyses. Differences between two means were assessed with unpaired two-tailed HMG onlineSupplementary Material is available at .Conflict of Interest statement. M.J. is an employee and shareholder of Acetylon Pharmaceuticals Inc., which provided ACY-738 for the studies described. All other authors have nothing to declare.Mayo Clinic Foundation (L.P.), National Institutes of Health/National Institute on Aging [5R01AG026251-04 (L.P.) and AG17216-10JP3 (L.P.)], National Institutes of Health/National Institute of Neurological Disorders and Stroke , BrightFocus FoundationA2013546S (L.P.), Sponsored Research – Acetylon Pharmaceuticals Inc. (L.P.), ADRC AG016574 . Funding to pay the Open Access publication charges for this article was provided by Mayo Clinic.This work was supported by


Among the plants studied, Euonymus sachalinensis, Rhododendron schlippenbachii, Astilbe chinensis and Juglans regia showed the strongest α-glucosidase inhibitory activity with IC50 values of 10, 20, 30 and 80 µg/mL, respectively. In addition, Meliosma oldhamii and Symplocos chinensis showed moderate α-glucosidase inhibition with IC50 values of 150 and 220 µg/mL, respectively. Therefore, they might prove to be a potential natural source for the treatment of metabolic ailments such as, diabetes, and need further investigations.Glycosidases are the enzymes involved in various biochemical processes related to metabolic disorders and diseases. Therefore, much effort has been focused on searching novel pharmacotherapy for the treatment of these ailments from medicinal plants due to higher safety margins. To pursue these efforts, the present study was performed to evaluate the  TheseDiabetes mellitus (DM) is a metabolic disorder characterized by chronic hyperglycemia that has a significant impact on the health, quality of life and life expectancy of patients, as well as health care system , 4. In cThus, many plants and crude drugs have recently been tested for their effects on α-glucosidase inhibition.To pursue the findings, in this research, we screened thirty Korean medicinal plants. The details of the plants’ scientific names and families are listed in Plant materialThe dried and matured plant parts of thirty Korean medicinal herbs were obtained from “Korean Collection of Herbal Extracts” a Biotech company in Korea. A collection of voucher specimen is available with the company .ExtractionThe dried plant parts individually were chopped into small pieces and pulverized into a fine powder. The powdered plant materials  were kept for extensive decoction in 80% methanol for 3 days at room temperature. The extracts were then concentrated using rotary vacuum evaporator at 20-30°C to obtain the dried crude extracts.Reagentsα-Glucosidase (from Saccharomyces cerevisiae type I) and 4-nitrophenyl α-D-glucopyranoside were purchased from Sigma-Aldrich . Other commercially available reagents and solvents were used as received.α-Glucosidase assayThe enzyme inhibition activity for α-glucosidase was evaluated according to the method previously reported by Shibano et al.  with minControl - ΔASample) / ΔAControlI α-glucosidase% = 100 X (ΔAControl = ΔATest - ΔABlankΔASample = ΔATest - ΔABlankΔAStatistical analyses50 values were only determined for the plant extracts with inhibition ≥ 50% at 5 mg/mL by plotting a percent inhibition versus concentration curve, in which the concentration of sample required for 50% inhibition was determined and expressed as IC50 value.All assays were performed at least three times with triplicate samples. All results are expressed as mean ± SD. ICPlants have always been used as an exemplary source of drugs and many of the currently available drugs have been directly or indirectly derived from them . Many heIn this study, the α-glucosidase inhibitory activity of the thirty plant extracts was evaluated at 5 mg/mL concentration at the preliminary level and the percentage inhibitions are shown in 50 values of 10 ± 1, 20 ± 1, 30 ± 2, 80 ± 2, 150 ± 3 and 220 ± 5 µg/mL, respectively. The traditional uses of these plants are listed in α-glucosidase inhibitory potency.The present data revealed that, six plant extracts demonstrated α-glucosidase inhibition ≥ 50%, namely, Euonymus sachalinensis, Rhododendron schlippenbachii, Astilbe chinensis, Juglans regia, Meliosma oldhamii and Symplocos chinensis with ICα-glucosidase inhibitory activity (In addition, in the present screening, eight plant extracts showed medium activity, ranging from 25 to 49%, 13 plant extracts showed less than 25% inhibition, and 3 plant extracts did not exhibit activity .α-glucosidase inhibitory potential of the identified potent crude extracts was lower as compared to PGG (Euonymus sachalinensis, Rhododendron schlippenbachii, Astilbe chinensis, Juglans regia, Meliosma oldhamii and Symplocos chinensis seem to be relatively potent inhibitors, where the inhibitory activity could further be improved by separation and purification of the active components.The d to PGG , since cα-glycosidase inhibition plays a key role, may prove fortune. However, further in-vivostudies needed to be confirmed to provide strong biochemical rationale.Thus, introduction of such innovative herbal extracts for the treatment of diabetes and other related metabolic disorders, where the


Total body irradiation (TBI) combined with chemotherapy is widely used as a pretreatment regimen of bone marrow transplantation (BMT) in hematologic disorders. Late complications related to TBI as part of the conditioning regimen for hematopoietic stem cell transplantation have been revealed. Acute transverse myelitis (ATM) is a neurological syndrome characterized by disorder of motor, sensorial, and autonomic nerves, and tracts at medulla spinalis, which is resulted from involvement of spinal cord. In this paper, we presented an ATM attack developed after TBI in a patient with acute lymphoblastic leukemia (ALL) as it is a rarely seen case. With the increasing use of bone marrow transplantation (BMT) for hematologic malignancies, total body irradiation (TBI) is becoming a more commonplace procedure. The goal from using TBI in the preparation regimens for BMT is threefold: destroying residual neoplastic cells, clearing the host marrow to allow repopulation with donor marrow cells, and providing sufficient immunosuppression to avoid allograft rejection by immunologically active cells in the host . Known a9/L, and platelet count (Plt), 354 × 109/L. Philadelphia (Ph) (+) chronic, stable-phase chronic myeloid leukemia (CML) diagnosis was made by peripheral blood smear and bone marrow evaluations including morphological and genetic studies. After cytoreduction by hydroxyurea, he was given 400 mg/day imatinib therapy; however, there was loss of hematological response for three times during 2004–2009 period; thus, his treatment was converted to dasatinib therapy at 2009. At 2010, dasatinib 1 × 70 mg plus induction chemotherapy protocol was given to Ph (+) patient with common ALL antigen (CALLA) (+) pre-B ALL: methotrexate 15 mg per day intrathecally on day 1, vincristine 2 mg per day intravenously on days 4, 11, and 18, dexamethasone 10 mg/m2 per day intravenously on days 4, 5, 11, 12, 13, and 14, daunorubicin 45 mg/m2 per day via intravenous infusion on days 4, 5, 11, and 12, and L-asparaginase 500 u/m2 per day via intravenous infusion over 2 h on day 18. As the consolidation therapy, the patient received methotrexate 1350 mg/m2 per day via intravenous infusion over 24 hours on day 1, cytarabine 2000 mg/m2 per day via intravenous infusion over 12 hours on day 5, vincristine 2 mg per day via intravenous route on day 1, dexamethasone 10 mg/m2 per day via intravenous route on days 1–5, etoposide 250 mg/m2 per day via intravenous infusion over 1 hour on days 4-5, and methotrexate 15 mg per day via intrathecal route on day 1. Allogeneic hematopoietic cell transplantation  decision was taken for patient who had bone marrow remission following remission induction and consolidation therapies and 10/10 fully compatible unrelated donor. According to allo-HCT preparation protocol, TBI  followed by 60 mg/kg/day over 2 days cyclophosphamide regime were planned; however, he reported that he could not move his arms on the day 3 of TBI therapy. He had no symptom other than neurological complaint. There was no abnormal finding in physical examination, but 3/5 weakness was detected in both upper extremities in neurologic examination, as being more prominent in distal. No pathological reflex was detected. Deep tendon reflexes were hypoactive at upper extremities, but there was no sensorial defect. Thus, TBI was discontinued and 1 mg/kg prednisolone intravenous treatment was initiated. Cerebrospinal fluid evaluation was negative for malignancy or infectious pathogens. On the cervical magnetic resonance imaging (MRI), there were T1-weighted isointense and T2-weighted hyperintense pathological signal changes, which begin at C2 level, involving all cervical spinal cord and extends to thoracic segments and expansion of spinal cord , 8.5 g/dL, white blood cells (WBC), 69.9 × 10nal cord . By thesnal cord . SteroidNo complication other than neutropenic fever was observed during transplantation period. On day 35, graft-versus-host-disease (GVHD) was developed involving skin (Grade I) and liver (Grade I). Thus, cyclosporine and steroid therapy was initiated. On day 91, chimerism result was found as 100% in the patient on dasatinib therapy (1 × 100 mg). The patient is being followed with hematological and molecular remission (Philadelphia PCR: negative). TBI is a complex technique used for treating hematologic diseases. The role of TBI is to destroy the recipient's bone marrow and tumor cells and to immunosuppress the patient sufficiently to avoid rejection of the donor's bone marrow transplant . Radiati


Benzalkonium chloride (BAK), the most commonly used preservative in eye drops, is known to induce ocular irritation symptoms and dry eye in long-term treated patients and animal models. As tear film hyperosmolarity is diagnostic of some types of dry eye disease, we determined in vitro on conjunctival epithelial cells the cytoxicity of BAK in hyperosmolar conditions through cell viability, apoptosis, and oxidative stress assays.−4%, 3.10−4%, and 5.10−4%), or in combination of both. We investigated cell viability through lysosomal integrity evaluation, cell death (cell membrane permeability and chromatin condensation), and oxidative stress  using spectrofluorimetry. Immunohistochemistry was performed for cytoskeleton shrinkage , mitochondrial permeability transition pore (cytochrome c release), the apoptosis effector active caspase-3, and the caspase-independent apoptosis factor AIF. We also observed early effects induced by the experimental conditions on the conjunctival cell layers using phase contrast imaging of live cells.The Wong Kilbourne derivative of Chang conjunctival epithelial cells were cultured for 24 h or 48 h either in NaCl-induced hyperosmolar conditions (400–425–500 mOsM), in low concentrations of BAK (10As compared to standard culture solutions, hyperosmolar stress potentiated BAK cytotoxicity on conjunctival cells through the induction of oxidative stress; reduction of cell viability; cell membrane permeability increase; cell shrinkage with cell blebbing, as shown in phase contrast imaging of live cells; and chromatin condensation. Like BAK, but to a much lesser extent, hyperosmolarity increased cell death in a concentration-dependent manner through a caspase-dependent apoptosis characterized by a release of cytochrome c in the cytoplasm from mitochondria and the activation of caspase-3. Moreover, the caspase-independent apoptosis factor AIF was found translocated from mitochondria to the nucleus in both conditions.This study showed increased cytotoxic effects of BAK in hyperosmotic conditions, with characteristic cell death processes, namely caspase-dependent and independent apoptosis and oxidative stress. As BAK is known to disrupt tear film, which could promote evaporative dry eye and tear hyperosmolarity, BAK could promote the conditions enhancing its own cytotoxicity. This in vitro hyperosmolarity model thus highlights the risk of inducing a vicious cycle and the importance of avoiding BAK in patients with dry eye conditions. The ocular surface is the most environmentally exposed mucosal surface of the body, protected only by the lids and tear film. The production and turnover of the tear functional unit are essential for maintaining the ocular surface in good health and efficiently preventing foreign particles, pathogens, allergens, or irritants from entering or injuring the eye. Dry eye is a multifactorial worldwide syndrome affecting one million people, with a prevalence in the general population over 50 years of 3%–15% -4. Dry eTo understand the mechanisms leading to or induced by dry eye disease, many experimental models have been developed in animals and cell lines. Mice, rats, rabbits, and even dogs have been investigated -15. PrimXiong et al.  and Lin The aim of the present study was to investigate the relationships between hyperosmolarity and BAK in vitro through their respective and combined proapoptotic and pro-oxidative effects on conjunctival epithelial cells, to determine whether BAK toxicity would be enhanced in hyperosmotic conditions, mimicking a frequent clinical condition in which dry eye patients receive artificial tears preserved with a chemical compound that may both promote and be potentiated by tear hyperosmolarity.2, 37 °C) in Dulbecco minimum essential medium  supplemented with 10% fetal bovine serum (FBS), 1% glutamine (200 mM stock solution), and 1% penicillin  and streptomycin  for 24h to reach confluence before challenges. This cell line has been used previously for toxicological in vitro studies despite the presence of a small amount of HeLa cells. A toxicological study aimed at comparing Wong Kilbourne cell line and IOBA conjunctival cell line confirmed that the two cell lines presented the same toxic responses [The Wong Kilbourne derivative of Chang conjunctival epithelial cell line  was cultured under standard conditions .−4%, 3.10−4%, or 5.10−4% (345 mOsM±5 mOsM); or (3) in a combination of hyperosmolarity and BAK, by crossing the three osmotic stresses with the three BAK concentrations.At confluence, cells were grown for 24 or 48 h: (1) in hyperosmolar media defined by different osmolarities, assessed using an osmometer , in accordance with previous studies ,18,20, r−2%, and a combination of HO90 mM and BAK10−2%, by adding 100 µl of HO900 mM, BAK10−1%, and HO900 mM associated with BAK10−1% solutions.Cells were cultured for 24 h on 0.17-mm-diameter Delta TPG uncoated culture dishes , then monitored at the microscope stage  every 5 s with a charge-coupled device (CCD) camera . Videos highlighted morphological changes of cell-size decrease, membrane blebbing, formation of apoptotic bodies, and cell detachment. Cells were first observed in 1 ml DMEM before being exposed for 96 s to the most challenging stresses, HO90 mM, BAK105 cells/ml in 96-well microplates  for 24 h incubation to reach confluence in standard medium before cell challenges for 24–48 h in hyperosmolar or toxic conditions. The neutral red uptake (NRU), YO-PRO-1, Hoechst 33342, 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA), and dihydroethidine (HE) assay methods were previously described and will be briefly explained [Cells were first seeded at 10xplained . Fluores2O) under agitation, and then fluorescence was measured .Viable cells incorporate NR (Fluka), a cationic dye that diffuses through the cell membrane to bind the lysosomal matrix. This lysosomal membrane integrity is correlated with CV. After challenges, cells were incubated in 200 µl of 50 µg/ml NR in DMEM for 3 h in standard conditions, washed in PBS , incubated for 15 min in a lysis solution (1% acetic acid/50% ethanol/49% HYO-PRO-1 (Fluka) binds DNA after opening specific membrane pore P2X7 (purinergic receptor) activated during early apoptosis through extracellular ATP (ATP) , making Caspase-3 activation was detected using the Fluorometric Assay Kit following the manufacturer’s instructions . Absorbance from cells with and without 24 h treatment was compared to determine relative caspase activation.Hoechst 33342 (Invitrogen) is a UV fluorescent probe used to evaluate cell chromatin condensation entering both apoptotic and living cells. PI is used to discriminate necrotic cells since this dye competes with Hoechst for binding to DNA. After challenges, 200 µl of a solution containing Hoechst 33342 (0.5 µg/ml) and PI (0.05 µg/ml) in PBS was added for a 30 min incubation at RT, in the dark before fluorometry analysis .2−. from H2O2. After challenges, 200 µl of a 5 µM (1.58 µg/ml) HE solution in PBS or a 20 µM (9.75 µg/ml) H2DCF-DA solution was added for a 20 min incubation at 37 °C in moist atmosphere with 5% CO2 before fluorometry analysis.Oxidative stress was assessed by reactive oxygen species (ROS) evaluation. Two different fluorometric methods were used: H2DCF-DA  detects hydroperoxide in cells , whereas HE (Interchim) detects superoxide anion in viable cells  distinguishing O® 488  before a 30 min incubation with phalloidin  and a 15 min incubation with DAPI . Cover glasses were then mounted with Mountant PermaFluor®  before observation with an epifluorescence microscope .Caspase-dependent and -independent apoptosis was explored by highlighting specific markers . Cells were grown on round sterile cover glasses , washed, fixed in 4% paraformaldehyde-PBS, and permeabilized in 0.3% Triton X-100 . This was followed by a 1% BSA  incubation for 30 min and a 1 h staining with the primary antibodies against AIF, cytochrome c , and active caspase-3 . The cells were then incubated for 1 with the secondary antibody Alexa FluorAll experiments were performed in triplicate, and the groups were compared using one-way ANOVA (ANOVA) followed by the Bonferroni adjustment .WKD cells exhibited morphological changes  with a c−4%, 3.10−4%, 5.10−4%; (4) HO60 mM with BAK10−4%, 3.10−4%, 5.10−4%; and (5) HO90 mM with BAK10−4%, 3.10−4%, 5.10−4%.These assays are illustrated in Cell viability was assessed at 24 h and 48 h following different challenges, hyperosmolarity, BAK and the associations of both conditions.−4%, which induced a slight CV increase (p<0.001). Incubation with BAK3.10−4% or BAK5.10−4% for 24 h decreased CV compared to DMEM (p<0.001). Whatever the concentration tested, BAK always decreased CV more than any HO condition alone. In HO associated with BAK, CV was further reduced compared to BAK alone, causing a significant cytotoxicity dependent on both HO and BAK concentrations. All BAK10−4% combinations were significantly more cytotoxic (p<0.001) than BAK10−4% alone. BAK3.10−4% combinations with HO50 mM, HO60 mM, or HO90 mM were significantly more cytotoxic (p<0.001) than BAK3.10−4% alone (p<0.001), but BAK5.10−4% combinations with all HO combinations did not differ from BAK5.10−4%, which was too cytotoxic for an additive effect to be observed.At 24 h, compared to DMEM, incubations in HO, BAK, and combinations induced a CV decrease (p<0.001), except for BAK10−4% combinations with HO60 mM and HO90 mM. All conditions were statistically different (p<0.001) from the 100% DMEM control.At 48 h, with a similar cytotoxicity profile, incubation in the same conditions  further induced a time-dependent CV decrease compared to 24 h. All combinations induced additive effects (p<0.001) compared to BAK solutions except for BAK 5.10We assessed apoptosis pathway at 24 h and 48 h using PMP assay in our experimental conditions, hyperosmolarity, BAK, and the associations of both.−4%, which induced a nonsignificant increase. Combinations of all BAK concentrations with HO solutions were statistically different (p<0.001) from their respective BAK concentrations alone, except for BAK5.10−4% combinations, in which no further cytotoxicity could be observed compared to BAK5.10−4% alone.At 24 h, compared to DMEM, incubation for 24 h in HO, BAK, and combinations induced a PMP increase (p<0.001), except for HO50 mM and BAK104% and BAK5.10−4% with all HO did not differ from the respective BAK solutions, as cytotoxicity was too high and the stress alone could not be distinguished from its combinations. All conditions differed (p<0.001) from isotonic DMEM except for BAK 10−4%, which still induced a nonsignificant increase at 48 h.At 48 h, incubations in the same conditions  induced a time-dependent increase in YO-PRO-1 incorporation compared to 24 h. Combinations of BAK3.10We evaluated the involvement of caspase-3 activation at 24 h to assess the caspase-dependent apoptosis pathway that could be stimulated by our different challenge.−4%, and combination of BAK3.10−4% with HO90 mM induced an increase (p<0.01) in caspase 3 activity of 1.99×, 3.17×, and 9.00×, respectively, compared to control.HO90mM, BAK3.10A late apoptosis process was evaluated at 24 h and 48 h using Hoechst 33342 to better-characterized apoptosis following different challenges, hyperosmolar, BAK and the association of both conditions.−4%, BAK3.10−4%, and for combinations of BAK10−4% in HO conditions. No combinations of BAK3.10−4% and BAK5.10−4% with HO50 mM, HO60 mM, and HO90 mM differed.At 24 h, compared to DMEM, incubations with HO, BAK, and combinations induced an increase (p<0.001) in fluorescence levels except for HO50 mM, BAK10−4%, BAK10−4% with HO50 mM, and HO60mM, which induced a nonsignificant increase in chromatin condensation at 48 h. No BAK5.10−4% combinations differed from the BAK solution alone because it was already too toxic for further toxicity to be observed at 48 h.At 48 h, incubations in the same conditions  induced a further increase in chromatin condensation compared to 24 h, higher in presence of BAK, and in associations with BAK than with HO solutions alone. All conditions differed (p<0.001) from DMEM for HO conditions except with BAK10As BAK is known as an inducer of oxidative stress, we wanted to evaluate at 24 h and 48 h the effects of hyperosmolarity alone and its association with BAK on this production.−4%, the combination of any HO with BAK3.10−4% or BAK5.10−4% induced a superoxide anion increase, BAK3.10−4% showing an increase only at 48 h (p<0.001). At 48 h, all HO conditions combined with BAK3.10−4% or BAK5.10−4% differed from their respective BAK and HO conditions (p<0.001), except for BAK5.10−4% combined with HO90 mM, which did not differ from BAK5.10−4%.Incubations for 24 h and 48 h induced a time-dependent increase of fluorescence. Compared to control, 24 h and 48 h incubations in HO60 mM, HO90 mM, BAK5.10−4% with HO60 mM. At 24 h and 48 h, compared to control, incubation in BAK5.10−4% and the combination of any HO with BAK3.10−4% and BAK5.10−4% induced a superoxide anion increase (p<0.001), except for HO50 mM and HO60 mM associated with BAK3.10−4% at 24 h. HO combinations with BAK3.10−4% or BAK5.10−4% were more cytotoxic (p<0.001) than their respective BAK and HO conditions, except for HO50 mM associated with BAK3.10−4% at 24 h where there was no difference from BAK3.10−4%. BAK10−4% alone or combined with HO did not differ from DMEM.Incubations for 24 h and 48 h induced a time-dependent fluorescence increase, higher at 48 h except for BAK5.10Hyperosmolarity, BAK, and both challenges combined induced caspase-dependent apoptosis. Cytochrome c was released from mitochondria  to the cHyperosmolarity induced the disruption of F-actin cytoskeleton, the decrease of several elongated bundles, and the disappearance of a ring-like structure  and alteThis study simulated various conditions encountered by patients having dry eye by studying the effects of hyperosmotic stress, separately or in combination with BAK, a commonly used eyedrop preservative, to test the hypothesis that BAK administered in an eye already submitted to hyperosmolar conditions would be more toxic than in a healthy, normal ocular surface. We therefore investigated conjunctival cell survival, apoptosis, and oxidative stress in these two conditions because BAK, whose cytotoxicity has been largely demonstrated , is stilWe used NaCl to obtain hyperosmolarity ranging from 430 to 500 mOsM, corresponding to high osmolarities compared to normal tear osmolarity (300.8±7.80 mOsM) or even dry eye tear osmolarity (343±32 mOsM) ,46 to ev−4% to 5.10−4%. BAK10−4% corresponds to the 1/100 dilution of the 0.01% concentration found in most eyedrops and to a noncytotoxic BAK concentration, as previously reported in similar in vitro models [−4% and BAK5.10−4% were chosen here to evaluate the impact of cytotoxic BAK concentrations, giving, respectively, 50% and 80% cell death as assessed with the CV assay. As BAK10−4% and BAK5.10−4% displayed extreme cytotoxic effects from very mild to very strong, respectively, we observed the most relevant findings with the intermediate BAK concentration of 3.10−4%.We tested three BAK concentrations from 10o models ,29,48,49−4%, exhibited increased toxic effects with all hyperosmolar solutions, compared to a normal osmolarity condition, showing hyperosmolarity-dependent cytotoxicity.Conjunctival epithelial cells were sensitive to hyperosmolarity in an osmolarity-dependent manner, but low hyperosmolarity caused limited changes, showing a relative capacity of cells to adapt to hyperosmolar conditions. Under higher hyperosmolar stress or associated toxic factors such as BAK, cells degenerated through an apoptotic mechanism associated with oxidative stress. Hyperosmolarity also induced a time- and dose-dependent increase of PMP. Interestingly, we observed additive cytotoxic effects of BAK and HO. For example, when added to cells submitted to HO90 mM, the lowest BAK concentration became highly cytotoxic. The intermediate concentration, BAK3.10BAK is known as an inducer of oxidative stress and hyperosmolarity was also shown in some in vitro  and in vWe also confirmed the involvement of two major apoptotic pathways, a caspase-dependent one, characterized by cytochrome c release from the mitochondria to cytoplasm and presence of the active caspase-3 and a caspase-independent one through the translocation of AIF from the mitochondria to the nucleus where AIF binds to DNA and leads to chromatin condensation and cell death ,53. HereThese results support the hypothesis and the frequent clinical observation that BAK, as a preservative contained in many eyedrops, when administered to a patient having dry eye, could dramatically affect conjunctival cells and maintain or aggravate ocular surface impairment. This model is a simple, reproducible in vitro model of NaCl-induced hyperosmolarity on a widely used conjunctival cell line, as already used in similar corneal models ,17,19,22In conclusion, both hyperosmolarity and BAK induce apoptosis via similar mechanisms, involving caspase-dependent and -independent pathways but with a much higher resistance to hyperosmolarity than to toxic stress. The noteworthy findings of this study are that the combination of a noncytotoxic challenge (hyperosmolarity) and a subtoxic stress (low preservative concentration) can induce substantial cell alterations and cell death, much greater than after each separated stress. This is important from a toxicology point of view, and this model could be valuable for testing the effects of many other non- or minimally cytotoxic compounds such as environmental pollutants or xenobiotics, alone or in combination with a condition mimicking dry eye. Concerning ocular surface diseases, this study highlights the importance of avoiding preservatives like BAK even at low concentrations in a dry eye patient because cytotoxic effects of BAK will act synergistically with hyperosmolarity.More broadly, the extensive use of BAK over the long-term, as in glaucoma, may progressively cause tear film instability that will enhance hyperosmolarity-induced responses. They were recently reported to increase in several BAK-containing medications . Hence,


Vibrio anguillarum is an important bacterial pathogen that causes vibriosis in marine fish. We present the complete genome sequence of V. anguillarum M3, a serotype O1 clinical strain isolated from Japanese flounder  in Shandong, China. Vibrio anguillarum causes vibriosis, a systemic disease of both wild and cultured marine fish characterized by hemorrhagic septicemia , striped bass (Morone saxatilis), and turbot , were released   . This stormation –7.V. anguillarum M3 was sequenced using the Roche 454 GS FLX Titanium system at SeqWrite, Inc. . A total of 23,745 reads were obtained from the sequencing (~20-fold coverage of the genome). The reads were assembled using DataAnalysis version 2.6 (Roche/454 Life Sciences), generating 59 contigs. Using the genome of V. anguillarum strain 775 as a reference, all gaps were filled by local assembly using the CLC Genomics Workbench (64-bit) 6.0.5 and ABI 3730 sequencing of the PCR products of the gap region. The coding sequences (CDSs) were predicted by the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) and the Rapid Annotations using Subsystems Technology (RAST) server (The genome of ) server . Noncodi) server  by searc) server . Functio) server  and the ) server .V. anguillarum M3 contains two circular chromosomes and a plasmid. Chromosome 1 has 3,063,587 bp containing 2,842 CDSs, chromosome 2 has 988,134 bp containing 918 CDSs, and the plasmid has 66,164 bp containing 64 CDSs. Overall, the M3 and 775 genomes share high levels of similarity, as >90% of the CDSs have the same annotation, length, and relative position. With respect to potential virulence traits, a majority of the virulence genes are conserved in both strains, such as those for hemolysins, metalloproteases, repeat in toxin (RTX) toxins, and vibriolysin (V. anguillarum and will contribute new insights into virulence studies of this particular species.The complete genome of riolysin . Both striolysin . In addiV. anguillarum M3 genome have been deposited in GenBank under accession no. CP006699 (M3 chromosome 1), CP006700 (M3 chromosome 2), and CP006701 (M3 plasmid).The sequence and annotation of the


Bacillus and Clostridium. Batch fermentation studies demonstrated that isolates had the ability to ferment glucose, xylose, or glycerol to industrially valuable products such as ethanol and 1,3-propanediol (PDO). Ethanol was detected as the major fermentation end product in glucose-fermenting cultures at pH 10 with yields of 0.205–0.304 g of ethanol/g of glucose. While a xylose-fermenting strain yielded 0.189 g of ethanol/g of xylose and 0.585 g of acetic acid/g of xylose at the end of fermentation. At pH 7, glycerol-fermenting isolates produced PDO (0.323–0.458 g of PDO/g of glycerol) and ethanol (0.284–0.350 g of ethanol/g of glycerol) as major end products while acetic acid and succinic acid were identified as minor by-products in fermentation broths. These results suggest that the deep biosphere of the former Homestake gold mine harbors bacterial strains which could be used in bio-based production of ethanol and PDO.Eight fermentative bacterial strains were isolated from mixed enrichment cultures of a composite soil sample collected at 1.34 km depth from the former Homestake gold mine in Lead, SD, USA. Phylogenetic analysis of their 16S rRNA gene sequences revealed that these isolates were affiliated with the phylum Firmicutes belonging to genera  Saccharomyces cerevisiae and Zymomonas mobilis that perform fermentation only under acidic pH (5.0) conditions . Considering these limitations, we have isolated glucose-, xylose-, or glycerol-fermenting bacteria from the extreme deep biosphere environment of the former Homestake gold mine, Lead, SD. There are very few reports on subsurface isolates utilizing glucose for ethanol production  is the deepest mine (2.4 km deep) in North America and had the largest gold deposits ever found in the Western Hemisphere , 0.05 g CaCl2·2H2O, 0.1 g MgSO4·7H2O, 0.005 g methionine, 1.8 g of 85% H3PO4, 0.05 g yeast extract, 0.01 g casamino acids, 0.01 g KCl, 0.3 g NH4Cl, and 1 ml of Nitsch's trace element solution 2SO4, 40 g glycerol, and 1 g yeast extract, and the pH of the medium was adjusted to 7. The glycerol fermentation medium was derived from the study of Zheng et al. (The glucose and xylose fermentation medium contained (per liter): 0.1 g nitriloacetic acid, 1 ml FeClg et al. .2(g) for 15 min to remove the dissolved and head space oxygen for creating anaerobic conditions  for 6 days. Samples were withdrawn periodically and analyzed for soluble intermediates and end products. Samples were prepared for high-pressure liquid chromatography (HPLC) by centrifuging 1-ml cultures at 10,000 rpm, and the resulting supernatant was further filtered using 0.2 μm pore size membrane filters (Gelman Acrodisc). HPLC employed a 300 mm Aminex HPX 87H column  on a HP 1100 Series HPLC system equipped with a refractive index detector . Samples (10 μl) were injected onto a heated column (65°C) and eluted at 0.6 ml/min using 5 mM HBacterial growth rate is a time-dependent variable in cultures. In each sample discrete growth rate measurements were calculated for each time step. Equation 1 was used to obtain each discrete growth rate datum point as described earlier  and “Xi” is the measured cell mass concentration (g/L). The “i” subscript is a counter variable for each time step. Similarly, “X0” represents cell concentration at time t = 0. The variable “t” is time (hour). The arithmetic mean of data obtained was then calculated and represented as specific growth rate, “μ”.In Equation 1 “μYield coefficients (g of biomass [X] or product [P] formed per g of substrate [S] consumed) were defined based on the amount of consumption of xylose, glucose, or glycerol. Growth yields were calculated from Equation 2.Product yields on substrate were calculated from Equation 3.Firmicutes and grouped within the Clostridiaceae and Bacillaceae families. A majority of isolates (7 out of 8) were closely related to Clostridium sp. except a single isolate (DUSELGlu2) that closely grouped with sequences from Bacillus sp. These findings were in agreement with earlier studies from the Homestake mine, where Clostridium sp. has been shown to be the most predominant member (up to 62.3%) followed by Bacillus sp. (32.7%) in cellulose-degrading enrichment cultures , acids , and PDO  was detected as the primary product at the end of fermentation with small amounts of acetic acid , and lactic acid . Interestingly, the concentration of lactic acid dropped below the limit of detection at the end of fermentation in DUSELGlu1 and DUSELGlu4 strains, while in DUSELGlu2 strain, it turned out to be the third fermentation product along with ethanol and acetic acid. All three glucose-fermenting isolates showed similar ethanol yields and specific growth rates Tables , the yieE. coli, Klebsiella oxytoca, and Zymomonas mobilis have been shown to produce very high amounts (23–63 g/L) of ethanol from xylose and glucose fermentations . Molecular characterization of deep-mine strains using techniques such as DNA–DNA hybridization or using other phylogenetic marker genes are also needed. Further elucidation of metabolic pathways leading to the formation of ethanol, acetic acid, or PDO will be desirable for the construction of recombinant strains for industrial applications.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.